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1.  A comprehensive analysis of deletions, multiplications, and copy number variations in PARK2 
Neurology  2010;75(13):1189-1194.
Objectives:
To perform a comprehensive population genetic study of PARK2. PARK2 mutations are associated with juvenile parkinsonism, Alzheimer disease, cancer, leprosy, and diabetes mellitus, yet ironically, there has been no comprehensive study of PARK2 in control subjects; and to resolve controversial association of PARK2 heterozygous mutations with Parkinson disease (PD) in a well-powered study.
Methods:
We studied 1,686 control subjects (mean age 66.1 ± 13.1 years) and 2,091 patients with PD (mean onset age 58.3 ± 12.1 years). We tested for PARK2 deletions/multiplications/copy number variations (CNV) using semiquantitative PCR and multiplex ligation-dependent probe amplification, and validated the mutations by real-time quantitative PCR. Subjects were tested for point mutations previously. Association with PD was tested as PARK2 main effect, and in combination with known PD risk factors: SNCA, MAPT, APOE, smoking, and coffee intake.
Results:
A total of 0.95% of control subjects and 0.86% of patients carried a heterozygous CNV mutation. CNV mutations found in 16 control subjects were all in exons 1–4, sparing exons that encode functionally critical protein domains. Thirteen patients had 2 CNV mutations, 5 had 1 CNV and 1 point mutation, and 18 had 1 CNV mutation. Mutations found in patients spanned exons 2–9. In whites, having 1 CNV was not associated with increased risk (odds ratio 1.05, p = 0.89) or earlier onset of PD (64.7 ± 8.6 heterozygous vs 58.5 ± 11.8 normal).
Conclusions:
This comprehensive population genetic study in control subjects fills the void for a PARK2 reference dataset. There is no compelling evidence for association of heterozygous PARK2 mutations, by themselves or in combination with known risk factors, with PD.
GLOSSARY
= autosomal recessive juvenile parkinsonism;
= confidence interval;
= copy number variation;
= moving average plots;
= multiplex ligation-dependent probe amplification;
= NeuroGenetics Research Consortium;
= odds ratio;
= Parkinson disease.
doi:10.1212/WNL.0b013e3181f4d832
PMCID: PMC3013490  PMID: 20876472
2.  Analysis of differential gene expression in colorectal cancer and stroma using fluorescence-activated cell sorting purification 
British Journal of Cancer  2009;100(9):1452-1464.
Tumour stroma gene expression in biopsy specimens may obscure the expression of tumour parenchyma, hampering the predictive power of microarrays. We aimed to assess the utility of fluorescence-activated cell sorting (FACS) for generating cell populations for gene expression analysis and to compare the gene expression of FACS-purified tumour parenchyma to that of whole tumour biopsies. Single cell suspensions were generated from colorectal tumour biopsies and tumour parenchyma was separated using FACS. Fluorescence-activated cell sorting allowed reliable estimation and purification of cell populations, generating parenchymal purity above 90%. RNA from FACS-purified and corresponding whole tumour biopsies was hybridised to Affymetrix oligonucleotide microarrays. Whole tumour and parenchymal samples demonstrated differential gene expression, with 289 genes significantly overexpressed in the whole tumour, many of which were consistent with stromal gene expression (e.g., COL6A3, COL1A2, POSTN, TIMP2). Genes characteristic of colorectal carcinoma were overexpressed in the FACS-purified cells (e.g., HOX2D and RHOB). We found FACS to be a robust method for generating samples for gene expression analysis, allowing simultaneous assessment of parenchymal and stromal compartments. Gross stromal contamination may affect the interpretation of cancer gene expression microarray experiments, with implications for hypotheses generation and the stability of expression signatures used for predicting clinical outcomes.
doi:10.1038/sj.bjc.6604931
PMCID: PMC2694425  PMID: 19401702
colorectal cancer; fluorescence-activated cell sorting; gene expression microarray analysis; humans
3.  Prophage-Like Elements in Bifidobacteria: Insights from Genomics, Transcription, Integration, Distribution, and Phylogenetic Analysis 
Applied and Environmental Microbiology  2005;71(12):8692-8705.
