Biomedical subjects

Michael Mitchell

Publications and source records attributed to Michael Mitchell.

10 recordsLinked to original sources

PubMed2003

Directed evolution of N-acetylneuraminic acid aldolase to catalyze enantiomeric aldol reactions.

Expanding the scope of substrate specificity and stereoselectivity is of current interest in enzyme catalysis. Using error-prone PCR for in vitro directed evolution, the Neu5Ac aldolase from Escherichia coli has been altered to improve its catalytic activity toward enantiomeric substrates including N-acetyl-L-mannosamine and L-arabinose to produce L-sialic acid and L-KDO, the mirror-image sugars of the corresponding naturally occurring D-sugars. The first generation variant containing two mutations (Tyr98His and Phe115Leu) outside the (alpha,beta)(8)-barrel active site exhibits an inversion of enantioselectivity toward KDO and the second generation variant contains an additional amino acid change Val251Ile outside the alpha,beta-barrel active site that improves the enantiomeric formation of L-sialic acid and L-KDO. The X-ray structure of the triple mutant epNanA.2.5 at 2.3A resolution showed no significant difference between the wild-type and the mutant enzymes. We probed the potential structural 'hot spot' of enantioselectivity with saturation mutagenesis at Val251, the mutated residue most proximal to the Schiff base forming Lys165. The selected variant had an increase in k(cat) via replacement with another hydrophobic residue, leucine. Further sampling of a larger sequence space with error-prone PCR selected a third generation variant with significant improvement in L-KDO catalysis and a complete reversal of enantioselectivity.

Aldehydes↗

PubMed2003

Evidence for a role for Galphai1 in mediating weak agonist-induced platelet aggregation in human platelets: reduced Galphai1 expression and defective Gi signaling in the platelets of a patient with a chronic bleeding disorder.

We have examined platelet functional responses and characterized a novel signaling defect in the platelets of a patient suffering from a chronic bleeding disorder. Platelet aggregation responses stimulated by weak agonists such as adenosine diphosphate (ADP) and adrenaline were severely impaired. In comparison, both aggregation and dense granule secretion were normal following activation with high doses of collagen, thrombin, or phorbol-12 myristate-13 acetate (PMA). ADP, thrombin, or thromboxane A2 (TxA2) signaling through their respective Gq-coupled receptors was normal as assessed by measuring either mobilization of intracellular calcium, diacylglycerol (DAG) generation, or pleckstrin phosphorylation. In comparison, Gi-mediated signaling induced by either thrombin, ADP, or adrenaline, examined by suppression of forskolin-stimulated rise in cyclic AMP (cAMP) was impaired, indicating dysfunctional Galphai signaling. Immunoblot analysis of platelet membranes with specific antiserum against different Galpha subunits indicated normal levels of Galphai2,Galphai3,Galphaz, and Galphaq in patient platelets. However, the Galphai1level was reduced to 25% of that found in normal platelets. Analysis of platelet cDNA and gDNA revealed no abnormality in either the Galphai1 or Galphai2 gene sequences. Our studies implicate the minor expressed Galphai subtype Galphai1 as having an important role in regulating signaling pathways associated with the activation of alphaIIbbeta3 and subsequent platelet aggregation by weak agonists.

Adenosine Diphosphate↗

PubMed2003

Usp9y (ubiquitin-specific protease 9 gene on the Y) is associated with a functional promoter and encodes an intact open reading frame homologous to Usp9x that is under selective constraint.

Sequences complementary to the X-linked ubiquitin-specific protease gene Usp9x (Dffrx) have been shown to map to the Sxr(b) interval of the mouse Y Chromosome (chr) and to be expressed in a testis-specific manner. In humans, ubiquitously expressed functional homologues (USP9Y and USP9X DFFRY/DFFRX) are present on both sex chromosomes, whereas in mouse it remains to be demonstrated that the Y-linked sequences encode a functional protein. In this paper, it is shown that the Usp9y gene encodes a potentially functional ubiquitin-specific protease possessing a core promoter region that shares several features characteristic of other testis-specific genes. Analysis of synonymous and nonsynonymous nucleotide changes suggests that there is constraint on the amino acid sequence of both the mouse Usp9x and Usp9y genes, a finding that mirrors similar analysis of the human orthologs. Thus, in both mouse and human, selection is acting to maintain the amino acid sequence of the X and Y-linked genes. This indicates that in both species the genes on each sex chromosome continue to encode an important function.

