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Biomedical subjects

J R Miller

Publications and source records attributed to J R Miller.

At least 91 records · Page 5Linked to original sources
PubMed1995

Amino-acid substitutions in the cleavage site of acyl-coenzyme A:isopenicillin N acyltransferase from Penicillium chrysogenum: effect on proenzyme cleavage and activity.

Site-directed mutagenesis of the penDE gene and expression in Escherichia coli has produced recombinant acylcoenzyme A:isopenicillin N acyltransferase (re-AT) containing amino-acid substitutions in the proenzyme cleavage site (decreases) region (Asp-Gly102 decreases Cys103-Thr-Thr). The effect of these substitutions on proenzyme cleavage and AT activity has been investigated. The re-AT with substitutions at Cys103 (Cys103-->Ser, Cys103-->Ala and Cys103-->Trp) were uncleaved and inactive. Substitutions at Asp101 and Gly102 (Asp101-->Gly, Gly102-->Ala, Gly102-->Val, Gly102-->Met, Gly102-->Val and Asp101Gly102-->GlyPhe) did not prevent proenzyme cleavage or abolish AT activity. Thr105-->Ser and Thr105-->Ala substitutions did not prevent proenzyme cleavage or AT activity; however, AT containing Thr105-->Val resulted in a significant inhibition of proenzyme cleavage.

Acyltransferases↗
PubMed1995

The accuracy of duplex ultrasonography in the planning of skin flaps in the lower extremity.

The septocutaneous perforators represent one of the major sources of the blood supply to the skin of the lower extremity. Despite several well-described anatomic accounts, the location of lower leg septocutaneous perforators, as they originate from each of the three main infrapopliteal vessels in the leg, remains inconsistent as a result of individual anatomic variations. With the aid of duplex ultrasonography (color Doppler imaging), preoperative, mapping and size determination of these perforators can be provided. The skin paddle can then be designed to lie exactly over these perforators, ensuring blood supply to the skin paddle. The location and distribution of medial septocutaneous perforators in the leg, which originate from the posterior tibial artery, were mapped using anatomic dissections (29 lower extremities). These findings were then compared with duplex ultrasonographic data in 9 living volunteers (18 lower extremities). The medial septocutaneous perforators were chosen for this study because they course directly over the posterior tibial artery, making their location difficult to assess with standard Doppler techniques. The hand-held Doppler is incapable of distinguishing flow originating from the perforators versus the posterior tibial artery. No significant difference existed between cadaver and duplex distributions. "Large" perforator vessels (> 1 mm outer diameter) were evenly distributed with a central tendency at 140 to 150 mm from the medial malleolus. The distribution of "small" perforator vessels (< 1 mm outer diameter) was skewed. Fifty percent were found within 80 mm of the medial malleolus and the remainder spread proximally in the leg.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Fast food arthritis--a clinico-pathologic study of post-Salmonella reactive arthritis.

OBJECTIVE: To study the clinical presentation, immunogenetics, and serum immune response to lipopolysaccharide (LPS) in a cohort of patients with post-Salmonella reactive arthritis (ReA). METHODS: A validate ReA screening questionnaire (Quest 2) was mailed to 919 individuals reporting symptoms of gastroenteritis to the health department after eating at a single restaurant. Three hundred twenty-one persons returned questionnaires; 170 reported symptoms outside the gastrointestinal tract; 23 of those 170 reporting persistent joint symptoms were seen 4 to 16 weeks after the outbreak and 5 of the 23 were seen in followup 12 to 20 weeks later. Clinical features, HLA Class I typing, serum soluble CD8 levels, and serum antibodies to gram negative LPS by ELISA were determined. RESULTS: Joint complaints were reported more frequently by individuals with a longer duration of diarrhea. Upper extremity joints were frequently involved, and 66% reported one or more extraarticular symptoms of Reiter's syndrome. Three of 5 typed individuals were HLA-B27 positive, including 3 of the 4 most severely involved. Serum soluble CD8 levels correlated poorly with disease activity measured either clinically or by C-reactive protein. Antibodies to Klebsiella and Shigella LPS rose over time, while antibodies to Salmonella LPS fell. CONCLUSION: The clinical picture of post-Salmonella ReA is less stereotyped than often assumed, although severity correlated with HLA-B27 status. The association of joint symptoms with duration of diarrhea and the kinetics of the anti-LPS antibody response support the hypothesis that abnormal gut permeability plays a role in the pathogenesis of post-Salmonella ReA.

