Alendronate medication possession ratio and the risk of second hip fracture: an 11-year population-based cohort study in Taiwan.

Alendronate is effective in preventing second hip fracture in osteoporotic patients. However, no consensus exists on the duration that is effective in preventing a second hip fracture. Our study demonstrated that risk can be reduced when the prescription is ≥ 6 months for the year following the index hip fracture. INTRODUCTION: Alendronate is effective in preventing second hip fracture in osteoporotic patients. However, no consensus exists on the accurate medication possession ratio (MPR) that is effective in preventing a second hip fracture. Our objective was to compare the risk of second hip fracture in patients treated with different MPR of alendronate. METHODS: In this population-based cohort study, data from National Health Insurance Research Database of Taiwan were analyzed. Patients 50 years and older who had an index hip fracture and were not receiving any osteoporotic medications before their fracture during 2000-2010 were included. The cohort consisted of 88,320 patients who were new alendronate users (n = 9278) and non-users (n = 79,042). Those without alendronate were matched 4:1 as the control group. Patients were subdivided into those with no medication, MPR < 25%, MPR 25-50%, MPR 50-75%, and MPR 75-100%. Cox proportional hazard models were used to calculate the adjusted hazard ratios for different MPRs of alendronate. RESULTS: After matching, 38,675 patients were included in this study; 20,363 (52.7%) were women, and 30,940 (80%) patients were without medication of alendronate. During follow-up on December 31, 2012, 2392 patients had a second hip fracture, for an incidence of 1449/100,000 person-years. Patients with alendronate MPR 50-75% had a lower risk of a second hip fracture compared to non-users (hazard ratio 0.66). When the MPR increased to 75-100%, the hazard ratio decreased to 0.61. CONCLUSIONS: In this population-based cohort study, risk of a second hip fracture can be reduced when the alendronate MPR is ≥ 50% for the year following the index hip fracture. As the MPR increases, the risk of a second hip fracture decreases.

Prognostic factors for the outcome of extracorporeal shockwave therapy for calcific tendinitis of the shoulder.

AIMS: We conducted a study to identify factors that are prognostic of the outcome of extracorporeal shockwave therapy (ESWT) for calcific tendinitis of the shoulder. PATIENTS AND METHODS: Since 1998, patients with symptomatic calcific tendinitis of the rotator cuff have been treated with ESWT using an electrohydraulic mode shockwave device. One year after ESWT, patients were grouped according to the level of resorption of calcification. RESULTS: Of 241 symptomatic shoulders, complete resorption (CR) of calcification occurred in 134 (CR group). The remaining 107 shoulders had incomplete resorption (ICR) (ICR group). Gartner type I calcification was most common (64.5%) in the ICR group. The mean duration of symptoms before ESWT was significantly longer in the ICR group. Overall, 81% of the CR group and 23.4% of the ICR group were symptom free. There was a strong relationship between subsidence of symptoms and remission of calcification. Poor prognosis was significantly related to Gartner type I calcification, calcification extent > 15 mm and duration of symptoms > 11 months. CONCLUSION: Patients with calcific tendinitis of the shoulder who have the factors identified for a poor outcome after ESWT should undergo a different procedure. Cite this article: Bone Joint J 2017;99-B:1643-50.

Race and health profiles in the United States: an examination of the social gradient through the 2009 CHIS adult survey.

OBJECTIVE: To examine the role of the social gradient on multiple health outcomes and behaviors. It was predicted that higher levels of SES, measured by educational attainment and family income, would be associated with positive health behaviors (i.e., smoking, drinking, physical activity, and diet) and health status (i.e., limited physical activity due to chronic condition, blood pressure, obesity, diabetes, BMI, and perceived health condition). The study also examined the differential effects of the social gradient in health among different racial/ethnic groups (i.e., non-Hispanic Whites, Blacks, Asian, Hispanics, and American Indians). STUDY DESIGN: Cross-sectional study. METHODS: The data were from the adult 2009 California Health Interview Survey (CHIS). Weighted multivariable linear and logistic regression models were conducted to examine trends found between SES and health conditions and health behaviors. Polynomial trends were examined for all linear and logistic models to test for the possible effects (linear, quadratic, and cubic) of the social gradient on health behaviors and outcomes stratified by race/ethnicity. RESULTS: Findings indicated that, in general, Whites had more favorable health profiles in comparison to other racial/ethnic groups with the exception of Asians who were likely to be as healthy as or healthier than Whites. Predicted marginals indicated that Asians in the upper two strata of social class display the healthiest outcomes of health status among all other racial/ethnic groups. Also, the social gradient was differentially associated with health outcomes across race/ethnicity groups. While the social gradient was most consistently observed for Whites, education did not have the same protective effect on health among Blacks and American Indians. Also, compared to other minority groups, Hispanics and Asians were more likely to display curvilinear trends of the social gradient: an initial increase from low SES to mid-level SES was associated with worse health outcomes and behaviors; however, continued increase from mid-SES to high SES saw returns to healthy outcomes and behaviors. CONCLUSION: The study contributes to the literature by illustrating unique patterns and trends of the social gradient across various racial/ethnic populations in a nationally representative sample. Future studies should further explore temporal trends to track the impact of the social gradient for different racial and ethnic populations in tandem with indices of national income inequalities.

