CAP 37, a 37 kD human neutrophil granule cationic protein shares homology with inflammatory proteinases

We have previously shown that a major granule-associated cationic protein CAP 37 (Mr = 37 kD) derived from human PMN is a monocyte-specific chemoattractant. The N-terminal amino acid sequence of this novel chemotactic protein shares significant homology with a number of inflammatory molecules with protease activity including elastase and cathepsin G. However, a critical substitution of a serine for a histidine at position 41, results in its lack of serine protease activity.     

Negative modulation of Epstein-Barr virus episomes by a human B-cell growth factor.

To study the effect of T-cell-derived BCGF-12kD on human B-cell autocrine growth-associated functions, we cultured Epstein Barr virus (EBV)-transformed normal B cells (LCL 72285) and Burkitt's lymphoma cells (Raji) in the presence or absence of BCGF-12kD. When cultured in media supplemented only with fetal calf serum, the LCL and Raji cell lines maintained relatively high levels of episomes. Although a similar level of proliferation could be maintained under defined culture conditions in media supplemented with BCGF-12kD, these conditions resulted in a time-dependent reduction of EBV sequences, as detected with EBV nuclear antigen (EBNA-1 and EBNA-2) gene probes. These results suggest that stimulation with T-cell-derived BCGF-12kD can alter a regulatory step which may be involved in the EBV transformation of B cells.     

Potent convulsant actions of the adenosine receptor antagonist, xanthine amine congener (XAC)

The convulsant properties of xanthine amine congener (XAC, 8-(4-(2-aminoethyl)-aminocarboxylmethyloxy)phenyl-1,3-dipropylxant hine) are compared to those of caffeine. Male Swiss albino mice were infused with convulsants through a lateral tail vein. Convulsion thresholds (i.e. the amount of convulsants required to elicit convulsions) of 39.8 +/- 2.0 mg/kg (n = 10) and 109.8 +/- 2.3 mg/kg (n = 10) were calculated for XAC and caffeine respectively. Pretreatment of animals with the adenosine receptor agonists 2-chloroadenosine, N6-cyclohexyladenosine or 5'-N-ethylcarboxamido-adenosine (1 mg/kg, i.p., 20 minutes prior to infusion) significantly decreased the seizure threshold of both XAC and caffeine. The adenosine uptake blockers, 6-nitrobenzylthioinosine or dipyridamole (0.25 mg/kg, i.p., 20 minutes prior to infusion) did not significantly affect the seizure threshold to either XAC or caffeine. The benzodiazepine agonist diazepam (5 mg/kg, i.p., 20 minutes prior to infusion) significantly increased the seizure threshold to both XAC (p less than 0.05) and caffeine (p less than 0.01), whereas the benzodiazepine antagonist Ro 15-1788 (10 mg/kg, i.p., 20 minutes prior to infusion) significantly increased the seizure threshold to caffeine (p less than 0.01), but not XAC. The results suggest that actions at benzodiazepine receptors may be a tenable hypothesis to explain the convulsant actions of caffeine, but not those of XAC.     

Genetic analysis of 24 French families with multiple endocrine neoplasia type 2A.

The gene for multiple endocrine neoplasia type 2A (MEN2A) has been mapped to the pericentromeric region of chromosome 10 by linkage analysis. Thirty-four families with multiple cases of medullary carcinoma of the thyroid (MTC), including 24 families with origins in France, have been typed with nine polymorphic markers spanning the centromere of chromosome 10. No recombination was observed between the MEN2A locus and either of the four loci D10Z1 (lod score 12.79), D10S102 (lod score 6.38), D10S94 (lod score 7.76), and D10S34 (lod score 5.94). There was no evidence for genetic linkage heterogeneity in the panel of 34 families. Haplotypes were constructed for a total of 11 polymorphisms in the MEN2A region, for mutation-bearing chromosomes in 24 French families and for 100 spouse controls. One haplotype was present in four MEN2A families but was not observed in any control (P less than .01). Two additional families share a core segment of this haplotype near the MEN2A gene. It is likely that these six families have a common affected ancestor. Because the incidence of pheochromocytoma among carriers varies from 0% to 74% within these six families, it is probable that additional factors modify the expression of the MEN2A gene.     

