Biomedical subjects
L Alexander
Publications and source records attributed to L Alexander.
Identification of the R1 oncogene and its protein product from the rhadinovirus of rhesus monkeys.
Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus that is most closely related to the human Kaposi's sarcoma-associated herpesvirus (KSHV). We have identified a distinct open reading frame at the left end of RRV and designated it R1. The position of the R1 gene is equivalent to that of the saimiri transforming protein (STP) of herpesvirus saimiri (HVS) and of K1 of KSHV, other members of the gamma-2 or rhadinovirus subgroup of herpesviruses. The R1 sequence revealed an open reading frame encoding a product of 423 amino acids that was predicted to contain an extracellular domain, a transmembrane domain, and a C-terminal cytoplasmic tail reflective of a type I membrane-bound protein. The predicted structural motifs of R1, including the presence of immunoreceptor tyrosine-based activation motifs, resembled those in K1 of KSHV but were distinct from those of STP. R1 sequences from four independent isolates from three different macaque species revealed 0.95 to 7.3% divergence over the 423 amino acids. Variation was located predominantly within the predicted extracellular domain. The R1 protein migrated at 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was extensively glycosylated. Tagged R1 protein was localized to the cytoplasmic and plasma membranes of transfected cells. Expression of the R1 gene in Rat-1 fibroblasts induced morphologic changes and focus formation, and injection of R1-expressing cells into nude mice induced the formation of multifocal tumors. A recombinant herpesvirus in which the STP oncogene of HVS was replaced by R1 immortalized T lymphocytes to interleukin-2-independent growth. These results indicate that R1 is an oncogene of RRV.
Induction of AIDS in rhesus monkeys by a recombinant simian immunodeficiency virus expressing nef of human immunodeficiency virus type 1.
A nef gene is present in all primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus of macaque monkeys (SIVmac). However, the nef genes of HIV-1 and SIVmac exhibit minimal sequence identity, and not all properties are shared by the two. Nef sequences of SIVmac239 were replaced by four independent nef alleles of HIV-1 in a context that was optimal for expression. The sources of the HIV-1 nef sequences included NL 4-3, a variant NL 4-3 gene derived from a recombinant-infected rhesus monkey, a patient nef allele, and a nef consensus sequence. Of 16 rhesus monkeys infected with these SHIVnef chimeras, 9 maintained high viral loads for prolonged periods, as observed with the parental SIVmac239, and 6 have died with AIDS 52 to 110 weeks postinfection. Persistent high loads were observed at similar frequencies with the four different SIV recombinants that expressed these independent HIV-1 nef alleles. Infection with other recombinant SHIVnef constructions resulted in sequence changes in infected monkeys that either created an open nef reading frame or optimized the HIV-1 nef translational context. The HIV-1 nef gene was uniformly retained in all SHIVnef-infected monkeys. These results demonstrate that HIV-1 nef can substitute for SIVmac nef in vivo to produce a pathogenic infection. However, the model suffers from an inability to consistently obtain persisting high viral loads in 100% of the infected animals, as is observed with the parental SIVmac239.
Evaluating evolutionary divergence with microsatellites.
We report the use of microsatellites (MS) to track the recent evolution of swine. Allelic frequencies for nine MS loci linked on swine chromosome 6 (SSC6) representing four western and one Chinese swine breeds were used to estimate genetic distances and times of breed divergence. A phylogenetic tree was constructed which partitioned into western and Meishan breed branches. Yorkshire and Hampshire breeds exhibited the most recent divergence with a calculated distance of 391 years. The oldest divergence, of 2,227 years, was between Meishan and Hampshire swine. Estimates of breed divergence are consistent with historical records. Additional analysis suggests that polymorphic MS linked on a single chromosome are sufficient to determine evolutionary relationships within a single species.
An audit of the management of thyroid cancer in a district general hospital.
