ALBERT

Notes: Summary Ultrastructural studies have been carried out on the innervation of the interstitial gland of the mouse ovary. In addition Falck's fluorescence method was applied. 1. Fluorescence microscopy: In the ovarian stroma green fluorescent nerve fibers are frequently to be found in the surroundings of large and small vessels. Seldom small fibers invade blocks of interstitial cells; however, their final ramification is not discernible. 2. Electron microscopy: Terminal fibers of the autonomic nervous system reach the cells of the interstitial gland from all sides. They may be independent from the course of the vessels. Many axons penetrate the basal membrane and come into close contact with interstitial cells, partly by forming large swellings (boutons), which may be deeply embedded into the cytoplasm of the innervated cell. The synaptic cleft is about 200 Å wide. Specialized pre- and postsynaptic membranes have not been found. The innervated cells show no peculiarities. The possible function of the synapses is discussed.

Notes: Summary The fine structure of the nerves of the ovarian stroma of the domestic fowl is described for the first time. In the fowl, the nerves are concentrated upon blood vessels, smoth muscles and mainly, the thecal gland with the steroid-producing cells. Myelinated as well as unmyelinated nerve fibers were observed. Numerous axon terminals representing adrenergic and also presumptive cholinergic nerve fibers are regularly seen in membranous contact with steroid-producing cells. In these axon terminals microvesicles are oriented towards the steroid-producing cells indicating a specialization of the surface from axon-to-cell contact. Evidence has been presented that there is a membranous neuro-humoral contact between the peripheral autonomie nervous system and the steroid-producing cells in the ovary. The present investigation has demonstrated that there is morphologic evidence for a nervous control of steroid-producing cells. The physiological importance of this neuro-humoral contact is discussed.

Notes: Summary Pituicytes of Rana pipiens could be classified into two types, pale and dense, according to their relative densities of cytoplasm and the populations of free ribosomes and cell organelles. An intermediate type of pituicyte was also recognized. Lipid droplet such as are typical in the cytoplasm of mammalian pituicytes, are not in the cytoplasm of either types of frog pituicyte. Both types have long cytoplasmic processes which run among the nerve fibers, and some of them end at the pericapillary space. Nerve endings making synapse-like contacts with the cell bodies or the processes of the pituicyte are frequent. According to the structures and sizes of granules and vesicles in the nerve endings, these endings are classified into one of three types: 1) A, which appears to be a peptidergic neuronal ending containing dense granules 1,200–2,000 Å in diameter and small clear vesicles 300–400 Å in diameter; 2) B, which appear to be monoaminergic endings containing cored vesicles 600–1,000 Å in diameter and small clear vesicles 300–500 Å in diameter; 3) C, which appear to be cholinergic endings containing only small clear vesicles. Type C endings are relatively rare. In the synaptic area the axonal membranes appose those of the pituicytes across a gap of about 200 Å and numerous “presynaptic” vesicles are clustered or accumulated near the presynaptic membranes.

Notes: Summary The pars tuberalis of the hypophysis of Rana temporaria presents the general structural and the cytological characteristics of an endocrine gland. It is composed of elongated cells with long, branching processes ending on the external basement membrane of the pericapillary space. The pars tuberalis cells produce secretory granules which are accumulated in the pericapillary endings of the processes. Corresponding to its separate localization, the pars tuberalis of Rana temporaria has a separate vascularization of which the efferent capillaries anastomose with the capillary plexus of the median eminence. The general direction of the blood flow of the pars tuberalis is towards the capillaries of the median eminence. Also, the secretory products of the pars tuberalis pass into the blood stream of the hypophysial portal system. Several characteristics of the pars tuberalis show that its function must be different from that of the pars distalis of the hypophysis. Moreover, in contrast with the pars distalis, the activity of the pars tuberalis is not regulated by neurohumoral factors. The results show that a role of the pars tuberalis in the regulation of the activity of the pars distalis of the hypophysis is not excluded.

Notes: Summary The innervation of the adrenal cortex of the rat and the pig is investigated with the electron microscope. Nerve fibers containing synaptic and two types of dense-cored vesicles come into contact with endocrine cells. There are no specialized pre- and postsynaptic membranes. The synaptic cleft is about 200 Å wide. Generally the basement membrane between nerve and cell is absent. These observations are discussed on the base of more recent experimental findings. Small fibers having an average diameter of about 0.2 to 0.5 μ and containing only tubules and filaments are considered to represent parts of an afferent nervous system.

Notes: Summary The innervation of the mesenteric microvasculature was studied in fetal and neonatal rabbits with the aid of methods demonstrating fluorescence of catecholamines and cholinesterase activity as well as a silver impregnation procedure. The results showed that: (1) adrenergic nerve fibers were present, coursing independently in the mesentery by day twenty-one of gestation, and were found routinely in the adventitia of arterioles and venules by day 25 of gestation; (2) cholinesterase positive cells and fibers of the myenteric plexus were present by day 18 of gestation but cholinergic fibers were not present in the mesentery until day 26; the latter not being associated with blood vessels; and (3) nerve fibers in the mesentery thought to be sensory stained positively with the Holmes silver method on day 18 of gestation.

Notes: Summary Quantitative electron microscopic studies have been carried out on the human thymus. According to the equation L v =(2n)/F (Hennig, 1963) we have calculated that there is less than 0.204 mm nerve per 1 mm3 thymus tissue inside the blood-thymus-barrier (level of significance of 0.95). This result is compared to the degree of innervation in brown adipose tissue, which contains more than 160 mm nerve per 1 mm3 tissue. The biological significance of the paucity of neuronal elements in the thymus is undetermined.

Notes: Summary 1. The Harderian gland in rabbits, representing the type of a tubulo-alveolar gland, is located on the medial and posterior aspect of the eyeball and consists of two different parts, a small white lobe and a larger red one. The secretory cells in the tubular endpieces of both lobes are lipids containing cells. The lipid droplets can be stained with Sudan IV and Sudan black B. The luminal surface of both cell types is characterized by an alcianophilia at pH 2,5. 2. The tubules of both lobes have a single layer of columnar epithelium. The lipid vacuoles in the cells of the red lobe are large, these of the white lobe small. The multilocular cytoplasm of all cells contains many free ribosomes and high amounts of mitochondria lying very closely together. All cells exhibit numerous and large Golgi-zones but only few ergastoplasm membranes. 3. The lateral surfaces of the secretory cells are connected by elaborate junctional complexes (Zonulae occludentes, zonulae adhaerentes, desmosomes). These lateral surfaces are increased by intercellular canaliculi. 4. Before being released into the glandular lumen, the limiting membranes of adjacent lipid droplets fuse, thus forming a large lipid vacuole. Extrusion generally is characterized by the coalescence of the limiting membrane with the plasmalemma, the formation of an opening at the cell surface and the discharge of the secretory lipid material. In the course of another mechanism of extrusion, the fat vacuoles are transported to the apical part of the cell where consequently the plasmamembrane bulges into the lumen. Eventually the fat vacuole is pinched off surrounded by a thin cytoplasmic envelope. 5. Terminal fibers of the autonomic nervous system penetrate the basal membrane and can be found closely attached to the secretory or myoepithelial cells, partly by forming large swellings, which may be deeply embedded into the cytoplasm of the innervated cell. These terminal parts of the axons contain groups of synaptic and dense-cored vesicles, mitochondria and neurotubuli. Specific pre- and postsynaptic membranes have not been observed. The possible function of the harderian gland is discussed.

Notes: Summary Electron microscopic studies have been carried out on the innervation of the mammalian anterior pituitary and parathyroids. The total area of grid squares (2.25·10−2mm2) examined was 2000 per gland and species. In the pituitary pars distalis and in the parenchyma of the parathyroid gland we did not observe a single axon profile. According to the equation $$L_V = \frac{{2n}}{F}$$ proposed by Hennig (1963) we have calculated that there might be—if any—0.133 mm of nerves per 1 mm3 tissue in those two endocrine glands (level of significance 0.95). Comparing these results to the degree of innervation in brown adipose tissue containing more than 160 mm nerve per 1 mm3 tissue we can not imagine that such a small degree of innervation is of any biological importance. In the pituitary pars tuberalis two types of axon terminals have been found both inside and outside the basement membrane surrounding the epithelial complexes. One type contains “synaptic” and two populations of smaller dense-cored vesicles, the other one contains a population of larger granules which have some properties of the classical elementary granules. Further investigations have to clarify the functional significance of those nerve endings.

