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Biomedical subjects

A A Smith

Publications and source records attributed to A A Smith.

At least 37 records · Page 2Linked to original sources
PubMed1996

Functional lower limb salvage with an osteocutaneous filet flap of the foot.

An osteocutaneous foot filet flap based on the posterior tibial vessels was successfully used to provide tibial coverage in a patient requiring a below-knee amputation following a high-voltage electrical injury. Addition of the calcaneus to the standard foot filet flap provided a vascularized bone graft that served to both lengthen the tibia and secure the flap via a tibial-calcaneal synostosis. The synostosis provided firm anchoring of the flap and allowed for a partial end-bearing, below-knee prosthesis.

Aged↗
PubMed1996

Muscle flap reconstruction aids in urethral regeneration.

Traditional methods of reconstructing full-thickness urethral defects have employed a cutaneous component utilized to replace the lining of the urethra. These methods have failed to take advantage of the regenerative ability of urethral epithelium. This epithelium is capable of regenerating, eliminating the need for urethral lining reconstruction. Muscle flap reconstruction provides an environment that allows for complete regeneration of the urethral epithelium. A 56-year-old male presented with a 12-cm defect of the bulbous and penile urethra involving 180 degrees of the urethral circumference secondary to Fournier's gangrene. A proximally pedicled gracilis muscle was used to reconstruct the urethral defect. This healed without stricture or leak. Urethral biopsies showed satisfactory migration of the uroepithelium across the urethral defect.

Epithelium↗
PubMed1995

Major contribution of a carboxymethyl group to transition-state stabilization by cytidine deaminase: mutation and rescue.

The crystal structure of an inhibitory complex formed between Escherichia coli cytidine deaminase and the transition-state analog 3,4-dihydrouridine indicates the presence of a short H-bond between Glu-104 and the inhibitor. To test the possibility that analogous H-bonds might play a significant role in stabilizing the hydrated substrate in the transition state for deamination, we replaced Glu-104 by alanine. Compared with the wild-type enzyme, the mutant enzyme's affinities for substrate cytidine and product uridine were found to have increased, whereas kcat for deamination of cytidine had been reduced by 8 orders of magnitude. By its presence, the carboxymethyl group of Glu-104 appears to minimize the activation barrier for deamination, not only by stabilizing the altered substrate in the transition state but also by destabilizing the enzyme-substrate and enzyme-product complexes. In the presence of added formate ion, but not in the presence of bulkier carboxylic acids, the low catalytic activity of the mutant enzyme was enhanced substantially.

Alanine↗
PubMed1995

Preliminary soft tissue distraction in congenital forearm deficiency.

Four patients (five limbs) were treated with preliminary soft tissue distraction using an Orthofix external distractor. The patient's ages ranged from 6 months to 14 years. Forearm deficiencies treated included partial absence of the radius (one patient), radial aplasia (two patients, three limbs), and partial absence of the ulna (one patient). Soft tissue distraction (mean, 55 days) was continued in radial deficiency until the hand could be passively centralized without residual radial deviation, allowing centralization to be accomplished through a single mid-dorsal incision. Mean measurement of distraction for the radial deficiencies was 1 cm. In the ulnar deficiency, soft tissue distraction was carried out until the cut radius was distal to the partially absent ulna (98 days) allowing for the creation of a one-bone forearm using the entire radius. Distraction (1 mm/day) was done by the children's parents with no distractor removed because of patient intolerance. All radial deficiency limbs remained centralized at a mean followup period of 14 months.

Adolescent↗
PubMed1995

Distal foot coverage with a reverse dorsalis pedis flap.

A reverse dorsalis pedis flap based on the proximal communicating branch of the dorsalis pedis artery was successfully used to close distal forefoot defects in two patients. The skin paddle was oriented transversely across the ankle crease, allowing for direct closure of the flap donor site. Both patients maintained full active ankle motion without bowstringing of the dorsal foot tendons and did not require special footwear. Both flaps maintained sensation to light touch and pinprick. We believe that this flap offers a viable alternative when faced with the challenge of a small soft-tissue defect requiring flap reconstruction in the distal foot.

Adult↗
PubMed1995

A vanadate hematoxylin stain for basic proteins.

Ammonium vanadate can act as both an oxidant and a mordant for hematoxylin. Lithium carbonate can remove vanadate hematoxylin from other structures so that only the most basic proteins are stained. Brief diazotization of the tissue sections restricts staining to the histone proteins of the nucleus.

Animals↗
PubMed1995

Postpericardiotomy syndrome: its diagnosis and treatment after muscle flap closure of an infected sternal wound.

