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Biomedical subjects

R Stein

Publications and source records attributed to R Stein.

At least 73 records · Page 4Linked to original sources
PubMed1999

Radical cystoprostatectomy combined with Mainz pouch bladder substitution to the urethra: long-term results.

OBJECTIVE: To analyse, in a retrospective study, the oncological outcome, pouch-related complications, continence and micturition after radical cystoprostatectomy combined with Mainz pouch orthotopic bladder substitution to the urethra for the treatment of bladder cancer. PATIENTS AND METHODS: Between 1986 and 1996, three urological departments contributed 108 male patients to the review. The same exclusion criteria from orthotopic bladder substitution were applied by all centres, i.e. multifocal or concomitant carcinoma in situ, tumour at the bladder neck, positive biopsy from the prostatic urethra, locally advanced tumour and lymph node involvement. In all, 103 patients were evaluable for follow-up, with a mean (range) follow-up of 42 (3-132) months. RESULTS: Pathological examination of the cystectomy specimen revealed 81% organ-confined tumours. During follow-up, 84% of patients remained free of tumour, 7% developed distant metastases, 5% local recurrences, 4% urethral recurrences, and 1% upper tract urothelial cancer; 85% of patients are capable of spontaneous voiding, with a mean pouch capacity of 720 mL. Daytime continence was achieved in 88%, including 17% wearing one safety pad; 9% had stress incontinence and 3% total incontinence; 67% could sleep through the night, with either complete continence (34%) or one safety pad (33%). Nocturnal incontinence occurred in 11%. Uretero-intestinal stenosis occurred in 15 of 205 (7%) renal units, requiring ureteric reimplantations in 11, nephrectomy in three and antegrade dilatation in one. Reflux was not noted in any patient. About half the patients were on anti-acidotic prophylaxis. CONCLUSION: The large bowel segment in the Mainz-pouch technique of orthotopic bladder substitution provides good reservoir capacity and continence rates, with less ileum used than in all-ileum pouches. The surgical technique is simple and reproducible, and in particular the antireflux ureteric implantation into the caecum protects the upper urinary tracts.

Adult↗
PubMed1999

[Pulmonary trichomoniasis: diagnosis based on identification of irritation in bronchoalveolar lavage].

Bronchopulmonary infections caused by trichomonads have been reported mainly in patients with pre-existing pulmonary or debilitating disease (e.g. bronchial carcinoma, lung abscess, bronchiectasis). Pulmonary trichomoniasis is most often due to infection with Trichomonas tenax, usually regarded as a harmless commensal of the human mouth, and may rarely be caused by other trichomonas species. A 45 year old female presented with a dry cough, exertional dyspnoea and malaise. These symptoms persisted for 6 months regardless of anti-inflammatory and anti-obstructive inhalative therapy. Sarcoidosis of the lungs, diagnosed 20 years prior, had been asymptomatic since and there was no coexistent disease. Laboratory data revealed increased ACE-levels (90 IE/ml) and lung function showed bronchial hyperreactivity on histamine challenge. No other abnormalities were found (chest x-ray, bronchoscopy, lung function test, blood count and serum calcium). The diagnosis was based on the cytological identification of numerous trophozoites of T. tenax in the bronchoalveolar lavage. Therapy with oral metronidazol for 40 days led to complete recovery from symptoms and normalisation of ACE serum levels. The patient has remained well for 12 months since. The pathogenicity of oral trichomonads in the non-immunocompromised host remains uncertain. Our patient had no known medical risk factors by comparison with published cases. The case illustrates the clinical relevance of pulmonary trichomoniasis in an otherwise healthy person.

Animals↗
PubMed1999

Carcinoembryonic antigen as a target for radioimmunotherapy of human medullary thyroid carcinoma: antibody processing, targeting, and experimental therapy with 131I and 90Y labeled MAbs.