Notes: Summary The penis retractor muscle of Helix pomatia is passed by thick nerve trunks, which probably are nerve nets. In the contracted muscle, they are folded meanderlike, in the extended they are pulled smooth. Four types of vesicles different in size and contents can be distinguished in the nerves. Varicose fibres accompany the muscle cells. Frequently the terminals are running in a groove of the muscle fibre. In certain axon types occur presynaptic accumulations of vesicles and thickenings of the terminal membrane. Terminal and muscle cell are separated by a cleft of 300 AE width. The muscle fibre membrane has no subsynaptic infoldings. Beside these axons thin, naked neurites are running through the connective tissue. They are characterized by a high content of neurotubuli. One part of the axons presumably possesses a monoaminergic transmitter. After the glutaraldehyde-dichromate-reaction they contain dense grana, whose diameters are mainly below 1000 AE. The nerve trunks fluoresce after exposure to paraformaldehyde vapour, excited with UV-light, green to green-yellow. The maximum of the excitation was determined at 413 nm and the maximum of emission at 496 respectively at 510 nm. It is concluded, that both, a catecholamine and 5-HT are responsible for the fluorescence. Extraction and paperchromatographic separation lead to the opinion, that the catecholamine is dopamine.

Notes: Summary Qualitative and quantitative studies were made to determine the amount of nerve fiber supplying corpora lutea (CL) of rats during the oestrous cycle and pregnancy and sow CL during days 4–6 after ovulation. Fluorescence microscopy of freeze-dried, paraformaldehyde treated (Falck-Hillarp method) rat ovaries reveals adrenergic nerve fibers which run along with vessels and form a network among interstitial gland cells. Nerve fibers do not enter the granulosa cell layer in follicles or CL. In the CL circumference both vascular and non-vascular nerves occur the latter being related to the fibromuscular layer and probably innervating smooth muscle cells. No striking differences exist between the innervation of the ovary in non-pregnant and pregnant rats. Bodian and methylene blue staining did not contribute to a more detailed knowledge of rat ovary nerve supply. Electron microscopic quantitative analysis of rat and pig CL (rat: day 18 of pregnancy; pig: day 4–6 after ovulation) revealed no axon profiles in 2.000 grid squares (one square measuring 2.25×10-2 mm2) of randomly taken CL sections. Thus it was possible to calculate an upper limit of 133 μm of nerve fibers per 1 mm3 CL tissue, in case there were any at all.

Notes: Summary The innervation of the pituitary gland of Carassius auratus was studied by light and electron microscopy under various physiological and experimental conditions to investigate whether or not neurosecretory fibres play a role in regulating pars distalis function. Two types of neurosecretory fibre (Type A and Type B) were distinguished. Prolactin, ACTH and TSH1 cells were innervated by Type B fibre terminals separated from the endocrine cells by a continuous basal lamina (“indirect contacts”). Gonadotropic, STH and TSH2 cells were innervated by Type A as well as Type B neurosecretory fibres, mostly without an intervening basal lamina (“direct contacts”). The assessment of the amount of neurosecretory granules and microvesicles in nerve terminals during the pre-spawning, spawning and postspawning seasons and following the administration of Oestradiol, Thyroxine, Thiourea and Metopiron respectively revealed convincing evidence for a participation in pars distalis control for Type A and Type B fibres innervating gonadotropic cells and STH cells and Type B fibres innervating TSH2 and ACTH cells. Immediately after spawning both nerve fibre types innervating gonadotropic cells and Type A fibres innervating STH cells showed a striking decrease in the amount of dense core vesicles. During the spawning season nerve fibres innervating somatotropic cells, TSH2 cells and ACTH cells also undergo changes suggesting that prior to spawning major changes in the endocrine system of the goldfish take place.—These results point to a dual control, by peptides and amines, of teleost pars distalis function.

Notes: Summary The olfactory mucosa of frog has been studied at an ultrastructural level to confirm previous light microscope observations in regard to the presence, in the sensory epithelium, of nerve fibres not belonging to the first cranial nerve proper. It has been observed that both myelinated and unmyelinated nerve fibres are present in the lamina propria and that eventually these fibres terminate inside the epithelium. Unmyelinated fibres usually contain dark core vesicles and similar content is seen in their intraepithelium terminals. Terminals containing only clear vesicles are also observed in the epithelium and they are believed to represent the terminals of the myelinated fibres. The significance of these ultrastructural findings is discussed in view of their functional meaning.

Notes: Summary The Octopus iris is composed of five different layers: A, the external epithelium; B, the chromatophore layer; C, the iridocyte layer; D, the layer of muscles and collagen strands; E, the pigment epithelium. The nerves innervating the sphincter and the chromatophore muscles are identified and their neuromuscular junction is described. The motor endings of chromatophore nerves have an additional ending in presynaptic position which probably functions as a modifier of neuromuscular transmission. The chromatophores are naked and exhibit a tubular channel system between plasmalemma and pigment container which looks similar to the T-system of muscle cells.

Notes: Summary In the tortoise Testudo graeca, the lizards Lacerta dugesi and Lacerta pityusensis, and the snake Natrix natrix, the innervation of the testicular interstitial tissue was studied by light and electron microscopy, the acetylcholinesterase (ache) technique, the Falck-Hillarp method for the detection of catecholamines, and the application of 6-hydroxydopamine. The intertubular spaces of the reptilian testes studied contain adrenergic nerve fibers the amount and distribution of which varies considerably both in various species and in various stages of the reproduction cycle. Nerve fibers do not enter the seminiferous epithelium. Fluorescence microscopy of the lizard testis reveals catecholaminergic varicosities which are mainly arranged around blood vessels, but do not show obvious connexions to Leydig cells. Ache-positive fibers are equally distributed in lizard testes surrounding each seminiferous tubule. In Natrix natrix ache-positive fibers are irregularly spread among groups of tubules, without showing a definite relation to Leydig cells either. By electron microscopy bundles of unmyelinated axons and axon terminals can be more easily detected in the testes of immature animals than in adult. Terminals of nerve fibers containing small (400–500 Å in diameter) and large (800–1400 Å) dense-cored vesicles and sometimes small clear vesicles establish contacts with Leydig cells. Three types of contact are described. 1. “Contacts” par distance at a distance of about 2000 Å and basal lamina interposed; 2. membranous contacts having a 200 Å gap only between axolemma and Leydig cell plasmalemma; 3. invaginations of terminals into Leydig cell perikarya. The latter may exhibit surface specialisations, which strongly resemble postsynaptic membrane thickenings. Experiments using 6-hydroxydopamine underline the adrenergic character of testicular nerve fibers, which can be regarded as another example of non-cholinergic, ache-positive neurons. In the testis of the immature tortoise profiles of axons occur which probably represent purinergic, ache-positive neurons.

Notes: Summary Terminal axons emerging from the inner plexiform layer of the cat retina reach the wall of the arteria centralis retinae, as revealed by electron microscopy. Numerous unusually large dense core vesicles (about 1000 Å in diameter), of different electron densities, occur in the varicosities of these axons. These observations may be compatible with the idea of an innervation of the central artery of the retina which is non-autonomic, possibly intrinsic in nature.

Notes: Summary Neural elements within the parenchyma of the sebaceous gland have not been reported previously. Nerve endings have been observed only in the connective tissue surrounding the gland or in close association with the undifferentiated basal cells. In this study, electron microscopy revealed the possible presence of nerve endings (or terminal portions of neural elements) in the suprabasal level of functional sebaceous glands of pinnae of white rats. Morphologically, there are two distinct types of nerve endings. Type 1 is bordered by a membrane of relatively irregular contour and contains a single mitochondrion, various-sized vesicles, numerous microtubules, fine neurofilament-like fibrils, and occasional ribosome-like granules. Type II is also bordered by a membrane, but its contour was relatively smooth and rounded. Moreover, Type II contains many mitochondria, varying in size, density, and the arrangement of cristae. While ribosome-like granules are scattered throughout the structure in relative abundance, there are scarcely any fine neurofilament-like fibrils or microtubules. Whether these two structures are sensory or autonomic fibers could not be determined by electron microscopic examination.

Notes: Summary The innervation of the brown, red and yellow chromatophore muscle cells in Loligo vulgaris shows quantitative and qualitative differences. The nerve bundles regularly contain axons with electronlucent vesicles of about 300 Å diameter, and occasionally axons with dense core vesicles of about 600 Å diameter. The myofibrils of the contractile cortex show a staggered arrangement and are wound in a spiral with respect to the axis of the muscle cell. In the axial sarcoplasm there is additionally a bundle of thin filaments of about 70 Å thickness. The bundle is orientated in parallel with the longitudinal axis of the muscle cell. Its function may be to maintain tonic contractions.

