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Biomedical subjects

D H Harrison

Publications and source records attributed to D H Harrison.

At least 19 recordsLinked to original sources
PubMed2000

Mirroring perfection: the structure of methylglyoxal synthase complexed with the competitive inhibitor 2-phosphoglycolate.

The crystal structure of the transition-state analogue 2-phosphoglycolate (2PG) bound to methylglyoxal synthase (MGS) is presented at a resolution of 2.0 A. This structure is very similar to the previously determined structure of MGS complexed to formate and phosphate. Since 2PG is a competitive inhibitor of both MGS and triosephosphate isomerase (TIM), the carboxylate groups of each bound 2PG from this structure and the structure of 2PG bound to TIM were used to align and compare the active sites despite differences in their protein folds. The distances between the functional groups of Asp 71, His 98, His 19, and the carboxylate oxygens of the 2PG molecule in MGS are similar to the corresponding distances between the functional groups of Glu 165, His 95, Lys 13, and the carboxylate oxygens of the 2PG molecule in TIM. However, these spatial relationships are enantiomorphic to each other. Consistent with the known stereochemical data, the catalytic base Asp 71 is positioned on the opposite face of the 2PG-carboxylate plane as Glu 165 of TIM. Both His 98 of MGS and His 95 of TIM are in the plane of the carboxylate of 2PG, suggesting that these two residues are homologous in function. While His 19 of MGS and Lys 13 of TIM appear on the opposite face of the 2PG carboxylate plane, their relative location to the 2PG molecule is quite different, suggesting that they probably have different functions. Most remarkably, unlike the coplanar structure found in the 2PG molecule bound to TIM, the torsion angle around the C1-C2 bond of 2PG bound to MGS brings the phosphoryl moiety out of the molecule's carboxylate plane, facilitating elimination. Further, the superimposition of this structure with the structure of MGS bound to formate and phosphate suggests a model for the enzyme bound to the first transition state.

Binding Sites↗
PubMed2000

Paralysis of the marginal mandibular branch of the facial nerve: treatment options.

Isolated paralysis of the marginal mandibular branch of the facial nerve results in an asymmetrical smile with elevation of the lower lip on the affected side. We discuss the surgical options for its correction and present a series of 26 patients who underwent either botulinum toxin injection, anterior belly of digastric transfer or free extensor digitorum brevis transfer as treatment. Botulinum toxin injection provided satisfactory results although these were temporary. Anterior belly of digastric transfer was the surgical procedure of choice. It yielded superior cosmetic results, less donor-site morbidity and required a shorter operating time. In more complex congenital facial hypoplastic syndromes, or following extensive surgery in the digastric triangle, the anterior belly of the digastric muscle may be absent or damaged. Extensor digitorum brevis transfer is the preferred option in these cases.

Adolescent↗
PubMed2000

Reducing postoperative morbidity after the insertion of gold weights to treat lagophthalmos.

Paralysis of the orbicularis oculi muscle leads to an unopposed action of the levator of the upper eyelid (lagophthalmos) in facial nerve palsy. The resultant exposure of the cornea may lead to keratitis, corneal ulceration, and eventual blindness. Although many surgical options exist in the treatment of lagophthalmos, upper lid loading with a gold weight implant has become one of the preferred methods to reduce the complications that may follow. The problems encountered after gold lid loading and methods to reduce postoperative morbidity are not well documented. The objective of this study was to determine the range of morbidity seen after gold weight insertion and to evaluate the effect of supratarsal fixation on subsequent morbidity. After retrospective reviews by questionnaire and case note analysis, supratarsal fixation was found to noticeably reduce the rate of implant ulceration and extrusion. This study demonstrates upper lid loading to be an effective method for the treatment of lagophthalmos, and it supports fixation of gold weights in reducing surgical morbidity.

Adolescent↗
PubMed2000

The abnormal nucleus as a cause of congenital facial palsy.

