Yanyan Li, Yuxiang Xu, Wenhao Li, Yong Yang, Lin Wang, Jun Yu, Changjun Wang, Xihong Li
In order to improve the spore yield of compound Bacillus spp. (B. amyloliquefaciens, B. laterosporus and B. megaterium), the effects of nutrient conditions including carbon source, nitrogen source, mineral salt and fermentation conditions including the inoculum age, inoculation amount, loading volume of liquid and initial pH on the spore yield were studied. The results indicated that the optimized medium was glucoses 20 g/L, soybean meal 30.0 g/L, K2HPO4 1.0 g/L; fermentation temperature is 37℃, the inoculum age 12 h, initial pH 7.0, 2% inoculation amount, loading volume of liquid 20 mL/250 mL. Under the optimized conditions of culture medium and fermentation for compound Bacillus spp., spore yield was 10.24 times more than the initial medium, and the spore formation rate reached more than 90%.
{"title":"Study on Optimizing Nutrition and Fermentation Conditions for Compound Bacillus spp.","authors":"Yanyan Li, Yuxiang Xu, Wenhao Li, Yong Yang, Lin Wang, Jun Yu, Changjun Wang, Xihong Li","doi":"10.4236/AJMB.2019.92007","DOIUrl":"https://doi.org/10.4236/AJMB.2019.92007","url":null,"abstract":"In order to improve the spore yield of compound Bacillus spp. (B. amyloliquefaciens, B. laterosporus and B. megaterium), the effects of nutrient conditions including carbon source, nitrogen source, mineral salt and fermentation conditions including the inoculum age, inoculation amount, loading volume of liquid and initial pH on the spore yield were studied. The results indicated that the optimized medium was glucoses 20 g/L, soybean meal 30.0 g/L, K2HPO4 1.0 g/L; fermentation temperature is 37℃, the inoculum age 12 h, initial pH 7.0, 2% inoculation amount, loading volume of liquid 20 mL/250 mL. Under the optimized conditions of culture medium and fermentation for compound Bacillus spp., spore yield was 10.24 times more than the initial medium, and the spore formation rate reached more than 90%.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44378692","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Fatima Aliyu Deba, S. Isma’il, M. Sahal, I. Okpanachi
Background: Crops of economic importance like Maize are preferred to other crops with capacity of giving the highest yields, having the ability to help alleviate poverty within the African continent. The consistent use of inorganic fertilizers has great adverse effect on soil structure. The quest to seek alternative method of boosting maize production is imminent to avoid further soil degradation. Most plants form symbiotic relationships with mycorrhizae and improve nutrients uptake by host plants. Objectives: The aim of this study is to examine the effect of mycorrhizae in local manure production on development of maize, in comparison with inorganic fertilizer application and its cost implication. Methods: The experiment involved samples of starter soil from 7 local governments. Organic waste was added to starter soil, each having three replicates. Nine other replicates served as non-inoculated controls and nine served for fertilizer application at 50 g, 100 g and 150 g. One [1] gram of Maize sown in 50 ml pot contains soil at different treatment levels 50 g, 100 g, and 150 g. After two weeks, samples were transplanted in an open field. Results: The result showed plots treated with locally produced fertilizer, significantly influenced plant height, culm diameter, number of leaves and leaf broadness in comparison to inorganic fertilizer, which showed 53%, 47% respectively. Conclusion: Cost production of a bag of organic fertilizer was one-fourth cheaper compared to an inorganic fertilizer price from an open market.