OBJECTIVE: Thyroid cancer is the commonest endocrine malignancy yet it appeared to present infrequently to the endocrinologists at this large District General Hospital. The management of well-differentiated thyroid cancer remains controversial with a wide variation in clinical practice. The aim of this survey was to determine the characteristics of the patients diagnosed with thyroid cancer and whether any deficiencies existed in the management of subjects diagnosed with thyroid cancer over a five-year period using standards of care based upon long-term outcome data and recently published USA guidelines. DESIGN AND PATIENTS: Retrospective case-note survey of all patients newly registered with thyroid cancer from 1990 to 1994 in North Staffordshire (estimated total population 450,000). RESULTS: The annual incidence of all thyroid cancer was two per 100,000 of which well-differentiated tumours comprised 70%. Medical records were obtained in 48 new cases (91% of total) identified. Fifteen subjects who presented as surgical emergencies received only palliative treatment and had a poor outcome. Two patients presented with metastatic medullary thyroid carcinoma (3% of total). Thirty-one patients (97% of whom presented with a thyroid nodule) were referred electively to either surgical (n = 22), ENT (n = 2) or endocrinology (n = 7) outpatients with well-differentiated papillary (n = 17) and follicular (n = 14) tumours. Thirteen patients (42%) had fine-needle aspiration cytology performed preoperatively. Of the 22 tumours (71%) greater than 1.5 cm, five (27%) had a total thyroidectomy and two (9%) also had radioiodine ablation. There was inadequate serum thyrotrophin suppression postoperatively in 12 patients (39%) and only five (16%) were being monitored for recurrence with serum thyroglobulin measurements. CONCLUSIONS: Deficiencies in the optimum management of small, well-differentiated thyroid cancers were identified. Improved communication between specialties has led to the development of an agreed management protocol to increase the quality of care offered to patients with thyroid cancer and for auditing the coordinated service in the future.
Livestock variation of linked microsatellite markers in diverse swine breeds.
A panel of nine framework microsatellites (MS) linked to the Calcium Release Channel (CRC) locus on swine chromosome 6 (SSC6) was developed from the consensus genetic map. MS were screened across groups of unrelated animals from Yorkshire, Hampshire, Duroc, Landrace and Meishan swine breeds. Unique MS alleles for Yorkshire, Duroc, Landrace and Meishan breeds, and statistically significant (P < .05) associations between breeds and allele frequencies were found for each MS. Although breed marker heterozygosities ranged from 0.0 (S0035 in Duroc) to 0.92 (S0087 in Meishan), Correspondence Analysis identified MS alleles uniquely associated with either the Meishan breed, western breeds or alleles common to all breeds. Furthermore, an overall marker heterozygosity of < 0.70 demonstrates the need for multiple MS panels to accommodate reduced within-breed differences for identification of quantitative trait loci (QTL), marker assisted selection (MAS) programs or parental identification in commercial breeds.
Substitution of ras for the herpesvirus saimiri STP oncogene in lymphocyte transformation.
STP-C488 (STP of herpesvirus saimiri [HVS] group C strain 488 [C488]) is the only virus-encoded protein found to associate with cellular ras and activate ras signal transduction pathways. To investigate an important role for ras signal transduction in STP-dependent growth transformation, we constructed recombinant strains of HVS C488 in which the STP-C488 oncogene was replaced with cellular normal ras (c-ras) or viral oncogenic ras (v-ras). Recombinant HVS deltaSTP/v-ras immortalized primary common marmoset T lymphocytes to interleukin-2-independent growth as efficiently as wild-type HVS C488 (wt HVS), while recombinant HVS deltaSTP/c-ras did so with low efficiency. Whereas wt HVS immortalized CD4- CD8+ single-positive T lymphocytes, HVS deltaSTP/c-ras- and HVS deltaSTP/v-ras-immortalized cells were principally CD4+ CD8+ double-positive T lymphocytes. In addition, HVS deltaSTP/v-ras-immortalized T cells showed a high level of ras expression and exhibited an adherent macrophage-like morphology. These phenotypes were likely caused by the drastic activation of AP-1 transcriptional factor activity. Finally, HVS deltaSTP/v-ras and HVS deltaSTP/c-ras each induced lymphoma in one of two common marmosets, although onset of disease was more rapid with the v-ras virus. These results demonstrate that ras can substitute for the STP oncogene of HVS C488 to allow immortalized growth of primary lymphoid cells and that an activated form of ras does so more efficiently than the normal cellular form of ras.
