Effects of treadmill running and limb immobilization on knee cartilage degeneration and locomotor joint kinematics in rats following knee meniscal transection.

OBJECTIVE: This study examined the effects of reduced and elevated weight bearing on post-traumatic osteoarthritis (PTOA) development, locomotor joint kinematics, and degree of voluntary activity in rats following medial meniscal transection (MMT). DESIGN: Twenty-one adult rats were subjected to MMT surgery of the left hindlimb and then assigned to one of three groups: (1) regular (i.e., no intervention), (2) hindlimb immobilization, or (3) treadmill running. Sham surgery was performed in four additional rats. Voluntary wheel run time/distance was measured, and 3D hindlimb kinematics were quantified during treadmill locomotion using biplanar radiography. Rats were euthanized 8 weeks after MMT or sham surgery, and the microstructure of the tibial cartilage and subchondral bone was quantified using contrast enhanced micro-CT. RESULTS: All three MMT groups showed signs of PTOA (full-thickness lesions and/or increased cartilage volume) compared to the sham group, however the regular and treadmill-running groups had greater osteophyte formation than the immobilization group. For the immobilization group, increased volume was only observed in the anterior region of the cartilage. The treadmill-running group demonstrated a greater knee varus angle at mid-stance than the sham group, while the immobilization group demonstrated greater reduction in voluntary running than all the other groups at 2 weeks post-surgery. CONCLUSIONS: Elevated weight-bearing via treadmill running at a slow/moderate speed did not accelerate PTOA in MMT rats when compared to regular weight-bearing. Reduced weight-bearing via immobilization may attenuate overall PTOA but still resulted in regional cartilage degeneration. Overall, there were minimal differences in hindlimb kinematics and voluntary running between MMT and sham rats.

Experiences of intimate-partner violence and contraception use among ever-married women in Jordan.

This study explored the relationship between intimate partner violence (IPV) and current contraception use among ever-married women in Jordan. Analysing a sample (n = 3434) from the 2007 Jordan demographic and health survey, women who reported ever experiencing severe physical violence from their husband were significantly less likely to use contraception than women who did not report severe physical violence (OR = 0.34). Conversely, women who reported ever experiencing sexual IPV were significantly more likely to use contraception (OR = 1.50). Emotional and less severe physical IPV were not significantly related to contraception use. Education, wealth, age, number of children, and fertility preferences were positively associated with contraception use, while residence in the Badia area and consanguineous marriages were negatively associated with contraception use. The findings have implications for the provision of IPV screening and contraception services in Jordan, as well as the specification of services for women most vulnerable to IPV.

Experiences of intimate-partner violence and contraception use among ever-married women in Jordan

This study explored the relationship between intimate partner violence [IPV] and current contraception use among ever-married women in Jordan. Analysing a sample [n = 3434] from the 2007 Jordan demographic and health survey, women who reported ever experiencing severe physical violence from their husband were significantly less likely to use contraception than women who did not report severe physical violence [OR = 0.34]. Conversely, women who reported ever experiencing sexual IPV were significantly more likely to use contraception [OR = 1.50]. Emotional and less severe physical IPV were not significantly related to contraception use. Education, wealth, age, number of children, and fertility preferences were positively associated with contraception use, while residence in the Badia area and consanguineous marriages were negatively associated with contraception use. The findings have implications for the provision of IPV screening and contraception services in Jordan, as well as the specification of services for women most vulnerable to IPV

Regulation of adrenal steroidogenesis by the high-affinity phosphodiesterase 8 family.

The main function of cyclic AMP phosphodiesterases (PDEs) is to degrade cAMP, a ubiquitous second messenger. Therefore, PDEs can function as prime regulators of cAMP/PKA-dependent processes such as steroidogenesis. Until recently, the roles of the PDE8 family have been largely unexplored, presumably due to the lack of a selective inhibitor. This review focuses on recent reports about the regulatory roles of the PDE8 family in adrenal steroidogenesis, as well as the inhibitory properties and specificity of a new PDE8-selective inhibitor, PF-04957325. We also describe a method of measuring urinary corticosterone levels in vivo as a minimally invasive way of monitoring the stress level in a mouse.

