Lymphocytic alveolitis after primary HIV infection with CMV coinfection

Herein is a report of an adult case of primary HIV infection with cytomegalovirus coinfection causing cough, fever, and lymphocytic alveolitis. Primary HIV infection has not been previously reported as a cause of lymphocytic alveolitis.     

The effect of number of days in culture and plasminogen activator inhibitor-1 (PAI-1) 4G/5G genotype on PAI-1 antigen release by cultured human umbilical vein endothelial cells

An insertion/deletion (4G/5G) polymorphism in what has been shown to be an enhancer/repressor binding site in the promoter region of the PAI-1 gene has been related to plasma PAI-1 activity. Transfection studies demonstrated increased interleukin-1 stimulated PAI-1 synthesis in cells containing the 4G sequence. To study this response in endothelial cells, first passage HUVEC from 26 umbilical cords were stimulated with interleukin-1 and tumor necrosis factor-alpha. PAI-1 antigen was measured in 24-hour conditioned medium and allele-specific PCR utilized to determine genotype at the 4G/5G locus. Analysis of covariance was used to determine whether the effect of a variable time in culture was masking a difference between genotypes. A trend towards higher PAI-1 levels with increasing time in culture was observed. The geometric mean (95% confidence interval) of the basal rate of PAI-1 release was, 4G/4G 9.7 (7.0, 13.5) ng/24 hours (n=11), 4G/5G 9.5 (6.5, 13.9) ng/24 hours (n=9), and 5G/5G 10.9 (7.8, 15.1) ng/24 hours (n=6). In cells of the same cultures, the interleukin-1 stimulated levels were 25.9 (23.1, 29.1), 27.2 (23.6, 31.3), and 23.1 (19.5, 27.3) ng/24 hours, respectively, corresponding to ratios of stimulated to basal levels of 2.68, 2.87, and 2.12. After adjustment for time in culture the basal PAI-1 release was 4G/4G 10.7, 4G/5G 9.1, and 5G/5G 9.7 ng/24 hours. For interleukin-1 stimulated release the adjusted levels were 26.3, 27.0, and 22.7 ng/24 hours, respectively. Adjusted levels in 4G/4G genotype cells were non-significantly greater than those in cells of 5G/5G genotype by a factor of 1.16 (0.95, 4.08). This study did not demonstrate a significant difference in basal or cytokine stimulated PAI-1 release from cells of different PAI-1 promoter (4G/5G) genotypes but does not exclude increased interleukin-1 stimulated PAI-1 release in the 4G/4G compared with the 5G/5G genotype.     

Comparative pharmacology of the novel cyclopropylpyrroloindole-prodrug carzelesin in mice, rats, and humans

Carzelesin is a novel cyclopropylpyrroloindole prodrug analogue that has recently been tested in Phase I clinical trials. To increase our understanding in the pharmacology of this new class of cytotoxic drugs, we have compared the pharmacology of this drug in mice, rats, and humans. The mouse was the most tolerant [10% lethal dose (LD10), 500 microg/kg], the rat was intermediate (LD10, 40 microg/kg), and humans were the least tolerant species in this series (maximum tolerated dose, 300 microg/m2 corresponding to 7.5 microg/kg). In both mice and humans, bone marrow toxicity was the primary toxic side effect. Pharmacokinetic studies, using a validated high-performance liquid chromatographic procedure, revealed that differences in drug clearance and conversion to the active drug (U-76,074) could not explain the substantial interspecies differences. The area under the plasma concentration time curve (AUCs) of carzelesin in mice and rats at their LD10s were about 80- and 20-fold higher, respectively, than in humans receiving the maximum tolerated dose, whereas the respective AUCs of U-76,074 in mice and rats were 50- and 10-fold higher. By using a colony-forming assay with bone marrow stem cells from mice and humans, we observed only a 3-fold higher toxicity in the latter. Although some of this discrepancy may be explained by the fact that the in vitro and the in vivo assays probably reflect the toxicity on different populations of colony-forming units, the tolerance of the mouse bone marrow in vivo against the very high drug levels in plasma suggest the presence of a protective mechanism, which is less active in humans. An important consequence of the much higher susceptibility of the human bone marrow for carzelesin is that the target plasma levels in humans are much below active concentrations achieved in mice, and it is clear that this may compromise the successful use of this agent in the clinic. Ultimately, however, the efficacy of this drug will be established in Phase II clinical trials.     

Characterization of the gene cassette required for biosynthesis of the (alpha1-->6)-linked N-acetyl-D-mannosamine-1-phosphate capsule of serogroup A Neisseria meningitidis

Apoptosis, the cellular mechanism of ovarian follicular atresia and luteal regression, is triggered by the activation of a proteolytic cascade of cysteine aspartate-specific proteases (caspases). The principle downstream effector of cell death is caspase-3, but little is known about the role or regulation of this enzyme in ovarian apoptosis. Two substrates of caspase-3, actin and poly(ADP-ribose) polymerase (PARP), are inhibitors of DNase I, which is the endonuclease responsible for ovarian apoptotic DNA degradation. We therefore investigated the proteolytic cleavage of actin and PARP as well as the localization of caspase-3 during follicular atresia (induced by gonadotropin withdrawal) and luteal regression (induced by prostaglandin F2alpha) in the rat ovary. Apoptotic DNA degradation was evident during both follicular atresia and luteal regression, but cleavage of PARP and actin was observed only during luteal regression. Caspase-3 was localized in luteal cells of healthy corpora lutea (CL) and in theca, but not in granulosa cells of healthy follicles. However, caspase-3 immunostaining was evident in granulosa cells of atretic follicles in a pattern similar to that of the localization of granulosa cell death. There was no difference between healthy and apoptotic CL in the distribution or intensity of caspase-3 staining. These results demonstrate that the cleavage of actin and PARP are not necessary for activation of apoptotic DNA degradation during ovarian apoptosis. In addition, the presence of caspase-3 in granulosa cells of atretic, but not healthy, follicles suggests that the expression of this enzyme is regulated by gonadotropin and may be up-regulated as part of the apoptotic process in granulosa cells.     

