Production and characterization of monoclonal antibodies specific for the murine T cell receptor ζ chain

The T cell receptor (TCR) comprises an antigen-specific β heterodimer non-covalently associated with the CD3 γδε and TCR ζ subunits. Both the CD3 and TCR ζ subunits are proposed to be responsible for the intracellular signal-transduction events. We report here the production of eight monoclonal antibodies (mAbs) that bind in an ELISA assay to a 113 amino acid synthetic peptide corresponding to the cytoplasmic domain of TCR ζ. Western blot analysis of anti-CD8 precipitates of lysates of transfectants expressing chimeric CD8/ζ constructs encoding increasing COOH-terminal truncations of TCR ζ indicates that four of these mAbs recognized the region of TCR ζ chain comprising the last 29 COOH-terminal residues. Thus, this region of TCR ζ may encode an immunodominant epitope. Furthermore, one of these mAbs, G3, is capable of precipitating both non-phosphorylated and tyrosine phosphorylated TCR ζ. The G3 mAb should be useful for elucidiating the structural and signalling characteristics of the TCR ζ chain.     

Localization of vasoactive intestinal polypeptide in penile erectile tissue and in the major pelvic ganglion of the rat

Vasoactive intestinal polypeptide was localized by immunocytochemical techniques in the major pelvic ganglion and penile erectile tissue of the rat. Vasoactive intestinal polypeptide fibers were concentrated in penile crura with the density of innervation decreasing distally. The helicine arteries were very densely innervated while fewer fibers surrounded the deep artery of the penis. Intrinsic smooth muscle of the cavernous bodies received a moderate supply of vasoactive intestinal polypeptide immunoreactive fibers. Dorsal vascular structures, including the deep dorsal vein were innervated by vasoactive intestinal polypeptide fibers. Vasoactive intestinal polypeptide immunoreactive cell bodies were found in the major pelvic ganglion, concentrated on one end of the ganglion. Rectrograde studies with a dye injected into the penile crura indicated that neurons in major pelvic ganglion projected to the penis. Combined dye and immunofluorescent studies showed that all the dye-labeled neurons were immunoreactive for vasoactive intestinal polypeptide.

It is concluded that all vascular beds in the penis of the rat are innervated by vasoactive intestinal polypeptide fibers and that the extent of the innervation is related to the occurrence of smooth muscle. Neurons in the major pelvic ganglion probably are the main source of vasoactive intestinal polypeptide fibers to the penis.     

Identification of a novel allele HLA-B*7805 in a Japanese female

A new HLA-B*78 allele, B*7805, was identified in a healthy Japanese female. The results of her serological HLA class I typing showed an unusual Bw4/Bw6 pattern with strongly positive reactivity to anti-Bw6, i.e. A24, -, B52, -, Bw4, Bw6. In DNA typing, she was typed as A*24, -, B*52, B*78-like, Cw1202, -, (Bw4, Bw6). Cloning and sequencing of exon 2 and exon 3 of her B locus genes revealed a new allele B*7805. The cloned B*7805 differed from B*78021 by three nucleotide substitutions in exon 2 at position 259 (A to G), 261 (C to G) and 272 (A to C), and contained sequences defining Bw6 motif in the region of codon 77 to 83.     

Quercetin inhibits the invasion and mobility of murine melanoma B16-BL6 cells through inducing apoptosis via decreasing Bcl-2 expression

Quercetin has been known to have anti-tumor and anti-oxidation activities. In the present study, we have investigated its in vitro anti-metastatic activity. Quercetin inhibited the invasion and mobility of murine melanoma B16-BL6 cells in a dose-dependent manner but did not affect their adhesion to either laminin, fibronectin, or type VI collagen. Moreover, quercetin significantly inhibited the proliferation of B16-BL6 cells only in the case of time incubation longer than 48 h. Quercetin dose-dependently decreased the cell rates in S and G2–M phases of cell cycle. The effect of quercetin to cause a remarkable apoptosis of B16-BL6 cells was also demonstrated by flow cytometric assay as well as DNA fragmentation with a typical 180-bp ladder band in agarose electrophoresis and a quantitative analysis. Furthermore, quercetin markedly inhibited the expression of anti-apoptotic protein Bcl-2 but hardly influenced Bcl-X_L. These results suggest that the inhibition of quercetin on invasiveness and migration of B16-BL6 cells are closely associated with the arrest of cell cycle as well as the induction of apoptosis by decreasing the Bcl-2 expression. This revised version was published online in July 2006 with corrections to the Cover Date.     

Primary and recurrent cytomegalovirus infections have different effects on human herpesvirus-6 antibodies in immunosuppressed organ graft recipients: absence of virus cross-reactivity and evidence for virus interaction.

