Biomedical subjects
N Cook
Publications and source records attributed to N Cook.
Repair and evolution of nef in vivo modulates simian immunodeficiency virus virulence.
Experimental evidence from the simian immunodeficiency virus (SIV) model of AIDS has shown that the nef gene is critical in the pathogenesis of AIDS. Consequently, nef is of considerable interest in both antiviral drug and vaccine development. Preliminary findings in two rhesus macaques indicated that a deletion of only 12 bp found in the overlapping nef/3' long terminal repeat (LTR) region (9501 to 9512) of the SIVmacC8 molecular clone was associated with reduced virus isolation frequency. We show that this deletion can be repaired in vivo by a sequence duplication event and that sequence evolution continues until the predicted amino acid sequence of the repair is virtually indistinguishable from that of the virulent wild type. These changes occurred concomitantly with reversion to virulence, evidenced by a high virus isolation frequency and load, decline in anti-p27 antibody, substantial reduction in the CD4/CD8 ratio, and development of opportunistic infections associated with AIDS. These findings clearly illustrate the capacity for repair of small attenuating deletions in primate lentiviruses and also strongly suggest that the region from 9501 to 9512 in the SIV nef/3' LTR region is of biological relevance. In addition, the ability of attenuated virus to revert to virulence raises fundamental questions regarding the nature of superinfection immunity.
Detection of human papillomavirus types in balanitis xerotica obliterans and other penile conditions.
OBJECTIVES: To determine the prevalence of human papillomavirus (HPV) types 6, 11, 16 and 18 in foreskin biopsies from patients with balanitis xerotica obliterans (BXO) and other penile conditions. MATERIALS AND METHODS: Foreskin biopsy specimens from 24 patients with penile lesions and 5 control patients were analysed by type-specific polymerase chain reaction (PCR). RESULTS: HPV6 or HPV16 were not detected in patients with BXO. HPV6 was detected in 2 controls. CONCLUSIONS: Genital papillomaviruses do not have a strong association with BXO.
The internal clock: electroencephalographic evidence for oscillatory processes underlying time perception.
It has been proposed that temporal perception and performance depend on a biological source of temporal information. A model for a temporal oscillator put forward by Treisman, Faulkner, Naish, and Brogan (1990) predicted that if intense sensory pulses (such as auditory clicks) were presented to subjects at suitable rates they would perturb the frequency at which the resulting pattern of interference between sensory pulse rates and time judgments would depend on the frequency of the temporal oscillator and so might allow the frequency to be estimated. Such interference patterns were found using auditory clicks and visual flicker (Treisman & Brogan, 1992; Treisman et al., 1990). The present study examines time estimation together with the simultaneously recorded electroencephalogram to examine whether evidence of such an interference pattern can be found in the EEG. Alternative models for the organization of a temporal system consisting of an oscillator or multiple oscillators are considered and predictions derived from them relating to the EEG. An experiment was run in which time intervals were presented for estimation, auditory clicks being given during those intervals, and the EEG was recorded concurrently. Analyses of the EEG revealed interactions between auditory click rates and certain EEG components which parallel the interference patterns previously found. The overall pattern of EEG results is interpreted as favouring a model for the organization of the temporal system in which sets of click-sensitive oscillators spaced at intervals of about 12.8 Hz contribute to the EEG spectrum. These are taken to represent a series of harmonically spaced distributions of oscillators involved in time-keeping.
Comparison of the polymerase chain reaction and Southern blot analysis in detecting and typing human papilloma virus deoxyribonucleic acid in tumors of the lower female genital tract.
To conduct studies on the clinical and pathologic significance of human papilloma virus (HPV) in genital malignancies, accurate detection and typing of the virus in clinical material are essential. Currently, Southern blotting and the polymerase chain reaction (PCR) are two of the most commonly used methods to identify HPV. This study was undertaken to compare these techniques in the detection and typing of HPV in 242 invasive malignancies of the lower female genital tract. BamHI and PstI restriction digests of tumor DNA were hybridized to 32P-labeled probes for HPV types 6, 16, and 18 at TM -20 degrees C after Southern transfer. Blots were then washed at Tm -20 degrees C and Tm -9 degrees C. The DNA was also amplified by PCR using both highly conserved consensus L1 primers that detect 25 different HPV genotypes and primers specific for HPV 6 E6, 16 E7, and 18 E6. All PCR products were hybridized to type-specific radiolabeled probes. In 202 of the 242 (83%) samples, HPV was detected, including 189 of 218 (87%) cervical cancers, 11 of the 20 (55%) vulvar cancers, and two of four tumors from the vagina, urethra, or anus. In 67% of the specimens, there was agreement between the Southern blot technique and both methods of PCR (consensus and type-specific primers), including 121 of the 202 HPV-positive specimens and 40 HPV-negative specimens. Of the 141 tumors with HPV detected by Southern blot analysis, the same HPV type was detected by PCR in 121 (86%).(ABSTRACT TRUNCATED AT 250 WORDS)
Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants.
