Gene therapy of erectile dysfunction in the rat with penile neuronal nitric oxide synthase

Gene transfer to the penile corpora cavernosa of constructs of the inducible and endothelial nitric oxide synthase (NOS) cDNAs ameliorates erectile dysfunction in aged rats. In this study, we investigated whether the neuronal NOS (nNOS) variant responsible for erection, penile nNOS (PnNOS), can exert a similar effect, and whether the combination of electroporation with a helper-dependent adenovirus (AdV) improves gene transfer. PnNOS and beta-galactosidase cDNAs were cloned in plasmid (pCMV-PnNOS; pCMV-beta-gal) and "gutless" AdV (AdV-CMV-PnNOS; AdV-CMV-beta-gal) vectors, and injected into the penis of adult (beta-gal) or aged (PnNOS) rats, with or without electroporation. Penile erection was measured at different times after PnNOS cDNA injection, by electrical field stimulation of the cavernosal nerve. The expression of beta-galactosidase or PnNOS was estimated in penile tissue by either histochemistry and luminometry or Western blot, and the effects of AdV-CMV-PnNOS on mRNA expression were examined by a DNA microarray. We found that electroporation increased pCMV-beta-gal uptake, and its expression was detectable at 56 days. In the aged rats treated with pCMV-PnNOS and electroporation, the maximal intracavernosal:mean arterial pressure ratios were elevated for 11 and 18 days when compared with those in controls. Electroporation intensified penile uptake of as few as 10(6) viral particles (vp) of AdV-CMV-beta-gal, and with 10(7) vp beta-galactosidase was still detectable at 60 days. Electroporated AdV-CMV-PnNOS (10(7) vp) was effective at 18 days in stimulating the erection of aged rats, without inducing the expression of cytotoxic genes. In conclusion, intracavernosal gene therapy with PnNOS cDNA corrected the aging-related erectile dysfunction for at least 18 days when given by electroporation in a helper-dependent AdV at low viral loads.     

Loss of caveolin-1 gene expression accelerates the development of dysplastic mammary lesions in tumor-prone transgenic mice

Caveolin-1 is the principal structural component of caveolae microdomains, which represent a subcompartment of the plasma membrane. Several independent lines of evidence support the notion that caveolin-1 functions as a suppressor of cell transformation. For example, the human CAV-1 gene maps to a suspected tumor suppressor locus (D7S522/7q31.1) that is frequently deleted in a number of carcinomas, including breast cancers. In addition, up to 16% of human breast cancers harbor a dominant-negative mutation, P132L, in the CAV-1 gene. Despite these genetic associations, the tumor suppressor role of caveolin-1 still remains controversial. To directly assess the in vivo transformation suppressor activity of the caveolin-1 gene, we interbred Cav-1 (-/-) null mice with tumor-prone transgenic mice (MMTV-PyMT) that normally develop multifocal dysplastic lesions throughout the entire mammary tree. Herein, we show that loss of caveolin-1 gene expression dramatically accelerates the development of these multifocal dysplastic mammary lesions. At 3 wk of age, loss of caveolin-1 resulted in an approximately twofold increase in the number of lesions (foci per gland; 3.3 +/- 1.0 vs. 7.0 +/- 1.2) and an approximately five- to sixfold increase in the total area occupied by these lesions. Similar results were obtained at 4 wk of age. However, complete loss of caveolin-1 was required to accelerate the appearance of these dysplastic mammary lesions, because Cav-1 (+/-) heterozygous mice did not show any increases in foci development. We also show that loss of caveolin-1 increases the extent and the histological grade of these mammary lesions and facilitates the development of papillary projections in the mammary ducts. Finally, we demonstrate that cyclin D1 expression levels are dramatically elevated in Cav-1 (-/-) null mammary lesions, consistent with the accelerated appearance and growth of these dysplastic foci. This is the first in vivo demonstration that caveolin-1 can function as a transformation suppressor gene.     