Inactivation of Pde8b enhances memory, motor performance, and protects against age-induced motor coordination decay.

Phosphodiesterases (PDEs) are critical regulatory enzymes in cyclic nucleotide signaling. PDEs have diverse expression patterns within the central nervous system (CNS), show differing affinities for cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), and regulate a vast array of behaviors. Here, we investigated the expression profile of the PDE8 gene family members Pde8a and Pde8b in the mouse brain. We find that Pde8a expression is largely absent in the CNS; by contrast, Pde8b is expressed in select regions of the hippocampus, ventral striatum, and cerebellum. Behavioral analysis of mice with Pde8b gene inactivation (PDE8B KO) demonstrate an enhancement in contextual fear, spatial memory, performance in an appetitive instrumental conditioning task, motor-coordination, and have an attenuation of age-induced motor coordination decline. In addition to improvements observed in select behaviors, we find basal anxiety levels to be increased in PDE8B KO mice. These findings indicate that selective antagonism of PDE8B may be an attractive target for enhancement of cognitive and motor functions; however, possible alterations in affective state will need to be weighed against potential therapeutic value.

Forensic Science in Support of Wildlife Conservation Efforts - Developments in Genetic Approaches in Taiwan.

To control illegal wildlife-product trade and protect endangered species of animals, unambiguous identification of the animal specimens is vitally important. Genetic approaches were adopted to identify animal species for conservation and to prevent their fraudulent misidentification in Taiwan, especially for samples of animal residues, powders, and processed products. PCR or nested PCR based on the nature of DNA was used for amplification of cyt b, COI, CHD, and D-loop DNA fragments. Sequences of these fragments were compared with those registered in DNA databases and phylogenetic analysis was performed. The established methods were applied in forensic cases for support of conservation efforts and they were proved to be robust. For conservation animal identification, various samples seized by law enforcement agents have been identified by our systems as rhinoceros horns, Indian sawback turtles, shahtoosh, ivories, dolphins, whales, etc. The systems were also successfully used in investigating the illegal trade of commercial turtle shells and the fraudulent misidentification of food contents on product labels in Taiwanese markets. This review summarizes the work conducted in our laboratory and describes the Taiwan experience.

Molecular cloning and characterization of full-length cDNAs encoding a novel high-molecular-weight Dermatophagoides pteronyssinus mite allergen, Der p 11.

BACKGROUND: Dermatophagoides pteronyssinus (Dp) and D. farinae (Df) mites are the most important source of indoor aeroallergens. Most Dp mite allergens identified to date have relatively low molecular weights (MWs). Identification of high-MW mite allergens is a crucial step in characterizing the complete spectrum of mite allergens and to provide appropriate tools for diagnostic and therapeutic application. METHODS: The full-length Der p 11 cDNA clone was isolated using cDNA library immunoscreening, the 5'-3' rapid amplification of cDNA ends (RACE) system and polymerase chain reactions (PCR). The whole cDNA insert and its PCR-derived DNA fragments (p1 to p4) were generated and expressed in the Escherichia coli expression system. The allergenicity of the recombinant protein and its peptide fragments was examined by IgE immunodot assays. The IgE-binding reactivity of rDer p 11 was analyzed in the serum of 50 asthmatic children with positive reactivity to Dp mite extract. Its recombinant peptide fragments were also examined by immunodot assays in 30 mite-allergic children. RESULTS: Der p 11 cDNA consists of a 2625-bp open reading frame encoding a 103-kDa protein with 875 amino acids. It exhibits significant homology with the paramyosin of other invertebrates. The protein sequence alignment of this newly identified Dp mite allergen (denominated as Der p 11) revealed over 89% identity with Der f 11 and Blo t 1. Among 50 Dp-sensitive asthmatic children, rDer p 11 showed positive IgE-binding reactivity to 39 patients (78%). Using immunodot assays, multiple human IgE-binding activities were demonstrated in all four fragments of Der p 11. Using immunoblot assays, the dominant IgG-binding epitope for monoclonal antibody (mAb642) was located in fragment p3 only. In immunoblot assays, cross-inhibition between rDer p 11 and rDer f 11 was up to 73-80% at concentrations of 100 microg/ml. CONCLUSIONS: This study confirms that the newly identified recombinant Der p 11 is a novel and important high-MW Dp mite allergen for asthmatic children. Our data also indicates that human IgE-binding major epitopes are scattered over the entire molecule of Der p 11.

