The bifunctional aminoadipic semialdehyde synthase in lysine degradation. Separation of reductase and dehydrogenase domains by limited proteolysis and column chromatography

The mammalian aminoadipic semialdehyde synthase is a bifunctional enzyme that catalyzes the first two sequential steps in lysine degradation in the major saccharopine pathway (Markovitz, P. J., Chuang, D. T., and Cox, R. P. (1984) J. Biol. Chem. 259, 11643-11646). We show here that limited proteolysis of the highly purified synthase from bovine liver with elastase, chymotrypsin, and papain resulted in separation of lysine-ketoglutarate reductase and saccharopine dehydrogenase activities as judged by activity stainings of the polyacrylamide gel. Enzyme assays showed no loss of the two activities after digestions with these proteases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis disclosed the presence of two limit polypeptides in the elastolytic digests, i.e. fragment A (Mr = 62,700) and fragment B (Mr = 49,200). These fragments were apparently derived from the same polypeptide (Mr = 115,000) of the parent synthase. The reductase and dehydrogenase activities of the elastase-digested synthase were completely resolved by DEAE-Bio-Gel column chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that fragment A and fragment B were associated with reductase and dehydrogenase activities, respectively. The bovine synthase showed Mr = 420,000 in sedimentation equilibrium, confirming a tetrameric structure for the enzyme. The above results establish that the reductase and dehydrogenase domains of the aminoadipic semialdehyde synthase are separately folded and functionally independent of each other.     

Fibular osteoseptocutaneous flap: anatomic study and clinical application

The vascularized fibular graft has been expanded to an osteoseptocutaneous flap by including a cutaneous flap on the lateral aspect of the lower leg. The cutaneous flap can serve not only for postoperative monitoring of the grafted fibula, but also as extra skin coverage to replace substantial skin defects or prevent tight closure of the wound. From anatomic studies of 20 cadaver legs and 15 clinical cases, it has been possible to demonstrate adequate circulation to the skin of the lateral aspect of the lower leg from the septocutaneous branches of the peroneal artery alone. This finding has allowed the development of a new concept and technique to elevate the fibula as an osteoseptocutaneous flap for reconstruction which provides the following advantages: Elevation of the fibular osteoseptocutaneous unit is easy and fast. The cutaneous flap of the fibular osteoseptocutaneous unit can slide almost freely while attached to the paper-thin posterior crural septum without being tethered by a bulky muscle cuff, facilitating the setting of the fibular osteocutaneous flap when the bone and skin are widely separated. Intraoperatively, in a situation in which it is necessary to change from originally selected recipient vessels to ones more suitable, the thin posterior crural septum can be folded around the fibula allowing more flexibility in choice of recipient vessels. The fibular osteoseptocutaneous flap meets the criteria outlined for composite tissue reconstruction of defects of the extremities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C does not regulate diacylglycerol metabolism in aortic smooth muscle cells

Summary The effect of a reduction in protein kinase C activity on the metabolism of exogenous [³H]diC8 by freshly isolated smooth muscle cells from rabbit aorta and cultured A10 smooth muscle cells was determined. The metabolism of [³H]diC8 by both smooth muscle cell preparations was predominantly by hydrolysis to yield monoC8 and glycerol (lipase pathway); very little radioactivity was incorporated into phospholipids. Diacylglycerol lipase activity measured in vitro with A10 cell homogenates was much greater than diacylglycerol kinase activity. The addition of the protein kinase C inhibitor H-7 to incubations of isolated aortic smooth muscle cells and cultured A10 cells had no significant effect on the metabolism of [³H]diC8. Protein kinase C activity in cultured A10 cells preincubated for 20 h with a phorbol ester was reduced to 14% of control as a consequence of down-regulation, but diC8 metabolism was not changed. Therefore, protein kinase C does not regulate the metabolism of diacylglycerols in aortic smooth muscle cells.Abbreviations IP₃ inositol 1,4,5-trisphosphate - DG diacylglycerol - MG monoacylglycerol - PL phospholipid(s) - diC8 dioctanoylglycerol - H-7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride - monoC8 monooctanoylglycerol - PS phosphatidylserine - PDBu phorbol 12,13-dibutyrate     