Notes: Summary The ultrastructure of the innervation of the anterior cerebral artery of the rat was studied in control animals and in animals after superior cervical ganglionectomy. Fluorescence histochemistry shows a periarterial network of intensely fluorescent fibers which are divided into two groups, adventitial and periadventitial. The fluorescence begins to decrease 26 hours after, and completely disappears about 32 hours after, ganglionectomy. Fine structural changes are first observed 18 hours after ganglionectomy, when the axoplasm of degenerating axons becomes electron dense. This density gradually increases up to about 32 hours. By 32 hours most axons with disintegrating axolemmas become inclusion bodies of the Schwann cells. At this stage, synaptic vesicles can still be distinguished as less dense areas, but the membrane structures of synaptic vesicles and mitochondria are difficult to recognize. The degenerating axons are gradually absorbed and by 38 hours dense, residual bodies are observed in the Schwann cells. Generally speaking, the degeneration occurs first in the adventitial fibers and then in the periadventitial fibers. The transient appearance of small, granular vesicles is noticed in axon terminals about 18 hours after denervation, although very few small, granular vesicles are seen in control tissue or at later stages of degeneration.

Notes: Summary Nerve fibers were demonstrated in the glycogen body of the avian lumbar spinal cord by using silver-impregnation and fluorescence microscopic (Falck-Hillarp) techniques. These nerve fibers originated from nuclear areas in lateral juxtaposition with the glycogen body. The fluorescence of the nuclear area was strongest near the polar regions of the glycogen body. It was suggested that the aminergic neurons of the avian lumbar spinal cord may influence the glycogen body. The avian glycogen body was compared with other storage sites of glycogen within the central nervous system.

Notes: Summary By means of electron microscopy “light” and “dark” pinealocytes can be distinguished in the guinea-pig pineal gland. Glial cells are rare. In the “light” pinealocyte. the most frequent cell type, some “vesicle-crowned rodlets” (VCR) show circumscribed thickenings. From these structures “vesicle-crowned balls” (VCB) have to be clearly distinguished. Furthermore “cylinders” occur, which, it is suggested, are precursors of VCB. “Dark” pinealocytes characterized by chromatin-rich nuclei and electron-dense cytoplasm are rare and contain fewer vesicles, VCR, VCB and “cylinders” than “light” pinealocytes. Numerous non-myelinated nerve fibres are situated within perivascular spaces, a few also in the parenchyma. Synapses between nerve fibres and pinealocytes were not observed. In the pineal gland of pregnant guinea-pigs the following changes can be observed in the second half of gestation. The “light” cells show many nuclear indentations and an increase of “active” zones, mitochondria, smooth ER, agranular vesicles, VCR, VCB, and “cylinders” respectively. The “dark” cells increase in number. After birth these changes reverse to normal within one week. Constant darkness leads to an activation of the “light” cells accompanied by an increase of the VCR and to an increase in number of the “dark” cells. Under constant illumination the “light” cells show a decrease of their organelles and a strong increase of the VCR. After 70 days the VCR also show a change in shape. Following reserpine treatment the VCR decrease in number and show signs of degeneration. It is discussed that the VCR function as pre- or postsynaptic structures and that they are involved either in transmitting impulses from nerve fibres to pinealocytes or from one pinealocyte to the other.

Notes: Summary This investigation is concerned with pineal organs of human embryos 60 to 150 days old. At every stage central nerve fibres enter the pineal organ by way of the habenular commissure, but are restricted to the pineal's proximal part. On about the 60th day of the development the sympathetic nervus conarii grows into the distal pole of the pineal organ from a dorso-caudal direction and plays the predominant part in the innervation of the pineal organ. After penetrating, it soon branches out and forms a network in the pineal tissue. Much later, not until the 5th embryonic month, sympathetic nerves appear accompanying the supplying vessels in the perivascular spaces. After a short time these nerves pierce the outer limiting basement membrane and penetrate the parenchyma. Towards the end of the 5th embryonic month the axons of the sympathetic nerves form varicosities containing clear and dense core vesicles. At this point large amounts of laminated granules appear primarily in cell processes, probably of pinealocytes. Isolated granules also occur in the varicosities of axons. The granules encountered here are most likely secretory granules.

Notes: Summary Nervous (sensory) pathways of the pineal complex were studied in Lampetra planeri. The following observations were made: (1) The pineal tract, containing more than 600 unmyelinated fibres, connects directly or indirectly to the posterior commissure. (2) Connections of the nervous region (NR) adjoining the parapinéal organ (“ganglion parapinéales” of Tretjakoff or “division of the left habenular ganglion” of Studnička) have been re-examined; our study shows that the rostral division of NR is connected to the parapineal organ by a short tract and to the left habenular ganglion (LHG) by a nervous layer (NL). The NR, NL, and LHG contain the same following elements: (1) A collection of typical neurones (which are rare in the NL) in an abundant meshwork of nerve fibres. (2) At least two main types of axons: (a) Type 1 axons, containing few granules (1,000–1,300 Å diameter). (b) Type 2 axons, containing numerous granules (900–2,500 Å diameter), and resembling neurosecretory axons. Type 2 axons are not observed in the pineal and parapinéal organs. (3) Axo-dendritic and axo-axonic(?) synapses. Our study concludes that NR and NL represent the rostral part of LHG; nevertheless, NR and NL may contain the parapinéal tract. The problem of possible incorporation of epithalamic elements (e.g., elements of the habenular ganglia) into the parapinéal and pineal organs in other animals has been considered phylogenetically.

Notes: Summary Umbilical vessels of guinea-pig fetuses were studied shortly before birth. In all umbilical cords investigated an innervation of the umbilical vessels is lacking. The intrafetal parts of the umbilical vessels on the other hand are richly innervated. A marked difference in the amount of nerve fibres and the pattern of innervation is found between artery and vein. The artery is supplied by a dense nerve plexus which spins around the media and which originates from nerve bundles within the outer adventitial layers. The comparatively scanty innervation of the vein exhibits a more coarsely meshed net pattern. The nerve bundles in the vein exhibit a close affinity to the vasa vasorum. Number and type of the close contacts between the muscle cells are different in the various sections of the umbilical vessels. Similar to the distribution of nerves they are almost absent in the vessels of the umbilical cord, numerously, however, in the intrafetal parts. Contrary to the innervation, the close contacts in the vein are developed more numerously and more broadly than in the corresponding artery.

Notes: Summary The development of adrenergic nerves to the anterior eye segment was studied in human and guinea-pig embryos. Adrenergic terminals had already appeared in the earliest human embryos available (4–6 cm). They first appeared mainly in nerve trunks in the primitive chorioid, especially in the region of the developing ciliary body. Adrenergic nerves then grow into different structures of the eye as these develop, but typical terminals in contact with effector cells appeared late during the development, about the 25–30 cm stage. No adrenergic nerves were observed in the chamber angle. Corneal adrenergic nerves (also intraepithelial terminals) appeared much more frequently in embryos than in adults. No adrenergic neurons were observed in the retina. In the guinea-pig, the first adrenergic fibres were observed at about gestation day 35. The general principle of the development was very similar to that of the humans. At gestation day 45 to 50, the supply of adrenergic fibres was essentially that of the adult animal, except that the corneal adrenergic fibres were increasing until just before birth and that the adrenergic terminals of the chamber angle appeared shortly before term.

Notes: Summary The intrinsic innervation of the anterior byssal retractor muscle in Mytilus edulis L. has been demonstrated using Maillet's osmium-zinc iodide technique (ZIO). Grouped and isolated osmiophilic cells, thought to be neurons, and nerve fibers forming neuromuscular contacts and nerve endings, have been observed. When the period of fixation is not overly prolonged, glio-interstitial cells may also be distinguished.

Notes: Summary The ultrastructure of the pars intermedia (PI) of the normal VII +/+ and hereditary nephrogenic diabetes inspidus DI Os/+ mice has been studied with particular reference to the morphology of the glandular cells and their innervation. Four types of cells were observed in both the genotypes of mice, 1) the light glandular cell, 2) the dark cell, 3) a type of cell similar to ependymal cells and 4) a small percentage of typical ACTH cells, observed mostly on the PI border of the cleft and rarely in the centre of PI. The predominant light glandular cells contain mainly two types of membrane bound granules: 1) electron dense core granules, which measure 1500–2500 Å and 2) electron lucent vesicles, which measure 3000–4000 Å in diameter. Granules of intermediate size with various density are also present in both types of mice. The electron dense core granules are predominant in DI Os/+ mice, whereas, electron lucent vesicles are predominant in the normal VII +/+ mice. Similar uniform size membrane bound electron dense granules have been observed in ACTH cells of PI and pars distalis. From earlier experimental evidences and the present observations, it is concluded that the dense core granules in PI may be synthesizing ACTH or ACTH-like substance. It is also discussed that these dense core granules may further mature and give rise to MSH in the form of electron lucent vesicles. If it is so, PI light glandular cells may have dual functions, of producing MSH and ACTH. One of the functions of ependymal-like cells, may be the transport of PI secretion. Three types of nerve endings are observed throughout the PI, making synaptic contact with the predominant cell type. The innervation is more in DI Os/+ mice than in normal mice. The classification of these nerves is according to Bargmann and co-workers 1) peptidergic neurosecretory fibers, contain mainly membrane bound dense core granules, measuring 1200 to 1800 Å, and are the classic neurosecretory granules; 2) adrenergic fibers, measuring 700–900 Å; 3) cholinergic fibers, measuring 300–400 Å. Adrenergic and cholinergic fibers are more towards the hypophysial cleft. The increased innervation, the synaptic contact, the extremely hypertrophied PI and the greater activity of its light glandular cells in the DI Os/+ mice show the PI is under the influence of the nervous system.