The proviral genome of the 32H reisolate of simian immunodeficiency of macaques (SIVmac32H) has been cloned and sequenced. Including both long terminal repeats, it is 10277 base pairs in length and contains open reading frames for all known SIV genes (gag, pol, vif, vpx, vpr, tat, rev, env and nef). This is the first report of an infectious SIVmac molecular clone which contains no premature termination codons. Three molecular clones of SIVmac32H have been constructed differing in sequence only within their last 1.2 kb. Two of the molecular clones, SIVmac32H(pJ5) and SIVmac32H (pC8), differ in the nef coding region by an in-frame deletion of four amino acids in pC8 and two conservative amino acid changes; other nucleotide changes in the 3' LTR were not associated with known functionally critical motifs. The third clone, SIVmac32H(pB1), contains the last 1.2 kb of the SIVmac251 clone pBK28. The biological properties of virus produced after electroporation of these clones into C8166 cells has been assessed by infection of rhesus and cynomolgus macaques, time to seroconversion and by induction of cytopathic effects upon co-cultivation of infected rhesus peripheral blood lymphocytes with C8166 cells. The viruses obtained from these clones have identical growth kinetics in vitro but differ in their ability to persist in macaques. Macaques infected with pJ5 derived virus remain viraemic longer than macaques infected with pC8-derived virus. PCR analysis of circulating provirus indicates that the nef gene evolved over time in pJ5 virus-infected macaques, whereas late in infection in pC8 virus-infected macaques the nef gene remained invariant in sequence. These results support the observation that a nef deletion mutant of SIVmac239 lost its pathogenic potential and resulted in low-level viraemia when rhesus macaques were infected. Virus challenge pools for vaccine studies have been prepared for pJ5 using both human and monkey cell substrates and these stocks have been titrated both in vitro and in vivo. Virus has also been prepared from pC8 and titrated in vitro. This virus pool is being assessed as an attenuated live-virus vaccine in macaques. Since only virus originating from the SIVmac239 molecular clone is known to cause AIDS-like symptoms in rhesus macaques consistently, the SIVmac32H molecular clones should tell us more about which viral sequence features are important for the pathogenesis of AIDS.
Anatomic subtype and survival after reconstructive operation for hypoplastic left heart syndrome.
We conducted a retrospective study of 78 patients who underwent palliative reconstructive operation for hypoplastic left heart syndrome representing an entire consecutive experience between 1983 and 1991 to identify predictors of mortality that might enable more appropriate triage of patients to either reconstruction or transplantation. Twenty-nine patients had aortic atresia, mitral atresia; 18 had aortic stenosis, mitral stenosis; 20 had aortic atresia and mitral stenosis; and 11 had miscellaneous forms of hypoplastic left heart syndrome. There were 29 hospital deaths (37%). A worst preoperative pH (p = 0.01) and immediate preoperative pH (p = 0.03) less than the median were predictors of hospital mortality. The anatomic subgroup aortic atresia, mitral stenosis (p = 0.06) had a possible increased hospital mortality. One patient was lost to follow-up. The Kaplan-Meier survival estimate among hospital survivors was 34% at 3 years and 25% at 5 years. The anatomic subgroup aortic atresia, mitral atresia (p = 0.02) had a worse late outcome (11% 3-year survival) whereas the subgroup aortic stenosis, mitral stenosis (p = 0.04; 76% 3-year survival) had a better late outcome. There were no other significant predictors of late survival other than immediate prerepair pH (p = 0.05). Interpretation of this experience is complicated by the large number of different surgical techniques used for both first-stage neonatal reconstruction and the Fontan procedure plus introduction of the bidirectional Glenn shunt as an intermediate step midway through the experience. Nevertheless in this time frame and with the variety of techniques used, this experience demonstrates that patients with aortic atresia, mitral atresia, particularly those who have been very acidotic in the neonatal period, are least likely to do well with the reconstructive approach to hypoplastic left heart syndrome and are the most appropriate subgroup to be directed to transplantation. Patients with aortic stenosis, mitral stenosis have an excellent late outcome with the reconstructive approach.
Human papillomavirus type 6 in squamous cell carcinoma of the bladder and cervix.
