Effects of different stocking densities on organ development, blood biochemical indices, and antioxidative status of breeder pigeons during the rearing period.

The present experiment was conducted to investigate the effect of different stocking densities on the organ development, blood biochemical indices, and antioxidative status of breeder pigeons during the rearing period. A total of 280 (half male and half female) 40-day-old young pigeons were allocated into 4 groups, including 3 experimental groups (in compartments of the flying room): the high stocking density (HSD) (0.308 m3/bird), standard stocking density (SD) (0.616 m3/bird), and low stocking density (LSD) (1.232 m3/bird) and a caged control (0.04125 m3/bird). The results showed that the contents of corticosterone and heat shock protein 70 in males and the corticosterone content in females were higher in the control than in the other groups. The relative weight of liver, lung, and gizzard in males of the HSD group was the highest among the 4 treatments, whereas the abdominal fat index in the control group was higher than those in the other 3 treatments. Body weight and the relative weight of liver and abdominal fat in female pigeons in HSD group increased significantly. The levels of serum urea nitrogen and uric acid in pigeons of LSD group increased significantly, while the concentration of total cholesterol and the activity of alanine aminotransferase were higher in the control group. Ion (K+, Ca2+, and Na+) concentrations in female pigeon serum were also elevated in the control. The activity of antioxidant enzymes, including the total antioxidant capacity, superoxide dismutase, and glutathione peroxidase in pigeon breast muscle and liver had different degrees of inhibition when the space room was crowded. Moreover, the level of malondialdehyde in the liver of male caged pigeons was higher than that in the other treatments. In summary, rearing in cages or at a high density caused stress responses in the breeder pigeons. The stocking density of breeder pigeons during the rearing period should be ranged from 0.616 m3/bird to 1.232 m3/bird.

Air pollution and global healthcare use for atopic dermatitis: A systematic review.

Increasing air pollution is common around the world, but the impacts of outdoor air pollution exposure on atopic dermatitis (AD) are unclear. We synthesized the current global epidemiologic evidence for air pollution exposure and associated medical visits for AD among adults and children. This review followed PRISMA guidelines, and searches were conducted on PubMed, MEDLINE, Web of Science and EMBASE databases. The searches yielded 390 studies, and after screening, 18 studies around the world assessing at least 5,197,643 medical visits for AD in total were included for the final analysis. We found that exposure to particulate matter ≤2.5 µm in diameter (PM2.5 ) [(10/11) of studies], particulate matter ≤10 µm in diameter (PM10 ) (11/13), nitrogen dioxide (NO2 ) (12/14) and sulfur dioxide (SO2 ) (10/13) was positively associated with AD visits. Results were equivocal for ozone [(4/8) of studies reported positive association] and limited for carbon monoxide [(1/4) of studies reported positive association]. When stratifying results by patient age, patient sex and season, we found that the associations with particulate matter, NO2 and O3 may be affected by temperature. Exposure to selected air pollutants is associated with AD visits, and increasingly poor worldwide air quality may increase global healthcare use for AD.

Dynamic changes in protein concentrations of keratins in crop milk and related gene expression in pigeon crops during different incubation and chick rearing stages.

1. The objective of this study was to examine the keratin composition of crop milk, the variation of epithelial thickness and keratin (K) gene expression in samples from young pigeon during incubation and chick rearing.2. Crop milk was collected from 1-, 3- and 5-day-old squab crops for keratin content analysis. Results showed that K4 accounted for the highest proportion of all detected keratins.3. In total, 42 pairs of adult pigeons were allocated to seven groups according to different stages to collect crop samples. Gene expression studies showed that the K3 gene expression was maximised at rearing Day 15 (15) and R1 in males and females, respectively. K6a gene level was the greatest at R15 in females, whereas it peaked at incubation Day 4 (I4) in males. The K12, K13, K23 and K80 gene levels were inhibited at the peak period of crop milk formation in comparison with I4. In females, K cochleal expression peaked at I10, whereas it was the greatest at R25 in males. K4 and K14 gene expression was the highest at I10 in females, while K4 and K14 were minimised at I17 and R7 in males, respectively. Gene expressions of K5, K8, K19 and K20 in males and K19 in females were maximised at R1. The K5, K20 and K75 gene levels in females peaked at R7. K75 and K8 expressions in males and females reached a maximum value at R25 and I17, respectively.4. The epithelial thickness of male and female crops reached their greatest levels at R1 and had the highest correlation with K19.5. These results emphasised the importance of keratinisation in crop milk formation, and different keratins probably play various roles during this period. The K19 was probably a marker for pigeon crop epithelium development. The sex of the parent pigeon affected keratin gene expression profiles.

