ALBERT

Notes: Summary Background: The knowledge of magnesium requirements of premature infants is still very limited, although it is essential for the optimal composition of suitable formulas. Aim of the study: The study concept was 1) to assess physiological magnesium balance data of healthy term infants and longitudinal results from formula-fed premature infants and 2) to deduce conclusions on the magnesium content of the formulas. Methods: Premature infants (n = 14, birth weight ≤ 1500 g, gestational age ≤ 32 weeks) were studied in conventional balance trials with 1) a semi-elemental diet (A), 2) preterm infant formula (B), and 3) infant formula (C). In addition, healthy term formula-fed (n = 11, D) and breast-fed (n = 14, E) infants were investigated. Analysis was performed by flame atomic absorption spectroscopy. Results: The median magnesium intake ranged between 4.84 mg/kg × d−1 (breast-fed infants) and 16.33 mg/kg × d−1 (premature infants). The term breast-fed infants retained nearly as much magnesium as term formula-fed infants (3.37 vs. 3.97 mg/kg × d−1), due to a low percental fecal and urinary excretion. A higher magnesium retention was observed in the premature group: A: 7.97 mg/kg × d−1, B. 5.3 mg/kg × d−1, 3.) 5.54 mg/kg × d−1. Conclusion: In view of the high percental magnesium retention in formula-fed premature infants, excessive supply should be avoided. The long-term effects of lower intakes have to be monitored.

Notes: Summary We reviewed 15 studies reporting on the fatty acid composition of colostrum lipids from 16 geographic regions: 11 European studies and one study each from Central America, the Caribbean, Australia and Asia. The contents of essential fatty acids, saturates and polyunsaturates were similar in the southern European countries Spain, Slovenia and France. Colostrum of St. Lucian women was high in saturates and low in oleic acid, reflecting a high-carbohydrate, low-fat diet. Abundant fish intake was reflected in high contents of docosahexaenoic acid and total n-3 long-chain polyunsaturated fatty acids in St. Lucia. Two French studies published with an interval of two years showed a very similar colostrum fatty acid composition, whereas two German studies obtained with an interval of 14 years showed higher docosahexaenoic acid and arachidonic acid contents in the later study, with an unchanged n-6/n-3 long-chain polyunsaturated fatty acid ratio. Studies from Spain reported a decline of α-linolenic acid in colostrum over a time period of 13 years. Colostrum of Australian women contained the lowest polyunsaturated/saturated and n-6/n-3 long-chain polyunsaturated fatty acids ratios (0.28 and 1.58) and the lowest contents of linoleic and α-linolenic acids (7.8 and 0.4 wt. %). In contrast, the contents of docosahexaenoic acid, eicosapentaenoic acid and total n-3 long-chain polyunsaturated fatty acids (0.6, 0.4 and 1.4 wt. %) were higher in Australian than in European samples. Fatty acid composition of human colostrum appears to be markedly influenced by geographic differences in maternal dietary composition.

Notes: Summary The metabolic strengths, weakness, opportunities and threats of the metabolic ability to split water brought about a proliferation of biological systems, produced a toxic oxygenic environment, and were responsible for the development of antioxidant defence mechanisms. Evolution is driven by heritable adaptions which improve environmental ‘fit’. Hence aerobic respiration, using oxygen as a nutrient, came to predominate in biological systems, and antioxidant defence mechanisms which prevent and neutralise toxic oxygen intermediates have become widespread, varied, coordinated and effective. Antioxidant defences are nor infallible however. In humans, reactive oxygen species-induced damage is associated with the ageing process, and with chronic diseases including cancer and coronary heart disease. Interestingly, some important antioxidants, including ascorbic acid and the tocopherols, cannot be synthesised by humans and must be taen in the diet. Another antioxidant, uric acid, is found in much higher concentrations in humans than in other mammals, and levels are also affected by diet. In humans, therefore, antioxidant defence against toxic oxygen intermediates is species specific and heavily influenced by nutrition. In this article, the atmospheric and metabolic changes which produced both the threat and opportunity offered by an oxygenic environment are outlined. An overview of oxygen toxicity, and adaptations to oxidative stress in terms of evolution of antioxidant defences, is presented. Finally, suggested benefits underlying our curious inability to manufacture ascorbic acid, and the possible role of uric acid in human antioxidant defence, are briefly discussed with particular reference to nutrition and toxicology.

Notes: Summary Background: Many epidemiological studies have identified a protection against cancer associated with consumption of fruit and vegetables. One factor is this protection may be the enhancement of cellular DNA repair activity by micronutrients, such as carotenoids, found in these foods. Aims of the study: To measure the capacity of lymphocytes isolated from volunteers supplemented with β-carotene, lutein or lycopene to recover from DNA damage induced in vivo by treatment with H2O2. Methods: Healthy volunteers were given supplements of lutein (15 mg/day), lycopene (15 mg/day) and βcarotene (15 mg/day), each for 1 week, the supplementation periods being separated by 3-week wash-out periods. Blood samples were taken at the beginning and end of each supplementation, and at 1 week and 3 weeks during the wash-out period. Carotenoid levels were measured in plasma. Lymphocytes were isolated and frozen. Subsequently, they were treated with 100 μM H2O2 and incubated for up to 24 h; DNA damage was measured with the comet assay (single cell gel electrophoresis) after 0, 2, 4, 8, and 24 h. Results: Increases of 2- to 3-fold in mean plasma lutein and β-carotene concentrations were seen at the end of the respective supplementation periods; they returned virtually to basal levels after wash-out. Lycopene concentrations were less affected by supplementation, and were more variable. H2O2-induced DNA strand breaks were apparently only slowly rejoined by the lymphocytes. The rejoining of breaks in the first few hours appeared substantially faster in lymphocytes following supplementation with β-carotene, but no such effect was seen with lutein. In those individuals who showed increases in lycopene concentrations, the recovery was significantly faster. Lymphocytes that were not treated with H2O2 showed a transient increase in DNA breakage to about double the background level in 2 h, presumably as a result of exposure to atmospheric oxygen; this effect, too, was relieved by supplementation with lycopene or β-carotene. Conclusions: While certain carotenoids appear to enhance recovery from oxidative damage, this is probably in fact an antioxidant protective effect against additional damage induced by atmospheric oxygen, rather than a stimulation of DNA repair.

Notes: Summary Obesity could well become the most common health problem of the 21st century. There are more opportunities to consume large quantities of food: big portions of tasty, varied food, at reasonable prices, are available everywhere. Moreover, our bodies are better adapted to combat weight loss than to combat weight gain, since for thousands of years our species evolved in circumstances where nutrients were in short supply. The response of each individual to diet and other environmental factors varies considerably, depending on the characteristics of his/her body weight control mechanism. The differentiating element in the future, especially as regards the dietary and pharmacological control of obesity, will be knowledge of an individual's possible response depending on his/her genetic background. Obesity can occur as a result of genetic or acquired changes in three main types of biochemical processes, which are the main focus of this review: a) feeding control, which determines the sensations of satiety and hunger through processes that depend on a interplay between internal signals (notably leptin) and environmental factors; b) energy efficiency, in particular the activation of thermogenesis mediated by uncoupling proteins (UCPs) that makes it possible to dissipate part of the energy contained in food as heat instead of accumulating it as fat, and c) adipogenesis, the process by which cells specialised in fat storage (adipocytes) are formed, which is controlled by an interplay of transcription factors, including memebers of the C/EBP, PPARγ and ADD families. The knowledge of a growing numbers of genes and molecules implicated in these three types of processes and of their metabolic relationships is leading toward a molecular understanding of the body weight regulatory system and is paving the way for new methods of obesity control, especially pharmacological but also nutritional and possibly involving genetic intervention.

Notes: Summary Background: Quantitative information on conversion of β-carotene to vitamin A in humans is limited. Aim of the study: Our laboratory has developed a stable isotope method for studying the conversion of β-carotene (β-C) to vitamin A. Methods: Two dosage levels (a pharmacological dose, 126.0 mg β-C-d 8, and a physiological dose, 6.0 mg β-D-d 8) were used 2.5 y apart in an adult female volunteer to study dose effects on the conversation of β-C to vitamin A. Blood samples were collected over 21 d. β-C and retinol were extracted from serum and isolated by high performance liquid chromatography. The retinol fraction was derivatized to a trimethylsilyl ether which was analyzed by gas chromatograph/mass spectrometry with electron capture negative chemical ionization. Results: The retinol-d 4 response in the circulation peaked at 24 hours after the β-C-d 8 dose, with a higher percent enrichment after the pharmacological dose than after the physiological dose. By using retinyl acetate-d 8 as the vitamin A reference, the retinol-d 4 formed from 6 mg of β-C-d 8 (11.2 μmol) was calculated to be equivalent to 1.6 mg of retinol (i. e., 3.8 mg of β-C was equivalent to 1 mg of retinol). However, the retinol-d 4 formed from 126 mg of β-C-d 8 (235 μmol) was equivalent to 2.3 mg of retinol (i. e., 55 mg β-C was equivalent to 1 mg retinol). Conclusion: These results provide evidence that it is feasibile to use stable isotope reference method to study retinol equivalence of β-C and that there may be a dose-dependence on bioconversion of β-carotene to retinol.

Notes: Summary The potsitive effect of glutamine on lymphocyte proliferation has previously been described. Its dipeptide glycyl-glutamine (GlyGln) is more stable than pure glutamine in aqueous solutions. The aim of our study was to investigate the relationship between lymphocyte proliferation and varying concentrations of glycyl-glutamine in vitro. Isolated human lymphocytes were stimulated with the mitogens phytohaemagglutinin (PHA), Concanavalin A (ConA), pokeweed mitogen (PWM), and Staphylococcus aureus (SAC). Glycyl-glutamine was added to yield final cincentrations of 0–2 mmol/l. Overall minimal concentrations of 0.01 mmol/l glycyl-glutamine were sufficient to enhance lymphocyte proliferation over baseline (glutamine-free) levels. No difference was found between concentrations in the “physiological” range of 0.4 mmol/l and very low concentrations (0.04–0.1 mmol/l) with SAC, ConA and PWM. Increasing the concentration beyond 0.4 mmol/l (up to 2.0 mmol/l) offered further gain with PHA-stimulation only. Lymphocyte proliferation under in vitro polyclonal stimulation is maintained even at very low concentrations of glycyl-glutamine. Raising the concentration above the equivalent of physiological levels does not seem to provide further benefit.