{"title":"Evaluation of Economic Importance of Locally Produced Manure over Inorganic Fertilizer for Maize Production: Vegetative Performance and Cost Implication","authors":"Fatima Aliyu Deba, S. Isma’il, M. Sahal, I. Okpanachi","doi":"10.4236/AJMB.2019.92006","DOIUrl":"https://doi.org/10.4236/AJMB.2019.92006","url":null,"abstract":"Background: Crops of economic importance like Maize are preferred to other crops with capacity of giving the highest yields, having the ability to help alleviate poverty within the African continent. The consistent use of inorganic fertilizers has great adverse effect on soil structure. The quest to seek alternative method of boosting maize production is imminent to avoid further soil degradation. Most plants form symbiotic relationships with mycorrhizae and improve nutrients uptake by host plants. Objectives: The aim of this study is to examine the effect of mycorrhizae in local manure production on development of maize, in comparison with inorganic fertilizer application and its cost implication. Methods: The experiment involved samples of starter soil from 7 local governments. Organic waste was added to starter soil, each having three replicates. Nine other replicates served as non-inoculated controls and nine served for fertilizer application at 50 g, 100 g and 150 g. One [1] gram of Maize sown in 50 ml pot contains soil at different treatment levels 50 g, 100 g, and 150 g. After two weeks, samples were transplanted in an open field. Results: The result showed plots treated with locally produced fertilizer, significantly influenced plant height, culm diameter, number of leaves and leaf broadness in comparison to inorganic fertilizer, which showed 53%, 47% respectively. Conclusion: Cost production of a bag of organic fertilizer was one-fourth cheaper compared to an inorganic fertilizer price from an open market.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44267962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
N. Pavliuchenko, Victoria Hazarnian, Marcel Bassil
Goat anti-Rabbit Horseradish Peroxidase (HRP) secondary conjugate was produced using a modified periodate oxidation method. The obtained conjugate was tested in the quality control techniques of therapeutic proteins. To determine the working dilution, titration of the prepared conjugate was performed in Indirect Enzyme-Linked Immunosorbent Assay (ELISA) and found to be 1:5000. This dilution was further tested in Western Blot analysis. The secondary conjugate was kept at 4°C for one month and its stability was verified by Western Blot and Indirect ELISA techniques.
{"title":"Modification of Periodate Oxidation Method to Produce HRP-IgG Conjugate and Test its Stability Overtime","authors":"N. Pavliuchenko, Victoria Hazarnian, Marcel Bassil","doi":"10.4236/AJMB.2019.92005","DOIUrl":"https://doi.org/10.4236/AJMB.2019.92005","url":null,"abstract":"Goat anti-Rabbit Horseradish Peroxidase (HRP) secondary conjugate was produced using a modified periodate oxidation method. The obtained conjugate was tested in the quality control techniques of therapeutic proteins. To determine the working dilution, titration of the prepared conjugate was performed in Indirect Enzyme-Linked Immunosorbent Assay (ELISA) and found to be 1:5000. This dilution was further tested in Western Blot analysis. The secondary conjugate was kept at 4°C for one month and its stability was verified by Western Blot and Indirect ELISA techniques.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46336186","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Molecular Cloning and Tissue Distribution of Troponin I from the Japanese Pearl Oyster, Pinctada fucata","authors":"D. Funabara, Yoshinori Urakawa, S. Kanoh","doi":"10.4236/ajmb.2019.92003","DOIUrl":"https://doi.org/10.4236/ajmb.2019.92003","url":null,"abstract":"","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70513368","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Ghani, M. Mehmood, A. Javed, F. Ansari, H. Anwar, Sana Noreen, Sajjad Hussain Shah