BACKGROUND: Congenital facial palsy (CFP) is clinically defined as facial palsy present at birth. It is associated with considerable disfigurement and causes functional and emotional problems for the affected child. The aetiology of the majority of cases however, remains elusive. AIMS: To investigate the role of a neuroanatomical abnormality as a cause of unilateral CFP. METHODS: Magnetic resonance imaging (MRI) scans were performed on 21 patients with unilateral CFP. Fifteen patients had unilateral CFP only; six suffered from syndromes which can include unilateral CFP. RESULTS: Of the 15 patients with unilateral CFP only, four (27%) had an abnormal nucleus or an abnormal weighting of this area on the MRI scan, compared to one (17%) of the remaining six patients. CONCLUSION: Developmental abnormalities of the facial nucleus itself constitute an important, and previously ignored, cause of monosymptomatic unilateral CFP.

Adolescent↗
PubMed1999

Identification of the allosteric regulatory site in bacterial phosphoribulokinase.

Bacterial phosphoribulokinases (PRKs) are octameric members of the adenylate kinase family of enzymes. The enzyme is allosterically activated by NADH and allosterically inhibited by AMP. We have determined the crystal structure of PRK from Rhodobacter sphaeroides bound to the ATP analogue AMP-PCP to a resolution of 2.6 A. The structure reveals that the ATP analogue does not bind to the canonical ATP site found in adenylate kinase family members. Rather, the AMP-PCP binds in two different orientations at the interface of three of the monomers in the octamer. This interface was previously characterized as having an unusually large number of arginine residues. Of the five arginine residues that are near the bound nucleotide, one (Arg 221) is highly conserved in both prokaryotic and eukaryotic (nonallosterically regulated) PRKs, two (Arg 234 and Arg 257) are on a second subunit and conserved in only prokaryotic PRKs, and two (Arg 30 and Arg 31) are on a third subunit with only one of them (Arg 31) conserved in prokaryotic PRKs. Each of these arginine residues was converted by site-directed mutagenesis to alanine. Fluorescence binding data suggest that none of these arginines are involved in active site ATP binding and that Arg 234 and Arg 257 on the second subunit are directly involved in NADH binding, while the other arginines have a minimal effect on NADH binding. While the wild-type enzyme exhibits low maximal activity and hyperbolic kinetics with respect to ATP in the absence of NADH and high maximal activity and sigmoidal kinetics in the presence of NADH, the R31A mutant exhibits identical hyperbolic kinetics with respect to ATP in the presence or absence of NADH. Thus, the transmission of allosteric information from one subunit to another is conducted through a single path that includes NADH and Arg 31.

Adenosine Triphosphate↗
PubMed1999

Rhodobacter sphaeroides phosphoribulokinase: identification of lysine-165 as a catalytic residue and evaluation of the contributions of invariant basic amino acids to ribulose 5-phosphate binding.

Rhodobacter sphaeroides phosphoribulokinase (PRK) is inactivated upon exposure to pyridoxal phosphate/sodium borohydride, suggesting a reactive lysine residue. Protection is afforded by a combination of the substrate ATP and the allosteric activator NADH, suggesting that the targeted lysine maps within the active site. PRK contains two invariant lysines, K53 and K165. PRK-K53M retains sensitivity to pyridoxal phosphate, implicating K165 as the target of this reagent. PRK-K165M retains wild-type structure, as judged by titration with effector NADH and the tight-binding alternative substrate trinitrophenyl-ATP. The catalytic activity of K165M and K165C mutants is depressed by >10(3)-fold. Residual activity of K165M is insensitive to pyridoxal phosphate, confirming K165 as the target of this reagent. The decreased catalytic efficiency of K165 mutants approaches the effect measured for a mutant of D169, which forms a salt-bridge to K165. K165M exhibits a 10-fold increase in S()1(/)()2 (ATP) and a 10(2)-fold increase in K(m) (Ru5P). To evaluate the contribution to Ru5P binding of K165 in comparison with this substrate's interaction with invariant H45, R49, R168, and R173, PRKs mutated at these positions have been used to determine relative K(i) values for 6-phosphogluconate, a competitive inhibitor with respect to Ru5P. Elimination of the basic side chain of K165, R49, and H45 results in increases in K(m) (Ru5P) which correlate well with the magnitude of increases in K(i) (phosphogluconate). In contrast, while mutations eliminating charge from R168 and R173 result in enzymes with substantial increases in K(m) (Ru5P), such mutant enzymes exhibit only small increases in K(i) (phosphogluconate). These observations suggest that K165, R49, and H45 are major contributors to Ru5P binding.

Amino Acid Sequence↗
PubMed1999

The crystal structure of methylglyoxal synthase from Escherichia coli.