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase

We generated replacement sets for three highly conserved residues, Pro196, Pro197 and His199, that flank the catalytic nucleophile, Cys198. Pro196 and Pro197 have restricted mobility that could be important for the structural transitions known to be essential for activity. To test this hypothesis we obtained and characterized 13 amino acid substitutions for Pro196, 14 for Pro197 and 14 for His199. All of the Pro196 and Pro197 variants, except P197R, and four of the His199 variants complemented TS-deficient Escherichia coli cells, indicating they had at least 1% of wild-type activity. For all His199 mutations, k(cat)/K(m) for substrate and cofactor decreased more than 40-fold, suggesting that the conserved hydrogen bond network co-ordinated by His199 is important for catalysis. Pro196 can be substituted with small hydrophilic residues with little loss in k(cat), but 15- to 23-fold increases in K(m)(dUMP). Small hydrophobic substitutions for Pro197 were most active, and the most conservative mutant, P197A, had only a 5-fold lower k(cat)/K(m)(dUMP) than wild-type TS. Several Pro196 and Pro197 variants were temperature sensitive. The small effects of Pro196 or Pro197 mutations on enzyme kinetics suggest that the conformational restrictions encoded by the Pro-Pro sequence are largely maintained when either member of the pair is mutated.     

Carbohydrate analysis of floral nectar using medium infrared

An instrumental method based on a chemometric model of the medium region of the infrared (MIR) was developed to analyse total sugar content and the proportions of glucose, fructose and sucrose. In order to construct the model, a set of 127 standard aqueous solutions of different sugars in the concentration range 0-20% (w/v) were prepared and analysed in the interval 4900-700 cm(-1). The MIR was transformed by normalisation, correction of baseline using the second derivative, and suppression of the signals of water and carbon dioxide. The region between 1150 and 950 cm(-1) showed the highest correlation between signal and concentration. The correlation coefficient for total sugar content was 0.956, whilst those for glucose, fructose and sucrose were 0.982, 0.972 and 0.992, respectively. The method was validated using a set of 28 samples of nectar which had been assayed by chromatographic and refractometric methods. The method shows potential utility for the prediction of nectar sugar components.     

A study of factors that may influence the determination of copper, iron, and zinc in human milk during sampling and in sample individuals

The aim of this study was to establish the possible effects of the sampling protocol (between-breast, within-feed, and diurnal differences) and the mother’s personal factors (age, parity, iron supple-mentation, smoking habits, and lactation period) on the copper, iron, and zinc contents in human milk. One hundred thirty-six human milk samples identified by their origin and sampling conditions were analyzed. The samples were obtained from the 2nd to 15th d postpartum from 62 women. The data on the individuals required for the study were available. Mineral determinations were analyzed by flame atomic absorption spectrometry following a standarized protocol. The results showed that iron contents were higher in hind-milk samples and at the nighttime feeding and depended on the breast from which the sample was taken. The copper and zinc concentrations showed no significant variations. There was no significant relationship among the mothers’ age, parity, smoking habits, iron supplementation, and copper content. Milk from older women had lower zinc contents than that of younger women. Increased amounts of iron were found in multiparous women. Between colostrum and transitional milk, a sharp decrease in zinc content was observed, whereas copper and iron contents remained constant. All of these results make it clear that standardized sampling protocols are needed in order to obtain comparable values.     

Black, Jewish, and Interracial: It's Not the Color of Your Skin but the Race of Your Kin, and Other Myths of Identity

Black, Jewish, and Interracial: It's Not the Color of Your Skin but the Race of Your Kin, and Other Myths of Identity. Katya Gibel Azoulay. Durham, NC: Duke University Press, 1997.220 pp.     

