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Biomedical subjects

L Greene

Publications and source records attributed to L Greene.

At least 19 recordsLinked to original sources
PubMed1999

ICU admission in patients infected with the human immunodeficiency virus - a multicentre survey.

We conducted a retrospective study to assess the reasons for admission to the intensive care unit, and subsequent outcome, in patients infected with the human immunodeficiency virus (HIV). Four hospitals in the south of England participated, all with specialist HIV units. Data were collected on 127 patients admitted to ICU on 133 separate occasions between June 1993 and October 1997. The mean age on admission was 38 years (range 23-60 years). Ninety-four patients (70.7%) were documented HIV-positive before admission and 36 (27%) were diagnosed HIV-positive for the first time during admission; 36.1% were admitted with Pneumocystis carinii pneumonia. Overall ICU mortality was 33%, in-hospital mortality was 56% and the eventual mortality at the end of follow-up (March 1998) was 72%. Survival was highest in those admitted with respiratory HIV-related disease or HIV-unrelated illness. Associations with poor outcome included a prior AIDS-defining illness, a CD4 cell count of less than 100 cells.ml-1 and admission secondary to sepsis.

AIDS-Related Opportunistic Infections↗
PubMed1998

Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins.

We recently have identified an antigen receptor in sharks called NAR (new or nurse shark antigen receptor) that is secreted by splenocytes but does not associate with Ig light (L) chains. The NAR variable (V) region undergoes high levels of somatic mutation and is equally divergent from both Ig and T cell receptors (TCR). Here we show by electron microscopy that NAR V regions, unlike those of conventional Ig and TCR, do not form dimers but rather are independent, flexible domains. This unusual feature is analogous to bona fide camelid IgG in which modifications of Ig heavy chain V (VH) sequences prevent dimer formation with L chains. NAR also displays a uniquely flexible constant (C) region. Sequence analysis and modeling show that there are only two types of expressed NAR genes, each having different combinations of noncanonical cysteine (Cys) residues in the V domains that likely form disulfide bonds to stabilize the single antigen-recognition unit. In one NAR class, rearrangement events result in mature genes encoding an even number of Cys (two or four) in complementarity-determining region 3 (CDR3), which is analogous to Cys codon expression in an unusual human diversity (D) segment family. The NAR CDR3 Cys generally are encoded by preferred reading frames of rearranging D segments, providing a clear design for use of preferred reading frame in antigen receptor D regions. These unusual characteristics shared by NAR and unconventional mammalian Ig are most likely the result of convergent evolution at the molecular level.

Amino Acid Sequence↗
PubMed1998

Characterization of D10S and K71E mutants of human cytosolic hsp70.

To determine the effect of mutations at the nucleotide-binding site of recombinant Hsp70 on its interaction with protein and peptide substrates, point mutations were made at D10 and K71, two residues at the active site. The D10S mutation weakened both ATP and ADP binding, while the K71E mutation weakened only ATP binding. In binding experiments using Hsp70 with no bound nucleotide, the mutated Hsp70s interacted with clathrin and peptide just like the wild-type Hsp70. However, the D10 mutation completely abolished the effects of both ATP and ADP on peptide and clathrin binding. The K71 mutation also abolished the effect of ATP on substrate binding, but ADP, which still bound tightly, had its normal effect on substrate binding. In addition, the D10S and K71E mutants had greatly reduced ability to uncoat clathrin-coated vesicles at pH 7.0, bind to clathrin baskets at pH 6.0, and undergo polymerization induced by YDJ1 in the presence of ATP. We conclude, first, that nucleotides must bind strongly to Hsp70 to affect substrate binding and, second, that interaction of Hsp70 with DnaJ homologues may also require a strongly bound ATP.

Adenosine Diphosphate↗
PubMed1997

Effect of yeast and human DnaJ homologs on clathrin uncoating by 70 kilodalton heat shock protein.

We recently found that the DnaJ homolog auxilin is required for Hsc70 to uncoat clathrin baskets. In the present study, we investigated the effect of two other DnaJ homologs, YDJ1 from yeast and HDJ1 from humans, on the uncoating activity of Hsc70. Neither YDJ1 nor HDJ1 substituted for auxilin in supporting uncoating. Rather, in the presence of auxilin, both YDJ1 and HDJ1 strongly inhibited uncoating at pH 7 and also prevented the binding of Hsc70 to clathrin baskets at pH 6. Both YDJ1, as shown previously, and HDJ1 catalytically induce polymerization of Hsc70 into large polymers in ATP, and the YDJ1 concentration required to inhibit uncoating was similar to the concentration required for polymerization. However, uncoating was almost completely inhibited even at low concentrations of Hsc70 where only partial polymerization occurs, suggesting that YDJ1 inhibits uncoating not only by polymerizing the Hsc70 but also by some other mechanism as well. The effects of YDJ1 and HDJ1 were completely reversible; when they were removed, the Hsc70 regained full activity. Since both YDJ1 and HDJ1 inhibited the uncoating of clathrin baskets by brain cytosol as well as by purified Hsc70, this could be a physiological phenomenon which could affect other activities of Hsc70 in addition to uncoating.