Notes: Summary The innervation of the salt gland of the goose, the duck and the swan was investigated by means of electron microscopy. Axonal swellings were observed in relationship to secretory cells as well as to central duct cells. The terminals contain synaptic and densecored vesicles. There are no specialized pre- and postsynaptic membranes.

Notes: Summary Morphological evidence is presented supporting the possibility of basal secretion into hypendymal capillaries of the adult rabbit subcommissural organ (SCO). The synthetic apparatus of the SCO cell is described as well as the heterogeneous granules and vesicles which are concentrated in the basal processes bordering a widened perivascular space. The origin of the electron dense granules, of which two fairly distinct subgroups are found, is discussed. A binding of secretory sacs to the lateral plasma membrane is seen. The possibility of a lateral secretion is supported by the presence of a system of extracellular channels between SCO cells which are filled with a flocculent material resembling that of the secretory sacs. Nerve perikarya which are separated from the SCO by only a few glial fibers are demonstrated. Synapses are described in nerve fascicles bordering on the hypendymal capillaries. The possibility of an innervation of the hypendymal region is discussed as well as possible nervous connections with the pineal gland.

Notes: Summary The salivary glands of the moth, Manduca sexta (Insecta: Sphingidae), are unlike most other salivary glands in that they are innervated from one source only. Vital staining of nerves with methylene-blue reveals numerous fine nerves extending to the glands from the oesophageal nerve, a part of the stomatogastric or visceral nervous system. Light and electron microscopy confirm that only the fluid-secreting cells, confined to a discrete region in these glands, are innervated. Axons with or without glial wrappings are found in intercellular spaces between fluid-secreting cells. Axons lacking a glial sheath contain, after glutaraldehyde-osmium tetroxide fixation, large granular and small agranular vesicles. In nerve endings in glands fixed with permanganate these smaller vesicles are granular, having the electron-dense cores characteristic of monoamine-containing neurons. These nerve endings with “synaptoid areas” are in close (“direct”) contact with the fluid-secreting cells.

Notes: Summary Electron microscope studies of axons, distributing singly or in small bundles in the human ventricular and atrial myocardium, indicate a few per-cent of the axon profiles to be significantly large in diameter (1.5–3.0 μ). They are characteristically packed with a profuse number of mitochondria along with large granular vesicles, glycogen rosettes, lysosomic bodies; and some of them terminate on a “specific terminal cell” (Knoche and Schmitt). These mitochondria-rich, large axons are assumed to be terminal portions of the cardiac afferents. About half of the axons encountered in the ventricle and 2/3 in the atrium are non-vesiculated, usually less than 0.5 μ. in diameter. The varicosities containing numerous vesicles are mostly 0.5–1.5 μ in diameter and are assumed to be terminal portions of the cardiac efferents. The ratio between the number of axon profiles containing small granular vesicles and that of axon profiles containing agranular vesicles without small granular vesicles is 2∶1 in the ventricular myocardium and 1∶1.7 in the atrial myocardium.

Notes: Summary The hilus cells in the ovary of the pig have been investigated with the electron microscope. These elements are identical with Leydig cells. The hilus cells contain an abundant agranular endoplasmic reticulum, which is either organized as a tubular network or as an onion like system of closely packed flattened cisternae sometimes filling up half of a cell and often being concentrated around lipid droplets and pigment granules. The mitochondria have cristae, tubuli and sacculi and a matrix of variable electron density. The golgi cisternae are poorly developed. The cells have centrioles, and there are hints of an amitotic cell division. The cytoplasm of the hilus cells contains lipid droplets, which have a diameter up to 5 μm. The pigment granules are extremely polymorphic, nearly always surrounded by a single membrane. They possibly may be derived from lipid droplets and mitochondria. Single axons of the hilus nerve occasionally penetrate the basement membrane and come into close contact with the hilus cells. Typical synapses were not observed. The results are discussed on the background of light microscopical, biochemical and pathological findings and compared with those obtained on Leydig cells of different species. Various cell images are interpreted to represent progressive stages of maturation.

Notes: Summary The rabbit SA-node was outlined electrophysiologically and its adrenergic and cholinergic innervation patterns were studied with the electron microscope. Differentiation between adrenergic and cholinergic terminals was achieved by fixation of the specimens in KMnO4 which produces dense-cored synaptic vesicles in adrenergic terminals, whereas synaptic vesicles in cholinergic terminals are empty. It was found that adrenergic and cholinergic nerve terminals often come in close apposition to each other, the distance between adjoining membranes being in the order of 250 Å. At times, faint membrane thickenings could be seen in these places. The available pharmacological, physiological and morphological evidence leaves little room for doubt that cholinergic terminal fibers can influence the adrenergic ones. From mainly morphological evidence it is also postulated that adrenergic terminals influence cholinergic terminals.

Notes: Summary The median eminence and the pars nervosa of Rana esculenta have a different structure. The median eminence has 2 different zones: the outer zone situated near the pars distalis and the inner zone under the ependyme. In the outer zone there are, according to the size and the shape of the granules, 5 types of nerve terminals. 1. Endings containing spherical fine dense granules of 800 to 1000 Å in diameter; 2. Endings with spherical granules from 1000 to 1200 Å in diameter; 3. Endings with granules of irregular shape which are bigger than the former (1200 to 1600 Å); 4. Endings with spherical dense granules of about 1200 to 1800 Å in diameter; 5. A few endings containing only clear vesicles. Type 3 and type 4 endings are probably neurosecretory. The inner zone contains numerous neurosecretory fibres. They are of two types: one with big granules (1600–2400 Å), the second with smaller granules (1300–2000 Å). Non-neurosecretory fibres have also been observed. The pars nervosa contains two principal types of neurosecretory fibres: one with dense granules of 1600 to 2400 Å in diameter, the other with lighter granules of about 1300 to 2000 Å. In the external zone lining the pars intermedia, aminergic fibres with fine granules have been observed.

Notes: Summary The innervation of the portal vein of the white rat was examined with light-, fluorescence-, and electronmicroscopic techniques. The results are as follows: 1. Paraldehyde treated vein preparations (Falck's fluorescence method) demonstrate a predominantly longitudinally orientated external nerve plexus, being situated on the outermost muscle cells. Near the liver the nerve net is characterized by broad meshes, near the intestinal tract by narrow ones. The circular subendothelial inner plexus originates in the outer plexus of the intestinal vascular bed. 2. Nerve bundles in the fibrous adventitia were demonstrated with Gomori's Acethylcholinesterase method. In other respects, the enzyme activity was only observed in the intercellular spaces of the muscle layer. The electronmicroscopic demonstration of Acetylcholinesterase (Karnovsky's method) further showed that the enzyme activity is restricted to the muscle cell membrane. 3. The electronmicroscopic examination verified the results obtained with fluorescence microscopic techniques. a) In the proximity of the liver, only isolated nerve bundles occur on the outer muscle layer. The individual nerves are entirely surrounded by Schwann cells. Only a few of the axons in the vicinity to the muscle cell have agranular vesicles. Evaginations of the vesicular axons occur infrequently. Their distance from the muscle cell amounts to 1000–2000 Å. b) In more than one thousand thin sections, no axons were found inside the thick muscular layer. c) Subendothelial axons (inner plexus) are either partially or totally evaginated from the Schwann cells. They are densely filled with empty vesicles (350–650 Å) and contain a few dense core vesicles of 800–1600 Å in diameter. Synaptic endings were not observed. d) A dense collection of vesicle-containing axons, that were partially in their entirety and partially only from the muscle cell proximal side evaginated from the Schwann cells, were observed in the single muscle layer at the junction of the superior mesenteric and the portal vein. From these bundles, smaller bundles and individual axons pass between the muscle cells and reach the endothelium. Typical synapses were not observed. No vesiclecontaining axon was nearer than 1000 Å to the muscle cell. 4. Those axons possessing vesicles and being evaginated are considered to be vegetative conducting pathways. The excitation of the effector structures by transmitter substances is discussed in connection with the post mortem autonomic vascular contractility.

