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.

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.

Dexamethasone suppresses apoptosis in a human gastric cancer cell line through modulation of bcl-x gene expression.

Treatment of human gastric cancer TMK-1 cells with transcription and translation inhibitors rapidly triggered cell apoptosis. Along with cell apoptosis, the Bcl-xS level was markedly upregulated suggesting a crucial role of this protein in promoting the apoptotic process. In the presence of dexamethasone, however, cell apoptosis was greatly attenuated as demonstrated by DNA histogram shift and DNA fragmentation. Studies using the glucocorticoid receptor antagonist RU486 indicated that attenuation of apoptosis was mediated through glucocorticoid receptors. Dexamethasone not only suppressed the apoptosis-associated upregulation of Bcl-xS but also enhanced the basal level of Bcl-xL in the cells. In addition, bcl-x mRNA stability was significantly extended in the presence of dexamethasone. These results indicate that dexamethasone exerted a protective effect and delayed apoptosis of TMK-1 cells by modulating bcl-x gene expression.

Effects of transcription and translation inhibitors on a human gastric carcinoma cell line. Potential role of Bcl-X(S) in apoptosis triggered by these inhibitors.

The effects of the macromolecular synthesis inhibitors 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), actinomycin D, and cycloheximide on the human gastric cancer TMK-1 cell line were studied. These agents inhibited DNA, RNA, or protein synthesis efficiently and induced cell death rapidly in a wide range of concentrations. After 8 hr of exposure to these agents, the cells exhibited morphological features of apoptosis, including cell shrinkage, nuclear condensation, DNA fragmentation, and formation of apoptotic bodies. Western blot analysis revealed that these inhibitors altered the protein levels of apoptosis-related gene products such as c-Myc, Bcl-X(S), and the mutant p53 (mp53) in TMK-1 cells markedly. The c-myc mRNA and protein levels were decreased initially and were then induced markedly to a new level after 4 hr of exposure to DRB, a RNA polymerase II inhibitor. The Bcl-X(S) levels were increased rapidly after treatment with all of these agents, whereas the levels of Bcl-X(L) and Bax remained largely unchanged. Northern blot analysis indicated that the c-myc overexpression is concomitant to DRB-induced DNA fragmentation and that the increased mp53 protein level was mainly a posttranscriptional event. Our observations suggest that the up-regulation of Bcl-X(S) may serve as an important mechanism for the apoptosis triggered by these inhibitors. This study also provides evidence for the notion that interference with the cellular survival pathway may lead to apoptosis.

A Common Allergenic Epitope of Bermuda Grass Pollen Shared by Other Grass Pollens.

The present study disclosed the cross-reactivity between Bermuda grass pollen (BGP) and other grass pollens using monoclonal antibodies (MAbs) and polyclonal antiserum. MAb 9-13, directed against a group of minor allergens of BGP (Cyn d Bd68K, 48K, 38K) was found to cross-react with extracts of ten other grass pollens. Immunoblotting assays illustrated that MAb 9-13 cross-reacted with multiple components of most of these pollens, and the major cross-reactive components had molecular weights of 29-36 kD. The crossreactivity between BGP and Lol pI, the group I allergen of rye grass pollen, was further evaluated; Lol pI was recognized by MAb 9-13, but not by our MAbs/polyclonal antiserum against Cyn dI, the major allergen of BGP. These results suggest that the epitope recognized by MAb 9-13 is a common (C) epitope shared by Lol pI and Cyn d Bd68K, 48K, 38K, and Cyn dI does not share significant antigenicity with Lol pI. In a modified radio-allergosorbent test, IgE antibodies in the serum of BGP-allergic patients reacted mildly with C-epitope-bearing components of both BGP and rye grass pollens, and this binding could be blocked specifically by MAb 9-13. This suggests that in addition to an antigenic cross-reaction, the C epitope can also lead to an allergenic cross-reaction. Copyright 1994 S. Karger AG, Basel

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.

Effects of tamoxifen and retinoic acid on cell growth and c-myc gene expression in human breast and cervical cancer cells.

