PCR-DGGE analysis of bacterial community dynamics in kava beverages during refrigeration.

AIMS: Kava beverages are highly perishable even under refrigerated conditions. This study aimed to investigate the bacterial community dynamics in kava beverages during refrigeration. METHODS AND RESULTS: Four freshly made kava beverages were obtained from kava bars and stored at 4°C. On days 0, 3 and 6, the aerobic plate count (APC), lactic acid bacteria (LAB) count and yeast and mould count (YMC) of the samples were determined. Meanwhile, bacterial DNA was extracted from each sample and subjected to the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Moreover, species-specific PCR assays were employed to identify predominant Pseudomonas spp. involved in kava spoilage. Over the storage period, the APC, LAB count and YMC of the four kava beverages all increased, whereas their pH values decreased. The DGGE profile revealed diverse bacterial populations in the samples. LAB, such as Weissella soli, Lactobacillus spp. and Lactococcus lactis, were found in the kava beverages. Species-specific PCR assays detected Pseudomonas putida and Pseudomonas fluorescens in the samples; Ps. fluorescens became dominant during refrigeration. CONCLUSIONS: LAB and Pseudomonas may play a significant role in the spoilage of kava beverages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides important information that may be used to extend the shelf life of kava beverages.

Rapid identification of bacterial isolates from aqueous kava (Piper methysticum) extracts by polymerase chain reaction and DNA sequencing.

AIM: Fresh kava beverages have a limited shelf life under refrigerated conditions. The objective of this study was to isolate and identify bacteria in aqueous extracts of kava rhizome. METHODS AND RESULTS: The internal part of kava rhizome was used to minimize soil contamination. Three kava extracts were prepared, serially diluted and plated on nutrient agar. Isolated colonies were identified by sequencing polymerase chain reaction amplicons targeting the eubacterial 16S rDNA and the tuf gene of Staphylococcus. Seventy-five bacterial isolates belonged to 16 genera. Bacillus, Cellulomonas, Enterococcus, Pectobacterium and Staphylococcus were identified in all kava extracts. CONCLUSIONS: Kava rhizome contains large amounts of starch and fibre, which justify the presence of polysaccharide-degrading bacteria in the extracts. Bacillus cereus group and Staphylococcus species may produce toxins and cause foodborne illness. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide fundamental information that may be used to enhance the microbial quality and safety of kava beverages.

Isolation compliance among university students during a mumps outbreak, Kansas 2006.

A large mumps outbreak occurred among students at a Kansas university in 2006. To reduce transmission, students with mumps were asked to isolate themselves. We describe isolation measures and student compliance with these measures. Questionnaires were administered to students suspected of having mumps. Of the 132 students instructed to stay isolated, 75% stayed isolated for the number of days recommended and were considered compliant. Case-students told to stay isolated for 1-4 days were more likely to be compliant [86% vs. 66%; adjusted odds ratio (aOR) 3.6, 95% CI 1.4-9.0] than those told to stay isolated for 5-9 days. Those who rated avoiding contact with others during isolation as very important were also more likely to be compliant (83% vs. 60%; aOR 3.6, 95% CI 1.5-8.4) than those who rated the importance lower. In a college setting, it may be difficult to achieve high compliance with guidelines recommending that persons stay isolated for much longer than 4 days.

Semaphorin-1a acts in concert with the cell adhesion molecules fasciclin II and connectin to regulate axon fasciculation in Drosophila.

Semaphorins comprise a large family of phylogenetically conserved secreted and transmembrane glycoproteins, many of which have been implicated in repulsive axon guidance events. The transmembrane semaphorin Sema-1a in Drosophila is expressed on motor axons and is required for the generation of neuromuscular connectivity. Sema-1a can function as an axonal repellent and mediates motor axon defasciculation. Here, by manipulating the levels of Sema-1a and the cell adhesion molecules fasciclin II (Fas II) and connectin (Conn) on motor axons, we provide further evidence that Sema-1a mediates axonal defasciculation events by acting as an axonally localized repellent and that correct motor axon guidance results from a balance between attractive and repulsive guidance cues expressed on motor neurons.

