Noise-modulated-microwave-induced response in snail neurons

Helix aspersa neurons were irradiated with noise-amplitude-modulated microwaves (carrier frequency 2450 MHz, 20% AM, 0-20 kHz, specific absorption rate 6.8 and 14.4 mW/g). It was found that such an exposure caused an appearance of high frequency bursts and a rise in membrane resistance.     

Cloning and characterization of a testis-specific thymosin beta 10 cDNA. Expression in post-meiotic male germ cells.

Thymosin beta 10 is one of a small family of proteins closely related in sequence to thymosin beta 4, recently identified as an actin-sequestering protein. A single molecular weight species of thymosin beta 10 mRNA is present in a number of rat tissues. In adult rat testis, an additional thymosin beta 10 mRNA of higher molecular weight was identified. Nucleotide sequencing of cDNA clones complementary to the testis-specific thymosin mRNA indicated that this mRNA differed from the ubiquitous thymosin beta 10 mRNA only in its 5'-untranslated region, beginning 14 nucleotides upstream of the translation initiation codon. These results, together with primer extension experiments, suggest that the two thymosin beta 10 mRNAs are transcribed from the same gene through a combination of differential promoter utilization and alternative splicing. The novel thymosin beta 10 mRNA could be detected only in RNA isolated from sexually mature rat testis. Both mRNAs were present in pachytene spermatocytes; only the testis-specific mRNA was detected in postmeiotic haploid spermatids. Immunoblot analysis using specific antibodies showed that the thymosin beta 10 protein synthesized in adult testis was identical in size to that synthesized in brain. Immunohistochemical analysis showed that the protein was present in differentiating spermatids, suggesting that the testis-specific thymosin beta 10 mRNA is translated in haploid male germ cells.     

FK-506 and cyclosporin A inhibit highly similar signal transduction pathways in human T lymphocytes.

This report compares the ability of cyclosporin A and FK-506 to inhibit human T cell activation triggered via cell surface molecules that utilize different intracellular processes. We stimulated highly purified peripheral blood T lymphocytes with mitogens (Con A and PHA), ionomycin + PMA, or monoclonal antibodies specific for cell surface antigens involved in activation (CD2, CD3, CD28) either in combination with each other or in conjunction with PMA. Using measurements of the proliferative response, IL-2 production, and changes in intracellular Ca2+ ([Ca2+]i), we demonstrate that FK-506 exerts its inhibitory effect on early events of T-cell activation in a manner indistinguishable from that of CsA. An important finding in this study is the strict correlation between those activation pathways that are inhibited by FK-506 and CsA and the requirement that the sensitive pathways induce a measurable rise in [Ca2+]i. This correlation held even for the CD28/CD2 pathway which was previously shown to be calcium-independent; however by employing FACS analysis of [Ca2+]i within individual cells, a subset of cells activated via CD28/CD2 was found to respond with a measurable rise in [Ca2+]i. We also noted that the proliferative response induced by certain stimuli, such as ionomycin + PMA and PHA + PMA, was partially resistant to FK-506 and CsA, while IL-2 production was completely suppressed. The partial FK-506/CsA-resistance of these responses was shown to be determined by the amount of PMA added to the cultures. We conclude from our investigations that FK-506 and CsA inhibit highly similar signal transduction pathways in human T lymphocytes.     

Differing Neurochemical and Morphological Sequelae of Global Ischemia: Comparison of Single- and Multiple-Insult Paradigms