The effects of estradiol, tamoxifen, and retinoic acid on the proliferation of breast and cervical cancer cells were investigated. Estrogen stimulated only MCF-7 cell growth, whereas tamoxifen and retinoic acid inhibited the proliferation of all cells studied. Northern blot analysis indicated that estradiol up-regulates c-myc mRNA level in all cell lines studied regardless of the estrogen receptor status in the cells. On the contrary, tamoxifen inhibits c-myc gene expression in all cell lines studied except in MCF-7 cells where the c-myc transcript was not affected. The inhibitory effect of tamoxifen on c-myc gene expression and cell proliferation in estrogen receptor-negative cells suggest an estrogen receptor-independent mechanism. The results also suggest that different mechanisms are involved in the regulation of cell growth and c-myc gene expression in different cancer cells by estrogen and tamoxifen.

Association between aminolevulinate dehydrogenase genotype and blood lead levels in Taiwan.

This study was designed to evaluate the association between the aminolevulinate dehydrogenase (ALAD) genotype and blood lead levels in a general population environmentally exposed to lead. This study population of 660 subjects was secondarily sampled from the 3000 random samples of Taiwanese general population to study the distribution of blood lead levels in the Taiwanese population. A simple assay based on the polymerase chain reaction-restriction fragment length polymorphism technique was used to determine the genotype of the ALAD gene. This study found that most of the Taiwanese population was ALAD 1-1 (95.4%). Only 4.6% (30 subjects) of population were found to be 1-2 or 2-2. It has been hypothesized that the ALAD2 allele is associated with increased absorption of lead. This study found that individuals with ALAD2 alleles had 20% higher blood lead levels than persons with ALAD1 alleles (7.83 +/- 5.95 vs 6.51 +/- 5.03 micrograms/dL). However, the difference was not statistically significant, even after adjustment for other risk factors of environmental exposure. The result supports the previous finding that individuals with ALAD2 allele had higher blood lead levels. The small sample size and large amount of variation in our study may account for the insignificant association.

Antimutagenic effect of Maillard browning products obtained from amino acids and sugars.

The antimutagenic effects of Maillard reaction products (MRPs) prepared by heating three sugars (fructose, glucose and xylose) and four amino acids (arginine, glycine, lysine and tryptophan) at 100 degrees C for 10 hr was evaluated in the Salmonella/microsome assay. The highest extent of browning was found in the MRPs of sugars-lysine and xylose-amino acids. The MRPs of xylose-amino acids showed stronger antioxidative activity and reducing power than did the other combinations. No mutagenicity or toxicity in Salmonella typhimurium TA98 was observed with any of the MRPs in the presence of S-9. Most MRPs, especially those of sugars-tryptophan and xylose-amino acids, strongly inhibited the mutagenicity of 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), 3-amino-1,4-dimethyl-5H-pyridol-(4,3-b)indole (Trp-P-1) and 2-amino-6-methyldipyrido(1,2-a:3',2'-d)imidazole (Glu-P-1) in the presence of S-9. However, the MRPs of fructose-glycine and fructose-arginine increased the mutagenicity of Trp-P-1. The antimutagenic effect of the MRPs was well correlated with their antioxidative activity and reducing power. The mutagenicity of benzo[a]pyrene was moderately inhibited by most MRPs, but was increased by the MRP of glucose-arginine. Aflatoxin B1 mutagenicity was increased greatly by all the MRPs except that of xylose-tryptophan. The findings suggested that MRPs might have a bifunctional property of co-mutagenicity and antimutagenicity in certain cases.

The screening of 13 short tandem repeat loci in the Chinese population.

Population studies of 13 short tandem repeat (STR) loci were carried out on Chinese in Taiwan. The STR loci included HUMF13B, HUMF13A01, HUMFES/FPS, HUMFABP, HUMPLA2A1, HUMTPOX, HUMTH01, HUMVWFA31/A, HUMCSFIPO, HUMLPL, HUMGPP3A09, HUMCYAR04 and HUMCD4. DNA samples from 100 unrelated individuals were screened. The STR allele patterns were detected by the fluorescence detector of an automated DNA sequencer. Two PCR amplifications were performed for each STR locus in this study. The first PCR amplification strategy used 26 base pairs of the T7 sequence extension in the 5' end of the forward primer of each STR locus. The second PCR amplification used a dye-labeled T7 primer instead of the forward primer in the first PCR amplification, and the first PCR products as template to produce fluorescent dye-labeled PCR products. PCR products of different STR loci with overlapping allele sizes could be detected in the same lane of the polyacrylamide gel on an automated DNA sequencer using different colored dye-labeled T7 primers. There was no need to directly conjugate the fluorescent dye to individual STR primers. The PCR products were obtained using 2 ng of template DNA in 25 microliters of PCR reaction mixture. No deviations from the Hardy-Weinberg equilibrium were observed for the 13 STR loci. The distributions of these STR alleles were different from those of Caucasians or Blacks. The probability of matching from the combination of the 13 STR loci was 5.9 x 10(-10) for our Chinese population. However, HUMF13B, HUMLPL and HUMCD4 loci were not as highly polymorphic as observed in other populations.

