Crystal structure of an archaeal DNA sliding clamp: proliferating cell nuclear antigen from Pyrococcus furiosus.

The proliferating cell nuclear antigen (PCNA) is now recognized as one of the key proteins in DNA metabolic events because of its direct interactions with many proteins involved in important cellular processes. We have determined the crystal structure of PCNA from a hyperthermophilic archaeon, Pyrococcus furiosus (pfuPCNA), at 2.1 A resolution. pfuPCNA forms a toroidal, ring-shaped structure consisting of homotrimeric molecules, which is also observed in the PCNA crystals from human and yeast. The overall structure of pfuPCNA is highly conserved with other PCNA proteins, as well as with the bacterial ss clamp and the bacteriophage gp45. This result shows that the three-dimensional structure of the sliding clamp is conserved in the three domains of life. pfuPCNA has two remarkable features compared with the human and yeast PCNA molecules: it has more ion pairs and fewer intermolecular main chain hydrogen bonds. The former may contribute to the thermal stability of pfuPCNA, and the latter may be the cause of the stimulatory effect of pfuPCNA on the DNA synthesizing activity of P. furiosus DNA polymerases in the absence of the clamp loader replication factor C in vitro.

Main ginseng saponin metabolites formed by intestinal bacteria.

Ginseng saponin metabolites produced by human intestinal bacteria and the urinary and blood compounds after oral administration of Ginseng extract and its saponins in human and specific pathogen-free rats were examined in order to elucidate their metabolites absorbed from the intestines. The main metabolites of ginsenosides Rb1, Rb2, Rc, Re, and Rg1 after anaerobic incubation with fecal flora were identified as 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (I) 20-O-[alpha-L-arabinopyranosyl (1-->6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol (II), 20-O-[alpha-L- arabinofuranosyl(1-->6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol+ ++ (III), and 20(S)-protopanaxatriol (IV), though the metabolic rate and mode were affected by fermentation media. Furthermore, metabolites I-IV and 20(S)-protopanaxadiol (XII) were detected in blood (0.3-5.1 micrograms/ml) and in urine (2.2-96 micrograms/ day) after the oral administration of Ginseng extract (150 mg/ kg/day) to human and of total saponin (1 g/kg/day) to rats.

Inhibitors of acyl-CoA: cholesterol acyltransferase. II. Preparation and hypocholesterolemic activity of optically active dibenz[b,e]oxepin-11-carboxanilides.

In a previous paper, we reported a novel inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT), 2-bromo-N-(2,6-diisopropylphenyl)-6,11- dihydrodibenz[b,e]oxepin-11-carboxamide (1). In this work, we prepared both enantiomers and tested them for ability to inhibit ACAT (liver microsomes from cholesterol-fed rabbits) in vitro and to decrease serum total cholesterol in cholesterol-fed golden hamsters in vivo. The precursor carboxylic acid 4 was optically resolved with cinchonidine. The obtained (-)- and (+)-4 were converted to (-)- and (+)-1 without racemization, respectively. The enantiomer (-)-1 showed potent ACAT inhibitory activity in vitro with an IC50 value of 8 nM and was approximately 10-fold more active than (+)-1. Furthermore, (-)-1 showed strong hypocholesterolemic activity in vivo, whereas (+)-1 was inactive. A molecular modeling study showed that the difference of ACAT inhibitory activity between the enantiomers was derived from the spatial alignment of the bromine. Compound (-)-1 was selected for further evaluation as KW-3033.

Reversal of daunomycin and vinblastine resistance in multidrug-resistant P388 leukemia in vitro through enhanced cytotoxicity by triterpenoids.

Examined in vitro were the effects of some triterpenoids from Panax (Araliaceae) and Glycyrrhiza (Leguminosae) spp. on the sensitivity to daunomycin (DAU) and vinblastine (VBL) of adriamycin (ADM)-resistant P388 leukemia cells (P388/ADM), which were resistant to multiple anticancer drugs. Quasipanaxatriol, 20(S)-protopanaxatriol, ginsenoside Rh2, and compound K greatly enhanced the cytotoxicity of the anti-cancer drugs in P388/ADM cells. The extent of enhancement was different among the triterpene compounds; the 4- to 46-fold increase in DAU cytotoxicity was observed in P388/ADM cells in the presence of non-toxic or marginally toxic concentrations of individual compounds, while those for VBL were in the ratios of 2- to 37-fold. The maximum increase in cytotoxicity was observed with 50 microM quasipanaxatriol; the resistance indices defined to be the ratios of the IC50 values for P388/ADM and P388 parental cells decreased from 79 to 1.7 and from 180 to 4.9 in the cases of DAU and VBL, respectively. The reversal of DAU resistance in P388/ADM by quasipanaxatriol could be explained by the effective accumulation of the drugs mediated by the DAU-efflux blockage.

Inhibitory effect of some triterpenoid saponins on glucose transport in tumor cells and its application to in vitro cytotoxic and antiviral activities.

