PCR method for genotyping and zygosity-testing of RasH2 transgenic mice.

In short-term carcinogenicity testing using CB6F1-TgrasH2 mice, sibling nonTgrasH2 mice are used as a negative control. However, selection of TgrasH2 and nonTgrasH2 mice has been performed by PCR with only transgene specific primers by the conventional method. Therefore, the conventional method involves the risk of false negative results due to reaction failure, and contamination with TgrasH2 mice in the control mice group. Based on the nucleotide sequence information around the pre-integration site, we developed a genotyping method for distinguishing not only TgrasH2 mice (hemizygous for the Tg allele) but also nonTgrasH2 (homozygous for the nonTg allele) in a positive manner.     

Phosphorylation of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) by Proline-Directed Protein Kinases and Its Dephosphorylation

Abstract: Excitatory amino acid (EAA) neurotransmitters may play a role in the pathophysiology of traumatic injury to the CNS. Although NMDA receptor antagonists have been reported to have therapeutic efficacy in animal models of brain injury, these compounds may have unacceptable toxicity for clinical use. One alternative approach is to inhibit the release of EAAs following traumatic injury. The present study examined the effects of administration of a novel sodium channel blocker and EAA release inhibitor, BW1003C87, or the NMDA receptor-associated ion channel blocker magnesium chloride on cerebral edema formation following experimental brain injury in the rat. Animals (n = 33) were subjected to fluid percussion brain injury of moderate severity (2.3 atm) over the left parietal cortex. Fifteen minutes after injury, the animals received a constant infusion of BW1003C87 (10 mg/kg, i.v.), magnesium chloride (300 µmol/kg, i.v.), or saline over 15 min (2.75 ml/kg/15 min). In all animals, regional tissue water content in brain was assessed at 48 h after injury, using the wet weight/dry weight technique. In saline-treated control animals, fluid percussion brain injury produced significant regional brain edema in injured left parietal cortex ( p < 0.001), the cortical area adjacent to the site of maximal injury ( p < 0.001), left hippocampus ( p < 0.001), and left thalamus ( p = 0.02) at 48 h after brain injury. Administration of BW1003C87 15 min postinjury significantly reduced focal brain edema in the cortical area adjacent to the site of maximal injury ( p < 0.02) and left hippocampus ( p < 0.01), whereas magnesium chloride attenuated edema in left hippocampus ( p = 0.02). These results suggest that excitatory neurotransmission may play an important role in the pathogenesis of posttraumatic brain edema and that pre- or post-synaptic blockade of glutamate receptor systems may attenuate part of the deleterious sequelae of traumatic brain injury.     

Partial characterization of the glucose transport activity in the golgi-rich fraction of fat cells

The glucose transport activity solubilized from the basal and plus insulin forms of the Golgi-rich fraction of adipocytes was partially characterized, and the results were compared with those of the activity obtained from the plus insulin form of the plasma membrane-rich fraction. The transport activity was determined in a cell-free, reconstituted, system. Prior to reconstitution, the activities in the three preparations were all (a) stable at 0°C for at least 4 h, but not at 37°C or above; (b) most stable at pH 7–9, and (c) less stable in Tes than in Tris buffer. After reconstitution, the three activities were all (d) stable at 0°C, (e) most active at pH 5.5, (f) mildly stimulated by divalent cations, (g) unaffected by insulin or 1 mM of several SH-blocking agents, (h) inhibited by heavy metal ions, 10–100 mM of monovalent salts, organic solvents, several sugar isomers, and specific sugar-transport inhibitors. The rates of d-glucose uptake by the three liposome preparations were all inhibited more strongly by 2-deoxy-d-glucose or $\text{3}\text{-O-}\text{methyl-}\text{d}\text{-glucose}$ than by d-glucose. These data indicate that the general properties of the glucose transport activity in the Golgi-rich fraction are similar to those of the activity in the plasma membrane-rich fraction.     

Activation of ATPase Activity in the Chromatin Fraction of Pea Nuclei by Calcium and Calmodulin

ATPase, especially the Ca^2$-dependent enzyme, in the chromatinfraction isolated from nuclei of pea seedlings was activatedby bovine brain calmodulin and by protein that was judged tobe calmodulin from its Ca^2$-dependent activation of NAD kinase.This calmodulin-dependent ATPase activity was blocked by calmodulin-specificinhibitors. (Received July 11, 1983; Accepted October 24, 1983)     

Antidiuretic activity of teleost urophysial extracts in the rat

Summary Extracts of the carp urophysis elicit a marked decrease in urine flow in the anaesthetized hydrated rat. Reproducible dose-dependent responses are obtained within the range of 2 to 16 g of acetonedried carp urophysis per 100 gBW of the rat. The carp urophysial antidiuretic substance is peptidic, and is different from the neurohypophysial peptides. The bulk of antidiuretic activity is located in the electrondense granules in the carp urophysis. The antidiuretic substance, probably urotensin I, is found generally in teleost urophyses. The activity per mg of acetonedried urophysis is higher in freshwater teleost species than in seawater species.Abbreviations AVP arginine vasopressin - AVT arginine vasotocin - LVP lysine vasopressin - US carp urophysial standard preparation     

Suppression by IL-2 of IgE production by B cells stimulated by IL-4.