Effect of ingestion of cow's milk hydrolysed formulas on whey protein-specific Th2 immune responses in naïve and sensitized mice.

BACKGROUND: For genetically predisposed atopic infants, cow's milk protein hydrolysed formulas have been widely used. OBJECTIVE: Whether hydrolysed formulas can induce oral tolerance to whey proteins will be extensively studied in naïve and sensitized mice. METHODS: Antigenicity of hydrolysed formulas was first studied using immunoblotting. Naïve mice fed hydrolysed formulas for 1-4 weeks were sensitized with whey allergens. In contrast, mice sensitized with whey allergens were fed hydrolysed formulas continually for 12 weeks. RESULTS: Whey allergens were found in Nan and Neoangelac FL. Large whey peptides with antigenicity were found in Nan-HA. Profound suppression of IgE, IgG1 and IgG responses to whey allergens were induced in those fed Nan for 1 week, or Nan-HA for 4 weeks. IgE responses to whey allergens were suppressed in those fed Neoangelac FL for 4 weeks, or Nan-HA for 1-2 weeks. In contrast, those fed extensively hydrolysed formulas for 1-4 weeks failed to show decreased responses. On the other hand, IgE responses to beta-lactoglobulin, but not to bovine serum albumin or alpha-lactalbumin, were decreased in sensitized mice fed Nan for 12 weeks. There was no suppression in sensitized mice fed hydrolysed formulas. CONCLUSION: Suppression of IgE responses to whey proteins was readily induced in naïve mice fed Nan or Nan-HA for 1 week. In contrast, it was hardly induced in sensitized mice even after prolonged feeding of Nan for 12 weeks, let alone hydrolysed formulas.

Protein sequence analysis of a novel 103-kDa Dermatophagoides pteronyssinus mite allergen and prevalence of serum immunoglobulin E reactivity to rDer p 11 in allergic adult patients.

BACKGROUND: House dust mites are regarded as important indoor allergens. While the most studies mite allergens are low molecular weight (mw), a high mw Dermatophagoides farinae mite paramyosin (Der f 11) has recently been cloned. We have also cloned a novel high mw Dermatophagoides pteronyssinus (Dp) mite allergen, Der p 11. OBJECTIVE: The aim of this study was to isolate and express a cDNA gene coding for a Der p 11 allergen, to compare the sequence of Der p 11 with other antigens and to evaluate the presence of IgE reactivity to the recombinant protein (rDer p 11) in the sera of allergic adult patients. METHODS: The full-length Der p 11 gene was isolated by cDNA library screening, 5'-3' rapid amplification of cDNA ends and PCR. The cDNA gene was expressed as a glutathione-S-transferase fusion protein in Escherichia coli. The allergenicity of rDer p 11 was tested by human IgE immunodot or immunoblot assay in a large panel of 100 allergic patients with bronchial asthma, allergic rhinitis or eczema. RESULTS: Der p 11 is a 2965 bp cDNA gene with a 2625 bp open reading frame coding for a 875 amino acid protein. The deduced amino acid sequence of the Der p 11 showed significant homology with various invertebrate paramyosins. The prevalence of serum IgE reactivity to rDer p 11 on immunodot assay ranged from 41.7% to 66.7% in different allergic patient groups, whereas it was rare in non-atopic patients with urticaria (18.8%) and in normal individuals (8%). A high frequency (five out of eight) of MAST(Dp)- allergic serum samples had specific IgE-binding activity to rDer p 11 or its fragments on immunoblot assay, even though their IgE-binding activity to Dp extract was either weak or negative. CONCLUSION: The 103-kDa Der p 11 appears to be major Dp mite allergen with a high frequency of IgE reactivity in sera of patients allergic to mites.

