Evaluation of sampling tools for environmental sampling of bacterial endospores from porous and nonporous surfaces.

AIMS: Having and executing a well-defined and validated sampling protocol is critical following a purposeful release of a biological agent for response and recovery activities, for clinical and epidemiological analysis and for forensic purposes. The objective of this study was to address the need for validated sampling and analysis methods called out by the General Accounting Office and others to systematically compare the collection efficiency of various swabs and wipes for collection of bacterial endospores from five different surfaces, both porous and nonporous. This study was also designed to test the collection and extraction solutions used for endospore recovery from swabs and wipes. METHODS AND RESULTS: Eight collection tools, five swabs and three wipes, were used. Three collection/preservation solutions were evaluated: an ink jet aerosol generator was used to apply Bacillus subtilis endospores to five porous and nonporous surfaces. The collection efficiencies of the swabs and wipes were compared using a statistical multiple comparison analysis. CONCLUSIONS: The ScottPure wipe had the highest collection efficiency and phosphate-buffered saline (PBST) with 0.3% Tween was the best collection solution of those tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Validated sampling for potential biological warfare is of significant importance and this study answered some relevant questions.

Dynamics of {beta}-amyloid reductions in brain, cerebrospinal fluid, and plasma of {beta}-amyloid precursor protein transgenic mice treated with a {gamma}-secretase inhibitor.

gamma-Secretase inhibitors are one promising approach to the development of a therapeutic for Alzheimer's disease (AD). gamma-Secretase inhibitors reduce brain beta-amyloid peptide (Abeta), which is believed to be a major contributor in the etiology of AD. Transgenic mice overexpressing the human beta-amyloid precursor protein (APP) are valuable models to examine the dynamics of Abeta changes with gamma-secretase inhibitors in plaque-free and plaque-bearing animals. BMS-299897 2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoic acid, a gamma-secretase inhibitor, showed dose- and time dependent reductions of Abeta in brain, cerebrospinal fluid (CSF), and plasma in young transgenic mice, with a significant correlation between brain and CSF Abeta levels. Because CSF and brain interstitial fluid are distinct compartments in composition and location, this correlation could not be assumed. In contrast, aged transgenic mice with large accumulations of Abeta in plaques showed reductions in CSF Abeta in the absence of measurable changes in plaque Abeta in the brain after up to 2 weeks of treatment. Hence, CSF Abeta levels were a valuable measure of gamma-secretase activity in the central nervous system in either the presence or absence of plaques. Transgenic mice were also used to examine potential side effects due to Notch inhibition. BMS-299897 was 15-fold more effective at preventing the cleavage of APP than of Notch in vitro. No changes in the maturation of CD8(+) thymocytes or of intestinal goblet cells were observed in mice treated with BMS-299897, showing that it is possible for gamma-secretase inhibitors to reduce brain Abeta without causing Notch-mediated toxicity.

[Markers of hepatitis C and its various genotypes in patients from Novosibirsk]. / Markery virusa gepatita C i razlichnky ego genotipov u patsientov Novosibirska.

Study of the prevalence of hepatitis C virus (HCV) markers in 153 patients of Municipal Infectious Diseases Clinical Hospital No. 1 in Novosibirsk revealed anti-HCV in 88.2% patients and viral RNA in 69.3% samples. For 93 Siberian HCV isolates 5'-UTR regions were amplified and sequenced. Comparison of these nucleotide sequences with databank showed that 63.4% HCV isolates belonged to subtype 1b, 7.5% to genotype 2, and 18.3% to genotype 3. In the rest HCV isolates the 5'-UTR sequences contained heretofore undescribed nucleotide substitutions, insertions, or deletions.

Behavioral effects of anti-muscarinic, anti-serotonergic, and anti-NMDA treatments: hippocampal and neocortical slow wave electrophysiology predict the effects on grooming in the rat.