The poor prognosis of patients with advanced medullary thyroid carcinoma (MTC) has prompted a search for new treatment modalities. In this report we explore the characteristics of carcinoembyronic antigen (CEA) as a target for radioimmunotherapy (RAIT) of MTC, with respect to antibody processing, targeting, and experimental therapy. In vitro studies showed a high level of CEA expression on the cell surface of the MTC cell line TT. MAbs bound to the cell were predominantly retained for several days, although there was also a significant level of internalization and catabolism. Immunohistology of frozen sections of tumor xenografts demonstrated that approximately half of the cells were darkly stained, however, some cells expressed little or no CEA. In biodistribution studies in nude mice bearing TT tumors, the mean percent injected dose per gram of tumor observed at three days post injection (time of maximum uptake) of 125I-MN-14 was 19.7%. When the MAb was labeled with 88Y, a residualizing label, a much higher accretion, 50.5%, was observed at the time of maximum uptake (7 days). Significant anti-tumor effects were seen at the maximum tolerated doses of 131I- and 90Y-MN-14, compared with relatively rapid tumor growth in untreated animals or those treated with the same dose of control MAbs. Importantly, it was observed that 90Y-MN-14 yielded significantly improved therapeutic efficacy in comparison to 131I-MN-14, which may have important implications for design and conduct of future clinical trials for the treatment of MTC.

Animals↗
PubMed1999

Intravenous cyclosporin in ulcerative colitis: a five-year experience.

OBJECTIVE: Cyclosporin (CSA) is a promising alternative for patients with severe steroid-refractory ulcerative colitis (UC) previously facing only surgical options. Concerns over the long term efficacy and side effects resulted in this investigation of the University of Chicago's 5-yr CSA experience in these patients. METHODS: All steroid-refractory severe ulcerative colitis (UC) patients treated with IV CSA from 1991 to 1995 were identified by using the university's IBD database, with additional information from patient charts and physician files. RESULTS: A total of 42 patients with severe UC unresponsive to IV steroids were treated with IV CSA (4 mg/kg/day). Of 42 patients, 36 (86%) responded; 31 were continued on oral CSA (8 mg/kg/day) for an overall mean of 20 wk. Ten initial CSA responders had colectomies after a mean of 6 months. Of the 36 initial responders, 25 (69%) also received 6-mercaptopurine (6-MP) or azathioprine (aza), and CSA and steroids were tapered. A total of 20% required colectomy, vs 45% of those not receiving 6MP/aza. In all, 62% of all patients, 72% of initial CSA responders, and 80% of initial CSA responders receiving 6MP/aza have avoided colectomy, with a life table analysis of "noncolectomy survival" of 58%, 70%, and 71%, respectively, at 5.5 yr. All colectomies occurred within 18 months of CSA initiation. Complications, resulting in CSA discontinuation in six patients, were all reversible, with complete recovery. CONCLUSIONS: CSA successfully allows most severe steroid resistant UC patients to retain their colons, and provides time for "elective" colectomy in others, especially if 6MP/aza are also given. Careful monitoring for side effects, including PCP prophylaxis, should be part of the treatment protocol.

Adolescent↗
PubMed1999

The NeuroD1/BETA2 sequences essential for insulin gene transcription colocalize with those necessary for neurogenesis and p300/CREB binding protein binding.

NeuroD1/BETA2 is a key regulator of pancreatic islet morphogenesis and insulin hormone gene transcription in islet beta cells. This factor also appears to be involved in neurogenic differentiation, because NeuroD1/BETA2 is able to induce premature differentiation of neuronal precursors and convert ectoderm into fully differentiated neurons upon ectopic expression in Xenopus embryos. We have identified amino acid sequences in mammalian and Xenopus NeuroD1/BETA2 that are necessary for insulin gene expression and ectopic neurogenesis. Our results indicate that evolutionarily conserved sequences spanning the basic helix-loop-helix (amino acids [aa] 100 to 155) and C-terminal (aa 156 to 355) regions are important for both of these processes. The transactivation domains (AD1, aa 189 to 299; AD2, aa 300 to 355) were within the carboxy-terminal region, as analyzed by using GAL4:NeuroD1/BETA2 chimeras. Selective activation of mammalian insulin gene enhancer-driven expression and ectopic neurogenesis in Xenopus embryos was regulated by two independent and separable domains of NeuroD1/BETA2, located between aa 156 to 251 and aa 252 to 355. GAL4:NeuroD1/BETA2 constructs spanning these sequences demonstrated that only aa 252 to 355 contained activation domain function, although both aa 156 to 251 and 300 to 355 were found to interact with the p300/CREB binding protein (CBP) coactivator. These results implicate p300/CBP in NeuroD1/BETA2 function and further suggest that comparable mechanisms are utilized to direct target gene transcription during differentiation and in adult islet beta cells.

Animals↗
PubMed1999

Strategies for reconstruction after unsuccessful or unsatisfactory primary treatment of patients with bladder exstrophy or incontinent epispadias.

