Biomedical subjects

A L Osterman

Publications and source records attributed to A L Osterman.

At least 19 recordsLinked to original sources

PubMed2003

Experimental determination and system level analysis of essential genes in Escherichia coli MG1655.

Defining the gene products that play an essential role in an organism's functional repertoire is vital to understanding the system level organization of living cells. We used a genetic footprinting technique for a genome-wide assessment of genes required for robust aerobic growth of Escherichia coli in rich media. We identified 620 genes as essential and 3,126 genes as dispensable for growth under these conditions. Functional context analysis of these data allows individual functional assignments to be refined. Evolutionary context analysis demonstrates a significant tendency of essential E. coli genes to be preserved throughout the bacterial kingdom. Projection of these data over metabolic subsystems reveals topologic modules with essential and evolutionarily preserved enzymes with reduced capacity for error tolerance.

Aerobiosis↗

PubMed2001

Scapholunate instability in athletes.

Sports medicine practitioners must be sensitive to even the smallest injuries that affect athletes. Often, less severe ligament injuries go undetected. Scapholunate dissociation is often overlooked in this manner. The authors provide an overview of presentation, diagnosis, and nonoperative and operative treatments of this condition.

Athletic Injuries↗

PubMed2001

Smoking and bony union after ulna-shortening osteotomy.

We examined the relation between cigarette use and occurrence of delayed union and nonunion after ulna-shortening osteotomy for ulnar impaction syndrome. After obtaining the smoking history of 39 patients (40 wrists), we found that, compared with nonsmokers, smokers experienced longer healing times and more of them experienced healing delay and nonunion. Mean union rates were 7.1 months in smokers and 4.1 months in nonsmokers. Six smokers (30%) and no nonsmokers experienced delayed union or nonunion. Given the adverse effects of smoking on bony union, we recommend that smoking history be considered when selecting patients for ulna-shortening procedures.

Adult↗

PubMed2000

Structure and mechanism of homoserine kinase: prototype for the GHMP kinase superfamily.

BACKGROUND: Homoserine kinase (HSK) catalyzes an important step in the threonine biosynthesis pathway. It belongs to a large yet unique class of small metabolite kinases, the GHMP kinase superfamily. Members in the GHMP superfamily participate in several essential metabolic pathways, such as amino acid biosynthesis, galactose metabolism, and the mevalonate pathway. RESULTS: The crystal structure of HSK and its complex with ADP reveal a novel nucleotide binding fold. The N-terminal domain contains an unusual left-handed betaalphabeta unit, while the C-terminal domain has a central alpha-beta plait fold with an insertion of four helices. The phosphate binding loop in HSK is distinct from the classical P loops found in many ATP/GTP binding proteins. The bound ADP molecule adopts a rare syn conformation and is in the opposite orientation from those bound to the P loop-containing proteins. Inspection of the substrate binding cavity indicates several amino acid residues that are likely to be involved in substrate binding and catalysis. CONCLUSIONS: The crystal structure of HSK is the first representative in the GHMP superfamily to have determined structure. It provides insight into the structure and nucleotide binding mechanism of not only the HSK family but also a variety of enzymes in the GHMP superfamily. Such enzymes include galactokinases, mevalonate kinases, phosphomevalonate kinases, mevalonate pyrophosphate decarboxylases, and several proteins of yet unknown functions.

Adenosine Diphosphate↗

PubMed2000

Altering the reaction specificity of eukaryotic ornithine decarboxylase.

Ornithine decarboxylase (ODC) catalyzes the first committed step in the biosynthesis of polyamines, and it has been identified as a drug target for the treatment of African sleeping sickness, caused by Trypanosoma brucei. ODC is a pyridoxal 5'-phosphate (PLP) dependent enzyme and an obligate homodimer. X-ray structural analysis of the complex of the T. brucei wild-type enzyme with the product putrescine reveals two structural changes that occur upon ligand binding: Lys-69 is displaced by putrescine and forms new interactions with Glu-94 and Asp-88, and the side chain of Cys-360 rotates into the active site to within 3.4 A of the imine bond. Mutation of Cys-360 to Ala or Ser reduces the k(cat) of the decarboxylation reaction by 50- and 1000-fold, respectively. However, HPLC analysis of the products demonstrates that the mutant enzymes almost exclusively catalyze a decarboxylation-dependent transamination reaction to form pyridoxamine 5-phosphate (PMP) and gamma-aminobutyraldehyde, instead of PLP and putrescine. This side reaction arises when the decarboxylated substrate intermediate is protonated at C4' of PLP instead of at the C(alpha) of substrate. For the reaction catalyzed by the wild-type enzyme, this side reaction occurs infrequently (<0.01% of the turnovers). Single turnover analysis and multiwavelength stopped-flow spectroscopic studies suggest that for the mutant ODCs protonation at C4' occurs either very rapidly or in a concerted reaction with decarboxylation and that the rate-limiting step in the steady-state reaction is Schiff base hydrolysis/product release. These studies demonstrate a role for Cys-360 in the control of the C(alpha) protonation step that catalyzes the formation of the physiological product putrescine. The results further provide insight into the mechanism by which this class of PLP-dependent enzymes controls reaction specificity.

