Phase I/II study of the toxicity, pharmacokinetics, and activity of the HIV protease inhibitor SC-52151

A significant restriction was demonstrated in the ability of herpes simplex virus type 1 virion host shutoff (vhs) mutant viruses to invade the corneal epithelium. Viral replication and invasion was confined to the areas of the cornea which were scarified prior to infection. Differences between wild-type and vhs mutant replication in corneas in vivo were 100- to 1000-fold at all timepoints postinfection. Smaller but still significant growth restrictions were observed in cultured corneal cells. This difference between in vitro and in vivo is not likely to be due to differences in cell cycle status since vhs-induced RNA degradation can occur in both cycling and noncycling cells in vitro. The vhs function is therefore important for invasion of the cornea and secondarily the nervous system and is thereby required for efficient establishment of latency.     

Low levels of follicle-stimulating hormone receptor-activation inhibitors in serum and follicular fluid from normal controls and anovulatory patients with or without polycystic ovary syndrome

In patients with normogonadotropic anovulation, either with or without polycystic ovary syndrome (PCOS), factors interfering with FSH action may be involved in arrested follicle development. The aim of this study is to assess whether factors inhibiting FSH receptor activation are elevated in serum or follicular fluid from anovulatory patients, as compared with regularly cycling women. For this purpose, a Chinese hamster ovary cell line, stably transfected with the human FSH receptor, has been applied. FSH-stimulated cAMP secretion in culture medium was measured in the presence of serum or follicular fluid. Chinese hamster ovary cells were stimulated with a fixed concentration of FSH (3 or 6 mIU/mL) to mimic FSH levels in serum or follicular fluid. Samples were added in concentrations ranging from 3-90% vol/vol to approach protein concentrations occurring in serum or follicular fluid. In the presence of 10% vol/vol serum from regularly cycling women (n = 8), FSH-stimulated cAMP production was inhibited to 42 +/- 2% (mean +/- SEM of 2 experiments, each performed in duplicate) of cAMP production in the absence of serum, whereas a similar cAMP level (up to 38 +/- 4% of the serum-free level) was observed at higher concentrations of serum (30-90% vol/vol). The inhibition of FSH-stimulated cAMP production in the presence of serum samples from normogonadotropic anovulatory patients, without (n = 13) or with (n = 16) PCOS, was similar to controls. Follicular fluid samples (n = 57) obtained during the follicular phase in 25 regularly cycling women and follicular fluid samples (n = 25) from 5 PCOS patients were tested in a slightly modified assay system. In the presence of 10 or 30% (vol/vol) follicular fluid, FSH-stimulated cAMP levels were decreased to 68 +/- 2% and 55 +/- 2% (mean +/- SEM of a single experiment in triplicate) of the cAMP levels in the absence of follicular fluid, respectively. There was no correlation between the degree of cAMP inhibition and follicle size, steroid content (androstenedione or estradiol concentrations), or menstrual cycle phase. Furthermore, no differences in inhibition were found, comparing PCOS follicles with size- and steroid content-matched follicles obtained during the normal follicular phase. It is concluded that inhibition of FSH receptor activation by proteins present in serum or follicular fluid is constant (60 and 40%, respectively) and independent from the developmental stage of the follicle, either during the normal follicular phase or in patients with normogonadotropic anovulation. Inhibition of FSH receptor activation may be of limited significance for normal and arrested follicle development.     

Typing of Pneumocystis carinii f. sp. hominis by single-strand conformation polymorphism of four genomic regions

To better investigate Pneumocystis carinii f. sp. hominis epidemiology, we have developed a molecular typing method. Because of the limited genetic variability of the P. carinii hominis genome, a multitarget approach was used. Four variable regions of the genome were amplified by PCR, polymorphism in each region was assessed by the single-strand conformation polymorphism (SSCP) technique, and the results for the four regions of each patient were combined. Bronchoalveolar lavage specimens collected from 11 patients were examined. Four patients were probably infected by a single strain, since their specimens yielded simple SSCP patterns (two bands corresponding to one allele). The combinations of these patterns were unique, suggesting that the strains which infected these patients were different. For the other seven patients, complex patterns were found (three or four bands corresponding to two alleles). The presence of more than one allele of a region in a patient is likely to be due to coinfection. Polymorphism was also assessed by sequencing, which revealed variations at nucleotide positions previously reported to vary. About half of the observed alleles had already been reported by laboratories in different countries. Multitarget typing of P. carinii hominis by PCR-SSCP should allow investigation of strain diversity and thus be useful for future epidemiological studies.     

