Random mutagenesis of human estrogen receptor ligand binding domain identifies mutations that decrease sensitivity to estradiol and increase sensitivity to a diphenol indene-ol compound: basis for a regulatable expression system

We have used low fidelity polymerase chain reaction amplification to generate mutations in the human estrogen receptor ligand binding domain (LBD). Screening of libraries of mutants in yeast revealed a variety of phenotypic changes including decreased responsiveness to estradiol and increased responsiveness to synthetic compounds. Identification of the mutations responsible for these phenotypic changes indicated discrete regions of the LBD that are important for human estrogen receptor function. Cumulative rounds of mutagenesis and screening allowed us to produce a mutant estrogen receptor that was of reversed specificity as compared with the wild type LBD, in that it was more responsive to a diphenol indene-ol than to estradiol. This mutant may form the basis of a useful regulatable expression system in mammalian cells.     

A nested PCR for detection of North American isolates of bluetongue virus based on NS1 genome sequence analysis of BTV-17

A nested polymerase chain reaction (PCR)-based assay, for detection of bluetongue virus (BTV) ribonucleic acid in cell culture and tissue samples, was developed. Two pairs of oligonucleotide primers (BTV1 and BTV4 and BTV2 and BTV3), selected from non-structural protein 1 (NS1) gene of BTV-17, were used for the nested PCR in two amplification steps. First a 826-bp product was amplified using an outer primer pair BTV1 and BTV4. The second amplification, using nested or internal primer pair BTV2 and BTV3, produced a 517-bp PCR product. RNA from North American prototype serotypes 2, 10, 11, 13 and 17, propagated in cell cultures, were detected by this nested PCR-based assay. The nested primers BTV2 and BTV3 increased the sensitivity of the BTV PCR assay, and as little as 0.1 fg of BTV RNA (equivalent to 5 viral particles) could be detected. Amplification products were not detected when the PCR-based assay was applied to RNA from a closely related orbivirus, epizootic hemorrhagic disease virus (EHDV) prototype serotypes 1 and 2; total nucleic acid extracts from uninfected BHK-21 cells; or whole blood from calves and deer that were BTV-seronegative and virus isolation negative. Application of this nested BTV PCR-based assay to clinical samples resulted in detection of BTV RNA from a variety of tissues collected from calves and deer with natural and experimental BTV infections. The described BTV PCR-based assay provides a valuable tool to study the epidemiology of BTV infection in susceptible wild ruminants and domestic livestock.     

Nitric oxide-dependent production of cGMP supports the survival of rat embryonic motor neurons cultured with brain-derived neurotrophic factor

Trophic factor deprivation induces neuronal nitric oxide synthase (NOS) and apoptosis of rat embryonic motor neurons in culture. We report here that motor neurons constitutively express endothelial NOS that helps support the survival of motor neurons cultured with brain-derived neurotrophic factor (BDNF) by activating the nitric oxide-dependent soluble guanylate cyclase. Exposure of BDNF-treated motor neurons to nitro-L-arginine methyl ester (L-NAME) decreased cell survival 40-50% 24 hr after plating. Both low steady-state concentrations of exogenous nitric oxide (<0.1 microM) and cGMP analogs protected BDNF-treated motor neurons from death induced by L-NAME. Equivalent concentrations of cAMP analogs did not affect cell survival. Inhibition of nitric oxide-sensitive guanylate cyclase with 2 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced the survival of BDNF-treated motor neurons by 35%. cGMP analogs also protected from ODQ-induced motor neuron death, whereas exogenous nitric oxide did not. In all cases, cell death was prevented with caspase inhibitors. Our results suggest that nitric oxide-stimulated cGMP synthesis helps to prevent apoptosis in BDNF-treated motor neurons.     

Single-drug therapy and reduction of left ventricular mass in hypertension

Echocardiography has provided most of what is understood today about the relationships between human hypertension, cardiac anatomic and functional responses. It has proven its value in determining the effects of antihypertensive therapy on cardiac structure and function. A growing body of research supports initial concerns that not all drugs effective for blood pressure reduction are effective for reduction of left ventricular mass and regression of LVH. It has been of interest that agents initially believed to be ineffective for left ventricular mass reduction (principally diuretics and beta blockers) on the basis of pathophysiological theory and inadequate clinical trials, may in fact be quite effective for LVH regression, as well as improved cardiac outcomes. Hence, supposed inefficacy of these agents for this purpose should no longer be used as a reason to disregard long-standing recommendations of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of Hypertension supporting the use of diuretics and beta blockers for the initial pharmacotherapy of hypertension.     