Circulating levels of leptin, adiposity and breast cancer risk.

The present case-control study was to investigate the relationships of plasma leptin level and anthropometric measures of adiposity with the risk of breast cancer. Questionnaire information, anthropometric measures and blood samples were taken before treatment from 297 incident cases with breast cancer and 593 controls admitted for health examination at the Tri-Service General Hospital, Taipei, between 2004 and 2006. Plasma levels of leptin were measured by RIA. Logistic regression analysis was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for assessing the associations. Overall, higher leptin concentrations were significantly associated with an increased risk of breast cancer (OR (95% CI) for top vs bottom tertile of leptin was 1.63 (1.07-2.49), P(trend)=0.009). Waist circumference was a significant anthropometric factor for breast cancer in both pre- and postmenopausal women. Furthermore, the associations of leptin with breast cancer risk remained after adjustment for obesity indices. These results suggest that leptin may have an independent role in breast tumorigenesis. Regardless of the impact of circulating leptin, more research is needed to elucidate molecular mechanisms and local leptin levels that are critical for the development of breast cancers.

Association of mu-opioid receptor gene polymorphism (A118G) with variations in morphine consumption for analgesia after total knee arthroplasty.

BACKGROUND: Morphine consumption after a given surgical procedure can vary considerably. Studies show that single nucleotide polymorphism involving the nucleotide position 118 at exon 1 of the mu-opioid receptor gene (OPRM1) may play a role in mediating the effects of opioids. This study was performed to correlate the A118G polymorphism at OPRM1 with morphine consumption in patients undergoing total knee arthroplasty. METHODS: Post-operative pain was relieved by patient-controlled analgesia (PCA). The analgesic effect was evaluated using a visual analogue scale. Side-effects, such as sedation, nausea and vomiting, and pruritus, were recorded systematically. The genotypes were determined by sequencing polymerase chain reaction-amplified DNA. The differences in demographics and consumed morphine from the PCA device between the different genotypes were tested using one-way analysis of variance. The prevalence of side-effects from morphine and sex distribution were compared using the Kruskal-Wallis test. RESULTS: One hundred and forty-seven patients were included in the study. Twenty-seven patients who required rescue analgesia were excluded; these patients did not differ demographically or genetically from the 120 who completed the study. Of the latter, 74 were A118 homozygous (AA), 33 were heterozygous (AG) and 13 were G118 homozygous (GG). Group GG consumed significantly more morphine (40.4 +/- 22.0 mg) than group AA (25.3 +/- 15.5 mg) and group AG (25.6 +/- 11.7 mg) during the first 48 h post-operatively. The groups did not differ with respect to reported pain, age, sex, weight and adverse effects. CONCLUSIONS: G118 homozygotes have a poorer response to morphine for post-operative pain control than A118 homozygotes or heterozygotes. The genotype may thus influence the post-operative response to pain and cause differences in the amounts of analgesic consumed by patients after total knee arthroplasty.

Electroporative alpha-MSH gene transfer attenuates thioacetamide-induced murine hepatic fibrosis by MMP and TIMP modulation.

