Molecular and functional analysis of a conserved CTL epitope in HIV-1 p24 recognized from a long-term nonprogressor: constraints on immune escape associated with targeting a sequence essential for viral replication

It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion.     

Enantioseparation of amfepramone (rac-diethylpropion): preparative separation of the enantiomers and enantioselective analysis

The enantiomers of the anorectic drug amfepramone [rac-diethylpropion, rac-2-(diethylamino)-1-phenyl-1-propanone; rac-DEP] were separated in the preparative scale by crystallization. With enantiopure di-O-benzoyltartaric acid as salt-forming chiral selector, diastereoisomeric salts of DEP enantiomers with a final purity of more than 97.5% were obtained. Analytical liquid chromatographic and electrophoretic methods for the control of the enantiomeric purity and the stoichiometry of the salts were developed. The enantioseparation of rac-DEP by capillary electrophoresis (CE) using hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral discriminator and phosphate buffer (pH 3.3) as run buffer led to good separations. HPLC methods were developed using polysaccharide chiral stationary phases (CSP). The separation of the two enantiomers and the two main degradation products (1-phenyl-1,2-propanedione and propiophenone), known from solid and liquid pharmaceutical preparations, was attained in one run on the silica-based CSP cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD). The conditions which might affect the enantioselectivity and the quality of the enantiomeric separation were investigated for Chiralcel OD and the related CSP amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD). Both CSPs showed very similar chromatographic properties. The separation factors could be influenced significantly by varying the polar organic modifier added to the mobile phase.     

Endogenous oxytocin excites phasic contraction of gallbladder in rabbits through oxytocin receptor

These experiments were performed to study the effect of oxytocin (OT) and it's specific receptor on gallbladder motility in rabbits. The fasted New Zealand white rabbits (2.0-2.5 kg) were anaesthetized by urethane (1 g/kg). The gallbladder pressure was recorded continuously to monitor the gallbladder motility. Systemic OT (0.01, 0.02, 0.04 mg/kg, iv) did not affect the gallbladder pressure, but dose-dependently increased the frequency of phasic contraction. Five min after OT administration (0.04 mg/kg, iv), the strength of phasic contraction increased to 0.23 +/- 0.08 mmHg/min (P < 0.01, n = 6). The gallbladder motility returned to normal 15 min later after OT treatment. Intravenous injection of atosiban (0.04 mg/kg, iv), an OT receptor antagonist, decreased the strength of gallbladder phasic contraction but did not affect gallbladder pressure. Pretreatment of atosiban (0.04 mg/kg, iv) completely abolished the systemic OT effect on gallbladder. Vasopressin (VP) (0.1 - 0.5 IU/kg, iv) dose-dependently decrease the gallbladder pressure but did not affect the phasic contraction. MK-329 (0.4 mg/kg, iv), the CCK-A receptor antagonist, L-365, 260 (0.4 mg/kg, iv), the CCK-B receptor antagonist and atropine (0.2 mg/kg, iv), the M receptor antagonist, did not affect the OT effect on gallbladder motility. We suggest that endogenous OT regulates gallbladder phasic contraction through specific OT receptor. This effect is independent of the peripheral CCK and M receptors.     

Role of testicular interstitial macrophages in regulating testosterone release in hyperprolactinemia