BACKGROUND: The reaction mechanism of methylglyoxal synthase (MGS) is believed to be similar to that of triosephosphate isomerase (TIM). Both enzymes utilise dihydroxyacetone phosphate (DHAP) to form an enediol(ate) phosphate intermediate as the first step of their reaction pathways. However, the second catalytic step in the MGS reaction pathway is characterized by the elimination of phosphate and collapse of the enediol(ate) to form methylglyoxal instead of reprotonation to form the isomer glyceraldehyde 3-phosphate. RESULTS: The crystal structure of MGS bound to formate and substoichiometric amounts of phosphate in the space group P6522 has been determined at 1.9 A resolution. This structure shows that the enzyme is a homohexamer composed of interacting five-stranded beta/alpha proteins, rather than the hallmark alpha/beta barrel structure of TIM. The conserved residues His19, Asp71, and His98 in each of the three monomers in the asymmetric unit bind to a formate ion that is present in the crystallization conditions. Differences in the three monomers in the asymmetric unit are localized at the mouth of the active site and can be ascribed to the presence or absence of a bound phosphate ion. CONCLUSIONS: In agreement with site-directed mutagenesis and mechanistic enzymology, the structure suggests that Asp71 acts as the catalytic base. Further, Asp20 and Asp101 are involved in intersubunit salt bridges. These salt bridges may provide a pathway for transmitting allosteric information.

Allosteric Regulation↗
PubMed1999

A technique for repairing massive ventral incisional hernias without the use of a mesh.

Incisional hernias are common. Those involving large fascial defects are usually repaired using a synthetic mesh. Complications of such repairs involving mesh pose particular problems in management. This paper describes a new technique for repairing large ventral incisional hernias which does not involve the use of a mesh. Our repair is anatomical in first reconstructing the posterior rectus sheath and then using a darn to approximate the recti. We have used this technique successfully in three cases of massive incisional hernias measuring 20 cm or more in diameter and have not experienced any complications. In particular, no recurrences of the hernias have occurred. The technique described can be used to repair primary incisional hernias as well as salvage cases when previous repairs have failed.

Adult↗
PubMed1998

Identification of catalytic bases in the active site of Escherichia coli methylglyoxal synthase: cloning, expression, and functional characterization of conserved aspartic acid residues.

Methylglyoxal synthase provides bacteria with an alternative to triosephosphate isomerase for metabolizing dihydroxyacetone phosphate (DHAP). In the present studies, the methylglyoxal synthase gene in Escherichia coli has been cloned and sequenced. The identified open reading frame (ORF) codes for a polypeptide of 152 amino acids, consistent with the 17 kDa purified protein. The sequence of this protein is not similar to any other protein of known function, including the functionally similar protein triosephosphate isomerase. The methylglyoxal synthase gene was amplified by PCR, subcloned into the pET16B expression vector, and expressed in the host E. coli BL21(DE3). Sequence comparison of the methylglyoxal protein and related ORFs from four different bacterial species revealed that four aspartic acid and no glutamic acid residues are absolutely conserved. The function of the four aspartic acid residues was tested by mutating them to either asparagine or glutamic acid. Thermal denaturation, CD spectroscopy, and gel filtration experiments showed that the mutant enzymes had the same secondary and quaternary structure as the wild-type enzyme. Kinetic characterization of both Asp 71 and Asp 101 mutant proteins shows reduced kcat/Km by 10(3)- and 10(4)-fold respectively, suggesting that they are both intimately involved in catalysis. A time-dependent inhibition of both Asp 20 and Asp 91 asparagine mutants by DHAP suggests that these two residues are involved with protecting the enzyme from DHAP or reactive intermediates along the catalytic pathway. In combination with the results of 2-phosphoglycolate binding studies, a catalytic mechanism is proposed.

Amino Acid Sequence↗
PubMed1998

The crystal structure of phosphoribulokinase from Rhodobacter sphaeroides reveals a fold similar to that of adenylate kinase.