Amino Acid Sequence↗

PubMed2003

Minimally invasive mitral valve repair suggests earlier operations for mitral valve disease.

OBJECTIVE: We began minimally invasive mitral valve surgery in August, 1996, to reduce hospital costs, to improve patient recovery, cosmetic appearance, and to decrease trauma, yet maintain the same quality of surgery. To validate this approach we reviewed our entire experience through May 2002. METHODS: From August 1996 to May 2002, we performed 413 minimally invasive mitral valve operations including 51 mitral valve replacements and 362 mitral valve repairs. Excluding 4 robotically assisted repairs, we evaluated 358 patients, using the mitral valve repairs as the basis for this retrospective survey. These operations were performed through a 6- to 8-cm minimally invasive incision, beginning with parasternal and, most recently, lower ministernotomy (181 patients). The mitral valve reparative techniques include repair of 94 prolapsed anterior leaflets, posterior leaflet resection, leaflet advancement, commissuroplasty, Polytetrafluoroethylene (PTFE; Gore-Tex, W. L. Gore & Associates, Inc, Flagstaff, Ariz) chordal placement, and ring annuloplasty. Cannulation sites varied but primarily utilized a miniaturized system of 24F catheters in both the inferior and superior venae cavae with assisted venous suction. The Cosgrove ring was used in 95% of the patients undergoing this procedure. RESULTS: The operative mortality was 0/358. Perioperative morbidity included a 26% incidence of new atrial fibrillation, 2% incidence of pacemaker implantation, 0.5% incidence of deep sternal wound infection, and 1.9% incidence of stroke after an operation. There were 10 arterial and 3 venous complications. The mean length of stay was 6 days and 208 patients stayed < or =5 days. Only 25% of the patients underwent homologous blood transfusion. The mean follow-up was 36 months with 1.4% lost to follow-up. There were 12 late deaths and a survival at 5 years of 95%. There were 21 valves requiring reoperation for structural valve failure of 5.8%. The probability of freedom from reoperation at 5 years was 92%. CONCLUSION: This study documents the safety of minimally invasive mitral valve repair surgery in 358 patients. It also documents a low incidence of homologous blood use, requirement for post-hospital rehabilitation, and general morbidity.

Adolescent↗

PubMed2003

Eighteen unrelated patients with factor XI deficiency, four novel mutations and a 100% detection rate by denaturing high-performance liquid chromatography.

Factor XI (FXI) deficiency is an autosomal bleeding disorder of variable severity. Inheritance is not completely recessive as heterozygotes may display a distinct, if mild, bleeding tendency. Eighteen unrelated FXI-deficient patients were screened blind by fluorescent single-stranded conformation polymorphism (F-SSCP) analysis and denaturing high-performance liquid chromatography (dHPLC). Mutations were detected in 14 of the 18 patients ( approximately 78%) by F-SSCP and in all 18 patients by dHPLC. Dideoxy sequencing confirmed the mutations in all 18 patients: eight of the mutations being novel (four of which were in previously reported patients). This showed dHPLC to be a highly sensitive, reliable technique for mutation screening in heterogeneous disorders.

Chromatography, High Pressure Liquid↗

PubMed2002

Absence of p16INK4a and truncation of ARF tumor suppressors in chickens.

The INK4b-ARF-INK4a locus on human chromosome 9p21 (Human Genome Organization designation CDKN2B-CDKN2A), and the corresponding locus on mouse chromosome 4, encodes three distinct products: two members of the INK4 cyclin-dependent kinase inhibitor family and a completely unrelated protein, ARF, whose carboxyl-terminal half is specified by the second exon of INK4a but in an alternative reading frame. As INK4 proteins block the phosphorylation of the retinoblastoma gene product and ARF protects p53 from degradation, the locus plays a key role in tumor suppression and the control of cell proliferation. To gain further insights into the relative importance of INK4a and ARF in different settings, we have isolated and characterized the equivalent locus in chickens. Surprisingly, although we identified orthologues of INK4b and ARF, chickens do not encode an equivalent of INK4a. Moreover, the reading frame for chicken ARF does not extend into exon 2, because splicing occurs in a different register to that used in mammals. The resultant 60-aa product nevertheless shares functional attributes with its mammalian counterparts. As well as indicating that the locus has been subject to dynamic evolutionary pressures, these unexpected findings suggest that in chickens, the tumor-suppressor functions of INK4a have been compensated for by other genes.