Antibodies, Bacterial↗
PubMed1994

Serine 228 is essential for catalytic activities of 85-kDa cytosolic phospholipase A2.

The Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) displays both a phospholipase A2 and a lysophospholipase activity. Numerous hydrolases, including lipases, catalyze the hydrolysis of ester bonds by means of an active site triad of amino acids that includes a serine or a cysteine residue. We have examined whether human cPLA2 belongs to this class of enzymes by using site-directed mutagenesis. Although chemical inactivation of cPLA2 by the sulfhydryl reagent N-ethylmaleimide made it appear that cysteine(s) may be essential for catalysis, all 9 cysteine residues of cPLA2 proved dispensable, allowing near-normal enzyme activity when substituted by alanine. We noted that cPLA2 contains a 110-amino-acid region with sequence homology to phospholipase B (PLB) from Penicillium notatum. Interestingly, one of the conserved serines of cPLA2, Ser-228, within this domain aligns with the lipase consensus sequence Gly-X(Leu)-Ser(137)-X(Gly)-Gly of PLB. Replacement of Ser-228 by alanine (or threonine or cysteine) yielded catalytically inactive cPLA2, even though the native conformation was maintained as determined by CD spectroscopy. Likewise, the lysophospholipase activity was completely abolished by the Ser-228 mutations. In contrast, substitution by alanine of three different serines of cPLA2 (Ser-195, Ser-215, or Ser-577) that also aligned with the PLB sequence allowed for substantial enzymatic activity of cPLA2. Our findings provide evidence that 1) Ser-228 participates in the catalytic mechanism of cPLA2 and that 2) both the phospholipase A2 and the lysophospholipase activities of cPLA2 are catalyzed by the same active site residue(s).

Amino Acid Sequence↗
PubMed1994

Acyl-coenzyme A: isopenicillin N acyltransferase from Penicillium chrysogenum: effect of amino acid substitutions at Ser227, Ser230 and Ser309 on proenzyme cleavage and activity.

Using a high level Escherichia coli expression system for the Penicillium chrysogenum penDE gene, we have produced acyl-coenzyme A: isopenicillin N acyltransferase (AT) enzymes containing amino acid substitutions at three conserved Ser residues. Chosen for study based on amino acid sequence homologies to other proteins, Ser227, Ser230 and Ser309 were changed to Cys or Ala to assess amino acid side chain involvement in proenzyme cleavage and AT enzyme mechanism. Substitutions at Ser230 had no effect on proenzyme cleavage, acyl-coenzyme A: IPN acyltransferase (IAT) or acyl-coenzyme A:6-aminopenicillanic acid acyltransferase (AAT) activities. While Ser227-->Cys had no effect, Ser227-->Ala produced uncleaved proenzyme lacking both AAT and IAT activities, suggesting that the presence of a nucleophilic side chain at this residue is required for proenzyme cleavage and AT activity. Substitution of Ser309-->Cys did not appreciably prevent proenzyme cleavage, IAT or AAT activity. Recombinant AT (recAT) proenzyme containing Ser309-->Ala was cleaved; however, IAT and AAT activities were not observed. This separation of proenzyme cleavage from IAT and AAT activities has not been previously observed, and suggests that Ser309 is involved in substrate acylation.

Acyltransferases↗
PubMed1994

Use of porcine interspersed repeat sequences in PCR-mediated genotyping.

PCR primers derived from porcine short and long interspersed repeat sequences were used to amplify DNA samples isolated from individual members of three-generation pig reference pedigrees. Subsequent high-resolution gel electrophoresis of both SINE and LINE-PCR products allowed direct visualisation of polymorphisms that segregated in a Mendelian manner. Additional polymorphisms were detected by Southern blotting of the gels described above followed by hybridization with simple sequence DNA. Genotyping by interspersed repeat-PCR exploits the natural architecture of the pig genome and allows the typing of polymorphisms by utilizing pre-existing sequence information.

Animals↗
PubMed1994

Characterization by electrospray mass spectrometry of human Ca(2+)-sensitive cytosolic phospholipase A2 produced in baculovirus-infected insect cells.