MiR-127-3p inhibits proliferation of ovarian cancer in rats through down-regulating MAPK4.

OBJECTIVE: To reveal the anti-tumor effect of micro ribonucleic acid (miR)-127-3p on epithelial ovarian cancer (EOC). PATIENTS AND METHODS: The expression of miR-127-3p in 7 kinds of EOC cell lines and 10 cases of clinical samples of EOC patients was detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). OVCAR-3 and Caov-3 cell lines were transfected with lentiviruses to overexpress endogenous miR-127-3p. Then, the anti-tumor effect of miR-127-3p on EOC cells was explored through the in vitro cell proliferation assay, bufalin sensitivity assay, wound healing assay, and invasion assay. In addition, whether the mitogen-activated protein kinase 4 (MAPK4) gene is a downstream target of miR-127-3p in EOC was verified via Dual-Luciferase reporter assay and qRT-PCR. The involvement of MAPK4 in regulating phenotypes of OVCAR-3 and Caov-3 cells was finally explored. RESULTS: MiR-127-3p was downregulated in both EOC cell lines and EOC tissues (p<0.05). After lentivirus-mediated overexpression of miR-127-3p, in vitro proliferation and invasion of EOC cells were inhibited, and the sensitivity to bufalin was enhanced (p<0.05). MiR-127-3p directly regulated MAPK4 gene in EOC. Moreover, the upregulation of MAPK4 inhibited the anti-tumor effect of miR-127-3p on EOC, manifested as the remarkably enhanced cell proliferation and migration (p<0.05), and the weakened sensitivity to bufalin (p<0.01). CONCLUSIONS: MiR-127-3p exerts an inhibitory effect on EOC cells via regulating MAPK4 level.

[Clinical efficacy of surgical opportunity of the modified transvaginal cervicoisthmic cerclage in treatment of the cervical incompetence].

Objective: To explore the surgical opportunity of the transvaginal cervicoisthmic cerclage in the treatment of the cervical incompetence and the effect on the pregnancy outcomes. Methods: A Retrospective controlled trial was carried out between January 2014 and December 2018 in the Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital in Zhejiang, China and a total of 1 027 patients with cervical incompetence underwent the transvaginal cerclage. According to the different surgical opportunity, the patients were divided into two groups: the conception cerclage (n=736) and the preconception cerclage (n=291), and the former were divided into two subgroups depending on the condition of the cervix, the history indicated conception cerclage (n=511) and the ultrasound indicated conception cerclage (n=225). Main outcome measures were the gestational age, term delivery rate, the fetal weight and the fetal survival rate. Results: After the cerclage, the gestational age was (36±4) weeks, the term delivery rate was 69.6% (710/1 020), the fetal weight was (3 038±624)g and the fetal survival rate was 94.7% (966/1 020). Compared with the ultrasound indicated conception cerclage subgroup, the gestational age, the term delivery rate, and the fetal weight were all significantly higher in the history indicated conception cerclage subgroup [(37±4) vs (36±5) weeks, t=2.691; 72.8% vs 62.7%,χ(2)=7.593; (3 091±594) vs (2 963±756) g, t=2.396; all P<0.05], but the fetal survival rate was comparable in these two groups(95.3% vs 92.9%, χ(2)=1.772, P>0.05). There were no significant differences in the gestational age, the term delivery rate, the fetal weight and fetal survival rate between the history indicated conception cerclage and the preconception cerclage (all P>0.05). Conclusion: The transvaginal cervicoisthmic cerclage is a promising and safe technique for improving obstetric outcomes in women with cervical incompetence, and the history indicated conception cerclage is better than the ultrasound indicated cerclage.