Amino Acid Sequence↗

PubMed2002

Micropenis with testicular regression, low LH levels, and poor androgen and HCG responses: a distinct syndrome?

We report three boys, including two brothers, with micropenis and poor phallic growth in response to both exogenous human chorionic gonadotropin (HCG) and testosterone therapy in the newborn period. They exhibited low neonatal testosterone levels that failed to respond to HCG stimulation. These boys displayed a unique gonadotropin profile with reduced luteinizing hormone levels and elevated follicle-stimulating hormone levels. They had small, cryptorchid testes with subsequent testicular regression and atrophy. Moreover, all three boys have developed microcephaly and mild learning delays. We review the hormonal profiles and phenotypes of known causes of micropenis, and compare them to the features of our three patients. Although individuals with similar features may have been described in past series of males with micropenis, the data presented previously were insufficient for classification; thus, we propose that our patients may represent a distinct, not previously recognized, syndrome with either X-linked recessive inheritance or autosomal recessive inheritance with male sex limitation.

Abnormalities, Multiple↗

PubMed2002

Fibroblast growth factor-10 is a mitogen for urothelial cells.

Fibroblast growth factor (FGF)-10 plays an important role in regulating growth, differentiation, and repair of the urothelium. This process occurs through a paracrine cascade originating in the mesenchyme (lamina propria) and targeting the epithelium (urothelium). In situ hybridization analysis demonstrated that (i) fibroblasts of the human lamina propria were the cell type that synthesized FGF-10 RNA and (ii) the FGF-10 gene is located at the 5p12-p13 locus of chromosome 5. Recombinant (r) preparations of human FGF-10 were found to induce proliferation of human urothelial cells in vitro and of transitional epithelium of wild-type and FGF7-null mice in vivo. Mechanistic studies with human cells indicated two modes of FGF-10 action: (i) translocation of rFGF-10 into urothelial cell nuclei and (ii) a signaling cascade that begins with the heparin-dependent phosphorylation of tyrosine residues of surface transmembrane receptors. The normal urothelial phenotype, that of quiescence, is proposed to be typified by negligible levels of FGF-10. During proliferative phases, levels of FGF-10 rise at the urothelial cell surface and/or within urothelial cell nuclei. An understanding of how FGF-10 works in conjunction with these other processes will lead to better management of many diseases of the bladder and urinary tract.

Amino Acid Sequence↗

PubMed2002

Perineal reconstruction of female conjoined twins.

BACKGROUND/PURPOSE: Conjoined twins are some of the most challenging patients faced by surgeons. Pygopagus and ischiopagus twins present particular gastrointestinal and genitourinary reconstructive challenges. This study reviews the authors' experience with the perineal reconstruction of these types of conjoined twins. METHODS: Retrospective analysis was performed for 3 sets of female conjoined twins undergoing separation between 1999 and 2001. Particular attention was given to the separation and reconstruction of the distal gastrointestinal and urogenital structures. RESULTS: Three sets of female conjoined twins underwent successful separation 2 pygopagus, one ischiopagus tripus) with 5 surviving infants. The sixth infant died of congenital anomalies incompatible with life. Four of the 5 surviving infants had diverting enterostomies. Two of these enterostomies have been closed. Perineal reconstruction consisted of anoplasty (5 of 5), vaginoplasty (4 of 5), and urethroplasty (4 of 5). Although fecal and urinary continence are not completely measurable at this age (<3 years), all 5 survivors void spontaneously. Three infants with intestinal continuity have apparently normal defecation without the need of a bowel regimen. CONCLUSIONS: With careful preoperative planning and a multidisciplinary team of pediatric surgeons and urologists, satisfactory reconstruction and functional outcome of the female perineum can be achieved in conjoined twins.

Feasibility Studies↗

PubMed2002

A HIPAA primer.

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Arkansas↗