Hepatic fibrosis represents a process of healing and scarring in response to chronic liver injury. alpha-Melanocyte-stimulating hormone (alpha-MSH) is a 13-amino-acid peptide with potent anti-inflammatory effects. We have previously demonstrated that alpha-MSH gene therapy protects against thioacetamide (TAA)-induced acute liver failure. Therefore, the aim of this study is to investigate whether alpha-MSH gene therapy possesses antihepatic fibrogenic effect. Liver fibrosis was induced by long-term TAA administration in mice. alpha-Melanocyte-stimulating hormone expression plasmid was delivered via electroporation after liver fibrosis was established. Our results showed that alpha-MSH gene therapy attenuated liver fibrosis in TAA-treated mice. Reverse transcription polymerase chain reaction revealed that alpha-MSH gene therapy attenuated the liver transforming growth factor-beta1, collagen alpha1 and cell adhesion molecule mRNA upregulation. Following gene transfer, the expression of alpha-smooth muscle actin and cyclooxygenase-2 were both significantly attenuated. Further, alpha-MSH significantly increased matrix metalloproteinase (MMP), while tissue inhibitors of matrix metalloproteinase (TIMPs) were inactivated. In summary, alpha-MSH gene therapy reversed established liver fibrosis in mice and prevented the upregulated fibrogenic and pro-inflammatory gene responses after TAA administration. Its collagenolytic effect might be attributed to MMP and TIMP modulation. Hence, alpha-MSH gene therapy may be an effective therapeutic modality against liver fibrosis with potential clinical use.

Anesthetic effect of epidural anesthesia with cephalad or caudad catheterization for ankle surgery or hemorrhoidectomy.

BACKGROUND: The larger size of the first sacral nerve root has been reported to be an unfavorable factor leading to sacral sparing in epidural anesthesia. Previous studies have shown that an adequate analgesic effect of the epidural block was achieved with the catheter placement in the caudal direction. In this study, the anesthetic effect of epidural anesthesia with catheter placement of a cephalic or caudad direction was compared in ankle and hemorrhoid surgery. METHODS: Twenty-one ASA physical status I or II patients undergoing surgery for ankle fractures with epidural anesthesia were enrolled and randomized into two groups. The epidural catheter was placed either to a cephalad (AU group) or caudal (AD group) direction. Another 21 patients undergoing hemorrhoidectomy were also randomized into two groups to receive epidural anesthesia in a similar way (HU and HD groups). The onset for, duration of, and recovery time from epidural anesthesia and the incidence of analgesic request were recorded. RESULTS: No significant differences were demonstrated when age, height, weight or sex were compared between the four study groups. The onset time of the block and the incidence of intrasurgical analgesic request were lower in the caudal subgroup when the ankle surgery patients were compared. Otherwise, there were no significant differences in the duration of anesthesia and time to recovery or level of anesthesia. CONCLUSION: Injection of local anesthetic solution through a caudally oriented epidural catheter produces faster onset and superior quality of anesthesia in comparison with the injection through the cephaladly oriented catheter in ankle surgery, but not hemorrhoidectomy.

Induction of connexin 37 expression in a rat model of neuropathic pain.

Activation of cutaneous C-fibers by capsaicin or sciatic nerve transection increases the number of astrocytic gap junctions as well as the levels of connexin 43 in the dorsal horn on the stimulated side. Changes in connexin 37 mRNA expression following nerve injury have not been previously documented. We examined the role of gap junction protein connexin 37 in neuropathic hypersensitivity following peripheral nerve injury. Study results showed ipsilaterally increased connexin 37 mRNA levels proximally and distally in rat sciatic nerves after injury and behavioral thermal hyperalgesia at 7 and 14 days. Proximal and distal connexin 37 mRNA levels returned to baseline by 21 days. Sciatic nerve connexin 37 mRNA increases were proportional to the extent of thermal hyperalgesia, but skin, muscle, and lumbar spinal cord connexin 37 mRNA showed no significant changes. Neuropathic pain relief correlated with downregulation of connexin 37 mRNA. Results indicate that upregulation of connexin 37 mRNA following sciatic nerve injury correlates with subsequent thermal hyperalgesia, which suggests that gap junctions (connexin 37) are responsible for the hyperexcitability following peripheral nerve injury.

Peroxisome proliferator-activated receptors (PPAR) and the mitochondrial aldehyde dehydrogenase (ALDH2) promoter in vitro and in vivo.