Notes: Summary 1. There is a gradual proximo-distal increase in the thickness of the muscle coat of the human ductuli efferentes, duetus epididymidis and ductus deferens. Circularly arranged smooth muscle bundles predominate in the ductuli efferentes and ductus epididymidis of the caput section. Scanty strands of longitudinally and obliquely oriented smooth muscle bundles form an additional, incomplete outer muscle layer around the ductus epididymidis of the corpus. Small smooth muscle-like cells constitute the muscle elements of the upper sections of the excretory ducts (from the ductuli efferentes to the midcauda). At the transition of the corpus and cauda epididymidis ordinary large smooth muscle cells join the small contractile cells to form—in more distal sections of the cauda—a composed, thick subepithelial muscle coat. In most distal portions of the cauda, the two-layered muscle coat of the ductus epididymidis is transformed into a three-layered coat, a pattern of construction which is retained in the vas deferens. 2. Electron microscopically, three types of contractile cells are distinguished in the human ductuli efferentes and ductus epididymidis: a) contractile cells of medium transparency containing exclusively thin myofilaments (60 Å in diameter), b) dark contractile cells containing bundles of thin myofilaments (60 Å in diameter) and single coarse filaments (140 Å in diameter), c) light contractile cells with loosely dispersed, interweaving thin and thick myofilaments. Commutual diameter changes at regular intervals are seen in individual myofilaments, giving the impression of structural periodicity not unlike that of filaments of striated muscle. Ordinary smooth muscle cells of the cauda epididymidis and vas deferens are characterized by uniformly sized, closely packed but evenly distributed thin myofilaments with numerous dense patches. 3. Fluorescence microscopy performed on formaldehyde treated freeze dried tissues reveals that the contractile cells of the ductuli efferentes in man and monkey receive a low number of single adrenergic terminal fibres penetrating the depth of the muscle coat. The adrenergic innervation of the ductus epididymidis is restricted to small peritubular nerve fascicles running contiguous to the most superficially located bundles of smooth muscle-like cells. The adrenergic ground plexus is rather wide-meshed in the proximal cauda, becomes increasingly dense in more distal cauda sections and in initial, funicular portions of the vas deferens, and reaches maximum density in abdominal parts of the ductus. Perivascular and adventitial adrenergic plexuses are well developed at arteries of the caput and corpus epididymidis in man, monkey, rabbit, guinea-pig and rat. 4. Electron microscopically, noradrenergic nerves have been identified by the presence of small granular vesicles in preterminal varicose axon dilatations. Nerve fibre swellings filled with small empty spherical vesicles have been considered to belong to “cholinergic” neurons whereas occasional varicosities equipped with some large membrane bound granules and abundant mitochondria may represent local expansions of sensory axons. 5. Neuromuscular relationships in the upper sections of excretory ducts comprise adrenergic synapses by distance (more than 1000 Å), and a few intimate, ensheated close contacts, whereas the main type of contact of nerves to ordinary smooth muscle cells in the lower duct section is by means of close but not intimate approach (500–2000 Å). 6. Adrenergic synapses in the ductus epididymidis and ductus deferens of the monkey resemble—what concerns their morphology, relationship to effectors and distribution pattern—those of man. 7. In accordance with the total number of vascular and non-vascular adrenergic nerves, visualized by fluorescence microscopy, the amount of noradrenaline varied considerably in different sections of the human male internal genital organs: The lowest amounts were estimated in the testis (0.12±0.03 μg/g). Medium to high concentrations were detected in various sections of the caput and corpus epididymidis (ductuli efferentes 0.60±0.09 μg/g; ductuli efferentes and caput 0.72±0.13 μg/g; corpus epididymidis 1.04±0.25 μg/g; proximal cauda 0.95±0.17 μg/g; distal cauda 0.97±0.19 μg/g). The highest noradrenaline content was found in the human vas deferens (prox. vas deferens 1.11±0.21 μg/g; interm. vas deferens 1.20±0.42 μg/g; distal portion 1.43±0.39 μg/g). 8. For comparison, the noradrenaline content of the testis and epididymis of the rhesus monkey, the epididymis of the rabbit and the vas deferens of the rabbit, mouse, guinea-pig and rat has been determined. 9. Adrenaline of exogenous origin was detected in the vas deferens, cauda epididymidis and plexus pampiniformis of two cases who received this catecholamine as part of the local anaesthetic drug mixture. Due to methodological reasons, the presence of small amounts of adrenaline of endogenous source in adrenergic nerves of the human and monkey internal male genital organs cannot be excluded. 10. The differences in motility behaviour of the ductus epididymidis (spontaneous, rhythmic contractions) and ductus deferens (absence of any spontaneous movements under conditions at rest) in vivo and in vitro have been correlated with the occurrence of specialized contractile cells in the upper segment (ductuli efferentes, ductus epididymidis of the caput, corpus and initial cauda) and ordinary large smooth muscle cells in the lower segment (ductus epididymidis of the distal cauda and the vas deferens) and furthermore correlated with differences in the pattern of the adrenergic innervation; the concept is advanced that progressive cytological differentiation of smooth muscle cells and the development of a dense direct adrenergic innervation suppresses autocontractility and, that the reverse condition may favour spontaneous motility of smooth muscle elements.

Notes: Summary The glandula infraorbitalis buccalis in rabbits represents the type of a tubuloacinar mucous secreting gland. The secretory product of gland cells is electron lucent. Extrusion generally is characterized by the formation of an opening at the cell surface and the discharge of the secretory material. Intercellular canaliculi are absent. Mitochondria and arrays of granular endoplasmic reticulum are more numerous than in other mucous glands of rabbits. Intercalated ducts are short and connect the tubules with intralobular ducts. Striated ducts are absent in the infraorbital gland. Nerve terminals occur in some instances between gland cells and between glandular and myoepithelial cells.

Notes: Summary 1. Three species of the genusGnathonemus (Gn. petersii, moorii andstanleyanus) as all species of the family Mormyridae possess two pairs of electric organs situated symmetrically on each side of the vertebral column between the caudal, dorsal and anal fins. Each organ is composed of a series of 70–170 electroplaques. 2. The stalks of the electroplaques contain no nerves. Unbranched motor nerve fibres innervate the end sections of the stalks by synaptic knobs inserting into cavities of the electroplasma. 3. The electroplasma membrane surrounds the stalk (also in the region where the stalk penetrates through holes in the body of the electroplaque) and the electroplaque. The membrane is deeply indented on the cranial side as well as on the caudal side of the electroplaque. Through this the surface is greatly enlarged. In the inside of the electroplaque are striated muscles. 4. The motor and sensory nerve fibres are clearly different in diameter. 5. The number of the nerve cells in the spinal ganglions is, in the electric organ, about a multiple larger than in the segments of the body muscular system situated in front of the electric organ. 6. The reconstruction of the peripheral sensory nerves of one segment (in view of electron microscopical analyses), reveals that these, with the exception of two free nerve endings in the integument and the free nerve endings in the dorsal myoseptum, mainly innervate four large tendons on the side of the electric organs. 7. The innervation of the electric organs leads to the hypothesis that the tendons, in connection with the function of the electric organs, possess sensory functions.

Notes: Summary The fine structure of the pancreatic nerves of the domestic fowl has been studied. Naked axon beadings were found in membranous contact with endocrine as well as exocrine cells. From an anatomical point of view it seems reasonable to suggest that the endocrine glands might be subjected to some influence of the autonomic nervous system.

Notes: Summary The innervation of the Purkinje fibres in the atrium of the heart of the adult fowl was investigated by light microscopy, using the Champy-Maillet OsO4-ZnI2 technique and the cholinesterase reaction and by electron microscopy. After impregnation of the tissue with OsO4-ZnI2, the dark-stained nerve fibres were clearly visible on the unstained Purkinje fibres. In the upper part of the posterior wall of the right atrium, the diffuse portion of the conducting system is especially richly innervated by varicose and smooth nerve fibres. Some of these fibres are cholinesterase-positive. The terminal axons run in the space between the Purkinje fibres and the fibrocytic envelope. They are either naked or accompanied by Schwann cell processes. In addition to varicosities containing granular vesicles, there are varicosities containing agranular vesicles with oval profiles. In the “en passant” synapses, the width of the synaptic cleft between a varicosity and the Purkinje cell is about 600 Å. The innervation of the Purkinje fibres appears more like the innervation of smooth muscle than that of striated skeletal muscle. The possible role of Purkinje fibres as mechanical receptors is discussed.

Notes: Summary The pathways of axonal transport of secretions from neurosecretory cells (NSC) in the medial group (viz. A-, A1, B-, and C-type NSC) and the lateral group (L-type NSC) are described. Individual axons can be recognized in the electron microscope by the kind of neurosecretory particles they contain. In general, the secretions from the medial NSC are carried to the contralateral nervi corporis cardiaci (NCC), those from the lateral NSC to the ipsilateral NCC. Some axons from the A-type NSC, in addition, may run to ipsilateral NCC. All A-type axons have collaterals which run to the ipsilateral NCC. The medial and lateral bundles of “mixed” axons run through one paired NCC but remain separated spatially. Release of secretion from the C-type NSC can take place before the corpus cardiacum is reached. A- and A1-type NSC have additional collaterals that branch from the proximal part of the axons and penetrate deeply into the neuropile of the protocerebral lobes. Local swellings appear to be closely associated with fibers from non-neurosecretory neurons. The sites of contact are characterized by the accumulation of microvesicles (400 Å) near an electron-dense cleft of 150–200 Å width, and resemble regular chemical synapses. The microvesicles prove to be present within the neurosecretory fiber in most of the specimens studied, and within the non-neurosecretory fiber in only a few cases. It seems most likely that the collaterals in some phases convey afferent signals to the NSC which inhibit the release of an efferent neurochemical “messenger” of unknown nature into the neuropile.