[Method for developing mouse model of endometrios is based on estrogen-releasing thermo-sensitive hydrogel system].

Objective: This study will explore and optimize the conditions for the construction of the mouse model of endometriosis using the estrogen-releasing thermo-sensitive hydrogel system. Methods: (1) The mice were injected subcutaneously with a gradient concentration of estrogen-releasing thermo-sensitive hydrogel systems, and the blood estrogen concentration was measured 3 days later. And then we draw the curve between mouse blood estrogen concentration and thermo-sensitive hydrogel systems concentration, selecting the most appropriate concentration of estrogen-releasing thermo-sensitive hydrogel systems. (2) To explore the release pattern of estrogen-releasing thermo-sensitive hydrogel system, the mice were injected subcutaneously with the optimal concentration of estrogen-releasing thermo-sensitive hydrogel system, and the blood estrogen concentration was measured on the 1st, 3rd, 5th, 7th and 10th day respectively. (3) We transplanted uteruses from donor mice into the abdominal cavities of recipient mice. The recipient mice were divided into three groups named a, b, and c. Group a was intraperitoneally injected with PBS everyday after transplantation, and group b was intraperitoneally injected with estrogen solution everyday after transplantation. And estrogen-releasing thermo-sensitive hydrogel systems mentioned above were injected subcutaneously to group c once a week. Mice were killed 21 days later to observe the success rate of the endometriosis models. Results: (1) The concentration of estrogen in mice was linear with the concentration of estrogenin the estrogen-releasing thermo-sensitive hydrogel systems. And 0.8 mg/ml was the most appropriate concentration of the estrogen-releasing thermo-sensitive hydrogel system. (2) The thermo- sensitive hydrogel systems including 0.8 mg/ml estrogen can release estrogen in mice for 7 days and can achieve effective blood estrogen concentration. (3) All mice in group a, b, and c survived. All mice in group b and c showed ectopic cysts, and no cyst was observed in group a. Conclusions: The construction of the mouse model of endometriosis requires exogenous estrogen. We have found that the novelestrogen-releasing thermo-sensitive hydrogel systems can slowly release estrogen after subcutaneously injection in mice, and the value of the subcutaneously injection of estrogen-releasing thermo-sensitive hydrogel system (0.8 mg/ml) per week to the construction of the mouse model is certain.The estrogen-releasing thermo-sensitive hydrogel system has high application value and is worthy of promotion.

Abdominal wall mixed malignant germ cell tumor: a case report and review of literature.

Abdominal wall mass is not uncommon in clinic, but it is very rare that germ cell tumors (GCTs) arise in the abdominal wall. The authors review the case of a 34-year-old female with abdominal wall mixed malignant GCT composed of embryonal carcinoma and teratoma and combine the relative literature to explain why GCTs originate from anterior abdominal wall.

New insights into implication of the SLIT/ROBO pathway in the prehierarchical follicle development of hen ovary.

The SLIT/Roundabout (ROBO) pathway is involved in follicle development of mammalian ovary, and 2 secreted hormones activin A and inhibin A have potential roles in modulation of the SLIT/ROBO system, but the related actions remain poorly understood in bird. The aims of the present study were to examine the spatial and temporal expression of the SLIT ligand genes (SLIT1, SLIT2, and SLIT3) and their receptor ROBO1, ROBO2, ROBO3, and ROBO4 genes in various-sized prehierarchical follicles during hen ovary development and the effects of activin A and inhibin A on the expression of these genes in the cultured hen follicles. Our result demonstrated that the transcripts of the 3 SLIT genes were highly expressed in the developing follicles and expression patterns of the SLIT transcripts were different from those of ROBO genes detected by real-time quantitative reverse transcriptase PCR. Both SLIT and ROBO transcripts were predominantly expressed in oocytes and granulosa cells from the prehierarchichal follicles examined by in situ hybridization. The localization for SLIT and ROBO proteins was revealed by immunohistochemistry similar to the spatial distribution of their transcript. In cultured follicles (4 to 8 mm in diameter), the expression levels of SLIT and ROBO members are hormonally regulated by activin A (10 ng/mL) and/or inhibin A (20 ng/mL) after treatment for 24 h. However, the expression of only SLIT2, SLIT3, and ROBO3 mRNA presented a directly opposite response to activin A and inhibin A hormones. These results indicate that SLIT/ROBO pathway is implicated in the prehierarchical follicular development of the hen ovary by an intrafollicular autocrine and/or paracrine action, and is influenced by activin A and inhibin A hormones.

