Regulation of the synthesis of total cellular proteins and monoclonal antibodies in a hybridoma cell culture

A study was made of the regulation of total protein synthesis in cells of the mouse hybridoma producing monoclonal antibodies (McAb) against lambda phage, and in the course of hybridoma growth and at the change of fetal bovine serum (FBS) concentration. FBS strictly affected proliferation of hybridoma cells, the specific production of McAb per cell being unchanged. The rate of total cellular protein synthesis does depend on FBS concentration in the medium, whereas the rates of protein degradation and secretion do not. Evidence is presented that the reduction in the protein-synthesis rate, after the removal of FBS from the medium, is caused by a coordinated decrease in both the rate of protein synthesis initiation and the rates of polypeptide chain elongation and translation termination. The decrease in the protein synthesis rate at the stationary phase of cell growth was shown to be related to the three main factors: 1) a 15-25% decrease in ribosome content per cell; 2) a two-fold decrease of the ribosome portion involved in mRNA translation; 3) a 5 to 15% decrease in the rate of mRNA translation. Evidence is presented that the decrease in the portion of mRNA translating ribosomes is due to the decrease in the rate of protein synthesis initiation.     

Barnase-barstar high affinity interaction phenomenon as the base for the heterogenous bioluminescence pseudorabies virus' immunoassay

The effective new variant of "sandwich" bioluminescent enzyme immunoassay (BEIA) for the sensitive detection of glycoprotein B (gB) of pseudorabies virus (PrV) was presently developed. The high affinity interaction of barnase-barstar protein pair and photoprotein obelin as bioluminescent marker were for the first time successfully applied to BEIA development. Preliminary the two monoclonal antibodies, 11/5 and 34/2, were raised against gB for ELISA PrV detection. Presently we used the same immuno-"sandwich" principle for BEIA. To do this the two different bioconjugates were elaborated. Recombinant barnase was chemically conjugated with monoclonal anti-PrV's gB IgG, and also barstar was fused in frame to obelin. The characteristics of BEIA method have been compared to ELISA PrV detection. We have shown the proposed here gB-BEIA was 40-fold more sensitive as opposed to gB-ELISA test. The construction might have a broad promise in multiple potential immunological applications.     

Level of neuroactive steroids in the brain, and gender-related differences in formation and decrease of conditioned reflex in rats

We investigated gender-related differences in dynamics of the brain sexual steroids during learning and the decrease of conditioned reflex in the modeling experiment. It was revealed that, before training to conditioned reflexes, females manifested a greater anxiety than males. Significant differences between males and females in formation of the conditioned reflex of passive avoidance were not revealed, whereas the conditioned response decrease were by 2-3 days faster in male rats than in females. It was revealed that there was an increase of testosterone content in various structures of the brain, especially in hippocampus and frontal cortex with its constant level in the blood plasma in learning conditioned response of passive avoidance in male adult rats. Also an increased estradiol concentration was found in females' amygdale, while increased value of estradiol was detected in hippocampus and the singular cortex in decrease of the conditioned response of passive avoidance. In blood plasma, the testosterone level was not changed, and the estradiol concentration was reduced significantly after the decreased conditioned reflex. Different dynamics of changes in the levels of sex steroids in the brain and the blood plasma can indicate a probability of their formation in nervous tissue. The correlation analysis confirms the conception about selective involvement of the brain's testosterone and estradiol in the specific structures in realization ofthe learning and memory processes in adult male and female rats.     

Three-dimensional ultrastructural and immunohistochemical study of immature neurons in the subgranular zone of the rat dentate gyrus

The present study is devoted to three-dimensional ultrastructural organization of mitotically dividing immature neurons in dentate gyrus using biophysical approaches. In adult vertebrate brain, cell proliferation persists throughout life mainly in dentate gyrus of the hippocampus (DG) and olfactory bulb. Neurogenesis has been demonstrated using tagged thymidine analogues incorporated into the S phase of the cell cycle, but these may also detect repaired DNA in postmitotic neurons. Recent retroviral labelling has shown that neuronal progenitors/neuroblasts divide and produce functional neurons. Providing ultrastructural evidence of mitotically active cells has proven problematical, not only because of technical issues of identifying dividing cells at electron microscope level, but also because it is difficult to demonstrate unequivocally that neurons identified in the electron microscope are really post mitotic. However by characterising post mitotic cells labelled with BrdU and doublecortin and comparing these with post mitotic cells reconstructed in 3-dimensions from ultrathin serial sections, we have been able to illustrate individual mitotic elements and phases of cells within the GC layer of adult rat dentate gyrus. Here we show dividing cells in metaphase within clusters of immature GCs in subgranular zone (SGZ). These reconstructions provide ultrastructural confirmation that cells expressing doublecortin (DCX), a microtubule-associated protein expressed in migrating neurons, localize as clusters in the subgranular zone (SGZ) of dentate gyrus (DG) in the hippocampus during all animal life. Such DG cells with clear synaptic specializations, somatic spines and basal dendrites are exclusive to immature GC that appear to re-enter the cell cycle, suggesting the possibility that newly generated neurons within the DG might arise not only from precursors, but also from clusters of immature GC.     