Notes: Summary Aim of the study. An allergen-reduced dietary intervention programme with strict dietary requirements was implemented over the first four months of life in an unselected population-based infant cohort and compared to a non-intervention cohort (the ZUFF study). Recommendations for the dietary programme in the intervention cohort were extended, but not strictly implemented, until the end of month six. The intervention was based on breastfeeding, a moderate whey hydrolysate formula (pHF), and delayed introduction of weaning foods with a high allergenicity. This study was a prospective, controlled, and unblinded study, the first to assess the effects of an allergen-reduced, pHF-based early nutritional programme in a broad unselected infant population. Because overall healthy development of the infant is a major objective of any nutritional programme, the study evaluated the effects of the dietary intervention on infant growth and general health status rather than specific allergic manifestations. Part I of this paper gave results for nutritional behaviour only, and Part II gives results for growth and general health status during the intervention period through the sixth month of life. Methods. Assignment of study infants was to demographically comparable intervention (Z) or control (FF) cohorts according to place of birth. In the intervention cohort (Z=564), the recommended dietary regimen was breastfeeding and – if exclusive breastfeeding was not possible – supplementation with a moderately hydrolysed, allergen-reduced infant formula (pHF). Weaning foods were delayed until four months of age or later in case of weaning foods with high allergenicity. In the control cohort (FF=566), there was no specific intervention. Imbalances between cohorts in confounding (adjuvant) factors that could influence health related outcomes were integrated as covariates into the logistic regression of the main analyses. Growth parameters included weight, length, head circumference, BMI, and Z scores (SDS). General health status was assessed by clinically significant findings in gastrointestinal, respiratory, or skin symptoms. Results. Growth at 6 weeks and at 3 and 6 months was similar for Z and FF. Significantly fewer Z than FF infants had clinically noteworthy health findings at 3 months (Z=27% versus FF=37%, odds ratio=0.63, CI=0.48–0.82) and 6 months (Z=33% versus FF=49%, odds ratio=0.51, CI=0.40–0.66). This corresponds to a 30% reduction in overall health concerns at 6 months for the intervention cohort. At 3 and 6 months, differences between cohorts in most measures of general health status were strongly influenced by a lower incidence of skin symptoms in the Z cohort. Within FF, there were fewer exclusively breastfed (eBF) infants with health problems at 3 months compared with those who were partially (pBF) or non-breastfed (nBF) (eBF=31%, pBF=40%, nBF=39%, p〈0.05). In contrast, in the Z intervention cohort, the number of infants with health concerns was similar for exclusively breastfed infants and for those in whom mother's milk was supplemented or replaced by pHF (eBF=29%, pBF=25%, nBF=26%, ns). In a subanalysis of overall health findings in infants without a family risk of allergies, there were again significantly fewer Z than FF infants with any health or any skin problem. Conclusion.An allergen-reduced dietary recommendation that includes a moderate whey hydrolysate infant formula (pHF) has no negative effects on growth parameters up to 6 months of life in an infant population unselected for atopic risk. The dietary intervention produced improvements in general health status when compared with a control cohort that received infant formula with unhydrolysed proteins (IF), and high allergenic weaning foods at an earlier age. The difference between cohorts was principally due to fewer adverse skin findings. In infants following our allergen-reduced feeding recommendation, 3-month general health status was comparable between those who were exclusively breastfed and those in whom breastfeeding was supplemented or replaced by pHF. Our results demonstrate that a pHF feeding recommendation during the first 4 to 6 months of life – when exclusive breestfeeding is not possible – is a safe and feasible regimen not only in high-allergic risk populations but in general unselected infant population. The general use of pHF formula in non- or partly breastfed infants could therefore be considered an important contribution to optimised infant nutrition.

Notes: Summary Background: The dietary supplementation with EPA (eicosapentaenoic adic; 20:5n3) and DHA (docosahexaenoic acid; 22:6n3) has been recommended because of their favourable effects on the cardiovascular system (including complications of NIDDM). Oleic acid (18:1n9) from olive oil has some analogous and complementary effects. Potential competitive relations between long-chain n-3 fatty acids (FAs) and the oleic acid would therefore mean a problem. Aim of the study: We focused primarily on the oleic acid changes in serum phospholipids (SPL) after a supplementation with EPA and DHA. Methods: Thirty-five patients with type 2 diabetes mellitus (NIDDM) were supplemented for 28 days with 1.7 g of EPA plus 1.15 g of DHA/day (as Maxepa® capsules, Seven Seas®, U. K.). After that, a 3-month wash-out control period with 21 patients followed. A fatty acid composition of serum phospholipids (SPL) was determined by capillary gas-chromatography. Values were calculated as relative percentages of all FAs. Results: After the supplementation with the Maxepa® capsules, there was a very strong increase in EPA, docosapentaenoic acid (22:5n3) and DHA content in SPL. It was followed by a stron decrease after the wash-out (all p〈0.0001). The oleic acid SPL content after the intervention significantly decreased from 10.105±0.307% (mean ±S.E.M.) to 9.082±0.276% (p〈0.0003). During the wash-out, the change was in the opposite direction (p〈0.0001). When the intervention and the wash-out periods were taken together, changes in the oleic acid were inversely correlated with changes in EPA, docosapentaenoic acid and DHA (r = −0.729; r = −0.552; r = −0.629, respectively; p〈0.0001; n = 56). On the background of the overall n-6 FA reduction, the decline in the arachidonic acid after the supplementation (p〈0.0001) and its rise after the wash-out (p〈0.0003) were similar. There were no significant changes in the saturared FA spectrum. Conclusions: Supplementation with long-chain n-3 FAs in NIDDM patients leads to the lowering of oleic acid SPL content. Whereas the reduction of the arachidonic acid may have some desirable aspects (e. g. suppression of thromboxane TxA2 or 4 series leukotriene production), the decline of the former is to be regarded as a potential problem. Therefore, the search for optimally balanced blends of n-3 polyunsaturated fatty acids (PUFAs) and monounsaturated fatty acids (MUFAs) seems to be more promising than a supplementation with only one type of FA.

Notes: Summary Background Present animal models used to emulate type 2 diabetes may not accurately reflect the metabolic changes that occur in humans. Aims of the study The purpose of this research was to evaluate diets reported to induce insulin resistance and impaired glucose metabolism in rats as a potentially useful model for studying type 2 diabets. Methods Three groups of male Sprague Dawley rats (n=7) were fed either a control diet, based on AIN recommendations (53% cornstarch, 10% sucrose and 7% soybean oil), a high fat diet (25% soybean oil, 35% cornstarch) or a high fructose diet (53% fructose, 10% sucrose) for a 3 month period. Glucose tolerance tests were carried out in week 3 and week 9 of the experiment. At the termination of the experiment, serum insulin, glucose, cholesterol and triacylglycerols were measured. Glucose incorporation into glycogen and glycogen synthase activity were measured in soleus muscles. Results Similar weight gain was observed for all three groups of rats. Glucose tolerance curves and fasting glucose levels were not significantly different at any time point in the experiment. Insulin levels were unchanged for the controls (171±21 pM), high fructose (164±16 pM) and high fat (181±30 pM) diets. Fasting serum triacylglycerols and cholesterol levels were not significantly elevated by dietary treatment. In soleus muscles, rats on all three diets had a significant increase in glycogen synthesis in response to insulin, but synthesis was similar in all three groups. Glycogen synthase activity was also not significantly affected by long-term dietary intervention. Conclusions In this study, healthy Sprague Dawley rats fed high fat or high fructose diets for 3 months adapted to the nutritional intervention without developing classical signs of insulin resistance and impaired glucose tolerance.

Notes: Summary Background Bone mineral density (BMD) is largely genetically determined and this influence is most powerful in the period of rapid skeletal development in childhood and late adolescence but environmental factors such as exercise and dietary calcium intake may influence up to 20%. Aims of the study The aims of the study were to examine healthy late adolescent females for the effects and benefits of a high calcium intake from dairy product foods on bone mineral density, body composition, lipids and biochemistry. The secondary aim is determine whether a high intake of dairy product foods in the diet is acceptable for this age group long term. Methods Ninety-one teenage girls who participated in a two-year randomised controlled study on the effect of dairy food supplementation on dietary patterns, body composition and bone density in post-pubertal teenage girls were approached one year after the cessation of the study to determine the effects of the cessation of dairy supplements on bone mineral density, dietary habits, biochemical markers, body composition and blood lipids. Bone mineral density and bone mineral content were assessed at the hip, spine and total body. Anthropometric data were collected, and exercise, Tanner, dietary assessment, preference and compliance questionnaires were administered. Lipid profiles, hydroxyproline excretion and urinary calcium and sodium excretion measurements were performed. Results There were no significant differences between the 2 groups for height, weight, lean and fat mass. The supplemented group had significantly higher calcium, phosphorus and protein intake during the supplementation period (p〈0.001). No differences were seen between the groups 12 months after supplementation finished. There were no significant differences in exercise level, preference or acceptability of dairy products or in the lipids and bone markers between baseline the end of supplementation and 1 year follow-up. There was a significant increase in trochanter (4.6%), lumbar spine (1.5%) and femoral neck (4.8%) BMD (p〈0.05) in the high calcium group at the end of supplementation. There was an increase in bone mineral content at the trochanter (p〈0.05) and lumbar spine; however the latter was not statistically significant, in the high calcium group at the end of supplementation. There was no difference in vertebral height or width at any stage of the study, indicating no influence on bone size. Conclusions In this 3 year study (2 years of supplementation, 1 year follow-up), teenage girls, aged 15–18 years, were able to significantly increase their BMD at the trochanter, femoral neck and lumbar spine when supplemented with dairy product foods to a mean calcium intake of 1160 mg/d. There was also an effect seen on the BMC particularly at the trochanter and to a lesser extent at the lumbar spine. The dietary calcium intake achieved did not adversely affect body weight, fat and lean mass or blood lipid profiles. Twelve months after the supplementation finished the girls had returned to their baseline diet, indicating self-selection of a high dairy product diet may be hard to achieve.

Notes: Summary Alpha-tocopherol occurs in nature as a single stereoisomer (RRR) while synthetic vitamin E is a mixture of eight stereoisomers (all-racemic, all-rac). The presently accepted ratio of biopotency (RRR: all-rac) is 1.36, based on the fetal resorption test in rats. This ratio has been disputed for humans. Clinical endpoint studies in humans are lacking, but plasma responses to RRR-and all-rac were measured in bioavailability studies. In nine studies comparing unlabeled forms, the ratio of plasma parameters (AUC, Cmax or steady-state concentration) concurred with the accepted ratio of biopotency within accepted bounds of equivalence. Four recent studies with simultaneous application of trideutero-RRR and hexadeutero-all-rac resulted in ratios of up to 2 for plasma, and of ≈ 2.7 and ≈ 3.4 for α-CEHC (a urinary metabolite) and umbilical cord plasma, respectively. Because these results have been widely assumed to reflect the difference in biopotency, this has prompted a proposal to the Food and Nutrition Board, National Academy of Sciences, USA to change the biopotency factor to 2:1. We challenge the validity of bioavailability data in lieu of clinical endpoints. Because RRR and all-rac are not chemically identical and differ in plasma and tissue kinetics and metabolism, the ratio of bioavailability parameters does not reflect the ratio of biopotency. This needs to be determined in adequately designed studies using clinical and biochemical endpoints. Until such studies have been performed it does not appear prudent to exchange the presently accepted ratio based on valid bioassays, albeit in a model animal, for another that is based on erroneous conclusions from human studies.

Notes: Summary Background Gastric emptying of non-nutrient liquids usually lacks the presence of an initial delay phase (lag phase), and so it has been considered to be monoexponential with an initial rapid phase followed by a slower emptying phase. However a lag phase in the gastric emptying of liquids can be found if there is a high caloric density in the liquid meal. Aims of the study To characterise with stable isotopes the presence of a lag phase in the gastric emptying of non-sold meals. Methods Healthy volunteers ingested a low caloric liquid meal (345 KJ/200 mL) (LCLM), a high caloric liquid meal (1135 KJ/180 mL) (HCLM) or a semisolid meal (1403 KJ/500 mL) (SSM). Test meals were labelled with 13C-acetate. Breath samples were collected for 13〉CO2 measurement and data were fitted to a power exponential function. Results Non-solid meals can have different behaviour related to the initial emptying. The presence of a lag phase in the gastric emptying of liquids was not masked by the processing of the tracer previous to its detection in breath. While the LCLM and SSM showed a rapid initial emptying phase (no lag phase), the HCLM has an initial slow emptying phase. The slower gastric emptying of the HCLM compared to the SSM was related to the presence of a lag phase in the gastric emptying of the HCLM. Conclusions The 13C-acetate breath test is very accurate to identify and study the lag phase if present of liquid meals.