Association of novel polymorphisms of forkhead box L2 and growth differentiation factor-9 genes with egg production traits in local Chinese Dagu hens.

Transcription factor forkhead box L2 (FOXL2) and growth differentiation factor-9 (GDF9) genes have critical roles in the regulation of hen ovarian development. In the present study, these genes were explored as possible molecular markers associated with BW, hen-housed egg production, and egg weight in Chinese Dagu hens. Samples were analyzed using the PCR-single strand conformation polymorphism (PCR-SSCP) technique followed by sequencing analysis, and two novel single nucleotide polymorphisms (SNPs) were identified within these candidate genes. Among them, an A/G transition at base position 238 in the coding region of the FOXL2 gene and a G/T transversion at base position 1609 in exon 2 of the GDF9 gene were found to be polymorphic and named SNPs A238G and G1609T, respectively. The SNP A238G (FOXL2) leads to a nonsynonymous substitution (isoleucine77-to-valine), and when the 360 Dagu hen samples were divided into genotypes AA and AB, allele A was found to be present at a higher frequency. Furthermore, the AA genotype correlated with significantly higher hen-housed egg production at 30, 43, 57, and 66 wk of age and with a higher egg weight at 43 wk (P<0.05). For the SNP G1609T (GDF9), the hens were typed into TT and TC genotypes, with the T allele shown to be dominant. The TC genotype was also markedly correlated with higher hen-housed egg production and a higher egg weight (P<0.05). Moreover, four haplotypes were reconstructed based on these two SNPs, with the AATC haplotype found to be correlated with the highest hen-housed egg production at 30 to 66 wk of age and with higher egg weights at 43 wk (P<0.05). Collectively, the two SNPs identified in this study might be used as possible genetic molecular markers to aid in the improvement of egg production traits in chicken breeding.

Herbal medicines for treatment of irritable bowel syndrome.

BACKGROUND: Traditional herbal therapies have been used for a long time to treat gastrointestinal disorders including irritable bowel syndrome, and their effectiveness from clinical research evidence needs to be systematically reviewed. OBJECTIVES: To assess the effectiveness and safety of herbal medicines in patients with irritable bowel syndrome. SEARCH STRATEGY: We searched the following electronic databases till July 2004: The Cochrane Library (CENTRAL), MEDLINE, EMBASE, AMED, LILACS, the Chinese Biomedical Database, combined with hand searches of Chinese journals and conference proceedings till end of 2003. No language restriction was used. SELECTION CRITERIA: Randomised controlled trials of herbal medicines compared with no treatment, placebo, pharmacological interventions were included. DATA COLLECTION AND ANALYSIS: Data were extracted independently by two authors. The methodological quality of trials was evaluated using the components of randomisation, allocation concealment, double blinding, and inclusion of randomised participants. MAIN RESULTS: Seventy-five randomised trials, involving 7957 participants with irritable bowel syndrome, met the inclusion criteria. The methodological quality of three double-blind, placebo-controlled trials was high, but the quality of remaining trials was generally low. Seventy-one different herbal medicines were tested in the included trials, in which herbal medicines were compared with placebo or conventional pharmacologic therapy. Herbal medicines were also combined with conventional therapy and compared to conventional therapy alone.Compared with placebo, a Standard Chinese herbal formula, individualised Chinese herbal medicine, STW 5 and STW 5-II, Tibetan herbal medicine Padma Lax, traditional Chinese formula Tongxie Yaofang, and Ayurvedic preparation showed significantly improvement of global symptoms. Compared with conventional therapy in 65 trials testing 51 different herbal medicines, 22 herbal medicines demonstrated a statistically significant benefit for symptom improvement, and 29 herbal medicines were not significantly different than conventional therapy. In nine trials that evaluated herbal medicine combined with conventional therapy, six tested herbal preparations showed additional benefit from the combination therapy compared with conventional monotherapy. No serious adverse events from the herbal medicines were reported. AUTHORS' CONCLUSIONS: Some herbal medicines may improve the symptoms of irritable bowel syndrome. However, positive findings from less rigorous trials should be interpreted with caution due to inadequate methodology, small sample sizes, and lack of confirming data. Some herbal medicines deserve further examination in high-quality trials.