Intramolecular electron transfer in cytochrome c oxidase: a cascade of equilibria.

Intramolecular electron redistribution in cytochrome c oxidase after photolysis of the partially reduced CO-bound enzyme was followed at a number of different wavelengths by absorption spectroscopy. Spectra were constructed for the first two phases of this process. The first phase (tau = 3 microseconds) has a spectrum essentially identical to the difference between the Fea and Fea3 reduced-minus-oxidized spectra, indicating a 1:1 stoichiometry between the amount of Fea3 oxidized and Fea reduced. It is not necessary to invoke reduction or oxidation of other redox carriers in this phase. The second phase (tau = 35 microseconds) spectrum appears to be a linear combination of the Fea3 and Fea reduced-minus-oxidized difference spectra, reflecting the oxidation of four parts of Fea3 for every part of Fea oxidized. This process can be described in terms of transfer to CuA of electrons from the Fea3<==>Fea equilibrium system established in the first phase. The relative contributions of Fea3 and Fea in the second phase allow us to calculate the equilibrium constant for Fea3<==>Fea electron exchange, which yields a delta Em of 36 mV for the two centers (Fea3 more positive). Together with the apparent rate constant for the fast phase, this equilibrium constant yields, in turn, the forward (kf) and reverse (kr) rates for electron transfer from Fea to Fea3 as follows: kf = 2.4 x 10(5) s-1 and kr = 6 x 10(4) s-1. kf is much faster than any observed step in the reaction of the reduced enzyme with O2. Thus, the catalytic mechanism of O2 reduction to water is not rate-limited by electron transfer from Fea to the binuclear Fea3/Cu(B) site.     

Isolation and characterization of a membrane-attack-complex-inhibiting protein present in human serum and other biological fluids.

Cytosolic, detergent-solubilized and membrane-bound growth hormone (GH) receptors from rabbit adipose tissue and liver were tested for reactivity with a panel of monoclonal antibodies (MAbs). The cytosolic and detergent-solubilized forms of adipose tissue and liver GH receptors were identically reactive with four precipitating and two hormone-binding-site-directed MAbs. However, the membrane-bound form of the adipose receptor was 1000-fold less reactive with one binding-site-directed MAb (MAb 7) than the membrane-bound liver GH receptor. Reactivity with another inhibitory MAb (MAb 263) was identical for adipose tissue and liver membrane GH receptors. The relative potency of 22,000-Mr and 20,000-Mr forms of human GH was identical in assays with liver and adipose tissue membrane receptors. Thus, contrary to earlier suggestions, the discrepancy between the growth-promoting and insulin-like activities of 20,000-Mr human GH cannot be rationalized by a difference in the affinity of this hormone for 'somatogenic' and 'metabolic' receptors when the comparison is made in the same species. Cross-linking studies showed that the major GH-binding subunit of liver and adipose tissue GH receptors had the same Mr (54,000 +/- 5000, reduced). The ligand-binding subunits of liver and adipose tissue receptors are identical by several criteria, but one epitope on the adipose tissue receptor appears to be masked upon membrane insertion, possibly by close association with a tissue-specific component. Tissue specificity may be determined by association of a ubiquitous GH-binding subunit with tissue-specific membrane components, rather than by differences in amino acid sequence.     

Relationship between arachidonic acid release and Ca2(+)-dependent exocytosis in digitonin-permeabilized bovine adrenal chromaffin cells.