So far, there is only fragmentary and unconfirmed information on bacteriophages infecting the genus Bifidobacterium. In this report we analyzed three prophage-like elements that are present in the genomes of Bifidobacterium breve UCC 2003, Bifidobacterium longum NCC 2705, and Bifidobacterium longum DJO10A, designated Bbr-1, Bl-1, and Blj-1, respectively. These prophagelike elements exhibit homology with genes of double-stranded DNA bacteriophages spanning a broad phylogenetic range of host bacteria and are surprisingly closely related to bacteriophages infecting low-G+C bacteria. All three prophage-like elements are integrated in a tRNAMet gene, which appears to be reconstructed following phage integration. Analysis of the distribution of this integration site in many bifidobacterial species revealed that the attB sites are well conserved. The Blj-1 prophage is 36.9 kb long and was induced when a B. longum DJO10A culture was exposed to mitomycin C or hydrogen peroxide. The Bbr-1 prophage-like element appears to consist of a noninducible 28.5-kb chimeric DNA fragment composed of a composite mobile element inserted into prophage-like sequences, which do not appear to be widely distributed among B. breve strains. Northern blot analysis of the Bbr-1 prophage-like element showed that large parts of its genome are transcriptionally silent. Interestingly, a gene predicted to encode an extracellular beta-glucosidase carried within the Bbr-1 prophage-like element was shown to be transcribed.
doi:10.1128/AEM.71.12.8692-8705.2005
PMCID: PMC1317369  PMID: 16332864
4.  Pharmacokinetics, Serum Inhibitory and Bactericidal Activity, and Safety of Telavancin in Healthy Subjects 
The pharmacokinetics, tolerability, and serum inhibitory and bactericidal titers of telavancin, a new rapidly bactericidal lipoglycopeptide with multiple mechanisms of action against gram-positive pathogens, were assessed in a two-part, randomized, double-blind, placebo-controlled, ascending-dose study with 54 healthy men. In part 1, single ascending intravenous doses of 0.25 to 15 mg/kg of body weight were studied. In part 2, multiple ascending doses (30-min infusions of 7.5 to 15 mg/kg/day) were studied over 7 days. Following the administration of multiple doses, steady state was achieved by days 3 to 4. At day 7 after the administration of telavancin at 7.5, 12.5, and 15 mg/kg/day, peak concentrations in plasma were 96.7, 151.3, and 202.5 μg/ml, respectively, and steady-state area-under-the-curve values were 700, 1,033, and 1,165 μg · h/ml, respectively. The elimination half-life ranged from 6.9 to 9.1 h following the administration of doses ≥5 mg/kg. Most adverse events were mild in severity. At 24 h postinfusion, serum from subjects given telavancin demonstrated potent bactericidal activity against methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae strains. The results suggest that telavancin may be an effective once-daily therapy for serious bacterial infections caused by these pathogens.
doi:10.1128/AAC.49.1.195-201.2005
PMCID: PMC538848  PMID: 15616296
5.  The Fatality Assessment and Control Evaluation program's role in the prevention of occupational fatalities 
Injury Prevention  2001;7(Suppl 1):i27-i33.
Objectives—The objective of the Fatality Assessment and Control Evaluation (FACE) program is to prevent traumatic occupational fatalities in the United States by identifying and investigating work situations at high risk for injury and formulating and disseminating prevention strategies to those who can intervene in the workplace.
Setting—The FACE program is a research program located in the Division of Safety Research, a division of the National Institute for Occupational Safety and Health (NIOSH). NIOSH is an agency of the United States government and is part of the Centers for Disease Control and Prevention. NIOSH is responsible for conducting research and making recommendations for prevention of work related illnesses and injuries. FACE investigators conduct traumatic occupational fatality investigations throughout the United States and provide technical assistance to 15 state health or labor departments who have cooperative agreements with NIOSH to conduct traumatic fatality surveillance, targeted investigations, and prevention activities at the state level.