Immunization is the most effective method still used against infectious agents. Although not always, vaccines ineffectiveness is reported enormously against many of the pathogens throughout the world in poultry, particularly in case of killed or sub unit vaccine. The current project is, therefore, carried out as a preliminary study on broiler chickens to investigate the modulation of immune system against avian influenza virus in association with purified Staphylococcus aureus toxoid. After isolation of Gram positive cocci bacteria on mannitol salt agar from raw milk, yogurt and chicken meat were subsequently biochemically characterized by using rapid diagnostic kit. Pure culture of S. aureus was inoculated into digitally controlled bio-fermentor containing mannitol salt broth for production of toxins. Enormous production of bacteria was passed through sequence of filtration system based on 0.45 μm followed by 0.22 μm size. The centrifugation of the filtrate was made at 10,000 rpm for 60 minutes at 5℃ followed by 56,100 rpm for 20 minutes and clear supernatant containing Staphylococcus enterotoxin (SEs) was obtained. Bradford estimation of proteins further provided 305 μg/ml of SEs toxoid. Four types of oil adjuvant avian influenza type H9N2 virus vaccines (without toxoid, 91.5 μg/0.3ml, 22.8 μg/0.3ml and 11.43 μg/0.3ml) were injected into healthy AI H9N2 susceptible broilers and anti-H9N2 HI antibody titers were measured in terms of hemagglutination inhibition test. It was observed that on the 8th day, post vaccination cumulative mean anti AIH9 HI antibody titer of G-1, G-2, G-3, G-4 and G-5 was 3.13 ± 0.406, 5.13 ± 0.246, 3.96 ± 0.159, 3.25 ± 0.237 and 0.78 ± 0.467 respectively. It was found that all the vaccines induced protective titers 18 days’ post vaccination, but vaccine containing 91.5 μg/dose of SEs toxoid showed significantly higher (P < 0.05) immune response as compared to vaccine containing 22.8 μg/dose and 11.43 μg/dose, whereas, vaccines containing SEs toxoid showed better (P < 0.05) anti AIH9 HI antibody titer as compared to vaccine without SEs toxoid. Thus, it is concluded that addition of super antigens of SEs in the form of toxoids, particularly in inactivated vaccines, could be the better choice for modulation of immediate and better immune response in future vaccines technologies.
{"title":"Immunomodulatory Effect of Purified Exotoxins of Staphylococcus aureus in Association with Bird Flu Virus Vaccine in Broilers","authors":"M. Ghani, M. Mehmood, A. Javed, F. Ansari, H. Anwar, Sana Noreen, Sajjad Hussain Shah","doi":"10.4236/ajmb.2019.91001","DOIUrl":"https://doi.org/10.4236/ajmb.2019.91001","url":null,"abstract":"Immunization is the most effective method still used against infectious agents. Although not always, vaccines ineffectiveness is reported enormously against many of the pathogens throughout the world in poultry, particularly in case of killed or sub unit vaccine. The current project is, therefore, carried out as a preliminary study on broiler chickens to investigate the modulation of immune system against avian influenza virus in association with purified Staphylococcus aureus toxoid. After isolation of Gram positive cocci bacteria on mannitol salt agar from raw milk, yogurt and chicken meat were subsequently biochemically characterized by using rapid diagnostic kit. Pure culture of S. aureus was inoculated into digitally controlled bio-fermentor containing mannitol salt broth for production of toxins. Enormous production of bacteria was passed through sequence of filtration system based on 0.45 μm followed by 0.22 μm size. The centrifugation of the filtrate was made at 10,000 rpm for 60 minutes at 5℃ followed by 56,100 rpm for 20 minutes and clear supernatant containing Staphylococcus enterotoxin (SEs) was obtained. Bradford estimation of proteins further provided 305 μg/ml of SEs toxoid. Four types of oil adjuvant avian influenza type H9N2 virus vaccines (without toxoid, 91.5 μg/0.3ml, 22.8 μg/0.3ml and 11.43 μg/0.3ml) were injected into healthy AI H9N2 susceptible broilers and anti-H9N2 HI antibody titers were measured in terms of hemagglutination inhibition test. It was observed that on the 8th day, post vaccination cumulative mean anti