The efficacy of the dicon screening field to detect eyes with glaucomatous field loss by Humphrey threshold testing.

PURPOSE: The authors compare the results of the Dicon suprathreshold, kinetic fixation perimeter with multiple stimulus presentation to automated threshold perimetry (Humphrey) in the same eye. METHODS: A Dicon screening visual field test and a Humphrey threshold visual field test were performed in 148 eyes of 148 persons with glaucoma or who were suspect for glaucoma. The number and pattern of missed points on the Dicon test were compared with Humphrey global indices in each eye. RESULTS: The median time to complete the 40-point, Dicon suprathreshold test was 2.7 minutes per eye. Regression analyses indicated that Dicon test parameters were modestly correlated with Humphrey corrected pattern standard deviation (CPSD) probability and mean deviation (R2 ranging from 0.21 to 0.46, p = 0.000). With glaucoma defined as a Humphrey Glaucoma Hemifield Test (GHT) result of outside normal limits, the best mix of sensitivity and specificity of Dicon results occurred at 2 or more missed points, with sensitivity of 64% and specificity of 83%. The specificity was maximum (90%) with a Dicon criterion of 3 or more adjacent missed points, but sensitivity at this level was 55%. With glaucoma defined by CPSD probability value less than 1%, sensitivity and specificity for two adjacent missed Dicon points were 69% and 87%, respectively. CONCLUSION: Dicon suprathreshold testing is a practical means to differentiate between some persons with glaucomatous damage and glaucoma suspects.

The efficacy of goniotomy/trabeculotomy in early-onset glaucoma associated with the Sturge-Weber syndrome.

PURPOSE: To assess the efficacy of goniotomy/trabeculotomy as the initial surgical procedure in early-onset glaucoma associated with Sturge-Weber syndrome. METHODS: We retrospectively analyzed 16 eyes of 14 consecutive patients with Sturge-Weber syndrome-associated glaucoma diagnosed before 4 years of age. All subjects were seen at a single institution from 1978 to 1996 and underwent goniotomy or trabeculotomy as their initial surgical procedure. RESULTS: Twelve eyes underwent initial goniotomy, and 4 eyes underwent initial trabeculotomy. One subject was lost to follow-up after surgery, resulting in 15 eyes for analysis. Of the initial goniotomy eyes, two thirds required a second surgical procedure. In the initial trabeculotomy eyes, half required a second procedure. Intraocular pressure was controlled (intraocular pressure < or = 22 mm Hg) in 66.7% of the eyes (10 of 15) after one or more goniotomy or trabeculotomy procedures for a median follow-up of 5.4 years (range, 1.4 to 15 years). For eyes with only one surgical procedure, 4 of 6 eyes had controlled intraocular pressure over a median follow-up of 3.4 years (range, 3 to 12 years). Seven of the 9 eyes that required more than one procedure had controlled intraocular pressure after all procedures over a median follow-up of 4.5 years (range, 1.4 to 15 years). CONCLUSION: Initial or repeated goniotomy or trabeculotomy may be an effective management choice for treatment of glaucoma associated with Sturge-Weber syndrome presenting in early childhood.

Temporal filter of the motion sensor in glaucoma.

Glaucoma reportedly affects motion perception. As an initial step in characterizing glaucoma-induced changes in the motion system, we determined the range of temporal frequencies that the motion system could process. A noise-masking paradigm was used to measure contrast energy thresholds of 26 glaucoma patients at various stages of the disease and 16 age-similar subjects with normal vision. Using a sinusoidal stimulus, thresholds were measured for the discrimination of motion direction and for the stimulus embedded within a pattern of dynamic spatial noise. The noise was filtered to contain only low spatial frequencies, and the temporal-frequency spectrum of the noise was manipulated across conditions to derive the temporal filter shape of the most efficient motion sensor. The results show that the range of temporal frequencies processed by the motion system is diminished in the glaucoma group. The filters of the glaucoma subjects have reduced bandwidths compared with the normal-vision group. In addition, the upper cut-off frequency of the filters of the glaucoma subjects is correlated with stage of disease as indexed by the mean deviation of the Humphrey Visual Field Analyzer program 24-2, as well as the cup-to-disk ratio.