Notes: Summary Background Animal studies have demonstrated dramatic changes in the intestinal flora during total enteral (TEN) or parenteral (TPN) nutrition. Aims of the study To assess the impact of TEN and TPN on human intestinal microflora. Methods Eight patients on fiber-free TEN, five patients on TPN, and ten controls were studied. Fecal bacteria were identified and numbered (logCFU/g feces), and fecal short-chain fatty acids (SCFAs) were measured in stool samples, by gas-liquid chromatography. Results In TEN patients, compared to controls (P〈0.01), aerobes were increased (8.46±0.24) while anaerobes were decreased (5.79±0.84). In TPN patients, both aerobes and anaerobes were decreased compared to controls (5.64±0.27 and 5.31±1.09 respectively, P〈0.01). Total SCFAs were lower in TPN patients than in TEN patients (48.3±16.6 vs 118.6±24.1 mmol/kg, P〈0.05). Conclusions Both TPN and TEN induce modifications in the intestinal microflora. During TPN, a homogeneous decrease occurs in both aerobic and anaerobic bacteria. TEN decreases only anaerobic bacteria, while aerobic bacteria are increased. This imbalance may play a role in the pathophysiology of TEN-induced diarrhea.

Notes: Summary The nutritional patterns of Paleolithic humans influenced genetic evolution during the time segment within which defining characteristics of contemporary humans were selected. Our genome can have changed little since the beginnings of agriculture, so, genetically, humans remain Stone Agers – adapted for a Paleolithic dietary regimen. Such diets were based chiefly on wild game, fish and uncultivated plant foods. They provided abundant protein; a fat profile much different from that of affluent Western nations; high fibre; carbohydrate from fruits and vegetables (and some honey) but not from cereals, refined sugars and dairy products; high levels of micronutrients and probably of phytochemicals as well. Differences between contemporary and ancestral diets have many pathophysiological implications. This review addressed phytochemicals and cancer; calcium, physical exertion, bone mineral density and bone structural geometry; dietary protein, potassium, renal acid secretion and urinary calcium loss; and finally sarcopenia, adiposity, insulin receptors and insulin resistance. While not, yet, a basis for formal recommendations, awareness of Paleolithic nutritional patterns should generate novel, testable hypotheses grounded in evolutionary theory and it should dispel complacency regarding currently accepted nutritional tenets.

Notes: Summary Background: Epidemiological studies report an inverse relationship between intake of the B vitamine folic acid and colon cancer. Folate is important for DNA synthesis and repair. Moreover, the production of S-adenosylmethionine (SAM), essential for normal DNA methylation and gene expression, is dependent on folic acid. Folate deficiency may increase the risk of malignant transformation by perturbing these pathways. Aims of the study: The principal aim of this study was to determine the effects of folate deficiency on DNA stability and DNA methylation in rat colonocytes in vivo. As the metabolic pathways of folate and other dietary methyl donors are closely linked, the effects of methionine and choline deficiency were also evaluated. Methods: Male Hooded-Lister rats were fed a diet deficient in folic acid, or in methionine and choline, or in folate, methionne and choline for 10 weeks. DNA strand breakage and misincorporated uracil were determined in isolated colonocytes using alkaline single cell gel electrophoresis. Global DNA methylation was measured in colonic scrapings. Folate was measured in plasma, erythrocyte and liver samples. Results: Methyl donor deficiency induced DNA strand breakage in colonocytes isolated from all experimental groups. Uracil levels in colonocytes DNA remained unchanged compared with controls. DNA methylation was unaffected either by folate and/or methionine and choline depletion. Rats fed a folate-deficient diet had less folate in plasma, red blood cells and liver than controls. Conclusions: Folate and methyl deficiency in vivo primarily afects DNA stability in isolated colonocytes of rats, without affecting overall DNA methylation.

Notes: Summary Background: Metallothionein (MT)# synthesis can be stimulated in many organs not only by various metals such as cadmium, zinc, and copper, but also by many nonmetalic compounds or experimental conditions such as oxidative stress. The latter lead to the hypothesis that MT is induced in response to free radicals formed in tissues and lipid peroxidation. Aims of the study: Whether the relationship between lipid peroxidation amd MT synthesis is a common phenomenon also valid for lipid peroxidation induced by dietary factors such as chronic vitamin E inadequacy and autoxidation products of polyenoic fatty acids derived from thermally oxidized oil was investigated in the presence study. Methods: The relationship between the induction of metallothionein isoforms I and II (MT-I and MT-II) in response to diet-induced lipid peroxidation using a rat model system in which lipid peroxidation was examined in vivo by chronic vitamin E inadequacy or by administration of lipid peroxidation products from a thermally treated polyenoicrich oil with either basal (dietary zinc concentration: 48 mg/kd; experiment 1) or Zn-stimulated MT levels (dietary zinc concentration: 305 mg/kd; experiment 2) was studied. In both experiments, growing male rats were fed diet containing either a fresh or a thermally treated soybean oil with deficient of sufficient amounts of vitamin E (14 and 11 vs. 648 and 560 mg α-tocopherol equivalents per kg diet) over 40 days according to a bifactorial experimental design. Plasma and liver concentrations of tocopherols and hepatic levels of thiobarbituric acid-reacitve substances (TBARS) were measured by high performance liquid chromatography. MT isoform concentrations in rat liver were isolated and quantified by ion-exchange high performance liquid chromatography and atomic absorption spectrometry. Results: Irrespective of the zinc supply, rats receiving inadequate amounts of vitamin E with the diet had markedly lower plasma and liver concentrations of α-tocopherol and total tocopherols than vitamin E-sufficient rats. ANOVA also revealed an interaction between the diet factors vitamin E and oil on tocopherols in plasma and liver of rats from both experiments. In experiment 1, where rats received normal amounts of dietary zinc, ingestion of the thermally treated oil impaired the tocopherol status compared to the treatment with the fresh oil, although this effect was only obvious in the vitamin E-deficient groups. In experiment 2, where rats received excessive amounts of zinc, the thermally treated oil did not contribute to a reduction of the tocopherol status in plasma and liver. In both experiments a significant increase in TBARS level, indicative of lipid peroxidation, was observed in the liver at chronic vitamin E inadequacy, but no effect of the oil was observed. Here, we show that the dietary treatment had some effects on the synthesis of liver metallothionein isoforms. In groups, receiving normal amounts of zinc, there was a significant interaction between the dietary treatments on the levels of MT-I and MT-II in liver. Chronic vitamin E inadequacy which was accompanied by diminisched tocopherol levels in liver induced the synthesis of MT-I and MT-II. When vitamin E inadequacy was combined with the ingestion of a thermally treated polyenoic acid-rich oil hepatic levels of MT-I and MT-II remained low. In experiment 2, where rats were fed the high zinc diet, vitamin E inadequacy caused an increase of hepatic MT-I level just as in experiment 1, although this MT stimulating effect was irrespective of the oil. For MT-II there was a 43% increase in the vitamin E-deficient group fed the fresh oil compared to all the other groups, although this effect was not statistically significant. The liver MT isoform response to stress was similar in rats with basal MT levels and Zn-induced liver MT levels. The failing effect of the thermally treated oil on MT levels which were stimulated by vitamin E deficiency in experiment 2 was possibly due to the low oxidation grade of the thermally treated oil. Conclusion: The present results are strongly indicative of an apparent induction of MT isoform synthesis in response to an impaired antioxidant defence system in the lipid regions of liver cells induced by vitamin E inadequacy. In contrast, thermally treated polyenoic-rich oils with a certain oxidation grade seem to restrain the induction of MT isoform synthesis under the present experimental conditions.

Notes: Summary Background Muscle triglycerides are important as a source of energy and in relation to metabolic sensitivity. However, the classic biopsy method does not distinguish intra- from extracellular fat, and their regulation by exercise and diet is largely unknown. Magnetic resonance spectroscopy (MRS) is available to assess the intramyocellular lipid (IMCL) pool non-invasively in humans. Aims of the study The aim of this work was to use sequential MRS measurements of IMCL and glycogen to explore the role of three levels of dietary fat on the replenishment of these energy stores after exercise. Methods Following 2h of exercise, two subjects (S1, S2) were fed one of three diets (15%, 40% or 70% fat energy), each on a separate occasion. IMCL and glycogen were measured by MRS in the tibialis anterior muscle before, after exercise, and at 10 and at 32 h of recovery. Results Initial IMCL concentration (mmol · kg−1:3.0 in S1 and 1.8 in S2) was reduced to 70% after exercise. The rate of replenishment was minimal with the low-fat (mmol · kg−1· 24 h−1: 0.7 and 0.0) and much higher with both higher fat diets (mmol · kg−1· 24 h−1: 3.1 and 3.2 in S1, 0.7 and 0.9 in S2). Glycogen and IMCL replenishments were inversely correlated. Conclusions IMCL and glycogen can vary acutely in response to diet after exercise. Studies are needed to determine if such variations occur within the range of ordinary diets and to clarify the functional significance of IMCL in differently active individuals.

Notes: Summary Background Recent data suggest that daily iron supplementation of iron-replete children could impair their growth. If verified for weekly iron supplementation these results would markedly complicate targeting and implementing school-based weekly iron supplementation programs. Aim of the study To ascertain the effect of weekly iron supplementation on the growth and hemoglobin status of non-anemic school-age children. Subjects and methods 73 Bolivian non-anemic school-age children randomly assigned to the treatment group (n=37; receiving supplements containing FeSO4 during 18 weeks) or the control group (n=36; receiving a placebo during the same period). Hemoglobin concentration and anthropometric measures were determined for each child at the beginning (T0) and the end (T18) of the study. Results The treatment group did not show any significant variation in hemoglobin concentration between T0 and T18 (−1.6±10.4 g/L; P=0.40) whereas the control group showed a significant decrease in hemoglobin concentration (−4.6±10.9 g/L; P=0.03). Anthropometric changes were not significantly different between the treatment and the control groups for weight, (1.63±1,11 kg vs 1.88±0.79 kg; P=0.30), height (2.35±0.94 cm vs 2.11±1.03 cm; P=0.34) or mid-upper arm circumference (0.29±0.57 cm vs 0.22±0.54 cm; P=0.64). Conclusion In our study, weekly iron supplementation of non-anemic school-age children had no negative effect on their growth while having a positive effect in preventing significant decreases in hemoglobin concentration. These results suggest that in regions where iron deficiency anemia (IDA) is prevalent, a simple and cost-effective way to control IDA in school-age children is to give weekly iron supplements to all children at school.

Notes: Summary Background: Diet restriction (DR) has been shown to extend the life spans of various laboratory animals, the mechanism may involve a decrease in oxidative stress. When determining if the total tissue defense has been altered, it is important to observe the overall direct antioxidant capacity, which consists of low molecular weight antioxidants (LMWA) and enzymes. Aim: To determine DR induced changes in total reducing power and overall direct antioxidant capacity of various mouse tissues. Methods: Young female Sabra mice were fed a 60% food restricted diet for 40 days (DR group). Organs of the DR group and of ad libitum (AL) fed controls were then dissected and examined. A cyclic voltammetry method was used to quantify the total reducing power, which correlates with the overall LMWA activity. Specific LMWA were identified by HPLC-ECD. Superoxide dismutase activity and H2O2 degrading ability were measured in order to include the enzymatic antioxidant component. Results: Short-term DR caused alterations in the total reducing power of various mouse tissues, indicating changes in the total scavenging ability of these tissues. Overall direct antioxidant capacity of heart, kidney and muscle was enhanced; liver and small intestine deteriorated; brain did not differ between DR and AL groups; lung and spleen exhibited a mixed response. Conclusions: We have shown for the first time that DR causes changes in the total reducing power of different mouse tissues, thus, affecting the overall direct antioxidant capacity. These findings support the suggestion that there may be a biological regulation of the antioxidant system.

