Oxygen consumption during embryonic development of the annual fish Nothobranchius korthausae with special reference to diapause

The oxygen consumption of Nothobranchius korthausae eggs in different developmental stages, including diapause II and III, was measured. Oxygen consumption increases exponentially during embryonic development. In diapause II and III there is a drop in oxygen consumption, which attains a minimal level in diapause II after 3 weeks and in diapause III after 2 weeks. During early development the embryos can escape from hypoxic stress by entering diapause I and II. During late embryogenesis embryos in diapause III can escape from hypoxic stress by hatching. We conclude that survival of annual fish embryos is enhanced during conditions of low oxygen concentration by reduced oxygen consumption rates during diapause.     

Intrinsic variability in the frequency of embryonic diapauses of the annual fish Nothobranchius korthausae,regulated by light:dark cycle and temperature

Synopsis The influence of the light:dark cycle and temperature on the embryonic development, especially diapause, of the annual fish Nothobranchius korthausae was investigated. The variability of the frequency of diapause II during constant L:D cycle and temperature, but at different times of the year, was also studied. In agreement with previous studies it appeared that diapause I does practically not occur in N. korthausae (and other Nothobranchius species), even under sub-optimal conditions which are known to induce diapause II and III. Only at very low temperatures, a first developmental arrest could be induced during the dispersed phase, as well as during the reaggregation phase. Diapause II and III can be induced more easily, by exposing the embryos to low (sub-optimal) temperatures and short light periods (or constant darkness). Both diapauses are inhibited or terminated at high temperature and long light periods (12L:12D). The occurrence of an indirect light:dark response via the adult fishes could not be demonstrated. At moderate temperatures and in constant darkness (or short light periods) considerable variability in diapause II-frequency was observed. This could be an intrinsic feature of annual fish development, constituting a strategy for better survival of the species.     

The relation between pituitary gland and thyroid growth during the lifespan of the annual fish Cynolebias whitei and Nothobranchius korthausae: gonadotropic and thyrotropic cells

Summary In the annual cyprinodont Cynolebias whitei the cell types responsible for the increase of pituitary growth at the onset of maturation and for pituitary hyperplasia in old specimens were identified as gonadotropic cells and thyrotropic cells, respectively. The gonadotropic cells showed a high affinity to anti-carp -GTH serum, both at light- and electron-microscopical levels. The allometric relation of total gonadotropic cell volume to body length, determined for fish from six weeks up to six months of age, showed no inflections. Therefore pituitary growth in maturing fish may be partly a result of proliferation of gonadotropes, although gonadotropic cells do not contribute to pituitary hyperplasia in old fish. Thyrotropic cells showed a weak affinity to anti-carp -GTH serum at light-microscopical level. Under the electron microscope thyrotropic cells displayed signs of activation in maturing fish and signs of proliferation in old fish. The allometric relation of thyroid gland volume to body length paralleled that of pituitary volume to body length. Histologically the thyroid gland showed signs of inactivity in adult fish and of hyperplasia in old fish. The possibility, that gonadal maturation, pituitary thyrotropic activity, and growth of the thyroid in maturing fish are related through the inhibitory action of gonadal steroids on thyroid hormone release, is discussed. Pituitary hyperplasia in old fish is the result of proliferation of thyrotropic cells. Similar hyperplasia of pituiary and thyroid glands was observed in old Nothobranchius korthausae.     

Contact inhibition and gene expression in HTC-L cell hybrid lines

Contact-inhibited somatic cell hybrids were formed between two malignant cell lines lacking contact inhibition of growth. One cell line was HTC-AR1, an azaguanine-resistant subline from the rat hepatoma line HTC +; the other line was the BUDR-resistant mouse L-cell subline L-B82. Hybrids were obtained from selective medium and characterized by chromosomal and enzymic analysis. The hybrids lacked the inducible rat enzyme tyrosine aminotransferase.     

Some factors affecting optimal differential staining of sister-chromatids in vivo in the fish Nothobranchius rachowi

In the past few years, routine studies of SCE induction in vivo in fish have been hampered by unreliable SCD techniques. This paper presents a number of modifications of the SCD technique in vivo in Nothobranchius rachowi. Major improvements were obtained by BrdU incorporation from aqueous solutions, short intervals between preparation and staining of slides and post-treatment with HCl. These improvements resulted in a highly reliable SCD procedure in Nothobranchius with a low level base-line SCE frequency (0.90 SCE/metaphase, 0.059 SCE/chromosome). Further research is now directed at gathering additional data on base-line SCE frequencies, establishing the sensitivity of the assay for aqueous solutions of known mutagens, and defining an experimental set-up for optimal statistical evaluation.     