RS-102221: a novel high affinity and selective, 5-HT2C receptor antagonist

The 5-HT2C receptor is one of three closely related receptor subtypes in the 5-HT2 receptor family. 5-HT2A and 5-HT2B selective antagonists have been described. However, no 5-HT2C selective antagonists have yet been disclosed. As part of an effort to further explore the function of 5-HT2C receptors, we have developed a selective 5-HT2C receptor antagonist, RS-102221 (a benzenesulfonamide of 8-[5-(5-amino-2,4-dimethoxyphenyl) 5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione). This compound exhibited nanomolar affinity for human (pKi = 8.4) and rat (pKi = 8.5) 5-HT2C receptors. The compound also demonstrated nearly 100-fold selectivity for the 5-HT2C receptor as compared to the 5-HT2A and 5-HT2B receptors. RS-102221 acted as an antagonist in a cell-based microphysiometry functional assay (pA2 = 8.1) and had no detectable intrinsic efficacy. Consistent with its action as a 5-HT2C receptor antagonist, daily dosing with RS-102221 (2 mg/kg intraperitoneal) increased food-intake and weight-gain in rats. Surprisingly, RS-102221 failed to reverse the hypolocomotion induced by the 5-HT2 receptor agonist 1-(3-chlorophenyl)piperazine (m-CPP). It is concluded that RS-102221 is the first selective, high affinity 5-HT2C receptor antagonist to be described.     

Simulated dipeptide recognition by vancomycin

The antimicrobial activity of vancomycin and related glycopeptide antibiotics is due to stereospecific recognition of polypeptide components in bacterial cell walls. To better understand how these antibiotics recognize polypeptide determinants, we have developed dynamic models of the complexes formed by the vancomycin aglycon and two different dipeptide ligands, Ac-D-ala-D-ala and Ac-D-ala-gly. Molecular dynamics simulations of the two complexes, initially conditioned with distance constraints derived from two-dimensional nuclear magnetic resonance (NMR) studies, are conformationally stable and propagate in a manner consistent with the NMR-derived constraints after the constraints are removed. Free energy calculations accurately predict the relative binding affinity of these two complexes and help validate the simulation models for detailed structural analysis. Although the two ligands adopt similar conformations when bound to the antibiotic, there are clear differences in the configuration of intermolecular hydrogen bonds, the overall shape of the antibiotic, and other structural features of the two complexes. This analysis illustrates how complex structural and dynamic factors interrelate and contribute to differences in binding affinity.     

Misinterpretation of body sensations in panic disorder

Cognitive accounts of panic predict that panic disorder patients will be particularly prone to misinterpret autonomic sensations. Several studies have produced results consistent with this prediction, but each is open to alternative interpretation. To clarify matters, 2 studies administered the Body Sensations Interpretation Questionnaire (BSIQ) to panic patients and controls. Panic patients were more likely to interpret ambiguous autonomic sensations as signs of immediately impending physical or mental disaster and were more likely than other anxiety disorder patients and nonpatients to believe these interpretations. In a 3rd study, a brief version of the BSIQ was shown to have satisfactory test-retest reliability, to change with treatment, and to discriminate treatments that varied in their effects on panic.     

Assessment of cerebral blood flow and CO2 reactivity after controlled cortical impact by perfusion magnetic resonance imaging using arterial spin-labeling in rats