BACKGROUND: The aldehyde dehydrogenase 2 (ALDH2) promoter contains a nuclear receptor response element (NRRE) that represents an overlapping direct repeat-1 (DR-1) and -5 (DR-5) element. Because DR-1 elements are preferred binding sites for peroxisome proliferator-activated receptors (PPARs), we tested the hypothesis that PPARs regulate ALDH2 expression. METHODS: We examined the ability of PPAR isoforms to bind to the ALDH2 NRRE in electrophoretic mobility shift assays, their ability to activate the transcription of promoter-reporter constructs containing this NRRE, the effect of PPAR ligands on ALDH2 expression in liver, and the role of the PPARalpha on the expression of ALDH2 by using PPARalpha-null mice. RESULTS: In vitro translated PPARs bound the ALDH NRRE with high affinity. Mutation of the NRRE indicated that binding was mediated by the DR-1 element. Cotransfection of PPAR expression plasmids showed that PPARalpha had no effect on expression of heterologous promoter constructs containing the NRRE. PPARgamma slightly induced expression, whereas PPARdelta repressed basal activity of the promoter and blocked induction by hepatocyte nuclear factor 4. Treatment of rats with the PPAR ligand clofibrate repressed expression of ALDH2 in rats fed either stock rodent chow or a low-protein diet. Consistent with the transfection data, expression of ALDH2 protein was not different in PPARalpha-null mice. Treatment of the mice with the PPARalpha agonist WY14643 slightly decreased the level of ALDH2 protein in both wild-type and PPARalpha-null mice, suggesting that the effect of WY14643 was not mediated by the receptor. CONCLUSIONS: These data indicate that ALDH2 is not part of the battery of lipid metabolizing enzymes and proteins regulated by PPARalpha

Protein oxidation and turnover.

All biomacromolecules are faced with oxidative stress. Oxidation of a protein molecule always induces inactivation of the molecule and introduces a tag to that molecule. These modified protein molecules are prone to degradation in vivo by the proteasome system. Coupling of protein modification and degradation of chemically modified proteins is one of the normal protein turnover pathways in vivo. We call this a 'chemical apoptosis' process, which is one of the early manifestations of programmed cell death. Impairment of the proteasome system leads to accumulation of modified nonfunctional proteins or 'aged proteins' that might cause various clinical syndromes including cataractogenesis, premature aging, neurological degeneration and rheumatoid disease. The metal-catalyzed oxidation of biomacromolecules provides an excellent artificial aging system in vitro. The system is very useful in the characterization of structure and function relationships of proteins (enzymes), especially in those containing metal binding domain(s), because the oxidation is always followed by an affinity cleavage at the metal binding site(s) that allows easy identification and further characterization.

The retinoid X receptor response element in the human aldehyde dehydrogenase 2 promoter is antagonized by the chicken ovalbumin upstream promoter family of orphan receptors.

Two tandem sites in the aldehyde dehydrogenase 2 promoter (designated FP330-5' and FP330-3') that bind members of the nuclear receptor superfamily were recently identified. Antibodies against apolipoprotein regulatory protein (ARP-1) altered DNA-protein interactions in electrophoretic mobility shift assays using oligonucleotides representing either promoter site and rat liver or cultured cell nuclear extracts. In vitro-translated chicken ovalbumin upstream promoter transcription factor (COUP-TFI), ARP-1, or ErbA-related protein 2 (Ear2) bound both sites. In addition, ARP-1/RXR, COUP-TFI/RXR, and ARP-1/COUP-TFI heterodimers bound the FP330-3' site. Mutagenesis of the FP330-3' site indicated that a DR-1 element was the preferred binding site for these factors. Transfected expression plasmids for these factors suppressed basal expression of reporter constructs containing the FP330-3' sites and the induction of the reporter by RXRalpha plus retinoic acid. Mutation of the two sites increased activity of a construct driven by 600 bp of the ALDH2 promoter in cell lines expressing COUP-TFs. The ALDH2 FP330-3' site appears to represent a complex nuclear receptor response element that is activated by RXRs and HNF-4 but repressed by members of the COUP-TF family.

Heat shock modulates prion protein expression in human NT-2 cells.

The pathological hallmarks of Prion disease are cortical spongiform changes and neuronal loss, which are induced by the accumulation of the scrapie-isoform prion protein (PrP(Sc)). PrP(Sc) is derived from a post-translational modification of the cellular form of prion protein (PrP(C)). Heat-shock proteins, a group of molecular chaperones, are involved in the degradation of denatured proteins and post-translational folding of newly synthesized polypeptides. In an attempt to examine any possible relationship between heat shock stress and an induction of prion protein (PrP), human NT-2 cells were treated with heat shock at 42 degrees C for 30 min. After heat-shock treatment, both the level of mRNA and PrP(C) protein were analyzed at various time points by Northern and Western blot, respectively. There was a 1.5- to 2.5-fold increase in PrP mRNA levels 1 and 3h following heat shock. In addition, a two-fold increase in protein level of PrP was found 3 h after heat-shock treatment. These results suggest that cellular stress induces the elevation of both PrP mRNA and protein synthesis. The up-regulation of prion-protein mRNA and protein, implies that PrP may play a role in cellular stress.