Overexpression of scatter factor and its receptor (c-met) in oral squamous cell carcinoma

HYPOTHESIS: Scatter factor (SF) is a pleiotropic growth factor that recently has been shown to induce epithelial cell proliferation, random motility, and invasion via interaction with its receptor, a tyrosine kinase encoded by the c-met proto-oncogene. Studies involving a variety of solid tumors have suggested that overexpression of the SF/c-met ligand-receptor pair is associated with the acquisition of a malignant phenotype. We hypothesize that SF and c-met are overexpressed in epithelial malignancies of the head and neck including squamous cell carcinoma (SCC) of the oral cavity. STUDY DESIGN: Immunohistochemical staining of randomly selected normal, dysplastic, and malignant oral tissues. METHODS: Formalin-fixed, paraffin-embedded tissues were obtained from the Department of Oral Pathology at Shands Hospital (University of Florida), Gainesville, Florida. Examples of mild dysplasia, severe dysplasia, well-differentiated SCC, moderately differentiated SCC, and poorly differentiated SCC were randomly selected from the dictated reports of one of two staff oral pathologists. Histologically normal margins of each specimen served as normal controls. The tissues were immunohistochemically stained using commercially available antibodies against SF and c-met. Appropriate negative controls were run with each batch to ensure staining specificity. Evaluation of staining intensity was carried out using a computerized image analysis system. A one-way analysis of variance (ANOVA) with pairwise multiple-comparison procedures (Fisher method) was used to analyze the data. RESULTS: Statistically significant differences (P < .0001) in the intensity of staining were noted between the malignant and normal and the malignant and dysplastic tissues for both SF and c-met. No differences were appreciated when staining of normal and dysplastic sections of the SF-stained tissue were compared. CONCLUSIONS: The results suggest that the SF/c-met ligand-receptor pair is overexpressed in SCC of the oral cavity.     

Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

The x-ray crystal structures of trans-cinnamoyl-subtilisin, an acyl-enzyme covalent intermediate of the serine protease subtilisin Carlsberg, have been determined to 2.2-A resolution in anhydrous acetonitrile and in water. The cinnamoyl-subtilisin structures are virtually identical in the two solvents. In addition, their enzyme portions are nearly indistinguishable from previously determined structures of the free enzyme in acetonitrile and in water; thus, acylation in either aqueous or nonaqueous solvent causes no appreciable conformational changes. However, the locations of bound solvent molecules in the active site of the acyl- and free enzyme forms in acetonitrile and in water are distinct. Such differences in the active site solvation may contribute to the observed variations in enzymatic activities. On prolonged exposure to organic solvent or removal of interstitial solvent from the crystal lattice, the channels within enzyme crystals are shown to collapse, leading to a drop in the number of active sites accessible to the substrate. The mechanistic and preparative implications of our findings for enzymatic catalysis in organic solvents are discussed.     

Opposing actions of prostaglandins and oxytocin determine the onset of murine labor

Prostaglandins (PGs) have been recently proven essential for parturition in mice. To dissect the contributions of the two cyclooxygenase (COX) isoforms to the synthesis of PGs during pregnancy, we have characterized the parturition phenotype of COX-1-deficient mice. We find that mice with targeted disruption of the COX-1 gene have delayed parturition resulting in neonatal death. Results of matings of COX-1-deficient females with COX-1 intact males, and blastocyst transfer of COX-1-deficient or -intact embryos into wild-type foster mothers, proved necessity and sufficiency of maternal COX-1 for the normal onset of labor. COX-1 expression is induced in gravid murine uterus and by in situ hybridization; this induction is localized to the decidua. Measurement of uterine PGs further confirmed that COX-1 accounted for the majority of PGF2alpha production. To evaluate the interaction of PGs with oxytocin during murine labor, we generated mice deficient in both oxytocin and COX-1. Surprisingly, the combined oxytocin and COX-1-deficient mice initiated labor at the normal time. COX-1-deficient mice demonstrated impaired luteolysis, as evidenced by elevated serum progesterone concentration and ovarian histology late in gestation, and delayed induction of uterine oxytocin receptors. In contrast, simultaneous oxytocin and COX-1 deficiency restored the normal onset of labor by allowing luteolysis in the absence of elevated PGF2alpha production. These findings demonstrate that COX-1 is essential for normal labor in the mouse, with a critical function being to overcome the luteotrophic action of oxytocin in late gestation.     

Identification of a retinoic acid responsive aldoketoreductase expressed in HL60 leukaemic cells

We evaluated the role of protein kinase C (PKC) in the regulation of apoptosis triggered by singlet oxygen. Activation of PKC by short-term 12-O-tetradecanoyl phorbol 13-acetate (TPA) treatment inhibited apoptosis, whereas inhibition of PKC with several inhibitors potentiated this process. The antiapoptotic effect of TPA was accompanied by phosphorylation of extracelluar signal-regulated kinase 1/2 (ERK1/2). Pretreatment of cells with MEK inhibitor, PD98059, inhibited TPA-induced phosphorylation of ERK1/2 and the cytoprotective ability of TPA. These results suggest that activation of PKC in HL-60 cells confers protection against apoptosis induced by singlet oxygen and that ERK1/2 mediates antiapoptotic signaling of PKC.