Hyperprolactinemia-induced hypogonadism has been linked to a dysfunction of the hypothalamus-pituitary-testis axis. The direct inhibitory effects of prolactin on the testicular release of testosterone have also been demonstrated, though their mechanisms remain unclear. Incubation of rat testicular interstitial cells (TICs) with prolactin stimulated the release of testosterone. TICs from rats with anterior pituitary-grafting-induced hyperprolactinemia release lower amounts of testosterone than controls. However, Leydig cells isolated from anterior pituitary-grafted rats release a greater amount of testosterone. These paradoxical observations have remained unexplained. This study examined the roles of testicular interstitial macrophages and of their product, tumor necrosis factor-alpha (TNF-alpha), in regulating Leydig cells under condition of hyperprolactinemia. Hyperprolactinemia was induced by grafting two anterior pituitary glands of rats under the renal capsule. Control animals were grafted with rat cortex tissue. The rats were sacrificed 6 weeks later. TICs and macrophages, and Leydig cells were isolated for in vitro incubation and drugs challenge. Testosterone released by testicular interstitial or Leydig cells was measured by radioimmunoassay. TNF-alpha concentration in the medium of TICs or macrophages was measured by enzyme-linked immunosorbent assay (ELISA). A dose-dependent stimulation of TNF-alpha secretion in the medium of TICs or macrophages by the prolactin challenge was observed. Higher amounts of TNF-alpha were released by TICs in the anterior pituitary-grafted rats than in the control group. In contrast, the release of TNF-alpha by testicular interstitial macrophages isolated from the anterior pituitary- and cortex-grafted groups was quantitatively similar. Challenge with human chorionic gonadotropin did not modify the TNF-alpha release by testicular interstitial macrophages in either group. Challenge of Leydig cells with TNF-alpha inhibited their release of testosterone stimulated by human chorionic gonadotropin, but not their basal testosterone release. These different patterns of testosterone release in TICs versus Leydig cells cultures in anterior pituitary-grafted rats may be due to the influence of testicular interstitial macrophages. These observations correlate with in vivo conditions, where prolactin increases the release of TNF-alpha by testicular interstitial macrophages, which, in turn, decreases the human chorionic gonadotropin-stimulated release of testosterone by Leydig cells. In summary, hyperprolactinemia-induced hypogonadism involves a mechanism of prolactin-originated, macrophage-mediated inhibitory regulation of testosterone release by Leydig cells. TNF-alpha, one of the cytokines secreted by macrophages, may play a key role in this mechanism.     

Effects of S-petasin on corticosterone release in rats

Petasites hybridus is used in Chinese herbal medicine. S-petasin is a bioactive compound isolated from leaves or roots of Petasites hybridus. S-petasin has been used to relieve gastrointestinal pain, lung disease, and spasms of the urogenital tract. However, the side effect of S-petasin on endocrine systems are still not clear. This study explored the effects of S-petasin on the release of corticosterone in vivo and in vitro. An intravenous injection of S-petasin (10 microg/kg) decreased both basal and adrenocorticotropin (ACTH)-induced plasma corticosterone concentration in male rats. In vitro, S-petasin (3 x 10(-6) - 10(-4) M) caused a significant reduction of basal and ACTH-stimulated release of corticosterone from the enzymatically dispersed rat zona fasciculata-reticularis (ZFR) cells in a dose-dependent manner. In order to study possible mechanisms, ZFR cells were incubated with S-petasin (10(-5) M) in the presence or absence of forskolin (adenylate cyclase activator, 10(-6) - 10(-4) M), 8-Br-cAMP (a cAMP analogue, 10(-6) 10(-4) M), 25-OH-cholesterol (pregnenolone biosynthesis precursor, 10(-5) M) combined with trilostane (a blocker of 3beta-hydroxysteriod dehydrogenase, 3beta-HSD, 10(-6) M) and deoxycorticosterone (corticosterone biosynthesis precursor, 10(-9) - 10(-6) M) at 37 degrees C for 1h. The concentration of pregnenolone and corticosterone in media were measured by radioimmunoassay. The stimulatory effects of corticosterone secretion induced by forskolin (10(-5) - 10(-4) M), 8-Br-cAMP (10(-5) - 10(-4) M) and deoxycorticosterone (10(-7) - 10(-6) M) were reduced by S-petasin at 10(-5) M. The stimulatory effects of pregnenolone secretion induced by 25-OH-cholesterol combined with or without trilostane was reduced by S-petasin at 10(-5) M. These results suggest that S-petasin inhibits the production of corticosterone from rat ZFR cells in part through decreasing the activities of adenylyl cyclase, P450scc and 11beta-hydroxylase.     

Inhibition of testosterone production by propylthiouracil in rat Leydig cells

Propylthiouracil (PTU) is a thioamide drug used clinically to inhibit thyroid hormone production. However, PTU is associated with some side effects in different organs. In the present study, the acute and direct effects of PTU on testosterone production in rat Leydig cells were investigated. Leydig cells were isolated from rat testes, and an investigation was performed on the effects of PTU on basal and evoked-testosterone release, the functions of steroidogenic enzymes, including protein expression of cytochrome P450 side-chain cleavage enzyme (P450(scc)) and mRNA expression of the steroidogenic acute regulatory protein (StAR). Rat Leydig cells were challenged with hCG, forskolin, and 8-bromo-cAMP to stimulate testosterone release. PTU inhibited both basal and evoked-testosterone release. To study the effects of PTU on steroidogenesis, steroidogenic precursor-stimulated testosterone release was examined. PTU inhibited pregnenolone production (i.e., it diminished the function of P450(scc) in Leydig cells). In addition to inhibiting hormone secretion, PTU also regulated steroidogenesis by diminishing mRNA expression of StAR. These results suggest that PTU acts directly on rat Leydig cells to diminish testosterone production by inhibiting P450(scc) function and StAR expression.     