The origins of the thenar and hypothenar muscles.

This paper presents an anatomical study of the origins of the thenar and hypothenar muscles and postulates the causes of weakness and pillar pain following carpal tunnel release.

Kainate receptor-mediated heterosynaptic facilitation in the amygdala.

Prolonged low-frequency stimulation of excitatory afferents to basolateral amygdala neurons results in enduring enhancement of excitatory synaptic responses. The induction of this form of synaptic plasticity is eliminated by selective antagonists of GluR5 kainate receptors and can be mimicked by the GluR5 agonist ATPA. Kainate receptor-mediated synaptic facilitation generalizes to include inactive afferent synapses on the target neurons, and therefore contrasts with other types of activity-dependent enduring synaptic facilitation that are input-pathway specific. Such heterosynaptic spread of synaptic facilitation could account for adaptive and pathological expansion in the set of critical internal and external stimuli that trigger amygdala-dependent behavioral responses.

LP-BM5 virus-infected mice produce activating autoantibodies to the AMPA receptor.

Autoantibodies to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors may contribute to chronic hyperexcitability syndromes and neurodegeneration, but their origin is unclear. We examined LP-BM5 murine leukemia virus-infected mice, which manifest excitotoxic brain lesions and hypergammaglobulinemia, for the presence of AMPA-receptor Ab's. Endogenous IgG accumulated upon neurons in the neocortex and caudate/putamen of infected mice and interacted with native and recombinant AMPA-receptor subunits with the following relative abundance: GluR3 > or = GluR1 > GluR2 = GluR4, as determined by immunoprecipitation. In a radioligand assay, IgG preparations from infected mice specifically inhibited [(3)H]AMPA binding to receptors in brain homogenates, an activity that was lost after preadsorbing the IgG preparation to immobilized LP-BM5 virus. These IgGs also evoked currents when applied to hippocampal pyramidal neurons or to damaged cerebellar granule neurons. These currents could be blocked using any of several AMPA receptor antagonists. Thus, anti-AMPA-receptor Ab's can be produced as the result of a virus infection, in part through molecular mimicry. These Ab's may alter neuronal signaling and contribute to the neurodegeneration observed in these mice, actions that may be curtailed by the use of AMPA-receptor antagonists.

Incidence of transitional cell carcinoma and arsenic in drinking water: a follow-up study of 8,102 residents in an arseniasis-endemic area in northeastern Taiwan.

A significant association between ingested arsenic and bladder cancer has been reported in an arseniasis-endemic area in southwestern Taiwan, where many households share only a few wells in their villages. In another arseniasis-endemic area in northeastern Taiwan, each household has its own well for obtaining drinking water. In 1991-1994, the authors examined risk of transitional cell carcinoma (TCC) in relation to ingested arsenic in a cohort of 8,102 residents in northeastern Taiwan. Estimation of each study subject's individual exposure to inorganic arsenic was based on the arsenic concentration in his or her own well water, which was determined by hydride generation combined with atomic absorption spectrometry. Information on duration of consumption of the well water was obtained through standardized questionnaire interviews. The occurrence of urinary tract cancers was ascertained by follow-up interview and by data linkage with community hospital records, the national death certification profile, and the cancer registry profile. Cox proportional hazards regression analysis was used to estimate multivariate-adjusted relative risks and 95% confidence intervals. There was a significantly increased incidence of urinary cancers for the study cohort compared with the general population in Taiwan (standardized incidence ratio = 2.05; 95% confidence interval (CI): 1.22, 3.24). A significant dose-response relation between risk of cancers of the urinary organs, especially TCC, and indices of arsenic exposure was observed after adjustment for age, sex, and cigarette smoking. The multivariate-adjusted relative risks of developing TCC were 1.9, 8.2, and 15.3 for arsenic concentrations of 10.1-50.0, 50.1-100, and >100 microg/liter, respectively, compared with the referent level of < or =10.0 microg/liter.