Pathogenesis of murine encephalitis limited by defective interfering particles. An immunohistochemical study.

To determine whether defective interfering (DI) particles alter viral encephalitis BALB/c mice were inoculated intranasally with standard vesicular stomatitis virus (VSV) and its DI particles. Addition of 10(7) PFU equivalents of DI particles to 10(5) PFU of VSV reduced morbidity but did not delay disease onset. Less mortality was also observed. When 10(3) PFU equivalents of DI particles or UV-irradiated DI particles were substituted, these effects were absent. Attempts to correlate mortality with virus recovered from the brain could not be made due to considerable variations in the few surviving mice. Immunohistochemical analysis obtained from 121 mice showed that inoculation of DI particles limited the specific pathways of VSV antigen dissemination within the central nervous system, and new pathways were not substituted. In the group of mice with reduced mortality due to DI particles, at day 4 post inoculation VSV antigen was limited to the outer layers of the glomeruli of the olfactory bulb and to the accessory olfactory bulb, whereas there was deeper invasion of the olfactory bulb and olfactory ventricular system with mice infected with standard VSV alone. Correlation between mortality and extent of invasion became more difficult to make from 8 days on, when VSV antigens were found in discrete areas of the brain. By 12 days, few surviving mice contained any detectable VSV antigen in their brains. These results demonstrate that DI particles have potential as therapeutic agents. Also, mortality resulting from VSV-induced encephalitis, although poorly understood, may be determined very early, possibly while the virus is replicating at the site of inoculation.

Inhibition of vesicular stomatitis virus RNA synthesis by protein hyperphosphorylation.

Vesicular stomatitis virus (VSV) RNA synthesis requires the template nucleocapsid, the polymerase (L) protein, and the cofactor phosphorylated (P/NS) protein. To determine whether the degree of phosphorylation regulated VSV RNA synthesis, infected Chinese hamster ovary cells were treated with okadaic acid (OKA), a serine/threonine phosphatase inhibitor. OKA reduced viral penetration and uncoating but had little or no effect on primary transcription or viral protein synthesis. However, approximately 80% of total viral RNA synthesis was inhibited when 2 microM or more OKA was added to infected cells after viral uncoating had taken place. Analysis of proteins and RNA species in infected cells labeled with 32P showed that OKA led to hyperphosphorylation of two viral phosphoproteins, the P/NS protein and matrix protein (M), resulting in inhibition of full-length RNA synthesis and subsequent secondary transcription. Pulse-chase experiments demonstrated that the hyperphosphorylated P/NS species was converted rapidly from the less phosphorylated form. Hyperphosphorylated P/NS as well as the less phosphorylated form, but not M, were found to be associated with nucleocapsids isolated from cytoplasmic extracts. These results suggest that phosphorylation played an important role in the regulation between viral transcription and viral RNA replication as well as the turning off of RNA replication. Thus, phosphatase inhibitors promise to be a valuable tool for dissecting the regulatory mechanisms involving phosphorylated viral proteins.

Replication-defective viruses modulate immune responses.

By immunizing inbred mice with purified replication-competent, defective virus particles, or an admixture of the two, differential effects on the cellular immune system have been uncovered. Defective virus, exemplified by the vesicular stomatitis virus (VSV) defective interfering particle (DI 0.33), induced in BALB/c mice low levels of proliferating, IL-2 secreting, and cytolytic Ag-specific T lymphocytes. This was not caused by a dominant suppressor cell response, or by a failure to stimulate lymphokine-secreting cells, but appeared to reflect a reduced efficiency of priming as compared with standard virus. Mice primed with a mixture of wt and DI virus showed reduced proliferation compared with mice primed with wt virus. When histocompatible target cells were sensitized by pure DI particles, they were neither recognized nor lysed by CD8+ CTL. Cells co-infected with wt and DI particles were not as readily lysed by CD8+ CTL as cells infected by VSV alone. The extent of this reduction was dependent on the concentration of DI particles. This suggests that DI particles may have prevented the proper presentation of endogenously synthesized Ag for recognition by CD8+ CTL. Metabolic labeling studies indicated that the presence of DI particles suppressed the synthesis of viral proteins in dually infected cells. However, CD4+ T lymphocyte clones recognized and efficiently lysed histocompatible Ia+ cells infected with DI particles alone or co-infected with replication-competent and defective virus.