PURPOSE: Following unsuccessful or unsatisfactory primary treatment in patients with the epispadias/exstrophy complex, the options for a surgical solution to preserve the upper urinary tract, to achieve complete continence, and to reconstruct the external and female internal genitalia are limited. We reviewed the records of the patients treated at our institution to determine a surgical compromise between ingenious operative constructions and patient desires, both of which are secondary to stabilization of renal function. MATERIALS AND METHODS: From 1967 to December 1997, 128 patients with bladder exstrophy/epispadias complex were treated, of 80 whom had received previous unsuccessful or unsatisfactory treatment. Of these patients 72 were followed for an average of 22.4 years after the first surgical intervention. There were 40 patients referred to our institution after primary bladder closure and bladder neck reconstruction, 10 after rectal bladder, 7 after ureterosigmoidostomy and 5 after incontinent diversion. RESULTS: At the latest followup 19 patients had a rectal reservoir, 38 an ileocecal pouch, 12 a conduit diversion and 3 an augmented Young-Dees procedure. The upper urinary tract remained stable in 95% of the renal units with rectal reservoir, 95% with ileocecal pouch, 96% with a colonic conduit and 100% with an augmented Young-Dees procedure. Day and night continence was achieved in 95% of the patients with a rectal reservoir and 97% of those with an ileocecal pouch were continent, whereas only 2 of the 3 patients with an augmented Young-Dees procedure were continent. Of the women 16 were satisfied with the cosmetic results and 6 delivered 8 children by cesarean section. Only 1 man was dissatisfied with the final cosmetic result. All adults but 1 engaged in sexual intercourse. CONCLUSIONS: The first operative intervention in patients with bladder exstrophy/epispadias complex determines their fate. After failure of primary treatment, the upper urinary tract must be stabilized. In patients with severely impaired renal function the colonic conduit is our method of choice while in those with a normal or slightly dilated upper urinary tract and intact anal sphincter we performed a rectal reservoir. In the remaining patients an ileocecal pouch guarantees continence day and night. The results of genital reconstruction are satisfactory.

Adult↗
PubMed1999

Outpatient laminotomy and discectomy.

This is a prospective study of 61 consecutive patients undergoing lumbar laminotomy and discectomy on an outpatient basis. The purpose of this study was to report on the feasibility of performing lumbar laminotomy and discectomy as an outpatient procedure and to assess perioperative complications, patient satisfaction, cost, and clinical results. Conventional lumbar laminotomy and discectomy traditionally requires a 1-3-day hospital stay. Recent advances in anesthesia and surgical techniques, as well as observation of patient progress after this procedure, has led the authors to believe that that this procedure may be performed on an outpatient basis without compromising patient satisfaction, outcome, or complications. Sixty-one consecutive patients underwent surgery for herniated nucleus pulposus in the lumbar spine. The procedure was performed under loupe magnification without the use of a microscope. Clinical outcome and patient satisfaction were assessed at an average follow-up of 12.5 months. The results showed 62% excellent, 31% good, 7% fair, and there were no reports of a poor outcome. During the time of the study, four patients (7%) were admitted to the hospital after the procedure for reasons of pain control, inability to void, or lack of caregiver at home. Overall cost savings were reflected in the cost of inpatient stay when compared to a representative group of inpatients. Laminotomy and discectomy for a hemiated nucleus pulposus has 93% good or excellent results as shown by this study and previous studies. Laminotomy and discectomy, which remains the gold-standard procedure for herniated disc surgery, can be performed safely and effectively as an outpatient procedure in the majority of patients.

Adult↗
PubMed1999

Enhancement of tumor-to-nontumor localization ratios by hepatocyte-directed blood clearance of antibodies labeled with certain residualizing radiolabels.

UNLABELLED: To increase tumor-to-nontumor localization ratios of injected radiolabeled antibodies (Abs), several interrelated methods were used. METHODS: The model systems used were two human carcinoma xenografts grown in nude mice, targeted by antibodies RS11 (antiepithelial glycoprotein-2) or MN-14 (anticarcinoembryonic antigen). The Abs were conjugated with biotin and 111In-benzyl diethylenetriamine pentaacetic acid, and, at various times after injection, were cleared by intraperitoneal injection of galactosylated streptavidin, which delivers the complexes to hepatocytes. The radiolabel used was selected because it is retained within tumors after catabolism of the Ab by the tumor cell but is quite rapidly excreted from hepatocytes into bile. RESULTS: With blood clearance induced at 24 h, and dissection 5 h later, high tumor-to-nontumor ratios were attained. Depending on the model used, tumor-to-blood ratios were 16:1 to 31:1, and tumor-to-nontumor ratios for the kidney, lungs and bone were also high and greatly increased by the clearance regimen. Despite clearance into the liver, tumor-to-liver ratios remained >1, due to fairly rapid biliary excretion of the label. The absolute antibody uptake by the tumors was also high, because 24 h was allowed for the Ab to penetrate and bind to cells within the subcutaneous tumors. CONCLUSION: The method described produced high tumor-to-nontumor ratios at 1 d after injection and may be advantageous for tumor imaging with antibodies. Radiation dosimetry calculations indicate that there is only a slight advantage with this approach for radioimmunotherapy.

