Functional complementation by electroporation of human BACs into mammalian fibroblast cells

Bacterial Artificial Chromosomes (BACs) have been used to complement a metabolic defect and to transfer a drug resistance marker into mammalian cells by electroporation. The selectable markers are stable and the recipient cells have BAC DNA integrated into the chromosomes as shown by fluorescent in situ hybridization, PCR and Southern hybridization.     

Immunoreactivity of neural crest-derived cells in thymic tissue developing under the rat kidney capsule

In order to study the functional development of a thymus in an experimental model, small pieces of adult rat thymic tissue were cultured for 9 days and implanted under the kidney capsule of littermates. The tissues were examined with a panel of antibodies raised against thymic and neural factors and neural crest cells at intervals from 5 to 13 days. At 5 days post-implantation, there were groups of L1+ cells within the implants that reacted with antibodies raised against neural and neural crest cell markers. L1+ cells were highly mitotic, rounded cells measuring 8.7 +/- 0.6 micrometer in diameter. Double immunostaining with different combinations of antibodies showed that 94% of the L1+ cells were also TH+, and many were HNK-1/NCAM+, PGP 9.5+, NGF+, chromogranin A+, VIP+, S100+, CGRP+, GAD+, and A2B5+. A few were also pan-cytokeratin+. These results indicate that these cells are derived from neural crest derived cells and belong to the neuroepithelial line of development. The L1+ cells were most numerous before nerves appeared (about Day 9) and reduced in number and extent as the thymus differentiated. The neural crest cells occasionally had long cytoplasmic extensions, but it was not possible to decide if they formed the nerves that appeared in the implants. Adult thymuses also contained a population of L1+ and HNK-1/NCAM+ cells, mainly in the subcapsular cortex, the septa, and the medulla. These cells could be a source of neural crest cells able to repopulate the implant. The adult thymus may always contain a reservoir of cells potentially capable of producing neuropeptides and transmitter factors required for thymic growth and regeneration.     

Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition

PURPOSE: In a rabbit model, transposition of a muscle pedicle flap to an ischemic hind limb has been shown to result in the development of new blood vessels that connect the arterial circulation of the flap to the circulation of the limb. The hypothesis that exogenous recombinant basic fibroblast growth factor (bFGF) would enhance the development of this new blood supply was examined and the regulation of bFGF in this process was investigated. METHODS: The right common iliac artery was ligated in 12 male New Zealand white rabbits. An abdominal wall muscle flap based on the left inferior epigastric artery was transposed to the right thigh. bFGF in phosphate-buffered saline (PBS) at 3 ng/h (n = 6), or PBS alone (n = 6), was infused for 7 days via mini-osmotic pumps with an infusion catheter positioned at the flap-muscle interface. The flap-muscle interface was immunostained with anti-alpha-actin antibody to determine blood vessel density (number of vessels/mm) and with anti-bFGF antibody to evaluate bFGF distribution. RNA was isolated from these sections, and polymerase chain reaction (PCR) was used to examine endogenous bFGF messenger RNA (mRNA) expression. RESULTS: Blood vessel density was significantly increased in animals receiving exogenous bFGF (22. 0 +/- 10.6 vessels/mm vs. 10.7 +/- 8.8 vessels/mm, P =.009). In the controls, neovessels were arranged in clusters with endogenous bFGF concentrated around these clusters. In bFGF-treated animals, vessels were diffusely scattered throughout the flap-limb interface, corresponding to the distribution pattern of infused bFGF. There was no difference in bFGF mRNA expression between the control and the bFGF-treated groups. CONCLUSION: Exogenous bFGF infusion significantly augmented new blood vessel development at the flap-limb interface. Endogenous bFGF was up-regulated around the newly developed microvessels in control animals, and vessel growth correlated with the diffuse distribution of exogenous bFGF, implicating bFGF as an important factor in angiogenesis. Exogenous bFGF did not affect bFGF mRNA expression, suggesting that the regulation of bFGF is not under autocrine control.     

