Reaction of young pigs to total gamma-irradiation

A study was made of clinical manifestations of radiation sickness of pigs exposed to gamma-radiation within a wide range of doses, LD50/30 was about 2.82 Gy after a short-term whole-body exposure: the nonuniformity coefficient was 1.2.     

Quantitative evaluation of changes in the microtubule content of lymphocytes

We modified a radioactive antibody binding assay in order to determine changes in the content of polymerised polymerised tubulin in lymphocytes of immunised animals is 2-4 times higher than in those of the control mice.     

On the total number of genes and their length distribution in complete microbial genomes

In sequenced microbial genomes, some of the annotated genes are actually not protein-coding genes, but rather open reading frames that occur by chance. Therefore, the number of annotated genes is higher than the actual number of genes for most of these microbes. Comparison of the length distribution of the annotated genes with the length distribution of those matching a known protein reveals that too many short genes are annotated in many genomes. Here we estimate the true number of protein-coding genes for sequenced genomes. Although it is often claimed that Escherichia coli has about 4300 genes, we show that it probably has only approximately 3800 genes, and that a similar discrepancy exists for almost all published genomes.     

Estimating the total number of protein folds.

Many seemingly unrelated protein families share common folds. Theoretical models based on structure designability have suggested that a few folds should be very common while many others have low probability. In agreement with the predictions of these models, we show that the distribution of observed protein families over different folds can be modeled with a highly-stretched exponential. Our results suggest that there are approximately 4,000 possible folds, some so unlikely that only approximately 2,000 folds existing among naturally-occurring proteins. Due to the large number of extremely rare folds, constructing a comprehensive database of all existent folds would be difficult. Constructing a database of the most-likely folds representing the vast majority of protein families would be considerably easier.     

Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration.

The electron transport system of respiring organisms reduces 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan. Respiring bacteria deposit accumulated INT-formazan intracellularly as dark red spots. Corresponding to electron transport system activity, these deposits attain a size and a degree of optical density which allows them to be examined by light microscopy. If polycarbonate filters and epifluorescence microscopy are applied to analyze an INT-treated water sample, it is possible to differentiate between respiring and apparently nonrespiring bacteria. This differentiation, which permits determinations of the total number of bacteria and the proportion thereof involved in respiration, is realized directly within one and the same microscopic image. Initial applications of the present method for hydrobiological purposes showed that the proportion of respiring aquatic bacteria ranged between 6 to 12% (samples taken from coastal areas of the Baltic Sea) and 5 to 36% (samples taken from freshwater lakes and ponds). Cells of 1.6 to 2.4 micrometer (freshwater) and 0.4 micrometer (Baltic Sea) account for the highest proportion of respiring bacteria.     

Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration.

The electron transport system of respiring organisms reduces 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan. Respiring bacteria deposit accumulated INT-formazan intracellularly as dark red spots. Corresponding to electron transport system activity, these deposits attain a size and a degree of optical density which allows them to be examined by light microscopy. If polycarbonate filters and epifluorescence microscopy are applied to analyze an INT-treated water sample, it is possible to differentiate between respiring and apparently nonrespiring bacteria. This differentiation, which permits determinations of the total number of bacteria and the proportion thereof involved in respiration, is realized directly within one and the same microscopic image. Initial applications of the present method for hydrobiological purposes showed that the proportion of respiring aquatic bacteria ranged between 6 to 12% (samples taken from coastal areas of the Baltic Sea) and 5 to 36% (samples taken from freshwater lakes and ponds). Cells of 1.6 to 2.4 micrometer (freshwater) and 0.4 micrometer (Baltic Sea) account for the highest proportion of respiring bacteria.     

Quantitative evaluation of radiation-induced changes in sperm morphology and chromatin distribution

Sperm head cytometry provides a useful assay for the detection of radiation-induced damage in mouse germ cells. Exposure of the gonads to radiation is known to lead to an increase of diploid and higher polyploid sperm and of sperm with head shape abnormalities. In the pilot studies reported here quantitative analysis of the total DNA content, the morphology, and the chromatin distribution of mouse sperm was performed. The goal was to evaluate the discriminative power of features derived by high resolution image cytometry in distinguishing sperm of control and irradiated mice. Our results suggest that besides the induction of the above mentioned variations in DNA content and shape of sperm head, changes of the nonhomogeneous chromatin distribution within the sperm may also be used to quantify the radiation effect on sperm cells. Whereas the chromatin distribution features show larger variations for sperm 21 days after exposure (dpr), the shape parameters seem to be more important to discriminate sperm 35 dpr. This may be explained by differentiation processes, which take place in different stages during mouse spermatogenesis.     

Quantitative studies of changes in cortical granule number and distribution in the mouse oocyte during meiotic maturation

Cortical granules (CGs) undergo a substantial change in distribution in the mouse oocyte cortex during meiotic maturation. In order to determine the mechanism of their change in distribution near the time of ovulation, CG density, total number per oocyte, and domain areas were quantitated. CGs were visualized microscopically by Lens culinaris agglutinin-biotin and Texas red-strepavidin fluorescence as well as by electron microscopy. Immature germinal vesicle stage (GV) oocytes from adult mice had a continuous cortical localization with some interior granules. Mature oocytes had an asymmetric cortical distribution with a CG-free domain, overlying the meiosis II metaphase spindle, occupying 40% of the cortex. The mean CG densities of the granule-occupied cortex of mature oocytes and the entire cortex of GV oocytes were 43 and 34 CGs/100 micron 2, respectively. The mean total numbers of CGs/oocyte were 4127 (mature) and 7440 (GV), and staining was absent in fertilized oocytes with two pronuclei. Calcium ionophore (A23187)-activated mature oocytes had a mean total number of 1235 CGs, some of which may have been in the process of exocytosis. The first polar body had few CGs, and thus was unlikely to account for the difference in CG number between GV and mature oocytes. The smaller total number and higher density of CGs in mature mouse oocytes suggests that both exocytosis and redistribution are plausible mechanisms for the development of the CG-free domain. Prefertilization exocytosis could account for the locus of sperm penetration which others have reported to occur in the hemisphere opposite the meiotic spindle in the mouse.     