Alanine↗

PubMed2000

Lunotriquetral injuries in the athlete.

The athlete with an LT injury typically presents with ulnar-sided wrist pain after a high-energy impaction of the wrist. Reagan's LT ballottement test and Kleinman's shear test help the examiner identify these injuries. A thorough radiographic examination includes standard PA and lateral radiographs. Magnetic resonance imaging or arthrography can be performed, but the sensitivity of these imaging studies varies. The palmar portion of the LT interosseous ligament, dorsal radiocarpal ligament, and dorsal intercarpal ligament play the most significant roles in LT stability. Lunotriquetral injuries without instability respond well to immobilization. Arthroscopy is valuable in staging and determining treatment but requires a thorough radiocarpal and midcarpal examination. Acute LT injuries with instability are treated with arthroscopic-assisted reduction and pinning. If desired, this procedure can be incorporated with an open ligament repair through a volar approach. Chronic LT tears without instability can also be treated arthroscopically. Treatment of the chronic LT tear with instability depends on the degree of collapse. Treatment in the athlete includes ligament reconstruction with capsulodesis or, rarely, intercarpal LT arthrodesis.

Athletic Injuries↗

PubMed1999

X-ray structure of ornithine decarboxylase from Trypanosoma brucei: the native structure and the structure in complex with alpha-difluoromethylornithine.

Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP) dependent homodimeric enzyme. It is a recognized drug target against African sleeping sickness, caused by Trypanosoma brucei. One of the currently used drugs, alpha-difluoromethylornithine (DFMO), is a suicide inhibitor of ODC. The structure of the T. brucei ODC (TbODC) mutant K69A bound to DFMO has been determined by X-ray crystallography to 2.0 A resolution. The protein crystallizes in the space group P2(1) (a = 66.8 A, b = 154.5 A, c = 77.1 A, beta = 90.58 degrees ), with two dimers per asymmetric unit. The initial phasing was done by molecular replacement with the mouse ODC structure. The structure of wild-type uncomplexed TbODC was also determined to 2.9 A resolution by molecular replacement using the TbODC DFMO-bound structure as the search model. The N-terminal domain of ODC is a beta/alpha-barrel, and the C-terminal domain of ODC is a modified Greek key beta-barrel. In comparison to structurally related alanine racemase, the two domains are rotated 27 degrees relative to each other. In addition, two of the beta-strands in the C-terminal domain have exchanged positions in order to maintain the location of essential active site residues in the context of the domain rotation. In ODC, the contacts in the dimer interface are formed primarily by the C-terminal domains, which interact through six aromatic rings that form stacking interactions across the domain boundary. The PLP binding site is formed by the C-termini of beta-strands and loops in the beta/alpha-barrel. In the native structure Lys69 forms a Schiff base with PLP. In both structures, the phosphate of PLP is bound between the seventh and eighth strands forming interactions with Arg277 and a Gly loop (residues 235-237). The pyridine nitrogen of PLP interacts with Glu274. DFMO forms a Schiff base with PLP and is covalently attached to Cys360. It is bound at the dimer interface and the delta-carbon amino group of DFMO is positioned between Asp361 of one subunit and Asp332 of the other. In comparison to the wild-type uncomplexed structure, Cys-360 has rotated 145 degrees toward the active site in the DFMO-bound structure. No domain, subunit rotations, or other significant structural changes are observed upon ligand binding. The structure offers insight into the enzyme mechanism by providing details of the enzyme/inhibitor binding site and allows for a detailed comparison between the enzymes from the host and parasite which will aid in selective inhibitor design.

Amino Acid Sequence↗

PubMed1999

Lysine-69 plays a key role in catalysis by ornithine decarboxylase through acceleration of the Schiff base formation, decarboxylation, and product release steps.