Murine infection by vesicular stomatitis virus: initial characterization of the H-2d system.

BALB/c mice and congenic H-2Ld-deficient BALB/c-H-2dm2 (dm2) mice were experimentally infected intranasally with isolates of vesicular stomatitis virus (VSV). The survival of infected hosts, viral replication in lungs and brains, and histopathologic in the two mouse strains were compared. In both strains of mice, mortality occurred during the period 7 to 10 days postinfection. However, dm2 mice were relatively resistant to lethal infections. Viral replication occurred at low levels in the lungs of both strains and did not evoke significant pathologic changes. In contrast, viral replication in the brains was much greater; in the BALB/c strain, this was accompanied by more frequent and more severe pathologic changes. In general, mice surviving at day 10 had effectively cleared virus from central nervous system but not respiratory sites. Evidence is presented that viral replication occurs first in the nasal cavity and is transmitted both to the lungs and to the olfactory bulb where focal cytopathology occurs. Virus enters the ventricles, causing encephalitis; necrosis occurs around the ventricles and in the lumbosacral region of the spinal cord. Necrotic lesions were accompanied by mononuclear infiltration. Mice immunized with virus of the same serotype or with a vaccinia virus hybrid encoding the VSV glycoprotein were protected from lethal infection; in contrast, mice immunized with heterotypic virus were susceptible to challenge.

The soluble viral glycoprotein of vesicular stomatitis virus efficiently sensitizes target cells for lysis by CD4+ T lymphocytes.

The soluble glycoprotein Gs of vesicular stomatitis virus (VSV), at approximately 10(4) molecules per cell, sensitized target cells for lysis by clones of CD4+ cytolytic T lymphocytes (CTL). In addition to lysis, the clones responded by proliferation and interleukin-2 release. Targets sensitized by Gs competed effectively with VSV-infected cells for recognition. Immune cytolysis by these CD4+ CTLs was restricted by class II major histocompatibility complex (MHC) antigens and was specific to VSV. The specific class II MHC antigen which was restricting for each clone remained the same whether the targets were sensitized by infection with VSV or by exogenously added soluble antigen. Sensitization by Gs appeared to require prior processing because the antigen-presenting cells that were fixed prior to exposure to Gs failed to be recognized by the CTL clones. The high efficiency of this uptake and processing was suggested by the inability of Gs at concentrations up to 10(7) per cell to block superinfection by VSV or to effect the RNA-synthetic machinery of uninfected cells. Also, Gs failed to hemolyze sheep erythrocytes when there was hemolysis by virions or an amino-terminal peptide of the VSV glycoprotein. Extrapolation of these results to viral diseases was possible because soluble viral glycoproteins were naturally synthesized during many viral infections and class II MHC antigens were inducible in cells of nonlymphoid origin. Therefore, CD4+ CTLs may be important participants in increasing virus-induced pathology, especially among adjacent uninfected cells.

Cytolytic T lymphocytes from the BALB/c-H-2dm2 mouse recognize the vesicular stomatitis virus glycoprotein and are restricted by class II MHC antigens.

