ALBERT

Notes: Previously, the precipitation of calcium carbonate by marine bacteria had been described in which bacteria occupy the nucleus of micro-aragonite crystals. The mechanism of the process of calcium-binding by the cells is the subject of this study.Sea-water cultures of Pseudomonas piscicida (Bein) Buck et al. (A.T.C.C. 15057) were grown under standardized conditions (1% peptone-artificial sea water, 25°C). Cells of various ages were collected and analyzed for calcium and magnesium content under various conditions. Material composing cell walls was fractionated and analyzed for low molecular weight components.Cells up to a week old (1% peptone-artificial sea water) increase their content of calcium from 0 to 55% of the dry weight. Furthermore the Ca/Mg ratio in young, actively growing cells (up to 24 hours old) is one-fifth (approximately that of sea water). By 72 hours, the ratio is 1/2.5. At one week, it is 1/1, and by two weeks calcium content exceeds that of magnesium.Concentration of the alkaline-earth elements is primarily by the cell envelope, particularly by the muco-polysaccharide fraction. This fraction constitutes 20-25% of the dry weight of cells during rapid growth but increases to 47-52% of dry weight after one week. Binding of alkaline earths to active sites of muco-polysaccharide is probably by hydrogen bonds and London interaction forces, as removal of the elements by washing is readily accomplished. In addition, a ready source of calciumactive ligands is available in the ammonia produced in quantity by the bacteria. The complex formation by ammonia and Ca, coupled with the increase in pH, favors binding of calcium over that of magnesium by the cells.

Notes: Rabbit psoas muscle fibers, extracted in 50% (w/v) glycerol for 20 hours, concentrate up to 300 μmol calcium per gm protein when placed in a suitable electrolyte environment at pH 6.5 containing ATP and 47Ca in the form of a Ca/EGTA metal buffer system. This property is retained at [Ca++] down to 2 × 10-8 M, the lowest studied. The kinetics of Ca pickup are consistent with a mathematical model based on diffusion of Ca as the rate-limiting factor. The pickup is dependent upon ATP and is increased by inclusion of CP or PEP. It is promoted by oxalate, fluoride, phosphate and pyrophosphate, which share the property of forming relatively insoluble calcium salts. Ability to concentrate calcium disappears on prolonged extraction in glycerol and after preincubation with desoxycholate.These properties are similar to those previously described for a granular fraction of muscle homogenates, and it is concluded that both are probably attributable to functional remnants of sarcoplasmic reticulum.

Notes: DNA synthesis was studied in vitro in lymphoid tissue taken from hibernating, cold adapted and active golden hamsters in an attempt to elucidate the mechanism of suppressed DNA synthesis during hibernation. The rates of DNA synthesis at temperatures varying from 4 to 37°C were greatest in cells from active animals, less from cold adapted and least from hibernating animals. The depression in cells from cold adapted animals was 70% reversible by returning the animals to a warm environment 40 minutes before they were sacrificed but the hibernating animals had to be aroused for more than 12 hours before their rates recovered, suggesting a different mechanism responsible for depression in these two instances.Radioautographic and biochemical measurements of DNA synthesis in cells taken from hibernating animals indicated that the depressed synthesis seen during hibernation is the result of a reduction in the percentage of cells engaged in DNA synthesis. This implies that the low body temperatures of hibernation produce a block in the cell cycle of lymphoid cells not unlike that observed when other mammalian cells are exposed to cold in vitro.

Notes: Addition of gibberellic acid to barley endosperm evokes the formation of α-amylase and of other hydrolases. Protein synthesis inhibitors-notably cycloheximide-inhibit the production of the hydrolases and the incorporation of labeled amino acids into proteins. Isolation, purification, and “fingerprinting” of the gibberellic acid-induced α-amylase indicate that it is formed by de novo synthesis. RNA synthesis inhibitors-notably actinomycin D-also inhibit the production of α - amylase and of the other hydrolases. Gibberellic acid enhances the incorporation of labeled RNA precursors into RNA. These data are consistent with the idea that gibberellic acid controls the level of α-amylase and of other enzymes by controlling the synthesis of specific RNA's which in turn control the synthesis of specific enzymes.

Notes: The fine structure of the thyroid of the newt, Notophthalmus viridescens, was studied with the electron microscope. Specimens were injected I.P. with 30 μc of I131 and sample thryoids were examined at 12 hour intervals thereafter. The ultrastructure of the normal thyroid gland is described, and compared with that of the irradiated glands. The first visible ultrastructural change observed after injection of the radioiodin was a striking alteration of nuclear morphology. This effect was followed by an increase in the frequency of whorled lamellar structures, a decrease in the number of microvilli, and degeneration of the Golgi apparatus and endoplasmic reticulum. Further effects observed included an increase in the number of large cytoplasmic granules and a decrease in the number of smaller ones, the presence of autophagic vacuoles, and finally, an increase in the number of degenerated mitochondria.

Notes: The histochemistry of the “sexual segment” granules of the kidney of male diamondback rattlesnakes has been studied to define the nature of these andro-genically responsive granules. The kidneys were variously fixed and sections stained with a number of acid dyes, as well as by a variety of carbohydrate, lipid, and protein histochemical methods. The results indicate that “sexual segment” granules bind acid dyes strongly, contain some lipids and neutral glyco- or mucoproteins, much tyrosine and lysine, and some tryptophan and cysteine. A resemblance, concluded to be superficial, is noted between the histochemical properties of zymogen granules and the predominantly proteinaceous “sexual segment” granules.