Complications associated with diabetes mellitus after guided tissue regeneration: case report

Dental practitioners need to be aware of the complications that can arise in the management and treatment of patients with diabetes mellitus. Patients with diabetes, and patients with a family history of diabetes, are at-risk dental patients. They are more likely to develop periodontal disease, and the periodontitis is more likely to be severe. Diabetes influences the progression and severity of periodontitis through changes in the small blood vessels, decreased collagen formation, and impairment of the host's defense mechanisms. Furthermore, complications associated with diabetes, such as impaired wound healing, can affect the patient's response to periodontal therapies like guided tissue regeneration (GTR). The case report in this article discusses the postsurgical complications that occurred during GTR treatment of a patient with non-insulin-dependent diabetes. The diabetic's susceptibility to periodontal disease and impaired wound healing can affect the progression of the disease and its treatment. Dental patients with diabetes require close supervision and frequent monitoring of their medical and dental health by the dental clinician.     

Regulation of insulin-stimulated GLUT4 translocation by Munc18c in 3T3L1 adipocytes

Insulin stimulates glucose transporter (GLUT) 4 vesicle translocation from intracellular storage sites to the plasma membrane in 3T3L1 adipocytes through a VAMP2- and syntaxin 4-dependent mechanism. We have observed that Munc18c, a mammalian homolog of the yeast syntaxin-binding protein n-Sec1p, competed for the binding of VAMP2 to syntaxin 4. Consistent with an inhibitory function for Munc18c, expression of Munc18c, but not the related Munc18b isoform, prevented the insulin stimulation of GLUT4 and IRAP/vp165 translocation to the plasma membrane without any significant effect on GLUT1 trafficking. As expected, overexpressed Munc18c was found to co-immunoprecipitate with syntaxin 4 in the basal state. However, these complexes were found to dissociate upon insulin stimulation. Furthermore, endogenous Munc18c was predominantly localized to the plasma membrane and its distribution was not altered by insulin stimulation. Although expression of enhanced green fluorescent protein-Munc18c primarily resulted in a dispersed cytosolic distribution, co-expression with syntaxin 4 resulted in increased localization to the plasma membrane. Together, these data suggest that Munc18c inhibits the docking/fusion of GLUT4-containing vesicles by blocking the binding of VAMP2 to syntaxin 4. Insulin relieves this inhibition by inducing the dissociation of Munc18c from syntaxin 4 and by sequestering Munc18c to an alternative plasma membrane binding site.     

The evolution of the role of radiation therapy in the management of mucocutaneous malignant melanoma

The role of radiation therapy in the treatment of malignant melanoma has evolved substantially over time. Years ago, malignant melanomas were generally considered radioresistant. Over time, the palliative value of radiation therapy was established. Most recently it also has become clear that judiciously applied therapy may be curative in either an adjuvant setting or for small-volume disease.     

Development of a questionnaire for measurement of vision-related quality of life

PURPOSE: To define vision-related quality of life, to outline the development of a vision-specific quality of life instrument and to present the characteristics of a 10-item 'core' questionnaire. METHOD: A standard method included: 1. Generation of relevant issues by individual interviews with 38 visually impaired adults, consultation with 37 professionals and support workers and literature review. 2. Operationalisation, involving 58 ophthalmic patients. 3. Pre-testing, to maximise face validity and content validity, involving 184 individuals with a variety of different visual problems and social backgrounds. 4. Adoption of a modular approach to item selection. 5. Formal piloting in 92 individuals to establish reliability and construct validity. RESULTS: More than 232 items were tested of which 139 were considered suitable for a final question pool or 'parent' questionnaire (the VQOL). From this parent questionnaire individual items or groups of items can be selected. Ten broadly applicable items referring to physical, social and psychological issues were selected for the core questionnaire (the VCM1). The VCM1 has high reliability (alpha = 0.93) and validity. CONCLUSIONS: Any self-reported problem relating to vision may constitute a quality of life issue. A modular approach to item selection may provide the flexibility to investigate vision-related quality of life in a wide range of clinical settings, allowing detailed assessment of specific problems and also cross-study comparisons where appropriate.