Notes: Summary Background: The body's nucleotide pool derives from three potential sources: de novo synthesis, salvage of preformednucleosides/bases or the diet. The relative contributions of these pathways of assimilation are poorly understood in vivo. Dietary nucleotides have been suggested to have beneficial effects an the development and repair of the gastrointestinal tract. Tissues with a rapid turnover, such as the gut and the immune system cells, may utilise preformed nucleotides (coming from the diet), in situation in which there is a high demand of nucleotides for nucleic acid synthesis. Therefore, nucleotides could be onsidered as conditionally essential nutrients. Aim of the work and methods: -Development of a method to measure synthesis de novo of RNA-purine nucleotides in Caco-2 cells, relying an the incorporation of 14C-glycine into the purine ring of the nucleotide. To establish the fractional synthesis rate of RNA purine nucleotides in Caco-2 cells, grown in culture medium containing different concentrations of glutamine, in the presence or abscence of added nucleotides. To investigate the degree to which tissue ribonucleosides are derived from the culture medium or from de novo synthesis in the presence of different concentrations of glutamine, using undifferentiated Caco-2 cells, stressed or not by the addition of IL-1β to the medium. Results and conclusions:The presence of high levels of glutamine in the culture medium is essential for cell proliferation (estimated by measurement of the fractional synthesis rate of purine nucleotides) and the presence of nucleotides cannot replace the glutaminedependence of Caco-2 cell proliferation. The incorporation or exogenous purine nucleotides into RNA of Caco-2 cells is rather limited, and it becomes important when cells are stressed by glutamine deprivation. Stress by addition of interleukin-1β resulted in the maintenance or the increase in de novo synthesised RNA-purine nucleotides, even in the presence of exogenous nucleotides. However, the addition of interleukin-1β to the culture medium led to an enhanced salvage of preformed pyrimidine nucleotides for nucleic acid synthesis when glutamine was present in the medium at a concentration of 0.5 mmol/L.

Notes: Summary Scientific evidence is accumulating that meat itself is not a risk factor for Western lifestyle diseases such as cardiovascular disease, but rather the risk stems from the excessive fat and particularly saturated fat associated with the meat of modern domesticated animals. In our own studies, we have shown evidence that diets high in lean red meat can actually lower plasma cholesterol, contribute significantly to tissue omega-3 fatty acid and provide a good source of iron, zinc and vitamin B12. A study of human and pre-human diet history shows that for a period of at least 2 million years the human ancestral line had been consuming increasing quantities of meat. During that time, evolutionary selection was in action, adapting our genetic make up and hence our physiological features to a diet high in lean meat. This meat was wild game meat, low in total and saturated fat and relatively rich in polyunsaturated fatty acids (PUFA). The evidence presented in this review looks at various lines of study which indicate the reliance on meat intake as a major energy source by pre-agricultural humans. The distinct fields briefly reviewed include: fossil isotope studies, human gut morphology, human encephalisation and energy requirements, optimal foraging theory, insulin resistance and studies on hunter-gatherer societies. In conclusion, lean meat is a healthy and beneficial component of any well-balanced diet as long as it is fat trimmed and consumed as part of a varied diet.

Notes: Abstract . Alterations of the cartilage matrix structure under non-destructive laser irradiation have been investigated by scanning force microscopy. Porcine nasal septum cartilage was irradiated with a CO2 laser with a power density of 50 W/cm2 under two different time regimes: for 3 s and for 30 s. Short-time irradiation had little effect on the structure of the cartilage matrix. In comparison with non-irradiated cartilage, small channels of 100–400 nm in cross-section appeared. This observation gives evidence that the underlying mechanism of laser-induced stress relaxation of cartilage is based on short-time depolymerisation and subsequent re-formation of proteoglycan units. The 30 s laser treatment results in melting and denaturation of the matrix. For the first time, small crystals, 100–800 nm, were found on cut sections of the laser treated cartilage. The crystals mainly consist of resolvable sodium carbonate. Thus, they cannot be responsible for the formation of a stable cartilage configuration after laser treatment.

Notes: Abstract Immune response after laser-photosensitiser application could be crucial in treatment of cancers, because without it there could be no systemic, long-term tumour control. Laser immunotherapy, a novel method for treatment of metastatic tumours, uses a near-infrared laser, a laser-absorbing dye indocyanine green, and an immunoadjuvant glycated chitosan. This modality has shown an induced antitumour immune response in treatment of rat mammary metastatic tumours. The influence of this new method on the cellular structure of the tumours and on the infiltrating immune cells was studied using optical and electron microscopes. The tumour samples were examined before and immediately after the treatment for acute effects, which appeared mainly photothermal. Two weeks after treatment, significant infiltrating lymphocytes and plasma cells were found around the surviving tumour cells. These morphological findings suggest that both cell-mediated and humoral immune responses could be responsible for the observed tumour eradication and induced long-term tumour resistance.

Notes: Abstract Topical or systemic administration of 5-aminolaevulinic acid results in biosynthesis of the photosensitiser protoporphyrin IX (PpIX) with some selectivity for malignant lesions. Excitation near 400 nm excites both intrinsic green tissue autofluorescence and red fluorescence from PpIX which may be exploited for the optical diagnosis of malignant and premalignant disease. In this work the utility of a cooled 12-bit single chip charge-coupled device (CCD) colour camera was investigated for photodiagnostic fluorescence ratio imaging. The red to green fluorescence intensity ratios were calculated for each pixel in real-time and fluorescence ratio images were displayed typically at a rate of 2 frames/s. Laboratory tests of fluorescence ratio imaging showed good contrast enhancement between control tissues and tissue phantoms and those containing porphyrin photosensitisers. In preliminary clinical tests, a clear demarcation between neoplastic/cancerous lesions and adjacent normal tissue was demonstrated. The extent of PpIX photobleaching during photodynamic therapy was also investigated using fluorescence ratio imaging.

Notes: Abstract . Three cell types including bovine pulmonary artery endothelium cells (CPAE), rat kangaroo kidney cells (PTK2), and human larynx epidermoid carcinoma cells (Hep-2) were used to study subcellular localisation and phototoxicity of Photofrin-II and lutetium texaphyrin (Lu Tex). Cells were examined for fluorescence after administration of the photosensitisers. Subcellular regions were exposed with a laser microbeam system that used an argon ion laser pumped dye laser generating a 630 nm for Photofrin-II and 730 nm for Lu Tex. Fluorescence detection suggests that the Photofrin-II is bound primarily to the mitochondria with some diffuse fluorescence in the rest of the cytoplasm. The fluorescence in Lu Tex treated cells appears to be localised to the lysosomes. The percentage of damaged cells following light exposure to the different subcellular regions after Photofrin-II or Lu Tex treatment demonstrates that the nuclear region was the most sensitive target followed by the perinuclear region and peripheral cytoplasm region.

Notes: Abstract. Pulsed infrared (IR) and ultraviolet (UV) lasers have been suggested for use in middle ear surgery due to potential fiberoptic delivery, decreased thermal trauma and precise ablation characteristics. Although attention has been focused on the thermal and photoacoustic events that occur during pulsed laser ablation of hard tissue, there are few studies which evaluate the acoustic energy generated from these devices from an audiological standpoint. In this study, the mastoid cavities of cadaveric human temporal bones were irradiated with a holmium: yttrium aluminium garnet (Ho:YAG) laser (λ=2.12 µm) using the following parameters: 5, 10, and 15 Hz pulse repetition rate and 1, 2, 4, 6, 8, and 10 W average power. During ablation, acoustic measurements were made using a sound level meter positioned 5 cm away from the target site. With each set of laser parameters, the sound intensity exceeded 85 dB (dBA scale). Peak intensities of 125 dB were measured, and a saturation effect noted above 4 W or 500 mJ/pulse. The clinical significance of these findings is discussed and the acoustical aspects of middle ear function and noise trauma are reviewed.

Notes: Abstract . A factor that might complicate the use of intraoperative photodynamic therapy (PDT) is a possible adverse effect on normal tissue recovery. In this study, rats with experimental skin incisions received intraoperative PDT (10 mg/kg haematoporphyrin derivative, 180 J/cm2 laser light), immediately followed by closure. Healing was evaluated by tensile strength assessment of the incisions 21 days after PDT. No significant differences between the PDT-treated group and control groups were found. We therefore concluded that with respect to healing of skin incisions in rats, intraoperative PDT is not contraindicated.

Notes: Abstract . Acupuncture is a form of traditional Chinese medicine that has developed over thousands of years. We studied the effects of laser puncture, needle acupuncture, and light stimulation on cerebral blood flow in 15 healthy volunteers (mean age 25.0±1.9 years, 5 female, 10 male) with non-invasive transcranial Doppler sonography. In addition 40-Hz stimulus-induced brain oscillations, heart rate, blood pressure, peripheral and cerebral oxygen saturation, and the bispectral index of the EEG were recorded. Stimulation with light significantly increased blood flow velocity in the posterior cerebral artery (p〈0.01, ANOVA). Similar but less pronounced effects were seen after needle acupuncture (p〈 0.05, ANOVA) and laserpuncture (n.s.) of vision-related acupuncture points. Furthermore both, laserpuncture and needle acupuncture, led to a significant increase in the amplitudes of 40-Hz cerebral oscillations. Stimulation of vision-related acupuncture points with laser light or needle acupuncture elicits specific effects in specific areas of the brain. The results indicate that the brain plays a key intermediate role in acupuncture. However, brain activity of itself does not explain anything about the healing power of acupuncture.

Notes: Abstract . This study had four main objectives (a) multicentre trial feasibility in nursing homes; (b) the applicability of registration methods; (c) the extent of wound size reduction and (d) the LLLT effect(s) on pressure sores stage III. The study was designed as a prospective randomised, single blind, multicentre clinical trial in four nursing homes, using the same prospective protocol. A total of 20 patients were enrolled into the study: 16 patients were randomised, and four patients were excluded. Treatment was the prevailing consensus decubitus treatment (n=8); one group (n=8) had 904 nm LLLT in addition, five times a week over a period of 6 weeks. The main outcome measure was the median wound size at six weeks after intervention. No statistical significant difference in wound size was found between the two groups (Mann Whitney U test; p=0.47). The sores in the LLLT group healed to a median of 83%, wounds in the control group to a median of 95% of their initial size. There was significant wound decrease within treatment arms (Friedman two-way analysis, p〈0.001). It was concluded that (a) a multicentre study is feasible in nursing homes; (b) the evaluation methods are accurate, adequate and reproducible; (c) there was significant sore area reduction within groups, but there were no significant differences between treatments; and (d) the efficacy of LLLT in healing of full thickness pressure sores stage III was not demonstrated and is still a matter of debate.

Notes: Abstract . Shape and deformation measurements of the eardrum are important both in fundamental and in clinical research on the function of the middle ear mechanics. We have developed an apparatus which allows high resolution full field shape measurements of the tympanic membrane. The method is based on Moiré topography, and makes use of the interference between the image of a straight line ruling and its image which is deformed by projecting it on to the object surface. Within a few seconds, our non-contacting method yields a full-field quantitative measurement of the eardrum shape on a matrix of 512 by 512 points, with a height resolution of 15 µm.