Simultaneous multiple toe transfers in hand reconstruction

Our experience with simultaneous transfer of two or more toe units to the same hand where multiple digits were missing is presented. Forty-six toes from 38 feet were transferred to reconstruct 19 hands in 19 patients. The transfers consisted of 7 combined second and third toe units and 32 single toes. Three patients had a primary and 16 patients had a secondary reconstruction. There was one complete and one partial failure. The two-point discrimination ranged from 6 mm to protective sensation. Total active movement averaged 57 degrees in the thumb and 127, 93, 71, and 68 degrees, respectively, in the fingers reconstructed at middle phalanx, proximal phalanx, metacarpophalangeal joint, and metacarpal head. Pulp-to-pulp pinch averaged 2.4 kg in patients who had thumbs reconstructed and averaged 3.0 kg in patients who had normal thumbs. There was no cold intolerance, and no significantly disabled foot occurred except one with scissoring deformity. Simultaneous multiple toe transfer in hand reconstruction is feasible without increased complications both in primary and secondary wound conditions. It is time-effective and cost-effective.     

Reconstruction of Achilles tendon and calcaneus defects with skin-aponeurosis-bone composite free tissue from the groin region

Eight patients had reconstructive surgery for soft-tissue, associated Achilles tendon, and calcaneus defects on the posterior aspect of the ankle. In group A, those patients with skin, soft-tissue, and Achilles tendon loss were treated with free groin flaps that included sheets of the external oblique aponeurosis based on the superficial circumflex iliac vessel. The groin flap provided skin coverage, and the aponeurosis was rolled to form a tendon-like structure to replace the Achilles tendon. In group B, those patients with an additional calcaneus bone loss were treated with free iliac osteocutaneous flaps, together with the external oblique aponeurosis based on the deep circumflex iliac vessel. The iliac bone was then utilized to reconstruct the calcaneus defect. All composite free tissue transfers were successful, except in two group B patients who suffered partial skin loss. The advantages of this technique are (1) a single, one-stage procedure, (2) faster wound healing with fewer adhesions of the reconstructed Achilles tendon, and (3) good cosmesis and minimal morbidity at the donor site.     

Resonance Raman spectra of bovine liver catalase: enhancement of proximal tyrosinate vibrations

Resonance Raman spectra of native bovine liver ferri-catalase have been obtained in the 200-1800 cm-1 region. Excitation at a series of wavelengths ranging from 406.7 to 514.5 nm has been used and gives rise to distinct sets of resonance Raman bands. Excitation within the Soret and Q-bands of the heme group produces the expected set of polarized and nonpolarized porphyrin modes, respectively. The frequencies of the porphyrin skeletal stretching bands in the 1450-1700 cm-1 region indicate that catalase contains only five-coordinate, high-spin heme groups. In addition to the porphyrin modes, bovine liver catalase exhibits bands near 1612 and 1520 cm-1 that are attributable to ring vibrations of the proximal tyrosinate that are enhanced via resonance with a proximal tyrosinate----Fe(III) change transfer transition centered near 490 nm. Similar bands have been observed in mutant hemoglobins that have tyrosinate axial ligands and in other Fe(III)-tyrosinate proteins. No resonance Raman bands have been observed that can be attributed to degraded hemes. The spectra are relatively insensitive to pH over the range of 5-10, and the same spectra are observed for catalase samples that do and do not contain tightly bound NADPH. Resonance Raman spectra of the fluoride complex exhibit porphyrin skeletal stretching modes that show it to be six coordinate, high spin, while the cyanide complex is six coordinate, low spin. Both the azide and thiocyanate complexes, however, are spin-state mixtures with the high-spin form predominant.     

Preparation and characterization of radioactive monoiodotyrosine and diiodotyrosine derivatives of parathyroid hormone

Highly purified native parathyroid hormone was iodinated by the enzymatic method and separated from unlabeled hormone by isocratic HPLC. The separation system used also resolved iodohistidine, monoiodotyrosine, and diiodotyrosine forms of the hormone from one another. A simplified procedure for direct bioassay of the carrier-free, high specific activity, mono- and diiodinated parathyroid hormone (PTH) by the renal membrane adenylyl cyclase method was also developed. Both labeled forms of the hormone are very potent in this assay, but the iodinated forms appeared to give a lower V_max than the native hormone. The methods for iodination, separation and biological characterization of this PTH tracer are exceptionally facile, inexpensive, and convenient.     

The Effect of Haemodialysis Access Types on Cardiac Performance and Morbidities in Patients with Symptomatic Heart Disease

Background

Little is known about whether the arteriovenous type haemodialysis access affects cardiac function and whether it is still advantageous to the uremic patient with symptomatic heart disease.

