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Biomedical subjects

L Hall

Publications and source records attributed to L Hall.

At least 37 records · Page 2Linked to original sources
PubMed1991

Conformational changes in yeast phosphoglycerate kinase upon ligand binding: fluorescence of a linked probe and chemical reactivity of genetically introduced cysteinyl residues.

The effects of ligands on the conformation of yeast phosphoglycerate kinase were explored by introducing cysteinyl residues at different positions in the molecule by site-directed mutagenesis. Thus several mutants were constructed, each containing a unique cysteinyl residue. Neither the conformation nor the enzyme activity was affected by the substitutions. The reactivity of the thiol groups and the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine covalently linked to these thiols were used to monitor the conformational changes induced upon ligand binding. It was found that the observed changes mainly involve the part of the protein located in the cleft, particularly the environment of residues 35 and 183. No alteration was observed on the external side of the protein. Only 3-Phosphoglycerate induced these conformational changes. However, when the fluorescent probe was attached to residue 377, the binding of the two substrates was required to induce a modification in the fluorescence of the probe. These results indicate that the substrates separately or together induce discrete molecular motions in phosphoglycerate kinase.

Adenosine Triphosphate↗
PubMed1991

Do children with severe head injury benefit from intensive care?

Intensive management of severe closed head injury is pursued in most neurosurgical units, as it no doubt reduces mortality. However, the fate of those salvaged from death is less clearly defined. The impact of aggressive management on the psychological recovery is even less clear. This retrospective study of children admitted to a regional paediatric neurosurgical service attempts to address these issues. Eighty-four children between the ages of 3 to 16 years with severe head injuries were studied and categorized according to conservative or intensive treatment groups. The availability of intensive management led to a greater number of such children referred. The mortality in those children sustaining more severe injury, i.e. Glasgow Coma Score (GCS) 3 or 4, was halved (44%) with intensive management from 80% in the conservative group. For those with less severe injury (GCS 5-8), it was unchanged at about 23%. The reduced mortality is at the expense of increased morbidity. Psychological testing showed that the cognitive recovery was comparable.

Adolescent↗
PubMed1991

A genetic linkage map of human chromosome 5 with 60 RFLP loci.

A genetic map of human chromosome 5 that contains 60 restriction fragment length polymorphism (RFLP) loci in one linkage group has been constructed. Segregation data using these markers and 40 large multigenerational families supplied by the Centre d'Etude du Polymorphisme Humain have been collected. Linkage analyses were performed with the program package CRI-MAP; using odds greater than 1000:1, 30 RFLP loci could be placed on the map. This genetic map spans 289 cM sex-equal, 353 cM in females, and 244 cM in males. While the relative rate of recombination for female meioses is nearly twice that of males over much of the chromosome, several instances of statistically significant excess male recombination were observed. The order of probes on the genetic map has been confirmed by their physical order as determined by somatic cell hybrid lines containing deletions of normal chromosome 5. There is concordance between the physical positions of markers and their genetic positions. Our most distal probes on the genetic map are cytologically localized to the most distal portions of the chromosome. This suggests that our genetic map spans most of chromosome 5.

Blotting, Southern↗
PubMed1991

Evaluation of a brief psychological treatment for emotional disorders in primary care.

A randomized trial in general practice compared: (i) a brief psychological treatment (problem-solving) given by a psychiatrist; (ii) any treatment of the GP's choice, whether psychological or pharmacological. The patients had recent onset emotional disorders of poor prognosis. Patients in the problem-solving group showed significantly greater reductions in symptoms. Problem-solving as given by a psychiatrist was feasible in primary care and acceptable to patients. Problem-solving is now being evaluated as given by general practitioners trained in the method.

Adolescent↗
PubMed1991

Relative activities of HIV-1-IIIB and HIV-1BaL LTR and tat in primary monocytes and lymphocytes.