Methods—Investigations are conducted at the worksite using the FACE model, an approach derived from the research conducted by William Haddon Jr. This approach reflects the public health perspective that the etiology of injuries is multifactorial and largely preventable. FACE investigators gather information on multiple factors that may have contributed to traumatic occupational fatalities. Information on factors associated with the agent (energy exchange, for example, thermal energy, mechanical energy, electrical energy, chemical energy), host (worker who died), and the environment (the physical and social aspects of the workplace), during the pre-event, event, and post-event time phases of the fatal incident are collected and analyzed. Organizational, behavioral, and environmental factors contributing to the death are detailed and prevention recommendations formulated and disseminated to help prevent future incidents of a similar nature.
Results—Between 1982 and the present, more than 1500 fatality investigations have been conducted and reports with prevention recommendations distributed. Findings have been published in scientific and trade journals; safety professionals and policy makers have used FACE findings for prevention efforts; and working partnerships have been formed to address newly emerging safety concerns.
Conclusions—FACE investigations identify multiple factors contributing to fatal occupational injuries, which lead to the formulation and dissemination of diverse strategies for preventing deaths of a similar nature.
doi:10.1136/ip.7.suppl_1.i27
PMCID: PMC1765407  PMID: 11565967
6.  Overlapping Antisense Transcription in the Human Genome 
Accumulating evidence indicates an important role for non-coding RNA molecules in eukaryotic cell regulation. A small number of coding and non-coding overlapping antisense transcripts (OATs) in eukaryotes have been reported, some of which regulate expression of the corresponding sense transcript. The prevalence of this phenomenon is unknown, but there may be an enrichment of such transcripts at imprinted gene loci. Taking a bioinformatics approach, we systematically searched a human mRNA database (RefSeq) for complementary regions that might facilitate pairing with other transcripts. We report 56 pairs of overlapping transcripts, in which each member of the pair is transcribed from the same locus. This allows us to make an estimate of 1000 for the minimum number of such transcript pairs in the entire human genome. This is a surprisingly large number of overlapping gene pairs and, clearly, some of the overlaps may not be functionally significant. Nonetheless, this may indicate an important general role for overlapping antisense control in gene regulation. EST databases were also investigated in order to address the prevalence of cases of imprinted genes with associated non-coding overlapping, antisense transcripts. However, EST databases were found to be completely inappropriate for this purpose.
doi:10.1002/cfg.173
PMCID: PMC2447278  PMID: 18628857
7.  Development, dissemination, implementation and evaluation of a clinical pathway for oxygen therapy 
BACKGROUND: Oxygen is commonly administered to patients in hospital, but prescribing and monitoring of such therapy may be suboptimal. The objective of this study was to develop, disseminate, implement and evaluate a multidisciplinary clinical pathway for the administration of oxygen. METHODS: The authors developed a clinical pathway for the ordering, titration and discontinuation of oxygen, which was disseminated through teaching sessions, in-service training sessions and information posters in a medical clinical teaching unit (CTU). Implementation of the pathway was ensured by means of reminders and patient-centred audit and feedback to CTU nurses and house staff. During a 3-month intervention phase, consecutive patients requiring supplemental oxygen were treated according to the pathway. During a 1-month "wash-out" phase followed by a 3-month non-intervention phase, patients were treated at the discretion of the CTU team. Clinical and economic data were collected in both phases. RESULTS: In the 2 phases, patient characteristics, the concentration and duration of oxygen prescribed, the frequency of oxygen saturation monitoring, the frequency of arterial blood gas testing and the clinical outcomes were similar. However, there were more discontinuation orders in the intervention phase (p < 0.001). In the intervention phase, costs were higher for monitoring of oxygen saturation ($44.95/patient v. $36.17/patient, p = 0.048) and for order transcription ($2.71/patient v. $1.28/patient, p < 0.001); total costs, including those for personnel, were also higher in the intervention phase ($76.93/patient v. $56.67/patient, p = 0.02). The cost of education about the oxygen pathway was $45.71/patient. When the education cost was included, the total cost of oxygen therapy during the intervention phase was $122.64/patient; this was significantly higher than the total cost of oxygen therapy during the non-intervention phase ($56.67/patient) (p < 0.001). INTERPRETATION: This multidisciplinary, multimethod oxygen pathway led to changes in oxygen-prescribing behaviour, consumed more resources than standard management and was not associated with changes in patient outcome. Appropriate management of oxygen prescribing and monitoring by physicians and nurses take time and costs money.
