Roles for both the tripeptide, GSH, and individual amino acids in modifying the cellular response to oxygen deprivation-induced injury have been suggested by prior work in kidney and other tissues, but the precise interrelationships have not been clearly defined. We have studied the effects of GSH, its component amino acids, and related compounds on the behavior of isolated renal proximal tubules in a well characterized model of hypoxic injury in vitro. GSH, the combination of cysteine, glutamate, and glycine and glycine alone, when present in the medium during 30 min hypoxia, a duration sufficient to produce extensive irreversible injury in untreated tubules, were protective. Significant effects were detected at 0.25 mM concentrations of the reagents, and protection was nearly complete at concentrations of 1 mM and above. Glutamate and cysteine alone were not protective. The exogenous GSH added to the tubule suspensions was rapidly degraded to its component amino acids. Treatment of tubules with GSH or cysteine, but not glycine, increased intracellular GSH levels. Oxidized GSH was protective. Serine, N-(2-mercaptopropionyl)-glycine, and a panel of agents known to modify injury produced by reactive oxygen metabolites were without benefit. These observations identify a novel and potent action of glycine to modify the course of hypoxic renal tubular cell injury. This effect is independent of changes in cellular GSH metabolism and appears to be unrelated to alterations of cell thiols or reactive oxygen metabolites. Further elucidation of its mechanism may provide insight into both the basic pathophysiology of oxygen deprivation-induced cell injury and a practical way to ameliorate it.

Glycine-treated, hypoxic, proximal tubules developed a progressive energetic defect that resulted in failure to restore ATP levels to greater than 10 to 20% of control values during reoxygenation after 60 minutes of hypoxia despite continued cytoprotection by glycine. The defect was not corrected by supplementation with exogenous purines and was not modified by lowering the pH during hypoxia or reoxygenation. In the continued presence of glycine, the failure to restore ATP was associated with impaired recovery of structural changes that developed during hypoxia and, if glycine was withdrawn, lethal membrane damage occurred. The lesion was significantly ameliorated by the presence during hypoxia of two agents known to suppress development of the mitochondrial permeability transition, cyclosporine A and butacaine, which were most effective when used in combination. The data suggest that development of the mitochondrial permeability transition in glycine-protected tubules during hypoxia contributes to continued metabolic and structural impairment and cell death that occur despite glycine replete conditions such as exist frequently during in vivo insults and may be a target for therapeutic maneuvers.

This study demonstrates that although anomalous origins of coronary arteries are rare in asymptomatic children, the prevalence is greater than that found in other prospective studies. Ischemia can occur with both ALMCA and ARCA even though patients remain asymptomatic. Because of the high risk of sudden cardiac death, aggressive surgical management and close follow-up are necessary.

To establish the connection between galaxies and UV-detected absorption systems in the local universe, a deep (g ≤ 20) and wide (∼ 20 radius) galaxy redshift survey is presented around 47 sight lines to UV-bright AGN observed by the Cosmic Origins Spectrograph (COS). Specific COS science team papers have used this survey to connect absorbers to galaxies, groups of galaxies, and large-scale structures, including voids. Here we present the technical details of the survey and the basic measurements required for its use, including redshifts for individual galaxies and uncertainties determined collectively by spectral class (emission-line, absorption-line, and composite spectra) and completeness for each sight line as a function of impact parameter and magnitude. For most of these sight lines the design criteria of > 90% completeness over a > 1 Mpc region down to 0.1 L * luminosities at z ≤ 0.1 allows a plausible association between low-z absorbers and individual galaxies. Lyα covering fractions are computed to approximate the star-forming and passive galaxy populations using the spectral classes above. In agreement with previous results, the covering fraction of star-forming galaxies with L ≥ 0.3 L * is consistent with unity inside one virial radius and declines slowly to > 50% at 4 virial radii. On the other hand, passive galaxies have lower covering fractions (∼ 60%) and a shallower decline with impact parameter, suggesting that their gaseous halos are patchy but have a larger scale-length than star-forming galaxies. All spectra obtained by this project are made available electronically for individual measurement and use.