Construction of a whole-genome radiation hybrid panel for high-resolution gene mapping in pigs.
We have developed a panel of 152 whole-genome radiation hybrids by fusing irradiated diploid pig lymphocytes or fibroblasts with recipient hamster permanent cells. The number and size of the porcine chromosome fragments retained in each hybrid clone were checked by fluorescence in situ hybridization with a SINE probe or by primed in situ labeling (PRINS) with SINE-specific primers. A strategy based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) was developed for selected clones to determine if the large fragments painted by the SINE probe corresponded to one pig chromosome or to different fragments of several chromosomes. This strategy was buttressed by a double PRINS approach using primers specific for alpha-satellite sequences of two different groups of swine chromosomes. Genome retention frequency was estimated for each clone by PCR with 32 markers localized on different porcine chromosomes. Of the 152 hybrids produced, 126 were selected on the basis of cytogenetic content and chromosome retention frequency to construct a radiation hybrid map of swine chromosome 8. Our initial results for this chromosome indicate that the resolution of the radiation hybrid map is 18 times higher than that obtained by linkage analysis.
A profile of sexual health behaviors among college women.
This study examined the risk taking and preventive behaviors related to sexually transmitted diseases among sexually active college women. Self-report questionnaires were distributed at two mid-Atlantic universities yielding a final sample of 556 students. Data were collected regarding frequencies of pelvic examinations, numbers of vaginal, oral, and anal sex partners, and number of partners who had forced sex against their will. Also, frequencies of tests for sexually transmitted disease and HIV before having sex with new partners, methods of protection and birth control, and types of relationships were assessed. Finally, subjects reported the types of questions asked before having sex with a new partner and diagnoses of sexually transmitted diseases. Because most college-aged women are sexually active and vulnerable to a host of short- and long-term complications from sexually transmitted infections, educational interventions, in addition to promoting condom use, must focus on the need for regular pelvic examinations, screenings for sexually transmitted disease/HIV (self and partner), and lower risk sexual activity.
Designing messages to reduce infant mortality: from talking posters to public service announcements.
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Prevalence of obesity with increased blood pressure in elementary school-aged children.
The objectives of this study were to estimate the prevalence of obesity in school-aged children in Jefferson County, Alabama; to learn when school-aged children become obese; to determine the susceptible groups; and to study the association between obesity and blood pressure. During the school year, 5,953 children, ranging in age from 5 years to 11 years, were screened for weight, height, and blood pressure, using standardized techniques. We found that obesity, defined as > or = 120% of ideal body weight for height, is prevalent in 5-year-old to 11-year-old children. The prevalence of obesity in girls at age 5 was 23% in blacks and 10% in whites, rising to 47% in blacks and 27% in whites by age 11. In boys, the prevalence of obesity at age 5 was 13% in blacks and 6% in whites, rising to 29% in blacks and 22% in whites by age 11. The prevalence of obesity is significantly greater in black than in white children and is also significantly greater in girls than boys. The systolic and diastolic blood pressures were significantly higher in obese than in non-obese children. We conclude that the significant prevalence of childhood obesity and an associated complication, increased blood pressure, emerge in school-aged children. Thus, we recommend investigations of prevention and intervention programs to be used in the school setting.
A role for natural simian immunodeficiency virus and human immunodeficiency virus type 1 nef alleles in lymphocyte activation.