ABO genotyping by mutagenically separated polymerase chain reaction.

ABO blood groups were determined by the mutagenically separated polymerase chain reaction (MS-PCR). The products from two sets of PCR reactions using the same program for the nucleotides at positions 261 and 703 from cDNA at the ABO locus were used to distinguish A, B and O alleles. Two forward mutagenic allele-specific primers of different lengths for the ABO polymorphic site were paired with the same reverse primer in each PCR reaction. The 216 bp fragment of the PCR products for the 261th nucleotide was A or B allele-specific and the 195 bp fragment was O allele-specific. The 126 bp fragment of the PCR products for the 703th nucleotide was B allele-specific and the 106 bp fragment was A or O allele-specific. The ABO genotypes were determined by the intersection of the predicted alleles from these two PCR reactions. The PCR products were obtained using 10 ng of DNA in 50 microL of PCR reaction mixture, and electrophoresed in 4% agarose gel. In this study, 265 ABO-phenotype known samples (A: 31, B: 48, AB: 6 and O: 180) in Chinese were used. The results of ABO genotypes were AA: 1, AO: 30, BB: 2, BO: 46, AB: 6 and OO: 180. These results were confirmed by the PCR-RFLP ABO genotyping method. This technique is a simple, rapid, and reliable method for ABO genotyping.

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.

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.

Radioimmunodetection of human cervical carcinoma xenograft by 111In-labeled monoclonal antibody MAb Cx-99.

Radioimmunodetection (RAID) is more sensitive and specific than conventional diagnostic methods. In this study, a monoclonal antibody against cervical carcinoma antigen, MAb Cx-99, was labeled with 111Indium (111In). This immunoconjugate was intravenously injected into athymic nude mice bearing cervical squamous cell carcinoma (SCC) xenografts. The tissue distribution study showed that the xenograft tumor had higher binding activity than most other tissues after 48 h from injection, demonstrated by localization ratio of tumor of tissues (c.p.m./g) against blood (c.p.m./g). However, this localization ratio was also high in the liver, spleen and kidney. The imaging study by immunoscintigraphy also showed that the tumor and liver were distinct from other background tissues 2 days after injection. This preliminary study showed that 111In-labeled MAb Cx-99 may have potential for RAID of cervical cancer, especially for tumors in the pelvis.

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.

Genotyping of the DQA1*4 alleles by restriction enzyme digestion of the PCR product from the AmpliType PM kit.

An earlier study has shown that the three DQA1*4 alleles (0401, 0501 and 0601) can be distinguished by restriction enzyme digestion of the polymerase chain reaction (PCR) product derived from the DQ alpha AmpliType kit (Perikin-Elmer, Norwalk, NJ). We have found that the AmpliType PM kit (Perkin-Elmer, Branchburg, NJ) can also be used to achieve the same goal. In this case, a Bio-Profil image analysis system (Vilber Lourmat, Marne La Vallee, France) is used for evaluating the restricted patterns. After typing the six alleles of DQA1 by the AmpliType HLA DQ alpha Detection Reagent Set (Perkin-Elmer, Branchburg, NJ), the PCR products from the PM kit with allele 4 were digested with Fok I and RsaI, separately. Since the other five fragments from PM kit will conceal the digested fragments of the HLA DQA1 PCR products, we measured the optical density of the pre- and post-digested 242 bp fragments in Fok I digestion, and 214/221 bp fragments in Rsa I digestion to decide the results of enzyme digestion. Out of 136 samples used in this study, 61 contain the DQA1 allele 4 determined by the DQ alpha AmpliType method. All 61 were typed with enzyme digestion, of which there are 2.3%, 19.8% and 8.1% in allele 0401, 0501 and 0601, respectively. Our procedure can thus extend the utilization of AmpliType PM kit and increase the discrimination power of the DQA1 system, especially in populations with high distribution of allele 4.

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.