Previous research has shown that hippocampal and neocortical activation accompanies the postural changes occurring during self-grooming in rats but is absent or reduced during the stereotyped components of grooming, including head-washing and licking or biting. Since electrocortical activation is dependent on ascending cholinergic and serotonergic projections, we hypothesized that central muscarinic and serotonergic blockade would disrupt grooming by degrading cerebral control of changes in posture. Consistent with this, we find that systemic injections of scopolamine: (a) markedly reduce the occurrence of adaptive changes in posture during grooming; (b) reduce the probability of transitions from head-washing to body grooming; (c) reduce both the probability and duration of sequences of body grooming; and (d) do not affect the duration of head-washing or the probability of transitions from washing the snout to washing over the top of the head. Destruction of central serotonergic neurons with intracerebral injections of 5,7-dihydroxytryptamine increases the tendency of scopolamine to shorten the duration and increase the number of separate sequences of grooming. Systemic injections of a NMDA antagonist (CGS 19755) also impair grooming behavior. The data show that blockade of muscarinic and glutamatergic transmission impairs instinctive behavior as well as learned behavior and that the behavioral effects of muscarinic and serotonergic blockade are consistent with data obtained from the study of cortical slow wave electrophysiology.

Functional conservations of the alkaline nuclease of herpes simplex type 1 and human cytomegalovirus.

The herpes simplex virus type 1 UL12 gene product, alkaline nuclease (AN), appears to be involved in viral DNA processing and capsid egress from the nucleus (Shao, L., Rapp, L. M., and Weller, S. K., Virology 196, 146-162, 1993). Although the HSV-1 AN is not absolutely essential for viral replication in tissue culture, conservation of the AN gene in all herpesviruses suggests an important role in the life cycle of herpesviruses. The counterpart of HSV-1 AN for human cytomegalovirus (HCMV) is the UL98 gene product. To examine whether the HCMV AN could substitute for HSV-1 AN, we performed trans-complementation experiments using a HSV-1 amplicon plasmid carrying the HCMV UL98 gene. Our results indicate (i) HCMV AN can complement the growth of the HSV-1 AN deletion mutant UL12lacZ virus in trans; (ii) a new recombinant virus, UL12laZcUL98/99, appears to be generated by the integration of the HCMV UL98 gene into the HSV-1 UL12lacZ viral genome; (iii) in contrast to its parental HSV-1 UL12lacZ virus, capsids formed in UL12lacZUL98/99-infected Vero cells were able to transport from the nucleus to the cytoplasm and mature into infectious viruses. Our results demonstrate a functional conservation of AN between HSV-1 and HCMV.

Na, an autoproteolytic product of the herpes simplex virus type 1 protease, can functionally substitute for the assembly protein ICP35.

The herpes simplex virus type 1 (HSV-1) protease and its substrate, the assembly protein ICP35, are involved in virion maturation. Both proteins are encoded by a single open reading frame but are translated independently from 3'-coterminal mRNAs of different sizes and are in frame. The herpesvirus shell assembles around an internal scaffold which is subsequently lost during packaging of the viral genome. The scaffold is composed of ICP35, which is the major component, and autoproteolytically processed forms of the viral protease containing sequences common to ICP35 (Nb). In the baculovirus system, HSV-1 intact capsids can be formed in the presence of the protease or ICP35, indicating that the protease may substitute for ICP35 (Thomsen et al., J. Virol. 68:2442-2457, 1994). This is further supported by the fact that ICP35, in contrast to the protease, is not absolutely essential for viral growth. The processed intermediate of the protease analogous to ICP35 is the 388-amino-acid (aa) protein, Na, which is an N-terminal 59-aa extension of the 329-aa ICP35. To directly examine whether Na can functionally substitute for ICP35 during viral replication, we first constructed a mutant virus, Na delta35, in which 35 aa from the N terminus of Na were deleted. Phenotypic analysis of the mutant showed that this deletion had no effect on protease function. The function of Na was further examined by construction of a plasmid expressing Na alone and testing its ability to complement the growth of the mutant Prb virus in the absence of ICP35. Our results demonstrate that Na can functionally substitute for ICP35 during viral replication.

Separate functional domains of the herpes simplex virus type 1 protease: evidence for cleavage inside capsids.