The effects of some triterpenoid saponins on glucose transport in Ehrlich ascites tumor (EAT) cells were examined by measuring 2-deoxy-D-glucose (2-DG) uptake. The correlation of the effects with those on the growth of a human T-cell line (MT-4) and the replication of human immunodeficiency virus in MT-4 cells was also studied. Chikusetsusaponin Ia isolated from rhizomes of Panax japonicus C. A. Meyer (Araliaceae) inhibited the 2-DG uptake (IC50 = 76.3 microM) in a competitive fashion with respect to 2-DG (Ki = 0.32 mM) and the growth of MT-4 cells with CC50 of 84.4 microM, whereas it did not show any significant anti-HIV-1 activity. In contrast, zingibroside R1 isolated from rhizomes of Panax zingiberensis Wu et Feng (Araliaceae) showed some anti-HIV-1 activity, which was found to be superior to that of glycyrrhizin, as well as the inhibitory effects on the 2-DG uptake by EAT cells (IC50 = 91.3 microM) and the growth of MT-4 cells (CC50 = 46.2 microM).

Interactions of ginseng extract, ginseng separated fractions, and some triterpenoid saponins with glucose transporters in sheep erythrocytes.

The effects of Panax ginseng extract, ginseng saponins, and some other triterpenoid saponins on glucose uptake were examined by using sheep erythrocytes. Initial rates of glucose transport were determined by measurements of 2-deoxy-D-glucose (2-DG) uptake. From kinetic analysis apparent Km and Vmax values of facilitated glucose transport in sheep erythrocytes were calculated as 2.3 +/- 0.08 mM and 1.4 +/- 0.05 nmol/min/10(9) cells. The results showed that ginseng extract stimulated glucose uptake in sheep erythrocytes dose-dependently. Ginseng saponins, in general, also stimulated glucose transport. The maximum effect was observed at 1 microM of ginsenoside Rb1 showing an increase of 24 +/- 5% above basal activity. However, ginsenoside Rg3, chikusetsusaponin Ia, and glycyrrhetic acid induced significant inhibitory effects on glucose transport in sheep erythrocytes.

Survey on testing criteria and reporting methods for human immunodeficiency virus serologic tests in Veterans Administration Medical Centers.

Guidelines for the indications for use, requirements for consent, and mechanisms for reporting of serologic tests for human immunodeficiency virus (HIV) infection are not standardized. In trying to establish such guidelines for our hospital, we surveyed all Veterans Administration Medical Centers regarding their current approach to testing both patients and employees. Infection control practitioners from 67 hospitals representing 37 states responded. Patients are likely to be tested for diverse reasons, unlikely to be counseled about the test or be required to consent to it, and test results are given no special precautions. Although 66% of the respondents do not use any extra precautions concerning patient confidentiality, 80% utilize more stringent criteria for testing and result-reporting with employees than patients. Thus, while the majority of hospitals maintain that current modes of confidentiality are acceptable for patients, practice suggests that these modes are considered inadequate for employees.

Evidence for the non-infectious etiology of early postoperative fever.

In a prospective study of infections in 871 general surgery patients, we identified 81 patients who developed unexplained postoperative fevers. The majority of these episodes (72%) occurred early (within the first 48 hours) following surgery. Patients who developed early, unexplained fevers differed significantly from patients who developed documented postoperative infections. Patients with unexplained fevers were younger, had less severe underlying disease and underwent less extensive surgeries than patients who subsequently developed infections. In these respects, they were more similar to non-infected, non-febrile patients. We concluded that episodes of early, unexplained postoperative fever occur frequently in a wide range of general surgery patients. Most of these episodes are non-infectious in origin. Patients with early postoperative fevers should be evaluated to identify any obvious sources of infection. If no focus is identified, empiric antibiotic therapy should not be initiated nor should prophylactic antibiotics be extended for prolonged durations. Unexplained fevers will resolve in time without specific therapeutic interventions.

Infections among patients in nursing homes: policies, prevalence, problems.

We performed one-day surveys in seven skilled-care nursing homes in order to evaluate their infection-control policies and to determine the prevalence of infections among their residents. Infection-control programs were not well developed at any of the home surveyed. We noted high patient-to-staff ratios, staffing by nonprofessional personnel, frequent job turnover, infrequent compensation for employee sick leave, and no general policies on immunization of patients or staff. The prevalence of infections among 532 patients was 16.2 per cent. Infected decubitus ulcers, conjunctivitis, symptomatic urinary-tract infections, and lower-respiratory tract infections were the most common types. Eight-five per cent of patients with indwelling urinary catheters had asymptomatic bacteriuria; many were colonized with antibiotic-resistant bacteria. Clustering of cases of upper-respiratory tract infections, diarrhea, conjunctivitis, and specific types of bacteriuria suggested that localized out-breaks of infectious occurred frequently. The high prevalence of infectious diseases and clustering of cases may reflect an increased susceptibility of patients in nursing homes to infections, high employee turnover, or lack of attention to infection-control practices.

Severity of underlying disease as a predictor of nosocomial infection. Utility in the control of nosocomial infection.

In a prospective study, we determined that severity of underlying disease at time of admission indicates medical patients at unusual risk of nosocomial infection. The nosocomial infection rate was 23.6% in patients with fatal underlying disease, 9.6% in those with ultimately fatal disease, and 2.1% in those with nonfatal disease. After an awareness program that promoted the use of established methods for prevention of nosocomial infections was established, there was a decline of overall incidence of endemic nosocomial infections from 9.2% to 4.8% (P less than .001) within an eight-month period. With subsequent discontinuation of the program, the infection rate rose to 8.1%. Reinstitution of the program resulted in a decline to 5.2% (P = .05).