AIH9 HI antibody titer of G-1, G-2, G-3, G-4 and G-5 was 3.13 ± 0.406, 5.13 ± 0.246, 3.96 ± 0.159, 3.25 ± 0.237 and 0.78 ± 0.467 respectively. It was found that all the vaccines induced protective titers 18 days’ post vaccination, but vaccine containing 91.5 μg/dose of SEs toxoid showed significantly higher (P < 0.05) immune response as compared to vaccine containing 22.8 μg/dose and 11.43 μg/dose, whereas, vaccines containing SEs toxoid showed better (P < 0.05) anti AIH9 HI antibody titer as compared to vaccine without SEs toxoid. Thus, it is concluded that addition of super antigens of SEs in the form of toxoids, particularly in inactivated vaccines, could be the better choice for modulation of immediate and better immune response in future vaccines technologies.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70513302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
We determined the full-length primary structure of the tropomyosin (TM)-1 and -2 proteins from the adductor muscle of the Japanese pearl oyster Pinctada fucata (Pifuc-TM-1 and Pifuc-TM-2), and found that they are each composed of 284 amino acid residues. We predicted the gene structure of P. fucata TM (Pifuc-TM) using Splign alignment of our cDNA with genomic sequences and elucidated that Pifuc-TM consists of 10 exons. Exons 1 - 3 and 5 - 10 are used to transcribe Pifuc-TM-1 mRNA, and exons 1 - 4 and 6 - 10 are used to transcribe Pifuc-TM-2 mRNA. Both genes share the same start and stop codons located in exon 1 and exon 10, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TM-1 gene was mainly expressed in adductor phasic muscle, and at a relatively weaker level in adductor catch muscle, whereas the Pifuc-TM-2 gene was expressed equally in both phasic and catch muscles. They were weakly expressed in gill and mantle. Immunoblot analysis using anti-Pifuc-TM-1 and anti-Pifuc-TM-2 antibodies revealed that adductor phasic muscle contained Pifuc-TM-1, while adductor catch muscle contained both Pifuc-TM-1 and Pifuc-TM-2. Differential scanning calorimetry (DSC) analysis was carried out for Pifuc-TM-1 and Pifuc-TM-2 expressed in bacteria, as well as TM purified from P. fucata phasic and catch muscle tissues (phasic-TM and catch-TM). The DSC data indicated that phasic-TM was mainly composed of Pifuc-TM-1, whereas catch-TM contained Pifuc-TM-1 and Pifuc-TM-2. These findings suggest that the distribution of Pifuc-TM-1 and Pifuc-TM-2 in adductor muscle is specific to the muscle fiber type, and reflects the properties of each.
{"title":"Tropomyosin Isoform Expression in the Adductor Muscle of the Japanese Pearl Oyster, Pinctada fucata","authors":"D. Funabara, A. Ohta, J. Sueyoshi, S. Kanoh","doi":"10.4236/AJMB.2019.91002","DOIUrl":"https://doi.org/10.4236/AJMB.2019.91002","url":null,"abstract":"We determined the full-length primary structure of the tropomyosin (TM)-1 and -2 proteins from the adductor muscle of the Japanese pearl oyster Pinctada fucata (Pifuc-TM-1 and Pifuc-TM-2), and found that they are each composed of 284 amino acid residues. We predicted the gene structure of P. fucata TM (Pifuc-TM) using Splign alignment of our cDNA with genomic sequences and elucidated that Pifuc-TM consists of 10 exons. Exons 1 - 3 and 5 - 10 are used to transcribe Pifuc-TM-1 mRNA, and exons 1 - 4 and 6 - 10 are used to transcribe Pifuc-TM-2 mRNA. Both genes share the same start and stop codons located in exon 1 and exon 10, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TM-1 gene was mainly expressed in adductor phasic muscle, and at a relatively weaker level in adductor catch muscle, whereas the Pifuc-TM-2 gene was expressed equally in both phasic and catch muscles. They were weakly expressed in gill and mantle. Immunoblot analysis using anti-Pifuc-TM-1 and anti-Pifuc-TM-2 antibodies revealed that adductor phasic muscle contained Pifuc-TM-1, while adductor catch muscle contained both Pifuc-TM-1 and Pifuc-TM-2. Differential scanning calorimetry (DSC) analysis was carried out for Pifuc-TM-1 and Pifuc-TM-2 expressed in bacteria, as well as TM purified from P. fucata phasic and catch muscle tissues (phasic-TM and catch-TM). The DSC data indicated that phasic-TM was mainly composed of Pifuc-TM-1, whereas catch-TM contained Pifuc-TM-1 and Pifuc-TM-2. These findings suggest that the distribution of Pifuc-TM-1 and Pifuc-TM-2 in adductor muscle is specific to the muscle fiber type, and reflects the properties of each.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70513314","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