The mechanisms determining the ability of some but not other strains of human immunodeficiency virus type 1 (HIV-1) to grow in peripheral blood monocyte-macrophages are presently unclear. The tat gene of HIV-1-IIIB which replicates poorly in human macrophages, and the tat gene of HIV-1-BaL, which replicates to high titers in the same cells in transient expression systems with their respective long terminal repeats (LTR) driving a reporter chloramphenicol acetyl transferase (CAT) gene were compared. The authors hypothesized that the tat gene and LTR of BaL might help account for its efficient growth in primary monocyte-macrophages by virtue of a high activity in these cells relative to that of the IIIB tat and LTR. Primary peripheral blood lymphocytes and monocytes were cotransfected with either the HIV-1BaL or HIV-1-IIIB LTR fused to the CAT gene and their respective tat genes. The IIIB tat and LTR were at least as active in primary lymphocytes as the BaL combination, and both tat-LTR pairs were more active in primary lymphocytes than monocytes. The same relative activities were also observed in primary monocytes after in vitro maturation to macrophages prior to transfection. These data strongly suggest that neither the tat gene nor the LTR of HIV-1-IIIB and HIV-1BaL can account for the great ability of the latter or the inability of the former to grow in monocyte-macrophages.

Amino Acid Sequence↗
PubMed1991

Very high affinity interaction of DPI 201-106 and BDF 8784 enantiomers with the phenylalkylamine-sensitive Ca2(+)-channel in Drosophila head membranes.

1. Piperazinylindoles (DPI 201-106, BDF 8784), drugs known to act on voltage-dependent Na(+)-channels, bind with very high affinity to a Ca2(+)-channel-associated phenylalkylamine receptor in Drosophila melanogaster head membranes. These compounds and (+)-tetrandrine, a naturally occurring Ca2(+)-antagonist, were the most selective inhibitors for phenylalkylamine-labelled Drosophila Ca2(+)-channels compared to mammalian L-type Ca2(+)-channels. 2. Replacement of the cyano group by a methyl group in (+)-DPI 201-106 ((+)-BDF 8784) increases the IC50 value for inhibition of phenylalkylamine labelling of Drosophila Ca2(+)-channels from 0.29 to 2.1 nM but decreases the IC50 value for inhibition of phenylalkylamine labelling of mammalian skeletal muscle Ca2(+)-channels from 3480 to 49 nM. 3. DPI 201-106 enantiomers completely block (at 0.1 microM) phenylalkylamine photolabelling of a 136 K polypeptide in Drosophila head membranes whereas 10 microM aconitine or lidocaine are without effect. 4. Assessment of the Ca2(+)-antagonist effects of the substituted DPI 201-106 enantiomers in K(+)-depolarized taenia strips from guinea-pig caecum yielded pA2 values of 6.33 +/- 0.07 for (-)-BDF 8784 and 6.99 +/- 0.17 for (+)-BDF 8784, respectively. 5. Piperazinylindoles, previously believed to act nonspecifically on voltage-dependent mammalian L-type Ca2(+)-channels, therefore have stereoselectivity for a novel binding site and chemical selectivity unrelated to local anaesthetic activity. 6. It is proposed that a very high affinity piperazinylindole-selective site is coupled to the phenylalkylamine receptor of Drosophila Ca2(+)-channels. These sites are still present on mammalian L-type Ca2(+)-channels but have lower affinity and/or are less tightly coupled to phenylalkylamine receptors on the alpha 1-subunit.

Affinity Labels↗
PubMed1991

Recognition of a single hsp-60 epitope by an entire subset of gamma delta T lymphocytes.