Adaptor Proteins, Vesicular Transport↗
PubMed1997

Auxilin-induced interaction of the molecular chaperone Hsc70 with clathrin baskets.

We previously reported that a 100-kDa cofactor, recently identified as auxilin, is a DnaJ homolog which is required for Hsc70 to uncoat clathrin baskets. In the present study we investigated the effect of auxilin on the interaction of Hsc70 with pure clathrin baskets at pH 6, where no uncoating occurs. In a reaction which required auxilin, the baskets activated the Hsc70 ATPase activity more than 100-fold with an apparent dissociation constant of about 0.2 microM. Maximal ATPase activity occurred at a 1 to 1 molar ratio of auxilin to clathrin triskelion independent of the Hsc70 concentration suggesting that auxilin is primarily complexed with the clathrin baskets. The binding of Hsc70 to baskets also required auxilin, but less auxilin was needed for maximum binding than for maximum ATPase activity showing that auxilin can catalytically induce binding of Hsc70. The binding also required ATP; Hsc70 dissociated from baskets with a 6 min half-life when ATP was hydrolyzed to ADP. In contrast to auxilin, the assembly proteins, AP-2 and AP180, did not support activation of the Hsc70 ATPase activity by clathrin baskets nor did soluble clathrin triskelions at pH 7 significantly activate the ATPase activity with auxilin present. Therefore, the interaction of auxilin, clathrin baskets, and Hsc70-ATP is highly specific with auxilin first binding to a clathrin triskelion in the baskets and then Hsc70-ATP strongly binding to the auxilin-clathrin complex; the auxilin can then migrate to another clathrin triskelion before the ATPase cycle is complete.

Adaptor Proteins, Vesicular Transport↗
PubMed1997

Interaction of auxilin with the molecular chaperone, Hsc70.

We have studied the direct interaction of the constitutive isoform of Hsp70 (Hsc70) with the DnaJ homolog, auxilin, a cofactor that binds to clathrin-coated vesicles and is required for their uncoating by Hsc70. Auxilin caused a 5-fold increase in Hsc70 ATPase activity and a corresponding increase in steady-state levels of bound ADP; the dissociation constant for this effect was 0.6 microM. Auxilin also induced polymerization of Hsc70 and bound to the resulting polymer at a 1:1 molar ratio; here too the dissociation constant was 0.6 microM. Both this binding and polymerization required ATP; the Hsc70 depolymerized with a 4-min half-life when ATP was completely hydrolyzed to ADP. Although auxilin induces polymerization stoichiometrically and other DnaJ homologs induce polymerization catalytically, these data show that auxilin is similar to other DnaJ homologs in its ability to activate the Hsc70 ATPase activity, to polymerize Hsc70, and in the nucleotide dependence of this polymerization. Furthermore, the 70-amino acid J-domain of auxilin polymerized Hsc70 with the same nucleotide dependence as intact auxilin. Therefore, although only auxilin and not other DnaJ homologs support uncoating, our data suggest that various DnaJ homologs share a common mechanism of interaction with Hsc70, perhaps because their J-domains interact similarly with Hsc70.

Adaptor Proteins, Vesicular Transport↗
PubMed1997

Cystic fibrosis, nutrition, and the health care team.

Because of the multiple systems involved in cystic fibrosis, the variability and chronicity of the disease, and the increased survival of this population, a specialty team of experts for care has evolved. A multidisciplinary approach is essential to assist patients and their families in adjusting to the disease and to optimize treatment interventions. The dietitian is responsible for assessment of nutritional status, including the determination of energy requirements and eating habits, interpretation of anthropometric data, and evaluation of nutritional adequacy. The nutrition care plan forms an integral part of the overall treatment objectives and is reported to other team members as it is devised, implemented, and monitored. A consensus report issued in April 1990 by the Cystic Fibrosis Foundation includes both general nutrition guidelines and detailed recommended treatment standards aimed at providing optimal nutrition care.

Child, Preschool↗
PubMed1996

Effect of constitutive 70-kDa heat shock protein polymerization on its interaction with protein substrate.