The 85-kD cytosolic phospholipase A2 (cPLA2) is a novel receptor-regulated phospholipase that is thought to initiate the production of inflammatory lipid mediators. Since cPLA2 is present only in minute amounts (less than 0.01% of total cellular protein) in various cells and tissues, we have used the baculovirus expression system to produce sufficient quantities of cPLA2 for structural and functional analysis. The cDNA for cPLA2 was cloned into a baculovirus expression vector and, upon infection of Spodoptera frugiperda Sf-21 cells with the recombinant virus, cPLA2 was produced at high levels (9% of total cellular soluble protein). Gel electrophoresis and immunoblot analysis demonstrated that the recombinant protein has properties indistinguishable from cPLA2 present in human monocytic U937 cells. Structural analysis of recombinant cPLA2, using electrospray mass spectrometry in conjunction with automated sequence analysis, confirmed the expected sequence and revealed two post-translational modifications of the protein, phosphorylation on at least one site, and acetylation of the N-terminal serine residue after removal of the initiating methionine. In spite of the presence of six potential N-glycosylation sites, there is no evidence that any of them is glycosylated. The baculovirus expression system should prove useful for production of cPLA2, and electrospray mass spectrometry is a rapid and accurate method for the analysis of post-translational modifications.

Amino Acid Sequence↗
PubMed1994

Substance abuse: the role of depression and trauma--a case report.

Substance abuse is associated diagnostically with existing depression, pathological mourning of the traumatic event itself, with the inevitable experience of object loss, and loss of love. Inability to form object representation insures primitive lack of object constancy, resulting in pre-oedipal longing and neediness and malignant affect. Ambivalence towards the sadistic love objective is recognized and allows for a healthier, more complete period of mourning (Freud, 1917). Indeed, depression is banished from family acknowledgement in the service of homeostatic denial. This only reinforces the identified patient's feeling of unreality, shame and guilt over verbalizing the depression and the frequent underlying traumatic history. The enormous role of socio-economic stress is pervasive, yet the focus here is on individuals with complex dynamic constellations which are often ignored in the face of external factors. Addiction as attempt to self-medicate depression and trauma remains the overarching theme.

Adult↗
PubMed1993

The requirement for subunit interaction in the production of Penicillium chrysogenum acyl-coenzyme A:isopenicillin N acyltransferase in Escherichia coli.

Subunit interaction in the formation of active acyl-coenzyme A:isopenicillin N acyltransferase (AT) has been investigated. Various AT derivatives were produced from altered Penicillium chrysogenum penDE genes placed in Escherichia coli expression systems. The regions of penDE encoding the alpha (11 kDa) and beta (29 kDa) AT subunits were separated at the DNA level by linker insertion at the region encoding Gly102/Cys103. Synthesis of AT from the resulting two-cistron mRNA resulted in active alpha,beta-heterodimeric recombinant AT (reAT), containing subunits of 11 and 29 kDa (similar to wild-type AT). Complete separation of the alpha and beta subunits was performed by placing the region of penDE encoding each subunit on different plasmids. Production of either subunit in the absence of the other did not form active reAT. However, cotransformation of E. coli with two plasmids, each encoding a different AT subunit, produced reAT having acyl-coenzyme A:6-aminopenicillanic acid (acyl-CoA:6-APA) AT activity. Mutation of penDE replacing Thr105 with Asn resulted in inactive and uncleaved reAT. Coexpression of this mutant penDE with a penDE derivative encoding the beta subunit in E. coli produced acyl-CoA:6-APA AT activity. These results suggest that the formation of reAT involves cooperative folding events between the subunits. In vitro transcription/translation was used to determine the origin of the AT hydrolase activity that cleaves the 40-kDa precursor polypeptide. The appearance of a 29-kDa protein (and presumably the corresponding 11-kDa protein, although not observable) from the 40-kDa in vitro translated protein provides further evidence that AT hydrolysis is an autocatalytic event.

Acyl Coenzyme A↗
PubMed1993

5' and 3' SINE-PCR allows genotyping of pig families without cloning and sequencing steps.

A microsatellite-containing clone, isolated from a pig Chromosome (Chr) 1-specific library was characterized by sequencing and computer analysis. The (CA)17 microsatellite motif was located at the 3' end of a short interspersed element (SINE) sequence at the position normally occupied by the oligo (A) stretch. Further computer analysis indicated that 12% of published pig SINE sequences contain dinucleotide repeat motifs adjacent to their 3' ends. By performing PCR with a single SINE primer in combination with a panel of arbitrarily selected unique primers, we have demonstrated that, as in human, polymorphisms can be detected and typed in pig family DNAs. A large number of SINE primer x unique primer combinations have been screened for the ability to detect polymorphisms in pig reference family DNAs. This approach does not require prior sequence information other than that of the pig SINE. We have also found polymorphisms at the 5' ends of pig SINE sequences by similar methods, but with a primer facing out to the 5' end of the SINE.

Animals↗