Overexpression of myoglobin and assignment of its amide, C alpha and C beta resonances.

Sperm whale apomyoglobin was expressed to high levels on minimal media and isotopically labeled with 13C and 15N nuclei. The isotopically labeled apoprotein was purified to homogeneity in a single step by reversed-phase chromatography and reconstituted with hemin and carbon monoxide gas for NMR analysis. Sequence-specific backbone 1HN, 15N and 13C alpha as well as side-chain 13C beta resonance assignments have been made for over 90% of the amino acids in the carbon monoxide complex of the protein.

Postnatal development of the calcium-dependency of glutamate release from rat cortical synaptosomes: comparison with 5-hydroxytryptamine release.

The ontogeny of the Ca(2+)-dependency of the depolarisation-induced release of preloaded [3H]glutamate and [3H]5-hydroxytryptamine [5-HT] from rat cortical synaptosomes was examined. 5-HT release was found to be exclusively Ca(2+)-dependent at all ages studied. In contrast, glutamate release only showed a significant Ca(2+)-dependent component from postnatal day 10 [PND 10] onwards.

In vitro activity of minimised hammerhead ribozymes.

A number of minimised hammerhead ribozymes (minizymes) which lack stem II have been kinetically characterised. These minizymes display optimal cleavage activity at temperatures around 37 degrees C. The cleavage reactions of the minizymes are first order in hydroxide ion concentration up to around pH 9.

Mammalian alanine/glyoxylate aminotransferase 1 is imported into peroxisomes via the PTS1 translocation pathway. Increased degeneracy and context specificity of the mammalian PTS1 motif and implications for the peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1.

Alanine/glyoxylate aminotransferase 1 (AGT) is peroxisomal in most normal humans, but in some patients with the hereditary disease primary hyperoxaluria type 1 (PH1), AGT is mislocalized to the mitochondria. In an attempt to identify the sequences in AGT that mediate its targeting to peroxisomes, and to determine the mechanism by which AGT is mistargeted in PH1, we have studied the intracellular compartmentalization of various normal and mutant AGT polypeptides in normal human fibroblasts and cell lines with selective deficiencies of peroxisomal protein import, using immunofluorescence microscopy after intranuclear microinjection of AGT expression plasmids. The results show that AGT is imported into peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) translocation pathway.

Epilepsy and children's social and psychological adjustment.

This research investigates the effects of epilepsy on the social and psychological adjustment of the children studied in the National Health Interview Survey of 1988. Analyses examine the effect of epilepsy on four measures of adjustment--home behavior problems, school behavior problems, depressed mood, and impulsiveness. For each outcome, we address five questions: (1) Do children with currently active epilepsy have poorer adjustment than children with inactive epilepsy? (2) Do children with epilepsy fare worse than other children? (3) Do demographic background and family structure moderate the apparent effects of epilepsy? (4) Do family processes mediate the apparent effects of epilepsy? and (5) Do cooccurring conditions produce the apparent effects of epilepsy? Generally, we find that: (1) Children with active and inactive epilepsy fare about equally; (2) Children with any history of epilepsy fare worse than children without epilepsy; (3) Demographic and family background moderate only a small part of epilepsy's effect; and (4) A combination of family processes and cooccurring conditions appears to produce epilepsy's apparent effects.

Popliteal vein thrombosis associated with femoral osteochondroma and popliteal artery pseudoaneurysm.

Deep vein thrombosis is a common condition thought to be caused by impaired venous blood flow or hypercoagulable blood states. However, often no predisposing cause can be found. We describe a deep vein thrombosis formed in association with femoral osteochondroma and popliteal artery pseudoaneurysm.

Calmodulin stabilizes an amphiphilic alpha-helix within RC3/neurogranin and GAP-43/neuromodulin only when Ca2+ is absent.

Two neuronal protein kinase C substrates, RC3/neurogranin and GAP-43/neuromodulin, preferentially bind to calmodulin (CaM) when Ca2+ is absent. We examine RC3.CaM and GAP-43.