Notes: Abstract . The study examines an Er:YAG laser (2940 nm) and different application systems of the CO2 laser (10 600 nm) with regard to their suitability for a one-shot laser myringotomy of an adequate perforation size (∼2 mm). The laser–tissue interaction of the Er:YAG laser and the CO2 laser in fresh tympanic membranes of horses (thickness: 80–100 µm) as well as in formalin-fixed human tympanic membranes (thickness: 100 µm) is studied correlating perforation diameters to the applied power/energy density and the effects demonstrated by light and scanning electron microscopy are analysed. Using the Er:YAG laser with a focused laser beam (spot diameter: 400 µm) or with a maximally defocused laser beam (spot diameter: 1600 µm) perforations of an adequte size (2 mm) can only be achieved with multiple laser pulses. Histological studies disclose only minimal thermic side effects in the adjacent tissue in both specimens. If the CO2 laser radiation is transmitted via a silver halide polycrystalline fibre (diameter: 900 µm) a maximal perforation diameter of 1300 µm is achieved with significant thermic side effects such as coagulation. Using an Acuspot™ 710 micromanipulator (focused beam diameter: 180 µm) combined with a SilkTouch™ scanner a maximal perforation diameters of 1700 µm can be achieved in horse tympanic membrane with one laser pulse. A prototype of a hand-held CO2 laser otoscope in combination with the SilkTouch™ scanner is suitable for performing laser myringotomies with a diameter of 2 mm with a single laser pulse in fresh horse tympanic membrane.

Notes: Abstract . The aim of this double-blind study was to examine the effects of helium–neon laser irradiation on the mechanical (pressure algometry) and electrical (1 ms monophasic square-wave pulses, 50 Hz) pain threshold. 32 pain-free subjects were randomly assigned to either the experimental group (helium–neon laser stimulation: 5 mW, 10 min) or the placebo group (sham stimulation). Laser or sham stimulation and pain threshold ascertainment were carried out on the dorsal aspect of the forearm area. The contralateral arm served as an untreated control. The groups were compared with each other and with the control arm. No significant differences were found between the laser stimulation and the sham stimulation in changes of either the mechanical or the electrical pain threshold. There were no changes in the mechanical pain threshold through laser stimulation and sham stimulation with respect to the untreated contralateral arm. After laser stimulation electrical pain threshold was significantly higher (p〈0.01) in the treated arm than in the untreated contralateral arm, because this threshold decreased in the contralateral arm. This was not the case in sham treatment. The data suggest that helium–neon laser stimulation does not raise the experimental pain threshold in healthy subjects compared to placebo treatment. After helium–neon laser stimulation there was a decrease of the electrical pain threshold in the contralateral arm. To disclose the mechanism of this effect further experimental investigations under strict electrophysiological conditions are required.

Notes: Abstract . Previous measurement of laser-induced fluorescence in bronchus tissue is simulated using an electromagnetic (EM) scattering model. The EM parameters derived indicate that the changes in the thickness of the epithelium associated with carcinoma in situ can be detected using an optical radar process, providing an early detection of cancer. Further simulation using the EM model indicates various practical methods of measuring the thickness, including frequency modulated continuous wave optical radar, multilaser continuous wave radar and low coherence interferometry. Initial optical experiments and calculations have demonstrated which of the methods is likely to lead to the development of an endoscopic instrument. The availability of suitable optical components is a major consideration. Some early interferometry results indicate that a resolution of at least 40 µm and a signal to noise of up to 45 dB can be readily reached for histological imaging.

Notes: Abstract . The purpose of this study was to determine the wavelength-response effects of low level laser irradiation (LLLI) on immunocompetence of mice in vivo and intracellular free calcium ([Ca2+]i) in human lymphocytes and cultured cortical neurogliocytes (CCN) in vitro. Mice were first immune compromised by cyclophosphamide (CTX) injection, and the immunological activities including the production of interleukin 2 (IL-2), the murine mixed lymphocyte reaction (MLR), the mitogenic response of murine thymocytes (MRT), the proliferation of murine bone marrow cells (BMC) and the natural killer (NK) cells activity, were investigated after intravascular low level laser irradiation (ILLLI) (1 mW, 1.1×104 J/cm2) at wavelengths of 532 nm, 632.8 nm, 650 nm and 1520 nm, respectively. In addition, using Ca2+ sensitive indicator Fura-2 AM with the Spex AR-CM-MIC cation measurement system ([Ca2+]i) in single human lymphocytes and CCN were measured after LLLI (7.5 J/cm2) at wavelengths of 532 nm, 632.8 nm, 650 nm, 810 nm and 1300 nm, respectively. Results showed that the ILLLI at wavelengths of 532 nm, 632.8 nm and 650 nm, produced a significant increase in the proliferation of BMC and the NK activity. The production of IL-2 was greatly promoted after irradiation at 632.8 nm and 650 nm. After irradiation at 532 nm and 650 nm, the murine MLR was evidently enhanced, and MRT was dramatically increased only after irradiation at 632.8 nm. In contrast, no significant effects were found on the above mentioned indexes by irradiation at 1520 nm in comparison to the control. In addition, [Ca2+]i in single human lymphocytes and CCN were increased after LLLI at wavelengths of 532 nm, 632.8 nm and 650 nm, respectively, whereas they were not significantly affected by the wavelengths of 810 nm and 1300 nm. Our results indicated that LLLI could induce significant and different effects on the immunological activities of the mice and cause an increase in [Ca2+]i in single human lymphocytes and CCN. Furthermore, these effects are dependent on the wavelengths, for example, more positive effects produced by the wavelengths of 532 nm, 632.8 nm and 652 nm than those produced by the wavelengths of 810 nm, 1300 nm and 1520 nm.

Notes: Abstract . Microvascular anastomosis compliance mismatch is a predictive factor for anastomosis patency rate. We compared the arterial wall compliance of manual and laser-assisted vascular micro-anastomosis. Twelve New Zealand white rabbits were operated with conventional manual microanastomosis (CMMA) on the left femoral artery (10-0 separated stitches) and laser-assisted microanastomosis (LAMA) on the right (diode laser, wavelength 988 nm, power output 500 mW). Diameter and compliance were immediately measured by echotracking with 20 MHz microprobes, on the anastomosis level, as well as 3 mm upstream and 3 mm downstream from the anastomosis. On the LAMA side, diameter decreased downstream from the anastomosis (from 1.94±0.41 to 1.65±0.21 mm; p=0.012), while compliance increased upstream from the anastomosis (from 0.0034±0.001 to 0.0042±0.0012 mm/mm Hg; p=0.0195). There were no significant diameter or compliance change on the CMMA side (p〈0.05). It was concluded that diode LAMA creates a transition site at the anastomosis level, increasing compliance mismatch.

Notes: Abstract . The aim of this study was to investigate the effects of low intensity laser therapy (LILT) on the conduction characteristics of peripheral sensory nerve. Fifteen healthy subjects participated in this experiment. Six points on the sural nerve of the left leg were irradiated with an 830 nm GaAlAs laser with different parameters. All 15 subjects received at random one of the following treatments: an average power of 400 mW at a dose of 1.5 J per point (7.65 J/cm2) (treatment A); an average power of 140 mW and a dose of 1.0 J per point (5.1 J/cm2) (treatment B); an average power of 30 mW, 0.5 J per point (2.55 J/cm2) (treatment C) and to evaluate possible placebo effects in a fourth moment, no power was given (sham). The influence of skin temperature on conduction characteristics of peripheral nerve was taken into account by using correction factors for conductance velocity (1.47 m/s per °C) and negative peak latency (0.2 ms per °C) to a reference temperature of 32°C. One way analysis of variation with repeated measurements of the data collected within 15 min postirradiation showed a significant decrease in conduction velocity and increase in negative peak latency in treatment B. Those results could explain possible laser-mediated pain relief.

Notes: Abstract . Cement removal at revision hip arthroplasty forms a critical step for a successful operation. The removal of polymethymethacrylate (PMMA) with curet and chisel can cause major damage to the femoral shaft. The use of ultrasound or lithotripsy can cause perforation and microfractures to bone tissue. The goal of our study was to evaluate the application and practicability of different laser systems for cement removal. We examined and compared the effects of a diode laser (wavelength λ=800 nm), a Nd:YAG laser (λ=1064 nm), and an Er:YSSG laser (λ=2780 nm) on PMMA and the PMMA–bone interface. Whereas the Nd:YAG laser with a high ablation rate led to severe bone damage with extensive carbonisation, the Er:YSSG laser with a low ablation rate produced a defined cut at the PMMA–bone interface (max. depth 3.70 mm at 6.0 W laser power). Using the diode laser a defined high quality ablation of PMMA at the PMMA–bone interface was possible without any visible damage of adjacent tissue (max. depth 2.75 mm at 2.9 W laser power). However, sufficient ablation in an adequate operating time could not be realised with this power. The use of a laser tool for cement removal is insufficient. It only facilitated the manual removal of the remaining cement with chisel and curet.

Notes: Abstract . Port wine stains (PWS) are congenital vascular malformations for which lasers are the treatment of choice. This article reviews laser optics in relation to PWS treatment and discusses the major laser and light source systems, including the flashlamp-pumped pulsed dye laser (FLPDL), cryogen spray cooling in combination with the FLPDL, the potassium titanyl phosphate (KTP) laser and the intense pulsed light source, currently utilised for this procedure. Future opportunities for therapeutic improvement are also considered.

Notes: Abstract . A device for the preventative photodynamic therapy of secondary cataract has been developed. Disturbance of the epithelial cells of the crystalline lens during the primary cataract surgery and/or their migration thereafter can frequently generate secondary cataract. This can generally be prevented by destroying these epithelial cells. One approach is to expose them to a phototoxic dye which is then activated by selective exposure to light. For this purpose we designed a light distributor furnishing a torus-shaped light intensity profile, allowing for selective destruction of these epithelial cells, while preventing any potentially damaging exposure to other parts of the eye. The size and geometry of the device are adapted to the eye's anatomy, providing a relatively homogeneous irradiation of the treated zone of up to about 2.7 J/cm2 in 1 min. The device is easy to handle and is sterilisable with ethylene oxide gas.

Notes: Abstract . Some lasers produce a loud photo-acoustic effect when treating skin. Although safety issues with regard to eye and skin protection are well known, we were unable to find any literature relating to noise production. We therefore measured the sound levels produced by three Erbium-YAG lasers (Continuum Biomedical 1J and 2J, and Laserscope). Measurements were made at distances of 50 and 5 cm, to represent levels reaching the laser-user and the patient, respectively, at a variety of energy (0.5, 1, 1.5 and 2 J) and spot settings (2, 3.5, 5 and 7 mm). Levels ranged from 72 to 99 decibels frequently exceeding safe limits, and increased with increasing energy setting, decreasing spot size and decreasing distance. We conclude that sound levels should be measured for lasers in medical use, so that where necessary both the patients and healthcare workers may avoid this potential hazard by wearing appropriate ear protection.

Notes: Abstract This article describes the design, fabrication and initial testing of an enzymatic microreactor using Si, glass, epoxy and Perspex components. The microreactor geometry was defined by a photostructured EPON SU-8 spacer. This gave two chambers, an upstream chamber (vol. ∼2.7 μl) which was packed with control pore glass (CPG) and a downstream chamber (vol. ∼1.5 μl) which contained the detectors. Eleven EPON SU-8 columns separated the chambers and prevented the CPG from passing. Glucose detection via glucose oxidase immobilized to the CPG was used as a model system. Conversion efficiencies between 15 and 60% at flow rates of 150 and 26 μ1 min−1 respectively were obtained. Overall the system was found to be robust and to have a lifetime of at least one month.

