Citations

Atherogenesis is enhanced in arterial segments exposed to disturbed blood flow, indicating the active participation of the hemodynamic environment in lesion formation. Turbulent shear stress selectively regulates responsive genes in the endothelium and increases the damage induced by free radicals. The purpose of the present study was to evaluate the effects of intervention with antioxidants and l -arginine on endothelial NO synthase (eNOS) and oxidation-sensitive gene perturbation induced by disturbed flow in vitro and in vivo . Both human endothelial cells exposed to shear stress and high atherosclerosis-prone areas of hypercholesterolemic low-density lipoprotein receptor knockout (LDLR −/− ) mice showed increased activities of redox-transcription factors (ELK-1, p -Jun, and p -CREB) and decreased expression of eNOS. Intervention with antioxidants and l -arginine reduced the activation of redox-transcription factors and increased eNOS expression in cells and in vivo . These results demonstrate that atherogenic effects induced by turbulent shear stress can be prevented by cotreatment with antioxidants and l -arginine. The therapeutic possibility to modulate shear stress-response genes may have important implications for the prevention of atherosclerosis and its clinical manifestations.

The effect of aging on long bone mechanical properties and bone formative capacity was characterized in the male Fisher 344 rat. The femurs of rats from three age groups (4 mo., 12 mo. and 28 mo.) were tested in three-point bending to determine their structural properties. The apparent material properties were then calculated by adjusting for bone geometry. Bone formation was assessed by dynamic histomorphometry of both cortical and cancellous bone as well as by Northern blot analysis for the expression of the osteoblast phenotypic proteins osteopontin (OP), osteocalcin (OC), type I collagen (COL) and alkaline phosphatase (AP). Aging resulted in a decline in the apparent material properties that was associated with a compensatory alteration of bone geometry that preserved structural strength and stiffness. Histomorphometric analysis revealed significant age-related decreases in cancellous bone volume, trabecular number and increased trabecular separation suggesting the existence of senile osteopenia in the proximal tibia of the male Fisher 344 rat. A significant decline in bone formation rate (BFR), but not mineral apposition rate, suggests that a reduction in osteoblast number, but not osteoblast activity, contributes to age-related bone loss. The decline in BFR with aging was reflected in a decreased mRNA expression for OP, OC and COL but not AP. Further, the pattern of mRNA expression was consistent with reduced osteoblast differentiation with aging. The present study indicates the age-related decline in material properties of long bones is paralleled by a decrease in osteogenesis.

In contrast to oncoretroviruses, lentiviruses such as human immunodeficiency virus 1 (HIV-1) are able to integrate their genetic material into the genome of nonproliferating cells that are metabolically active. Likewise, vectors derived from HIV-1 can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T cells. Completion of reverse transcription, nuclear import, and subsequent integration of the lentivirus genome do not occur in these cells unless they are activated via the T-cell receptor (TCR) or by cytokines or both. However, to preserve the functional properties of these important gene therapy target cells, only minimal activation with cytokines or TCR-specific antibodies should be performed during gene transfer. Here we report the characterization of HIV-1–derived lentiviral vectors whose virion surface was genetically engineered to display a T cell-activating single-chain antibody polypeptide derived from the anti-CD3 OKT3 monoclonal antibody. Interaction of OKT3 IgGs with the TCR can activate resting peripheral blood lymphocytes (PBLs) by promoting the transition from G0 to G1 phases of the cell cycle. Compared to unmodified HIV-1–based vectors, OKT3-displaying lentiviral vectors strongly increased gene delivery in freshly isolated PBLs by up to 100-fold. Up to 48% transduction could be obtained without addition of PBL activation stimuli during infection. Taken together, these results show that surface-engineered lentiviral vectors significantly improve transduction of primary lymphocytes by activating the target cells. Moreover these results provide a proof of concept for an approach that may have utility in various gene transfer applications, including in vivo gene delivery.

Background and Purpose — Post-subarachnoid hemorrhage (SAH) cerebral vasospasm is a potentially devastating condition whose pathogenesis involves impaired nitric oxide (NO) bioavailability. We aimed to determine whether recombinant endothelial NO synthase (eNOS) gene expression may protect vasomotor function and prevent vasospasm in a canine experimental SAH model. Methods — Recombinant adenoviral vectors (5×10 9 plaque-forming units/animal) encoding genes for eNOS (AdeNOS) and β-galactosidase (AdLacZ) or vehicle were injected into the cerebrospinal fluid (CSF) of dogs on day −1 (ie, 24 hours before the first intra-CSF injection of blood on day 0). Cerebral angiography was performed at day 0 (baseline) and day 7 (immediately before death), and tissues were harvested for additional studies. Results — Western analysis and immunohistochemistry detected recombinant eNOS exclusively in cerebral arteries isolated from AdeNOS-transduced dogs, and in this group of animals CSF NO concentrations were significantly elevated by day 2. Analysis of day 7 versus day 0 cerebral angiograms for each group revealed significant spasm at the basilar artery midpoint in AdLacZ-transduced and nontransduced dogs but not in AdeNOS-transduced dogs. Isometric force recording of basilar arteries isolated from AdeNOS-transduced dogs showed significantly augmented relaxations to bradykinin and reduced contractions to endothelin-1. Conclusions — Our results suggest that expression of recombinant eNOS in the adventitia of cerebral arteries may contribute toward protection against post-SAH vasospasm.