Neuroactive steroids in the brain and age and sex specificity of behavior and anxiety: An experimental study

Changes in the brain’s neuroactive steroid levels, behavior in the open field, and the anxious-phobic status of male and female rats in the course of development have been studied. An increase in the motor and exploratory activity and emotionality in rats of both sexes in the pubertal period and a decrease in their values in mature and old animals have been detected. Anxiety has no sexual dimorphism in adult animals; it is significantly higher in males than in females in the prepubertal and pubertal periods of development and is higher in old females than in males of the same age. An increase in the level of corticosterone in some brain structures in maturing and old rats has been found; the testosterone concentration increases in one-month-old and adult animals but decreases in old individuals, while the estradiol concentration in all studied brain structures of male and female rats was low in all periods of postnatal life. Correlation analysis has shown modulation by steroid hormones of the changes in behavioral responses during development.     

The role of membrane-bound heat shock Hsp90 proteins in the migration of tumor cells in vitro and the involvement of cell surface heparan sulfate proteoglycans in protein binding to the plasma membrane

The heat-shock protein Hsp90, which is found in the extracellular space and on the cell membrane, plays an important role in cell motility, migration, invasion, and metastasis of tumor cells. Currently, the functional role and molecular mechanisms of Hsp90 binding to the plasma membrane are not clear. Using isoform-specific antibodies against Hsp90, Hsp90α, and Hsp90β, we showed that the membrane-bound Hsp90α and Hsp90β proteins play a significant role in the migration of human fibrosarcoma (HT1080) and glioblastoma (A-172) cells in vitro. Impairment of sulfonation of cellular heparan sulfates, cleavage of cellular heparan sulfates by heparinase I/III, as well as the treatment of cells with heparin, lead to a dramatic reduction in the expression levels of the Hsp90 isoforms. Furthermore, heparin significantly inhibits tumor cell migration. These results demonstrate that two isoforms of membrane-bound Hsp90 are involved in tumor-cell migration in vitro. As well, the cell surface heparan sulfate proteoglycans are shown to play a pivotal role in the “anchoring” of Hsp90α and Hsp90β to the plasma membrane.     

Hybridoma cultures using porcine serum not containing immunoglobulin and dialysis fractions

The whole swine serum was treated with ammonium sulphate to precipitate immunoglobulins. The remained IgG was removed with the use of protein A-sepharose. The hybridoma cells producing monoclonal antibodies to lambda phage (class IgG) were cultured in Dalbecco's modified Eagle medium with addition of a 5% whole swine serum or of a treated unwhole one (final concentration of the protein being 3 mg/ml). Upon these conditions, hybridoma cells had similar growth rate and population density (1-1.3 X 10(6) cells/ml). Maximal antibody concentration was almost similar (80-90 mcg/ml). Purity of a sample of monoclonal antibodies isolated by the method of chromatography with the use of protein A-sepharose from supernatant containing the unwhole serum was no less than 99%, whereas it was considerably lower (12-15%) in the case of the whole serum.     

Responses and properties of receptive fields of neurons in the visual projection zone of the pigeon hyperstriatum

Unit responses in the hyperstriatal region of the pigeon forebrain to the action of various visual stimuli were investigated. Particular attention was paid to the discovery of retinotopic projection in the Wulst region. It was shown that as the electrode was advanced in the caudal direction in the zone of visual projection of the hyperstriatum the receptive fields of the neurons recorded shifted in the opposite direction in the visual field. The receptive fields of neurons of the ventral and dorsal hyperstriatum lie higher in the visual field and are larger in diameter than those of neurons of the accessory hyperstriatum. Unit responses in the visual projection zone of the Wulst depend on the intensity of illumination, size, and speed and direction of movement of the test objects across the receptive field. The functional role of the retino-thalamo-telencephalic system in visual interpretation in birds is discussed and it is suggested that the Wulst region is comparable with the striatal and also with the frontal regions of the mammalian cortex.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 8, No. 3, pp. 230–236, May–June, 1976.