Notes: Abstract In this paper, fluid coupled metallic micromachined needle arrays are designed, fabricated, packaged, and characterized. The described hollow metallic needle arrays include design features such as dual structural supports and needle coupling channels. The supports and needle walls are formed by micro-electroformed metal to provide increased structural integrity. The needle coupling channels are used to fluidically interconnect the needles and allow pressure equalization and balance of fluid flow between needles. In addition, the needle coupling channels minimize the effects of restricted needle passages by providing a redistribution point for fluid flow between them. The optimum design for the needle coupling channels is investigated using an ANSYS finite element numerical model. The significance of this work includes the development of hollow, metallic micromachined needle arrays for biomedical applications, as well as, a discussion of structural, fluidic, and biological design considerations

Notes: Abstract In this paper a low cost, multi-channel separation and detection system is reported for use in fast, high throughput screening of bioanalytes. Applications include genetic engineering and drug discovery, particularly combinatorial chemistry. A prototype detection system is presented which is comprised of an array of 50 microchannels fabricated on 25 mm×75 mm glass substrates with planar, thin film metal electrodes for electrophoresis and electrochemiluminescence excitation. Fluidic interconnects to the microchannels via standard tubing provide quick and facile interfacing to external macro components, controllers and/or other microsystems. The fabrication process is readily scalable to higher density arrays.

Notes: Abstract Micromachining technologies were established to fabricate microelectrode arrays and devices for interfacing parts of the central or peripheral nervous system in case of neuronal disorders. The devices were part of a neural prosthesis that allows simultaneous multichannel recording and multisite stimulation of neurons. Overcoming the brittle mechanics of silicon, we established a process technology to fabricate light-weighted and highly flexible polyimide based devices. Concerning the challenging housing demands close to the nerve to prevent mechanical induced nerve traumatization, we integrated interconnects to decouple the nerve interface from plugs and signal processing electronics. Hybrid integration with a new assembling technique—the MicroFlex interconnection (MFI)—has been applied for the connection of the flexible microsystems to silicon microelectronics. In this paper, we present different shapes and applications of the flexible electrodes: sieve electrodes for regeneration studies, cuff electrodes for interfacing peripheral nerves, and a retina implant for ganglion cell stimulation. The discussion is focused on electrode and material properties and the hybrid assembly of a fully implantable neural prosthesis.

Notes: Abstract The increasingly global capacity of civilisation to manipulate natural and human capital has fuelled faith in the economic conceit that humankind can be freed from its dependence on nature. In addition, enormous wealth, enjoyed by a small proportion of the world's richest people, in large part derived from exploitation of its poor and least empowered populations, is justified by doctrines of 'wealth and health for all,' in the face of mounting contrary evidence.

Notes: Abstract The Amazon is a vast region of about 7 million km2, encompassing nine countries in the northern part of South America. Around 70% of the Amazon, or approximately 5 million km2, are within the Brazilian National boundary (Figure 1). It is, in many aspects, a unique region characterised by the largest stock of biological diversity in the world, an extensive network of rivers, the persistence of traditional Indian population — some still isolated from contacts with modern society — and significant mineral reserves. Around 65% of the Brazilian Amazon region is covered by the different sub-types of the tropical rainforest, with some 100.00 km2 of it periodically flooded every year. Another 1.3 million km2 are covered with other types of vegetation, especially tropical savannahs. The Brazilian Amazon is home to about 20 million inhabitants of which only 0.8% are Indian; the urbanisation rate in the region is about 60%. Although industrial exploitation of the natural resources of the region has been under way since the last quarter of the 19th century, it was only in the last 30 years that human intervention in the Amazonian ecosystems has been followed by major negative impacts. This has happened for economic, social and geopolitical reasons and included the opening of new roads, the fiscal incentives for industrialisation in urban areas, building of dams for the generation of hydroelectricity, intensive mining schemes, settlements for agricultural development and cattle raching. This has brought profound social, cultural as well as environmental changes, often resulting in benefits for only a small part of the population. Among the adverse environmental impacts, deforestation stands as the most important one due to its large extension, rapid progress and global and multi-factorial consequences. All these processes — social as well as environmental — have been changing the disease profile in the region (Figure 2). With the intense and widespread exploitation of the Amazon, new diseases are emerging, others are being introduced and the old ones are becoming out of control.

Notes: Abstract. Avian species, particularly waterfowl, are the natural hosts of influenza A viruses. Influenza viruses bearing each of the 15 hemagglutinin and nine neuraminidase subtypes infect birds and serve as a reservoir from which influenza viruses or genes are introduced into the human population. Viruses with novel hemagglutinin genes derived from avian influenza viruses, with or without other accompanying avian influenza virus genes, have the potential for pandemic spread when the human population lacks protective immunity against the new hemagglutinin. Avian influenza viruses were thought to be limited in their ability to directly infect humans until 1997, when 18 human infections with avian influenza H5N1 viruses occurred in Hong Kong. In 1999, two human infections with avian influenza H9N2 viruses were also identified in Hong Kong. These events established that avian viruses could infect humans without acquiring human influenza genes by reassortment in an intermediate host and highlighted challenges associated with the detection of human immune responses to avian influenza viruses and the development of appropriate vaccines.

Notes: Abstract. The amyloid β-peptide (Aβ) is a 4-kDa species derived from the amyloid precursor protein, which accumulates in the brains of patients with Alzheimer’s disease. Although we lack full understanding of the etiology and pathogenesis of selective neuron death, considerable data do imply roles for both the toxic Aβ and increased oxidative stress. Another significant observation is the accumulation of abnormal, ubiquitin-conjugated proteins in affected neurons, suggesting dysfunction of the proteasome proteolytic system in these cells. Recent reports have indicated that Aβ can bind and inhibit the proteasome, the major cytoslic protease for degrading damaged and ubiquitin-conjugated proteins. Earlier results from our laboratory showed that moderately oxidized proteins are preferentially recognized and degraded by the proteasome; however, severely oxidized proteins cannot be easily degraded and, instead, inhibit the proteasome. We hypothesized that oxidatively modified Aβ might have a stronger (or weaker) inhibitory effect on the proteasome than does native Aβ. We therefore also investigated the proteasome inhibitory action of Aβ 1–40 (a peptide comprising the first 40 residues of Aβ) modified by the intracellular oxidant hydrogen peroxide, and by the lipid peroxidation product 4-hydroxynonenal (HNE). H2O2 modification of Aβ 1–40 generates a progressively poorer inhibitor of the purified human 20S proteasome. In contrast, HNE modification of Aβ 1–40 generates a progressively more selective and efficient inhibitor of the degradation of fluorogenic peptides and oxidized protein substrates by human 20S proteasome. This interaction may contribute to certain pathological manifestations of Alzheimer’s disease

Notes: Abstract. Selenoprotein P (SeP) is an extracellular, monomeric glycoprotein containing up to 10 selenocysteine residues in the polypeptide chain. It is ubiquitously expressed in mammalian tissues, and in human plasma it accounts for at least 40% of the total selenium concentration. SeP binds to heparin and cell membranes, and is associated with endothelial cells. SeP in human plasma protects against peroxynitrite-mediated oxidation and reduces phospholipid hydroperoxide in vitro, in accordance with the presumption that it has a function as an extracellular oxidant defense. Immunochemical assays have demonstrated that its concentration in plasma varies much with selenium intake, but other factors also have an influence.

Notes: Abstract. The type III inositol 1,4,5-trisphosphate receptor (InsP3R) is an important intracellular calcium (Ca2+) release channel in the pancreatic β cell. Pancreatic β cells secrete insulin following a characteristic change in membrane potential that leads to an increase in cytoplasmic Ca2+. Both extracellular Ca2+ and Ca2+ mobilized from InsP3-sensitive stores contribute to this increase. RIN-m5F cells, an insulin-secreting β cell line, preferentially express the type III InsP3R . These cells have been useful in determining the regulatory properties of the type III InsP3R and the role of this isoform in an intact cell. The type III InsP3R is ideal for signal initiation because high cytoplasmic Ca2+ does not inhibit its activity . Altered insulin secretion, the result of changes in Ca2+ handling by the β cell, has significant clinical consequences.

Notes: Abstract. Ferrochelatase (E.C. 4.99.1.1, protoheme ferrolyase) catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme (heme). In the past 2 years, the crystal structures of ferrochelatases from the bacterium Bacillus subtilis and human have been determined. These structures along with years of biophysical and kinetic studies have led to a better understanding of the catalytic mechanism of ferrochelatase. At present, the complete DNA sequences of 45 ferrochelatases from procaryotes and eucaryotes are available. These sequences along with direct protein studies reveal that ferrochelatases, while related, vary significantly in amino acid sequence, molecular size, subunit composition, solubility, and the presence or absence of nitric-oxide-sensitive [2Fe-2S] cluster.

Notes: Abstract. Replication of linear genomes is incomplete and leaves terminal gaps. Solutions to this ‘end replication’ problem can be traced back to the prebiotic RNA world: ‘fossils’ of the presumptive archetypes of telomere structure and of the telomerase enzyme are retained in the terminal structures of some RNA viruses. Telomerase expression in mammals is ubiquitous in embryonic tissues but downregulated in somatic tissues of adults. Exceptions are regenerative tissues and, notably, tumor cells. Telomerase activation is controlled by cellular proliferation, and it is an early step in the development of many tumors. In contrast to mammals, indeterminately growing multicellular organisms, such as fish and crustaceae, maintain telomerase competence in all somatic tissues. In human tumor diagnostics, detection of proliferation markers with monoclonal antibodies is well established, and in this review, the significance of additional telomerase assays is evaluated. Telomerase inhibitors are attractive goals for application in tumor therapy, and telomerase knockout mice have proven that telomere erosion limits the lifespan of cells in vivo. In contrast, telomerase stimulation can be used to expand the potential of cellular proliferation in vitro, with possible applications for transplantation of in vitro expanded human cells, for immortalizing primary human cells as improved tissue models and for the isolation of otherwise intractable products, such as genuine human monoclonal antibodies.

Notes: Abstract. The methanol extract of the stem of Sinofranchetia chinensis inhibited the activity of xanthine oxidase in vitro. Bioassay-guided purification led to the isolation of liquiritigenin and isoliquiritigenin as the main xanthine oxidase inhibitors. This inhibition of enzyme activity was found to be dose dependent, with an IC50 value of approximately 49.3 μM for liquiritigenin and 55.8 μM for isoliquiritigenin. Lineweaver-Burk transformation of the inhibition data indicated that the inhibition was of a mixed type for both liquiritigenin and isoliquiritigenin. For liquiritigenin, the Ki and KI were determined to be 14.0 μM and 151.6 μM, respectively. For isoliquiritigenin, the Ki and KI were determined to be 17.4 μM and 81.9 μM, respectively. These results suggest that these natural products could be used to treat conditions where the inhibition of xanthine oxidase is warranted.

Notes: Abstract. The topic of nuclear Ca2+ signalling is beset by discrepant observations of substantial nuclear/cytoplasmic gradients. The reasons why some labs have recorded such gradients, whilst other workers see equilibration of Ca2+ cyt and Ca2+ nuc using the same cells and techniques, is unexplained. Furthermore, how such gradients could arise across the NE that possesses many highly-conductive NPCs is a mystery. Although nuclei may have the capacity to be autonomous signalling entities, with functional Ca2+ release channels and an inositide cycle, the balance of evidence suggests that Ca2+ release on the inner NE does not occur during physiological stimulation. Our work suggests that elementary Ca2+ release events originating in the cytoplasm can give rise to Ca2+ nuc signals without causing elevation of the bulk cytoplasm. Clearly, the many Ca2+ signalling mechanisms that may impinge on Ca2+ nuc will remain a topic of controversy and debate for some time.