PMCID: PMC1232226  PMID: 11216195
8.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. 
Nucleic Acids Research  1997;25(24):4876-4882.
CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for x86 PCs, and Macintosh PowerMac.
PMCID: PMC147148  PMID: 9396791
9.  RAGA: RNA sequence alignment by genetic algorithm. 
Nucleic Acids Research  1997;25(22):4570-4580.
We describe a new approach for accurately aligning two homologous RNA sequences when the secondary structure of one of them is known. To do so we developed two software packages, called RAGA and PRAGA, which use a genetic algorithm approach to optimize the alignments. RAGA is mainly an extension of SAGA, an earlier package for multiple protein sequence alignment. In PRAGA several genetic algorithms run in parallel and exchange individual solutions. This method allows us to optimize an objective function that describes the quality of a RNA pairwise alignment, taking into account both primary and secondary structure, including pseudoknots. We report results obtained using PRAGA on nine test cases of pairs of eukaryotic small subunit rRNA sequence (nuclear and mitochondrial).
PMCID: PMC147093  PMID: 9358168
10.  Developing community networks to deliver HIV prevention interventions. 
Public Health Reports  1996;111(Suppl 1):41-49.
Outreach has a long history in health and social service programs as an important method for reaching at-risk persons within their communities. One method of "outreach" is based on the recruitment of networks of community members (or "networkers") to deliver HIV prevention messages and materials in the context of their social networks and everyday lives. This paper documents the experiences of the AIDS Community Demonstration Projects in recruiting networkers to deliver HIV prevention interventions to high-risk populations, including injecting drug users not in treatment; female sex partners of injecting drug users; female sex traders; men who have sex with men but do not self-identify as gay; and youth in high-risk situations. The authors interviewed project staff and reviewed project records of the implementation of community networks in five cities. Across cities, the projects successfully recruited persons into one or more community networks to distribute small media materials, condoms, and bleach kits, and encourage risk-reduction behaviors among community members. Networkers' continuing participation was enlisted through a variety of monetary and nonmonetary incentives. While continuous recruitment of networkers was necessary due to attrition, most interventions reported maintaining a core group of networkers. In addition, the projects appeared to serve as a starting point for some networkers to become more active in other community events and issues.
PMCID: PMC1382042  PMID: 8862156
11.  Using formative research to lay the foundation for community level HIV prevention efforts: an example from the AIDS Community Demonstration Projects. 
Public Health Reports  1996;111(Suppl 1):28-35.
The AIDS Community Demonstration Projects provided community-level HIV prevention interventions to historically hard-to-reach groups at high risk for HIV infection. The projects operated under a common research protocol which encompassed formative research, intervention delivery, process evaluation, and outcome evaluation. A formative research process specifically focusing on intervention development was devised to assist project staff in identifying, prioritizing, accessing, and understanding the intervention target groups. This process was central to the creation of interventions that were acceptable and unique to the target populations. Intended to be rapid, the process took 6 months to complete. Drawn from the disciplines of anthropology, community psychology, sociology, and public health, the formative research process followed distinct steps which included (a) defining the populations at high-risk for HIV; (b) gathering information about these populations through interviews with persons who were outside of, but who had contact with, the target groups (such as staff from the health department and alcohol and drug treatment facilities, as well as persons who interacted in an informal manner with the target groups, such as clerks in neighborhood grocery stores and bartenders); (c) interviewing people with access to the target populations (gatekeepers), and conducting observations in areas where these high-risk groups were reported to gather (from previous interviews); (d) interviewing members of these groups at high risk for HIV infection or transmission; and (e) systematically integrating information throughout the process. Semistructured interview schedules were used for all data collection in this process. This standardized systematic method yielded valuable information about the focal groups in each demonstration project site. The method, if adopted by others, would assist community intervention specialists in developing interventions that are culturally appropriate and meaningful to their respective target populations.