Comparative allergenicity studies of native and recombinant Blomia tropicalis Paramyosin (Blo t 11).

BACKGROUND: The complementary DNA (cDNA) encoding for Blo t 11, a 102 kD allergen from Blomia tropicalis (Bt) was isolated, expressed and characterized previously. This study aimed to isolate the native Blo t 11 allergen and compare its allergenicity with the recombinant forms. METHODS: Native Blo t 11 (nBlo t 11) was isolated from crude Bt extract by immuno-affinity chromatography, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and verified by MALDI-TOF MS. Recombinant full-length Blo t 11 (rFL-Blo t 11) and its immunodominant peptide (fD) were expressed as glutathione S-transferase (GST)-fusion proteins in Escherichia coli. Immunoglobulin E (IgE) reactivity of the Blo t 11 allergens were determined by enzyme-linked immunosorbent assay (ELISA) and skin prick test. The inhibition capacity of the nBlo t 11 against fD and vice versa was determined by absorption studies. RESULTS: Affinity purified nBlo t 11 was susceptible to degradation with the major degraded product resolved at approximately 66 kD. The nBlo t 11 was confirmed by immunoblot analysis and MALDI-TOF MS that generated 13 peptides with complete identity to the deduced amino acid sequence of Blo t 11. Comparative in vitro and in vivo allergenicity tests and the cross inhibition studies between the native and recombinant Blo t 11 showed that recombinant fD, but not the rFL-Blo t 11, has comparable IgE reactivity with the native counterpart. CONCLUSIONS: This comparative study confirmed that the recombinant peptide fD contains the main immunodominant region of Blo t 11. This recombinant peptide, instead of the full-length protein, is a good candidate for diagnostic and therapeutics development for mite allergy.

Both TPA and SCC-Ag levels are prognostic even in high-risk stage Ib-IIa cervical carcinoma as determined by a stratification analysis.

OBJECTIVE: To determine the prognostic values of tissue polypeptide antigen (TPA), squamous cell carcinoma antigen (SCC-Ag), and carcinoembryonic antigen (CEA) in the sera of cervical carcinoma patients, especially in those with a poor prognosis. METHODS: In this retrospective study, the preoperative serum SCC-Ag, TPA, and CEA were analyzed in 779 patients with cervical squamous cell carcinoma of stage Ib-IIa who received radical hysterectomy and pelvic lymph node dissection (RAH-PLND) between 1984 and 1994. RESULTS: Due to poor predictive value and poor correlation between serum CEA and clinico-pathological factors, CEA was abandoned in this study. Elevated TPA and SCC-Ag levels, pelvic lymph node metastasis (PLNM), lymphvascular space involvement (LVSI) and deep stromal invasion (DSI) were associated with poor survival time by univariate analysis. The correlation study showed that elevated serum TPA was significantly related to PLNM, LVSI, and DSI (p = 0.004, 0.008, and 0.021, respectively), and SCC-Ag was related to PLNM and bulky tumor size (p = 0.001 and 0.02, respectively). In the multivariate analysis, only PLNM and LVSI remained independently significant indicating poor survival. Further stratification studies by PLNM and LVSI showed that elevated TPA levels could even indicate higher recurrence rates in patients with PLNM (p = 0.045), as well as SCC-Ag in patients with LVSI (p = 0.038). CONCLUSIONS: The results suggest that both elevated TPA and SCC-Ag levels depicting poor prognosis in stage Ib-IIa cervical SCC, especially indicates a group of high-risk patients who may need more aggressive therapy.