D. Funabara, D. Ishikawa, Yoshinori Urakawa, S. Kanoh
Troponin is a thin filament-associated regulator of vertebrate striated muscle contraction. Troponin changes its structure upon Ca2+ binding to troponin C, one of the subunits of troponin, allowing myosin to interact with actin. We recently elucidated the molecular characteristics of the Japanese pearl oyster Pinctada fucata troponin C (Pifuc-TnC), revealing the possibilities that Pifuc-TnC and vertebrate muscle TnC play dissimilar roles in muscle contraction. Pifuc-TnC has four EF-hand motifs, but, unlike vertebrate TnC, only one (site IV) was predicted to bind Ca2+. To confirm the number of Ca2+-binding sites in Pifuc-TnC and whether Ca2+ binding induces a conformational change, we purified the full-length protein and a variant, Pifuc-TnC-E142Q (that has a mutation in the predicted Ca2+-binding site of site IV), following their expression in laboratory E. coli. Isothermal titration calorimetry demonstrated Ca2+ binding to Pifuc-TnC, whereas Pifuc-TnC-E142Q was unable to bind Ca2+, confirming that site IV is the only Ca2+-binding site in Pifuc-TnC. Pifuc-TnC eluted in a later fraction from a gel filtration column in the presence of Ca2+ compared with the condition when Ca2+ was absent. In contrast, the elution profiles of Pifuc-TnC-E142Q were equivalent in both the presence and absence of Ca2+, suggesting that Ca2+ binding to Pifuc-TnC induces a conformational change that delays its elution from the column. UV-absorption spectral analysis revealed that binding of Ca2+ to Pifuc-TnC caused an increase in absorption at a wavelength of approximately 250 nm, possibly because phenylalanine residues had been exposed on the surface of the molecule as a result of a conformational change. Differential scanning calorimetric analyses of Pifuc-TnC showed aggregation in the presence of Ca2+ in accordance with an increase of temperature, but no aggregation was seen in the absence of Ca2+. In combination, these findings suggest that Ca2+ binding to site IV induces a conformational change in Pifuc-TnC.
肌钙蛋白是脊椎动物横纹肌收缩的细丝相关调节因子。肌钙蛋白在Ca2+与肌钙蛋白C（肌钙蛋白的亚基之一）结合时改变其结构，使肌球蛋白与肌动蛋白相互作用。我们最近阐明了日本珍珠牡蛎Pinctada fucata肌钙蛋白C（Pifuc TnC）的分子特征，揭示了Pifuc T nC和脊椎动物肌肉T nC在肌肉收缩中发挥不同作用的可能性。Pifuc-TnC有四个EF手基序，但与脊椎动物TnC不同，只有一个（位点IV）被预测与Ca2+结合。为了确认Pifuc-TnC中Ca2+结合位点的数量以及Ca2+结合是否诱导构象变化，我们纯化了全长蛋白和变异体Pifuc-TnC-E142Q（其在位点IV的预测Ca2+结合点中具有突变），随后它们在实验室大肠杆菌中表达。等温滴定量热法证明Ca2+与Pifuc-TnC结合，而Pifuc-TnC-E142Q不能结合Ca2+，证实了位点IV是Pifuc-TnC中唯一的Ca2+结合位点。与不存在Ca2+时的条件相比，在存在Ca2+的情况下，Pifuc-TnC在来自凝胶过滤柱的稍后级分中洗脱。相反，在存在和不存在Ca2+的情况下，Pifuc-TnC-E142Q的洗脱图谱是等效的，这表明Ca2+与Pifuc-TnC的结合诱导了构象变化，从而延迟了其从柱中的洗脱。UV吸收光谱分析显示，Ca2+与Pifuc-TnC的结合导致在大约250nm的波长下的吸收增加，这可能是因为苯丙氨酸残基由于构象变化而暴露在分子表面。Pifuc-TnC的差示扫描量热分析显示，随着温度的升高，在存在Ca2+的情况下发生聚集，但在不存在Ca2+时未观察到聚集。总之，这些发现表明Ca2+结合位点IV诱导Pifuc-TnC的构象变化。
{"title":"Ca 2+ -Induced Conformational Change of Troponin C from the Japanese Pearl Oyster, Pinctada fucata","authors":"D. Funabara, D. Ishikawa, Yoshinori Urakawa, S. Kanoh","doi":"10.4236/ajmb.2018.84018","DOIUrl":"https://doi.org/10.4236/ajmb.2018.84018","url":null,"abstract":"Troponin is a thin filament-associated regulator of vertebrate striated muscle contraction. Troponin changes its structure upon Ca2+ binding to troponin C, one of the subunits of troponin, allowing myosin to interact with actin. We recently elucidated the molecular characteristics of the Japanese pearl oyster Pinctada fucata troponin C (Pifuc-TnC), revealing the possibilities that Pifuc-TnC and vertebrate muscle TnC play dissimilar roles in muscle contraction. Pifuc-TnC has four EF-hand motifs, but, unlike vertebrate TnC, only one (site IV) was predicted to bind Ca2+. To confirm the number of Ca2+-binding sites in Pifuc-TnC and whether Ca2+ binding induces