PMCID: PMC1382040  PMID: 8862154
12.  Building a peer network for a community level HIV prevention program among injecting drug users in Denver. 
Public Health Reports  1996;111(Suppl 1):50-53.
As part of a multi-site Centers for Disease Control and Prevention-funded initiative, a community-level HIV prevention project targeting injection drug users was implemented in the FivePoints community in Denver, Colorado. The protocol for the initiative included the use of peer networks to conduct outreach and disseminate intervention materials to injecting drug users. Since April 1993, project staff established a peer network of 119 participants who distribute approximately 3,000 materials per month.
PMCID: PMC1382043  PMID: 8862157
13.  Non-gay-identifying men who have sex with men: formative research results from Seattle, Washington. 
Public Health Reports  1996;111(Suppl 1):36-40.
Non-gay-identifying men who have sex with men are at risk for human immunodeficiency virus (HIV) infection. To understand these men and to develop interventions to reduce their HIV risks, the authors interviewed staff at agencies that serve non-gay-identifying men who have sex with men, business people who interact with them, and the men themselves. Interviews were augmented with focus groups of non-gay-identifying men who have sex with men and field observations at sites identified as places where they meet to negotiate or have sex. These qualitative data suggested 73 possible groups, which were consolidated into 16 broader "sectors," and then formally ranked by level of HIV risk, ease of access to the sector, psychosocial risks, and influence of other local interventions or research activities. The authors identified six priority groups of non-gay-identifying men who have sex with men (and sites where members of these groups could be approached): hustlers, closeted men, experimenters, incarcerated or formerly incarcerated men, men of color, and heterosexually identified bisexuals. Masturbation and oral sex were reportedly common, but anal and vaginal sex were also noted; condom use was rarely reported. Risk behaviors among non-gay-identifying men who have sex with men persist for a variety of reasons and may require a variety of intervention approaches.
PMCID: PMC1382041  PMID: 8862155
14.  SAGA: sequence alignment by genetic algorithm. 
Nucleic Acids Research  1996;24(8):1515-1524.
We describe a new approach to multiple sequence alignment using genetic algorithms and an associated software package called SAGA. The method involves evolving a population of alignments in a quasi evolutionary manner and gradually improving the fitness of the population as measured by an objective function which measures multiple alignment quality. SAGA uses an automatic scheduling scheme to control the usage of 22 different operators for combining alignments or mutating them between generations. When used to optimise the well known sums of pairs objective function, SAGA performs better than some of the widely used alternative packages. This is seen with respect to the ability to achieve an optimal solution and with regard to the accuracy of alignment by comparison with reference alignments based on sequences of known tertiary structure. The general attraction of the approach is the ability to optimise any objective function that one can invent.
PMCID: PMC145823  PMID: 8628686
15.  Genetic analysis of the interaction between Vibrio cholerae transcription activator ToxR and toxT promoter DNA. 
Journal of Bacteriology  1996;178(4):1080-1087.
Expression of many virulence genes in Vibrio cholerae is under the control of the ToxT protein. These include genes whose products are required for the biogenesis of the toxin-coregulated pilus, accessory colonization factor, and cholera toxin. ToxT is a member of the AraC family of transcriptional activators and is part of the ToxR regulatory cascade. ToxR is a transmembrane DNA-binding protein that is required for transcription of toxT and also can directly activate transcription of the cholera toxin operon (ctxAB). The sequences upstream of ctxAB and toxT to which ToxR binds show no obvious similarity, which implies that ToxR may be recognizing a degenerate sequence or, alternatively, a common structural motif within both binding sites. Data presented in this report demonstrate that nucleotides within the upstream half-site of an inverted repeat element in the toxT promoter are critical for ToxR-regulated activation of transcription in V. cholerae. In addition, gene fusion and DNA-binding studies with mutant ToxR proteins indicate that residues of ToxR required for binding to the ctx promoter are also required for binding to the toxT promoter. These data suggest that ToxR is not recognizing an inverted repeat sequence per se in the activation of toxT but, rather, some motif composed in part of sequences within the upstream half-site of the inverted repeat and that ToxR recognizes similar motifs within the ctxAB and toxT promoters.