We measured CBF and CO2 reactivity after traumatic brain injury (TBI) produced by controlled cortical impact (CCI) using magnetic resonance imaging (MRI) and spin-labeled carotid artery water protons as an endogenous tracer. Fourteen Sprague-Dawley rats divided into TBI (CCI; 4.02 +/- 0.14 m/s velocity; 2.5 mm deformation), sham, and control groups were studied 24 hours after TBI or surgery. Perfusion maps were generated during normocarbia (Paco2 30 to 40 mm Hg) and hypocarbia (PaCO2 15 to 25 mm Hg). During normocarbia, CBF was reduced within a cortical region of interest (ROI, injured versus contralateral) after TBI (200 +/- 82 versus 296 +/- 65 mL.100 g-1.min-1, P < 0.05). Within a contusion-enriched ROI, CBF was reduced after TBI (142 +/- 73 versus 280 +/- 64 mL.100 g-1.min-1, P < 0.05). Cerebral blood flow in the sham group was modestly reduced (212 +/- 112 versus 262 +/- 118 mL.100 g-1.min-1, P < 0.05). Also, TBI widened the distribution of CBF in injured and contralateral cortex. Hypocarbia reduced cortical CBF in control (48%), sham (45%), and TBI rats (48%) versus normocarbia, P < 0.05. In the contusion-enriched ROI, only controls showed a significant reduction in CBF, suggesting blunted CO2 reactivity in the sham and TBI group. CO2 reactivity was reduced in the sham (13%) and TBI (30%) groups within the cortical ROI (versus contralateral cortex). These values were increased twofold within the contusion-enriched ROI but were not statistically significant. After TBI, hypocarbia narrowed the CBF distribution in the injured cortex. We conclude that perfusion MRI using arterial spin-labeling is feasible for the serial, noninvasive measurement of CBF and CO2 reactivity in rats.     

A comparison of methods of phenotypic and genotypic fingerprinting of Exophiala dermatitidis isolated from sputum samples of patients with cystic fibrosis

A 77-year-old man was admitted because of massive pericardial effusion and cardiac tumor. Cytological examination of the effusion and histological examination of a subcutaneous tumor in the chest wall revealed diffuse large B cell lymphoma. The immunophenotype of tumor cells was CD5+ CD20+ CD22+ CD38+ HLA-DR+ CD19-. Chromosome analysis revealed complex abnormal karyotypes containing t(8;14) (q24;q32). C-myc gene rearrangement was shown by Southern blotting. Chemotherapy with pirarubicin, cyclophosphamide, vincristin, and prednisolone (THP-COP) was not effective for his lymphoma. He suffered from cardiac tamponade and died at 5 months after diagnosis. Autopsy revealed a large cardiac tumor, extensive epicardial infiltration, tiny tumors in the lung and pancreas, but no lymphadenopathy, the combination of which suggested a primary cardiac lymphoma. Immunohistochemistry for p53 protein showed nuclear staining of more than 50% of the lymphoma cells. In situ hybridization for EBER-1 was negative. Rearrangement of c-myc gene and overexpression of p53 protein are usually observed in Burkitt's lymphoma and some cases of high grade lymphomas including AIDS-associated non-Hodgkin lymphomas. In this case the association of these molecular findings and resistance to chemotherapy is suggested.     

The catalytic domain of protein kinase C chimeras modulates the affinity and targeting of phorbol ester-induced translocation

Emerging evidence suggests important differences among protein kinase C (PKC) isozymes in terms of their regulation and biological functions. PKC is regulated by multiple interdependent mechanisms, including enzymatic activation, translocation of the enzyme in response to activation, phosphorylation, and proteolysis. As part of our ongoing studies to define the factors contributing to the specificity of PKC isozymes, we prepared chimeras between the catalytic and regulatory domains of PKCalpha, -delta, and -epsilon. These chimeras, which preserve the overall structure of the native PKC enzymes, were stably expressed in NIH 3T3 fibroblasts. Their intracellular distribution was similar to that of the endogenous enzymes, and they responded with translocation upon treatment with phorbol 12-myristate 13-acetate (PMA). We found that the potency of PMA for translocation of the PKCalpha/x chimeras from the soluble fraction was influenced by the catalytic domain. The ED50 for translocation of PKCalpha/alpha was 26 nM, in marked contrast to the ED50 of 0.9 nM in the case of the PKCalpha/epsilon chimera. In addition to this increase in potency, the site of translocation was also changed; the PKCalpha/epsilon chimera translocated mainly into the cytoskeletal fraction. PKCx/epsilon chimeras displayed twin isoforms with different mobilities on Western blots. PMA treatment increased the proportion of the higher mobility isoform. The two PKCx/epsilon isoforms differed in their localization; moreover, their localization pattern depended on the regulatory domain. Our results emphasize the complex contributions of the regulatory and catalytic domains to the overall behavior of PKC.