a conformational change, we purified the full-length protein and a variant, Pifuc-TnC-E142Q (that has a mutation in the predicted Ca2+-binding site of site IV), following their expression in laboratory E. coli. Isothermal titration calorimetry demonstrated Ca2+ binding to Pifuc-TnC, whereas Pifuc-TnC-E142Q was unable to bind Ca2+, confirming that site IV is the only Ca2+-binding site in Pifuc-TnC. Pifuc-TnC eluted in a later fraction from a gel filtration column in the presence of Ca2+ compared with the condition when Ca2+ was absent. In contrast, the elution profiles of Pifuc-TnC-E142Q were equivalent in both the presence and absence of Ca2+, suggesting that Ca2+ binding to Pifuc-TnC induces a conformational change that delays its elution from the column. UV-absorption spectral analysis revealed that binding of Ca2+ to Pifuc-TnC caused an increase in absorption at a wavelength of approximately 250 nm, possibly because phenylalanine residues had been exposed on the surface of the molecule as a result of a conformational change. Differential scanning calorimetric analyses of Pifuc-TnC showed aggregation in the presence of Ca2+ in accordance with an increase of temperature, but no aggregation was seen in the absence of Ca2+. In combination, these findings suggest that Ca2+ binding to site IV induces a conformational change in Pifuc-TnC.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":"08 1","pages":"205-214"},"PeriodicalIF":0.0,"publicationDate":"2018-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43070752","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Dr. Okoth Patrick Kirsteen, Muoma John, O. Dennis, Barasa Mustafa, Angienda Paul
While the mutational processes that subsume biological diversity can be revealed in great detail through phylogenetic inferencing using plastid markers, few studies document their use. Accurate phylogenic inference can provide a framework for addressing a host of important evolutionary questions including a context to reconstruct molecular evolution of an organism. Despite the obvious utility of plastid markers in illuminating biological enquiry, many important questions still abound. The use of cp-DNA gene sequence data for phylogenetic inference can have an enormous impact on plant phylogenetics and systematics. The repertoire of genetic diversity of Kenya’s Gene Bank repositories can be explored based on cp-genome signatures. This is because cp-DNA-based mutational changes are an important additional tool to the previous evidence available on plant evolution yet to be explored in biodiversity studies in Kenya. Taken together, these evolutionary changes can inspire development of realistic algorithms for phylogenetic inferencing based on molecular data. Phylogenetic reconstructions are at the very core of molecular evolution. Comparative sequence analyses of plastid markers can have utility beyond the study of phylogeny. The pattern of nucleotide substitution observed over evolutionary time can reflect functional constraints imposed due to natural selection. In line with this, it is possible to detect subtle anatomical variations associated with small fitness effects that can account for genetic diversity at varietal level. The lack of sequence information in Kenyan cowpea has limited the robust advancement of molecular markers use in dissecting diversity based on the putative plastid markers [1]. The present study sought to generate and upscale novel technologies such as genomics, DNA barcoding and bio-informatics in understanding molecular diversity of cowpea accessions from the Gene Bank of Kenya and ecotypes. A total of 298 sequences of cowpea germplasm conserved as in situ and ex situ in Kenya but sourced from phylogeographically diverse settings were examined and their genetic profiles were characterized and evaluated using molecular tools. The Gene Bank materials were purposefully sampled to develop subsets representative of the diversity in the genepool’s collection. We present an extensive study on characterizing the genetic diversity of cp-DNA gene sequence data for the cowpea accessions from the Nation Gene Bank of Kenya. The comparative sequence analyses and phylogenetic clustering of seven plastid markers widely used in the DNA barcoding of land plants provide insights on the molecular evolution of this vascular plant. The detailed and in-depth genome characterization herein greatly enriches the genetic profile of this important crop, which can help in reconstructing realistic models of mutational process during plant evolutionary history. This study addressed this gap by employing a DNA barcode library for cowpea to determine the