Mengovirus leader is involved in the inhibition of host cell protein synthesis.

The presence of a leader peptide in picornaviruses is restricted to the Cardiovirus and Aphthovirus genera. However, the leader peptides of these two genera are structurally and functionally unrelated. The aphthovirus leader is a protease involved in viral polyprotein processing and host cell translation shutoff. The function of the cardiovirus leader peptide is still unknown. To gain an insight into the function of the cardiovirus leader peptide, a mengovirus leader peptide deletion mutant was constructed. The deletion mutant was able to grow at a reduced rate in baby hamster kidney cells (BHK-21). Mutant virus production in mouse fibroblasts (L929 cells), however, could be demonstrated only after inoculation of BHK-21 cells with the transfected L929 cells. Analysis of cellular and viral protein synthesis in mutant virus-infected cells showed a delayed inhibition of host cell protein synthesis and a reduced production of viral proteins. In a single-cycle infection, mutant virus produced only 1% of wild-type virus yield at 8 h postinfection. Host cell translation shutoff in L929 cells infected with mutant virus was restored by the addition of the kinase inhibitor 2-aminopurine. Mutant virus production in 2-aminopurine-treated L929 cells was increased to 60% of wild-type virus yield at 8 h postinfection. Our results suggest that the cardiovirus leader peptide is involved in the inhibition of host cell protein synthesis.     

Contact inhibition, polyribosomes, and cell surface membranes in cultured mammalian cells

An “overlay” method for rapidly and synchronously inducing contact inhibition in normal cultured cells has been developed. Using this method, disaggregation of cytoplasmic polyribosomes has been observed to occur within a matter of hours after overlay, followed by a decrease in cellular ribosomal RNA. Polysome disaggregation was influenced by the extent of cell-cell interaction and was inhibited by pretreatment of overlay cells with cycloheximide. Treatment of underlay cells with cytosine arabinoside also induced polysome disaggregation, but only after an appreciable lag as compared to that observed in overlaid cultures. Disaggregation could be induced by this method in cultured cells derived from normal tissue but not in cells derived from cancerous tissue. Polysome synthesis in growing “normal” cells (as measured by incorporation of tracer uridine into RNA) was markedly decreased when a cell surface membrane preparation was added to cultures.     

Contact inhibition of division: involvement of the electrical transmembrane potential

Measurements of simultaneous mitotic activity, electrical transmembrane potential (E_m), and cell density levels in both 3T3 and Chinese hamster ovary (CHO) cell cultures reveal that a 5- to 6-fold increase in the E_m level is associated with development of mitotic arrest at saturation densities. This rise occurs both in confluent monolayers and in interior areas of isolated colonies, and is independent of the rate at which confluence is attained. The E_m rise is accompanied by a substantial decrease in intracellular Na. Electron microscopy of saturated CHO monolayer sections shows from 46 to 63% of the cell surfaces to be in close apposition (<300 Å spacing). These results for contact inhibited cultures support the hypothesis that mitotic activity may be functionally coupled with the E_m level and associated ionic concentration levels. It is suggested that contact inhibition of mitosis may result from a reduction in synthesis of mitogenically essential RNA following a decrease in intracellular Na produced by contact-induced alteration of surface ion-transport activity.     

Arabidopsis membrane steroid binding protein 1 is involved in inhibition of cell elongation

A putative Membrane Steroid Binding Protein (designated MSBP1) was identified and functionally characterized as a negative regulator of cell elongation in Arabidopsis thaliana. The MSBP1 gene encodes a 220-amino acid protein that can bind to progesterone, 5-dihydrotestosterone, 24-epi-brassinolide (24-eBL), and stigmasterol with different affinities in vitro. Transgenic plants overexpressing MSBP1 showed short hypocotyl phenotype and increased steroid binding capacity in membrane fractions, whereas antisense MSBP1 transgenic plants showed long hypocotyl phenotypes and reduced steroid binding capacity, indicating that MSBP1 negatively regulates hypocotyl elongation. The reduced cell elongation of MSBP1-overexpressing plants was correlated with altered expression of genes involved in cell elongation, such as expansins and extensins, indicating that enhanced MSBP1 affected a regulatory pathway for cell elongation. Suppression or overexpression of MSBP1 resulted in enhanced or reduced sensitivities, respectively, to exogenous progesterone and 24-eBL, suggesting a negative role of MSBP1 in steroid signaling. Expression of MSBP1 in hypocotyls is suppressed by darkness and activated by light, suggesting that MSBP1, as a negative regulator of cell elongation, plays a role in plant photomorphogenesis. This study demonstrates the functional roles of a steroid binding protein in growth regulation in higher plants.     