We can conclude that a large subset of gamma delta cells, present in both murine newborn thymus and in adult spleen, respond to the stress protein, hsp60. hsp60 seems to be stimulatory whether it is derived from a foreign pathogen such as mycobacteria, or whether it originates from the mouse's own cells. The gamma delta cells that respond to this antigen bear very similar receptors, all expressing V gamma 1 and most expressing V delta 6, although their junctional variations indicate that not all members of the subset stem from clonal expansion of only one or a few cells. The hsp60-reactive subset has not at this time been shown to "home" to an epithelial location, in contrast to other known gamma delta cell subsets, and may rather carry out its functions while in circulation. Whether the hsp60 antigen requires a "presenting" molecule remains at this point unclear, but because the gamma delta cells all respond to a synthetic peptide representing an epitope of hsp60, presentation is implied. Human gamma delta cells that respond to PPD from mycobacteria, as do the mouse hsp60-reactive gamma delta cells, have also been described, many as members of a major subset in peripheral blood, although only rarely have these been reported to respond to mycobacterial hsp60. The antigenic source in PPD for these cells has not yet been determined, but as for the mouse, a low molecular weight peptide appears to be sufficient for stimulation (P. Brennan and R. Modlin, personal communication). The PPD-reactive gamma delta cells, when their receptors have been characterized, have been found to express a V gamma 9+ chain. Some evidence indicates that these cells can also recognize self hsp60; hence, in several ways, this human subset has characteristics similar to the mouse hsp60-reactive subset. Perhaps gamma delta cells that respond to hsp60 play an important role, in both mice and humans, in the detection of transformed self cells or cells containing intracellular pathogens, that escape detection by alpha beta T cells.

Amino Acid Sequence↗
PubMed1991

Expandable venous stents for treatment of the Budd-Chiari syndrome.

The goals of treatment of the Budd-Chiari syndrome are relief of portal hypertension, relief of inferior vena cava syndrome, if present, and preservation of hepatic function. This study presents a patient with clinical resolution of the Budd-Chiari syndrome after placement of expandable metallic stents in the inferior vena cava and hepatic veins. A 26-year-old man with severe ascites and lower extremity edema but with relatively preserved hepatic function had a small gradient across a suprahepatic caval web, large gradients across an intrahepatic caval stenosis and the left hepatic vein, and an occluded right hepatic vein. Under angiographic control, web and caval stenosis were balloon-dilated, and modified Gianturco expandable metallic stents were placed in the intrahepatic vena cava. The left hepatic vein was dilated twice and a stent was placed. All gradients were completely eliminated. There were no complications and after 1 year, the stents have fully expanded without migration, edema and ascites have resolved, hepatic function has normalized, and the patient has returned to work. This new technique provides a simple, safe, effective, relatively inexpensive, and potentially long-lasting treatment for selected patients with the Budd-Chiari syndrome.

Adult↗
PubMed1990

Analysis of major androgen-regulated cDNA clones from the rat epididymis.

Four abundant cDNA clones have been isolated from a rat epididymal cDNA library. Northern blot analysis has shown that these clones partially encode 4.5 kb, 2.8 kb, 1.2 kb and 0.85 kb mRNAs and that their expression is not detectable in total RNA preparations from heart, kidney, liver or testis. Fourteen days after castration the levels of the 2.8 kb, 1.2 kb and 0.85 kb transcripts were greatly reduced whereas the 4.5 kb mRNA was undetectable. Subsequent treatment of castrated rats with testosterone for 1 day resulted in a complete restoration of the pre-castration steady-state levels of the 2.8 kb and 0.85 kb mRNAs, restoration of the 4.5 kb mRNA to 70% of pre-castration levels, and a slight over-induction of the 1.2 kb mRNA. Analyses of separate regions of the epididymal tract showed that expression of the 2.8 kb and 1.2 kb mRNAs increased towards the distal end of the epididymis, while the 4.5 kb and 0.85 kb transcripts were primarily synthesised in the caput region.

Amino Acid Sequence↗
PubMed1990

Recognition of a peptide antigen by heat shock--reactive gamma delta T lymphocytes.