PMCID: PMC177768  PMID: 8576041
16.  AIDS Community Demonstration Projects for HIV prevention among hard-to-reach groups. 
Public Health Reports  1991;106(6):714-720.
The AIDS Community Demonstration Projects are multicenter prevention projects directing community-based interventions to members of hard-to-reach groups at risk of infection from human immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome (AIDS). The projects are supported by the Centers for Disease Control (CDC). Interventions are derived from theories of behavior change and have as their goal reducing HIV and other sexually transmitted diseases in the communities. The current objectives, intentionally narrow to improve the project's specificity and clarity, are to increase the use of condoms in sexual activity and the use of bleach to clean injecting drug equipment. Additional objectives may be added. The impact of the interventions is seen in increases in the use of HIV counseling and testing services, decreases in all or specific sexual and drug-use risk behaviors, and requests for related social and public health services. A quasi-experimental research design is being used to evaluate the projects. Multiple evaluation measures are used, including a street-based interview with randomly identified respondents in both intervention and control communities. Success in facilitating HIV and AIDS risk reduction is being measured using a model of behavior change describing stages of change. Upon successful completion of these projects in 1994, CDC may be able to offer models of effective, feasible, and easy-to-monitor State and local health departments and community-based organizations.
PMCID: PMC1580338  PMID: 1659721
17.  Molecular characterization of a second abortive phage resistance gene present in Lactococcus lactis subsp. lactis ME2. 
Journal of Bacteriology  1992;174(22):7463-7469.
The fifth phage resistance factor from the prototype phage-insensitive strain Lactococcus lactis subsp. lactis ME2 has been characterized and sequenced. The genetic determinant for Prf (phage resistance five) was subcloned from the conjugative plasmid pTN20, which also encodes a restriction and modification system. Typical of other abortive resistance mechanisms, Prf reduces the efficiency of plaquing to 10(-2) to 10(-3) and decreases the plaque size and burst size of the small isometric-headed phage p2 in L. lactis subsp. lactis LM0230. However, normal-size plaques occurred at a frequency of 10(-4) and contained mutant phages that were resistant to Prf, even after repeated propagation through a sensitive host. Prf does not prevent phage adsorption or promote restriction and modification activities, but 90% of Prf+ cells infected with phage p2 die. Thus, phage infections in Prf+ cells are aborted. Prf is effective in both L. lactis subsp. lactis and L. lactis subsp. cremoris strains against several small isometric-headed phages but not against prolate-headed phages. The Prf determinant was localized by Tn5 mutagenesis and subcloning. DNA sequencing identified a 1,056-nucleotide structural gene designated abiC. Prf+ expression was obtained when abiC was subcloned into the lactococcal expression vector pMG36e. abiC is distinct from two other lactococcal abortive phage resistance genes, abiA (Hsp+, from L. lactis subsp. lactis ME2) and abi416 (Abi+, from L. lactis subsp. lactis IL416). Unlike abiA, the action of abiC does not appear to affect DNA replication. Thus, abiC represents a second abortive system found in ME2 that acts at a different point of the phage lytic cycle.
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PMCID: PMC207445  PMID: 1429469
18.  The virulence gene activator ToxT from Vibrio cholerae is a member of the AraC family of transcriptional activators. 
Journal of Bacteriology  1992;174(21):6974-6980.
