Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.     

Identification of spherical virus particles in digitized images of entire electron micrographs

Interferon-gamma (IFN-gamma) has the most potent immunomodulatory activity of all the interferons. This phase II-B study was performed to define time- and dose-dependent immunomodulatory effects mediated by IFN-gamma in a subset of patients with melanoma treated in the dose-seeking therapeutic trial conducted by the Eastern Cooperative Oncology Group E4687 (13). The effects of IFN-gamma (Genentech, San Francisco, CA) were evaluated for phenotype and function of peripheral blood lymphocytes obtained twice prestudy, and on days 2, 9, and 29 of IFN-gamma therapy for 50 patients. Early significant increases in CD4/CD8 ratio (p = 0.001) were noted, largely due to a rise in CD4+ and fall in CD8+ T-cell populations sustained through day 29 at only the lowest dosage. Increased natural killer cell (NK) activity (p = 0.001 on day 9; p = 0.01 on day 29) was accompanied by durable increases in circulating activated NK cells (CD56+DR+% p = 0.001, day 9; p = 0.001, day 29). After initial depression of CD56+ and CD16+ cells on day 2, the total percent of CD56+ and CD16+ cells increased significantly by day 29. Increases in NK cell activity were maximal at doses > or =0.1 mg. Monocyte CD14+ expression of DQ+ rose early (p = 0.011 and 0.001 on days 2 and 9), accompanied by elevation in CD14+DR+ cells that was less significant. Immunomodulatory effects of IFN-gamma reported in this trial have major implications for interpretation of past and current clinical trials, and the design of future trials. This is the first trial in which IFN-gamma has been shown to have significant effects on the T-cell compartment of the immune system.     

Iodized oil enhances the thermal effect of high-intensity focused ultrasound on ablating experimental liver cancer

The influence of the biological medium on high-intensity focused ultrasound (HIFU) therapy for ablating experimental liver cancer was studied. In study 1, the temperature rise in the focal zone in the presence of iodized oil or castor oil was observed in vitro. The results showed that HIFU with iodized oil produced a higher and faster temperature rise than did HIFU with castor oil, whether high-power (500 W/cm2) or relatively low-power (136 W/cm2) conditions were used (P = 0.0008 and P = 0.0004 respectively). With the excised liver samples, the temperature also rose higher and more rapidly after injection of iodized oil into the liver than when castor oil was injected (P = 0.0239), and the target liver tissue revealed more radically and extensive destruction with iodized oil than with castor oil. In study 2, 48 nude mice, bearing primary liver cancer LTNM4 implanted subcutaneously, were randomly divided into four groups. Group I (n = 12) were the controls, group II (n = 12) were injected with iodized oil alone, group III (n = 12) received HIFU treatment, and group IV (n = 12) were exposed to HIFU after iodized oil injection. Significant inhibition of tumor growth was seen in groups III and IV as compared with group I or group II (P < 0.05), the tumor growth inhibition rate on the 28th day after treatment being 87% and 93% respectively. Significantly improved survival was noted in groups III and IV compared with groups I and II (P < 0.05). Histologically, group IV showed more complete tumor necrosis than did group III. These data suggest that HIFU combined with iodized oil might have achieve of synergism, location and targeting in the treatment of liver cancer.     

Uric acid, a natural scavenger of peroxynitrite, in experimental allergic encephalomyelitis and multiple sclerosis

Uric acid, the naturally occurring product of purine metabolism, is a strong peroxynitrite scavenger, as demonstrated by the capacity to bind peroxynitrite but not nitric oxide (NO) produced by lipopolysaccharide-stimulated cells of a mouse monocyte line. In this study, we used uric acid to treat experimental allergic encephalomyelitis (EAE) in the PLSJL strain of mice, which develop a chronic form of the disease with remissions and exacerbations. Uric acid administration was found to have strong therapeutic effects in a dose-dependent fashion. A regimen of four daily doses of 500 mg/kg uric acid was required to promote long-term survival regardless of whether treatment was initiated before or after the clinical symptoms of EAE had appeared. The requirement for multiple doses is likely to be caused by the rapid clearance of uric acid in mice which, unlike humans, metabolize uric acid a step further to allantoin. Uric acid treatment also was found to diminish clinical signs of a disease resembling EAE in interferon-gamma receptor knockout mice. A possible association between multiple sclerosis (MS), the disease on which EAE is modeled, and uric acid is supported by the finding that patients with MS have significantly lower levels of serum uric acid than controls. In addition, statistical evaluation of more than 20 million patient records for the incidence of MS and gout (hyperuricemic) revealed that the two diseases are almost mutually exclusive, raising the possibility that hyperuricemia may protect against MS.     