Notes: Summary The nerve supply to the iridic melanophores of the rat was studied with the electron microscope. The adrenergic and cholinergic terminals were identified with the aid of 5-hydroxydopamine, which produces dense-cored 400–800 Å synaptic vesicles in adrenergic axon varicosities, whereas the synaptic vesicles of cholinergic axons remain empty. It was found that both adrenergic and cholinergic terminal axons come in close apposition (200–250 Å) with the melanophores. The appositions have the same appearance as synapses in peripheral tissues. It seems likely that the murine iridic melanophores have a double innervation, although its functional significance is obscure.

Notes: Repetitive mass spectrometric scanning of gas-liquid chromatographic effuents at short (6 to 15 s) intervals, with a digital computer for data acquisition, reduction and display, allows plots (mass chromatograms) to be made of the intensities of specific ion fragments. Plotting of m/e values characteristic for certain classes of compounds and/or individual components reveals the location of constituents in the gas chromatogram. This technique was applied to the identification of trimethylsilyl derivatives of unsubstituted and 2-hydroxy fatty acid methyl esters in mixtures obtained from acid catalyzed methanolysis of cat brain galactocerebrosides. The plot of total ionization intensity vs scan number generated the gas-liquid chromatogram. Mass chromatograms of m/e 73 and [M - 59]+ gave the locations in the gas-liquid chromatogram of the hydroxy fatty acid derivatives, and mass chromatograms of m/e 74 and [M]+ located the unsubstituted fatty acid derivatives. Integrated ion intensities were used to determine the composition of the mixture of fatty acids.

Notes: Complete mass spectra of the pertimethylsilylated derivatives of three high molecular weight polyene macrolide antibiotics are reported for the first time. The fragmentation pathways which are proposed have been corroborated by the stable isotope derivatives d9-TMS and d3-acetyl. Accurate mass measurements and metastable transitions confirm the proposed fragmentation mechanisms in the low mass range. Nystatin - the macrolide of highest molecular weight in this study - expels several TMSOH molecules and a TMS radical. Amphotericin B underwent extensive rearrangements preliminary to eliminating a series of TMSOH molecules. An apparent equilibrium between the keto and ketal forms of amphotericin B facilitated the rearrangements. Pimaricin fragmented in a manner parallel to that of the other two macrolides. The sugar portion of the molecules dominated the fragmentation in the low mass region in the spectra of all compounds. The transfer of a TMS group from the sugar amine to the glycosidic oxygen was observed when the amino sugar was eliminated from the intact molecule. The resulting sugar ion then expelled ammonia.

Notes: A method is described for the separation, identification and quantitation of endogenous hydrogenated metabolities of corticosterone in rat liver Corticosterone, 5α- and 5β-dihydrocorticosterone, 3α,11β,21-trihydroxy-5α-pregnane-3,20-dione, 3β,11β,21-trihydroxy-5α-pregnane-3,20-dione, 3α,11β,21-trihydroxy-5β-pregnane-3,20-dione, 3β,11β,21-trihydroxy-5α-pregnane-3,20-dione, 5α-pregnane-3α,11β,20α,21-tetrol, 5α-pregnane-3α,11β,20β,21-tetrol and 5α-pregnane-3β,11β,20β,21-tetrol are specifically quantitated in one analysis by mass fragmentography all as their O-methyloxime pertrimethylsilyl derivatives. A pronounced difference in the amounts of these nine metabolites between male and female adult rats was found, while in newborn rats only the two tetrahydro-compounds bearing a 5α-hydrogen were detected in both sexes. On the other hand 3α,11β,1 5α,21-tetrahydroxy-5α-pregnan-20-one was detected only in the adult female as its second prominent metabolite of corticosterone. Corticosterone metabolites being biochemical markers of the sex-linked differentiation of the hepatocytes, their mass fragmentographic assay is proposed as a tool for the quantitative analysis of this type of gene expression.

Notes: The separation and structural determination of ten partially methylated methyl glucosides by gas chromatography and gas chromatography-mass spectrometry are described. These glucosides were four possible structures for methyl tri-O-methylglucosides and six for methyl di-O-methylglucosides. The positions of free hydroxyl groups in four isomers of methyl tri-O-methylglucosides could be fairly distinguished from each other by their mass spectra. Mass spectra of methyl di-O-methylglucosides were similar to each other, though their spectra were different from those of fully methylated or methyl tri-O-methylglucosides. After introduction of a trimethylsilyl group to the free hydroxyl group, mass spectra of methyl di-O-methylglucosides showed a characteristic pattern for the position of the trimethylsilylated hydroxyl group, and O-trimethylsilyl ethers of methyl tri-O-methylglucosides also showed distinguishable mass spectra. The structures of fragment ions of partially methylated methyl glucosides were confirmed by high resolution mass spectrometry.

Notes: Monitoring of solutes eluted from a liquid chromatograph is possible by continuous introduction of the effluent solution into the ion source of a chemical ionization mass spectrometer. Detection sensitivities in the nanogram range are possible for a wide variety of solutes and solvents. Auxiliary techniques which have proved valuable for g.c.-m.s. also appear to be applicable to l.c.m.s.

Notes: The determination of drug metabolites is very important in the biochemical field. However, the procedure for doing this has been very complicated in many cases. The major reason is because of the need for fairly large amounts of the samples. We have been studying procedures for the determination and identification by use of mass spectrometry, which usually gives a lot of information rapidly from very small amounts of the samples (below μg). In this paper, the metabolites excreted in urine and bile following oral administration of 2-(diethyamino)ethyl tetrahydro-α-(1-naphthylmethyl)-2-furanpropionate oxalate (1) in rats, were investigated by means of mass spectrometry and other techniques. The drug was not excreted unchanged in urine. Of the eight metabolites suspected to be present in urine and bile, the structures of three metabolites were elucidated to be tetrahydro-α-(1-naphthylmethyl)-2-furanpropionic acid (3) and α-(1-naphthylmethyl)-2-perhydro-5-oxofuranpropionic acid (4) in urine, and diethylaminoethanol (2) in bile. These metabolites might also be present as their structural isomers or conjugates.

Notes: The use of the integrated ion current technique for the analysis of biogenic amines and phenols as their 1-dimethylaminonaphthalene-5-sulphonyl (dansyl) derivatives is described, particularly as regards to the linearity and sensitivity of detection. The mono derivatized amines gave a linear response over the range 10-9 to 10-14 mol, with a few being detected in quantities as small as 5 × 10-15 mol. The bisdansyl derivatives have a lower sensitivity and the response is linear over a more restricted range, terminating at 10-13 mol. Deuterated analogues of some of the amines were prepared and the feasibility of their use as internal standards was investigated. In this way, the problems associated with quantitative biochemical analyses caused by losses during extractions, chromatographic separations and thermal decomposition in the mass spectrometer lon source are overcome. The differentiation of isomers of biogenic amines as their dansyl derivatives has been accomplished by fractional sublimation and by examination of differences in their fragmentation patterns.

Notes: The amino acids, arginine, citrulline, cysteine, cystine, histidine, hydroxyproline, methionine, ornithine, proline, serine, threonine, tryptophan and tyrosine, have been analysed in the form of their N-(N′, N′-dimethylaminomethylene) methyl esters. formed in a single step by reaction of the free amino acid or its hydrochloride with N,N-dimethylformamide dimethylacetal in acetonitrile/methanol (2:1). A reaction time of 15 minutes at 100 °C ensures complete derivatization, the derivatives being volatile at source temperatures below 150 °C, and giving electron impact spectra characterized by abundant (or moderately abundant) peaks at or near the mass of the molecular ion.The analysis of amino acid mixtures by mass fragmentography on characteristic high mass peaks of their N-(N′,N′-dimethylaminoethylene) methyl ester derivatives, introduced on a direct insertion probe and volatilized during temperature programming of the source, is qescribed. The method is applied to the analysis of a mixture containing arginine, citrulline, cystine, histidine and tryptophan, traditionally difficult to analyse by other methods.