Virulence gene expression in Vibrio cholerae is postulated to involve ToxR-dependent activation of the toxT gene followed by ToxT activation of virulence genes, including several of those involved in biogenesis of the toxin-coregulated pilus. ToxR is a transmembrane, DNA-binding protein which is a member of the OmpR subclass of two-component activator systems in bacteria. Data presented in this report demonstrate that ToxT is similar to the AraC family of transcriptional activators identified in a variety of gram-negative bacteria. The toxT open reading frame begins approximately 200 nucleotides from the end of the tcpF gene, which is part of a cluster of genes responsible for production of the toxin-coregulated pilus. Accumulation of toxT specific mRNA is ToxR dependent and is modulated by environmental conditions that modulate expression of the regulon. Within the intergenic region between tcpF and toxT is a potential stem-loop structure of an unusual nature which may play a role in regulating expression of toxT mRNA. Experiments with tcpF and toxT cloned behind a strong, constitutive promoter suggest that the two genes can be cotranscribed, but Northern (RNA) blot analysis of V. cholerae suggests that if they are, steady-state levels of their messages may be controlled by a posttranscriptional mechanism. Possible mechanisms for ToxR-dependent expression of toxT are discussed.
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PMCID: PMC207377  PMID: 1400247
19.  Human immunodeficiency virus type 1 cellular host range, replication, and cytopathicity are linked to the envelope region of the viral genome. 
Journal of Virology  1990;64(8):4016-4020.
Human immunodeficiency virus type 1 (HIV-1) isolates vary in their in vitro biologic characteristics such as cellular host range, replication kinetics, and cytopathicity. In this study, we molecularly exchanged equivalent regions between two cloned HIV-1 isolates with differing replicative and cytopathic properties. To facilitate generation of recombinant viruses, we used a method involving cotransfection of human monolayer cells with plasmid constructs containing half of the biologically active viral genome. The two halves of the genome were subsequently ligated by intracellular processes to form the complete proviral genome. This method simplifies plasmid construction, since new infectious virus particles can be produced easily from the individual constructs that are correctly ligated in vivo. Results obtained by using recombinant viruses generated in this manner indicate that the ability of HIV to replicate in specific cell types and cytopathicity segregate with the env region of the viral genome.
PMCID: PMC249703  PMID: 2370688
22.  Bleeding diathesis due to decreased functional activity of type 1 plasminogen activator inhibitor. 
Journal of Clinical Investigation  1989;83(5):1747-1752.
We evaluated an elderly patient with a lifelong history of severe bleeding after surgery or trauma and with evidence of persistent hyperfibrinolysis. Routine coagulation studies were normal. Serum plasminogen (40%, normal 72-128%) and alpha 2-antiplasmin (55%, normal 70-145%) activities were decreased. Euglobulin clot lysis was abnormally shortened (50 min) and normalized in vitro with epsilon-aminocaproic acid (EACA). The patient was treated with EACA with prompt cessation of bleeding. Patient tissue-plasminogen activator (t-PA) levels in serum were normal (4.7 ng/ml, control 3.5-7.2) as detected by a two-site immunoradiometric assay (IRMA). Patient fibrinolytic inhibitor activities were assessed by incubating 125I-labeled t-PA with either whole blood or serum followed by SDS-PAGE and autoradiography to identify the resultant protease/protease inhibitor complexes. In comparison to blood samples obtained from normal donors, patient plasma and serum demonstrated reduced binding of a fast-acting plasminogen activator inhibitor to 125I-labeled t-PA. Immunoprecipitation experiments indicated diminished complex formation between type 1 plasminogen activator inhibitor (PAI-1) in patient serum and 125I-labeled t-PA. Low patient PAI-1 activity was confirmed in serum (0.36 U/ml, control 0.87-1.81; n = 3) and in platelet lysates using a functional IRMA to quantitate PAI-1 binding to immobilized t-PA. However, patient serum PAI-1 antigen was within the normal range when analyzed by IRMA (31.8 ng/ml, control 19.6-42.2); this result was confirmed in both serum and platelets by Western blot (n = 3). Mixing experiments using purified PAI-1 as well as patient and control sera did not show evidence for an inhibitor against PAI-1. We conclude that this patient's bleeding diathesis was due to hyperfibrinolysis and defective PAI-1. This patient provides the first demonstration of a link between decreased in vivo PAI-1 activity and disordered hemostasis, and supports a role for PAI-1 in control of vivo fibrinolysis.
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PMCID: PMC303885  PMID: 2496147
23.  Replication and segregational stability of Bacillus plasmid pBAA1. 
Journal of Bacteriology  1989;171(2):1166-1172.