Ornithine decarboxylase (ODC) is a pyridoxal-5'-phosphate-dependent (PLP) enzyme that catalyzes the biosynthesis of the polyamine putrescine. Similar to other PLP-dependent enzymes, an active site Lys residue forms a Schiff base with PLP in the absence of substrate. The mechanistic role of this residue (Lys-69) in catalysis by Trypanosoma brucei ODC has been studied by analysis of the mutant enzymes, in which Lys-69 has been replaced by Arg (K69R ODC) and Ala (K69A ODC). Analysis of K69A ODC demonstrated that the enzyme copurified with amines (e.g. putrescine) that were tightly bound to the active site through a Schiff base with PLP. In contrast, on the basis of an absorption spectrum of K69R ODC, PLP is likely to be bound to this mutant enzyme in the aldehyde form. Pre-steady-state kinetic analysis of the reaction of K69R ODC with L-Orn and putrescine demonstrated that the rates of both the product release (k(off.Put) = 0.0041 s(-)(1)) and the decarboxylation (k(decarb) = 0.016 s(-)(1)) steps were decreased by10(4)-fold in comparison to wild-type ODC. Further, the rates of Schiff base formation between K69R ODC and either substrate or product have decreased by at least 10(3)-fold. Product release remains as the dominant rate-limiting step in the reaction (the steady-state parameters for K69R ODC are k(cat) = 0.0031 s(-)(1) and K(m) = 0.18 mM). The effect of mutating Lys-69 on the decarboxylation step suggests that Lys-69 may play a role in the proper positioning of the alpha-carboxylate for efficient decarboxylation. K69R ODC binds diamines and amino acids with higher affinity than the wild-type enzyme; however, Lys-69 does not mediate substrate specificity. Wild-type and K69R ODC have similar ligand specificity preferring to bind putrescine over longer and shorter diamines. Kinetic analysis of the binding of a series of diamines and amino acids to K69R ODC suggests that noncovalent interactions in the active site of K69R ODC promote selective ligand binding during Schiff base formation.

Animals↗

PubMed1998

Carbon-13 isotope effect studies of Trypanosoma brucei ornithine decarboxylase.

Carbon isotope effect studies were undertaken with the wild-type pyridoxal 5'-phosphate (PLP)-dependent enzyme ornithine decarboxylase (ODC) from Trypanosoma brucei and with several active site mutants of the enzyme. For the decarboxylation of the optimal substrate, L-ornithine, by wild-type ODC, the observed carbon isotope effect (k12/k13) is 1.033 at pH 7.3. In comparison to the expected intrinsic isotope effect (k12/k13 = 1.06) for decarboxylation, this value suggests that both the rate of decarboxylation and the rate of Schiff base interchange with L-ornithine are partially rate-limiting for the reaction steps up to decarboxylation. In contrast, with the alternate substrate L-Lys, which shows lower catalytic efficiency, the carbon isotope effect increased to 1.063, demonstrating that decarboxylation has become the rate-limiting step. For the mutant enzymes, E274A ODC and C360A ODC, with L-ornithine as substrate the carbon isotope effect also approaches the intrinsic limit. Glu-274 was previously demonstrated to play a direct role in carbanion stabilization, and thus the large carbon isotope effect (k12/k13 = 1.055) is consistent with an impaired rate of decarboxylation compared to wild-type ODC. In contrast, for K69A ODC, the isotope effect is almost entirely suppressed, suggesting that Schiff-base formation (which now must occur from enzyme-bound PLP, rather than from an enzyme-bound PLP-Schiff base) has become rate-determining.

Animals↗

PubMed1998

Revision of the ununited one-bone forearm.

Between 1975 and 1995, 7 patients were surgically treated for persistent forearm pain following previously unsuccessful attempts of constructing a radioulnar synostosis using interosseous bone grafting with either cross screw or pin fixation. These prior reconstructive procedures were salvage operations to address symptomatic radioulnar instability. The time interval between index operation and revision surgery was a mean of 12.6 months (range, 4-36 months). The postoperative follow-up period averaged 29.7 months (range, 11-61 months). To address these failed radioulnar arthrodeses, plate osteosynthesis and aggressive bone grafting were used in 5 of 7 patients (group 1) (4 with autologous graft and 1 with demineralized bone matrix). Union was achieved in all 5 patients after a single revision operation. Radiographic evidence of solid union was seen at approximately 4 months. In the remaining 2 patients (group 2), revision synostosis procedures were performed; these involved local bone grafting and repeat transverse screw fixation in 1 patient and iliac crest bone grafting alone in the other. One of these 2 patients progressed to union; the other had a persistent symptomatic fibrous union. The 5 patients in group 1 reported symptomatic relief, while the 2 patients in group 2 were dissatisfied. There were no postoperative complications in our series. Based on our findings, we advocate abundant bone grafting and plating for rigid internal fixation in failed radioulnar synostosis procedures.