BALB/c-H-2dm2 mice (H-2KdI-AdI-EdDd), a congenic strain of BALB/c mice, have a deletion of the class I MHC Ag, H-2Ld. This gene encodes the exclusive class I MHC-restricting gene product for vesicular stomatitis virus-specific cytolytic T lymphocytes. When dm2 mice were immunized with infectious vesicular stomatitis virus, a specific CTL response was generated. These CTL lysed VSV-infected targets that expressed Iad gene products, but not VSV-infected Iad- targets. The CTL were used initially as long term cytolytic lines; 13 CTL clones were derived by limit dilution. All of the clones expressed the phenotype CD3+, CD4+, CD8-; some clones expressed TCR that are members of the V beta 8 family, others did not. The clones were restricted by class II MHC Ag, both I-Ad and I-Ed serving as restricting elements for individual clones of the panel. All of the clones derived from dm2 mice were specific for the immunizing serotype, Indiana, of VSV and did not lyse syngeneic cells infected with VSV of the New Jersey serotype. Studies using defective interfering virus particles, UV light-inactivated virus, and purified micelles of the viral glycoprotein indicated that infectious virus was not required for sensitization of target cells for immune recognition by the class II MHC-restricted CTL clones. Additional studies using recombinant vaccinia virus vectors to sensitize targets confirmed the specificity of the clones for the viral glycoprotein. These studies also demonstrated a cryptic population of class II-restricted CTL in BALB/c lines specific for VSV G. Naturally occurring variant viruses and mutant viruses, selected for escape from neutralization by mAb, were used in an effort to map the determinant(s) recognized; on the basis of patterns of target cell lysis, three groups of epitopes recognized by the clones were defined. Therefore, in the absence of the class I MHC Ag required for a CTL response to VSV, dm2 mice generated CTL with the CD4+ phenotype that recognized different epitopes on the viral glycoprotein, and lysed cells in a class II-MHC restricted, Ag-specific manner.

Coupling efficiencies of amino acids in the solid phase synthesis of peptides.

The "classical" Merrifield method was used to synthesize over 500 peptides using Boc-benzyl strategy. The peptides were prepared either manually or on a Beckman 990B synthesizer or an Applied Biosystems 430A synthesizer. Each coupling of Boc amino acid to the growing peptide on the resin was monitored with the ninhydrin reaction. Couplings were considered "incomplete" if there was 99% or less coupling and "high incomplete" if there was 98% or less coupling. The efficiency of coupling was evaluated in regard to the specific amino acids involved in the coupling reaction and to the length of the peptide at the time of the coupling. The most difficult carboxyl-reacting amino acids were histidine, threonine, arginine, valine, isoleucine and glutamine; the most difficult amine reacting residues were glutamine, leucine, alanine, arginine and isoleucine. The number of "incomplete" and "high incomplete" couplings and the total number of monitored couplings of each of the 20 carboxyl-reacting amino acids when reacting with each of the 20 amine-reacting residues were tabulated. Coupling efficiencies decreased with the length of the peptide. The conclusion of this study is that, with the chemistries and methods used in this group of peptides, no amino acid coupling can be predicted to be complete with a single coupling reaction. The study points to the need for on-line determination of coupling efficiency during the synthesis in which a recoupling step is initiated when the first coupling is incomplete.

A comparative study of virus isolation, polymerase chain reaction, and antigen detection in children of mothers infected with human immunodeficiency virus.

We report on an investigation designed to compare the polymerase chain reaction (PCR) with culture and p24 measurement for the diagnosis of human immunodeficiency virus (HIV) infection in infants and children. Forty-five children born of mothers with antibodies to HIV type 1 were studied; P24 antigen was measured in plasma, and HIV-1 proviral DNA was sought in peripheral blood mononuclear cells after amplification by PCR. In 26 cases, blood specimens were cultured for HIV; in all but two instances cultures were established at the same time that the PCR test was performed. Primer pairs in three regions of the proviral genome were used for the PCR test. There was good agreement between the results obtained from PCR tests and from cultures; of 24 children in whom both tests were done at the same time, 10 had positive results on both the culture and the PCR test, 1 had positive results on the PCR test but negative culture results, and 13 had negative results on both tests (concordance 96%). Measurement of p24 antigen in plasma was, in contrast, an insensitive marker of infection: 6 of 12 infants with positive cultures had positive p24 test results, and 8 of 18 infants had positive PCR test results. Sixteen children with subsequent seronegativity for HIV-1 had negative PCR results. This study provides further evidence that the PCR test is a valid alternative to viral culture for the diagnosis of pediatric HIV infection.