Quantitative proteomics of lymphocytes

Lymphocytes are the best-studied higher eukaryote cells. In this report, quantitative relationships of the protein components in resting cell, blast cell and plasma cell types are evaluated. The comparison of these cell types leads to the conclusion that resting cells synthesize about one-twentieth of the protein species as compared to blast cells. Blast cells seem to be metabolically the most robust lymphocyte type. Plasma cells are geared towards synthesis of one main product (antibody in B plasma cells), while most of the synthesis of other protein species (including those for housekeeping and repair) decreases as the messages decay. Although the data presented in this communication allow a meaningful comparison of three cell populations, they are far from providing a full picture. Both silver staining and radiofluorography depict only proteins of high or intermediate abundance. Silver staining misses most proteins present at <10 000 copies/cell, while radiofluorography misses all those proteins with slow turnover (and those with no methionine residue in their sequence). The detection of 1100 spots in the blast cell-related radiofluorograph includes visualization of some 97-99% of protein mass, but some 3900 polypeptide species in the remaining 1-3% of protein mass will pass undetected. This protein mass (0.7-2 pg) reflects some 2500-7500 copies of each of those 3900 polypeptide species that are present in the cell below the detection limit. The work emphasizes that full understanding of cellular function can be achieved only if quantitative aspects of cell inventory are considered.     

Micronuclei in lymphocytes of young patients under antileukemic therapy.

The micronucleus test in peripheral blood lymphocytes was employed in the cytogenetic monitoring of children with acute lymphocytic leukemia (ALL), who had undergone chemotherapy and radiotherapy. Patients were treated with a variety of drugs, which included vincristine, methotrexate, daunomycin and prednisone; they also underwent cranial irradiation at the end of the first intensive phase of therapy. The first group under study consisted of 15 subjects on therapy, who showed a marked increase in micronucleated lymphocytes (mean: 19.96 +/- 12.96%) as a consequence of treatment compared with the control group (mean: 3.67 +/- 1.55%), while lower average values were obtained from 15 other subjects at the end of treatment (mean: 13.16 +/- 8.44%). A group of 6 patients was monitored during the entire period of therapy, namely at diagnosis, after 3 months of therapy, throughout maintenance therapy and at the end of it. The whole treatment lasted about 2 years. The results revealed a marked increase in basal micronucleus frequency, due to therapy: the micronucleated lymphocyte frequency remained significantly high throughout the treatment for almost all patients. These data clearly suggest the validity of the methodology in pointing out the role played by antileukemic agents in inducing somatic genetic damage.     

Effects of atherogenic diet on young farm pigs.

This study was designed to assess the usefulness of young farm pigs as an experimental model for hypercholesterolemia. In order to test this, we investigated both serologic and electrocardiographic effects of atherogenic diet. Four-week-old pigs were fed an atherogenic diet for 8 weeks. No arrhythmia was observed on ECG in all animals. There were no significant difference between control and atherogenic diet group on the values of ECG parameters. However, plasma lipids values of atherogenic diet group were significantly (p less than 0.05) higher than those of control diet group. Thus, hypercholesterolemia was induced in young farm pigs by feeding atherogenic diet in a relatively short time. This fact suggests that young farm pigs may be an useful model for further studies of the effect of hypercholesterolemia on cardiovascular function and the early pathogenesis of atherosclerosis.     

Quantitative distribution of lysosomal hydrolases in the rat nephron.

The activities of N-acetyl-beta, D-glucosaminidase (NAG, EC 3.2.1.30), beta, D-galactosidase (beta-gal, EC 3.2.1.23) and acid phosphatase (ac-Pase, EC 3.1.3.2) were measured in the glomeruli, five segments of the proximal and four segments of the distal tubule of normal male Wistar rats. The activities of NAG and beta-gal are 3- to 5-fold higher in the first part of the proximal tubule than in other segments and very low in glomeruli. We propose that the distribution of these two glycosidases reflects the contribution of the different tubular segments to the reabsorption of glycoproteins. The maximal activity of ac-Pase was found in the straight part of the proximal tubule. It was only 1.5-fold higher than in the distal tubule. Moreover, the activity in glomeruli is rather high. We conclude that ac-Pase is not primarily involved in the handling of reabsorbed molecules.     

Influence of amino acids on gastric emptying in young pigs.

The effects of amino acids on gastric emptying and secretion were studied in four young conscious pigs provided with a chronic gastric cannula. A basal test meal of 500 ml containing 10 g citrus pectin, 17.5 g sucrose, 100 mg phenol red alone or with glycine, L- or DL-tryptophan, DL-methionine or L-glutamic acid was poured into the stomach and recovered 20 min later. Glycine at concentrations of 26.7--106.8 mM/l did significantly affect gastric emptying, although at the highest concentration gastric emptying appeared to slow down. L-lysine (41.0 mM/l), DL-methionine (40.3 mM/l or L-tryptophan (29.4 mM/l did not significantly affect the rate of stomach emptying; L-tryptophan increased the amount of Cl- and H+ secreted as compared with the basal diet alone. The effects of addition of the essential amino acids, L-lysine and DL-methionine, were compared with those of the nonessential glycine and L-glutamic acid. There was no significant difference in the rate of gastric emptying or secretion between them. The L and DL isomers of lysine and trypotphan were not found to differ significantly in their effects on gastric emptying and secretion.