Herpes simplex virus glycoproteins gC-1 and gC-2 bind to the third component of complement and provide protection against complement-mediated neutralization of viral infectivity.

Cells infected with herpes simplex virus type 1 (HSV-1) form rosettes with C3b-coated erythrocytes, whereas cells infected with herpes simplex virus type 2 (HSV-2) or other herpes viruses do not. It was reported that glycoprotein C of HSV-1 (gC-1) mediates the binding of C3b-coated erythrocytes to infected cells and has regulatory (decay-accelerating) activity for the alternative pathway C3 convertase of human complement. We show here that solubilized gC-1 binds to iC3-Sepharose affinity columns. We also report that solubilized gC-2, the genetically related glycoprotein specified by HSV-2, binds to iC3-Sepharose. mAb specific for gC-1 or gC-2 and mutant viral strains were used to identify the C3-binding glycoproteins. In other experiments, HSV-1 mutant strains and recombinants, differing only in their expression of gC, were tested for sensitivity to neutralization by human complement in the presence or absence of antibodies specific for HSV gD. In either case the gC- strain was most sensitive. Expression of gC-1 or gC-2 by isogenic insertion mutants provided protection against complement-mediated neutralization. These results indicate that the genetically and structurally related gC-1 and gC-2 share the functional activity of binding to human C3 and enhance viral infectivity.

Conformation and activity of chymotrypsin: the pH-dependent, substrate-induced proton uptake.

Hydrogen ion uptake by chymotrypsin during reversible binding of specific substrate is shown to be due to an ionizing group of the enzyme with a pK(apparent) approximately 9 in the free enzyme. This pK(apparent) is shifted to higher value in the enzyme-substrate complexes. Previous results indicating an equilibrium, controlled by this ionizing group, between active and inactive conformational forms of chymotrypsin are confirmed.

Levels of proteolytic activities as intermediate marker endpoints in oral carcinogenesis.

It is essential to identify intermediate marker endpoints of carcinogenesis for the evaluation of the effectiveness of cancer-chemopreventive agents. We have observed that levels of proteolytic activities (as detected by 4 different substrates) are increased 2-3-fold (P < 0.003) in oral buccal mucosa cells of smokers and patients with oral leukoplakia or erythroplakia as compared to a nonsmoking comparison group. In addition, proteolytic activity levels in the buccal cells were increased nearly 3-fold in patients with oral trauma (P < 0.01) or diabetes (P < 0.02), as well as pregnant women (P < 0.04). Excluding these subgroups of patients in epidemiological studies increase the differences in levels of proteolytic activities between both the nonsmoking comparison group and smokers and between the comparison group and patients with oral leukoplakia or erythroplakia. Evaluation of prerandomization levels of proteolytic activities of patients in cancer chemoprevention trials will increase the statistical power by allowing stratified randomization based on levels of proteolytic activities. The observed increases in levels of proteolytic activities in tissues at higher than normal risk of cancer development suggest that levels of proteolytic activities should be used as immediate marker endpoints in human cancer prevention trials using protease inhibitors as potential anticarcinogenic agents.

Monoclonal antibodies differentially reactive with native and reductively modified Bowman-Birk protease inhibitor.

Bowman-Birk protease inhibitor (BBI) is a potent anticarcinogen that suppresses malignant transformation at nanomolar concentrations. Small amounts of BBI in its native form can be measured by immunoassay using specific monoclonal antibodies (MAbs); however, the MAbs currently available are not capable of detecting BBI metabolites in human body fluids. To develop new reagents for the study of BBI exposure and pharmacokinetics, we produced four MAbs, designated 3B6, 3E3, 4H8 and 5G2, from hybridomas derived from a mouse immunized with reductively modified BBI. The epitopes recognized by the four MAbs were characterized using BBI in its native form or modified by different methods. MAb 3B6 reacted with native BBI. Partial reduction of BBI with 720 Gy of gamma radiation in an oxygen-free solution of 100 mM formate increased the reactivity of BBI with 3B6; however, extensive reduction of BBI with 100 mM DL-dithiothreitol (DTT) completely abolished this antigenic reactivity. In contrast, the other three MAbs reacted with BBI molecules that had been reduced either with 720 Gy of radiation in formate solution or with DTT. Alkylation of the radiochemically reduced BBI with N-ethylmaleimide further increased the reactivity of BBI with 3E3, 4H8 and 5G2, possibly by preventing the formation of new disulfide bonds within the BBI molecules. The binding of 4H8 and 5G2 to BBI antigen was inhibited by the binding of 3E3, and vice versa. Thus, the epitopes recognized by 3E3, 4H8 and 5G2 are probably located close to one another on the reduced BBI molecules. These three MAbs were able to react with BBI metabolites in urine samples collected from volunteers after oral administration of BBI. The ability of these MAbs to detect BBI metabolites indicates that BBI may be reductively modified in vivo and these MAbs may be useful reagents for monitoring the uptake of BBI into human tissues in cancer chemoprevention studies with BBI.

