Astrocyte-Derived Vascular Endothelial Growth Factor Stabilizes Vessels in the Developing Retinal Vasculature

Vascular endothelial growth factor (VEGF) plays a critical role in normal development as well as retinal vasculature disease. During retinal vascularization, VEGF is most strongly expressed by not yet vascularized retinal astrocytes, but also by retinal astrocytes within the developing vascular plexus, suggesting a role for retinal astrocyte-derived VEGF in angiogenesis and vessel network maturation. To test the role of astrocyte-derived VEGF, we used Cre-lox technology in mice to delete VEGF in retinal astrocytes during development. Surprisingly, this only had a minor impact on retinal vasculature development, with only small decreases in plexus spreading, endothelial cell proliferation and survival observed. In contrast, astrocyte VEGF deletion had more pronounced effects on hyperoxia-induced vaso-obliteration and led to the regression of smooth muscle cell-coated radial arteries and veins, which are usually resistant to the vessel-collapsing effects of hyperoxia. These results suggest that VEGF production from retinal astrocytes is relatively dispensable during development, but performs vessel stabilizing functions in the retinal vasculature and might be relevant for retinopathy of prematurity in humans.     

Balance between two transpeptidation mechanisms determines the expression of beta-lactam resistance in Enterococcus faecium

The d,d-transpeptidase activity of high molecular weight penicillin-binding proteins (PBPs) is essential to maintain cell wall integrity as it catalyzes the final cross-linking step of bacterial peptidoglycan synthesis. We investigated a novel beta-lactam resistance mechanism involving by-pass of the essential PBPs by l,d-transpeptidation in Enterococcus faecium. Determination of the peptidoglycan structure by reverse phase high performance liquid chromatography coupled to mass spectrometry revealed that stepwise selection for ampicillin resistance led to the gradual replacement of the usual cross-links generated by the PBPs (d-Ala(4) --> d-Asx-Lys(3)) by cross-links resulting from l,d-transpeptidation (l-Lys(3) --> d-Asx-Lys(3)). This was associated with no modification of the level of production of the PBPs or of their affinity for beta-lactams, indicating that altered PBP activity was not required for ampicillin resistance. A beta-lactam-insensitive l,d-transpeptidase was detected in membrane preparations of the parental susceptible strain. Acquisition of resistance was not because of variation of this activity. Instead, selection led to production of a beta-lactam-insensitive d,d-carboxypeptidase that cleaved the C-terminal d-Ala residue of pentapeptide stems in vitro and caused massive accumulation of cytoplasmic precursors containing a tetrapeptide stem in vivo. The parallel dramatic increase in the proportion of l-Lys(3) --> d-Asx-Lys(3) cross-links showed that the enzyme was activating the resistance pathway by generating the substrate for the l,d-transpeptidase.     

Localization of Proanthocyanidins Using in Situ-Hydrolysis with Sulfuric Acid

A valuable method for specific localization of proanthocyanidins in plant tissues is given. The specificity is based on the acid hydrolysis of condensed tannins in situ via hot sulfuric acid, yielding bright red anthocyanidins. This treatment did not cause noticeable damage of tissue embedded in glycol methacrylate prior to sectioning. Further proof of specificity was achieved by control staining with the flavan-3-01-selective p-dimethylaminocinnamaldehyde (DMACA) reagent. In addition, comparison of adjacent sections stained with the DMACA reagent may give coarse information about the degree of polymerization of the proanthocyanidins.     

Regeneration and transplantation of muscles in old rats and between young and old rats.

In order to compare the regenerative ability of skeletal muscle between young (5 month) and old (26 month) rats, sliced or intact extensor digitorum longus muscles were freely autografted into young and old rats and also reciprocally grafted from young to old inbred animals and vice versa. Sixty days after grafting, the transplants were analyzed for contractile and histochemical properties. There was a relative similarity between the contraction times of both normal control muscles and of all groups of transplants, although the contraction time tended to be prolonged and histochemical fiber pattern was more often found to be uniform in grafts of senescent animals. All groups of transplants possessed histochemically heterogeneous fiber types at 60 days. The experiments demonstrate that skeletal muscle in old rats possesses a substantial degree of regenerative ability and that the free tranpllantation of entire muscles in old animals is feasible.     