The herpes simplex virus type 1 (HSV-1) protease (Pra) and related proteins are involved in the assembly of viral capsids and virion maturation. Pra is a serine protease, and the active-site residue has been mapped to amino acid (aa) 129 (Ser). This 635-aa protease, encoded by the UL26 gene, is autoproteolytically processed at two sites, the release (R) site between amino acid residues 247 and 248 and the maturation (M) site between residues 610 and 611. When the protease cleaves itself at both sites, it releases Nb, the catalytic domain (N0), and the C-terminal 25 aa. ICP35, a substrate of the HSV-1 protease, is the product of the UL26.5 gene. As it is translated from a Met codon within the UL26 gene, ICP35 cd are identical to the C-terminal 329-aa sequence of the protease and are trans cleaved at an identical C-terminal site to generate ICP35 e,f and a 25-aa peptide. Only fully processed Pra (N0 and Nb) and ICP35 (ICP35 e,f) are present in B capsids, which are believed to be precursors of mature virions. Using an R-site mutant A247S virus, we have recently shown that this mutant protease retains enzymatic activity but fails to support viral growth, suggesting that the release of N0 is required for viral replication. Here we report that another mutant protease, with an amino acid substitution (Ser to Cys) at the active site, can complement the A247S mutant but not a protease deletion mutant. Cell lines expressing the active-site mutant protease were isolated and shown to complement the A247S mutant at the levels of capsid assembly, DNA packaging, and viral growth. Therefore, the complementation between the R-site mutant and the active-site mutant reconstituted wild-type Pra function. One feature of this intragenic complementation is that following sedimentation of infected-cell lysates on sucrose gradients, both N-terminally unprocessed and processed proteases were isolated from the fractions where normal B capsids sediment, suggesting that proteolytic processing occurs inside capsids. Our results demonstrate that the HSV-1 protease has distinct functional domains and some of these functions can complement in trans.

Kaiser Colorado's Cooperative Health Care Clinic: a group approach to patient care.

A Kaiser Permanente pilot project demonstrates that managing health status with patients on a group basis versus one-on-one office visits can improve health care delivery services and reduce unnecessary resource utilization. Kaiser's Cooperative Health Care Clinic (CHCC) in Denver, Colorado, uses a multidisciplinary team to successfully provide care to elderly members with chronic conditions.

Release of the catalytic domain N(o) from the herpes simplex virus type 1 protease is required for viral growth.

The herpes simplex virus type 1 (HSV-1) protease and its substrate, ICP35, are involved in the assembly of viral capsids and required for efficient viral growth. The full-length protease (Pra) consists of 635 amino acid (aa) residues and is autoproteolytically processed at the release (R) site and the maturation (M) site, releasing the catalytic domain No (VP24), Nb (VP21), and a 25-aa peptide. To understand the biological importance of cleavage at these sites, we constructed several mutations in the cloned protease gene. Transfection assays were performed to determine the functional properties of these mutant proteins by their abilities to complement the growth of the protease deletion mutant m100. Our results indicate that (i) expression of full-length protease is not required for viral replication, since a 514-aa protease molecule lacking the M site could support viral growth; and that (ii) elimination of the R site by changing the residue Ala-247 to Ser abolished viral replication. To better understand the functions that are mediated by proteolytic processing at the R site of the protease, we engineered an HSV-1 recombinant virus containing a mutation at this site. Analysis of the mutant A247S virus demonstrated that (i) the mutant protease retained the ability to cleave at the M site and to trans process ICP35 but failed to support viral growth on Vero cells, demonstrating that release of the catalytic domain No from Pra is required for viral replication; and that (ii) only empty capsid structures were observed by electron microscopy in thin sections of A247S-infected Vero cells, indicating that viral DNA was not encapsidated. Our results demonstrate that processing of ICP35 is not sufficient to support viral replication and provide genetic evidence that the HSV-1 protease has nuclear functions other than enzymatic activity.

Sexual behaviour and risk of exposure to HIV among 18-25-year-olds in Scotland: assessing change 1988-1993.

