In utero delivery of adeno-associated viral vectors: intraperitoneal gene transfer produces long-term expression.

Recombinant adeno-associated viruses (rAAV) are promising gene transfer vectors that produce long-term expression without toxicity. To investigate future approaches for in utero gene delivery, the efficacy and safety of prenatal administration of rAAV were determined. Using luciferase as a reporter, expression was assessed by whole-body imaging and by analysis of luciferase activity in tissue extracts, at the time of birth and monthly thereafter. Transgene expression was detected in all injected animals. Highest levels of luciferase activity were detected at birth in the peritoneum and liver, while the heart, brain, and lung demonstrated low-level expression. In vivo luciferase imaging revealed persistent peritoneal expression for 18 months after in utero injection and provided a sensitive whole-body assay, useful in identifying tissues for subsequent analyses. There was no detectable hepatocellular injury. Antibodies that reacted with either luciferase or rAAV were not found. AAV sequences were not detected in germ-line tissues of injected animals or in tissues of their progeny. In utero AAV-mediated gene transfer in this animal model demonstrates that novel therapeutic vectors and strategies can be rapidly tested in vivo and that rAAV may be developed to ameliorate genetic diseases with perinatal morbidity and mortality.

Highly cooperative homodimerization is a conserved property of neural POU proteins.

POU-domain proteins have been shown to play important roles in the development of the nervous, endocrine, and immune systems. However, the distinctive DNA recognition properties of the six major POU subclasses have not been well defined. Here, we have used random oligonucleotide selection and competitive binding assays to determine the optimal DNA recognition elements for the POU-III and POU-VI protein classes, represented by Brn-2 and Brn-5, respectively. The optimal Brn-5 consensus binding sequence GCATAA(T/A)TTAT strongly resembles that previously determined for the POU-IV (Brn-3) class, whereas Brn-2 exhibits highest affinity for non-octamer sites of the form ATG(A/C)AT(A/T)0-2ATTNAT and for octamer sites that contain a full associated heptamer sequence. Brn-2, Brn-3.0, and their invertebrate homologues all exhibit highly cooperative homodimerization on the Brn-2 consensus sequence, demonstrating that cooperative dimerization is a general property of these neural POU proteins. However, modified sites to which Brn-2 binds only as a monomer mediate the transcriptional effects of Brn-2 better than the consensus sequence, demonstrating that dimerization on these sites diminishes the transactivation ability of the protein. Together with the findings of our prior studies these data greatly facilitate the identification of functional POU recognition elements in the regulatory regions of neural genes.

POU domain factors of the Brn-3 class recognize functional DNA elements which are distinctive, symmetrical, and highly conserved in evolution.

To better understand the diversity of function within the POU domain class of transcriptional regulators, we have determined the optimal DNA recognition site of several proteins of the POU-IV (Brn-3) subclass by random oligonucleotide selection. The consensus recognition element derived in this study, ATAATTAAT, is clearly distinct from octamer sites described for the POU factor Oct-1. The optimal POU-IV site determined here also binds Brn-3.0 with significantly higher affinity than consensus recognition sites previously proposed for this POU subclass. The binding affinity of Brn-3.0 on its optimal site, several variants of this site, and several naturally occurring POU recognition elements is highly correlated with the activation of reporter gene expression by Brn-3.0 in transfection assays. The preferred DNA recognition site of Brn-3.0 resembles strongly the optimal sites of another mammalian POU-IV class protein, Brn-3.2, and of the Caenorhabditis elegans Brn-3.0 homolog Unc-86, demonstrating that the site-specific DNA recognition properties of these factors are highly conserved between widely divergent species.

Supplemental vitamin A prevents the acute radiation-induced defect in wound healing.

Acute radiation injury leads to thymic involution, adrenal enlargement, leukopenia, thrombocytopenia, gastrointestinal ulceration, and impaired wound healing. The authors hypothesized that supplemental vitamin A would mitigate these adverse effects in rats exposed to acute whole-body radiation. This hypothesis was based on previous experiments in their laboratory that showed that supplemental vitamin A is thymotropic for normal rodents and lessens the thymic involution, lymphopenia, and adrenal enlargement that follows stress, trauma, and neoplasia, largely obviates the impaired wound healing induced by the radiomimetic drugs streptozotocin and cyclophosphamide, lessens the systemic response (thymic involution, adrenal enlargement, leukopenia, lymphocytopenia) to local radiation, and shifts the median lethal dose (LD50/30) following whole-body radiation to the right. To test their hypothesis, dorsal skin incisions and subcutaneous implantation of polyvinyl alcohol sponges were performed in anesthetized Sprague-Dawley rats at varying times following sham radiation or varying doses of whole-body radiation (175-850 rad). In each experiment, the control diet [which contains about 18,000 IU vit. A/kg chow (3 X the NRC RDA for normal rats)] was supplemented with 150,000 IU vit. A/kg diet beginning at, before, or after sham radiation and wounding or radiation and wounding. The supplemental vitamin A prevented the impaired wound healing and lessened the weight loss, leukopenia, thrombocytopenia, thymic involution, adrenal enlargement, decrease in splenic weight, and gastric ulceration of the radiated (750-850 rad) wounded rats. This was true whether the supplemental vitamin A was begun before (2 or 4 days) or after (1-2 hours to 4 days) radiation and wounding; the supplemental vitamin A was more effective when started before or up to 2 days after radiation and wounding. The authors believe that prevention of the impaired wound healing following radiation by supplemental vitamin A is due to its enhancing the early inflammatory reaction to wounding, including increasing the number of monocytes and macrophages at the wound site; possible effect on modulating collagenase activity; effect on epithelial cell (and possible mesenchymal cell) differentiation; stimulation of immune responsiveness; and lessening of the adverse effects of radiation.