Analysis of synergism/antagonism between HIV-1 antibody-positive human sera and soluble CD4 in blocking HIV-1 binding and infectivity.

We tested human immunodeficiency virus type 1 (HIV-1) antibody-positive human sera and sCD4, alone and in combination, for synergistic, additive, or antagonistic effects on blocking of HIV binding and infectivity. Data were analyzed by an application of the median effect principle derived from the law of mass action. This allows the assessment of synergism/antagonism at any desired level of effect. Using three assays (whole virus binding to CD4 cells, neutralization of HIV infectivity, and binding of purified gp120 to solid-phase sCD4), we generally observed additive effects or slight synergism between antibody and sCD4 in inhibiting gp120-CD4 interaction. We used a fourth assay to measure the irreversible inactivation of HIV infectivity by sCD4, a property that can also be mediated by antibody but with considerably less potency than sCD4. The reduction in HIV infectivity mediated by mixtures of sCD4 and antibody was always equal to or greater than the arithmetic sum of the reductions by either agent alone. The relevant antiviral effects of sCD4 and anti-HIV sera may include reversible blockage of receptor binding, irreversible inactivation of HIV infectivity, and in the case of antibody, additional reactions that are independent of receptor binding. Although predictions concerning the in vivo situation are speculative, we find no evidence in vitro for antagonism between sCD4 and antibody with respect to the net effect of the two in blocking HIV binding and infectivity.

Myelodysplastic syndrome and acute myeloid leukemia after treatment for solid tumors of childhood.

Three cases of secondary (therapy-related) hematologic malignant conditions were identified among 95 children as old as 18 years of age; the cases were diagnosed between 1984 and 1990 and consisted of acute lymphoblastic leukemia, acute myeloid leukemia (AML), and myelodysplastic syndrome (MDSs). They constituted 10% of all new cases of AML and MDS seen at the University Hospitals of Cleveland during this time and were not related to congenital factors. The primary malignant conditions were malignant thoracopulmonary tumor (Askin tumor), neuroblastoma, and Burkitt's lymphoma. The secondary hematologic disorders all showed a prominent monocytic component: acute monocytic leukemia, MDSs evolving to acute myelomonocytic leukemia, and chronic myelomonocytic leukemia. The mean interval between treatment for the primary malignant condition and the onset of secondary disease was 36 months. All had received cyclophosphamide and an epipodophyllotoxin for the primary tumor; two were treated with radiation therapy. Cytogenetic abnormalities included del(5), del(13), t(1;6), and t(9;11)(p22[symbol:see text]3). The survival time after the onset of secondary disease was short.

Human IgG antibody to group b Streptococcus type III: comparison of protective levels in a murine model with levels in infected human neonates.

We determined the serum concentration of human IgG antibody to the native capsular polysaccharide of group B Streptococcus (GBS) type III needed to passively protect mice against lethal homologous challenge. Antibody was measured by an ELISA, standardized by two methods, and corrected for nonprecipitating antibody. A concentration of 1.3 micrograms of IgG antibody to GBS type III/ml protected 126 (97%) of 130 mice from an 80%-96% lethal dose bacterial challenge. Concentrations of IgG antibody to GBS type III in sera from 42 infected infants were less than or equal to 0.3 micrograms/ml. Concentrations of antibody ranged from less than 0.02 to 21.7 micrograms/ml in sera from 102 unselected pregnant women (median, 0.05 microgram/ml); 13% had concentrations greater than or equal to 1.3 microgram/ml. Levels in 25 women colonized with GBS type III who gave birth to normal infants were significantly higher and ranged from 0.1 to 10.7 microgram/ml (median, 0.78 micrograms/ml). In a study of transplacental passage of antibody, protective levels were found in a number of infants with gestational ages between 28 and 36 weeks.

Lowry protein determination by automated flow injection analysis for bovine serum albumin and hepatitis B surface antigen.

The Lowry method for quantitation of protein was adapted to automated flow injection analysis. The procedure was developed using two different pure proteins: bovine serum albumin and hepatitis B surface antigen. The system was optimized for reagent concentration, pH, gain, temperature, sample volume, and output. The response of each protein was affected differently by temperature. The reaction slopes and absorbance values of the proteins were similar at 90 degrees C to allow quantitation of hepatitis surface antigen against bovine serum albumin. Advantages of the automated flow injection analysis Lowry procedure include: rapid analyses (90 samples/h), small sample volume (30 microliters, 100 microliters), fast response (20 s), reproducibility (less than or equal to 2% CV within an assay and 3 to 6% CV among assays), sensitivity (5 micrograms), and high correlation (99.8%) with manual assay. After a 30-min set-up period, the analyzer was available to assay protein on demand throughout the day, making it suitable for process and quality control testing.