{"title":"Molecular Footprint of Kenya’s Gene Bank Repositories Based on the cp -Genome Signatures","authors":"Dr. Okoth Patrick Kirsteen, Muoma John, O. Dennis, Barasa Mustafa, Angienda Paul","doi":"10.4236/ajmb.2018.84019","DOIUrl":"https://doi.org/10.4236/ajmb.2018.84019","url":null,"abstract":"While the mutational processes that subsume biological diversity can be revealed in great detail through phylogenetic inferencing using plastid markers, few studies document their use. Accurate phylogenic inference can provide a framework for addressing a host of important evolutionary questions including a context to reconstruct molecular evolution of an organism. Despite the obvious utility of plastid markers in illuminating biological enquiry, many important questions still abound. The use of cp-DNA gene sequence data for phylogenetic inference can have an enormous impact on plant phylogenetics and systematics. The repertoire of genetic diversity of Kenya’s Gene Bank repositories can be explored based on cp-genome signatures. This is because cp-DNA-based mutational changes are an important additional tool to the previous evidence available on plant evolution yet to be explored in biodiversity studies in Kenya. Taken together, these evolutionary changes can inspire development of realistic algorithms for phylogenetic inferencing based on molecular data. Phylogenetic reconstructions are at the very core of molecular evolution. Comparative sequence analyses of plastid markers can have utility beyond the study of phylogeny. The pattern of nucleotide substitution observed over evolutionary time can reflect functional constraints imposed due to natural selection. In line with this, it is possible to detect subtle anatomical variations associated with small fitness effects that can account for genetic diversity at varietal level. The lack of sequence information in Kenyan cowpea has limited the robust advancement of molecular markers use in dissecting diversity based on the putative plastid markers [1]. The present study sought to generate and upscale novel technologies such as genomics, DNA barcoding and bio-informatics in understanding molecular diversity of cowpea accessions from the Gene Bank of Kenya and ecotypes. A total of 298 sequences of cowpea germplasm conserved as in situ and ex situ in Kenya but sourced from phylogeographically diverse settings were examined and their genetic profiles were characterized and evaluated using molecular tools. The Gene Bank materials were purposefully sampled to develop subsets representative of the diversity in the genepool’s collection. We present an extensive study on characterizing the genetic diversity of cp-DNA gene sequence data for the cowpea accessions from the Nation Gene Bank of Kenya. The comparative sequence analyses and phylogenetic clustering of seven plastid markers widely used in the DNA barcoding of land plants provide insights on the molecular evolution of this vascular plant. The detailed and in-depth genome characterization herein greatly enriches the genetic profile of this important crop, which can help in reconstructing realistic models of mutational process during plant evolutionary history. This study addressed this gap by employing a DNA barcode library for cowpea to determine the ","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":"8 1","pages":"215-244"},"PeriodicalIF":0.0,"publicationDate":"2018-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43902511","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic human pathogen that can lead to severe diseases in immunocompromised patients. The