Small synthetic peptides that correspond to different portions of the 65-kilodalton mycobacterial heat shock protein (Hsp65) were used to identify a putative antigenic epitope for gamma delta cells. Weaker gamma delta responses to the equivalent portion of the autologous homolog, mouse Hsp63, were also seen. The stimulatory epitope overlaps with an epitope recognized by arthritogenic alpha beta T cell clones. The data suggest that gamma delta cells have a role in autoimmune disorders and imply that these cells recognize ligands by a mechanism similar to that of alpha beta T lymphocytes, that is, in the form of small processed protein fragments bound to antigen-presenting molecules.

Amino Acid Sequence↗
PubMed1990

Metallothionein gene expression and resistance to cisplatin in human ovarian cancer.

Intracellular thiols have been proposed as mediators of resistance to alkylating agents and cisplatin. As metallothionein is the predominant protein thiol, we examined its relationship to cisplatin resistance in human ovarian cancer cell lines. A human ovarian carcinoma cell line, A2780, derived from an untreated patient, was treated with cisplatin in several ways and the induced resistance to cisplatin ranged from 13- to 68-fold. The degree of resistance was dependent upon the method of selection. The drug-resistant cell lines also developed low levels of cross-resistance to cadmium. Additional cell lines established from untreated patients or ovarian cancer patients refractory to cisplatin- and/or carboplatin-containing combination chemotherapy were studied. The most cisplatin-resistant cell lines, OVCAR-8 and -10, were from patients previously treated with intensive chemotherapy. OVCAR-8 was relatively cross-resistant to cadmium while OVCAR-10 appeared relatively sensitive. Cell lines were examined for expression of metallothionein mRNA to evaluate the relationship between cisplatin resistance, cadmium cross-resistance and metallothionein expression. Only two of the cell lines with in vitro-induced resistance to cisplatin, 2780E80 and 2780CP70B3, had detectable metallothionein mRNA. The other cell lines selected in vitro for cisplatin resistance, as well as the parental A2780 ovarian cancer cell line, showed no expression at our level of detection. There was variable expression of metallothionein among the OVCAR cell lines. Cell lines from untreated patients, OVCAR-5 and -7, did express metallothionein, while the most cisplatin-resistant cell lines, OVCAR-8 and -10, did not. We also examined cisplatin induction of metallothionein mRNA in the cell lines. Only 2780CP70B3 among the cell lines with in vitro-induced cisplatin resistance showed increased expression after short-term exposure to cisplatin. OVCAR-4 also had a slight increase in expression after exposure to cisplatin. Mouse C127 cells transfected with a bovine papilloma virus-metallothionein gene construct were compared for cisplatin sensitivity to the same cell type transfected with bovine papilloma virus alone. In this model system, metallothionein expression did not influence cisplatin cytotoxicity. On the basis of these studies, we conclude that there is no causal relationship between metallothionein expression and cisplatin resistance.

Cadmium↗
PubMed1990

Introduction of internal cysteines as conformational probes in yeast phosphoglycerate kinase.

Several mutants of yeast phosphoglycerate kinase, each containing only one internal cysteine residue, were constructed from a single mutant devoid of cysteine. These cysteines were introduced as local conformational probes in selected buried positions. The enzyme activity, conformational characteristics and stability indicated that the mutations introduced only small perturbations in the molecule. The folding-unfolding process mediated by guanidine hydrochloride under equilibrium conditions was studied by following the variations in ellipticity and the reactivity of the cysteine residue towards 5,5'-dithiobis(nitrobenzoate). The process was found to be reversible except for mutant C97A, V49C, suggesting that this region located in helix I might be crucial in determining an intermediate on the folding pathway. The transitions obtained by the two signals did not coincide, indicating that the local structures, in several parts inside the molecule, are more sensitive to the denaturant than the overall conformation.

Catalysis↗
PubMed1990

Site-directed mutagenesis of aspartic acid 372 at the ATP binding site of yeast phosphoglycerate kinase: over-expression and characterization of the mutant enzyme.