The relationship between Ca2(+)-dependent arachidonic acid release and exocytosis from digitonin-permeabilized bovine adrenal chromaffin cells was investigated. The phospholipase A2 inhibitors mepacrine, nordihydroguaiaretic acid and indomethacin had no effect on either arachidonic acid release or secretion. The phospholipase A2 activator melittin had no effect on secretion. The specific diacylglycerol lipase inhibitor RG80267 had no effect on secretion, but decreased basal arachidonic acid release to such an extent that the level of arachidonic acid in treated cells in response to 10 microM-Ca2+ was equivalent to that of control cells in the absence of Ca2+. Staurosporine, a protein kinase C inhibitor, was found to abolish Ca2(+)-dependent arachidonic acid release completely, but had only a slight inhibitory effect on Ca2(+)-dependent secretion. It is concluded that arachidonic acid is not essential for Ca2(+)-dependent exocytosis in adrenal chromaffin cells.     

Relationships between follicular fluid steroid hormone concentrations, oocyte maturity, in vitro fertilization and embryonic development in the rhesus monkey

Oocytes and matched samples of follicular fluid (FF) were obtained from 70 follicles of five rhesus monkeys stimulated with either pregnant mare serum gonadotropin or human menopausal gonadotropin. Follicular aspiration was performed 30-32 h after human chorionic gonadotropin administration. The concentrations of estradiol (E2), progesterone (P), testosterone (T), and dihydrotestosterone (DHT) in FF were measured. Twenty-six percent of oocytes were classified as mature (M), 41% matured in vitro (Miv), 13% were dysmature, and 20% atretic. M oocytes were associated with significantly higher levels of P and a higher P:E2 ratio. There were no differences in hormone levels associated with fertilized and nonfertilized oocytes. Thirty-five embryos developed to the six- to eight-cell stage in vitro, of which 13 exhibited optimal cleavage rates. Significantly lower levels of E2 and higher P:E2 ratios were associated with the more rapidly cleaving embryos. Proportionally more embryos showing optimal cleavage rates developed from M compared to Miv oocytes, and only embryos derived from M oocytes developed to blastocysts in culture. Optimal cleavage rates to the six- to eight-cell stage in vitro, rather than fertilization rates, are a better indicator of (subsequent) developmental capacity, and, in this study, embryonic development was closely associated with the maturity of the oocyte at recovery.     

A recombinant adenovirus expressing an Epstein-Barr virus (EBV) target antigen can selectively reactivate rare components of EBV cytotoxic T-lymphocyte memory in vitro.

While the bulk of a virus-induced cytotoxic T-lymphocyte (CTL) response may focus on a few immunodominant viral antigens, in certain tumor virus systems the detectability of clones recognizing other, subdominant antigens can assume particular importance. By using the human CTL response to Epstein-Barr virus (EBV) as a model system, here we show that even rare components of virus-specific memory can be selectively reactivated in vitro when the relevant target antigen is expressed in autologous stimulator cells from a recombinant adenovirus (RAd) vector. We generated a replication-deficient adenovirus, RAd-E3C, which in skin fibroblast cultures expressed the EBV nuclear antigen EBNA3C at a 10- to 100-fold-higher level than that naturally present in EBV-transformed lymphoblastoid cell lines (LCLs). Initial experiments with a donor whose polyclonal CTL response to LCL stimulation contained a strong EBNA3C-specific component showed that these CTLs could be efficiently reactivated by in vitro stimulation either with RAd-E3C-infected fibroblasts or with RAd-E3C-infected peripheral blood mononuclear cells. Then we studied donors whose responses to LCL stimulation contained little if any detectable EBNA3C reactivity but were dominated by clones recognizing other EBV target antigens; in vitro stimulation with RAd-E3C-infected peripheral blood mononuclear cells selectively reactivated EBNA3C-specific CTL clones from these individuals, with the epitope specificities of responses subsequently identified at the peptide level. This RAd-based approach could be applied more generally to screen for human CTL responses against any candidate target antigen expressed by tumor cells.