A phenotypic host range alteration determines RD114 virus restriction in feline embryonic cells

We have characterized the restriction mechanism for RD114 virus replication in embryonic feline cells (FeF). By comparing growth properties of the virus in FeF cells with its behavior in a fetal feline glial cell line (G355) permissive for RD114, we showed that both cell lines were readily infectible by virus grown in permissive cells and that no significant differences in viral integration or viral RNA expression could be detected. However, analysis of viral protein expression revealed differences in viral env gene processing in the two cell types. Envelope precursor pR85 was produced, but the expected processed gp70 product was detectable only in permissive (G355) cells. An envelope product of 85 kDa was packaged into virions produced by FeF cells, while virions produced by G355 cells contained the expected RD114 gp70. While the gp85 env-containing virions were infectious for permissive G355 cells, they were unable to infect FeF cells. The block to infection by the gp85-containing particles in FeF cells could be abrogated by treatment with the glycosylation inhibitor tunicamycin. Our results indicate that restriction of RD114 virus involves a novel mechanism dependent on two factors: altered glycosylation of the envelope to a gp85 form and an altered RD114 receptor in FeF cells.     

Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. Implications for atherosclerotic lesion development

The subendothelial aggregation and retention of low density lipoprotein (LDL) are key events in atherogenesis, but the mechanisms in vivo are not known. Previous studies have shown that treatment of LDL with bacterial sphingomyelinase (SMase) in vitro leads to the formation of lesion-like LDL aggregates that become retained on extracellular matrix and stimulate macrophage foam cell formation. In addition, aggregated human lesional LDL, but not unaggregated lesional LDL or plasma LDL, shows evidence of hydrolysis by an arterial wall SMase in vivo, and several arterial wall cell types secrete a SMase (S-SMase). S-SMase, however, has a sharp acid pH optimum using a standard in vitro SM-micelle assay. Thus, a critical issue regarding the potential role of S-SMase in atherogenesis is whether the enzyme can hydrolyze lipoprotein-SM, particularly at neutral pH. We now show that S-SMase can hydrolyze and aggregate native plasma LDL at pH 5.5 but not at pH 7.4. Remarkably, LDL modified by oxidation, treatment with phospholipase A2, or enrichment with apolipoprotein CIII, which are modifications associated with increased atherogenesis, is hydrolyzed readily by S-SMase at pH 7.4. In addition, lipoproteins from the plasma of apolipoprotein E knock-out mice, which develop extensive atherosclerosis, are highly susceptible to hydrolysis and aggregation by S-SMase at pH 7.4; a high SM:PC ratio in these lipoproteins appears to be an important factor in their susceptibility to S-SMase. Most importantly, LDL extracted from human atherosclerotic lesions, which is enriched in sphingomyelin compared with plasma LDL, is hydrolyzed by S-SMase at pH 7.4 10-fold more than same donor plasma LDL, suggesting that LDL is modified in the arterial wall to increase its susceptibility to S-SMase. In summary, atherogenic lipoproteins are excellent substrates for S-SMase, even at neutral pH, making this enzyme a leading candidate for the arterial wall SMase that hydrolyzes LDL-SM and causes subendothelial LDL aggregation.     

Pharmacology and toxicology of artelinic acid: preclinical investigations on pharmacokinetics, metabolism, protein and red blood cell binding, and acute and anorectic toxicities