A new phosphoglycerate kinase over-expression vector, pYE-PGK, has been constructed which greatly facilitates the insertion and removal of mutant enzyme genes by cleavage at newly introduced BamHI sites. This vector has been used to prepare mutant protein in appreciable (100 mg) quantities for use in kinetic, crystallographic and NMR experiments. Aspartate 372 is an invariant amino acid residue in genes known to code for a functionally active PGK. The function of this acidic residue appears to be to help desolvate the magnesium ion complexed with either ADP or ATP when this substrate binds to the enzyme. Both crystallographic and nuclear magnetic resonance experiments show that the replacement of the residue with asparagine has only minimal effects on the overall structure. The substitution of the charged carboxyl group with that of the neutral amide affects the binding of the nucleotide substrate as predicted but not, as might have been expected, the binding of 3-phosphoglycerate. The overall velocity of the enzymic reaction (Vmax) is reduced 10-fold by the substitution of aspartic acid 372 by an asparagine residue (D372N). This reduction in Vmax is considerably less than one would expect from its known position within the structure of the enzyme. This result therefore poses questions about our understanding of charged groups at the active centres of enzymes and of the reason for their apparent conservation.

Adenosine Triphosphate↗
PubMed1990

An 18-kDa androgen-regulated protein that modifies galactosyltransferase activity is synthesized by the rat caput epididymidis, but has no structural similarity to rat milk alphalactalbumin.

Galactosyltransferase and alphalactalbumin-like activities have been reported to be present in the post-testicular fluids of the male reproductive tract. In the lactating mammary gland, these activities constitute the lactose synthetase complex. Kinetic parameters and acceptor specificities previously reported, along with recent amino acid sequence analysis argue against the mammary gland and epididymal activities being products of the same gene. In this paper we present cell-free translation of rat epididymal mRNA and Northern blot analysis of epididymal mRNA hybridized with authentic rat alpha-lactalbumin cDNA supporting this lack of identity and describe the differential synthesis and secretion of the androgen-regulated 18 kDa component of the so-called rat epididymal alphalactalbumin-like complex along the length of the epididymis. We conclude that although the 18 kDa component of the so-called epididymal alphalactalbumin moiety (E alpha LA) is capable, in common with a number of unrelated molecules, of modifying galactosyltransferase acceptor specificity in vitro, there is no primary structural similarity between it and authentic rat mammary alphalactalbumin. In view of the fact that the activity of E alpha LA is 1/100th that of authentic milk alphalactalbumin, we suggest that it may not be of physiological importance and that modification of galactosyltransferase activity may not be the function of the 18 kDa molecule.

Amino Acid Sequence↗
PubMed1990

Identification of the Epstein-Barr virus terminal protein gene products in latently infected lymphocytes.

The terminal protein (TP) gene produces two overlapping mRNAs in latently infected lymphocytes that are predicted to encode the similar polypeptides TP1 (497 amino acids) and TP2 (378 amino acids), with TP1 exon 1 providing 119 extra unique residues at the N terminus. Rabbit antisera were raised to procaryotic fusion proteins and used to detect expression of a predicted 53-kilodalton (kDa) TP product in transfected 293 cells and latently infected lymphocytes. Fractionation of transfected 293 cells showed this protein to be localized to an integral membrane preparation. The same fraction of latently infected lymphocytes contained proteins of 53 and 27 to 39 kDa as determined by Western immunoblotting with the TP-specific rabbit antisera. Immunoprecipitation of TP products from 35S-labeled human lymphoblastoid cells (CR/B95-8) was used in pulse-chase experiments and showed that TP1 was a labile protein with a half-life of approximately 2 to 4 h. The anti-fusion protein serum detected a 53-kDa TP1 and degradation products in the range of 25 to 35 kDa. A panel of Burkitt's lymphoma cell lines and cell lines established with virus recovered from the BL cells were analyzed by Western immunoblotting and found to contain the 53-kDa TP1 product, its degradation products, or both. Only two EBV-positive BL cell lines (BL72 and Wewak II) were negative in this assay. The results suggest that a labile TP1 protein may be expressed by most, if not all, EBV-infected cell lines.

Antibodies, Monoclonal↗