Inhibition of HIV-1 gp160-dependent membrane fusion by a furin-directed alpha 1-antitrypsin variant

Furin is a membrane-associated calcium-dependent serine endoprotease that cleaves proproteins on the carboxyl side of the consensus sequence -Arg-X-Lys/Arg-Arg-. Using site-directed mutagenesis, a variant alpha 1-antitrypsin (alpha 1-AT) was constructed which contains in its reactive site -Arg-X-X-Arg-, the minimal sequence required for efficient processing by furin (Molloy, S. S., Bresnahan, P. A., Leppla, S. H., Klimpel, K. R., and Thomas, G. (1992) J. Biol. Chem. 267, 16396-16402). This alpha 1-AT variant, [Arg355 Arg358]alpha 1-AT (alpha 1-PDX), is greater than 3,000-fold more effective than [Arg358]alpha 1-AT (alpha 1-AT Pittsburgh, alpha 1-PIT) at inhibiting furin in vitro (K0.5 = 0.03 microgram/ml). Furthermore, the P4 Arg in alpha 1-PDX greatly attenuates the thrombin inhibitory properties of this serpin (> 300-fold) compared with alpha 1-PIT (which contains a P4 Ala), thus increasing the selectivity of alpha 1-PDX for furin. Expression studies show that alpha 1-PDX, and not alpha 1-PIT, blocks the processing of two furin substrates, pro-beta-nerve growth factor and human immunodeficiency virus (HIV)-1 gp160 in transfected cells. In addition, a syncytium assay shows that alpha 1-PDX blocks the membrane fusogenic properties of HIV-1 gp160. The potential use of alpha 1-PDX in manipulating the activation of proproteins in a tissue- and time-specific manner is discussed.     

Treatment of advanced or recurrent endometrial carcinoma with single-agent carboplatin

Cisplatin and the combination of cisplatin, doxorubicin, and cyclophosphamide have documented activity in women with advanced or recurrent endometrial adenocarcinoma. However, response duration has been short and toxicity is substantial. To determine if similar activity could be obtained with less morbidity, we prospectively treated 33 patients with 360 mg/m2 carboplatin given intravenously every 28 days. Mean patient age was 69 years (range 40-86); all had a Zubrod functional status of 2 or less. Seventeen patients had advanced primary tumors, and 16 had recurrent disease. Prior treatment included surgical resection in 29 cases, hormonal agents in 7, and radiotherapy in 22. No patient had received prior chemotherapy. Mean treatment was 5.7 cycles. Nine of 27 patients (33%) with measurable disease had objective responses, including three complete and six partial responses. Nonresponders included 10 patients with stable disease and 8 whose disease progressed while on treatment. Median time to response was 3 months. Median progression-free survival for responders and nonresponders was 5 and 4 months, respectively. At analysis, 20 patients had died of disease, 7 were alive with disease, and 6 were clinically free of disease. Disease-free patients include 1 with a complete response and 5 who began treatment without measurable disease. Median follow-up for surviving patients was 18 months (range 4-32). Treatment toxic effects were minimal and largely limited to myelosuppression; 3 patients had grade 3 thrombocytopenia, 1 had grade 3 neutropenia, and 5 had grade 3 anemia. Carboplatin has define activity in endometrial carcinoma and offers a well-tolerated palliative therapeutic alternative.     

Quantitation of three-month intraindividual variability and influence of sex and menstrual cycle phase on CYP1A2, N-acetyltransferase-2, and xanthine oxidase activity determined with caffeine phenotyping

OBJECTIVE: To evaluate intraindividual variability and the effects of sex and menstrual cycle phase on the activity of cytochrome P450 1A2 (CYP1A2), N-acetyltransferase 2 (NAT2), and xanthine oxidase. METHODS: Ten white men were given 2 mg/kg caffeine orally every 14 days for 3 months. The same dosage of caffeine was given to 10 premenopausal white women during the midfollicular and midluteal phases of three complete menstrual cycles. Phenotype was determined with urinary caffeine metabolite ratios. RESULTS: For CYP1A2, mean metabolic ratio (+/- SD) was 5.97 +/- 2.78 during the midfollicular phase and 5.32 +/- 1.99 during the midluteal phase (p = 0.2). For extensive and poor metabolizer of NAT2. Mean midfollicular phase metabolite ratios were 0.71 +/- 0.060 and 0.37 +/- 0.030, and mean midluteal phase metabolite ratios were 0.69 +/- 0.076 and 0.39 +/- 0.053 (p = 0.9). For xanthine oxidase, mean midfollicular phase metabolite ratio was 0.63 +/- 0.06 and mean midluteal phase metabolite ratio was 0.63 +/- 0.05 (p = 0.3). Among the men, mean CYP1A2, NAT2 rapid and slow acetylator, and xanthine oxidase indices were 9.42 +/- 10.18, 0.66 +/- 0.021, 0.31 +/- 0.056, and 0.64 +/- 0.03. There were no differences in metabolite ratios between men and women for CYP1A2, NAT2 extensive metabolizers, or xanthine oxidase. A statistically significant sex difference was found for poor metabolizers of NAT2 (p < 0.05). Median coefficients of variation for CYP1A2, NAT2 extensive and poor metabolizers, and xanthine oxidase ratios were 16.8% (range, 4.5% to 49.3%), 2.9% (range, 2.2% to 4.7%), 13.4% (range, 7.5% to 27.2%), and 4.5% (range, 2.3% to 13.0%). CONCLUSION: Stratification by menstrual cycle phase or sex need not be performed for pharmacokinetic or clinical investigations of substrates for CYP1A2, NAT2, or xanthine oxidase in which the subject are adults.     