The pharmacokinetics, metabolism, protein binding, red blood cell (RBC) binding, stability in vitro, and acute and anorectic toxicity of artelinic acid (ARTL) were investigated in various animal species and human blood samples. Absorption and distribution following 10 mg/kg intramuscular or oral administration in dogs and rats were very rapid with t1/2 0.12-0.54; there were also a high AUC (11,262 ng/h/mL) and Vss (9.5 L/kg), low CL (15 mL/min/kg) and long elimination time (t1/2 = 2.6 h), compared with rat data. Oral bioavailability of ARTL was 79.7% in dogs and 30.1% in rats. The conversion of ARTL to dihydroartemisinin (DART) in dogs (0.1-0.5% of total dose) after 3 routes of administration (intravenous, intramuscular and oral) was 10-fold lower than that in rats. In rats dosed with [14C]ARTL, unchanged ARTL accounted for less than 13% of the total radioactivity after all 3 administration routes, suggesting that ARTL was extensively biotransformed. The half-lives of total radioactivity (21-49 h) in urine were much longer than that of unchanged ARTL in plasma (1.4-3.7 h), indicating that some long-lasting metabolites of ARTL were formed in rats. The mass balance data showed that 77-83% of total radioactivity was recovered in urine and faeces. High binding capacity (79-95%) and low binding affinity (1.1-9.3 x 10-7 M) of ARTL were measured in rat, rabbit, dog, monkey and human plasma. The RBC/plasma ratios of [14C]ARTL were 0.35 and 0.44 for dog and human plasma, respectively. ARTL was much more stable than artesunic acid (ARTS) in rat and dog plasma, and both ARTL and ARTS were more stable in dog plasma than in rat plasma in vitro. The 50% lethal dose (LD50) of ARTL in rats was about 535 mg/kg. Multiple intramuscular dosing for 7 d of 50 mg/kg/d of ARTL caused mild anorectic toxicity compared to ARTS in rats. In contrast to 4 other artemisinin derivatives, ARTL seems to be a good antimalarial candidate as it has the highest plasma concentration, the highest binding capacities in RBC, the highest oral bioavailability, the longest elimination half-life, the lowest metabolism rate and the lowest toxicity at equivalent dose levels.     

Production and purification of the heavy-chain fragment C of botulinum neurotoxin, serotype B, expressed in the methylotrophic yeast Pichia pastoris

Information on the turnover and lifespan of murine gamma/delta cells was obtained by administering the DNA precursor, bromodeoxyuridine (BrdU), in the drinking water and staining lymphoid cells for BrdU incorporation. For TCR-gamma/delta (Vgamma2) transgenic mice, nearly all gamma/delta thymocytes became BrdU+ within 2 d and were released rapidly into the peripheral lymphoid tissues. These recent thymic emigrants (RTEs) underwent phenotypic maturation in the periphery for several days, but most of these cells died within 4 wk. In adult thymectomized (ATx) transgenic mice, only a small proportion of gamma/delta cells survived as long-lived cells; most of these cells had a slow turnover and retained a naive phenotype. As in transgenic mice, the majority of RTEs generated in normal mice (C57BL/6) appeared to have a restricted lifespan as naive cells. However, in marked contrast to TCR transgenic mice, most of the gamma/delta cells surviving in ATx normal mice had a rapid turnover and displayed an activated/memory phenotype, implying a chronic response to environmental antigens. Hence, in normal mice many gamma/delta RTEs did not die but switched to memory cells.     

Actinomyces naeslundii genospecies 1 and 2 express different binding specificities to N-acetyl-beta-D-galactosamine, whereas Actinomyces odontolyticus expresses a different binding specificity in colonizing the human mouth

Postoperative thromboembolism is a potentially lethal complication. Its diagnosis may be difficult, as the classic clinical signs and symptoms are often absent, making a high index of suspicion imperative for diagnosis. Anticoagulant therapy is effective in reducing morbidity and mortality due to thromboembolism, but is associated with a substantial rate of bleeding complications in the immediate postoperative period. Inferior vena cava filters constitute an alternative to anticoagulant therapy, but are also associated with a substantial complication rate. The appropriate use of diagnostic tests combined with clinical suspicion can guide the orthopaedic surgeon in deciding which patients require treatment for thromboembolism.     