Characterization of the polymerase activity associated with cultured peripheral blood mononuclear cells from patients with Kawasaki disease.

The particulate fractions of culture supernatants from peripheral blood mononuclear cells from 39 patients with Kawasaki disease (KD) were examined for the presence of particle-associated reverse transcriptase activity. The peak polymerase activity was significantly higher in cultures from KD patients compared to controls (mean = 6.4 versus 3.6 pmol of dTMP incorporated, p = 0.001). PBMC cultured between the 3rd and 9th wk after onset of fever were most likely to be associated with reverse transcriptase activity. Peak polymerase activity was positively associated with older age (r = 0.41, p = 0.01) and greater magnitude of the serum IgA response at 7-14 d after onset of fever (r = 0.45, p = 0.01) and IgM response at 6-9 wk after onset of fever (r = 0.46, p = 0.01). The appearance of enzyme activity was not associated with a decrease in viability of the cultured cells. A purified enzyme preparation showed radiolabel incorporation only with an RNA template with DNA primer. These data suggest that circulating mononuclear cells from KD patients may harbor a polymerase-associated agent and that these cells can be most readily detected in the early convalescent phase of KD from older patients who mount a marked humoral immune response.

Phenotypic mixing between human immunodeficiency virus and vesicular stomatitis virus or herpes simplex virus.

Superinfection of H9 cells persistently infected with human immunodeficiency virus (HIV) with thermolabile vesicular stomatitis virus (VSV) or herpes simplex virus (HSV) led to the synthesis of hybrid progeny. These phenotypic mixtures were able to infect HeLa or Chinese hamster ovary cell lines, leading to the production of HIV p24 antigen and infectious HIV. This production was abrogated by prior incubation of the phenotypic mixtures with antiserum against VSV or HSV, as well as by incubation of the mixtures at 39 degrees C for 10 h. These results demonstrate that during coinfection of cells with either a RNA or DNA virus, HIV forms hybrid virions composed of the genetic information of HIV and the envelope glycoproteins of the coinfecting viruses.

Human cervical carcinoma cell lines contain an antigen identical to the tumor-specific 75 kDa antigen of HeLa cells: detection by viral acquisition.

Purified vesicular stomatitis virus grown in the human cervical carcinoma HeLa cell line, VSV(HeLa), contains a 75 kDa tumor-specific antigen, detectable by immunoblotting of electrophoretically separated proteins with rabbit antiserum made against whole HeLa cells. Nearly identical results were obtained with VSV grown in the tumorigenic human hybrid ESH-5L cells, but not with the matched non-tumorigenic ESH-5E cells. Growth of VSV in 4 other independently isolated human cervical carcinoma cell lines led to the concentration of the same 75 kDa tumor-specific antigen by VSV. Infection of 2 other human cervical carcinoma cell lines did not lead to the detection of this antigen. The expression of the tumor-specific antigen correlated directly with the amount of RNA expression from human papillomavirus integrated in the DNA of these cells, irrespective of whether the papillomavirus was type 16 or 18.

Multiple thermolabile and temperature-sensitive lesions in mutant tl17 of vesicular stomatitis virus.

The vesicular stomatitis virus thermolabile mutant tl17 contains multiple lesions. The RNA-dependent RNA polymerase is temperature sensitive during primary transcription. The glycoprotein develops Endo H sensitivity more slowly at the nonpermissive temperature. Maturation or incorporation of the glycoprotein into progeny virions is also reduced. When virions of tl17 made at the permissive temperature are incubated in buffered medium at 39 degrees, their glycoprotein is cleaved, resulting in a product that resembles soluble glycoprotein. Compared to another glycoprotein mutant, ts 045, the glycoprotein of tl17 is only partially degraded to soluble G intracellularly and is more thermolabile. These properties of tl17 make it potentially useful for studies on glycoprotein synthesis, processing, and transport as well as for studies on pseudotype formation and viral maturation.