Notes: The metabolism of cyclophosphamide-4-d2 (2-[bis(2-chloroethyl)amino]-tetrahydro-4,4-dideuterio-2H-1,3,2-oxazaphosphorine 2-oxide), was studied. The first detectable metabolite was a hydroxy derivative which was trapped with ethanol. Mass spectrometry of the resulting two ethoxy derivatives and of the deuterated acrolein 2,4-dinitrophenylhydrazones formed therefrom via reaction with acidic 2,4-dinitrophenylhydrazine, afforded evidence that the ethoxy substituents were at C-4, which was therefore the position of the original hydroxy substituent. The mass spectrum of the deuterated acrolein 2,4-dinitrophenylhydrazone obtained from the total reactive metabolites was used to estimate the ratio of 4- to 6-hydroxylation. The rate of metabolism and the antitumour activity of cyclophosphamide and its 4-d2 analogue were compared.

Notes: The organic material in airborne particulate matter is being studied by high resolution mass spectrometry to determine if hydrocarbons typical of major sources of pollution can be detected. Carbon number distributions and hydrogen-to-carbon ratios of components found in airborne particulate samples and the corresponding data for compounds detected in fuel combustion products show great similarity. Samples collected near large power stations and major airports were compared with typical urban particulate samples. Particulates collected from jet engine exhaust and a condensate from the stack effluent from a coal-fired combustion system were studied. Mass spectral data were obtained for an auto exhaust tar, a coal tar pitch and several fuels. The mass spectra of two total particulate samples, compared to the spectrum of an ether extract of the organic matter, indicate that direct vaporization can be used and extraction is not essential for qualitative mass spectral studies.

Notes: The first application of field desorption mass spectrometry to the analysis of intact diacyl glycerophosphorylcholines (lecithins) has led to the detection of prominent molecular ions. This technique enables positive identification of the components of a mixture of phosphatidylcholines, which will provide valuable information on the phospholipid composition of biological fluids.

Notes: The high resolution mass spectrometric integrated ion current procedure has been used to identify and quantitate amphetamine, isolated as its dansyl derivative, as it is found after addition to urine and in tissues of the rat following intraperitoneal injection. A linear relationship between amphetamine and ion current response has been established at least in the range 5 × 10-12 to 5 × 10-7 g, and as little as 8 × 10-13 mol of amphetamine in urine may be detected.

Notes: A method is described for the identification of the sapogenins of several Digitalis species. The steroids are separated by repeated development thin-layer chromatography and identified by means of high resolution mass spectrometry. The spectra of tigogenin, gitogenin, digalogenin and digitogenin are recorded. These spectra are of particular use in establishing the position of extra hydroxy moieties in the steroid nuclei. Several species of Digitalis were screened for their sapogenin content. D. purpurea, D. mertonensis and D. lutea were found to contain tigogenin and digitogenin in small amounts, the principal steroid being gitogenin, whilst D. ambigua and D. lanata contained all four steroids.

Notes: Acetic acid isolated from cider vinegar and inorganically synthesized glacial acetic acid have markedly different intramolecular isotopic distributions of the stable carbon isotopes. Carbon-12 is concentrated in the methyl group relative to the carboxyl group in the biologically produced acid. The reverse distribution is observed in the particular sample of glacial acetic acid examined here.

Notes: An analysis has been made of a sample of rain water collected in Washington, D.C. following a sustained photo-oxidant smog alert which was accompanied by haze and low visibility. The analysis was effected by purging the organic content from the water with helium gas and identifying the constituents by means of gas chromatography - mass spectrometry. The predominant compound was found to be 3-methylfuran, a possible intermediate or end product of the atmospheric decomposition of terpenoid hydrocarbons. The analysis suggests that the smog resulted from the air oxidation of hydrocarbons evolved from Appalachian forestation rather than from manassociated activities.

Notes: Combined gas chromatography-mass spectrometry and isotope labeling techniques have been employed to demonstrate that the carboxamide nitrogen atom in 2-carboxamido-5-isopropoxycarbonylaminobenzimidazole, a metabolite of 2-thiazolyl-5-isopropoxycarbonylaminobenzimidazole (cambendazole), is derived wholly from the parent drug. [15N] Thiazole-labeled cambendazole was administered to a rat and the metabolite isolated from urine. Trideutero-2-carboxamido-5-isopropoxycarbonylaminobenzimidazole was employed as a carrier to isolate the metabolite in sufficient purity for isotope ratio measurements using multiple ion detection. The metabolite was converted to its tetramethyl derivative by on-column reaction with trimethylanilium hydroxide to permit satisfactory gas chromatography.

Notes: Twelve derivatives of bilirubin were evaluated from the point of view of providing intense molecular ion peaks on electron impact and unambiguous molecular weights. The tetrakis-(trimethylsilyl) derivative of bilirubin was judged to be the most suitable because its molecular ion carries 22% of the total ion current and is the base peak in the spectrum. Molecular ions were found to carry 10% of the total ion current in the spectrum of the trimethylsilylated methyl ester of the phenylazo derivative of bilirubin. Fragmentation patterns of these two compounds were studied using deuterium labeling.

Notes: Mass spectrometric analysis of peptide mixtures may lead to ambiguous results when it is possible to change from one peptide chain to another during the interpretation of the spectra. This kind of ambiguity can be eliminated by submission of part of the sample to one cycle of Edman degradation, followed by comparison of the results obtained for this preparation with those obtained for the nondegraded sample. Combination of this procedure with the use of deuterated derivatives has allowed an unambiguous interpretation for two mixtures of synthetic peptides.

Notes: The field desorption mass spectra of ten medicinal carbamates - meprobamate, mebutamate, carisoprodol, emylcamate, bethanechol chloride, styramate, hydroxyphenamate, mephenesin carbamate, methocarbamol and chlorphenesin carbamate - were run and compared with their published electron impact spectra. Of the ten compounds run only emylcamate did not give a field desorption mass spectrum. All others gave abundant molecular ions, with the exception of bethanechol chloride which has a quarternary ammonium group and gave a field desorption spectrum characteristic of compounds possessing such a functional group. No general characteristic fragmentation was attributed to the carbamate function in the field desorption mode. However, the ease of obtaining abundant molecular ions or quasimolecular ions makes the field desorption method a valuable complementary technique to electron impact studies of carbamates.

Notes: An azurin, a small respiratory copper protein from the bacterium Pseudomonas fluorescens biotype G, has been studied by mass spectrometry to determine sequence information. The study of homologously related proteins by mass spectiometry is particularly attractive, since the correct nature of major parts of the deduced sequences can be confirmed by comparison with the sequences of the protein from related organisms. An oxidized tryptohan residue has been identified amongst the products from a cyanogen bromide digest of this wild type azurin. In the same digest, a product is also found to arise from cleavage of the peptide chain at the C-terminal side of the same tryptophan residue. These results are rationalized.

Notes: The mass spectral data for the primary fragmentation of partially ethylated alditol acetates have been tabulated in order to allow easy reference for the identification of these polysaccharide derivatives. Sodium borodeuteride is used in all the aldose to alditol reductions, since the presence of a single deuterium label on C-1 greatly increases the information available in the mass spectrum and allows some identifications to be made which would not otherwise be possible. The primary fragmentations of these derivatives are analogous to those of the partially methylated alditol acetates, with each fragment shifted to a higher m/e value by fourteen mass units for each ether linkage contained in the fragment. The secondary fragmentation is also very similar, being characterized by the loss of acetic acid or ketene, or, less frequently, by the loss of ethanol or acetaldehyde. Coupled with the chromatographic retention time data for the partially ethylated alditol acetates tabulated elsewhere, the unambiguous mass spectral identification makes this derivative an excellent choice as a complementary derivative to the partially methylated alditol acetates for polysaccharide analysis. The utility of the partially ethylated alditol as a routine analytical derivative is further enhanced by the almost identical procedures required for synthesis of the ethyl and methyl derivatives. Through the combined use of these derivatives, most of the possible linkage isomers of the seven common aldoses of plant cell wall polysaccharides can be resolved, identified and quantitated.

Notes: The mass spectra of most nucleosides of cytosine exhibit molecular ions of low abundance, rearranged ions base + 2H in greater abundance than the companion species base + H, and a characteristic and abundant ion which corresponds to base + C2HO from the sugar. Deuterium, oxygen-18 and substituent labeling showed the presence of C-1′, 2′, H-2′ and O-4′ in the latter ion, which led to a proposed mechanism of formation involving opening of the sugar ring and recyclization between O2 and C-2′ with expulsion of water. Primary driving force for the reaction is due to resonance stabilization promoted by unshared electrons from N4, which results in diminished abundance in the case of imino isomers and derivatives which contain electron-withdrawing groups at N4, and enhanced abundance resulting from methylation at N4. Data for 24 nucleosides are presented and discussed in terms of the determination of nucleoside structure.

Notes: The metabolism of sodium dipropylacetate (a newly developed anticonvulsant) was investigated in rats, and two metabolites were found in urine of rats which received dipropylacetate. The metabolites were identified as 2-n-propyl-5-hydroxypentanoic acid and 2-n-propylglutaric acid, respectively, by low and high resolution mass spectrometry. Synthetic 2-n-propylglutaric acid, when administered to rats, was not oxidized further and was excreted intact in urine. These data clearly show that ω-oxidation is involved in the metabolism of sodium dipropylacetate and that 2-n-propylglutaric acid, the end product of ω-oxidation, does not serve as a substrate for β-oxidation.