Adult↗

PubMed1998

Reconstruction of the scapholunate ligament in a cadaver model using a bone-ligament-bone autograft from the foot.

This study is an investigation of a new procedure in which the scapholunate interosseous ligament (SLIL) is reconstructed using a bone-ligament-bone autograft from the foot. After investigation, the dorsal medial portion of the navicular-first cuneiform ligament (NFCL) was chosen for testing as a potential donor since it is similar in length and thickness to the SLIL and it is easily harvested with minimal potential donor site morbidity. Eight SLILs and NFCLs were harvested from fresh-frozen cadavers. Biomechanical extensometry testing was performed using an Instron 1000 machine. A 5-mm-wide central portion of the NFCL was tested since this width was compatible with the technical aspects of reconstructing the SLIL. Both ligaments were tested for elastic properties, including stiffness, load to failure, and deformation to failure. Mean length of the NFCL was 7.6 mm (range, 5.5-8.5 mm). Stiffness of the NFCL was 10.6 x 10(5) Nm (range, 8.0-13.0 Nm) compared with 14.4 x 10(5) Nm for the SLIL (range, 10.0-19.5 Nm). Peak load to failure for the NFCL was 1,980 N (range, 1,530-2,940 N) compared with 2,940 N for the SLIL (range, 1,780-4,050 N). Total elongation to failure for the NFCL was 2.50 mm (range, 1.7-3.2 mm) compared with 3.2 mm for the SLIL (range, 2.1-5.2 mm). Thus, the biomechanical characteristics of the NFCL were found to be very similar to those of the SLIL. Having established the biomechanical similarities of the 2 ligaments, we are currently using the NFCL to reconstruct the sectioned SLIL in a fresh-frozen cadaver model. Early results suggest that this procedure is feasible for restoration of normal kinematics of the wrist.

Aged↗

PubMed1998

The effect of shear stress on fibroblasts derived from Dupuytren's tissue and normal palmar fascia.

This study examines the real-time intracellular calcium changes of palmar fascia from normal and Dupuytren's diseased fibroblasts in response to shear stress. The real-time cytosolic calcium changes were measured using fluorescence microscopy image processing. The preconfluent primary cultured cells were exposed to 1 minute of flow at 25 dyne/cm2 after a 2-minute baseline of no flow. Additionally, the cells were exposed to an influx of Hank's buffered saline solution with 2% newborn bovine serum to examine the response to serum-born (chemical) agonists. Cytosolic calcium changes were measured as the percentage change over the 2-minute baseline of the mean [Ca2+]i peak. The mean change of the peak [Ca2+]i response of the normal palmar fascia was significantly greater than that of the cells from the Dupuytren's nodular and perinodular tissue. The response to the chemical agonist showed a robust but not statistically different response between the 3 cell types. Our work supports the hypothesis that palmar fascia responds to mechanical stress, specifically laminar fluid flow. These findings may help to explain that an underlying abnormality in the cells of the palmar fascia may be expressed by exposure to laminar fluid flow, a physical signal, rather than a chemical agonist.

Adult↗

PubMed1998

Workers compensation. Effect of state law on treatment cost and work status.

Workers compensation legislation is regulated at a state level resulting in different benefits based on the state of employment. The negative effect of workers compensation on the results of surgical treatment has been established previously. A comparison of the results of treatment of patients receiving workers compensation from different states has not been reported. This study prospectively evaluated the effect of the difference in state workers compensation laws on the number of postoperative visits, amount of postoperative therapy, time off from work, and return to work status. The study group consisted of 275 patients. The results indicated a significant increase in the number of postoperative visits, amount of therapy, time off from work, and number of patients who remained out of work in the compensation versus the noncompensation group. A significant increase in these parameters also was present in the Pennsylvania compensation group as compared with the New Jersey compensation group. This study shows that differences in workers compensation benefits have a significant effect on the results of medical treatment, return to work status, and the cost of medical care.

Absenteeism↗

PubMed1997

Role of Arg-277 in the binding of pyridoxal 5'-phosphate to Trypanosoma brucei ornithine decarboxylase.