Human papillomavirus (HPV) types 16 and 18 are the most frequent genotypes identified in genital malignancies, while HPV types 6 and 11 are found predominantly in condylomas and low-grade dysplasias. It is thought that HPV types 16 and 18 represent high-risk genotypes, while HPV types 6 and 11 rarely, if ever, participate in the development of malignant tumors. In a series of over 300 invasive tumors of the lower genital tract analyzed for the presence of HPV three have been found to contain HPV type 6 DNA: two invasive squamous cell carcinomas of the cervix and one squamous cell carcinoma of the bladder. Human papillomavirus type 6 was the only HPV type detected in these tumor DNAs by Southern blot hybridization and by the polymerase chain reaction using both consensus and type-specific primers. In situ hybridization using whole genomic RNA probes localized viral DNA to tumor cells. Although extensive virologic and epidemiologic studies conducted in the last decade indicate that HPV types 16 and 18 are more likely to be associated with high-grade dysplasias and invasive cancer, HPV type 6 may not be as innocuous as previously supposed.
Studies on the specificity of the vaccine effect elicited by inactivated simian immunodeficiency virus.
Inactivated, partially purified simian immunodeficiency virus (SIVmac) protected macaques from intravenous challenge with homologous and heterologous strains of SIV that had been grown on human cells but no protection against challenge with monkey peripheral blood mononuclear cell-grown SIVmac was afforded. Human immunodeficiency virus type 1 prepared in an analogous way to the SIVmac vaccine on the C8166 human T cell line protected macaques against challenge with human cell-grown SIVmac. These results suggest that protection may be mediated by xenoimmunization with the vaccine cell substrate proteins. All vaccinated macaques had anti-cell antibodies. Major reactivity to MHC class I antigens was found as well as to a 70-kD protein detectable only under nonreducing conditions.
Comparison of serum antibody reactivities to a conformational and to linear antigenic sites in the external envelope glycoprotein of simian immunodeficiency virus (SIVmac) induced by infection and vaccination.
Forty-six overlapping peptides (20-mers) representing the amino acid sequence of the external envelope glycoprotein of simian immunodeficiency virus (SIVmac; 32H isolate) were used to investigate linear antigenic sites recognized by antibodies in sera from SIV-infected rhesus macaques and in animals vaccinated with formalin-inactivated SIV. The reactivity to a discontinuous antigenic site as defined by a neutralizing monoclonal antibody was measured by competition assay. The majority of infected macaques recognized three linear antigenic determinants within the V1, V3 and C5 regions of the external glycoprotein. Animals infected with virus derived from the molecular clone SIVmac 32H (pJ5) showed broader reactivity to peptides with half of these animals having antibodies to the V2 region in addition to the V1, V3 and C5 regions. The majority of animals produced antibodies in response to the discontinuous epitope although these responses were weaker in animals infected with molecularly cloned virus. Seven of eight animals given vaccine in syntex adjuvant formulation (saf-1) produced antibodies in response to the discontinuous epitope and all reacted with peptides from the V1, V2 and V3 regions but only half recognized the C5 region. Animals receiving vaccine in alum adjuvant generally showed weaker responses to the discontinuous and linear determinants than those receiving saf-1 adjuvanted vaccine.
Construction and detection of bioluminescent strains of Bacillus subtilis.
Bioluminescence (lux) genes from Vibrio fischeri and V. harveyi were introduced into Bacillus subtilis on a plasmid vector and by chromosomal integration. The plasmid-bearing strain was highly luminescent and stable under antibiotic selection, but luminescence was lost in the absence of selection and following sporulation and germination. The chromosomally marked strains emitted less light but were found to be stable without the requirement for antibiotic selection and following sporulation and germination. Individual luminescing colonies of both B. subtilis strains could be detected against a high background of non-bioluminescent indigenous soil microbial colonies on agar plates using a charge-coupled device camera. These bioluminescent Gram-positive strains could be of value in studies concerning the survival and spread of genetically-modified micro-organisms in soil environments.
'Meaningful' patterns in visual noise: effects of lateral stimulation and the observer's belief in ESP.
Visual noise subjectively contains more meaningful patterns (1) when tachistoscopically presented to the left visual field, and (2) for persons who believe in extrasensory perception (ESP). These results indicate a possible right hemisphere mediation of delusional perception and suggest some delusional component in the belief in ESP.
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Role of the cyclic AMP response element in rat fibronectin gene expression.