IgE production was obtained from B cells of BALB/c or nude mice when these cells were cultured with IL-4 plus LPS. IL-2 added to these cultures at the start (day 0), 1 or 2 days later completely suppressed the production of IgE. The production of IgG1 was also inhibited, but only if IL-2 was added on day 0. The production of other isotypes (IgM, IgG2a, IgG2b) was only slightly decreased by addition of IL-2. No suppression of IgE or IgG1 production was observed if monoclonal anti-IL-2 was added, whereas anti-IFN-gamma had no effect on the suppression of the production of these isotypes. The expression of CD23 on the third day of culture on B cells stimulated with LPS and IL-4 was markedly decreased when IL-2 was added to the cultures on day 0. Addition of monoclonal anti-IL-2 suppressed all effects produced by IL-2, whereas addition of anti-IFN-gamma had no effect. These results show that the suppression by IL-2, at least for the first signaling processes, are different from the suppression produced by IFN-gamma.     

Reversal of CD45R isoform switching in CD8+ T cells.

Both CD4+ and CD8+ T cells express either CD45RA or CD45R0 isoform of CD45R in an exclusive way. Recent reports have shown that CD45RA+ T cells lose CD45RA and gain CD45R0 upon activation. This switching has been suggested to be irreversible although more recently, examples of reversal of CD45R isotype switching in CD4+ T cells have been reported. We report here that freshly isolated unprimed CD8+ T cells, when activated with PHA, temporarily lose CD45RA but reexpress an intermediate level of CD45RA 2-3 weeks after activation with PHA. This reversal seems to take place much more slowly in unprimed CD4+ T cells: the majority of CD4+ T cells that had lost CD45RA and gained CD45R0 remained CD45RA-CD45R0+ in 3 weeks after the stimulation. Also, long-term CD8+ CD45RA+ T cell lines stimulated with PHA or OKT3 showed even more rapid recovery of CD45RA while PPD-specific CD4+ T cell clones retained the original CD45R0 phenotype 3 weeks after stimulation with PPD or PHA.     

Generation propagation, and targeting of human CD4+ helper/killer T cells induced by anti-CD3 monoclonal antibody plus recombinant IL-2. An efficient strategy for adoptive tumor immunotherapy.

We developed a culture system for the rapid generation of CD4+ T cells that have both helper and killer functions. CD4+ T cells isolated from human PBL did not proliferate or develop significant cytotoxicity when treated with rIL-2 because of the lack of p75 IL-2R expression. However, culture of isolated CD4+ T cells with immobilized anti-CD3 mAb plus rIL-2 resulted in a marked proliferation (500-fold increase in 14 days) of CD4+ T cells. The proliferating CD4+ T cells produced IL-2 (92 U/ml) and showed strong cytotoxicity against OKT3 hybridoma cells and Daudi, K562, and U937 tumor cells in an anti-CD3 mAb-dependent manner. The CD4+ T cells contained significant amounts of cytolytic granule-related proteins such as serine esterase and perforin. Activated CD4+ helper/killer cells can be generated from both healthy donors and tumor patients and can be propagated in vitro for 14 to 35 days by biweekly restimulation with immobilized anti-CD3 mAb plus rIL-2. This culture yielded about 20,000-fold increase in cell number after a 21-day culture. Bispecific antibody containing anti-CD3 and anti-glioma Fab components enhanced the cytotoxicity of activated CD4+ helper/killer cells against IMR32 glioma cells. Moreover, the activated CD4+ helper/killer cells showed both helper and antitumor activity in vivo and prevented growth of anti-CD3 hybridoma cells in nude mice whether or not IL-2 was administered. These results indicate that anti-CD3 mAb plus IL-2-activated CD4+ helper/killer cells may provide an effective strategy for adoptive tumor immunotherapy of cancer.     

Sigma (sigma) antagonist BMY 14802 prevents methamphetamine-induced sensitization.

We have demonstrated for the first time that the sigma antagonist BMY 14802 prevents the development of behavioral sensitization induced by repeated administration of methamphetamine. Rats received an intraperitoneal injection of 15 or 30 mg/kg BMY 14802 followed by 2 mg/kg methamphetamine 30 min later. Unlike dopamine antagonists, BMY 14802 did not induce major changes in the acute motor effects of 2 mg/kg methamphetamine. Repeated administration of methamphetamine induced progressive augmentation of stereotyped behaviors and resulted in behavioral sensitization. However, repeated administration of methamphetamine in combination with BMY 14802 at either dose produced no increase in the intensity of stereotypy when compared with the first treatment. After a 7-day abstinence period, a challenge test with methamphetamine alone revealed supersensitivity of methamphetamine-sensitized rats to subsequent methamphetamine, whereas rats pretreated with repeated methamphetamine in combination with BMY 14802 exhibited no difference in the intensity of stereotypy from rats pretreated with repeated saline. These results suggest that sigma receptors play a crucial role in the induction of methamphetamine-induced sensitization.