After nearly any form of cardiac surgery, an acute febrile illness characterized by leukocytosis, pericarditis with pericardial effusion, and pleural effusion may occur. In some instances, this postpericardiotomy syndrome may suggest the presence of mediastinal infection. Treatment of postpericardiotomy syndrome is conservative, and symptoms typically resolve with nonsteroidal antiinflammatory medication. We report a case of postpericardiotomy syndrome that mimicked recurrent mediastinal infection and developed after muscle flap closure of an infected sternal wound. Pericardial, pleural, and periflap fluid accumulated postmuscle flap closure and was sterile on culture. A diagnosis of postpericardiotomy syndrome was made, and the patient improved while receiving oral indomethacin. She has remained free of infection as of the 2-year follow-up.

Diagnostic Errors↗
PubMed1995

Supplemental oxygen: ensuring its safe delivery during facial surgery.

Electrosurgical coagulation in the presence of blow-by oxygen is a potential source of fire in facial surgery. A case report of a patient sustaining partial-thickness facial burns secondary to such a flash fire is presented. A fiberglass facial model is then used to study the variables involved in providing supplemental oxygen when an electrosurgical unit is employed. Oxygen flow, oxygen delivery systems, distance from the oxygen source, and coagulation current levels were varied. A nasal cannula and an adapted suction tubing provided the oxygen delivery systems on the model. Both the "displaced" nasal cannula and the adapted suction tubing ignited at a minimum coagulation level of 30 W, an oxygen flow of 2 liters/minute, and a linear distance of 5 cm from the oxygen source. The properly placed nasal cannula did not ignite at any combination of oxygen flow, coagulation current level, or distance from the oxygen source. Facial cutaneous surgery in patients provided supplemental oxygen should be practiced with caution when an electrosurgical unit is used for coagulation. The oxygen delivery systems adapted for use are hazardous and should not be used until their safety has been demonstrated.

Accident Prevention↗
PubMed1995

The effect of acyl CoA: cholesterol acyltransferase inhibition on the uptake, esterification and secretion of cholesterol by the hamster small intestine.

Acyl CoA: cholesterol acyltransferase (ACAT) inhibitors are known to inhibit cholesterol absorption and are under investigation to reduce hypercholesterolemia. These studies examine the effect of an ACAT inhibitor 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)-dodecanamide (PD128042) on the uptake, metabolism and secretion of cholesterol by the hamster intestinal wall in a short-term model. Preliminary studies in this model indicated that the uptake of 14C-cholesterol and its subsequent esterification 2 hr postoral dosing occurs primarily in the duodenal and jejunal segments of the small intestine and most of the radiolabeled cholesterol and cholesteryl ester in the plasma was associated with chylomicrons. In both single- and multiple-dose studies, PD128042 (50 mg kg-1 day-1) did not inhibit intestinal uptake of [14C]-cholesterol but [14C]-cholesteryl ester formation was inhibited. The free [14C]-cholesterol appearing in plasma was not affected despite a large reduction in [14C]-cholesteryl ester. In contrast, cholestyramine (1 g kg-1 day-1) inhibited the uptake of the radiolabeled free cholesterol and the appearance of cholesteryl ester in the intestine and plasma. The effects of PD128042 on cholesterol and cholesteryl ester mass associated with scraped intestinal mucosa were consistent with the effects observed with the use of the radiolabeled cholesterol. In addition, PD128042 did not affect the uptake of appearance of radiolabeled triglyceride in the intestinal wall after oral gavage of 3H-trioleoylglycerol. Taken together, the data suggest that ACAT inhibition reduces cholesterol absorption by limiting cholesteryl ester incorporation into chylomicrons and has no effect on the intestinal processing of free cholesterol to be secreted into plasma.

Anilides↗
PubMed1994

Mutations affecting transition-state stabilization by residues coordinating zinc at the active site of cytidine deaminase.

