Purine metabolizing enzyme activities in radiosensitive tissues of mice after sublethal whole-body irradiation

The activities of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were determined between days 1-14 in the spleen, thymus and femoral bone marrow of mice subjected to whole-body gama irradiation with a dose of 5.5 Gy. In control animals, the highest activity of ADA (as related to 10(6) cells) was recorded in the thymus (58.9 pmol.s-1), the lowest one in the femur (34.8 pmol.s-1), the PNP activity was the lowest in the thymus (14.5 pmol.s-1) and the highest in the femur (96.0 pmol.s-1). In the spleen, an elevation of ADA activity (up to 379%) was observed during the first postirradiation days; PNP activity was reduced (to 58%) on postirradiation day 3, followed by the return and even elevation on day 14 (265%). In the thymus, a parallel reduction of the activities of both enzymes appeared during the first postirradiation days, with a subsequent increase during the regeneration phase. In the femoral bone marrow, ADA and PNP activities were increased on postirradiation day 1 (275% and 201%, respectively). Reference is made to the possible relationship between the observed characteristic changes in activities and the degree of damage and/or renewal of cell population in the hemopoietic tissues after irradiation.     

Immunostimulating properties of synthetic derivatives of muramyl dipeptide and glucosaminyl muramyl dipeptide in vitro

Production of tumor necrosis factor (TNF) and interleukin-1 (IL-1) by macrophages of the spleen and peritoneal exudate of mice as well as cytotoxic factors (CFs) by murine splenocytes after in vitro activation was estimated. All the derivatives of muramyldipeptide (MDP) and glucosaminylmuramyldipeptide (GMDP) were able to induce production of TNF and CFs. In the presence of lipopolysaccharide (LPS), the effect was always higher. The response of the spleen macrophages to the effect of the preparations was higher than that of the peritoneal ones and ++non-fractionated splenocytes. GMDP and GMDP4 especially in the presence of LPS had the highest effect on induction of IL-1 by the murine peritoneal macrophages. On the contrary, MDP induced higher IL-1 synthesis by the spleen macrophages. The most active substances with respect to production of TNF, CFs and IL-1, i.e. MDP3 and GMDP4, might be recommended for immunotherapy of syngeneic tumors in animals.     

Activity of "nonspecific pancreatic carboxylesterase" in rat serum in experimentally induced acute pancreatitis (preliminary results)

The aim of this study was to obtain more information on the serum level of "nonspecific pancreatic carboxylesterase" (PCE) in experimentally induced acute pancreatitis in rats. The effects of caerulein stimulation, hepatic duct ligation, bile-pancreatic duct ligation or the effect of retrograde injection of saline, 5% taurocholate and sunflower oil were investigated. The activity of PCE and amylase was measured in the serum, pancreatic tissue, pancreatic juice and ascitic fluid. The changes in PCE activity were greater (both in directions to increase or decrease) than that of amylase, produced by different experimental procedures. The results confirm the thesis that the serum activity of PCE is a more sensitive diagnostic method than that of amylase to detect the inflammatory process in the pancreas or the effect of obstruction of the pancreatic duct.     

Study of the sensitivity of the L-forms of group A Streptococcus to antibiotics in artificial nutrient media and in human cell cultures

Sensitivity of L-forms of group A streptococci to 5 antibiotics such as erythromycin, lincomycin, tetracycline, gentamicin and chloramphenicol was studied in an artificial nutrient medium and cell cultures i.e. human fibroblast diploid cells and transplantable human heart cells (Girardi). In vitro investigation of the antibiotic effect on the streptococcal L-forms revealed their sensitivity to erythromycin (MIC, 0.4 micrograms/ml), lincomycin (MIC, 0.08 microgram/ml) and tetracycline (MIC, 2 micrograms/ml). The streptococcal L-forms were slightly sensitive to gentamicin (MIC, 6 micrograms/ml) and chloramphenicol (MIC, 30 micrograms/ml). Complete inhibition of the growth of the L-forms in the Girardi cells on the 1st day of the experiment after the antibiotics administration in single doses was induced by lincomycin, 5 micrograms/ml, erythromycin, 10 micrograms/ml, and tetracycline, 100 micrograms/ml. In the diploid cells, the respective figures were 50, 100 and 200 micrograms/ml. Chloramphenicol and gentamicin had an inhibitory effect on the growth of the L-forms but produced no sanative effect.     

Ultrastructural studies of scrapie-associated fibrils and prion protein from hamster brains infected with scrapie

We report here about the purification of prion protein 27-30 (PrP 27-30) and scrapie-associated fibrils (SAF) from hamsters infected with the 263K strain of scrapie. Ultrastructural analysis of fractions from scrapie-infected brains revealed numerous fibrils measuring approximately 20 nm in diameter and 100-200 nm in length. The substructure of these fibrils consisted of protofilaments which were usually straight and rarely helically arranged. We conclude that the electron microscopic appearance of SAF depends much on the purification scheme.     