Notes: Ribonucleic acid polymerase and deoxyribonucleic acid polymerase have been partially purified from bovine lymphosarcoma, lymph node, and thymus. An examination of the deoxyribonucleic acid requirements of the two enzymes indicates that “native” deoxyribonucleic acid is the preferred template for ribonucleic acid synthesis; heat-denatured deoxyribonucleic acid is considerably less active. The primer requirements for deoxyribonucleic acid synthesis differ: “native” deoxyribonucleic acid is usually inactive, while denatured deoxyribonucleic acid is active. The two enzymes also differ in pH optima and in their requirements for metal cofactors.

Notes: The developmental features of the pancreas are reviewed as an example of cytodifferentiation and organogenesis. Attention is directed to the regulatory characteristics of the specific proteins synthesized and secreted by the endocrine and exocrine cells. The following topics are discussed: (1) number of specific protein species and, inferentially, the number of genes involved in differentiated function. (2) The stringent regulation of the concentration of these specific proteins and the probable restriction of their synthesis to exocrine and endocrine cells. (3) The multiphasic pattern of accumulation of these specific proteins during pancreatic development and the synchronized but noncoordinate regulation of individual protein species. Synthetic rates of specific exocrine proteins in vitro correlate closely with measurements of the accumulation of proteins during development. (4) A model postulating three regulatory transitions. The primary transition (related to organ “determination”) denotes the conversion of a “predifferentiated” cell to the “protodifferentiated” state in which low but significant levels of specific proteins are present. The secondary transition is viewed as an amplification of this specific protein synthesis and is associated with typical pancreatic histogenesis. In the third regulatory transition, the synthesis of specific proteins in the “differentiated state” is modulated by diet, or hormonal states, etc. The third regulatory transition may be similar to some types of “enzyme induction” as studied in multicellular systems. (5) The differentiative fidelity in an organotypic culture system; the role of mesenchymal tissue or a particle fraction derived therefrom in supporting the protodifferentiated state and the secondary regulatory transition. (6) The possible mechanisms of the secondary regulatory transition in exocrine cells. Effects of actinomycin D, bromodeoxyuridine, and other mitotic inhibitors suggest the requirement for a critical cell division prior to the loss of proliferative capacity. (7) The synthesis of pro-insulin and insulin during primary and secondary regulatory transitions; the possible interrelationships of endocrine and exocrine cells in pancreas development.

Notes: The development of a virus is programmed by a series of negative and positive controls which determine the timing and the segment on either of the two DNA strands (l or r) to be transcribed into specific messenger RNA's. Bacteriophage λ provides one of the most deeply studied systems for following the development of lysogenic viruses. In the lysogenic repressed state, only 2-4% of the λ genome is expressed. This pc-cI-rex region is transcribed leftward to produce a repressor protein which prevents any further transcription by blocking the oL and oR operators flanking the cI-rex operon (figs. 1, 2). This negative control is relieved by destruction of the repressor, and the result is the “induction” of viral development. The earliest post-induction or postinfection events are the leftward transcription of the pLoL N region from strand l and the rightward transcription mainly of the pRoR-x segment from strand r. The N product acts as a positive control, permitting a leftward transcription beyond gene N and a rightward transcription of genes cII-O-P and also Q. The int-xis system controls the excision of the λ genome, whereas the act of rightward transcription and the products of genes O and P initiate the replication of λ DNA. The product of gene Q, still another positive control, stimulates rightward transcription of the late genes which control the synthesis and assembly of the phage heads and tails as well as cell lysis. Among other types of negative control are the possible competition between the two divergent transcriptions originating in region x, the “antirepressor” effect of the x product, and the interference between the two convergent transcriptions which collide in the central b2 region. The majority of controls are based on protein-DNA interactions and can be modified by mutations. For instance, transcription can be rendered independent of negative repressor control either by constitutive, v, mutations which decrease or abolish the affinity of the o operators for the repressor or by insertion of new promoters-e.g., c17 or ric- on the “downstream” side of the operator. The need for the positive N and Q controls may also be obviated by mutations in the N- or Q-dependent promoter or terminator elements.The specific DNA structure within the controlling sites is not known. However, a remarkable coincidence was observed; namely, the occurrence of pyrimidine-rich clusters in those segments of the individual DNA strands acting as templates for RNA synthesis. This observation, which pertains to all studied DNA's, including those of phages T2, T3, T4, T5, T6, T7, λ, and φ 80, formed the basis for a proposal that implicates pyrimidine-rich clusters in the initiation, control and/or termination of transcription, and also in the determination of the preferred strand and, consequently, the orientation of transcription. General considerations regarding the possible role of the structural singularities, especially those represented by the pyrimidine clusters, in the bipartite structure of the recognition regions in DNA are discussed.

Notes: The cellular composition of individual hemopoietic spleen colonies has been studied using techniques which tested primarily for cell function rather than cell morphology. Erythroblastic cells were recognized by their capacity to incorporate radioiron, granulocytic cells by their content of peroxidase-positive material, and hemopoietic stem cells by their ability to form spleen colonies in irradiated hosts. It was found that, 14 days after the initiation of spleen colonies, the distribution of these cell types among individual colonies was very heterogeneous, but that most colonies contained detectable numbers of erythroblasts, granulocytes and colony-forming cells. An appreciable proportion of the cells in the colonies could not be identified as any of these three cell types. No strong correlations between numbers of erythroblasts, granulocytes and colony-forming cells in individual colonies were observed, though there was a tendency for colonies containing a high proportion of erythroblasts to contain a low proportion of granulocytes, and for colonies containing a high proportion of granulocytes to contain a higher proportion of colony-forming cells. An analysis of colonies which contained cells bearing radiation-induced chromosomal markers indicated that 83-98% of the dividing cells within 14-day spleen colonies were derived from single precursors.