Cytidine deaminase from Escherichia coli contains 1 mol of tightly bound zinc per enzyme subunit (Yang, C., Carlow, D., Wolfenden, R., & Short, S.A. (1992) Biochemistry 31, 4168-4174). When the metal liganding residues Cys-129 and Cys-132 were replaced by Ala, and His-102 was replaced by Ala, Asn, or Gln, deaminase activities of cell extracts containing these mutant enzymes were decreased by several orders of magnitude relative to that of the wild-type enzyme. After purification, each mutant protein was found to contain less than 0.2 mol of zinc per enzyme subunit, except mutant H102Q, which contained 1 mol of zinc per subunit. The activity of each mutant enzyme increased in the presence of added zinc but never attained wild-type activity. Mutant H102N was unique in that this protein could be purified as a stable apoenzyme, activated by added zinc, and then inhibited by EDTA. This mutant enzyme bound zinc with an apparent Kd value of 6.0 x 10(-10) M and regained maximal activity in the presence of 1 mol of zinc per subunit. Affinities of the mutant cytidine deaminases for the transition-state analogue, 5-fluoropyrimidin-2-one ribonucleoside (3,4) hydrate, were found to decrease in rough proportion to kcat/Km over a range spanning several orders of magnitude. This variation in catalytic efficiency arose mainly from effects on kcat, indicating the involvement of zinc coordination in the catalytic process rather than in substrate binding.

Binding Sites↗
PubMed1994

Demonstration of a direct effect on hepatic acyl CoA: cholesterol acyl transferase (ACAT) activity by an orally administered enzyme inhibitor in the hamster.

Orally active inhibitors of acyl CoA:cholesterol acyl transferase (ACAT), such as Lederle CL277082 (LE), are known to reduce plasma and hepatic cholesteryl ester levels, although the mechanisms are not well understood. Several groups have reported the inhibition of cholesterol absorption upon oral ACAT inhibitor administration. In this study, we used 7-day dietary and drug treatments of hamsters to examine the possible effects of LE on hepatic ACAT. ACAT assays were performed using liver homogenates in the absence and presence of a saturating level of exogenously added cholesterol. LE (100 mg/kg/day) treatment of chow or 0.5% cholesterol-fed animals caused reductions in ACAT activity without additional cholesterol as compared with non-treated animals. When a saturating level of cholesterol was added to the assays, reductions in ACAT activity upon LE treatment of chow- or cholesterol-fed animals were also observed. Treatment of cholesterol-fed animals with cholestyramine in the diet reduced ACAT activity in the absence of added cholesterol. However, ACAT activities similar to those of non-treated animals were observed at a saturating level of cholesterol. This latter effect demonstrates that inhibition of cholesterol absorption reduces cholesterol delivery to the liver but does not reduce cholesterol esterifying capacity since cholestyramine is not absorbed and has no direct effect on the liver. The decreased ACAT activity in homogenates from LE-treated animals could also be mimicked in a dose-dependent manner by the addition of exogenous LE to liver homogenates from non-treated animals. These results indicate that hepatic ACAT activity is regulated by the availability of free cholesterol, and that orally administered LE has a direct effect on hepatic ACAT activity in the liver. In addition, the data are consistent with LE activity in the liver as being responsible, in part, for the reduced hepatic and plasma cholesteryl esters in treated animals.

Animals↗
PubMed1994

Characterization of leucocin B-Ta11a: a bacteriocin from Leuconostoc carnosum Ta11a isolated from meat.

Leuconostoc (Lc.) carnosum Ta11a, isolated from vacuum-packaged processed meats, produced a bacteriocin designated leucocin B-Ta11a. The crude bacteriocin was heat stable and sensitive to proteolytic enzymes, but not to catalase, lysozyme, or chloroform. It was active against Listeria monocytogenes and several lactic acid bacteria. Leucocin B-Ta11a was optimally produced at 25 degrees C in MRS broth at an initial pH of 6.0 or 6.5. An 8.9-MDa plasmid in Leuconostoc carnosum Ta11a hybridized to a 36-mer oligonucleotide probe (JF-1) that was homologous to leucocin A-UAL187. A 4.9-kb Sau3A fragment from a partial digest of the 8.9-MDa plasmid was cloned into pUC118. The 8.1-kb recombinant plasmid (pJF8.1) was used for sequencing and revealed the presence of two open reading frames (ORFs). ORF1 codes for a protein of 61 amino acids comprising a 37-amino-acid bacteriocin that was determined to be the leucocin B-Ta11a structural gene by virtue of its homology to leucocin A-UAL 187 (Hastings et al. 1991. J. Bacteriol 173:7491-7500). The 24-amino-acid N-terminal extension, however, differs from that of leucocin A-UAL187 by seven residues. The predicted protein of the ORF2 has 113 amino acids and is identical with the amino acid sequence of the cognate ORF of the leucocin A-UAL 187 operon.

Amino Acid Sequence↗
PubMed1994

Donor site deficit of the osteocutaneous radial forearm flap.