Effect of Staphylococcus aureus serine proteinase on human lymphocyte impairment

Assessment of lymphocyte surface membrane integrity by means of trypan exclusion test and longer term survival studies based on 3HTdR uptake and Con A stimulation test were used to estimate effect of Staphylococcus aureus serine proteinase on human lymphocytes in vitro. At concentration of proteinase as high as 10 micrograms/ml, the percentage of trypan-stained lymphocytes increase with enzyme level. In contrast, decrease of 3HTdR level and Con A stimulation is already apparent at concentrations of proteinase ten times lower. Autologous serum exerts strong protective effect. Observed impairment is discussed as significant for local response in inflammation.     

Both wound-inducible and tuber-specific expression are mediated by the promoter of a single member of the potato proteinase inhibitor II gene family

A chimeric gene consisting of 1.3 kb of the 5' regulatory region of a member of the potato proteinase inhibitor II gene family, the coding region of the bacterial β-glucuronidase (GUS) gene and 260 bp of the proteinase inhibitor II 3'-untranslated region containing the poly(A) addition site was introduced into potato and tobacco by Agrobacterium tumefaciens mediated transformation. Analysis of transgenic plants demonstrates systemic, wound-inducible expression of this gene in stem and leaves of potato and tobacco. Constitutive expression was found in stolons and tubers of non-wounded potato plants. Histochemical experiments based on the enzymatic activity of the GUS protein indicate an association of the proteinase inhibitor II promoter activity with vascular tissue in wounded as well as in systemically induced non-wounded leaves, petioles, potato stems and in developing tubers. These data prove that one single member of the proteinase inhibitor II gene family contains cis-active elements, which are able to respond to both developmental and environmental signals. Furthermore they support the hypothesis of an inducing signal (previously called proteinase inhibitor inducing factor), which is released at the wound site and subsequently transported to non-wounded parts of the plant via the vascular system from where it is released to the surrounding tissue.     

A physical genome map of Pseudomonas aeruginosa PAO.

A complete macrorestriction map of the 5.9 Mb genome of Pseudomonas aeruginosa PAO (DSM 1707) was constructed by the combination of various one- and two-dimensional pulsed field gel electrophoresis techniques. A total of 51 restriction sites (36 SpeI sites, 15 DpnI sites) were placed on the physical map yielding an average resolution of 110 kb. Several genes encoding virulence factors and enzymes of metabolic pathways were located on the anonymous map by Southern hybridization. Distances between the gene loci were similar on the genetic and physical maps, suggesting an even distribution of genome mobility throughout the bacterial chromosome. The four rRNA operons were organized in pairs of inverted repeats. The two-dimensional macro-restriction techniques described herein are generally applicable for the genome mapping of any prokaryote and lower eukaryote which yields resolvable fragment patterns on two-dimensional pulsed field gels.     

Glucocorticoid receptor binds cooperatively to adjacent recognition sites.

In order to define the mechanism of synergistic induction mediated by multiple glucocorticoid response elements (GRE), the affinity of the glucocorticoid receptor to a single or duplicated GRE was analyzed by gel retardation, nitrocellulose filter binding and by footprinting experiments. Direct measurement of the relative affinity and indirect determination by competition showed greater than 10-fold higher affinity of the glucocorticoid receptor to a duplicated GRE when compared to a single element. Maximal stability of the GRE-receptor complex was obtained using two closely spaced GREs positioned on the same side of the DNA helix. Increasing the distance or changing the helical position of the GREs considerably increased the off rate of the receptor. DNase I footprinting shows in addition to the protection of the GRE region, an altered pattern in the nonprotected intervening DNA indicating structural alteration of the DNA helix by the receptor bound to adjacent GREs.     

Dual translational initiation sites control function of the lambda S gene.

Lysis gene S of phage lambda has a 107 codon reading frame beginning with the codons Met1-Lys2-Met3. Genetic data have suggested that translational initiation occurs at both Met1 and Met3, generating two polypeptides, S107 and S105 respectively. We have proposed a model in which the proper scheduling of lysis depends on the partition of translational initiations between the two start codons. Here, using in vitro methods, we show that two stem-loop structures, one immediately upstream of the reading frame and a second approximately 10 codons within the gene, control the partitioning event. Utilizing primer-extension inhibition or 'toeprinting', we show that the two S start codons are served by two adjacent Shine-Dalgarno sequences. Moreover, the timing of lysis supported by the wild-type and a number of mutant alleles in vivo can be correlated with the ratio of ternary complex formation over Met1 and Met3 in vitro. Thus the regulation of the S gene is unique in that the products of two adjacent in-frame initiation events have opposing function.