Methods

We conducted a retrospective comparative study. Patients with heart disease and end-stage renal disease that had a new chronic access created between January 2007 and December 2008 and met the inclusion criteria were assessed. The endpoint was major adverse event (MAE)-free survivals of arteriovenous access (AVA) and tunneled cuffed double-lumen central venous catheter (CVC) groups. Whether accesses worsened heart failure was also evaluated.

Results

There were 43 CVC patients and 60 AVA patients. The median follow-up time from access creation was 27.6 months (IQR 34.7, 10.9~45.6). Although CVC patients were older than AVA patients (median age 78.0, IQR 14.0 vs. 67.5, IQR 16.0, respectively, p = .009), they manifested non-inferior MAE-free survival (mean 17.1, 95% CI 10.3~24.0 vs. 12.9, 95% CI 8.5~17.4 months in CVC and AVA patients, respectively, p = .290). During follow-up, more patients in the AVA group than in the CVC group deteriorated in heart failure status (35 of 57 vs. 10 of 42, respectively, odds ratio 5.1, p < .001). Preoperative-postoperative pairwise comparison of echocardiographic scans revealed an increased number of abnormal findings in the AVA group (Z = 3.91, p < .001), but not in the CVC group.

Conclusions

In patients with both symptomatic heart disease and end stage renal disease (ESRD), CVC patients showed non-inferior MAE-free survival in comparison to those in the AVA group. AV type access could deteriorate heart failure. Accordingly, uremic patients with symptomatic heart disease are not ideal candidates for AV type access creation.     

A Patient Friendly Corifollitropin Alfa Protocol without Routine Pituitary Suppression in Normal Responders

The release of corifollitropin alfa simplifies daily injections of short-acting recombinant follicular stimulating hormone (rFSH), and its widely-used protocol involves short-acting gonadotropins supplements and a fixed GnRH antagonist regimen, largely based on follicle size. In this study, the feasibility of corifollitropin alfa without routine pituitary suppression was evaluated. A total of 288 patients were stimulated by corifollitropin alfa on cycle day 3 following with routine serum hormone monitoring and follicle scanning every other day after 5 days of initial stimulation, and a GnRH antagonist (0.25 mg) was only used prophylactically when the luteinizing hormone (LH) was ≧ 6 IU/L (over half of the definitive LH surge). The incidence of premature LH surge (≧ 10 IU/L) was 2.4% (7/288) before the timely injection of a single GnRH antagonist, and the elevated LH level was dropped down from 11.9 IU/L to 2.2 IU/L after the suppression. Two hundred fifty-one patients did not need any antagonist (87.2% [251/288]) throughout the whole stimulation. No adverse effects were observed regarding oocyte competency (fertilization rate: 78%; blastocyst formation rate: 64%). The live birth rate per OPU cycle after the first cryotransfer was 56.3% (161/286), and the cumulative live birth rate per OPU cycle after cyrotransfers was 69.6% (199/286). Of patients who did and did not receive GnRH antagonist during stimulation, no significant difference existed in the cumulative live birth rates (78.4% vs. 68.3%, p = 0.25). The results demonstrated that the routine GnRH antagonist administration is not required in the corifollitropin-alfa cycles using a flexible and hormone-depended antagonist regimen, while the clinical outcome is not compromised. This finding reveals that the use of a GnRH antagonist only occasionally may be needed.     

Repeated Activation of Lung Invariant NKT Cells Results in Chronic Obstructive Pulmonary Disease-Like Symptoms

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation, mucus hypersecretion, and emphysema, which lead to reduced lung function and breathlessness. The pathologies of COPD are due to an abnormal immune response. Invariant natural killer T (iNKT) cells are an important population of innate lymphocytes and have been implicated in the regulation of immune responses associated with a broad range of diseases including COPD. We have here analyzed the role of iNKT cells in a model of COPD induced by repeated intranasal administration of iNKT cell agonist α-galactosylceramide (α-GalCer). Our results demonstrated that mice that received repeated intranasal administration of α-GalCer had molecular and inflammatory features of COPD including airway inflammation with significant increases in infiltration of macrophages and lymphocytes, CD8⁺ T cells, as well as proinflammatory cytokines IL-6 and TNF-α. In particular, these mice also showed the presence of pulmonary emphysema, mucus production, and pulmonary fibrosis. Furthermore, neutralization of IL-4 reduced α-GalCer induced emphysema. This study indicates the importance of iNKT cells in the pathogenesis of COPD by an IL-4 dependent mechanism.