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ATP hydrolysis and polypeptide binding, the two key activities of Hsp70 molecular chaperones, are inherent properties of different domains of the protein. The coupling of these two activities is critical because the bound nucleotide determines, in part, the affinity of Hsp70s for protein substrate. In addition, cochaperones of the Hsp40 (DnaJ) class, which stimulate Hsp70 ATPase activity, have been proposed to play an important role in promoting efficient Hsp70 substrate binding. Because little is understood about this functional interaction between domains of Hsp70s, we investigated mutations in the region encoding the ATPase domain that acted as intragenic suppressors of a lethal mutation (I485N) mapping to the peptide-binding domain of the mitochondrial Hsp70 Ssc1. Analogous amino acid substitution in the ATPase domain of the Escherichia coli Hsp70 DnaK had a similar intragenic suppressive effect on the corresponding I462T temperature-sensitive peptide-binding domain mutation. I462T protein had a normal basal ATPase activity and was capable of nucleotide-dependent conformation changes. However, the reduced affinity of I462T for substrate peptide (and DnaJ) is likely responsible for the inability of I462T to function in vivo. The suppressor mutation (D79A) appears to partly alleviate the defect in DnaJ ATPase stimulation caused by I462T, suggesting that alteration in the interaction with DnaJ may alter the chaperone cycle to allow productive interaction with polypeptide substrates. Preservation of the intragenic suppression phenotypes between eukaryotic mitochondrial and bacterial Hsp70s suggests that the phenomenon studied here is a fundamental aspect of the function of Hsp70:Hsp40 chaperone machines.

Targeted cellular delivery of drugs to specific tissues is an important goal in biomedical chemistry. Achieving this requires harnessing and applying molecular-level recognition events prevalent in (or specific to) the desired tissue type. Tissues rich in estrogen receptors (ERs), which include many types of breast cancer, accumulate molecules that have high binding affinities for these receptors. Therefore, molecules that (i) bind to the ER, (ii) have favorable cellular transport properties, and (iii) contain a second functionality (such as a center that may be used for diagnostic imaging or medical therapy) are exciting synthetic targets in the field of drug delivery. To this end, we have prepared a range of metallo-estrogens based on 17alpha-ethynylestradiol and examined their binding to the ER both as isolated receptor and in whole cell assays (ER positive MCF-7 cells). Estrogens functionalized with metal binding units are prepared by palladium-catalyzed cross-coupling reactions and a wide range of metal centers introduced readily. All the compounds prepared and tested exhibit effective binding to the estrogen receptor and are delivered across the cell membrane into MCF-7 cells. In the whole cell assays, despite their monocationic nature, the palladium and platinum complexes prepared exhibit similar (and even enhanced) receptor binding affinities compared to their corresponding neutral free ligands. It is unprecedented for a higher ER binding affinity to be observed for a cationic complex than for its metal-free ligand.

Abstract. SSH1, a newly identified member of the heat shock protein (hsp70) multigene family of the budding yeast Saccharomyces cerevisiae, encodes a protein localized to the mitochondrial matrix. Deletion of the SSH1 gene results in extremely slow growth at 23°C or 30°C, but nearly wild-type growth at 37°C. The matrix of the mitochondria contains another hsp70, Sscl, which is essential for growth and required for translocation of proteins into mitochondria. Unlike SSC1 mutants, an SSH1 mutant showed no detectable defects in import of several proteins from the cytosol to the matrix compared to wild type. Increased expression of Sscl partially suppressed the cold-sensitive growth defect of the SSHI mutant, suggesting that when present in increased amounts, Sscl can at least partially carry out the normal functions of Sshl. Spontaneous suppressors of the cold-sensitive phenotype of an SSH1 null mutant were obtained at a high frequency at 23°C, and were all found to be respiration deficient. 15 of 16 suppressors that were analyzed lacked mitochondrial DNA, while the 16th had reduced amounts. We suggest that Sshl is required for normal mitochondrial DNA replication, and that disruption of this process in sshl cells results in a defect in mitochondrial function at low temperatures. HEAT shock proteins of the hsp70 class are found in all organisms and play essential roles as molecular chaperones (for reviews see 7,18,20). Related hsp70s are found in cellular compartments such as the cytosol, ER, and mitochondria (5). The cytosol of yeast contains two functionally distinct classes of hsp70s, the SSA and SSB proteins (9). The SSA proteins, besides being involved in the regulation of expression of heat-inducible genes, are required for normal rates of translocation of some proteins into the ER or mitochondria. The SSB proteins associate with translating ribosomes, and they are required for normal protein translation. The presence of both SSA and SSB classes of hsp70s in the same cellular compartment illustrates the evolution of related but functionally distinct types of hsp70s.To date, Sscl has been the only hsp70 identified in the mitochondria matrix, and Kar2 has been the sole family member identified in the ER (8,30,36). In the yeast Saccharomyces cerevisiae, Sscl is required for the translocation of proteins from the cytosol into the matrix and is involved in their subsequent folding, maturation, and proteolysis (16,21,22,40,49). Sscl is proposed to bind to