A functional evaluation was made of osteocutaneous radial forearm flap donor upper extremities at a mean 19.4 months postoperatively. Donor deficits were evaluated as follows: (1) skin: subjective appearance and durability; (2) skeleton: range of motion of adjacent joints; (3) vessels: cold intolerance, digital temperature, digital--brachial index; (4) muscle: key pinch; (5) nerve: two-point discrimination of dorsal first web space. Ten consecutive patients with a mean donor site area of 51.5 cm2 and mean length of harvested radius of 10.9 cm were studied. Incomplete skin graft take at the donor site (7 of 10 patients) and appearance of fair or poor (6 of 10 patients) were frequent cutaneous complications. Wrist range of motion was decreased with pronation (90%), flexion (90%), and extension (89%). Mean digital temperature, comparing extremities that did (operated) and did not (nonoperated) undergo surgery, or comparing the radial artery-supplied fingers to the ulnar artery-supplied fingers within the same operated hand showed no effect from the lost radial artery. Mean digital-brachial index was 1.24 for the nonoperated and 1.15 for the operated extremity. Key pinch on the operated side was a mean of 74% of the nonoperated side. Although raising the flap creates multiple tissue deficits, each with the potential to alter upper extremity function, detailed functional evaluation failed to demonstrate significant alteration in upper extremity function after flap harvest.

Forearm↗
PubMed1992

The abolition of collagen gene expression in SV40-transformed fibroblasts is associated with trans-acting factor switching.

The goal of this study was to determine whether alpha 2(1) procollagen gene expression is modulated by positive or negative trans-acting DNA-binding proteins. Previous studies have shown that a clone of SV40-transformed WI-38 fibroblasts (SVWI-38) does not produce any alpha 2(1) procollagen mRNA (Parker et al (1989), J. Biol Chem. 264, 7147-7152). In order to elucidate the mechanism(s) responsible for such inactivation, we have examined the activity of a transfected wild type COL1A2 promoter in SVWI-38 cells. A set of 5' promoter deletions was linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into SVWI-38 and other cell lines expression type I collagen. The resulting CAT assays confirmed the importance of several upstream regions for promoter activity and documented the decreased transcriptional activity from an exogenous COL1A2 promoter in the SVWI-38 cell line. Competition experiments with an excess of COL1A2 promoter DNA fragment and a constant amount of COL1A2/CAT construct displayed a linear relationship between excess COL1A2 fragment and CAT activity in SVWI-38 cells, suggesting the involvement of a titratable negative effector. Electrophoretic mobility shift assays revealed the presence of a specific DNA-protein complex which was present in SVWI-38 cells and almost absent in control fibroblasts. Methylation interference analysis mapped the region of binding of this factor between nucleotides -80 and -72, relative to the transcription start site. Thus the data presented provide strong evidence for the existence of a negative trans-acting factor that may play a role in the repression of COL1A2 expression in SVWI-38 fibroblasts.

Base Sequence↗
PubMed1991

Use of site-directed mutagenesis to enhance the epitope-shielding effect of covalent modification of proteins with polyethylene glycol.

Modification by covalent attachment of polyethylene glycol (PEG) can reduce the immunogenicity and prolong the circulating life of proteins, but the utility of this approach for any protein is restricted by the number and distribution of PEG attachment sites (e.g., epsilon-amino groups of lysine residues). We have developed a strategy for introducing additional sites for PEG attachment by using site-directed mutagenesis to selectively replace arginine with lysine codons and tested it with purine nucleoside phosphorylase (PNP) from Escherichia coli, an extremely stable but immunogenic enzyme, that could potentially be used to treat an inherited deficiency of PNP. A triple mutant, RK3, possessing three Arg----Lys substitutions was constructed that increased the number of lysines per PNP subunit from 14 to 17, providing an additional 18 potential PEG attachment sites per hexameric enzyme molecule. The wild-type and RK3 enzymes had similar catalytic activity, antigenicity, and immunogenicity. After PEG modification, both enzymes retained catalytic activity, the plasma half-life of both enzymes in mice increased from approximately 4 hr to 4 days, and the binding of both enzymes by antisera raised against each unmodified enzyme was markedly diminished. However, antibody raised against wild-type PEG-PNP did not bind the PEG-RK3 enzyme. PEG-RK3 PNP was also substantially less immunogenic than wild-type PEG-PNP. Accelerated antibody-mediated clearance of PEG-PNP occurred in 2 of 12 mice treated with PEG-RK3 PNP, compared with 10 of 16 mice treated with the modified wild-type enzyme. This combined use of directed mutagenesis and PEG modification is aimed at permitting the widest choice of proteins, including products of genetic and chemical "engineering," to be used for therapy of inherited and acquired disorders.

Amino Acid Sequence↗