Physiological and morphological characteristics of stationary phase vibrio cells able to support phase growth

Growth of phase alpha 3a on stationary phase Vibrio cultures requires micro-aerophilic conditions and is inhibited by aeration. Since pre-conditioning of the bacteria by allowing them to stand for 24 h after shaking for 3 d is an important aspect of the stationary phase phage growth system, various physiological and morphological characteristics of the stationary phase cells during the transition from shaking to standing were investigated. Shaken stationary phase cells were less viable and more sensitive to ultraviolet irradiation and heat than standing stationary phase cells. During pre-conditioning the small, non-flagellated cells present in shaken stationary phase cultures underwent morphological changes and became large, flagellated rods which resembled exponential phase cells. The transition of stationary phase cells from shaking to standing was associated with a marked increase in total RNA synthesis but a rapid and large decrease in total protein synthesis. Intracellular concentrations of ATP in shaken stationary phase cells were 53% lower than those in standing stationary phase cells. Studies on leucine uptake indicated that its transport was inhibited by isoleucine and that the major part (90%) of the total leucine uptake was due to a shared system for uptake of both amino acids. Shaken stationary phase cells transported less leucine than standing stationary phase cells. Inhibition of phage growth in aerated stationary phase cultures was not due to the prevention of phase absorption by shaking. It is suggested that the observed differences between shaken and standing stationary phase cells could be due to aeration affecting the template specificity of the Vibrio RNA polymerase.     

Studies on the development of the eye cup and optic nerve in normal mice and in mutants with congenital optic nerve aplasia.

With the use of quantitative histological techniques, we have described, in normal mice, the formation of a system of intercellular channels within the embryonic retina and continuing without interruption into the optic stalk. The channels develop in advance of the morphological differentiation of the retinal ganglion cells and their neurites. Moreover, they appear at predictable times during gestation and are localized along the potential route to be taken by the earliest developing fibers of the optic nerve. A functional relationship may exist between the development of the channels and the subsequent outgrowth of the optic nerve from the eye. We have also examined a series of mouse embryos homozygous for the mutant gene ocular retardation (or^J), which causes optic nerve aplasia. In the or^J mutant, there is a reduction in area of these extracellular spaces and the optic nerve fails to exit from the eye. The lack of intercellular space within the mutant retina is associated with an increased number of cells which, in turn, may result from a continuing absence of normal cell death during earlier stages.     

An ultrastructural and cytochemical study of the interaction between latex particles and the haemocytes of the wax moth Galleria mellonella in vitro

Summary The plasmatocytes are the major phagocytic blood-cell type in the haemolymph of the wax-moth, Galleria mellonella. In the present study, these cells were allowed to attach to tissue culture dishes for 1 h, rinsed and then incubated with latex beads for up to 72 h. These cells were then fixed for routine transmission electron microscopy and acid phosphatase cytochemistry. Intracellular latex particles were found in tight, ill-defined phagosomes, which were often clearly associated with the Golgi complexes of the plasmatocytes. Fusion of both primary lysosomes and multivesicular bodies with the phagosomes occasionally occurred and this resulted in the accumulation of an acid phosphatase positive reaction product around the test particles. Subsequent experiments showed that this acid phosphatase activity was mainly associated with the primary lysosomes. The results of the lysosome/latex interactions are compared with those obtained from similar studies on the digestive mechanisms in other phagocytes.We are grateful to Professor E.W. Knight-Jones in whose department this work was carried out and to Mrs. M. Colley for help in insect rearing. This work was supported by grants from the Royal Society and the Science Research Council (B/73/0176. and B/RG/2286.0)     

Changes in the immunoglobulin levels of the mouse gut and serum during conventionalisation and following administration of Salmonella typhimurium.

Increasess in all immunoglobulin classes, except IgM, were observed in the sera of specific pathogen-free (SPF) mice beginning 10 days after their removal from barrier conditions. Concentrations of serum immunoglobulins, comparable with those of conventional mice, were obtained in these animals between 21 and 35 days. Following the removal of germ-free mice from their sterile isolaters, their intestinal IgA levels increased over 35 days to attain levels found in conventional animals. A marked increase in serum immunoglobulin occurred within one day following intravenous administration of live Salmonella typhimurium organisms to SPF animals, and this persisted for longer than 7 weeks (the duration of the study), This rapid elevation in serum immunoglobulin was not elicited by nonbacterial antigens, killed Salmonellae, or viable Vibrio cholerae. Negligible amounts of this immunoglobulin increase could be attributed to specific antibody.     

Influence of postirradiation incubation temperature on recovery of radiation-injured Clostridium botulinum 62A spores.