Fibronectin expression is of considerable importance in normal and fibrotic liver. Plasma fibronectin levels are correlated with good prognosis in liver failure, and cellular fibronectin plays a crucial role in fibrogenesis. In this study, we observed that the H4II rat hepatoma cell line does not express fibronectin. Furthermore, a recombinant vector (pFGH) containing the promoter elements of the fibronectin gene showed no promoter function when transfected into this cell line. However, pFGH was actively expressed in L-cells and rat skin fibroblasts, cell types that express large amounts of endogenous fibronectin. To study the mechanisms regulating fibronectin expression, we evaluated the transcriptional regulatory elements of the rat fibronectin gene by mutational analysis and DNA-protein binding studies. Deletional mutation analysis showed that the sequences between positions -164 and -90 are essential for promoter activity. This region contains the consensus binding sites for CCAAT and the cyclic AMP-responsive element. Gel retardation assays demonstrated that although the binding activity to the CCAAT site at -140 was essentially the same as that in extracts from L-cells, hepatoma cells and rat livers, substantially greater amounts and different patterns of binding to the adjacent cyclic AMP-responsive element were observed in the extracts from the expressing L-cells and rat livers compared with those in the nonexpressing hepatoma cell nuclear extracts. Furthermore, mutagenesis of the cyclic AMP-responsive element site dramatically reduced promoter activity in transient transfection assays. The cyclic AMP-responsive element at position -160 appears to play an important role in the constitutive expression of the rat fibronectin gene.
Intrarectal challenge of macaques vaccinated with formalin-inactivated simian immunodeficiency virus.
Macaques can be protected from intravenous infection with simian immunodeficiency virus (SIV) by vaccination with chemically inactivated virus. However, protection against infection via a mucosal surface has not been demonstrated. We vaccinated four rhesus macaques with formalin-inactivated SIV given intramuscularly. These monkeys, which had remained virus free for 10 months after intravenous challenge with SIV, were given a further dose of vaccine and together with four unvaccinated controls were challenged intrarectally with SIV. Subsequently, virus was isolated from all control animals on five successive occasions, but the vaccinated animals remained free of virus. Proviral DNA could not be detected in peripheral blood mononuclear cells from the vaccinated animals. Preliminary data indicate that vaccinated animals make a local antibody response.
Transmission studies with simian immunodeficiency virus of macaques; persistent infection of baboons.
The host range of SIVmac was investigated in three monkey species. Blood-borne and cell-adapted virus inocula obtained from a rhesus macaque infected with SIVmac251 were compared. African green monkeys were not susceptible to infection, whereas baboons and rhesus macaques became persistently infected and showed similar patterns of seroconversion. However, in contrast to the macaques, no clinical or histopathological evidence of disease was seen in the baboons 2 years after virus inoculation. Thus baboons could be used as an alternative to macaques in vaccine development studies with this particular isolate of SIVmac. Furthermore, this system may be useful for the investigation of factors responsible for disease progression.
Salivary testosterone levels and major depressive illness in men.
Studies of testosterone levels in patients with major depressive illness have produced varying results, plasma levels usually being reported. Saliva levels reflect 'free' plasma testosterone, and a pilot study of 11 men with major depression with melancholia, who underwent a standard dexamethasone suppression test, is described. There were no significant differences in testosterone levels compared with an age-matched control group. In the patient group, pre-dexamethasone levels correlated significantly and negatively with depression ratings on the 21-item Hamilton and the Montgomery and Asberg depression scales, and also with state anxiety measured on the Spielberger scale.
Interferon-gamma stimulates the survival and influences the development of bipotential granulocyte-macrophage colony-forming cells.
The effects of interferon-gamma (IFN-gamma) on a highly enriched population of granulocyte-macrophage colony-forming cells (GM-CFC) were assessed. When added with myeloid growth factors (interleukin-3 [IL-3], granulocyte-macrophage colony-stimulating factor [GM-CSF], or macrophage-CSF [M-CSF]), IFN-gamma inhibited the formation of colonies in soft agar assays. Furthermore IFN-gamma stimulated an increase in the number of macrophages present in colonies formed in the presence of IL-3. IFN-gamma also inhibited M-CSF-, GM-CSF-, or IL-3-stimulated [3H]-thymidine incorporation in highly enriched GM-CFC. However, when added in the absence of hematopoietic growth factors, IFN-gamma promoted the survival of GM-CFC and had a modest stimulatory effect on DNA synthesis. The direct interaction of the IFN with GM-CFC was confirmed by showing its ability to rapidly activate the sodium/hydrogen antiport in GM-CFC, as do the mitogens GM-CSF, M-CSF, and IL-3. However, the effect of IFN-gamma on intracellular pH and DNA synthesis was transient and pretreatment with IFN markedly inhibited the ability of GM-CSF, M-CSF, and IL-3 to activate the sodium/hydrogen antiport. IFN-gamma has a dual effect on GM-CFC, decreasing the rate of cell death but also limiting the proliferative response to CSFs.