The purpose of this investigation was to investigate pathomechanisms responsible for the deleterious effects of repeated episodes of brief forebrain ischemia. Halothane-anesthetized male Wistar rats were subjected to either (a) a single 15-min period or (b) three 5-min periods (separated by 1 h) of global forebrain ischemia by bilateral carotid artery occlusions plus hypotension (50 mm Hg), followed by various periods of recirculation. Brain temperature was normothermic throughout. In one series of rats, extracellular levels of glutamate, glycine, and gamma-aminobutyric acid (GABA) were measured in the dorsolateral striatum (n = 6-8 per group) and lateral thalamus (n = 4-6 per group) by microdialysis and HPLC before and during ischemia and during 3-5 h of recirculation. In a parallel series of rats (n = 6 per group), ischemic cell change was quantified at 2 (dark neurons), 24, or 72 h following either single or multiple ischemic insults. A single 15-min ischemic period led to massive glutamate release (13-fold increase; p = 0.001), which returned to normal by 20-30 min of recirculation and remained normal thereafter. By contrast, in rats with three 5-min periods of ischemia, the glutamate level rise with each repeated insult (four- to 4.5-fold; p < or = 0.02) was smaller than that observed during the single 15-min insult, but a late sustained rise (five- to six-fold; p < 0.05) occurred at 2-3 h of recirculation. Brief ischemia-induced elevations of glycine and GABA levels were detected in both the single- and multiple-insult groups, with normalization during recirculation. In contrast, the excitotoxic index, a composite measure of neurotransmitter release ([glutamate] x [glycine]/[GABA]), differed markedly following single versus multiple insults (p = 0.002 by repeated-measures analysis of variance) and increased by seven- to 12-fold (p < 0.05) at 1-3 h following the third insult. The total amount of glutamate released was 3.3-fold higher in the multiple-insult than in the single-insult group (p < 0.02). At 2 h of recirculation, histopathological analysis of dorsolateral striatum showed a significantly greater frequency of dark neurons in the multiple- than in the single-insult group (p < 0.05 by analysis of variance). In the thalamus, a higher frequency of ischemic neurons was seen in the multiple-than in the single-insult group at all intervals studied. Thus, in rats with multiple ischemic insults, accelerated ischemic damage was found in the striatum, and severe ischemic injury was documented in the thalamus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and partial characterization of immobilization antigens from Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis, a parasitic ciliate of freshwater fishes, was found to have surface antigens (Ag) which elicited immobilizing antibodies (Ab) when injected into rabbits. An effort was made to purify and characterize these Ag (referred to as immobilization Ag) because of their potential role in protective immunity in fishes. Mice immunized with theront cilia were used for production of immobilizing monoclonal antibodies (MAb). Hybridomas were screened by indirect immunofluorescent light microscopy and immobilization of live parasites. Six hybridomas producing immobilizing MAb were cloned. Immobilizing MAb were used to affinity purify Ag solubilized with Triton X-114 and Na deoxycholate. Two membrane protein Ag of approximately 48 and 60 kDa were identified. Immobilizing MAb failed to react with these Ag on Western blots and, conversely, MAb that reacted with the Ag on Western blots did not immobilize live organisms. These results suggest that immobilization required native conformational epitopes which were altered by Western blotting procedures. Individual MAb reactive on Western blots recognized both the 48- and 60-kDa proteins indicating the presence of common epitopes. Affinity purified Ag elicited immobilizing antisera when injected into rabbits, mice, and channel catfish.     

Primary listerial infections are exacerbated in mice administered neutralizing antibody to macrophage colony-stimulating factor.

The serum and tissue levels of macrophage colony-stimulating factor (M-CSF) are elevated in mice during a primary immunologic response to infection by Listeria monocytogenes. Experiments were performed to determine the specific role of M-CSF in the resolution of listerial infections. The bulk of Listeria injected into a mouse i.v. is deposited in the liver. The expression of M-CSF mRNA in the liver increased markedly within 2 h postinfection. Maximum expression was dependent upon the dose of Listeria inoculated. The administration of anti-M-CSF mAb reduced the percentage of Mac-1+ mononuclear phagocytes subsequently found in the livers of infected animals. This reduction correlated inversely with an increase in the number of Listeria associated with both the parenchymal and NPC populations. These results suggest that M-CSF may play an important role in the primary immunologic response to Listeria in the liver by stimulating the production, mobilization, and/or biologic activity of Mac-1+ mononuclear phagocytes.     

Selectable plasmid vectors with alternative and ultrasensitive histochemical marker genes

Three different histochemical marker genes--E. coli beta-galactosidase gene (lacZ), Drosophila alcohol dehydrogenase gene (ADH) and human placenta alkaline phosphatase gene (ALP)--were cloned into a eukaryotic expression vector also containing the neomycin resistance gene. After calcium phosphate transfection and G418 sulfate selection of recipient BALB/c 3T3 cells, stable transfectants were pooled for histochemical staining. The lacZ-bearing cells produce aqua blue staining for beta-galactosidase; ADH-bearing cells, blue-black staining for alcohol dehydrogenase; and ALP-bearing cells, red staining for alkaline phosphatase. Cells carrying different marker genes can be easily differentiated by double-staining protocols. In addition, various photographic films can be used to enhance the colors of specific histochemically tagged cell classes. These plasmid vectors, providing selectability with the neomycin resistance gene and ultrasensitivity of alternative histochemical marker genes, will be very effective in virtually any biological system requiring analyses of multiple cell clones or classes in culture model systems or in situ.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Preparation of enteric-coated microspheres of Mycoplasma hyopneumoniae vaccine with cellulose acetate phthalate: The effect of swine serum on micromeritic properties

Summary Cellulose acetate phthalate was used to prepare the Mycoplasma hyopneumoniae vaccine (MHV) microspheres using a solvent evaporation method. Swine serum was used as an additive in the antigen to form the core materials. The addition of serum had a significant effect on surface topography of the MHV microspheres. By using this modified solvent evaporation method, the recoveries of antigens in the MHV microspheres were generally over 90% of the weight and antigenicity of antigens originally added in the formulation.