Structural and functional characterization of mutants of the bovine leukemia virus transactivator protein p34

Mutants of the bovine leukemia virus (BLV) transactivator protein (tat, tax, p34, the XLOR gene product) were constructed by site-directed deletions, in-phase linker insertions, or fragment replacements (swapping) among BLV variants. The mutant constructs were transfected into cos cells and transiently expressed. Western blot analysis using a mixture of monoclonal antibodies to wild-type p34 revealed the presence of mutated XLOR gene products in all the mutants tested. The transactivating activity of 11 tax mutants containing site-directed deletions and in-phase linker insertions was completely abolished. Only the swapping mutant tested, a hybrid between two BLV variants, transactivated LTR-directed gene expression at wild-type levels. These data illustrate the narrow range of structural variations that allow full activity of the BLV tax product and suggest that the present molecular structure of the transactivator protein results from heavy evolutionary constraints.     

Electric activity in the neocortex of freely moving young and aged rats

Electroencephalographic activity of the neocortex was evaluated in young (5-7 months) and aged (26-28 months) rats. All animals in the aged group showed behavioral impairment in a spatial task (water maze). A neocortical electroencephalogram was derived simultaneously from 16 different neocortical locations and was subjected to spectral analysis. The frequency of occurrence and duration of high-voltage spindles was determined in two sessions, each involving a total of 30 min alert immobility. Changes in spectral characteristics and high-voltage spindles in response to scopolamine administration were also evaluated. The power of high-frequency activity (8-20 Hz) was significantly reduced in the aged subjects. This was greatest in the temporo-occipital regions, while no significant changes were seen in the mediofrontal region. Scopolamine resulted in a large power increase in all frequency bands, but the increase in the higher-frequency range (8-20 Hz) was significantly less in the aged group. The incidence of high-voltage spindles was 6 times higher and their total duration was 9 times longer in aged rats, with virtually no overlap with the young group. In young rats, scopolamine increased the incidence and total duration of high-voltage spindles, while it decreased both parameters in the aged subjects. Cholinergic neurons in the nucleus basalis appeared shrunken in the aged animals. These findings demonstrate that reliable electroencephalographic changes are present in the neocortex of the aged rat, and that some of the physiological alterations may be due to the pathological changes in the cholinergic nucleus basalis.     

Secondary sulphonylurea failure: what pathogenesis is responsible?

Sulphonylurea (SU) stimulates insulin secretion by pancreatic beta-cells and is generally used as a first-line treatment for type 2 diabetes. However, after long-term SU treatment (six months or over), some patients begin to show an increase in blood glucose once again (secondary SU failure). Two theories have been put forward to explain this failure--dysfunction of the proinsulin conversion machinery or insulin resistance. However, the primary pathogenesis behind secondary SU failure still needs to be investigated. Using a reliable technique that specifically identifies intact proinsulin (IPI), total proinsulin (TPI) and specific insulin (SI), this study aims to discover if a defect in the proinsulin converting mechanism plays a role in SU failure. Three groups were recruited for this study: healthy controls (n=8), SU responders (n=38) and secondary SU failures (n= 46). Serum concentrations of insulin-related molecules released in response to a standard glucose challenge test were compared between the groups. It was found that total SI was lower in the patient groups (P<0.05 compared to the control group), while TPI and IPI showed no distinct difference between the three groups (P>0.05). TPI:SI ratio and IPI:SI ratio showed marked increases in the patient groups (P<0.05 compared to control group), with no obvious quantitative difference between SU responders and secondary SU failures (P>0.05). Similar results for the Homa Insulin Resistant Index were found between the two patient groups. Interestingly, blood glucose at 180 mins after glucose challenge was significantly higher in the secondary SU failure group (P<0.05), with no correlation to SI, while the SU responder group showed good correlation between the parameters (P<0.05). We conclude that type 2 diabetes is associated with obvious dysfunction in the proinsulin-converting process and shows severe SI deficiency in responding to glucose challenge. Dysfunction of the proinsulin conversion mechanism was not an extra cause responsible for SU failure.     

Specific heat of bone

The specific heat of dry bone, as well as decalcified bone, obtained from bovine femur samples are measured as a function of temperature in the range 200 to 390K, using a differential-scanning-calorimetry technique. Special sample pans for volatile materials were used to provide a uniform thermal environment and to eliminate errors due to the evaporation of the moisture contained in the bone samples; the rate of heating was 10 K/min. From measurements of the constant pressure values, and using the Nernst-Lindermann equation, the constant-volume specific heat of both the collage and hydroxyapatite components are evaluated in the given temperature range.     

Enhanced striatal opioid receptor-mediated G-protein activation in L-DOPA-treated dyskinetic monkeys

Long-term l-3,4-dihydroxyphenylalanine (L-DOPA) treatment in Parkinson's disease leads to dyskinesias in the majority of patients. The underlying molecular mechanisms for L-DOPA-induced dyskinesias (LIDs) are currently unclear. However, the findings that there are alterations in opioid peptide mRNA and protein expression and that opioid ligands modulate dyskinesias suggest that the opioid system may be involved. To further understand its role in dyskinesias, we mapped opioid receptor-stimulated G-protein activation using [35S]guanylyl-5'-O-(gamma-thio)-triphosphate ([35S]GTPgammaS) autoradiography in the basal ganglia of normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned squirrel monkeys administered water or L-DOPA. Subtype-selective opioid receptor G-protein coupling was investigated using the mu-opioid agonist [D-Ala, N-Me-Phe, Gly-ol]-enkephalin, delta-agonist SNC80 and kappa-agonist U50488H. Our data show that mu-opioid receptor-mediated G-protein activation is significantly enhanced in the basal ganglia and cortex of L-DOPA-treated dyskinetic monkeys, whereas delta- and kappa-receptor-induced increases were limited to only a few regions. A similar pattern of enhancement was observed in both MPTP-lesioned and unlesioned animals with LIDs suggesting the effect was not simply due to a compromised nigrostriatal system. Opioid receptor G-protein coupling was not enhanced in non-dyskinetic L-DOPA-treated animals, or lesioned monkeys not given L-DOPA. The increases in opioid-stimulated [35S]GTPgammaS binding are directly correlated with dyskinesias. The present data demonstrate an enhanced subtype-selective opioid-receptor G-protein coupling in the basal ganglia of monkeys with LIDs. The positive correlation with LIDs suggests this may represent an intracellular signaling mechanism underlying these movement abnormalities.     