The essential photosynthetic enzyme phosphoribulokinase (PRK) is responsible for the conversion of ribulose 5-phosphate (Ru5P) to ribulose 1,5-bisphosphate, the substrate for the CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). We have determined the structure of the octameric bacterial form of PRK to a resolution of 2.5 A. The protein is folded into a seven-member mixed beta-sheet surrounded by alpha-helices, giving the overall appearance of the nucleotide monophosphate family of kinases. Homology with the nucleotide monophosphate kinases suggests a number of amino acid residues that are likely to be important in catalysis and suggests the roles of some amino acid residues that have been mutated prior to the determination of the structure. Further, sequence identity across eukaryotic and prokaryotic species and a calculation of the buried surface area suggests the identity within the octamer of a dimer conserved throughout evolution. The width of the groove leading to the active site is consistent with an oriented molecule of thioredoxin controlling the oxidation state of two cysteines that regulate activity in the eukaryotic enzymes. Although neither Asp 42 nor Asp 169 can be definitively assigned as the catalytic base, the crystal structure suggests the location of a ribulose 5-phosphate binding site and suggests a role for several of the conserved basic residues.

Adenylate Kinase↗
PubMed1998

Functional evaluation of invariant arginines situated in the mobile lid domain of phosphoribulokinase.

Rhodobacter sphaeroides phosphoribulokinase contains four invariant arginines (R49, R168, R173, and R187). The high-resolution structure of this enzyme [Harrison, D. H. T., Runquist, J. A., Holub, A., and Miziorko, H. M. (1998) Biochemistry (submitted for publication)] reveals that it folds in a manner similar to that of adenylate kinase. Three invariant arginines (R168, R173, and R187) as well as arginine-186, which is conserved in prokaryotic phosphoribulokinases, have not been previously functionally evaluated. These arginine residues map within the mobile lid domain that is a distinctive feature of the adenylate kinase family of proteins. Precedent for the significant function of arginines in phosphotransferase reactions prompted substitution of glutamine for each of these three invariant arginines. Solution state characterization of the isolated mutant proteins indicated that they retained a high degree of structural integrity, as indicated by their stoichiometric binding of an alternative nucleotide substrate (trinitrophenyl-ATP) as well as the allosteric effector (NADH). Kinetic characterization indicated > 10(4)-fold diminution in V/KRu5P for R168Q, attributable to a > 300-fold decrease in catalytic efficiency and an increase (approximately 50-fold) in Km Ru5P. For R173Q, a 15-fold diminution in Vmax and a 100-fold increase in Km Ru5P were observed. These observations implicate new components of the ribulose 5-phosphate binding site. Additionally, they confirm assignment of the mobile lid domain as part of the phosphoribulokinase active site, even though this region is well separated from other active site elements in the structure of the open form of the protein. Characterization of R186Q and R187Q mutants suggests that they influence the cooperativity of substrate binding.

Amino Acid Sequence↗
PubMed1997

The alrestatin double-decker: binding of two inhibitor molecules to human aldose reductase reveals a new specificity determinant.

It is generally expected that only one inhibitor molecule will bind to an enzyme active site. In fact, specific drug design theories depend upon this assumption. Here, we report the binding of two molecules of an inhibitor to the same active site which we observed in the 1.8 A resolution structure of the drug Alrestatin bound to a mutant of human aldose reductase. The two molecules of Alrestatin bind to the active site in a stacked arrangement (a double-decker). This stack positions the carboxylic acid of one drug molecule near the NADP+ cofactor at a previously determined anion binding site and the carboxylic acid of the second drug molecule near the carboxy-terminal tail of the enzyme. We propose that interactions of inhibitors with the carboxy-terminal loop of aldose reductase are critical for the development of inhibitors that are able to discriminate between aldose reductase and other members of the aldo-keto reductase superfamily. This finding suggests a new direction for the introduction of specificity to aldose reductase-targeted drugs.

Aldehyde Reductase↗
PubMed1997

Urethral advancement and glanuloplasty (UGPI): a modification of the MAGPI procedure for distal hypospadias.

A modification of the meatal advancement and glanuloplasty technique (MAGPI), the urethral advancement and glanuloplasty (UGPI), is described. Forty-seven patients, who had this operation from 1985 to 1994, have been reviewed. The overall complication rate was low with a 2.1% fistula rate and a 6.4% incidence of meatal retraction. Only one patient required secondary surgery.

Child, Preschool↗
PubMed1997

The distally based ulnar artery island flap in hand reconstruction.