Differential distribution of factors involved in pre-mRNA processing in the yeast cell nucleus.

The yeast cell nucleus has previously been shown to be divided into two regions by a variety of microscopic approaches. We used antibodies specific for the 2,2,7-trimethylguanosine cap structure of small nuclear ribonucleic acids (snRNAs) and for a protein component of small nuclear ribonucleoprotein particles to identify the distribution of small nuclear ribonucleoprotein particles within the yeast cell nucleus. These studies were performed with the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae. By using immunofluorescence microscopy and immunoelectron microscopy, most of the abundant snRNAs were localized to the portion of the nucleus which has heretofore been referred to as the nucleolus. This distribution of snRNAs is different from that found in mammalian cells and suggests that the nucleolar portion of the yeast nucleus contains functional domains in addition to those associated with RNA polymerase I activity.     

Host cell factors and functions involved in vesicular stomatitis virus entry

Vesicular stomatitis virus (VSV) is an animal virus that based on electron microscopy and its dependence on acidic cellular compartments for infection is thought to enter its host cells in a clathrin-dependent manner. The exact cellular mechanism, however, is largely unknown. In this study, we characterized the entry kinetics of VSV and elucidated viral requirements for host cell factors during infection in HeLa cells. We found that endocytosis of VSV was a fast process with a half time of 2.5 to 3 min and that acid activation occurred within 1 to 2 min after internalization in early endosomes. The majority of viral particles were endocytosed in a clathrin-based, dynamin-2-dependent manner. Although associated with some of the surface-bound viruses, the classical adaptor protein complex AP-2 was not required for infection. Time-lapse microscopy revealed that the virus either entered preformed clathrin-coated pits or induced de novo formation of pits. Dynamin-2 was recruited to plasma membrane-confined virus particles. Thus, VSV can induce productive internalization by exploiting a specific combination of the clathrin-associated proteins and cellular functions.     

Low molecular weight protein-tyrosine phosphatase is involved in growth inhibition during cell differentiation

Low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an enzyme involved in mitogenic signaling and cytoskeletal rearrangement after platelet-derived growth factor (PDGF) stimulation. Recently, we demonstrated that LMW-PTP is regulated by a redox mechanism involving the two cysteine residues of the catalytic site, which turn reversibly from reduced to oxidized state after PDGF stimulation. Since recent findings showed a decrease of intracellular reactive oxygen species in contact inhibited cells and a lower tyrosine phosphorylation level in dense cultures in comparison to sparse ones, we studied if the level of endogenous LMW-PTP is regulated by growth inhibition conditions, such as cell confluence and differentiation. Results show that both cell confluence and cell differentiation up-regulate LMW-PTP expression in C2C12 and PC12 cells. We demonstrate that during myogenesis LMW-PTP is regulated at translational level and that the protein accumulates at the plasma membrane. Furthermore, we showed that both myogenesis and cell-cell contact lead to a dramatic decrease of tyrosine phosphorylation level of PDGF receptor. In addition, we observed an increased association of the receptor with LMW-PTP during myogenesis. Herein, we demonstrate that myogenesis decreases the intracellular level of reactive oxygen species, as observed in dense cultures. As a consequence, LMW-PTP turns from oxidized to reduced form during muscle differentiation, increasing its activity in growth inhibition conditions such as differentiation. These data suggest that LMW-PTP plays a crucial role in physiological processes, which require cell growth arrest such as confluence and differentiation.     

Contact inhibition in the failure of mammalian CNS axonal regeneration

Anamniote animals, such as fish and amphibians, are able to regenerate damaged CNS nerves following injury, but regeneration in the mammalian CNS tracts, such as the optic nerve, does not occur. However, severed adult mammalian retinal axons can regenerate into peripheral nerve segments grafted into the brain and this finding has emphasized the importance of the environment in explaining regenerative failure in the adult mammalian CNS. Following lesions, regenerating axons encounter the glial cells, oligodendrocytes and astro-cytes, and their derivatives, respectively myelin and the astrocytic scar. Experiments to investigate the influence of these components on axon growth in culture have revealed cell-surface and extracellular matrix molecules that inhibit axon extension and growth cone motility. Structural and functional characterization of these ligands and their receptors is underway, and may solve the interesting neurobiological conundrum posed by the failure of mammalian CNS regeneration. Simultaneously, this might allow new possibilities for treatment of the severe clinical disabilities resulting from injury to the brain and spinal cord.