The pyridoxal 5'-phosphate (PLP) binding site in Trypanosoma brucei ornithine decarboxylase (ODC) has been studied by site-directed mutagenesis and spectroscopy. The beta/alpha barrel model proposed for the eukaryotic ODC structure predicts that the phosphate group of PLP is stabilized by interactions with a Gly-rich loop (residues 235-237) and by a salt bridge to Arg-277 [Grishin, N. V., Phillips, M. A., & Goldsmith, E. J. (1995) Protein Sci. 4, 1291-1304]. Mutation of Arg-277 to Ala increases the K(m) for PLP by 270-fold compared to that of wild-type ODC while reducing k(cat) by only 2-fold at pH 8. PLP binding affinity was measured directly by ultrafiltration; the K(d) for PLP is at least 20-fold higher in the mutant enzyme at pH 8. In addition, R277A ODC also has weaker binding affinities for a series of cofactor analogs than the wild-type enzyme. These results demonstrate that Arg-277 is necessary for high-affinity PLP binding by ODC. The 31P NMR spectra of ODC suggest that the phosphate is bound in a strained conformation as a dianion to both wild-type and R277A ODC. However, the 31P chemical shift for R277A ODC (6.7 ppm) is 0.5 ppm downfield from that observed for the wild-type enzyme, indicating that the environment of the enzyme-bound phosphate is altered in the mutant enzyme. The binding affinity of PLP for both wild-type and R277A ODC is weaker at high pH, corresponding to the titration of a protonated species with a pK(a) of approximately 8.5. Concomitant with these changes are a decreased k(cat) and an altered absorption spectra which arises from bound PLP. PLP bound to wild-type ODC has a 31P chemical shift and a CD signal observable over the entire tested pH range (7-9). In contrast, for R277A ODC between pH 8 and 9, the 31P chemical shift becomes solution-like and the CD signal is abolished. The data suggest that for R277A ODC the rigid PLP binding mode which characterizes the wild-type enzyme is lost at high pH. Thus, multiple interactions between the wild-type active site and PLP maintain the cofactor in a constrained conformation that is essential for efficient catalysis, tempering the consequence of the removal of any single interaction.

Animals↗

PubMed1997

Pediatric hand injuries due to exercise bicycles.

METHODS: This is a 5-year retrospective review of hand injuries caused by exercise bicycles among 34 children. RESULTS: Mean age was 3 years and 5 months. Accidents were caused most commonly by impingement of the fingers between the chain and sprocket wheel (77%), but injuries caused by spinning wheel spokes (23%) were generally more severe. Injuries were usually of the avulsion type and ranged in severity from lacerations (43%) to fractures (19%) to amputations (38%). The central digits (87%) were involved more frequently than the border digits (13%). Replantation was possible for 33% of the amputated digits, with 50% of the fingers surviving. CONCLUSION: The avulsive nature of these injuries should be taken into consideration when attempting replantation. Increased community awareness and equipment safety modifications are needed to eliminate this preventable problem.

Accidents, Home↗

PubMed1997

Arthroscopic repairs of triangular fibrocartilage complex tears.

Technical advancements in arthroscopic wrist procedures have improved our knowledge of normal and abnormal intraarticular wrist function. Triangular fibrocartilage complex (TFCC) tears from trauma injuries are a common source of ulnar-sided wrist pain. Fortunately, the TFCC is a structure that can be evaluated and treated arthroscopically with results that are comparable to open surgical procedures. Successful arthroscopic repairs of TFCC tears depend on a coordinated team effort between perioperative nurses, orthopedic surgeons, nurse practitioners, and occupational hand therapists, as well as cooperation from patients and family members. This article reviews the anatomy and physiology of the TFCC, the biomechanics of the wrist and mechanisms of injury, and arthroscopic repairs of TFCC tears.

Arthroscopy↗

PubMed1997

Dorsal capsulodesis using suture anchors.

Scapholunate instability can be treated by stabilizing the scaphoid with the dorsal wrist capsule, which acts as a tether to control palmar rotation. We began using suture anchors instead of pull-out wires to secure the dorsal capsule to the scaphoid. Thirty-eight patients were treated with this method, and 35 were followed an average of 24 months. Sixteen were pain free, 12 had pain with activity, and 7 required additional surgical treatment. Patients regained 85% of expected grip strength, with 25 of the 35 returning to their preinjury job. The scapholunate gap, reduced at the time of surgery, recurred in 31 of the 35 patients, but this alone did not correlate with symptoms. This procedure improves symptoms in a majority of patients, but like other procedures described for scapholunate instability, it does not completely cure the problem.

Adult↗

PubMed1996

Crystallization and preliminary X-ray studies of ornithine decarboxylase from Trypanosoma brucei.

Trypanosoma brucei ornithine decarboxylase, expressed and purified from E. coli, has been crystallized by the vapor diffusion method using PEG 3350 as a precipitant. The crystals belong to the monoclinic space group P2(1) and have cell constants of a = 66.3 angstroms, b = 151.8 angstroms, c = 83.7 angstroms, and beta = 101.2 degrees. While larger crystals are twinned, smaller crystals (0.4 x 0.3 x 0.05 mm3) are single.

Animals↗