The number of colonies formed by unirradiated Clostridium botulinum 62A spores was independent of temperature, in the range from 20 to 45 degrees C (in 5 degrees C increments); no colonies developed at 50 degrees C. Spores irradiated at 1.2 or 1.4 Mrads produced more macrocolonies at 40 degrees C than at higher or lower temperatures. Apparently, radiation-injured spores were capable of repair of 40 degrees C than at the other temperatures studied. More than 99% of the radiation (1.2 Mrads) survivors were injured and were unable to form macrocolonies in the presence of 5% NaCl. The germinated radiation-injured spores were also sensitive to dilution, resulting in the loss of viability of 77 to 79% of the radiation survivors. At 30 and 40 degrees C, the irradiated spores did not differ significantly in the extent of germination (greater than 99% at both 30 and 40 degrees C), emergence (64% at 30 degrees C and 67% at 40 degrees C), and the maximum number of emerged cells that started to elongate (69% at 30 degrees C and 79% at 40 degrees C). However, elongation was remarkably more extensive at 40 degrees C than at 30 degrees C. Many elongated cells lysed within 48 h at 30 degrees C, indicating an impaired repair mechanism. If the radiation-injured spores were incubated at 40 degrees C in the recovery (repair) medium for 8 to 10 h, they germinated, emerged, and elongated extensively and were capable of repair. If, after 8 to 10 h at 40 degrees C, these cultures were shifted to 30 degrees C, the recovery at 30 increased by more than eightfold, resulting in similar colony counts at 30 and 40 degrees C. Thus, repair appeared to be associated with outgrowth. Repair did not occur in the presence of chloramphenicol at 40 degrees C, whereas penicillin had no effect, suggesting that the repair involved protein synthesis but did not require multiplication.     

Oxidation of compounds metabolized through folate coenzyme pathways in vitamin B12-deficient rats

The effects of vitamin B₁₂ deficiency in rats and dietary supplementation with vitamin B₁₂ and/or l-methionine plus folate on the oxidation of compounds metabolized through folate coenzyme pathways were investigated. Rats fed a vitamin B₁₂-deficient diet oxidized significantly lower amounts in 60 min of l-histidine, glycine, sarcosine, formate, and l-serine to CO₂ than vitamin B₁₂-supplemented controls. Supplementation of the deficient diet with l-methionine plus folate restored the ability to oxidize the ring-2-carbon of l-histidine, the methyl group of sarcosine, and formate to the same level as that observed in animals receiving vitamin B₁₂. In contrast, oxidation of the 1-carbon of glycine and the 3-carbon of l-serine was not restored to control levels by addition of methionine plus folate to the vitamin B₁₂-deficient diet. Inhibition of the metabolism of the 2-carbon of glycine to CO₂ was partially overcome by additional dietary methionine and folate. Glycine synthase activity in homogenates paralleled the in vivo pattern of oxidation of the 1-carbon of glycine to CO₂, whereas sarcosine dehydrogenase activity appeared to increase 2-fold in vitamin B₁₂ deficiency.     

Improvements in a Non-Proprietary Radiometric Medium to Allow the Detection of Some Pseudomonas Species and Alcaligenes faecalis

The addition of [5-¹⁴C]glutamate and [¹⁴C]formate to a non-proprietary medium containing [¹⁴C]glucose, Trypticase, yeast extract, thiotone, and salts enabled the radiometric detection of the presence of nonfermenters of glucose. It did not interfere with the rapid detection of the presence of aerobic and anaerobic sporeforemers and nonsporeformers.     

Cellular defense reactions of insect hemocytes in vitro: phagocytosis in a new suspension culture system.

In a newly developed short-term culture system the plasmatocytes of Galleria mellonella, Pieris brassicae, Calliphora erythrocephala, and Periplaneta americana are the most active cell types in the phagocytosis of latex, chick erythrocytes, and certain bacteria. The granular cells of G. mellonella and the spherule cells of P. brassicae also phagocytose these test particles to a limited extent. This culture system is described, together with the appearance of ingested particles in preparations of living cells, and in fixed and stained monolayers. In culture, the hemocytes clump together due to cell instability in vitro and to the presence of uningested particles. This clumping reaction may be similar to nodule formation observed in vivo in these insects.     

Hepatic clearance of adenosine 3:5-cyclic monophosphate from plasma in the rat.

Rats were given intravenous injections of cyclic [3H]AMP and the disappearance of radioactivity from plasma and its appearance in bile were followed. Livers were removed and the cyclic [3H]AMP content was measured. The binding of radioactivity to soluble proteins was measured after preparations of a cytoplasmic fraction. Experiments in vitro to determine the ability of hepatic cytoplasmic proteins to bind cyclic [3H]AMP were also carried out. A role for cytoplasmic proteins in the clearance of cyclic AMP from plasma is discounted.     

Ultrastructure of the grasshopper proximal femoral chordotonal organ

Summary Calpasoma dactyloptera, a tentacled form of minute, freshwater coelenterate, has been investigated by light and electron microscopy and time-lapse cinematography. Each tentacle consists of a protrusion from a single ectodermal epithelial cell termed a tentaculocyte. hin tentaculocyte vesicles which represent invaginations of the plasma membrane. A cnidocil protrudes into the external medium. The bottom of each nematocyte is elongated as a stalk which extends to the tentacle base, coursing through tubular membrane lined channels within the tentaculocyte. A network of fibers and microtubules, originating in the cnidocil, extends to the base of the nematocyte stalk.Supported by PHS Research Career Development Award 1-K04-GM42595 and NSF Research Grant GB 29284.