OBJECTIVE: To study changes in sexual behaviour related to HIV transmission in young people between 1988 and 1993. DESIGN: Data collected by computer-assisted telephone interview with 4081 individuals aged 18-25 years resident in central Scotland. The data were collected on a monthly basis between 1988 and 1993 and aggregated into 11 6-month periods. The data were examined for changes relating to condom use, numbers of sexual partners and perceived risk of contracting HIV. METHODS: The statistical significance of observed changes over time in reported behaviours was assessed using a general linear modelling programme. RESULTS: Increasing numbers of young men and women reported behaviour change because of HIV, using condoms, and concern that they or someone close to them would get AIDS. There is no evidence of a reduction in the number of sexual partners reported by men or women or a change in the number of individuals perceiving that they are at risk of HIV infection. CONCLUSIONS: Significant changes in sexual behaviour related to HIV transmission are taking place among young people in the general population; most notably an increase in the reported use of condoms by both men and women.

Perceived risk of becoming infected with HIV by donating blood and changes in reported blood donation practice among the Scottish general public 1989-1992.

A total of 17,537 respondents aged 18-60 and resident in Edinburgh and Glasgow were interviewed between January 1989 and May 1992 as part of a large scale continuous monitoring survey of lifestyles and health. A computer assisted telephone interview (CATI) system was used. Respondents were chosen randomly from households with telephones. The objective was to see whether concern about the risks of becoming infected with HIV by donating blood led to a change in the blood donating habits of existing blood donors. Results showed no change in the percentage of donors, ex-donors and non-donors between 1989 and 1992, but a recent decrease in the percentage of respondents who thought that you could become infected with HIV by donating blood was observed. The percentage of new donors and ex-donors balanced each other out, but in all years respondents reporting a decreased frequency of donation outweighed those reporting an increased frequency. The belief that you can become infected with HIV by donating blood was most prevalent among non-donors followed by ex-, current and new-donors in that order. There was some evidence that the belief that you can become infected with HIV by donating blood was adversely affecting blood donation habits.

Defective processing of the insulin receptor in an endoprotease-deficient Chinese hamster cell strain is corrected by expression of mouse furin.

Characterization of an endoprotease-deficient mutant Chinese hamster ovary (CHO) cell, designated RPE.40, revealed that it bound less than 10% as much insulin as did its parent, CHO-K1. We examined processing of the endogenous insulin receptor in CHO-K1 and RPE.40 cells, and processing of the human insulin receptor expressed in these cells. RPE.40 cells did not process the endogenous insulin proreceptor to its subunit forms, and processed the human insulin proreceptor inefficiently. Accumulation of the proreceptor form of the insulin receptor was seen in both cases. Furin is a mammalian endoprotease that cleaves proproteins at a consensus sequence of basic amino acids found in the insulin proreceptor. Expression of mouse furin in RPE.40 cells restored normal processing of the endogenous and the human insulin receptor in these cells. In addition, expression of mouse furin corrected the reduced binding of insulin in RPE.40 cells, indicating that receptor function as well as processing was restored.

Conceptions and misconceptions about transmission of HIV/AIDS among the Scottish general public, 1988-1992.

Knowledge levels among the Scottish general public about so called 'casual' transmission of the AIDS virus were analysed using data collected by our Unit's survey using computer-assisted telephone interviewing methods. Five cross-sections of data from the period March to May of each year were analysed and compared to provide overall estimates of knowledge on four items relating to casual transmission between respondent groups and to provide an estimate of changes over time in knowledge levels from 1988 to 1992. The data show that significant differences in knowledge exist, with the lowest levels of knowledge found among the older and among the less educated respondents. There is evidence for a continuing increase in knowledge for all items studied and for most respondent groups, but little evidence that disparities in knowledge between respondent groups are lessening over time. Misconceptions about potential risks from donating blood and kissing persist at quite high levels.

Expression of mouse furin in a Chinese hamster cell resistant to Pseudomonas exotoxin A and viruses complements the genetic lesion.

RPE.40 is a strain of mutated CHO-K1 cells with elevated resistance to Pseudomonas exotoxin A, Sindbis virus, and Newcastle disease virus. Virus resistance is due to an inability to cleave precursor viral membrane glycoproteins and produce infectious virions. Transfection of RPE.40 cells with cDNA for mouse furin causes them to lose all resistance and become as sensitive as wild-type cells to the toxin and viruses. Transfection of RPE.40 cells with cDNA for the related yeast protease Kex2 reduces their resistance to the toxin and viruses, but does not completely eliminate it.

Influence of menstrual cycle, parity and oral contraceptive use on steroid hormone receptors in normal breast.