The splicing determinants of a regulated exon in the axonal MAP tau reside within the exon and in its upstream intron.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 6 of the gene is an alternatively spliced cassette whose expression profile is distinct from that of the other tau regulated exons, implying the utilization of distinct regulatory factors. Previous work had established the use of cryptic splice sites within exon 6 and the influence of flanking exons on the ratio of exon 6 variants. The present work shows that, in addition to the previously identified participants, the ratio of exon 6 isoforms is affected by: (1) suppression of the cryptic sites, (2) deletions of the upstream intron, and (3) the splicing regulators PTB and U2AF, both of which act on the branch point/polypyrimidine tract region. These results strongly suggest that factors binding immediately upstream of exon 6 are involved in regulation of this exon. They also lead to the conclusion that splicing of exon 6 is primarily governed by multiple branch points.

Splicing of a regulated exon reveals additional complexity in the axonal microtubule-associated protein tau.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 6 of the gene is an alternatively spliced cassette whose expression pattern and splicing regulation had not been previously analyzed in the human. The expression profile of exon 6 is completely different from that of the better-analyzed exons 2, 3, 4A, and 10, implying the utilization of distinct regulatory factors. The default splicing behavior of the exon had demonstrated the existence of what were initially considered cryptic splice sites. However, analysis of the expression pattern of exon 6 suggests that these splice sites are utilized in certain human tissues and, if translated, would result in a radically altered tau protein. Lastly, expression of exon 6 minigene constructs in cells indicates that its flanking exons are involved in its inclusion and in the modulation of the ratio of its variants.

GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells.

The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750-12754.). In this study, we demonstrate that at 37 degrees C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37 degrees C, cell surface-labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15 degrees C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15 degrees C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37 degrees C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15 degrees and 37 degrees C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

Genetic engineering of Serratia marcescens with bacterial hemoglobin gene: effects on growth, oxygen utilization, and cell size.

The bacterial hemoglobin from Vitreoscilla has been shown to increase growth yield and yield of genetically engineered product in Escherichia coli. To test the generality of this phenomenon, the approximately 560-bp bacterial (Vitreoscilla) hemoglobin gene (vgb) (including the native promoter), cloned into the vector pUC8 in two constructs containing about 1650 and 850 bp, respectively, of Vitreoscilla DNA downstream of vgb, was transformed into Serratia marcescens. After several transfers of the transformants on selective media, both plasmids became stable in this host and the resulting strains produced hemoglobin. Both transformants were compared, regarding growth in liquid Luria-Bertani (LB) medium, with untransformed S. marcescens and S. marcescens transformed with pUC8. The vgb-bearing strains had about 5 times lower maximum viable cell numbers than the strains without hemoglobin, but the former also had late log or early stationary phase cells that were 5-10 times larger than those of the latter. Further, on a dry cell mass basis the presence of vgb inhibited cell growth in liquid media. In contrast, growth of the vgb-bearing strains on LB plates based on cell mass (determined from colony size) was markedly enhanced compared with that of the pUC8 transformant. Respiration of the vgb-bearing strains was lower than that of the strains without vgb on a cell mass basis. These results show that the presence of vgb can have idiosyncratic effects and is not always an aid to cell growth so that its use for genetic engineering must be tested on a case by case basis.

Metabolic engineering of Serratia marcescens with the bacterial hemoglobin gene: alterations in fermentation pathways.