A T-lymphoid cell line termed 221 was derived from a rhesus monkey infected with herpesvirus saimiri. Growth of 221 cells was dependent on the addition of interleukin-2 (IL-2) to the culture medium. In the absence of IL-2, 221 cells arrested in G0-G1 but did not die. Simian immunodeficiency virus (SIV) replicated efficiently in IL-2-stimulated 221 cells whether or not the nef gene was present. In the absence of IL-2, nef-containing SIV replicated 8 to 100 times more efficiently in 221 cells than did the same virus lacking nef. nef-containing virus preferentially stimulated the production of IL-2 from 221 cells. HIV-1 nef and v-ras genes, but not the c-ras gene, were shown to substitute functionally for SIV nef when tested as recombinant viruses in this assay system. These results demonstrate a role for natural nef in causing lymphoid cell activation, and they provide a system for delineating the biochemical mechanisms responsible for this activation.
EGFP-containing vector system that facilitates stable and transient expression assays.
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The Workers Compensation legislation.
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Internal ribosomal entry site substitution eliminates neurovirulence in intergeneric poliovirus recombinants.
Neuropathogenicity of poliovirus can be attenuated by mutations in the internal ribosomal entry site (IRES) within the 5' nontranslated region of its genome. The Sabin vaccine strains used in prevention of poliomyelitis carry such mutations in their IRES elements. In addition, mutations within the structural and nonstructural proteins of Sabin strains may equally contribute to the attenuation phenotype. Despite their effectiveness as vaccines, the Sabin strains retain a neuropathogenic potential in animal models for poliomyelitis and, at a very low rate, they can cause poliomyelitis in vaccine recipients. The elimination of the neurocytopathic phenotype was achieved through the exchange of the entire poliovirus IRES with its counterpart from human rhinovirus type 2 without affecting growth properties in nonneuronal cells. The attenuating effect of the human rhinovirus type 2 IRES within the context of a poliovirus genome has been mapped to the 3' portion of this genetic element.
An unassigned porcine microsatellite linkage group maps to chromosome 6.
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Feasibility study of the use of bilevel positive airway pressure for respiratory support in the emergency department.
STUDY OBJECTIVE: To determine the feasibility of bilevel positive airway pressure (BiPAP) support for acute respiratory distress (ARD) in the emergency department. METHODS: A convenience sample of patients in ARD as a result of any nontraumatic cause was recruited for a prospective, noncontrolled clinical trial in the ED of an urban tertiary care teaching hospital. Hemodynamically unstable patients and those requiring immediate endotracheal intubation were excluded. After an initial arterial blood gas (AGB) analysis was obtained, the patient was placed on BiPAP (Bi-PAP S/T noninvasive ventilator; Respironics, Incorporated) by nose mask or face mask to provide noninvasive pressure support at 5 cm H20. Settings were titrated to patient tolerance and satisfactory pulse oximetry. After at least 30 minutes on a stable setting, arterial blood gases were remeasured. The cause of respiratory distress, vital signs, assessment of need for intubation, arterial blood gas results, and patient disposition were recorded. Success of noninvasive support was defined as the presence of (1) improvement in ABG parameters, (2) clinical improvement and decrease in evident dyspnea, and (3) avoidance of endotracheal intubation and mechanical ventilation. RESULTS: Fifty patients were studied. Causes of ARD included acute congestive heart failure (CHF; n=16), exacerbation of chronic obstructive pulmonary disease (COPD; n=9), mixed COPD/CHF (n=3), pneumonia (n=10), status asthmaticus (n=6), and other causes of acute respiratory failure (eg, stroke, overdose; n=6). Noninvasive management was successful in 43 patients (86%), with patients in all etiologic categories being equally likely to respond favorably to therapy. All patients were admitted to the hospital, but 52.5% of those who ordinarily would have required ICU beds were admitted to lower (and less costly) levels of care. Three patients were eventually intubated, all after admission to the ICU on BiPAP. Two patients did not tolerate BiPAP, and two others were considered ED treatment failures but were not intubated because of advance directives. CONCLUSION: As has been reported from other critical care settings, use of BiPAP is feasible and has potential utility in the management of ARD in the ED.