Constitutive 70-kDa heat shock protein (hsc70) is a mixture of monomers and oligomers in ADP, while in ATP it is monomeric unless certain DnaJ homologs are present which induce hsc70 to form large polymers in an ATP-dependent reaction. A key question regarding polymerized hsc70 is whether it is able to bind protein substrates. Polymerized BiP, the hsc70 present in the endoplasmic reticulum, has been found to bind substrates in vitro although substrates appear to bind only to monomeric BiP in vivo. In this study, we investigated whether substrate binds to polymerized cytoplasmic hsc70 in vitro. Although both stoichiometric ATP and high concentrations of cytochrome c peptide monomerized hsc70, direct binding studies provided no evidence that cytochrome c peptide binds to polymerized hsc70. Furthermore, the time course of cytochrome c peptide and clathrin binding to hsc70 suggested that rather than binding to polymerized hsc70, they monomerized it by reducing free monomer, thereby shifting the monomer-polymer equilibrium toward monomer. We conclude that peptide and protein substrates bind at least an order of magnitude more weakly to polymerized hsc70 than to monomer, suggesting that polymerization of hsc70 in vivo, perhaps by DnaJ homologs, may store it in an inactive form.

Adenosine Triphosphate↗
PubMed1995

Polymerization of 70-kDa heat shock protein by yeast DnaJ in ATP.

DnaK, the Escherichia coli hsp70 protein, interacts with DnaJ, a protein cofactor that appears to be involved in presenting protein substrates to DnaK. The yeast DnaJ homolog, YDJ1, has also been shown to interact with yeast hsp70, although the function of this interaction is unknown. In the present study, we investigated the interaction of YDJ1 with both yeast and bovine brain hsp70. We found that, in the presence of ATP, where hsp70 is normally monomeric, YDJ1 induced almost all of the yeast and bovine brain hsp70 to form large polymers, which are readily sedimentable. These polymers were much larger than the dimers and trimers of hsp70, which normally form in the presence of ADP. YDJ1 appeared to be acting catalytically since very little YDJ1 copolymerized with the hsp70, and maximum polymerization occurred at low ratios of YDJ1 to hsp70. The polymerization required ATP and was completely reversed when ATP was replaced by ADP. These data suggest that, in the presence of ATP, YDJ1 may present one hsp70 to another just as under other conditions DnaJ is able to present protein substrates to DnaK.

Adenosine Triphosphate↗
PubMed1995

Interaction of nucleotide-free Hsc70 with clathrin and peptide and effect of ATP analogues.

The functions of the 70 kDa heat-shock proteins (hsp70s) are regulated by their bound nucleotide. We previously observed major differences in the effect of bound ATP and ADP on the interaction of hsc70 (constitutive hsp70) with its protein substrates. In the present study, we investigated the interaction of protein substrates with nucleotide-free hsc70 and with hsc70 with bound ATP analogues. We found, first, that nucleotide-free hsc70 appeared to interact differently with different substrates. Specifically, nucleotide-free hsc70 behaved much more like hsc70-ATP than hsc70-ADP in that clathrin very rapidly bound to and dissociated from nucleotide-free hsc70 in contrast to its very slow binding to and dissociation from hsc70-ADP. On the other hand, nucleotide-free hsc70 behaved more like hsc70-ADP than hsc70-ATP in that cytochrome c peptide dissociated very slowly from nucleotide-free hsc70 compared to its rapid dissociation from hsc70-ATP. Second, binding of the ATP analogues AMP-PNP, dATP, and ATP gamma S to nucleotide-free hsc70 had very little further effect on the properties of the nucleotide-free hsc70. Therefore, previously observed effects of ATP analogues may have been due to removal of the bound ADP rather than to the presence of analogues.

Adenosine Diphosphate↗
PubMed1994

Complex formation between clathrin and uncoating ATPase.

The bovine brain uncoating ATPase, a constitutive 70-kDa heat shock protein, uncoats clathrin-coated vesicles in an ATP-dependent reaction. The uncoating ATPase-clathrin complex formed from the uncoating reaction was compared to the complex formed by directly binding free clathrin to uncoating ATPase. The amount of the latter complex shows a simple hyperbolic dependence on either free clathrin or free uncoating ATPase concentration, whichever is in excess, with a binding stoichiometry of one uncoating ATPase per clathrin heavy chain. ATP markedly increases the rates of formation and dissociation of this complex while ADP profoundly inhibits these rates. At low uncoating ATPase concentrations, much more complex is formed by uncoating than by directly binding clathrin to enzyme. However, during column chromatography or dilution for electron microscopy, both types of complex dissociate in ATP but not ADP, and electron microscopy of both types of complex diluted into ADP shows binding only to the vertex of the clathrin triskelion. We conclude that the uncoating ATPase forms only one type of complex with clathrin and has only one site for nucleotide; ADP at this site prevents either formation or dissociation of complex, whereas ATP at this site allows both processes to occur rapidly.