Deficient transport of dehydroascorbic acid in the glucose transporter protein syndrome

The glucose transporter protein syndrome (GTPS) is caused by defective transport of glucose across the blood-brain barrier via the glucose transporter GLUT1, resulting in hypoglycorrhachia, infantile seizures, and developmental delay. Recent reports indicated that GLUT1 is a multifunctional transporter. We investigated the transport of vitamin C in its oxidized form (dehydroascorbic acid) via GLUT1 into erythrocytes of 2 patients with GTPS. In both patients, uptake of oxidized vitamin C was 61% of the mothers' values. Our findings are consistent with recent observations that vitamin C is transported in its oxidized form via GLUT1. We speculate that impaired transport of this substrate and perhaps other substrates in GTPS might contribute to the pathophysiology of this condition.     

Positive affectivity and negative outcomes: the role of tenure and job satisfaction

In this study, the authors proposed and tested a 3-way interaction among positive affectivity (PA), job satisfaction, and tenure in predicting negative employee outcomes. Specifically, the authors predicted that the relationship between job satisfaction and negative outcomes would be stronger for high PAs and that this relationship would be more pronounced for longer tenured employees. Results support this 3-way interaction in predicting job search behavior, physical health complaints, and counterproductive employee behavior. In particular, the relationship between job satisfaction and negative outcomes was most strongly negative for high-PA individuals with longer tenure. The authors discuss the implications of these results and some directions for future research.     

Investigation of spectral reproducibility in direct analysis of bacteria proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) can be used for rapid detection of bacteria proteins in a crude mixture. It can potentially be used as a tool for bacterial identification based on the mass spectral patterns or the appearance of some characteristic mass peaks. However, there are many experimental parameters that can potentially have a strong effect on the observed mass spectra. The objective of this work is to address the mass spectral reproducibility issue. Several experimental parameters that may affect the MALDI spectra are systematically investigated. Results of spectral comparison from two laboratories with different operators and instrumentation are presented. It is demonstrated that minor variations in the sample/matrix preparation procedures for MALDI and in the experimental conditions used for bacterial protein extraction can result in a significant change in the observed spectra, though a number of peaks are conserved in the spectra obtained under different experimental conditions from the same bacterial sample. These conserved peaks may potentially be used as the biomarkers for bacterial identification. It is stressed that this type of investigation on spectral reproducibility should be carried out for different bacterial species in order to identify the mass spectral peaks that are consistently detected regardless of operator and nominal variations in sample preparation approach.     

Homocamptothecins: synthesis and antitumor activity of novel E-ring-modified camptothecin analogues

Homocamptothecin (hCPT), a camptothecin (CPT) analogue with a seven membered beta-hydroxylactone which combines enhanced plasma stability and potent topoisomerase I (Topo I)-mediated activity, is an attractive template for the elaboration of new anticancer agents. Like CPT, hCPT carries an asymmetric tertiary alcohol and displays stereoselective inhibition of Topo I. The preparation and biological screening of racemic hCPT analogues are described. The 10 hCPTs tested were better Topo I inhibitors than CPT. Fluorinated hCPTs 23c, d,f,g were found to have potent cytotoxic activity on A427 and PC-3 tumor cell lines. Their cytotoxicity remained high on the K562adr and MCF7mdr cell lines, which overexpress a functionally active P-glycoprotein. Fluorinated hCPTs were more efficacious in vivo than CPT on HT-29 xenografts. In this model, a tumor growth delay of 25 days was reached with hCPT 23g at a daily dose of 0.32 mg/kg, compared to 4 days with CPT at 0.625 mg/kg. Thus difluorinated hCPT 23g warrants further investigation as a novel Topo I inhibitor with high cytotoxicity toward tumor cells and promising in vivo efficacy.     

Tertiary structure of the major house dust mite allergen Der p 2: sequential and structural homologies

Sensitization to indoor allergens, especially those of the house dust mite, is strongly correlated with the development of asthma. We report the tertiary structure of the major house dust mite allergen, Der p 2, determined by NMR methods. The structure of Der p 2 is a beta-barrel and is composed of two three-stranded antiparallel beta-pleated sheets. This arrangement of beta-strands is similar to the immunoglobulin fold with respect to the orientation of the two sheets and the interactions of the strands. However, the three-dimensional structure of Der p 2 aligns equivalently with a number of proteins from different families within the immunoglobulin superfamily. The structural homology with the highest significance score from analysis by DALI is to Der f 2. Although Der p 2 and Der f 2 are 87% identical in amino acid sequence, they align in three dimensions rather poorly (4.85 A RMSD; Z-score, 8.58). This unexpected finding is likely due to the different solution conditions used during structure determination by NMR for both proteins. While the structural comparisons did not elucidate a clear homologue for the function of Der p 2 in mites, we report that Der p 2 is sequentially homologous to esr16. This is a protein from moths that is expressed coincident with molting. Thus, this homology has important ramifications for the study of mite allergy. The structure of Der p 2 provides a useful tool in the design of recombinant immunotherapeutics for the group 2 allergens.