Serratia marcescens was transformed with plasmid vector pUC8 or pUC8 containing the bacterial (Vitreoscilla) hemoglobin gene (vgb) on either a 2.3-kb fragment (pUC8:15) or 1.4-kb fragment (pUC8:16) of Vitreoscilla DNA. The vgb-bearing strains were compared with the pUC8 transformant and untransformed S. marcescens with respect to growth in Luria-Bertani (LB) broth supplemented with glucose or casein acid hydrolysate. Growth (on a viable cell basis) was similar to that in unsupplemented LB. Total acid excretion (as estimated by medium pH) was similar for all strains in both LB plus 2% casein acid hydrolysate and LB without additions. Acid excretion in LB plus 2% glucose was somewhat greater at up to 10 h in culture for the two vgb-bearing strains; from 10 to 26 h in culture, the pHs of these cultures continued to decrease (to 4.1-4.2), whereas those of the non-vgb-bearing strains returned to near the starting pH (7.4-7.8). Concomitantly, after 26 h of culture in LB plus 2% glucose, the non-vgb-bearing strains had produced about 15 times as much acetoin and about three to four times as much 2,3-butanediol as the vgb-bearing strains. In general, for all strains, much more acetoin and 2,3-butanediol were produced in LB plus 2% glucose than in unsupplemented LB. The exception was acetoin production by the strain bearing vgb on plasmid pUC8:15; after 26 h of culture in LB without supplementation it was between three and four times that of the other strains, and about 50% higher than its level in LB plus 2% glucose. When grown with the 2% casein acid hydrolysate supplement, the strain bearing vgb on plasmid pUC8:15 produced much more acetoin and 2,3-butanediol than the other strains after 26 hours in culture. The results confirm that vgb can significantly alter carbon metabolism and suggest that the use of vgb technology for directed metabolic engineering may be a complicated process, depending in part on medium composition.

Arsenic methylation capacity, body retention, and null genotypes of glutathione S-transferase M1 and T1 among current arsenic-exposed residents in Taiwan.

In order to elucidate the relationships among arsenic methylation capacity, body retention, and genetic polymorphisms of glutathione S-transferase (GST) M1 and T1, a total of 115 study subjects were recruited from Lanyang Basin located on the northeast coast of Taiwan. Specimens of drinking water, blood, urine, hair and toenail were collected from each study subject. Urinary inorganic and methylated arsenic were speciated by high performance liquid chromatography combined with hydride-generation atomic absorption spectrometry. Arsenic concentration in hair and toenail were quantitated by atomic absorption spectrophotometry. The polymerase chain reaction was used to determine genetic polymorphisms of GST M1 and T1. Arsenic concentrations in urine, hair, and toenail of study subjects were positively correlated with arsenic levels in their drinking water. Percentages of various arsenic species in urine (mean +/- standard error (SE) were 11.8 +/- 1.0, 26.9 +/- 1.2 and 61.3 +/- 1.4, respectively, for inorganic arsenic, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). Men and women had similar arsenic methylation capability. No associations were observed between arsenic methylation capability and arsenic content in either drinking water or urine. Ratios of arsenic contents in hair and toenail to urinary arsenic content (mean +/- standard error) were 6.2 +/- 0.7 and 16.5 +/- 1.7, respectively. Genetic polymorphisms of GST M1 and T1 were significantly associated with arsenic methylation. Subjects having the null genotype of GST M1 had an increased percentage of inorganic arsenic in urine, while those with null genotype of GST T1 had an elevated percentage of DMA in urine. Arsenic contents in hair and toenail were significantly correlated with the increase in arsenic concentrations of drinking water and urine, while no significant associations were observed between arsenic contents in hair and toenail and polymorphisms of GST M1 and T1. The relationship between arsenic methylation capability and body retention was modified by genetic polymorphisms of GST M1 and T1. Arsenic contents in hair and toenail were negatively associated with MMA percentage and positively associated with DMA percentage among subjects having null genotypes of GST M1 and T1, but not among those with non-null genotypes.