Physiological and biochemical characterization of glyoxalase I,a general marker for cell proliferation,from a soybean cell suspension

Using a strictly auxin-dependent soybean (Glycine max (L.) Merr.) cell suspension, we studied the correlation of auxin-dependent cell proliferation and the activity of glyoxalase I (S-lactoylglutathione-lyase EC 4.4.1.5.), an enzyme generally associated with cell proliferation in animal, microbial and, as reported recently, also plant systems. We found the activity of glyoxalase I to be modulated during the proliferation cycle, with a maximal activity between day 2 and day 4 of culture growth. After starving the culture of auxins for three subsequent periods, both the enzyme activity and cell growth could be re-initiated with auxin. Enzyme activity reached its maximum 1 d before cell number was at a maximum. The enzyme was purified to homogeneity and characterized.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GSH reuced glutathione - Mr relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate The authors thank Dr. K. Palme, Max-Planck-Institute, Cologne, for reverse-phase chromatography. Part of this work was done by C. Paulus at the Department of Biotechnology, New Delhi, India under the Bundesministerium für Forschung und Technologie (BMFT)-funded Indo-FRG collaboration programme. Thanks are due to Professors S. Guha-Mukherjee and S.K. Sopory, New Delhi, for introduction into glyoxalase research. The research was funded by a BMFT-DECHEMA fellowship to C. Paulus, a BMFT grant to H.-J. Jacobsen and a Graduierten Förderung des Landes Nordrhein-Westfalen fellowship to B. Köllner.     

Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation.

Protein splicing involves the excision of an internal domain from a precursor protein and the ligation of the external domains so as to generate two new proteins. Study of this process has recently been facilitated by the isolation of a precursor and a branched intermediate from a thermophilic protein splicing element expressed in a foreign protein context. Two aspects of protein splicing are examined in this paper. We demonstrate a succinimide at the C-terminus of the spliced internal protein, implicating cyclization of asparagine in resolution of the branched intermediate, and we identify an alkali-labile bond in the branched intermediate. A revised protein splicing model based on these experimental results is presented.     

Characterization of pyrimidine-repressible and arginine-repressible carbamyl phosphate synthetases from Bacillus subtilis.

The number and properties of carbamyl phosphate synthetases in Bacillus subtilis have been uncertain because of conflicting genetic results and instability of the enzyme in extracts. The discovery of a previously unrecognized requirement of B. subtilis carbamyl phosphate synthetases for a high concentration of potassium ions for activity and stability permitted unequivocal demonstration that this bacterium elaborates two carbamyl phosphate synthetases. Carbamyl phosphate synthetase A was shown to be repressed by arginine, to have a molecular weight of about 200,000, and to be coded for by a gene that maps near argC4. This isozyme was insensitive to metabolites of the arginine and pyrimidine biosynthetic pathways. Carbamyl phosphate synthetase P was found to be repressed by uracil, to have a molecular weight of 90,000 to 100,000, and to be coded for by a gene that maps near the other pyr genes. This isozyme was activated by phosphoridine nucleotides. Other kinetic properties of the two isozymes were compared. Bacillus thus resembles eucaryotic microbes in producing two carbamyl phosphate synthetases, rather than the enteric bacteria, which produce a single carbamyl phosphate synthetase.     

Comparison of the effect of linear gramicidin analogues on bacterial sporulation, membrane permeability, and ribonucleic acid polymerase.

Various analogues of linear gramicidin were tested for their biological activity in restoring the normal spore phenotype of gramicidin-negative mutants of Bacillus brevis and for their ability to increase cation conductivity of black lipid membranes and to inhibit bacterial RNA polymerase. Whereas many biologically active gramicidin analogues had no effect on membrane permeability, all biologically active peptides were able to inhibit ribonucleic acid (RNA) polymerase. These observations make it unlikely that membranes are the site of action of gramicidin during bacterial sporulation, but they are consistent with the notion that gramicidin functions to control RNA synthesis during the transition from vegetative growth to sporulation (Sarkar & Paulus, 1972). The relationship between peptide structure and the ability to restore normal sporulation and inhibit RNA polymerase showed that the eight amino-terminal residues have little influence on the function of gramicidin, whereas the highly nonpolar repeating sequence D-leucyl-L-tryptophan is essential for biological activity and may represent the site of interaction with RNA polymerase.