Notes: The precise isotopic analysis of carbon by means of differential comparison of CO2 samples is applied to neutral, acid c and basic extract fractions of human urine. It is shown that the standard deviation of the analytical procedure, including sample preparation steps, is about 1% or 0.001 atom % excess carbon-13, but depends some what on the fraction considered. Day to day variations (expressed as standard deviations) in the isotopic composition of the urine fractions are generally less than 2.8%, 1.4% and 3.9% for the neutral, acidica and basic fractions, respectively, although the effect of unusual dietary inputs can be recognized. The ingestion of 23 μg excess carbon-13 in the form of isotopically labelled aspirin is shown to perturb significantly the isotopic composition of the acidic urine fraction which, for a 24 hour collection period, had a mass of 570 mg C. Because only 0.01% of the fraction was consumed by isotopic analysis, further extensive analysis would be possible. It is concluded that carbon-13 tracer experiments generally should be arranged to provide at least 5 × 10-5 g excess 13C/g carbon in any fraction which is to be used for lable detection.

Notes: The mass spectra of a mixture twenty dansyl derivatives of biogenic amines were run using low energy electron impact. This method appears to be useful for the analysis of amines present in biological materials.

Notes: The mass spectra of the trimethylsilyl derivatives of thyroxine and sixteen related compounds are reported. In addition to deiodinated analogues and iodinated tyrosines, the latter include a series of thyroacetic and thyropropionic acids and one thyroformic acid compound. Their spectra are discussed in terms of their fragmentation and utility for qualitative and quantitative estimation by gas chromatography - mass spectrometry.

Notes: The mass spectra of synthetic methoxychlorobiphenyls have been investigated. These substances are of interest as reference compounds in studies of the metabolism of chlorobiphenyls. Some characteristic fragmentation patterns have been found, useful for differentiation between the 2-, 3- and 4-methoxychlorobiphenyl isomers. Thus, mass spectra of 2-methoxychlorobiphenyls show large fragments at [M - CH3Cl]+., while those of 4-methoxy compounds differ from 3-methoxy isomers by the presence of significant [M - CH3]+ fragments in the former.

Notes: Chemical ionization mass spectra of four explosive compounds have been obtained by using water as reagent gas. The compounds are 1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane, 1,3,5-trinitor-1,3,5-triazacyclohexane, pentaerythritol tetranitrate, and an explosive which has been used in letter bombs, containing the two latter compounds and a plasticizer. The chemical ionization H2O mass spectra were recorded at an ion source temperature range of 170 to 225°C, a probe temperature range of 160 to 265°C and a reagent gas (H2O) pressure range of 0.07 to 0.10 Torr. Unlike in chemical ionization using methane or isobutane, chemical ionization H2O spectrum of these compounds provides a method for obtaining quasimolecular peaks. Adduct ion peaks like [M + H3O]+, [M + H5O2]+, [M + NO]+ and [M + NO2]+ are also observed in the chemical ionization H2O mass spectra of these compounds. Chemical ionization H2O mass spectra provide thus a better means of explosive identification.

Notes: Acetobacter suboxydans, strain 8.3, was grown using ethanol, made by the hydration of ethylene, as the sole energy source. After the microorganism had oxidized some of the ethanol to acetic acid, the unassimilated alcohol and produced acetic acid were isolated from the supernatant. Carbon isotopic analyses of these compounds and the starting ethanol show: (1) the methyl carbon of the starting alcohol is enriched in carbon-13 by 4.6% relative to the hydroxyl carbon, (2) the starting alcohol contains 2% less carbon-13 than the unassimilated ethanol and (3) the carboxyl carbon of the acetic acid excreted by the A. suboxydans is enriched by 3.8% in carbon-13 relative to the methyl carbon. These results support earlier findings which indicate that organisms preferentially utilize compounds enriched in carbon-12 and tend to concentrate carbon-12 in the reduced carbons relative to the oxidized carbons of metabolic products.

Notes: The electron impact, chemical ionization and field desorption mass spectra of seven steroids showing varying stability to electron impact have been compared.

Notes: The 3′- and 5′-O-t-butyldimethylsilyl derivatives of thymidine and deoxyadenosine have characteristically different mass spectra and can be separated by gas chromatography.

Notes: The difficulty of collecting volatile hydrocarbons, such as benzene, from street air in a form suitable for subsequent analysis is overcome by the use of a time-based elution procedure. By linking this technique to a gas chromatograph-mass spectrometer-data system, a rapid analysis is possible for low concentrations of collected material. Among advantages of this novel sample introduction procedure are the facility for collecting organic materials without interference from water vapour and their subsequent introduction into the g.c.-m.s. system without the use of any auxiliary solvents. Consequently the mass spectrometer can be used continuously on-line enabling the more volatile materials, normally masked by solvents, to be examined. This procedure is illustrated by the measurement of benzene and other hydrocarbon levels in street air (London). Following gas chromatographic separation the hydrocarbons can be identified by library search facilities attached to the mass spectrometer.

Notes: Several reagent gases and mixed reagent gases for high pressure ion-molecule reaction ionization of samples have been investigated for use in gas chromatography-mass spectrometry analyses. The common g.c. carrier gases and their mixtures with other gases such as isobutane, ammonia, tetramethylsilane, hexafluoroacetone and nitric oxide, were studied. These reagents were used to enhance the molecular ion intensity and to simplify the spectra of steroidal alcohols and the trimethylsilyl ethers of biologically important compounds.

Notes: The presence of epinine in adrenal medulla and in reaction mixtures of enzymic in vitro experiments is proven by mass fragmentography. Derivatization of catecholamines, suitable for gas chromatographic analysis is discussed. The perfluoropropionates were selected for mass fragmentography.

Notes: Using methylsulphinyl carbanion as base. methylation of 17-ketosteroids with methyl iodide not only converts hydroxy into methoxy groups, but also replaces both C-16 hydrogen atoms by methyl groups. These permethyl derivatives are very stable, have excellent properties for gas chromatographic separation and fragment characteristically upor electron impact. The mass spectra of a number of these derivatives are discussed.

Notes: The high resolution field desorption mass spectra of a number of glycosides are described. In all cases the spectra enabled molecular weights and elemental formulae to be established, and the presence of fragment ions due to cleavage of the sugar groups provides considerable assistance in the identification of sugar and aglycone moieties. The [M + 23Na]+ ion was the base peak in the spectrum of the sodium salt of testosterone glucuronide, and the presence [M + 39K]+, [M + 41K]+ and [M + 23Na]+ ions in the spectra of other glycosides are attributed to the presence of their sodium or potassium salts as impurities.

Notes: Preliminary results are reported for the quantitative determination of the antiarrhythmic agents quinidine and lidocaine in human plasma by combined isobutane chemical ionization mass spectrometry and stable isotope labeling. The concentration of monoethylglycinexylidide, a known metabolite of lidocaine, was also determined using this method. In the procedure used, stable isotope analogs of the materials to be determined were added to serial plasma samples. The plasma was then made basic and extracted with benzene. The residue after evaporation of the solvent was placed directly into the mass spectrometer via the direct insertion probe and the spectrum determined. In this manner the above named compounds could readily be quantitated without recourse to either derivitization or further purification.

Notes: The urinary excretion of butobarbitone and its metabolites has been studied quantitatively using combined gas chromatography-mass fragmentography, tuned to ions at m/e 169 and 184. After a single oral dose (200 mg) to healthy male volunteers, unchanged drug (7 to 9%), 3′-hydroxybutobarbitone (22 to 27%), 3′-oxobutobarbitone (14 to 18%) and the 3′-carboxylic acid (4 to 8%), were found. In all cases, the maximum rate of excretion was in or near the third 12 hour urine.

Notes: The high resolution, electron impact mass spectrum of 1,6-anhydro-3,4-O-isopropylidene-β-D-talopyranose, together with those of its derivatives having specific deuterium substitution at C-2, at C-3, at C-2 and C-3, and in the isopropylidene group, is considered in detail and compared with the ammonia and isobutane mediated chemical ionization spectra of these five compounds. From the elemental compositions of the fragment ions and mass number shifts upon deuterium incorporation, the origin of the hydrogen atoms in each of the fragments is traced, and a detailed scheme of the main fragmentation. pathways is presented.

Notes: The methyl derivative of tolbutamide, obtained by the action of dimethyl sulfate, has been subjected to g.c.-m.s. at high resolution. From the mass spectrum it is deduced that methylation of the sulfonamide nitrogen atom has occurred. During the loss of SO2 from the molecular ion, aryl rearrangement to both oxygen and nitrogen occurs in the ratio of 1:2.