Animals↗
PubMed1999

Targeting human cancer xenografts with monoclonal antibodies labeled using radioiodinated, diethylenetriaminepentaacetic acid-appended peptides.

A new nonmetabolizable peptide approach to the production of residualizing radioiodine was evaluated in nude mice bearing xenografts of human lung adenocarcinoma (Calu-3) and B-cell lymphoma (Ramos). Monoclonal antibodies (MAbs) RS7 (anti-epithelial glycoprotein-1) and LL2 (anti-CD22) were radioiodinated using the thiol-reactive diethylenetriaminepentaacetic acid-D-peptide adducts IMP-R1 and IMP-R2. 125I-IMP-R1- and 125I-IMP-R2-labeled MAbs were compared to the MAbs iodinated by the conventional chloramine-T approach, (111)In, and 131I-dilactitoltyramine (DLT). In vivo biodistribution studies demonstrated a significant improvement in the tumor accretion of radiolabel using the 125I-IMP-R1 labeled MAbs compared with the conventionally iodinated antibodies. For example, at day 7, the percentage of injected dose per gram of tissue in Calu-3 was 7.9 +/- 4.1% and 18.1 +/- 7.9% (P < 0.05) for the conventional 131I- and 125I-IMP-R1-RS7, respectively, and tumor:nontumor ratios were 2.6-4.5-fold higher with the 125I-IMP-R1-RS7. It is estimated that 131I-IMP-R1-RS7 would deliver a dose to tumor (at the estimated maximum tolerated dose) 3.9 times greater than conventional 131I-labeled RS7, 1.4 times greater than 90Y-labeled RS7, and 0.7 times that of 131I-DLT-labeled RS7. Tumor accretion of 125I-IMP-R2-RS7 was also improved compared with conventionally iodinated antibody. However, this label also caused a large increase in kidney accretion. Similar improvements in tumor accretion and tumor:nontumor ratios were observed when 125I-IMP-R1-LL2 was used in the Ramos model. IMP-R1 offers a practical and useful residualizing radioiodine label because labeling efficiency is at least 10 times greater than that of the residualizing label DLT, without MAb aggregation. Structural modifications can be envisioned for further improvements in radioiodine incorporation, specific activity, and tumor dosimetry, and efforts along these lines are under way.

Animals↗
PubMed1999

Assessment of combined radioimmunotherapy and chemotherapy for treatment of medullary thyroid cancer.

We have shown previously significant antitumor effects using 90Y-MN-14 anti-CEA monoclonal antibody (MAb) for radioimmunotherapy (RAIT) of human medullary thyroid cancer (MTC) xenografts using the TT cell line. The purpose of this investigation was to determine the effect of combining chemotherapy and RAIT with 90Y-MN-14 in MTC. In particular, the toxicity and efficacy of various dose schedules of RAIT and doxorubicin were examined and compared with that at the maximum tolerated dose (MTD) of each single modality treatment. The MTD of RAIT of 105 microCi of 90Y-MN-14 was given alone and combined with 100 and 75% of the MTD of doxorubicin (60 mg/m2); and the MTD of doxorubicin was given alone and combined with 100 and 75% of the MTD of RAIT. In addition, 75% of each agent was also administered in combination. The MTD of RAIT was also evaluated in combination with 58 and 78% of the MTD of Taxol. Whereas 90Y-MN-14 (105 microCi) led to significant antitumor effects (P < 0.0001), doxorubicin at 60 mg/m2 or Taxol at 225 mg/m2 yielded only a slight tumor growth delay. The combinations of 100% of the MTD of RAIT and 75% of the MTD of doxorubicin and 100% of the MTD of doxorubicin and 75% of the MTD of RAIT were equitoxic to the MTD of RAIT alone and appear to result in improved efficacy compared with either RAIT or doxorubicin alone. For the 100% RAIT and 75% doxorubicin combination, the therapeutic efficacy was similar when doxorubicin was administered on the same day or 1 day after RAIT, but the treatment was less effective when doxorubicin was administered 2 days after RAIT (P < 0.03). Prolonged retardation of tumor progression was also observed in animals treated with the MTD of RAIT combined with 175 mg/m2 of Taxol, without increases in toxicity above that observed with RAIT alone. In conclusion, the combination of RAIT and chemotherapy appears to augment the antitumor effects of either treatment alone without a significant increase in toxicity. In addition, the timing of drug administration relative to RAIT in the combined therapy appears to be important.