Lysine residues 162 and 340 are involved in the catalysis and coenzyme binding of NADP(+)-dependent malic enzyme from pigeon.

Alanine-scanning site-directed mutagenesis was carried out on all conserved lysine residues of pigeon cytosolic NADP(+)-dependent malic enzyme. Only two mutant enzymes, K162A and K340A, showed significant effect on their kinetic parameters. Both mutant enzymes have K(m) values for Mn(2+) and l-malate similar to those of wild-type. The K(m) value for NADP(+) of K162A is identical to that of wild-type. However, K162A demonstrated a 235-fold decrease in the k(cat) value (0.17 +/- 0.01 vs 40.0 +/- 1.3 s(-1)). These data suggested that the side chain of K162 is important for the enzyme catalytic reaction. We propose that the epsilon-amino group of K162 may serve as a general acid to protonate the 3-carbon of enolpyruvate after decarboxylation. The K340A mutant demonstrated no effect on the k(cat) value. However, its K(m) value for NADP(+) was increased by a factor of 65 (225.7 +/- 5.07 vs 3.49 +/- 0.05 microM). We propose that the NADP(+) specificity is determined by the electrostatic interaction between the epsilon-amino group of K340 and 2'-phosphate of NADP(+).

Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-L-malate binding of pigeon liver malic enzyme.

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns. The mutants showed similar apparent Km,NADP values to that of the WT. The Km,Mal value was increased in the D141N and D194N mutants. The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy. Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [k(cat)/(Kd,Mn Km,Mal Km,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.

Creutzfeldt-Jakob disease: heat shock protein 70 mRNA levels in mononuclear blood cells and clinical study.

Prion diseases such as Creutzfeldt-Jakob disease (CJD) are associated in most cases with the accumulation of an unusual isoform of prion protein (PrPSC). PrPSC is derived from the abnormal folding of the cellular isoform of prion protein (PrPC). On the other hand, heat shock protein is known to ensure proper protein assembly and folding and to facilitate proteolytic digestion of abnormal or denatured proteins. Many studies have therefore hypothesized that heat shock protein is linked to prion disease. We examined the relationship between heat shock protein HSP70 and prion disease in CJD patients. HSP70 mRNA levels in mononuclear blood cells (MBCs) were compared in 14 CJD patients (10 confirmed by histo-pathological study), 12 vascular dementia (VD) patients, 16 patients with Parkinson's disease and dementia (PD) and 14 nondemented control subjects. The possible correlation between HSP70 mRNA expression levels and clinical findings was also evaluated. HSP70 mRNA expression levels in MBCs were measured by northern blotting. HSP70 mRNA levels in MBCs from patients with CJD were significantly higher than those from patients with VD or PD and in nondemented controls. Age at symptom onset, dementia severity, disease duration and neuroimaging grade of CJD patients were not correlated with relative HSP70 mRNA levels. No significant relationship between HSP70 mRNA levels and ageing was found. These results suggest that measurement of HSP70 mRNA in MBCs might provide an auxiliary tool for the diagnosis of CJD.

Crystallization and preliminary x-ray diffraction analysis of malic enzyme from pigeon liver.

Recombinant pigeon-liver malic enzyme was expressed in Escherichia coli and purified to homogeneity. Two different crystal forms were grown by the hanging-drop vapour-diffusion method. Both types of crystals belong to the tetragonal space group P4(2)22, with unit-cell dimensions a = b = 163.8, c = 174.3 A for the octahedral crystals and a = b = 124.5, c = 179.2 A for the rod-like crystals. X-ray diffraction data were collected at 100 K using a synchrotron-radiation X-ray source. The Matthews parameter suggests that there are four and two molecules per asymmetric unit for the larger and the smaller tetragonal unit cells, respectively.

An A/G polymorphism in the promoter of mitochondrial aldehyde dehydrogenase (ALDH2): effects of the sequence variant on transcription factor binding and promoter strength.