Cytochrome b gene for species identification of the conservation animals.

A partial DNA sequence of cytochrome b gene was used to identify the remains of endangered animals and species endemic to Taiwan. The conservation of animals species included in this study were: the formosan gem-faced civets, leopard cats, tigers, clouded leopards, lion, formosan muntjacs, formosan sika deers, formosan sambars, formosan serows, water buffalo, formosan pangolins and formosan macaques. The control species used included domestic cats, domestic dogs, domestic sheeps, domestic cattles, domestic pigs and humans. Heteroplasmy was detected in the formosan macaque, domestic pig and domestic cats. The frequencies of heteroplasmy in these animals were about 0.25% (1 in 402bp). Sequences were aligned by Pileup program of GCG computer package, and the phylogenetic tree was constructed by the neighbor-joining method. The results of sequence comparison showed that the percentage range of sequence diversity in the same species was from 0.25 to 2.74%, and that between the different species was from 5.97 to 34.83%. The results of phylogenetic analysis showed that the genetic distance between the different species was from 6.33 to 40.59. Animals of the same species, both the endangered animal species and domestic animals, were clustered together in the neighbor-joining tree. Three unknown samples of animal remains were identified by this system. The partial sequence of cytochrome b gene adopted in this study proved to be usable for animal identification.

Serum specific IgE reactivity to recombinant Der f 11 in asthmatic children.

This study was designed to examine the prevalence of positive serum IgE reactivity to the recombinant group 11 Dermatophagoides farinae allergen (rDer f 11) in asthmatic children in Taiwan. Using immunoblot analysis in a preliminary study of 18 asthmatic children, 13 (72.2%) reacted positively to rDer f 11 and 16 (88.9%) showed positive reactivity to D. farinae extracts. The allergenicity of rDer f 11 was further evaluated with in vivo skin tests and in vitro IgE immunodot assays in 24 mite skin-test-positive asthmatic children. Whereas 17 (70.8%) had positive skin tests to rDer f 11, 18 (75.0%) had positive serum IgE reactivity to rDer f 11. A good coincidence (87.5%) between the immunodot assay and the skin test was confirmed in these asthmatic children. Moreover, the prevalence of serum IgE reactivity to rDer f 11 was further investigated in a large panel of 49 mite skin-test-positive asthmatic children. Again, 38 (77.6%) had positive serum IgE reactivity to rDer f 11 in immunodot assays. Taken together the positive IgE reactivity to rDer f 11 in immunodot analysis ranged from 75 to 77.6% in two groups of 73 mite skin-test-positive asthmatic children. High incidence of serum IgE antibodies specific for rDer f 11 in the present study suggests that Der f 11 is a novel major allergen of house dust mites.

Crystallization and preliminary X-ray diffraction analysis of the 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes.

The truncated 1,3-1,4-beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase; E.C. 3.2.1.73) from Fibrobacter succinogenes was crystallized in four different forms by the vapour-diffusion method. Form A crystals have the largest trigonal P321 unit cell, diffracting to 3.0 A resolution with four to six molecules per asymmetric unit. Form B and C crystals belong to the same monoclinic space group P2(1), but the form B unit cell is twice as large as the unit cell of form C. Form B crystals diffract to 2.5 A resolution and contain four molecules per asymmetric unit. Form C crystals diffract to 2.1 A resolution and contain two molecules per asymmetric unit. Form D crystals have the smallest orthorhombic P2(1)2(1)2(1) unit cell, containing only one molecule per asymmetric unit, and diffract beyond 2.1 A resolution. The crystallization conditions for form B and C crystals are almost identical, except that form C crystals were grown in the presence of 2 mM Ca(2+) ions. It is likely that Ca(2+) directly binds to the glucanase, leading to unit-cell shrinkage as observed in other Bacillus glucanase crystals. A self-rotation search identified non-crystallographic twofold axes that combine with the crystallographic twofold dyads to give 222 symmetry for both form A and form B crystals, indicating that the glucanase has a tendency to pack in 222 symmetry.