Animals↗
PubMed1998

Role of the proteasome and NF-kappaB in streptococcal cell wall-induced polyarthritis.

The transcription factor NF-kappaB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-kappaB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IkappaB is degraded by the ubiquitin-proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-kappaB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin-proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-kappaB and the subsequent transcription of genes that are regulated by NF-kappaB. Significantly, PS-341 displayed similar effects in vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IkappaBalpha degradation and NF-kappaB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin-proteasome pathway and NF-kappaB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect.

Animals↗
PubMed1998

Human phosphoinositide 3-kinase C2beta, the role of calcium and the C2 domain in enzyme activity.

The cDNA for a human Class II phosphoinositide 3-kinase (PI 3-kinase C2beta) with a C2 domain was cloned from a U937 monocyte cDNA library and the enzyme expressed in mammalian and insect cells. Like other Class II PI 3-kinases in vitro, PI 3-kinase C2beta utilizes phosphatidylinositol (PI) and PI 4-monophosphate but not PI 4, 5-biphosphate as substrates in the presence of Mg2+. Remarkably, and unlike other PI 3-kinases, the enzyme can use either Mg-ATP or Ca-ATP to generate PI 3-monophosphate. PI 3-kinase C2beta, like the Class I PI 3-kinases, but unlike PI 3-kinase C2alpha, is sensitive to low nanomolar levels of the inhibitor wortmannin. The enzyme is not regulated by the small GTP-binding protein Ras. The C2 domain of the enzyme bound anionic phospholipids such as PI and phosphatidylserine in vitro, but did not co-operatively bind Ca2+ and phospholipids. Deletion of the C2 domain increased the lipid kinase activity suggesting that it functions as a negative regulator of the catalytic domain. Although presently it is not known whether PI 3-kinase C2beta is regulated by Ca2+ in vivo, our results suggest a novel role for Ca2+ ions in phosphate transfer reactions.

Animals↗
PubMed1998

NGF induces transient but not sustained activation of ERK in PC12 mutant cells incapable of differentiating.

Activation of receptor tyrosine kinases stimulates a diverse array of cellular responses such as proliferation and differentiation. The first events in the signal transduction pathways mediated by different receptor tyrosine kinases are similar and include activation of the mitogen-activated protein kinase (MAPK) pathway and the induction of immediate early genes. The precise signaling pathways leading to each of the cellular responses mediated by receptor tyrosine kinases are still unknown, although it has been proposed that sustained activation of the MAPK pathway by receptor tyrosine kinases such as the nerve growth factor (NGF) receptor TrkA is sufficient to induce differentiation in PC12 cells. In the present study we examined the effect of NGF on mutant PC12 cells that were derived spontaneously in our cultures. NGF induced normal activation of immediate early genes in these cells, whereas the activation of some delayed response genes, as well as neurite outgrowth, was impaired. Furthermore, activation of the NGF-induced extracellular signal-regulated kinase (ERK) in these cells was transient, not sustained. These results support the hypothesis that sustained activation of ERK plays an important role in activating the induction of delayed response genes. However, sustained ERK activation is not a mandatory condition for the promotion of all the features of differentiated PC12 cells, as NGF could induce transcription of the delayed response gene, transin, in PC12 mutant cells. Taken together, our results suggest that NGF induces differentiation of PC12 cells via several signaling pathways, an important one of which is the MAPK pathway.

Animals↗
PubMed1998

Bcl-xL inhibits different apoptotic pathways in rat PC12 cells.