A cryptic plasmid, pBAA1, was identified in an industrial Bacillus strain. The plasmid is 6.8 kilobases in size and is present in cells at a copy number of approximately 5 per chromosome equivalent. The plasmid has been maintained under industrial fermentation conditions without apparent selective pressure and so is assumed to be partition proficient. The minimal replicon was localized to a 1.4-kilobase fragment which also contains the functions required for copy number control. The very low level of segregational instability of the minimal replicon suggests that it also contains functions involved in plasmid maintenance. Comparison with other plasmids indicates that pBAA1 belongs to the group of small gram-positive plasmids which replicate by a rolling cycle-type mechanism. A sequence was identified which is required for the efficient conversion of the single plus strand to the double-stranded form during plasmid replication. Deletion of this sequence resulted in a low level of segregational plasmid instability.
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PMCID: PMC209715  PMID: 2492507
24.  Restriction and modification activities from Streptococcus lactis ME2 are encoded by a self-transmissible plasmid, pTN20, that forms cointegrates during mobilization of lactose-fermenting ability. 
Journal of Bacteriology  1988;170(8):3435-3442.
A self-transmissible (Tra+) plasmid encoding determinants for restriction and modification activities (R+/M+) from Streptococcus lactis ME2 was isolated and characterized. The 28-kilobase (kb) plasmid (pTN20) was detected in lactose-fermenting (Lac+) transconjugants generated from matings between S. lactis N1, and ME2 variant, and a plasmid-free recipient, S. lactis LM2301. The plaquing efficiencies of prolate- and small isometric-headed phages were reduced on transconjugants containing either pTN20 (R+/M+ Tra+) or 100-kb plasmids encoding Lac+, R+/M+, and Tra+. Lac+ transconjugants which harbored pTR1040 (Lac+) and pTN20 (R+/M+) were phenotypically R-/M- and transferred Lac+ at low frequency in subsequent matings to give rise to 100-kb R+/M+ plasmids. R+/M+ activities and high-frequency conjugal transfer ability were detected in Lac+ transconjugants that contained pTR1041 (Lac+) and pTN20 (R+/M+). No 100-kb R+/M+ plasmids were recovered after these matings, suggesting that pTR1041 was mobilized by pTN20 through a process that resembled plasmid donation. pTR1041 was identical to pTR1040 but contained an additional 3.3-kb DNA fragment. These data suggested that phenotypic expression of R+/M+ and Tra+ is affected by coresident Lac+ plasmids. Restriction enzyme analysis and hybridization reactions demonstrated that the 100-kb R+/M+ plasmid was formed by a cointegration event between pTR1040 (Lac+) and pTN20 (R+/M+ Tra+) during conjugal transfer via a conductive-type process. This is the first report that defines self-transmissible restriction and modification plasmids in the lactic streptococci.
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PMCID: PMC211312  PMID: 2841286
25.  The cerebellum and initiation of movement: the stretch reflex. 
Studies of the stretch reflex in decerebrate cats indicate a phase advance of peak sinusoidal tension in steady-state cycles between 0.1 and 10 Hz. This phase advance is reduced in acute and chronic cerebellectomy, as shown in previous investigations. Also, the augmentation of muscle peak tension in initial sinusoidal stretch cycles at 0.5-5 Hz has been found to be reduced during the time of reflex and motor instability in the several months following cerebellar ablation. This report shows the increased amplitude and phase lead of integrated electromyographic activity in initiating sinusoidal stretch cycles in the decerebrate cat. These reflex aspects are demonstrated in relation to the discharge of neurons in the dorsal spinocerebellar tract and of cerebellar cortical Purkinje cells in initial sinusoidal cycles. The intensity and phase advance of the discharge in dorsal spinocerebellar tract neurons is altered little, but these features are usually increased in Purkinje cells during initial stretches compared to continuous cycling. In terms of overall motor control, these findings are compatible with concepts of movement control, modulated by the cerebellum, in which the discharge of antagonist motor neurons is regulated in concert with that of agonist muscles upon initiation and termination of movement.
PMCID: PMC2590329  PMID: 3577209

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