Erythropoietin and renal function in sickle-cell disease.

The relation between haemoglobin concentration, creatinine clearance, and the serum concentration of erythropoiesis-stimulating factor were assessed in 31 patients with homozygous sickle-cell disease. Haemoglobin concentrations fell significantly with decreasing creatinine clearance (r = 0.58, p less than 0.001) and were positively correlated with the concentration of erythropoiesis-stimulating factor (r = 0.65, p less than 0.001). These observations suggest that erythropoietin concentration is the factor limiting production of red cells in sickle-cell disease with renal insufficiency and have implications for treatment.

Tabagismo entre academicos de Ribeirao Preto - USP. / Smoking habit among universitary students of Ribeirao Preto University of Sao Paulo

Os autores estudam a prevalencia de fumantes entre academicos de algumas Faculdades da USP (ligadas a area de saude) do Campus de Ribeirao Preto. De um total de 1327 academicos (sendo 491 da medicina; 304 da enfermagem; 201 da farmacia e 331 da odontologia) levantaram os dados atraves de um questionario em 1146 (ou seja 86,4% da populacao) sobre o habito de fumar e caracteristicas pessoais.Concluiram que: 1. a prevalencia instantanea de fumantes nesta populacao era de 26,9% (sendo de 28,4%; 20,7%; 36,1% e 28,4% respectivamente para academicos de medicina, enfermagem, farmacia e odontologia); 2. Ha pequena diferenca entre prevalencias observadas entre homens e mulheres, (exceto na farmacia); nos primeiros anos academicos as mulheres fumam mais que os homens; 3. a maioria dos academicos fumantes iniciaram com o habito antes dos 17-18 anos de idade; 4. os academicos destas 4 areas tem um gasto anual com cigarros de cerca de Cr$ 3.735.698,40, pagando por isso um tributo ao governo de Cr$ 2.614.988,88, oferecendo um lucro aos varejistas de Cr$ 410.926,82 e contribuem para as companhias produtoras com um valor de Cr$ 709.782,70

Quantitation of hemoglobin A1a+b and hemoglobin A1c by automated "high-performance" liquid chromatography.

The glycosylated hemoglobins A1a+b and A1c have been rapidly and precisely quantitated in 5-micro L samples of human blood hemolysate (approximately 240 micrograms of hemoglobin) by cation-exchange column chromatography. Total chromatographic is 22.0 min. Proportions of Hb A1c range from 3.85 to 6.71% in normal individuals and from 4.23 to 19.90% in diabetic subjects. Within-day variation was 1.58 and 1.10% for mean Hb A1c proportions of 4.92 and 10.32%, respectively. Hb A1c and Hb A1 are stable in hemolysates stored at 4 degrees C for as long as seven days, and indefinitely under liquid nitrogen.

The effect of prostaglandin E2-induced echinocytic transformation on the potassium loss, viscosity and osmotic fragility of normal and sickle cell erythrocytes.

Prostaglandin E2 (PGE2)-induced discocyte leads to echinocytic transformation has no effect on the viscosity or osmotic fragility of normal or sickle cell erythrocytes. Membrane permeability, reflected as potassium efflux, is significantly affected in normal erythrocytes when greater than 90% of the cells are morphologically transformed to the echinocytic III stage (PGE2 concentration of 1--2x10(6) ng/ml blood). This potassium loss is significant in sickle erythrocytes when 50-70% of the cell population has been transformed (PGE2 concentration, 5x10(5) ng/ml blood). This change in membrane permeability represents one-half to one-third the flux that occurs with sickling (i.e., greater than 80% of the erythrocytes sickled).

Prostaglandin E2-hemoglobin AA and SS erythrocyte interaction (prostaglandin-erythrocyte interaction).

Prostaglandin E2 (PGE2), at concentration larger than or equal to 5 x 10(4) ng/ml, induced discocyte leads to echinocyte transformation of saline-suspended hemoglobin (Hb) AA and SS erythrocytes. This erythrocyte transformation is concentration-dependent and is reversible at room temperature after 90-120 min. The Hb SS erythrocytes treated with PGE2 did not exhibit accelerated sickling or increased formation of sickled echinocytes. Erythrocytes suspended in autologous plasma treated with PGE2, 2 X 10(6) ng/ml, did not exhibit echinocytic transformation probably because of drug binding to the plasma proteins. Other in vitro studies showed that PGE2 of concentrations of 10-500 ng/ml had no adverse effects on intact, plasma-suspended Hb SS erythrocytes. These Hb SS erythrocytes were examined for changes in morphology, potassium and calcium flux, and blood viscosity under oxygenated and hypoxic conditions.