Effectiveness of a program of early hospital discharge of cardiac surgery patients.

Managed care was the impetus for a program designed to move adult patients from acute care to the lowest level of appropriate services after cardiac surgery. Clinical pathways and a home care cardiac specialty team were the major components of the Early Discharge Program. The program was evaluated based on both financial and clinical outcomes. A convenience sample of 119 pretest patients was compared with 101 posttest patients 3 months after program implementation. Hospital length of stay decreased only 0.34 days on average, but inpatient direct variable costs decreased by an average of $1,790 per patient. Based on the 101 patients in the posttest group, $180,790 in direct variable hospital costs were saved. The largest decrease in resource use was in those patients who were discharged to home care. Complications and home caregiver burden after discharge were no higher in patients discharged early. Early discharge of cardiac surgery patients appears to be safe and cost-effective.

Epidermal growth factor in mouse ocular tissue: effects of thyroxine and exogenous epidermal growth factor.

Using a specific and sensitive epidermal growth factor (EGF) radioimmunoassay, we identified radioimmunoassayable EGF both in developing and adult mouse ocular tissues. In neonatal animals ocular EGF concentrations increase during the first 4-9 days and then decline between 9 and 15 days. Thyroxine (T4) administration (0.4 micrograms/g body weight/day from day 0) increased local EGF concentrations in eye and skin of 7-day-old neonatal mouse pups. However, this treatment did not affect submandibular gland EGF concentrations during the 1st wk of life. Both EGF and T4 are known to accelerate eye opening in the neonatal mouse. Exogenous EGF administration (2 micrograms/g body weight/day) during the first 8 days of life elicited precocious eyelid opening as expected but did not alter the serum T4 concentration, suggesting that EGF does not mediate eye opening by T4 dependent mechanism(s). Tissue EGF measurements revealed that the exogenous EGF was localized in skin and eye; however, other tissues including lung, liver, heart and submandibular gland also contained exogenous EGF. Kidney-EGF concentrations did not rise while brain-EGF levels were significantly decreased after exogenous EGF, suggesting that different EGF uptake and regulatory mechanism(s) exist in different tissues during the neonatal period. T4 administration (0.4 micrograms/g body weight/day) for 10 days to adult mice also increased ocular-EGF concentrations. However, this increase was abolished by sialoadenectomy, suggesting in contrast to the newborn, that submandibular gland is an important source of ocular-EGF in adult mice. These studies indicate that ocular EGF in the mouse is thyroxine responsive only during the neonatal period.

The nucleotide sequence of Saccharomyces cerevisiae chromosome IX.

Large-scale systematic sequencing has generally depended on the availability of an ordered library of large-insert bacterial or viral genomic clones for the organism under study. The generation of these large insert libraries, and the location of each clone on a genome map, is a laborious and time-consuming process. In an effort to overcome these problems, several groups have successfully demonstrated the viability of the whole-genome random 'shotgun' method in large-scale sequencing of both viruses and prokaryotes. Here we report the sequence of Saccharomyces cerevisiae chromosome IX, determined in part by a whole-chromosome 'shotgun', and describe the particular difficulties encountered in the random 'shotgun' sequencing of an entire eukaryotic chromosome. Analysis of this sequence shows that chromosome IX contains 221 open reading frames (ORFs), of which approximately 30% have been sequenced previously. This chromosome shows features typical of a small Saccharomyces cerevisiae chromosome.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XIII.

Systematic sequencing of the genome of Saccharomyces cerevisiae has revealed thousands of new predicted genes and allowed analysis of long-range features of chromosomal organization. Generally, genes and predicted genes seem to be distributed evenly throughout the genome, having no overall preference for DNA strand. Apart from the smaller chromosomes, which can have substantially lower gene density in their telomeric regions, there is a consistent average of one open reading frame (ORF) approximately every two kilobases. However, one of the most surprising findings for a eukaryote with approximately 6,000 genes was the amount of apparent redundancy in its genome. This redundancy occurs both between individual ORFs and over more extensive chromosome regions, which have been duplicated preserving gene order and orientation. Here we report the entire nucleotide sequence of chromosome XIII, the sixth-largest S. cerevisiae chromosome, and demonstrate that its features and organization are consistent with those observed for other S. cerevisiae chromosomes. Analysis revealed 459 ORFs, 284 have not been identified previously. Both intra- and interchromosomal duplications of regions of this chromosome have occurred.

The genome sequence of Schizosaccharomyces pombe.

We have sequenced and annotated the genome of fission yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly reflecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have significant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identified, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.