Adenosine Diphosphate↗
PubMed1994

Characterization of nucleotide-free uncoating ATPase and its binding to ATP, ADP, and ATP analogues.

The interactions of the 70-kDa heat-shock proteins (hsp70s) with their protein substrates appear to be regulated by bound nucleotide. Previous work has shown that the nucleotide binding site of the bovine brain uncoating ATPase, a constitutive member of the hsp70 family, crystallographically resembles the nucleotide binding site of actin and, like actin, the uncoating ATPase has a strongly bound ADP which cannot be removed by dialysis or treatment with ethylenediaminetetraacetic acid (EDTA). This suggests that, like the bound nucleotide of actin, it may be required for the enzyme to retain its native structure. In this study, the strongly bound ADP was removed by first replacing it with 5'-adenylyl imidodiphosphate (AMP-PNP) and then removing the bound AMP-PNP by dialysis. Following this treatment, more than 95% of the uncoating ATPase becomes nucleotide-free. The nucleotide-free uncoating ATPase retains its ability to bind and hydrolyze ATP and to uncoat clathrin-coated vesicles, even after 10 days of storage at 4 degrees C. Therefore, in contrast to actin, the bound nucleotide of the uncoating ATPase is not required to prevent denaturation of the enzyme. Using nucleotide-free uncoating ATPase, we were able to accurately measure the dissociation constants of ATP, ADP, and the nucleotide analogues AMP-PNP and 2'-deoxyadenosine 5'-triphosphate (dATP). The dissociation constants of both ATP and ADP are about 10(-8) M, more than 1-2 orders of magnitude stronger than previously reported, while AMP-PNP and dATP bind 2-3 orders of magnitude more weakly than ATP.

Adenosine Diphosphate↗
PubMed1993

A protein cofactor is required for uncoating of clathrin baskets by uncoating ATPase.

Immediately after clathrin-coated pits pinch off from the cell membrane to form clathrin-coated vesicles, clathrin dissociates from the vesicles. In vitro studies suggest that this dissociation is carried out by the uncoating ATPase, a constitutive member of the 70-kDa heat shock family. Aside from the requirement for ATP, nothing is known about the regulation of the uncoating process. We now show that clathrin baskets prepared from highly purified clathrin and AP2, the assembly protein associated with plasma membrane coated vesicles, cannot be uncoated by the bovine brain uncoating ATPase alone. A 100-kDa protein cofactor, which was isolated from coated vesicles, is essential for uncoating by the uncoating ATPase. This cofactor restores normal uncoating when present at a molar ratio of about 1 to 10 to clathrin and uncoating ATPase.

Adenosine Triphosphatases↗
PubMed1993

Nucleotide binding properties of bovine brain uncoating ATPase.

Many functions of the 70-kDa heat-shock proteins (hsp70s) appear to be regulated by bound nucleotide. In this study we examined the nucleotide binding properties of purified bovine brain uncoating ATPase, one of the constitutively expressed members of the hsp70 family. We found that uncoating ATPase purified by ATP-agarose column chromatography retained one ADP molecule bound per enzyme molecule which could not be removed by extensive dialysis. Since this bound ADP exchanged rapidly with free ADP or ATP, the inability to remove the bound nucleotide was not due to slow dissociation but rather to strong binding of the nucleotide to the uncoating ATPase. In confirmation of this view, equilibrium dialysis experiments suggested that the dissociation constants for both ADP and ATP were less than 0.1 microM. Schmid et al. (Schmid, S. L., Braell, W. A., and Rothman, J. E. (1985) J. Biol. Chem 260, 10057-10062) suggested that the uncoating ATPase had two sites for bound nucleotide, one specific for ATP and one binding both ATP and ATP analogues but not ADP. In contrast, we found that enzyme with bound ADP did not bind further adenosine 5'-(beta,gamma-imino)triphosphate or dATP, nor did more than one ATP molecule bind per enzyme even in 200 microM free ATP. These results strongly suggest that the enzyme has only one binding site for nucleotide. During steady-state ATP hydrolysis, 85% of the bound nucleotide at this site was determined to be ATP and 15% ADP; this is consistent with the rate of ADP release determined in the exchange experiments noted above, where ADP release was found to be six times faster than the overall rate of ATP hydrolysis.

Adenosine Diphosphate↗