Development of nonpolar surfaces in the folding of Escherichia coli dihydrofolate reductase detected by 1-anilinonaphthalene-8-sulfonate binding.

The development of nonpolar surfaces during the folding of Escherichia coli dihydrofolate reductase (DHFR) was studied by monitoring the time-dependent fluorescence of 1-anilinonaphthalene-8-sulfonate (ANS) included in the refolding solution. Stopped-flow refolding experiments demonstrated a rapid increase in fluorescence intensity within the dead time of mixing (5 ms), indicating that the earliest detectable folding intermediate contains hydrophobic surfaces which are capable of binding ANS. A further increase in fluorescence intensity over the next 300 ms coincides with the formation of a set of four intermediates which are known to contain a specific tertiary contact [Kuwajima, K.

U.S. military working dogs with Vietnam service: definition and characteristics of the cohort.

We verified and corrected inaccuracies in descriptive profile information on military working dogs (MWDs) that died from 1965 to 1980 and were reported in the Armed Forces Institute of Pathology Registry of Veterinary Pathology. Using other available military records, we determined which dogs served in Vietnam. Identified were 3,895 MWDs with a unique identity tattoo that served in Vietnam, of which 2,389 served exclusively with U.

Using linkers to investigate the spatial separation of the conserved nucleotides A9 and G12 in the hammerhead ribozyme.

Two series of hammerhead-derived ribozymes, or 'minizymes', in which helix II has been replaced by linkers of non-nucleotidic moieties, have been synthesised by solid-phase methods. In the first series, the minizymes had linkers containing one, two, three, four or five repeated units of phosphopropanediol, so that the number of atoms in the chain connecting the 3'O of the conserved A9 to the 5'O of the conserved G12 varied from 7 to 31. In the second, more-limited series, the minizymes contained linkers of either tetra- or hexa-ethyleneglycol.

From hemobiology to vascular disease: a review of the potential of gliclazide to influence the pathogenesis of diabetic vascular disease.

Patients with type II diabetes commonly die from thrombotic vascular disease. Large vessel occlusion due to thrombosis or atherosclerotic stenosis is a process accelerated by diabetes and results in premature death. Diabetic small vessel disease, with its unique microangiopathic process, underlies many of the large vessel changes as well as causing retinopathy and nephropathy.

Evolution of alanine:glyoxylate aminotransferase 1 peroxisomal and mitochondrial targeting. A survey of its subcellular distribution in the livers of various representatives of the classes Mammalia, Aves and Amphibia.

As part of a wider study on the molecular evolution of alanine:glyoxylate aminotransferase 1 (AGT1) intracellular compartmentalization, we have determined the subcellular distribution of immunoreactive AGT1, using postembedding protein A-gold immunoelectron microscopy, in the livers of various members of the classes Mammalia, Aves, and Amphibia. As far as organellar distribution is concerned, three categories could be distinguished. In members of the first category (type I), all, or nearly all, of the immunoreactive AGT1 was concentrated within the peroxisomes.

Learning through experience: an evaluation of 'Hospital at Home'.

A Hospital at Home service for children was begun in April 1991 in a rural health authority in England. After 18 months a mainly qualitative evaluation has shown that the children and parents concerned have been very satisfied with the service and would choose the same type of care again. General practitioners and health visitors felt it was to the benefit of the whole family for the child to be at home while ill.

A new library of alpha-lytic protease S1 mutants generated by combinatorial random substitution.

Four positions in the S1 site of alpha-lytic protease (positions 190, 192, 213 and 218) were subjected to targeted random mutagenesis. In the resulting library we found active mutant proteases whose cleavage preferences could be grouped into three distinct families. Some potentially useful enzymes (such as one that prefers to cleave at Asn and Cys residues) were identified.

Cytosolic compartmentalization of hepatic alanine:glyoxylate aminotransferase in patients with aberrant peroxisomal biogenesis and its effect on oxalate metabolism.