Notes: Abstract. Ca2+ is a uniquely important messenger that penetrates into cells through gated channels to transmit signals to a large number of enzymes. The evolutionary choice of Ca2+ was dictated by its unusual chemical properties, which permit its reversible complexation by specific proteins in the presence of much larger amounts of other potentially competing cations. The decoding of the Ca2+ signal consists in two conformational changes of the complexing proteins, of which calmodulin is the most important. The first occurs when Ca2+ is bound, the second (a collapse of the elongated protein) when interaction with the targeted enzymes occurs. Soluble proteins such as calmodulin contribute to the buffering of cell Ca2+, but membrane intrinsic transporting proteins are more important. Ca2+ is transported across the plasma membrane (channel, a pump, a Na+/Ca2+ exchanger) and across the membrane of the organelles. The endoplasmic reticulum is the most dynamic store: it accumulates Ca2+ by a pump, and releases it via channels gated by either inositol 1,4,5-trisphosphate (IP3) and cyclic adenosine diphosphate ribose (cADPr). The mitochondrion is more sluggish, but it is closed-connected with the reticulum, and senses microdomains of high Ca2+ close to IP3 or cADPr release channels. The regulation of Ca2+ in the nucleus, where important Ca2+-sensitive processes reside, is a debated issue. Finally, if the control of cellular Ca2+ homeostasis somehow fails (excess penetration), mitochondria ‘buy time’ by precipitating inside Ca2+ and phosphate. If injury persists, Ca2+-death eventually ensues.

Notes: Abstract. Results from several laboratories have suggested that peptide factors known as neurotrophins may play roles coupling changes in synaptic activity to lasting changes in synaptic function. Consistent with this idea, increases in synaptic activity and intracellular calcium induce the expression of the gene that encodes the neurotrophin, brain-derived neurotrophic factor. Recently, a pathway has been elucidated in neurons by which the influx of extracellular calcium evokes brain-derived neurotrophic factor transcription (BDNF). Calcium activates BDNF transcription through two adjacent calcium response elements within one of the promoters of the BDNF gene. One of the two elements binds to the cyclic adenosine monophosphate (AMP) response element binding protein (CREB) transcription factor, and interfering with CREB or related family members inhibits calcium-dependent BDNF transcription. This review focuses on the mechanisms by which calcium influx regulates brain-derived neurotrophic factor expression and the implications that these results have for potential roles of neurotrophins in synaptic function.

Notes: Abstract. In the nervous system, calcium signals associated with electrical activation of neurons induce gene transcription that may be important for long-lasting adaptation. The type of transcriptional response is determined by the properties of the calcium signal that include subcellular localisation, amplitude, duration and the physical site of entry. Here we review calcium-regulated protein kinase cascades and discuss potential mechanisms through which they propagate calcium signals to and within the nucleus and control the activity of transcription factors and transcriptional coactivators.

Notes: Abstract. Deposition of basic calcium phosphate (hydroxyapatite, octacalcium phosphate and tricalcium phosphate) (BCP) and crystalline calcium pyrophosphate dihydrate (CPPD) is associated with a variety of aging-related pathologies, including osteoarthritis, cartilage degeneration and pseudogout. These diseases of calcium deposition serve as some of the best-studied examples of how calcium-regulated changes in gene expression can directly lead to pathogenic consequences. Tissue damage can result when crystals stimulate cells to release matrix-degrading molecules or secrete cytokines that stimulate the release of matrix-degrading molecules. Exposure of cultured cells to crystals induces expression of cellular proto-oncogenes such as c-fos, c-myc and c-jun, by a calcium-dependent mechanism, and this response can be blocked by a potential therapeutic compound, phosphocitrate. Activation of the c-fos and c-jun genes is directly involved in expression of metalloproteinases such as collagenase and stromelysin, suggesting that crystal-mediated activation of these genes is directly involved in pathogenesis. In this review recent advances in the molecular mechanisms responsible for crystal-mediated cell activation are discussed.

Notes: Abstract. Water has been recognized as one of the major structuring factors in biological macromolecules. Indeed, water clusters influence many aspects of biological function, and the water-protein interaction has long been recognized as a major determinant of chain folding, conformational stability, internal dynamics, binding specificity and catalysis. I discuss here several themes arising from recent progress in understanding structural aspects of ‘direct’ and ‘indirect’ ligands in terms of enzyme-substrate interactions, and the role of water bridges in enzyme catalysis. The review also attempts to illuminate issues relating to efficiency, through solvent interactions associated with enzymic specificity, and versatility. Over the years, carbonic anhydrase (CA; carbonate hydro-lyase, EC 4.2.1.1) has played a significant role in the continuing delineation of principles underlying the role of water in enzyme reactions. As a result of its pronounced catalytic power and robust constitution CA was transformed into a veritable ‘laboratory’ in which active site mechanisms were rigorously tested and explored.

Notes: Abstract. Water often acts as a critical reactant in cellular reactions. Its role can be detected by modulating water activity with osmotic agents. We describe the principles behind this ‘osmotic stress’ strategy, and survey the ubiquity of water effects on molecular structures that have aqueous, solute-excluding regions. These effects are seen with single-functioning molecules such as membrane channels and solution enzymes, as well as in the molecular assembly of actin, the organization of DNA and the specificity of protein/DNA interactions

Notes: Abstract. The complete sequence of the 3-kb cDNA and the 5′ genomic structure are reported for the gene encoding the shrimp alpha-glucosidase. Alpha-glucosidase cDNA was isolated from a shrimp digestive gland cDNA library. The 2830-base pair cDNA contains an open reading frame that encodes 919 amino acids. The shrimp alpha-glucosidase cDNA shows a high level of identity with that of the human sucrase-isomaltase, human maltase-glucoamylase, and human acid lysosomal alpha-glucosidase, indicating that the protein shares the same structural domains. The similarities among these proteins are found as clusters and characterize the glycosyl hydrolase family 31. To our knowledge, this is the first report to describe a satellite sequence in the 5′ genomic structure before the TATA box in an invertebrate sequence.

Notes: Abstract. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are produced during cell activation and have multiple effects on cells. A family of seven transmembrane-spanning domain G-protein-coupled receptors, named Edg, mediate these effects of LPA and S1P. In this study, transient overexpression of Edg-2 sensitized MG63 human osteosarcoma cells to both LPA- and S1P-mediated stimulation of fibronectin matrix deposition and actin stress fiber formation. Both lipids were active in the 1–20 nM concentration range on cells transfected with Edg-2 as compared to the 10–200 nM range on mock-transfected cells. The signaling pathway for matrix deposition by Edg-2-transfected cells was Rho dependent. Overexpression of Edg-2 also caused a tenfold decrease in the concentration of either LPA or S1P that activated MAPKinase (Erk1/2) in MG63 cells. LPA- or S1P-stimulated activation of Erkl/2 was Gi dependent. These results indicate that, in MG63 cells, Edg-2 mediates actin stress fiber formation, fibronectin matrix assembly, and MAPKinase activation in response to either LPA or S1P.

Notes: Abstract. To investigate changes in BRCA1 following DNA damage, we exposed MCF-7 cells to increasing doses of ultraviolet C. We observed an increase in BRCA1 protein levels above 78 J/m2. This increase was observed as early as 5 min after irradiation. BRCA1 levels were then observed to decrease after 2 h, consistent with the previously published data. By pretreating with cycloheximide prior to irradiation, we observed a decrease in the protein half-life, from 3.5 h to 53 min, suggesting that a decrease in protein half-life may cause the lower levels of BRCA1 after irradiation. We also observed an increase in BRCA1 mRNA within 15 min of irradiation, followed by a decrease after 4 h. These data suggest that newly translated protein may contribute to increases in BRCA1 protein levels. The very rapid changes in BRCA1 support its role as a sensor of DNA damage, as opposed to being a repair gene.

Notes: Abstract. Na+-dependent D-glucose and D-galactose transport were studied in brush-border membrane vesicles (BBMVs) from kidney cortex isolated from both spontaneously hypertensive rats (SHR) and their normotensive genetic control Wistar-Kyoto (WKY) rats. Initial rates and accumulation ratios of Na+-dependent D-glucose and D-galactose transport were significantly lower in SHR compared with WKY, the observed decreases being similar for both substrates. To explain the reduction in sugar transport by renal BBMVs, the density of Na+-dependent sugar cotransporters was studied in BBMVs from kidney cortex isolated from SHR and WKY rats. Phlorizin-specific binding and Western blot analysis indicated a reduction in the density of the cotransporters in SHR relative to WKY rats. This reduction was similar to those found for the initial rates and accumulation ratios for D-glucose and D-galactose in SHR. Na+ uptake, studied using 22Na+, was significantly increased in SHR, so the observed reduction in sugar transport could be due to disruption of the Na+ gradient between renal BBMVs in SHR. Furthermore, a significant decrease in the activity of Na+-K+-ATPase was observed in SHR. In conclusion, changes in the density of the Na+-dependent sugar cotransporter and in the Na+ gradient across the brush-border membranes might be involved in the observed reduction in sugar transport by renal BBMVs from SHR.

Notes: Abstract. Enzymatic radical catalysis is defined as a mechanism of catalysis by which enzymes catalyze chemically difficult reactions by utilizing the high reactivity of free radicals. Adenosylcobalamin (coenzyme B12) serves as a cofactor for enzymatic radical reactions. The recent structural analysis of adenosylcobalamin-dependent diol dehydratase revealed that the substrate 1,2-propanediol and an essential potassium ion are located inside a (β/α)8 barrel. Two hydroxyl groups of the substrate coordinate directly to the potassium ion which binds to the negatively charged inner part of the cavity. Cobalamin bound in the base-on mode covers the cavity to isolate the active site from solvent. Based on the three-dimensional structure and theoretical calculations, a new mechanism for diol dehydratase is proposed in which the potassium ion plays a direct role in the catalysis. The mechanisms for generation of a catalytic radical by homolysis of the coenzyme Co-C bond and for protection of radical intermediates from undesired side reactions during catalysis are discussed based on the structure. The reactivating factors for diol and glycerol dehydratases have been identified. These factors are a new type of molecular chaperone which participate in reactivation of the inactivated holoenzymes by mediating ATP-dependent exchange of the modified coenzyme for free intact coenzyme.

Notes: Abstract. This review focuses on the biochemical, biophysical, and catalytic properties of terminase, an enzyme involved in bacteriophage λ genome packaging. The holoenzyme possesses ATPase, DNA strand-separation, and site-specific nuclease activities that work in concert to insert a viral genome into the confines of a preformed capsid. Moreover, the terminase subunits are part of a series of nucleoprotein complexes involved in genome packaging, including remarkably stable intermediates that transition to a highly mobile DNA packaging ‘machine.’ Models for the assembly and interconversion of these complexes are presented. Interactions between the catalytic sites in the enzyme complex, and modulation of these catalytic activities as it relates to the assembly and relative stability of the packaging intermediates are discussed. This ordered progression of nucleoprotein intermediates is a common theme in biology as demonstrated by mechanistic similarities between viral DNA packaging, the initiation of chromosomal replication, and the initiation of transcription. Terminase is thus part of a growing number of examples of biological ‘machines’ or molecular ‘motors.’