INTRODUCTION: The strong protective effect of the ALDH2*2 mutation on risk of alcoholism suggests that other mutations that reduce mitochondrial aldehyde dehydrogenase (ALDH) activity in the liver might also deter drinking. This study describes a polymorphic locus found in the promoter of the ALDH2 gene that affects expression of reporter constructs. METHODS: Polymerase chain reaction (PCR)-based sequencing was used to search for polymorphisms. The ability of the promoter variants to bind transcription factors apolipoprotein A regulatory protein 1 (ARP-1) and chicken ovalbumin upstream promoter-transcription factor (COUP-TF) was tested in gel retardation assays using in vitro synthesized transcription factors. The variant promoters were tested for transcriptional activity using a heterologous promoter system and transient transfection assays. RESULTS: A common polymorphism (A or G) in the human ALDH2 promoter region was found at -361 base pair (bp) from the translation start site. This polymorphism was found at different frequencies in African Americans, Caucasians, and Asians. The polymorphism occurs adjacent to the core binding motif for the transcription factors COUP-TF and ARP-1. Competition and binding affinity determinations did not show differences in the ability of these two sequences to bind the factors. Reporter genes containing these elements upstream of a basal thymidine kinase promoter had similar activity when transfected into a fibroblast (CV-1) cell line. However, the reporter containing the G allele was more active than that containing the A allele in hepatoma (H4IIEC3) cells. CONCLUSIONS: The -361 bp A/G polymorphism is common in all racial groups tested. The G allele was more active than the A allele in a transfection assay. The basis for this difference is not known. If the differences in activity of the promoter constructs were paralleled by differences in ALDH2 enzyme activity in the liver, this polymorphism could affect risk of alcoholism.

Conformational stability of the N-terminal amino acid residues of mutated recombinant pigeon liver malic enzymes.

Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met-Lys-Lys-Gly-Tyr-Glu-Val-Leu-Arg-. Our previous results indicated that the N-terminus of the enzyme is located at or near the enzyme's active center involved in Mn(II)-L-malate binding and is also near to the subunits' interface. In the present study, the conformational stability of the various deletion (delta) and substitution mutants at Lys2/Lys3 of the enzyme was investigated with chemical and thermal sensitivities. The lysine residue at position 2 or 3 seems to be crucial for the correct active site conformation, probably through an ion-pairing with Glu6. Deletion at Lys2 or Lys3, delta(K2/K3), and the double mutant K(2,3)E were much less stable than the wild-type enzyme towards chemical denaturation. Kinetic analysis of the thermal inactivation at 58 degrees C of the recombinant enzymes indicated that mutation at position 3 to alanine (K3A) endows the protein with extra stability compared with the wild-type enzyme. K3A is also stable towards chemical denaturation. The concentration of urea that causes half unfolding, [urea]0.5, for K3A is 3.25 M compared with 2.54 M for the wild-type enzyme. The K3A mutant of malic enzyme might therefore have potential practical applications.

Parathyroid hormone regulates the rat collagenase-3 promoter in osteoblastic cells through the cooperative interaction of the activator protein-1 site and the runt domain binding sequence.

Parathyroid hormone induces collagenase-3 gene transcription in rat osteoblastic cells. Here, we characterized the basal, parathyroid hormone regulatory regions of the rat collagenase-3 gene and the proteins involved in this regulation. The minimal parathyroid hormone-responsive region was observed to be between base pairs -38 and -148. Deleted and mutated constructs showed that the activator protein-1 and the runt domain binding sites are both required for basal expression and parathyroid hormone activation of this gene. The runt domain site is identical to an osteoblast-specific element-2 or acute myelogenous leukemia binding sequence in the mouse and rat osteocalcin genes, respectively. Overexpression of an acute myelogenous leukemia-1 repressor protein inhibited parathyroid hormone activation of the promoter, indicating a requirement of acute myelogenous leukemia-related factor(s) for this activity. Overexpression of c-Fos, c-Jun, osteoblast-specific factor-2, and core binding factor-beta increased the response to parathyroid hormone of the wild type (-148) promoter but not with mutation of either or both the activator protein-1 and runt domain binding sites. In summary, we conclude that there is a cooperative interaction of acute myelogenous leukemia/polyomavirus enhancer-binding protein-2-related factor(s) binding to the runt domain binding site with members of the activator protein-1 transcription factor family binding to the activator protein-1 site in the rat collagenase-3 gene in response to parathyroid hormone in osteoblastic cells.