The relationship between serum antibodies to human herpesvirus-6 (HHV-6) and cytomegalovirus (CMV) infection was studied in immunosuppressed adult organ graft recipients all of whom had IgG to both HHV-6 and Epstein-Barr virus capsid antigen (EBVCA) before operation and who had received an organ or organs from HHV-6 seropositive donors. In primary CMV infection the titre of IgG to HHV-6 rose substantially (between 32- and 512-fold) in eight out of eight patients whereas IgG to EBVCA only rose 32-fold in two patients. Moreover, the HHV-6 responses coincided closely with the CMV seroconversion. Serum absorption studies gave no evidence for antibody cross-reaction between CMV and HHV-6 because the CMV antibody titre could be reduced specifically without affecting HHV-6 antibody titres and vice versa. In recurrent CMV infection, HHV-6 antibody levels rose (32-fold) in three out of eight patients but these changes did not coincide with the CMV antibody response. Similarly, in the complete absence of CMV infection, five out of eight patients showed antibody rises to HHV-6 (between four- and 16-fold). IgG titres to EBVCA were stable in both these groups of patients. It is concluded that there is serological evidence (rising titre greater than or equal to four-fold) for genuine HHV-6 reactivation or, alternatively, for reinfection in 16 out of the 24 patients. This phenomenon was most frequent in primary CMV infection where the largest HHV-6 antibody responses were seen probably because of an, as yet, undetermined interaction between the two viruses.     

Prolonged vitamin C supplementation and recovery from eccentric exercise

We have previously shown that vitamin C supplementation affects recovery from an unaccustomed bout of demanding exercise, with the most pronounced effect being that on plasma interleukin-6 concentration. However, because of the proposed role of interleukin-6 in the regulation of metabolism, it was unclear whether this represented a reduced response to muscle damage or some form of interaction with the metabolic demands of the activity. Therefore, the aim of the present study was to investigate the effect of the same form of supplementation on a bout of exercise that initiated similar muscle damage but had a low metabolic cost. Fourteen male subjects were allocated to either a placebo (P) or a vitamin C (VC) group. The VC group consumed 200 mg of ascorbic acid twice a day for 14 days prior to a bout of exercise and for the 3 days after exercise. The P group consumed identical capsules that contained 200 mg lactose. Subjects performed 30 min of downhill running at a gradient of –18% and recovery was monitored for up to 3 days after exercise. Plasma VC concentrations in the VC group increased following supplementation. Nevertheless, downhill running provoked a similar increase in circulating markers of muscle damage (creatine kinase activity and myoglobin concentration) and muscle soreness in P and VC groups. Similarly, although downhill running increased plasma interleukin-6, there was no effect from VC supplementation. These results suggest that vitamin C supplementation does not affect interleukin-6 concentrations following eccentric exercise that has a low metabolic component.     

NMDA receptor mediated dendritic plasticity in cortical cultures after oxygen-glucose deprivation

Dendrites and spines undergo dynamic changes in physiological and pathological conditions. Dendritic outgrowth has been observed in surviving neurons months after ischemia, which is associated with the functional compensation. It remains unclear how dendrites in surviving neurons are altered shortly after ischemia, which might reveal the mechanisms underlying neuronal survival. Using primary cortical cultures, we monitored the dendritic changes in individual neurons after oxygen-glucose deprivation (OGD). Two to four hours of OGD induced approximately 30–50% cell death in 24 h. However, the total dendritic length in surviving neurons was significantly increased after OGD with a peak at 6 h after re-oxygenation. The increase of dendritic length after OGD was mainly due to the sprouting rather than the extension of the dendrites. The dendritic outgrowth after 2 h of OGD was greater than that after 4 h of OGD. Application of NMDA receptor blocker MK-801 abolished OGD-induced dendritic outgrowth, whereas application of AMPA receptor antagonist CNQX had no significant effects. These results demonstrate a NMDA receptor-dependent dendritic plasticity shortly after OGD, which provides insights into the early response of surviving neurons after ischemia.     

A graded dose assessment of the efficacy of beclomethasone dipropionate aerosol for severe chronic asthma.

In a 26-wk double-blind controlled study of 34 patients whose asthma had been poorly controlled despite oral steroids, valuable clinical and pulmonary function improvement was derived by adding beclomethasone aerosol to the prednisone regimen. The amount of improvement correlated linearly with beclomethasone dosage over the range 200 to 1,600 microng/day. These patients required relatively high dosage. Success in achieving asymptomatic status was only 26% with the conventional 400 microng/day and 60% at 1,600 microng/day. Oropharyngeal candidiasis was also dose-related but did not prohibit the use of high-dosage beclomethasone. Respiratory infections, physical signs, blood glucose, and electrolytes were unaffected by the drug. A dose-related suppression of cortisol secretion was demonstrated, but about 1/4 of the group had normal plasma cortisol even at 1,600 microng/day plus the oral prednisone. An individualized risk-benefit assessment seems a better basis for choosing an optimal beclomethasone regimen for each patient than adherence to a conventionalized fixed dosage of 400 microng/day. This requires definition of: (1) a specific goal of treatment in the individual patient and the beclomethasone dosage required to achieve it; (2) the adrenocortical functional response of that particular patient to the desired dose of beclomethasone; and (3) the presence and degree of any dose-limiting constraints such as preexisting complications of steroid use.