Endocytosis by the corneal endothelium. I. Regulation of binding and transport of hemeproteins and peroxidase-conjugated lectins across the tissue

 Binding, internalization, and movement of hemeproteins and peroxidase-conjugated lectins across organ cultured rat corneal endothelia has been investigated. Horseradish peroxidase (HRP) type II, bound to the surface, was minimally internalized and was easily washed off. In contrast, HRP-VI bound and was rapidly internalized. Reaction product was observed in vesicles, endosomes, multivesicular bodies, and extended along the length of the intercellular space (ICS) to Descemet’s membrane. Studies at 4° C indicated HRP-VI bound uniformly along the surface in a punctate fashion. Exposure to polylysine or mannose significantly decreased uptake. Other tracers such as HRP-VIII, -IX, catalase, and microperoxidase exhibited limited uptake by the tissue. However, endothelia vigorously internalized soybean agglutinin (SBA)–HRP, and reaction product was found intracellularly and within the ICS at the cell/Descemet’s membrane interface. Internalization and the appearance of SBA–HRP within the ICS was diminished following polylysine or mannose treatment. Experiments at 4° C indicated that SBA–HRP binding and uptake were temperature sensitive. Wheat germ agglutinin (WGA)–HRP was also strongly endocytosed and reaction product was visualized within vesicles, endosomes, and multivesicular bodies. Although WGA-HRP reaction product was observed within the ICS, none was detected at the level of Descemet’s membrane. The WGA competitive sugar N-acetyl-d-glucosamine, reduced endocytosis, whereas exposure to unlabeled WGA and mannose together reduced uptake. These results indicate endothelia exhibit differential uptake of various hemeproteins and lectins which is dependent on charge, mannose receptors, and appropriate surface sugars. Accepted: 3 Mach 1998     

The taxonomic treatment of agamosperms in the genusLimonium mill. (Plumbaginaceae)

Agamospermous species account for a large proportion of the species ofLimonium. Agamospermy is indicated by uneven polyploidy or aneuploidy, low pollen stainability and by the presence of monomorphic self-incompatible populations. The taxonomic treatment of agamospermous taxa varies from recognition of all of them at the same specific rank or by utilising a range of ranks in the taxonomic hierarchy. The influence of evolutionary hypotheses on taxonomic systems is considered. Molecular data provide a means of measuring the genetical relationships of taxa and establishing groups in a taxonomic hierarchy.     

The Pleckstrin Homology and Phosphotyrosine Binding Domains of Insulin Receptor Substrate 1 Mediate Inhibition of Apoptosis by Insulin

Insulin and insulin-like growth factor 1 (IGF-1) evoke diverse biological effects through receptor-mediated tyrosine phosphorylation of insulin receptor substrate (IRS) proteins. We investigated the elements of IRS-1 signaling that inhibit apoptosis of interleukin 3 (IL-3)-deprived 32D myeloid progenitor cells. 32D cells have few insulin receptors and no IRS proteins; therefore, insulin failed to inhibit apoptosis during IL-3 withdrawal. Insulin stimulated mitogen-activated protein kinase in 32D cells expressing insulin receptors (32D^IR) but failed to activate the phosphatidylinositol 3 (PI 3)-kinase cascade or to inhibit apoptosis. By contrast, insulin stimulated the PI 3-kinase cascade, inhibited apoptosis, and promoted replication of 32D^IR cells expressing IRS-1. As expected, insulin did not stimulate PI 3-kinase in 32D^IR cells, which expressed a truncated IRS-1 protein lacking the tail of tyrosine phosphorylation sites. However, this truncated IRS-1 protein, which retained the NH₂-terminal pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains, mediated phosphorylation of PKB/akt, inhibition of apoptosis, and replication of 32D^IR cells during insulin stimulation. These results suggest that a phosphotyrosine-independent mechanism mediated by the PH and PTB domains promoted antiapoptotic and growth actions of insulin. Although PI 3-kinase was not activated, its phospholipid products were required, since LY294002 inhibited these responses. Without IRS-1, a chimeric insulin receptor containing a tail of tyrosine phosphorylation sites derived from IRS-1 activated the PI 3-kinase cascade but failed to inhibit apoptosis. Thus, phosphotyrosine-independent IRS-1-linked pathways may be critical for survival and growth of IL-3-deprived 32D cells during insulin stimulation.