Isolation and partial characterization of a 46-kd allergen of Bermuda grass pollen.

Cyn d Bd46K, a 46-kD component of Bermuda grass (Cynodon dactylon) pollen, had been identified as an allergenic constituent. In the present study two-dimensional (2D) gel electrophoresis illustrated the presence of five acidic isoforms in Cyn d Bd46K, and this molecule was purified by monoclonal antibody (MAb) affinity chromatography for further characterization. Using a digoxigenin-labeled lectin-binding assay, the elucidating protein was disclosed to be a glycoprotein with terminal mannose. The involvement of a carbohydrate moiety in the allergenicity and antigenicity of the elucidated molecule was demonstrated with sodium-periodate-treated Cyn d Bd46K, which reduced binding to its specific MAb and human IgE. We were unable to identify the N-terminal amino acid sequences of Cyn d Bd46K, but some internal amino acid sequences were disclosed by microsequencing some fragments cleaved by Achromobacter protease I and fractionated by reversed-phase column chromatography. The amino acid sequences of 4 identified Cyn d Bd46K internal peptide fragments were found to be 25-71% identical with that of cytochrome c oxidase III from corn grass pollen. The present study provided important information for future experiments on the molecular cloning of the elucidated allergen.

Differential regulation of placental and germ cell alkaline phosphatases by glucocorticoid and sodium butyrate in human gastric carcinoma cell line TMK-1.

The expression and regulation of alkaline phosphatase (AP) was studied in the human gastric cancer cell line TMK-1. Biochemical analysis, reverse transcription-polymerase chain reaction, and Northern blot analysis demonstrated that the cells express placental, germ cell, and intestinal AP isozymes constitutively. Dexamethasone (Dex), a synthetic glucocorticoid, was shown to specifically induce the placental AP activity to about 10-fold and sodium butyrate (NaBu) induced germ cell AP activity to about 4-fold, respectively. In contrast, these two agents showed little effect on the level of intestinal isozymes. Dex and NaBu also differentially induced the mRNA levels of the placental and germ cell APs. Northern blot analysis of the placental AP transcript in the presence of the transcription inhibitor, 5, 6-dichloro-1-beta-D-ribofuranosyl benzimidazole, revealed that the half-life of placental AP mRNA is about 27 h for both the Dex-treated and untreated cells. Nuclear run-on transcription analysis indicated an apparent increase in the rate of placental AP gene transcription in Dex-treated cells. These results indicated that the effect of Dex occurred primarily by activation of the placental AP gene transcription in the cells. In order to study the direct Dex and NaBu effect on AP gene expression, the proximal promoter regions of AP genes were fused to luciferase reporter vectors. Despite the high similarity in nucleotide sequences of these two genes, transient transfection analysis demonstrated that Dex and NaBu exerted a specific stimulation only through the respective placental and germ cell AP gene promoter. Taken together, this study indicates that the expression of PAP and GCAP isozymes have specific regulatory mechanisms that can be differentially controlled by signals including glucocorticoid and NaBu.

A novel nomenclature for the hypervariable short tandem repeat APOAI1.

A novel nomenclature for the hypervariable microsatellite DNA, APOAI1 locus, is proposed. The complex nature of the repeat unit in this locus results in alleles separated by a single base. Polymerase chain reaction (PCR) products amplified from this locus were separated by single-strand conformation polymorphism (SSCP) electrophoresis. All the single-stranded DNA bands on the SSCP gel were removed from the gel and a second amplification performed. Homozygous DNA fragments amplified from single-stranded DNA were sequenced. From the 100 individuals studied, 30 alleles and 73 genotypes were found. A system of nomenclature for the APOAI1 locus is provided that is logical and in line with previous models. Using the primers described, the locus can be amplified and alleles designated on the basis of size. This system of nomenclature will assist in the exchange of data between laboratories for this locus.