Steroid receptor was assessed immunohistochemically in 158 samples of normal breast for variation through the menstrual cycle. Patterns and intensity of reaction were used in a semi-quantitative scoring system to examine the influence of cycle phase, cycle type, parity and age. The changes in oestrogen receptor for natural cycle and oral contraceptive (OC) cycles indicated down-regulation by progestins. Progesterone receptor did not vary significantly in natural cycles, but increased steadily through OC cycles. This study provides strong evidence that both oestrogen and progesterone influence breast epithelium, but dissimilarities from the endometrium are apparent. The interval since pregnancy had a significant negative effect on frequency and score of oestrogen receptor and score of progesterone receptor. Multivariate analysis established the phase of cycle and OC use as independent significant influences on oestrogen receptor. The interval since pregnancy was an independent significant factor for both oestrogen and progesterone receptor presence.

PIP: Presence, distribution, and quantity of estrogen and progesterone receptors (ER, PR) were determined by immunohistochemical techniques in 158 breast tissue samples, and results scored and analyzed for age, cycle phase, and oral contraceptive use. Frozen specimens fixed by standard histologic methods were analyzed with the ER-ICA kit using rat monoclonal antibody for ER (Abbott), or the mouse monoclonal antibody against rabbit uterine PR. One section from each case was scored, counting all terminal duct lobular units (TDLU), and accounting for staining intensity, percentage of positive TDLUs, and staining pattern. Most of the sections showed mixed positive and negative areas, sometimes a sporadic pattern, and less often a ring pattern. 38% head positive ER, and 72% were positive for PR. ER scores ranged from 0-513 (median 0), and PR scores from 0-600 (median 186). ER appeared in 47% of cycling women, significantly more often on days 1-13, while there were more low and moderate scores but fewer negatives on days 14-28. 70% of cycling women and 70% of pill users had PR. 26% of the oral contraceptive users were positive for ER, with scores ranging from 9-417, significantly lower than those seen in the natural cycle. There were no significant variations in ER throughout the cycle; PR scores were significantly higher on days 14-28 of the oral contraceptive cycle. There were no effects of age, breast age, or parity on ER or PR. Among parous women, however, ER and PR were detected much less frequently in women naturally cycling and 5 years postpartum. In multivariate analysis, controlling for cycle phase, oral contraceptives significantly lowered frequency of staining, and time postpartum also lowered ER and PR staining significantly. In the discussion it was noted that the decline in ER in the 2nd half of the cycle in breast parallels that in endometrium, but PR rise in breast is in contrast to falling in endometrium in the later half of the natural or pill cycle. These data show a blunted response in numbers of steroid receptors after pregnancy, as has been reported in other indicators of breast proliferation.

Role of vesicular traffic in the transport of surface transferrin receptor to the Golgi complex in cultured human cells.

We have previously shown that transferrin receptor (TfR) recycles from the cell surface through the Golgi complex in K562 human leukemia cells. However, little is known about the transport pathway that carries these receptors to the Golgi complex. To learn more about this transport, we studied the effects of treatments that block specific types of vesicular traffic. K562 cells were cultured in test media and the transport of surface TfR to the Golgi complex was assessed by measuring the entry of asialo-TfR into the sialyltransferase compartment of the Golgi complex. Depletion of cellular potassium, which blocks formation of coated vesicles at the cell surface, stimulated asialo-TfR resialylation by 60% over controls, suggesting that coated vesicle formation is not the rate-limiting step in cell surface-to-Golgi transport. Similarly, culture in sodium-free medium, which blocks transport from endosomes to lysosomes, increased asialo-TfR resialylation by 40%, arguing that lysosomes do not lie on the transport pathway. In contrast, incubation of cells in hypertonic medium, which blocks many vesicular transport steps, inhibited TfR resialylation by 40%, confirming the importance of vesicular traffic in transport of asialo-TfR from the cell surface to the Golgi complex. These results are consistent with two possible pathways for cell surface-to-Golgi transport. Receptor could be transported via an endosomal intermediate, with the rate-limiting step occurring at a post-endosomal site. Alternatively, receptor could be transported directly to the Golgi via a pathway that does not involve endosomes.