The distally based ulnar artery island flap is a highly versatile flap for hand reconstruction. It fulfils all the necessary criteria required for a flap to the hand providing thin pliable hairless skin. It can be used as a composite flap including tendon and bone and provides an ideal tunnel for tendons to glide in. It can be used as a fasciocutaneous flap or as a fascial flap and can easily be rotated to the dorsum of the hand by opening Guyon's canal. The palm or dorsum of the hand and even the fingertips can be reached easily. A superficial venous anastomosis should be made if marked venous congestion is noted intraoperatively. Six cases utilizing a distally based ulnar island flap in hand reconstruction are presented.

Adolescent↗
PubMed1996

Regeneration through a long nerve graft used in the correction of facial palsy. A qualitative and quantitative study.

A surgical technique has been developed for the correction of established unilateral palsy in man. A long (20 cm or more) sural nerve graft is anastomosed to a facial nerve branch on the unaffected side and its distal end left lying free in the cheek of the affected side. After regeneration times of 5.5-14.5 months, the distal end of the graft is joined to a free (pectoralis minor) muscle graft. In due course the muscle graft contracts in unison with the unaffected side giving near normal symmetry to facial movements. In 30 cases (ages 6-52 years) qualitative and quantitative examination was made of the distal end of grafts taken at the time of joining the graft to the muscle. Total axon counts, myelinated plus non-myelinated, confirmed abundant regeneration when compared with total axons in the supplying facial nerve; myelinated fibres remained small (mean diameter 2.5 micrometers) over the range of regeneration times. Quantitation included non-myelinated axons because they probably have the potential to become myelinated once the nerve is functional. Numbers of regenerating axons were not correlated with age, nor with regeneration time. Lack of a distal connection did not appear to lead to secondary degeneration of the regenerated myelinated fibres. These were maintained in an 'immature' state for many months. This observation is of practical interest since it has been suggested that delayed connection to the distal target may have a deleterious effect on the outcome of the procedure.

Adolescent↗
PubMed1996

Is permanent congenital facial palsy caused by birth trauma?

OBJECTIVE: To study the relation between traumatic birth and the development of permanent facial palsy in the newborn. DESIGN: Retrospective case control study of children with 'congenital' facial palsy. SETTING: Two tertiary referral centres for patients with facial palsy. SUBJECTS: 61 children with established facial palsy. MAIN OUTCOME MEASURES: Odds ratios of recognised factors for birth injury: maternal primiparity, high birth weight, and the use of obstetric forceps at delivery. RESULTS: 13.2% of those studied had forceps assisted delivery compared to 10.2% in the normal population (odds ratio 1.34; 95% confidence intervals 0.61 to 2.97) 39.6% were born to primiparae compared to a national rate of 36.7% (1.13; 0.65 to 1.96) and only 18.9% weighed more than 3500 g at birth (0.37; 0.19 to 0.74). CONCLUSIONS: There is no association between the development of permanent 'congenital' facial palsy and recognised risk factors for birth injury. These data suggest an intrauterine rather than a traumatic aetiology.

Birth Injuries↗
PubMed1996

Hypospadias repair: the influence of postoperative care and a patient factor on surgical morbidity.

More than 200 surgical techniques have been advocated for hypospadias repair and perfection is still eluding us. The purpose of this paper is not to comment on a surgical technique, but to highlight the importance of postoperative care on the eventual surgical outcome. Two hundred six children undergoing primary hypospadias surgery from 1984 to 1994 at Mount Vernon Hospital were analyzed. The mean age of surgery was 3.8 years (range, 8 months-17 years) and our mean follow-up was 6.1 years. Various surgical techniques were used. During 1989, we changed our postoperative regime and patients were mobilized early (within 48 hours) compared to the strict-bed rest-till-catheter-out regime before 1988. This significantly reduced our fistula rate from 22% to 9.8% (chi squared p = 0.0016). Patient factors such as pulling on the catheter, urinary obstruction/blocked catheter, erections, straining due to constipation, and interference with dressings influence surgical outcome negatively. One of these patient factors was documented in all of our 33 patients who developed fistulas. In 6 patients (3.5%), a patient factor was present without any complication. This is highly significant statistically (chi squared p < 0.0001). The type of urinary diversion, period of urinary diversion, type of dressing, catheter size, and anesthetic regime did not influence outcome significantly.

Adolescent↗