Two patients with atypical manifestations of aberrant peroxisomal biogenesis are described. Contrary to previous studies, which had shown that Zellweger syndrome patients usually have normal levels of urinary oxalate excretion, the patients in the present study had evidence of abnormal oxalate metabolism in the form of hyperoxaluria and, in one of the patients, calcium oxalate urolithiasis. Activity of the liver-specific peroxisomal enzyme alanine:-glyoxylate aminotransferase (AGT), which is a major determinant of the level of endogenous oxalate synthesis in humans, was normal in one patient and markedly supranormal in the other.

Primary hyperoxaluria type 1: genotypic and phenotypic heterogeneity.

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disease caused by a deficiency of the liver-specific peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT). The disease is notable for its extensive heterogeneity at the clinical, biochemical, enzymic and molecular genetic levels. A study of 116 PH1 patients over the past 8 years has revealed four main enzymic phenotypes: (1) absence of both AGT catalytic activity and immunoreactive AGT protein (approximately 40% of patients); (2) absence of AGT catalytic activity but presence of immunoreactive protein (approximately 16% of patients); (3) presence of both AGT catalytic activity and immunoreactive protein (approximately 41% of patients), in most of which cases the AGT is mistargeted to the mitochondria instead of the peroxisomes; and (4) a variation of the mistargeting phenotype in which AGT is equally distributed between peroxisomes and mitochondria, but in which that in the peroxisomes is aggregated into matrical core-like structures (approximately 3% of patients).

Multiple domains in a ribozyme construct confer increased suppressive activity in monkey cells.

An expression vector designed to express a long (approximately 1 kb) RNA containing multiple ribozyme domains was cotransfected into mammalian cells with a plasmid encoding, as target, messenger RNA for chloramphenicol acetyltransferase (CAT). In comparative studies the multimeric ribozyme construct proved to be significantly more effective at suppressing CAT expression than either the corresponding antisense RNA, or a transcript carrying a single ribozyme domain. Suppression of gene activity was apparently specific because expression of an independently expressed gene for human growth hormone was unaffected.

The potential of gliclazide, a sulphonylurea to influence the oxidative processes within the pathogenesis of diabetic vascular disease.

The clinical studies have demonstrated that patients showed significant improvements in their oxidative status after 3 months treatment. This improvement in oxidative status which was associated with a reduction in platelet reactivity, remained constant for the rest of the study period. The effect was independent of glycaemic control.

Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin.

Hydrogen exchange pulse labeling and stopped-flow circular dichroism were used to establish that the structure of the earliest detectable intermediate formed during refolding of apomyoglobin corresponds closely to that of a previously characterized equilibrium molten globule. This compact, cooperatively folded intermediate was formed in less than 5 milliseconds and contained stable, hydrogen-bonded secondary structure localized in the A, G, and H helices and part of the B helix. The remainder of the B helix folded on a much slower time scale, followed by the C and E helices and the CD loop.

An engineered PGK promoter and lac operator-repressor system for the regulation of gene expression in mammalian cells.

Previous reports have demonstrated that the Escherichia coli lac repressor can operate effectively in mammalian cells to repress expression of genes driven by modified viral or metallothionein (MT) promoters. We have developed a more general expression system using the promoter from the PGK1 gene (encoding murine 3-phosphoglycerate kinase) which is widely expressed in almost all cell types, including early embryonic and ES (embryonic stem) cells. Firstly, we engineered the lac repressor to include a nuclear localisation signal and placed it under control of the PGK1 promoter.

Enzymological and mutational analysis of a complex primary hyperoxaluria type 1 phenotype involving alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting and intraperoxisomal aggregation.

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disease caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). Three unrelated PH1 patients, who possess a novel complex phenotype, are described. At the enzymological level, this phenotype is characterized by a complete, or nearly complete, absence of AGT catalytic activity and reduced AGT immunoreactivity.