Notes: Abstract. The advent of the polymerase chain reaction (PCR) has revolutionised the way in which molecular biologists view their task at hand, for it is now possible to amplify and examine minute quantities of rare genetic material: the limit of this exploration being the single cell. It is especially in the field of prenatal diagnostics that this ability has been readily seized upon, as it has opened up the prospect of preimplantation genetic analysis and the use of fetal cells enriched from the blood of pregnant women for the assessment of single-gene Mendelian disorders. However, apart from diagnostic applications, single-cell PCR has proven to be of enormous use to basic scientists, addressing diverse immunological, neurological and developmental questions, where both the genome but also messenger RNA expression patterns were examined. Furthermore, recent advances, such as optimised whole genome amplification (WGA) procedures, single-cell complementary DNA arrays and perhaps even single-cell comparative genomic hybridisation will ensure that the genetic analysis of single cells will become common practice, thereby opening up new possibilities for diagnosis and research.

Notes: Abstract. The heart is an important target organ for cholinergic function. In this study, muscarinic receptor subtype(s) in the human heart were determined using reverse transcription-polymerase chain reaction. Our results demonstrated muscarinic receptor M2 and M3 subtype RNA in left/right atria/ventricles of donor hearts. Receptor autoradiography analysis using selective muscarinic ligands indicated an absence of M1 receptor subtype in the human heart. The level of muscarinic receptor binding in atria was two to three times greater than in ventricles. Our results suggest that muscarinic receptors in the human heart are of the M2 and M3 subtypes. This is the first report of M3 receptors in the human myocardium.

Notes: Abstract. Female reproductive tissues possess a unique ability to accommodate a remarkable amount of cell turnover and extracellular matrix (ECM) remodeling following puberty. Cellular structures within ovary, uterus, and mammary tissue not only change cyclically in response to ovarian hormones but also undergo differentiation during pregnancy, and eventually revert to that resembling the pre-pregnant stage. Cell proliferation, apoptosis, invasion, and differentiation are integral cellular processes that are precisely regulated in reproductive tissues, but become dysregulated in pathologies such as cancer. Explicit reorganization of ECM and basement membranes is also critical to preserve the form and function of these tissues. Here we review the evidence that coordinated spatiotemporal expression patterns of matrix metalloproteinase (MMP) genes and their tissue inhibitors (TIMPs) are important in cell and ECM turnover of the ovary, uterus, and mammary tissues. We discuss how perturbation in these gene families may impact the biology of these reproductive tissues and the factors implicated in the control of MMP and TIMP gene expression. The observed trends in MMP and TIMP expression involved in ovarian and mammary carcinomas are also presented.

Notes: Abstract. Cell migration in three-dimensional (3-D) extracellular matrix (ECM) is not a uniform event but rather comprises a modular spectrum of interdependent biophysical and biochemical cell functions. Haptokinetic cell migration across two-dimensional (2-D) surfaces consists of at least three processes: (i) the protrusion of the leading edge for adhesive cell-substratum interactions is followed by (ii) contraction of the cell body and (iii) detachment of the trailing edge. In cells of flattened morphology migrating slowly across 2-D substrate, contact-dependent clustering of adhesion receptors including integrins results in focal contact and stress fiber formation. While haptokinetic migration is predominantly a function of adhesion and deadhesion events lacking spatial barriers towards the advancing cell body, the biophysics of the tissues require a set of cellular strategies to overcome matrix resistance. Matrix barriers force the cells to adapt their morphology and change shape and/or enzymatically degrade ECM components, either by contact-dependent proteolysis or by protease secretion. In 3-D ECM, in contrast to 2-D substrate, the cell shape is mostly bipolar and the cytoskeletal organization is less stringent, frequently lacking discrete focal contacts and stress fibers. Morphologically large spindle-shaped cells (i.e., fibroblasts, endothelial cells, and many tumor cells) of high integrin expression and strong cytoskeletal contractility utilize integrin-dependent migration strategies that are coupled to the capacity to reorganize ECM. In contrast, a more dynamic ameboid migration type employed by smaller cells expressing low levels of integrins (i.e., T lymphocytes, dendritic cells, some tumor cells) is characterized by largely integrin-independent interaction strategies and flexible morphological adaptation to preformed fiber strands, without structurally changing matrix architecture. In tumor invasion and angiogenesis, migration mechanisms further comprise the migration of entire cell clusters or strands maintaining stringent cell-cell adhesion and communication while migrating. Lastly, cellular interactions, enzyme and cytokine secretion, and tissue remodeling provided by reactive stroma cells i.e. fibroblasts and macrophage contribute to cell migration. In conclusion, depending on the cellular composition and tissue context of migration, diverse cellular and molecular migration strategies can be developed by different cell types.

Notes: Abstract. Rieger syndrome (RIEG 1; MIM 180500) is an autosomal dominant disorder of morphogenesis. It is a phenotypically heterogeneous disorder characterized by malformations of the eyes, teeth, and umbilicus. RIEG belongs to the Axenfeld-Rieger group of anomalies, which includes Axenfeld anomaly and Rieger anomaly (or Rieger eye malformation), which display ocular features only. Recently, mutations in the homeodomain transcription factor, PITX2, have been shown to be associated with Rieger syndrome. This review discusses the clinical manifestations of Rieger syndrome and how they correlate with the current molecular and biochemical studies on this human disorder.

Notes: Abstract. The Ras family of small GTPases includes three closely related proteins: H-, K-, and N-Ras. Ras proteins are involved in the transduction of signals elicited by activated surface receptors, acting as key components by relaying signals downstream through diverse pathways. Mutant, constitutively activated forms of Ras proteins are frequently found in cancer. While constitutive Ras activation induces oncogenic-like transformation in immortalized fibroblasts, it causes growth arrest in primary vertebrate cells. Induction of p53 and cyclin-dependent kinase inhibitors such as p15INK4b, p16INK4a, p19ARF, and p21WAF1 accounts for this response. Interestingly, while ras has usually been regarded as a transforming oncogene, the analysis of Ras function in most of the cellular systems studied so far indicates that the promotion of differentiation is the most prominent effect of Ras. While in some cell types, particularly muscle, Ras inhibits differentiation, in others such as neuronal, adipocytic, or myeloid cells, Ras induces differentiation, in some cases accompanied by growth arrest. Several possible mechanisms for the pleiotropic effects of Ras in animal cells are discussed.

Notes: Abstract. Lyme disease is a multisystem illness initiated upon infection with the spirochete Borrelia burgdorferi. Whereas the majority of patients who develop Lyme arthritis may be successfully treated with antibiotic therapy, about 10% go on to develop arthritis which persists for months to years, despite antibiotic therapy. Development of what we have termed treatment-resistant Lyme arthritis has previously been associated with both the presence of particular major histocompatibility complex class II alleles and immunoreactivity to the spriochetal outer surface protein A (OspA). Recently, we showed that patients with treatment-resistant Lyme arthritis, but not patients with other forms of arthritis, generate synovial fluid T cell responses to an immunodominant epitope of OspA and a highly homologous region of the human-lymphocyte-function-associated antigen-1α L chain. Identification of a bacterial antigen capable of propagating an autoimmune response against a self-antigen provides a model of molecular mimicry in the pathogenesis of treatment-resistant Lyme arthritis.

Notes: Abstract. Appropriate targeting and clustering of ionotropic glutamate receptors (iGluRs) is critical for the formation and maintenance of excitatory synapses. Recent studies have demonstrated that the synaptic localization of iGluR subtypes is remarkably heterogeneous and subject to regulation over time scales ranging from minutes to months. These findings, together with the identification of key protein binding partners of iGluRs, have opened a window onto the complex cell biology of iGluR membrane trafficking. In this article, we review recent findings on the cellular and molecular mechanisms involved in localizing iGluRs at synapses and discuss their implications for synaptogenesis and synaptic plasticity.

Notes: Abstract. Synaptic plasticity is the foremost candidate mechanism to explain the rapid acquisition of memories. In the mammalian brain, the NMDA subclass of glutamate receptors plays a central role in the induction of several forms of use-dependent plasticity. The finding that modifications in synaptic strength are largely expressed by receptors of the AMPA subclass has focused attention on molecular mechanisms that affect their function and targeting. Receptor plasticity has also been reported in pathological situations, notably in animal and human forms of epilepsy. Which of these changes are causally implicated in the generation of seizures, and which may be compensatory or neuroprotective adaptations, has not been fully resolved.

Notes: Abstract. Long-term potentiation (LTP) and long-term depression (LTD) are two electrophysiological models that have been studied extensively in recent years as they may represent basic mechanisms in many neuronal networks to store certain types of information. In several brain regions, it has been shown that these two forms of synaptic plasticity require sufficient dendritic depolarization, with the amplitude of the calcium signal being crucial for the generation of either LTP or LTD. The rise in calcium concentration mediated by the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors has been proposed to stimulate various calcium-dependent enzymatic processes that could convert the induction signal into long-lasting changes in synaptic structure; protein kinases and phosphatases have so far been considered predominantly with regard to LTP and LTD formation. According to several lines of experimental evidence, changes in synaptic function observed with LTP and LTD are thought to be the result of modifications of postsynaptic currents mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtype of glutamate receptors. Moreover, it has become apparent recently that activation of the calcium-dependent enzyme phospholipase A2 (PLA2) could be part of the molecular mechanisms involved in alterations of AMPA receptor properties during long-term changes in synaptic operation. In the present review, we will first describe the results that indicate a critical role of the phospholipases in regulating synaptic function. Next, sections will be devoted to the effects of PLA2 and phospholipids on the binding properties of glutamate receptors, and a revised biochemical model will be presented as an attempt to integrate the PLA2 enzyme into the mechanisms (in particular kinases and phosphatases) that participate in adaptive neural plasticity. Finally, we will review data relevant to the issue of selective changes in AMPA binding after environmental enrichment and LTP.

Notes: Abstract. Nuclear receptors are DNA-binding factors which regulate the transcription of sets of specific genes in response to cognate ligands, usually small lipophilic molecules, thus controlling numerous physiological events in development, procreation, homeostasis, and cellular life. Their ligand-dependent activity makes nuclear receptors obvious targets for drug design in many therapeutic areas. Crystallographic studies have revealed the structure of isolated domains but not, yet, of a whole protein, probably due to an intrinsic flexibility at work in nuclear receptor action. The structure of DNA-binding domain dimers in complex with an oligonucleotide has brought insights into how nuclear receptors recognize and bind to their target sequences (‘response elements’). The structure of several ligand-binding domains in different ligation states has provided evidence for a ligand-dependent transcriptional switch and a molecular basis for the mode of action of agonists and antagonists.

Notes: Abstract. Evidence is clear that each melatonin-producing cell in the chick pineal gland contains a circadian oscillator that continues to function in vitro, resulting in a prominent day/night rhythm of melatonin secretion. The aim of the present investigation was to examine whether the circadian organization of the gland has an electrophysiological correlate. To this end, single-cell recordings were made from isolated chick pineal glands kept in vitro under a light/dark cycle of 12:12 h, identical to that of the donors, or under continuous light or darkness. In all the glands investigated, a very small percentage of cells exhibited sodium-dependent spontaneous spike activity with a mean frequency below 10 Hz. The cells revealed rhythms with periods in the 15- to 60-min range and, additionally, exhibited ultradian and circadian rhythms in firing, with periods of 10.75 ± 1.06 h and 26.25 ± 1.26 h (mean ± standard deviation), respectively. Most of the cells exhibited circadian rhythms with higher activity during daytime than at night, showing that the electrical activity and melatonin rhythm were out of phase. Under constant light or darkness, the circadian rhythm persisted. When the light/dark cycle of the donors was phase-advanced by 5 h, the cells revealed complete entrainment. We discuss whether the cells, albeit small in number, could function as a secondary ultradian/circadian oscillator contributing to the ultradian/circadian organization of the gland.