Distribution of type II diabetes in nuclear families

Type II diabetes has a substantial genetic component, but the mode of inheritance and the molecular basis of this inheritance are uncertain. This study documents the familial distribution of the disease in the parents and siblings of a consecutive series of type II diabetic subjects. We studied 66 first-degree relatives of 20 white subjects with type II diabetes and both parents alive. They were tested with a continuous infusion of glucose (5 mg.kg IBW-1.min-1) (n = 49) or FPG and hemoglobin A1c (n = 17). Seven probands had neither parent affected with diabetes or IGT, 10 had one parent affected (6 with diabetes and 4 with IGT), and 3 had both parents affected. The probands with affected and those with unaffected parents were phenotypically similar. These findings indicate that a sizable subgroup of type II diabetic subjects may have neither parent affected with a demonstrable abnormality of glucose tolerance. The assumption of autosomal dominance with complete penetrance is not supported, although it remains possible that a dominant gene of low penetrance may play a role in some pedigrees. Polygenic inheritance would appear likely, and genetic heterogeneity may occur. The inheritance of diabetic traits from phenotypically normal parents needs to be considered in the analysis of genetic linkage with type II diabetes.     

Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid

The N-linked oligosaccharides of frog (Rana pipiens) rhodopsin were analysed by sequential exoglycosidase digestion and gel filtration chromatography, following reductive tritiation. In addition, selected tryptic glycopeptides obtained from frog retinal rod outer segment membranes were examined by electrospray mass spectrometry (ES-MS), fast atom bombardment mass spectrometry (FAB-MS), amino acid sequence and composition analysis, and carbohydrate composition analysis. The amino acid sequence data demonstrated that the glycopeptides were derived from rhodopsin and confirmed the presence of two N-glycosylation sites, at residues Asn2 and Asn15. The predominant glycan (approximately 60% of total) had the structure GlcNAc beta 1-2Man alpha 1-3(Man alpha 1-6) Man beta 1-4GlcNAc beta 1-4GlcNAc-(Asn), with the remaining structures containing 1-3 additional hexose residues, as reported previously for bovine rhodopsin. Unlike bovine rhodopsin, however, a sizable fraction of the total glycans of frog rhodopsin also contained sialic acid (NeuAc), with the sialylated oligosaccharides being present exclusively at the Asn2 site. FAB-MS analysis of oligosaccharides released from the Asn2 site gave, among other signals, an abundant quasimolecular ion corresponding to a glycan of composition NeuAc1Hex6HexNAc3 (where Hex is hexose and HexNAc is N-acetylhexosamine), consistent with a hybrid structure. The potential biological implications of these results are discussed in the context of rod outer segment membrane renewal.     

Risk factors for low birth weight and intrauterine growth retardation in Santiago, Chile

An epidemiologic case-control study to ascertain the determinants of low birthweight was carried out in Santiago, Chile, from January to December 1989. The cases were defined as livebirths < 2500 g. The controls were livebirths > or = 2500 g of birthweight. All cases and a random sample (1:1) of controls were selected among 8,254 singleton births occurring at the El Salvador Hospital in the Eastern area of Santiago. These deliveries represented 50% of institutional deliveries in the area. Home deliveries (2%) and private hospital deliveries were not included in the study. Information was obtained from hospital medical records by six trained medical students. Some information could not be obtained from the hospital medical records. Thus the second step in data collection was the tracking of all the selected subjects to their referring neighborhood health centers. For the analysis, the data were divided into 3 case (outcome) categories: 453 subjects were the total case group. From these, 153 were the IUGR case group and 300 were the LBW preterm case group. The general control group consisted of 605 normal birthweight infants. 565 were the IUGR control group and 40 were the preterm control group. A total of 25 risk factors showed a significant crude odds ratio for at least one of the groups. In the multivariate logistic regression analysis eight variables: No. of pregnancies, previous adverse outcomes, previous LBW, pregnancy maternal weight, No. of visits, month of first prenatal care visit, maternal smoking and intrahepatic cholestasis of pregnancy, were significantly associated with LBW after adjustment by confounding. Eight risk factors: IUGR in previous pregnancies, Previous adverse outcome, Maternal smoking, intrahepatic cholestasis, maternal pregnancy weight, maternal height, month first prenatal visit, No. of visit, were significant to IUGR. Only two variables: pregnancy weight, divorced mother, were significantly associated with low birth weight in the preterm group. The most relevant risk factors were included in stepwise logistic regression models carried out for the outcome LBW for the general group, term group and preterm group, in order to adjust by confounding. Adjusted odds ratios were then obtained. Prenatal care related factors and maternal adverse obstetric factors were at higher significance for LBW in the general and IUGR groups. Only nutritional factors were related to LBW in preterm group. Women who delivered a LBW or IUGR infant were more likely to have fewer pregnancies, a history of previous LBW, lower prepregnancy weight and lower gestational weight gain. ICP was associated with an elevated risk of LBW that was independent of gestational age.