Bcl-xL, a member of the bcl-2 family of proteins is required for the survival of neurons early in development. To study the mechanism of action of Bcl-xL in a neuronal context, we generated rat PC12 cells overexpressing Bcl-xL and examined their susceptibility to apoptotic stimuli that induce apoptosis through different pathways involving trophic-factor deprivation, staurosporine, tumor necrosis factor alpha or cisplatin. Overexpression of Bcl-xL in both naive and neuronal PC12 cells inhibited apoptosis induced by the different pathways. However, the extent of this protective effect varied, suggesting that the contribution of the Bcl-xL-controlled step to apoptosis differs in the different pathways. Our findings also showed that TNF alpha-induced activation of caspase-3 is inhibited by overexpression of Bcl-xL, suggesting that Bcl-xL acts upstream of caspase activation.

Animals↗
PubMed1998

Glucose stimulates the activation domain potential of the PDX-1 homeodomain transcription factor.

Glucose-stimulated expression of the insulin gene in beta cells is mediated by the PDX-1 transcription factor. In this report, we show that stimulation results from effects on activation and DNA-binding potential. Thus, glucose specifically stimulated expression in MIN6 beta cells from chimeras of PDX-1 and the GAL4 DNA-binding domain which spanned the N-terminal PDX-1 activation domain located between amino acids 1 to 79. GAL4:PDX activity was induced over physiological glucose concentrations and was also regulated by effectors of this response. The level of endogenous PDX-1 binding and phosphorylation were also induced under these conditions. We discuss how changes in PDX-1 phosphorylation may influence activity in glucose-treated beta cells.

Base Sequence↗
PubMed1998

Effects of changes in three catalytic residues on the relative stabilities of some of the intermediates and transition states in the citrate synthase reaction.

This work reports the relative importance of the interactions provided by three catalytic residues to individual steps in the mechanism of citrate synthase. When the side chains of any of the residues (H320, D375, and H274) are mutated, the data indicate that they are involved in the stabilization of one or more of the transition/intermediate states in the multistep citrate synthase reaction. H320 forms a hydrogen bond with the carbonyl of oxaloacetate and the alcohols of the citryl-coenzyme A and citrate products. Enzymes substituted at H320 (Q, G, N, and R) have reaction profiles for which the condensation reaction is cleanly rate determining. None of these mutants can activate the carbonyl of oxaloacetate by polarization. All these mutants catalyze the necessary proton transfer from the methyl group of acetyl-coenzyme A only poorly, a process which occurs in a structurally separate site. Furthermore, all H320 mutants hydrolyze the citryl-coenzyme A intermediate significantly more slowly than does the wild-type. D375 is the base removing the proton of acetyl-coenzyme A. D375E and D375G have greatly diminished ability to catalyze proton transfer from acetyl-CoA. The D375 mutants polarize the oxaloacetate carbonyl as well as wild-type. For D375E, the hydrolysis of citryl-CoA is rate determining. D375G, having no side chain capable of acid-base chemistry in either the condensation or hydrolysis reactions is nearly completely devoid of activity in any of the reactions catalyzed by the wild-type. H274 hydrogen bonds to the carbonyl of acetyl-coenzyme A but also forms the back wall of the oxaloacetate-binding site. H274G cannot properly activate either oxaloacetate or acetyl-coenzyme A, and the condensation reaction is overwhelmingly rate determining. Nonetheless, hydrolysis of the intermediate is impaired. All the enzymes except H320R and H274G show kinetic cooperativity with CitCoA as substrate, indicating changes in the subunit interactions with these latter two mutants. The energetics of citrate synthase are surprisingly tightly coupled. All changes affect more than one step in the catalytic cycle. Within the condensation reaction, the intermediate of proton transfer must occupy a shallow well between transition states close in free energy so that perturbations of one have substantial effects on that of the other.

Acetyl Coenzyme A↗
PubMed1998

Need for caspase-2 in apoptosis of growth-factor-deprived PC12 cells.

Previous studies have shown that caspases (proteases related to interleukin-1beta converting enzyme) are needed for the death of trophic factor-deprived PC12 cells. However, the protease involved in this process has not been identified. The results presented here strongly suggest that caspase-2 (Nedd2/Ich-1) plays a major role in the death of serum-deprived PC12 cells. We show that in PC12 cells overexpression of caspase-2 induces cell death, serum deprivation induces processing (i.e., activation) of the 48-kDa pro-caspase-2, and stable expression of caspase-2 antisense RNA inhibits apoptosis induced by serum deprivation. In addition, overexpression of bcl-2, which prevents this death process, also inhibits the processing of pro-caspase-2, suggesting that bcl-2 acts upstream of pro-caspase-2 activation.

Animals↗