Psychosocial predictors of life satisfaction among persons living with HIV infection and AIDS

As AIDS becomes a more chronic but manageable illness, understanding quality of life issues among persons living with this disease has become an important goal of health care researchers. However, most quality of life investigations of persons living with HIV disease have relied heavily on clinical samples (e.g., hospitalized patients, psychiatric outpatients). The present study sought to identify psychosocial predictors of general life satisfaction in a community sample of 275 persons living with HIV/AIDS in a large midwestern state. Principal components and multiple regression analyses revealed that improved physical/functional well-being, increased social support, more frequent use of active coping strategies, and fewer incidents of AIDS-related discrimination and stigma predicted higher levels of general life satisfaction (R2 = 39). Intervention strategies likely to produce higher levels of life satisfaction among persons living with HIV disease are discussed.     

Fibrinogen receptor (GPIIb-IIIa) antagonists derived from 5,6-bicyclic templates. Amidinoindoles, amidinoindazoles, and amidinobenzofurans containing the N-alpha-sulfonamide carboxylic acid function as potent platelet aggregation inhibitors

A series of highly potent and specific fibrinogen receptor antagonists have been discovered and optimized through structural modification of the novel amidinoindole and benzofuran compounds, I and II. Systematic linker optimization afforded the amidinobenzofuran-containing inhibitor 29, which displayed an IC50 value of 250 nM in platelet aggregation assays. Attempts to enhance activity by modification of the beta-position of the beta-alanyl carboxylate group of 29 had only a modest effect on inhibitory activity in aggregation assays. Analogues prepared to enhance the activity by conformational restriction were also found to be equally or less potent. In contrast, modification at the alpha-position of the beta-alanyl carboxylate group resulted in the identification of extremely potent and novel amidinobenzofuran-containing derivatives 46-49. Reexamination of 5,6-bicyclic aromatic nucleus led to the further identification of amidinoindole- and amidinoindazole-containing derivatives 53-55. These analogues, 46-49 and 53-55, exhibited potent in vitro activity with IC50 values of 25-65 nM in platelet aggregation assays and an IC50 value of 2 nM in fibrinogen binding assays and demonstrated a selectivity of > 50,000-fold for GPIIb-IIIa versus the most closely related integrin, the vitronectin receptor, alpha v beta 3.     

Correction of hypertriglyceridemia and impaired fat tolerance in lipoprotein lipase-deficient mice by adenovirus-mediated expression of human lipoprotein lipase

Humans homozygous or heterozygous for mutations in the lipoprotein lipase (LPL) gene demonstrate significant disturbances in plasma lipoproteins, including raised triglyceride (TG) and reduced HDL cholesterol levels. In this study we explored the feasibility of adenovirus-mediated gene replacement therapy for LPL deficiency. A total of 5 x 10(9) plaque-forming units (pfu) of an E1/E3-deleted adenovirus expressing either human LPL (Ad-LPL) or the bacterial beta-galactosidase gene (Ad-LacZ) as a control were administered to mice heterozygous for targeted disruption in the LPL gene (n = 57). Peak expression of total postheparin plasma LPL activity was observed at day 7 in Ad-LPL mice versus Ad-LacZ controls (834 +/- 133 vs 313 +/- 89 mU/mL, P < .01), and correlated with human-specific LPL activity (522 +/- 219 mU/mL) and mass (9214 +/- 782 ng/mL), a change that was significant to 14 and 42 days, respectively. At day 7, plasma TGs were significantly reduced relative to Ad-LacZ mice (0.17 +/- 0.07 vs 1.90 +/- 0.89 mmol/L, P < .01) but returned to endogenous levels by day 42. Ectopic liver expression of human LPL was confirmed by in situ hybridization analysis and from raised LPL activity and mass in liver homogenates. Analysis of plasma lipoprotein composition revealed a marked decrease in VLDL-derived TGs. Severely impaired oral and intravenous fat-load tolerance in LPL-deficient mice was subsequently corrected after Ad-LPL administration and closely paralleled that observed in wild-type mice. These findings suggest that liver-targeted adenovirus-mediated LPL gene transfer offers an effective means for transient correction of altered lipoprotein metabolism and impaired fat tolerance due to LPL deficiency.