The electrochemical interactions of heparin and the aminoglycoside antibiotics

Various aminoglycoside antibiotics—tobramycin "analytical standard"; tobramycin, gentamycin, kanamycin, and amikacin, all containing preservatives added in pharmaceutical preparations; and streptomycin—without preservatives—yield in aqueous solutions a conductance peak when titrated against a heparin aqueous solution, indicating the formation of a charge transfer complex, which subsequently dissociates. At the volume ratio corresponding to the peak, a colloidal suspensoid forms which is possibly a micellar complex stabilized by the ions produced by the dissociation of the complex. The clinical significance of this interaction is suggested by preliminary microbiological tests. The possible effects of plasma protein binding and dissociation of the complexes here reported in tissues, however, were not studied.In the interaction with heparin, the aminoglycoside antibiotics appear to act as electron acceptors, though heparin is reported to act as an acceptor against chlorpromazine, which is well known to be a strong electron donor. There appears to be an interaction between nonpreserved tobramycin and the preservative added for the pharmaceutical preparation, which would explain the difference in titration behavior between the antibiotic alone and the antibiotic with preservative.Conductance titrations of heparin against penicillin and cloxacillin, which are not aminoglycosides, show no evidence of complexation or any other interaction. No suspensoid forms.     

Contribution of the ATP binding site of ParE to susceptibility to novobiocin and quinolones in Streptococcus pneumoniae

In Streptococcus pneumoniae, an H103Y substitution in the ATP binding site of the ParE subunit of topoisomerase IV was shown to confer quinolone resistance and hypersensitivity to novobiocin when associated with an S84F change in the A subunit of DNA gyrase. We reconstituted in vitro the wild-type topoisomerase IV and its ParE mutant. The ParE mutant enzyme showed a decreased activity for decatenation at subsaturating ATP levels and was more sensitive to inhibition by novobiocin but was as sensitive to quinolones. These results show that the ParE alteration H103Y alone is not responsible for quinolone resistance and agree with the assumption that it facilitates the open conformation of the ATP binding site that would lead to novobiocin hypersensitivity and to a higher requirement of ATP.     

HRS inhibits EGF receptor signaling in the RT4 rat schwannoma cell line

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is required for trafficking of cell surface receptors to the lysosome. Previously, we identified HRS as a protein that interacts with the neurofibromatosis 2 tumor suppressor schwannomin. In the present study, we established modified RT4 schwannoma cell lines that inducibly express HRS and constitutively express epidermal growth factor receptor (EGFR) fused to the green fluorescent protein. We demonstrated that HRS expression reduced EGFR abundance and EGF-mediated Stat3 activation. HRS expression also targeted EGFR to late endosomes. Schwannomin inhibited EGF-mediated Stat3 activation, consistent with HRS and schwannomin interacting in the same signaling pathway. Paradoxically, past studies have shown that HRS overexpression blocked EGFR trafficking to the late endosome and EGFR downregulation contrary to predictions of HRS function in HRS knockout studies. This study is the first to show that HRS can reduce the abundance of total and active EGFR and may reflect cell type-specific HRS function.     

Comparison of vancomycin-inducible proteins from four strains of Enterococci

Vancomycin-inducible proteins of 39.5 and 39 kDa from respectively, low-level and high-level resistant Enterococci were compared. Electrophoretic, immunoblot and peptide analysis revealed three types of protein, one in a low-level resistant strain of E. faecium, one in 2 high-level-resistant strains of E. faecium, and one in a high-level resistant strain of E. faecalis. The inducible proteins of E. faecium and E. faecalis, of 39.5 and 39 kDa respectively, which may function in a similar fashion (Al-Obeid et al. (1990) Antimicrob. Agents Chemother. 34, 252-256), are not related immunologically.     

Adaptive Lipid Packing and Bioactivity in Membrane Domains

Lateral compositional and physicochemical heterogeneity is a ubiquitous feature of cellular membranes on various length scales, from molecular assemblies to micrometric domains. Segregated lipid domains of increased local order, referred to as rafts, are believed to be prominent features in eukaryotic plasma membranes; however, their exact nature (i.e. size, lifetime, composition, homogeneity) in live cells remains difficult to define. Here we present evidence that both synthetic and natural plasma membranes assume a wide range of lipid packing states with varying levels of molecular order. These states may be adapted and specifically tuned by cells during active cellular processes, as we show for stimulated insulin secretion. Most importantly, these states regulate both the partitioning of molecules between coexisting domains and the bioactivity of their constituent molecules, which we demonstrate for the ligand binding activity of the glycosphingolipid receptor GM1. These results confirm the complexity and flexibility of lipid-mediated membrane organization and reveal mechanisms by which this flexibility could be functionalized by cells.