Commentary on the new Guide to Clinical Preventive Services

Condoms are one of the oldest form of contraceptive and the best recognized form of protection against sexually transmitted diseases. Their use, however, is limited by both behavioral factors and device-related factors, including complaints about decreased sensitivity and sexual enjoyment. To address these limitations, a male condom made of polyurethane was developed. Polyurethane is a strong impermeable material with good heat transfer characteristics that is less susceptible to deterioration during storage than latex. Because little information is available comparing polyurethane and latex condoms in terms of consumer preferences as well as breakage and slippage, we reviewed four pre-marketing studies of polyurethane condoms, one of which included comparison to latex. No significant differences in slippage and breakage rates between latex and polyurethane condoms were reported in the study that included a latex comparator, and other studies of polyurethane condoms alone resulted in rates in the same range as published for latex condoms. Subjectively, consumers expressed significantly greater preference for the polyurethane condom over latex in regard to appearance, lack of smell, likelihood of slippage, comfort, sensitivity, natural look, natural feel, and overall. While additional testing is needed, these preliminary results suggest that the male polyurethane condom reviewed performed at least as well as latex condoms and is preferred by consumers. If preference translates to greater use, the male polyurethane condom may address important barriers that have been linked with inadequate condom use in the past. These results, however, may not be generalizable to other brands of polyurethane condom currently under development.     

A cytokine mRNA-destabilizing element that is structurally and functionally distinct from A+U-rich elements

The control of mRNA stability is crucial to the regulation of cytokine expression. We describe here a novel, potent destabilizing element found in the 3' untranslated region of granulocyte colony-stimulating factor mRNA. This element, which appears to require at least one stem-loop structure, we term the stem-loop destabilizing element (SLDE). Functionally equivalent elements appear to also exist in the interleukin 2 and interleukin 6 mRNAs. The SLDE is functionally distinct from the A+U-rich elements, which are also present in these and other cytokine mRNAs, because it destabilizes a chimeric mRNA in a tumor cell line in which A+U-rich elements do not function. In addition, the effect of the SLDE is insensitive to calcium ionophore and is therefore regulated independently of A+U destabilizing elements. The existence of two distinct mRNA-destabilizing elements provides an additional mechanism for the differential regulation of cytokine expression.     

Human melanocytes and keratinocytes exposed to UVB or UVA in vivo show comparable levels of thymine dimers

Epidemiology shows a relationship between solar exposure and all types of skin cancer. Understanding the mechanisms of skin cancer requires knowledge of the photomolecular events that occur within the relevant epidermal cell types in vivo. Studies to date have focused on UVR-induced DNA lesions in keratinocytes, the majority epidermal cell population which gives rise to most skin cancers. Malignant melanoma, arising from melanocytes (5%-10% of epidermal cells), accounts for most skin cancer deaths. We report on new techniques to detect DNA photolesions in human epidermal melanocytes in situ. Previously nonexposed buttock skin of volunteers of skin types I/II was exposed to clinically relevant doses of narrow bandwidth UVB (300 nm) and UVA (320 nm, 340 nm, 360 nm) radiation. Biopsies were taken immediately afterwards and processed for routine histology. Microscope sections were prepared and double-stained with fluorescent-tagged monoclonal antibodies for thymine dimers and melanocytes. UVR dose-response curves for dimer levels within melanocyte nuclei were determined by image analysis and compared with dimer levels in adjacent basal cell keratinocytes. Our data show that UVB and UVA readily induce thymine dimers in melanocytes at levels that are comparable with those found in adjacent keratinocytes. This new technique will enable melanocyte specific studies, such as DNA repair kinetics, to be done in vivo.     

The effect of various treatments in vitro and in vivo on the binding of 125I-labeled anti-rat serum albumin Fab'' to rat tissue polyribosomes

With 125I-labeled Fab' specific for rat liver serine dehydratase it has been possible to localize polyribosomes synthesizing the enzyme under several different environmental conditions. Evidence is presented to show that, following the administration of amino acids in vivo, the relative synthetic capabilities of free and membrane-bound polyribosomes synthesizing serine dehydratase vary with time. Early during the period of induction of the enzyme by administration of amino acids or by feeding a high protein diet the majority of the newly synthesized enzyme is derived from membrane-bound polyribosomes. Later in the induction process an increasing proportion of the enzyme is synthesized by the free polyribosomes. Subcellular localization studies clearly show that serine dehydratase is synthesized by both subclasses of hepatic membrane-bound polyribosomes, the loose and tight membrane-bound polyribosomes, as well as by the free polyribosomes. It was found that the membrane-bound polyribosomes are the preferential sites of synthesis of the majority of serine dehydratase molecules in the Morris hepatomas 5123C and 7800. It is concluded that the synthesis of the enzyme, serine dehydratase, in rat liver is not discretely compartmentalized in either class of free or membrane-bound polyribosomes. Rather, the relative proportions of the serine dehydratase synthesizing polyribosomes within these two classes of polyribosomes can vary depending on the metabolic and physiologic state of the liver cell.