Sepsis-induced SOCS-3 expression is immunologically restricted to phagocytes

We have shown that immune cells from septic mice exhibit a suppressed response to exogenous stimuli in vitro. The suppressors of the cytokine signaling (SOCS) family are proteins that block intracellular signaling and can be induced by inflammatory mediators. Therefore, we hypothesized that SOCS-3 is up-regulated in immune cells in response to a septic challenge induced by cecal ligation and puncture (CLP). Mice were subjected to CLP or sham-CLP, and 2-48 h later, the blood, thymus, spleen, lung, and peritoneal leukocytes were harvested and examined. SOCS-3 was undetectable in thymocytes or blood leukocytes. In contrast, SOCS-3 was up-regulated in the spleen, lung, and peritoneal leukocytes in a time-dependent manner. Further examination revealed that only the macrophages and neutrophils expressed SOCS-3. These data suggest that cytokines and bacterial toxins present during sepsis have the ability to suppress the cytokine and/or lipopolysaccharide response and the function of immune cells by up-regulating SOCS-3.     

Association of the CTLA-4 gene with rheumatoid arthritis in Chinese Han population

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is important for downregulation of T-cell activation, and CTLA-4 gene polymorphisms have been implicated as risk factors for rheumatoid arthritis (RA). Previous studies of the association between the +49 polymorphism of the CTLA-4 gene in RA have provided conflicting results. In order to determine association of the CTLA-4 gene with RA in Chinese Han population, we used denaturing gradient gel electrophoresis (DGGE) to genotype polymorphisms of four SNPs (MH30, +49, CT60 and JO31) of the CTLA-4 gene in 326 RA patients and 250 healthy controls. Furthermore, meta-analysis of all available studies relating +49 polymorphism to the risk of RA was performed to confirm the disease association. Among the SNPs examined, the genotype frequencies of CTLA-4 +49 and CT60 in RA patients differed significantly from controls (P=0.028 and 0.007). In addition, the distribution of four haplotypes constructed by these two SNPs was significantly different between patients and controls (chi(2)=10.58, d.f. =3, P=0.014). The meta-analysis also revealed that in both European and Asian populations, the CLTA-4 +49 G allele was associated with the risk of RA. These results suggested that the CTLA-4 gene might be involved in the susceptibility to RA in the Chinese Han population and both +49 and CT60 of CTLA-4 gene might be the causal variants in RA disease.     

Interferon: a cytotoxic T lymphocyte differentiation signal

Human T cell clones which were able to proliferate in response to specific stimuli but could not kill even in the presence of lectins were found to acquire the specific lytic function when interferon alpha or gamma was added on day 1 of the 7-day restimulation culture. These results demonstrate that interferon may act as a cytotoxic T lymphocyte differentiation signal. This signal can be blocked by the monoclonal antibody LeoA1 which recognizes a 70-kDa cell surface structure, involved in cytotoxic T lymphocyte differentiation.     

Resistance of severe combined immunodeficient mice to infection with Cryptosporidium parvum: the importance of intestinal microflora.

Cryptosporidium parvum is a protozoan parasite which colonizes intestinal epithelium, causing transient diarrheal illness in immunocompetent hosts and severe chronic disease in immunocompromised hosts. We examined the resistance of severe combined immunodeficient mice, either bearing intestinal flora or germfree, to intestinal infection with C. parvum. Infection was not readily detected in flora-bearing adult severe combined immunodeficient mice until 5 to 7 weeks following oral challenge with C. parvum. In contrast, germfree adult severe combined immunodeficient mice were heavily infected 3 weeks following challenge. These data support the hypothesis that resistance of adult mice to C. parvum infection does not require a specific immune response but can be mediated by nonspecific mechanisms associated with the presence of intestinal flora.     

A rapid screening assay for detection of IgE-binding factors in humans

During the last few years studies in rats and mice have demonstrated IgE-binding factors, some of which have IgE-selective regulatory activities. This prompted us to develop a rapid, sensitive screening assay for measuring IgE-binding factors in humans. The principle of the assay is to measure the degree of inhibition of binding between anti-human IgE antibodies and human IgE. Thus, 200 pg IgE plus testing samples were added to each well precoated with anti-human IgE antiserum. After an overnight incubation, the wells were washed and radiolabeled anti-IgE antibodies were added to the wells. Under the optimum conditions, the assay can detect 10(-11)M anti-human IgE antibodies. With this assay, we have been able to detect IgE-binding factors in the supernatants of 2 human B cell lines which bear Fc receptors for IgE (FcR epsilon) on their surface membranes (e.g., WIL-2 and RPMI 8866), but not in the supernatants of DAUDI cells (a human cell line without FcR epsilon). Furthermore, the IgE-binding factors of WIL-2 cells were specifically adsorbed to, and eluted from, IgE-coupled Sepharose, but not BSA-Sepharose. These findings prove that the inhibition factors are indeed human IgE-binding factors, and that the assay described herein is a specific and sensitive screening assay for detecting human IgE-binding factors.