ABSTRACT We have previously identified, in Paenibacillus popilliae , a 708-bp sequence which has homology to the sequence of the enterococcal vanA gene. We have performed further studies revealing five genes encoding homologues of VanY, VanZ, VanH, VanA, and VanX in P. popilliae . The predicted amino acid sequences are similar to those in VanA vancomycin-resistant enterococci: 61% identity for VanY, 21% for VanZ, 74% for VanH, 77% for VanA, and 79% for VanX. The genes in P. popilliae may have been a precursor to or have had ancestral genes in common with vancomycin resistance genes in enterococci. The use of P. popilliae biopesticidal preparations in agricultural practice may have an impact on bacterial resistance in human pathogens.

ABSTRACTPneumocystis cariniiremains an important opportunistic fungal pathogen causing life-threatening pneumonia in patients with AIDS and malignancy. Currently, little is known about how the organism adapts to environmental stresses and maintains its cellular integrity. We recently discovered an open reading frame approximately 600 bp downstream of the region codingGSC-1, a gene mediating β-glucan cell wall synthesis inP. carinii. The predicted amino acid sequence of this new gene, termedP. carinii PHR1,exhibited 38% homology toSaccharomyces cerevisiae GAS1, a glycosylphosphatidylinositol-anchored protein essential to maintaining cell wall integrity, and 37% homology toCandida albicans PHR1/PHR2, pH-responsive genes encoding proteins recently implicated in cross-linking β-1,3- and β-1,6-glucans. In view of its homology to these related fungal genes, the pH-dependent expression ofP. carinii PHR1was examined. As inC. albicans,P. carinii PHR1expression was repressed under acidic conditions but induced at neutral and more alkaline pH.PHR1-related proteins have been implicated in glucan cell wall stability under various environmental conditions. Although difficulties withP. cariniiculture and transformation have traditionally limited assessment of gene function in the organism itself, we have successfully used heterologous expression ofP. cariniigenes in related fungi to address functional correlates ofP. carinii-encoded proteins. Therefore, the potential role ofP. carinii PHR1in cell wall integrity was examined by assessing its ability to rescue anS. cerevisiae gas1mutant with absent endogenous Phr1p-like activity. Interestingly,P. carinii PHR1DNA successfully restored proliferation ofS. cerevisiae gas1mutants under lethal conditions of cell wall stress. These results indicate thatP. carinii PHR1encodes a protein responsive to environmental pH and capable of mediating fungal cell wall integrity.

ABSTRACT In a previous study which evaluated the BACTEC 9240 automated blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.), we noted a 1.3% “instrument false-positive” rate. That is, the BACTEC system signaled that a bottle (BACTEC Plus Aerobic/F bottle or BACTEC Anaerobic Lytic/10 bottle) culture was positive but a Gram stain was negative and there was no growth of bacteria or yeasts on subculture to chocolate agar. Furthermore, from the same sample of blood, cultures for fungi using the Isolator blood culture system (Wampole Laboratories, Cranbury, N.J.) were negative for growth. For the present study, we evaluated 76 instrument false-positive samples for the presence of 16S ribosomal DNA using the MicroSeq 500 kit (PE Biosystems, Foster City, Calif.). These samples also were negative for fungi by the Isolator method. This kit has a PCR module and sequencing module for the amplification and sequencing of the 16S RNA gene and provides a database for sequence alignment and identification of bacteria. To optimize the assay, we evaluated the effect of adding 0.5% bovine serum albumin to the sample from blood culture bottles and found that it decreased the effects of inhibitors on the PCR. Two control groups of blood culture specimens were also evaluated. One group ( n = 45) were “instrument true positives”; the instrument signaled positive, and subsequent Gram stains were positive and subcultures on chocolate agar grew bacteria. The other group ( n = 20) were “instrument true negatives”; the instrument signaled negative, the Gram stain was negative, and subcultures on chocolate agar and from the Isolator tube on fungal media showed no growth. None of the 76 instrument false-positive samples had evidence for 16S rRNA gene sequences. All of the instrument true-positive samples and all of the instrument true-negative specimens were positive and negative, respectively, using the MicroSeq 500 kit. Total peripheral white blood cell counts were statistically significantly higher for patients who had instrument false-positive results than for patients who had instrument true-positive or true-negative results ( P = 0.001). We conclude that instrument false positives signaled by the BACTEC 9240 system are not due to bacteria in the blood culture samples.