Sequence polymorphism of mitochondrial D-loop DNA in the Taiwanese Han population.

In order to demonstrate the sequence diversity of mitochondrial D-loop DNA in the Taiwanese Han population, we established a database of 155 unrelated individuals. For each individual, the complete 980bp DNA region from the 5' end of HVI to 3' end of HVII segment was sequenced. In these 155 sequence data, 149 different haplotypes were observed, amongst these haplotypes, 144 were unique, 4 were found in 2 individuals and 1 was found in 3 individuals. When compare to the Anderson sequence, 144 transitions, 24 transversions, 5 insertions and 5 deletions were found. Eight positions exhibited more than one polymorphic sequence, six exhibited two variants while two exhibited three variants. Over the 1024bp that was analysed, pairwise differences between the sequences were 11.35+/-3.53bp. The sequence and nucleotide diversity were 0.9994 and 0.0116, respectively. The probability of two individuals randomly matching over the entire control region was 0.007. The diversity in the mitochondrial D-loop indicates the value of this locus for casework within Taiwan.

Directed mutagenesis of apecific active site residues on Fibrobacter succinogenes 1,3-1,4-beta -D-glucanase significantly affects catalysis and enzyme structural stability.

The functional and structural significance of amino acid residues Met(39), Glu(56), Asp(58), Glu(60), and Gly(63) of Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase was explored by the approach of site-directed mutagenesis, initial rate kinetics, fluorescence spectroscopy, and CD spectrometry. Glu(56), Asp(58), Glu(60), and Gly(63) residues are conserved among known primary sequences of the bacterial and fungal enzymes. Kinetic analyses revealed that 240-, 540-, 570-, and 880-fold decreases in k(cat) were observed for the E56D, E60D, D58N, and D58E mutant enzymes, respectively, with a similar substrate affinity relative to the wild type enzyme. In contrast, no detectable enzymatic activity was observed for the E56A, E56Q, D58A, E60A, and E60Q mutants. These results indicated that the carboxyl side chain at positions 56 and 60 is mandatory for enzyme catalysis. M39F, unlike the other mutants, exhibited a 5-fold increase in K(m) value. Lower thermostability was found with the G63A mutant when compared with wild type or other mutant forms of F. succinogenes 1,3-1,4-beta-d-glucanase. Denatured wild type and mutant enzymes were, however, recoverable as active enzymes when 8 m urea was employed as the denaturant. Structural modeling and kinetic studies suggest that Glu(56), Asp(58), and Glu(60) residues apparently play important role(s) in the catalysis of F. succinogenes 1,3-1,4-beta-d-glucanase.

Apoptosis induced by the sodium butyrate in human gastric cancer TMK-1 cells.

The effects of sodium butyrate on cell proliferation, gene expression, and apoptosis were investigated. Upon exposure to sodium butyrate the cells exhibited marked morphological changes, reduced cell proliferation and most cells died through apoptosis within 48 hours. In the presence of dexamethasone, however, the sodium butyrate-triggered apoptosis was markedly reduced. Studies using the glucocorticoid receptor antagonist RU486 indicated that the protective effect of dexamethasone was mediated through glucocorticoid receptor. Sodium butyrate markedly induced the c-jun proteins level, whereas the c-Myc protein was down-regulated rapidly. c-Jun protein may play an important role in the action of sodium butyrate since its induction preceded the onset of DNA fragmentation. In addition, preincubation of the cells with dexamethasone markedly delayed the induction of c-jun levels by sodium butyrate. Analysis of the expression of bel-2-related genes indicated that the Bcl-xS protein level was increased in the presence of sodium butyrate and the up-regulation of Bcl-xS by sodium butyrate was also blocked by dexamethasone. Taken together, these results indicate that c-myc, c-jun and Bcl-xS proteins may be involved in the mechanism of sodium butyrate-triggered apoptosis in these cells.