insulin-cleaving membrane protease (ICMP) of P. aeruginosa plays a vital role in the pathogenesis of the bacterium and is therefore characterized as an important bacterial virulence factor. In addition, ICMP also serves as a founding member of the M75 peptidase family and represents a prototype of the imelysin/imelysin-like proteins. Despite of its functional importance in the pathogenesis of P. aeruginosa and of a root position as the prototypic imelysin/imelysin-like member, the structural features of the protein remain uninvestigated. Since preparation of homogeneous and crystallizable protein species is the prerequisite for structural studies by crystallography, we reported the successful expression, purification, and crystallization of P. aeruginosa ICMP in this study. The protein was over-expressed in Escherichia coli as a GST-fusion protein, cleaved to remove the fusion tag, and then purified to homogeneity. Diffractable crystals were obtained using the sitting-drop vapour-diffusion method. The crystals diffracted to 2.5 A resolution and belonged to space group P212121, with unit-cell parameters a = 54.47, b = 158.98, c = 162.84 A, α = β = γ = 90°. Preliminary analysis of the diffraction data revealed high-quality crystallographic statistics with a Matthews coefficient of about 2.61 A3.Da-1 and a solvent content of about 52.58%, indicating the presence of three ICMP molecules in the asymmetric unit. The current work therefore paved the way for future studies aiming to delineate the characteristics of ICMP at the atomic level.
{"title":"Cloning, Expression, Purification, and Crystallization of P. aeruginosa ICMP","authors":"Ruliang Pi, J. Gu, G. Lu","doi":"10.4236/AJMB.2018.84017","DOIUrl":"https://doi.org/10.4236/AJMB.2018.84017","url":null,"abstract":"Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic human pathogen that can lead to severe diseases in immunocompromised patients. The insulin-cleaving membrane protease (ICMP) of P. aeruginosa plays a vital role in the pathogenesis of the bacterium and is therefore characterized as an important bacterial virulence factor. In addition, ICMP also serves as a founding member of the M75 peptidase family and represents a prototype of the imelysin/imelysin-like proteins. Despite of its functional importance in the pathogenesis of P. aeruginosa and of a root position as the prototypic imelysin/imelysin-like member, the structural features of the protein remain uninvestigated. Since preparation of homogeneous and crystallizable protein species is the prerequisite for structural studies by crystallography, we reported the successful expression, purification, and crystallization of P. aeruginosa ICMP in this study. The protein was over-expressed in Escherichia coli as a GST-fusion protein, cleaved to remove the fusion tag, and then purified to homogeneity. Diffractable crystals were obtained using the sitting-drop vapour-diffusion method. The crystals diffracted to 2.5 A resolution and belonged to space group P212121, with unit-cell parameters a = 54.47, b = 158.98, c = 162.84 A, α = β = γ = 90°. Preliminary analysis of the diffraction data revealed high-quality crystallographic statistics with a Matthews coefficient of about 2.61 A3.Da-1 and a solvent content of about 52.58%, indicating the presence of three ICMP molecules in the asymmetric unit. The current work therefore paved the way for future studies aiming to delineate the characteristics of ICMP at the atomic level.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":"08 1","pages":"195-204"},"PeriodicalIF":0.0,"publicationDate":"2018-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41849643","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Introduction: Non-Syndromic Clefts Lip-Palates (NSCLP/CP) are most common congenital malformation in the world, with very important psychic and social impact. Formation of NSCLP/CP arises from the interaction of environmental and genetic factors. This paper provides a review of recent progress in defining the genetic causes of NSCLP. Methods: A literature review was conducted on the Medline data by searching for the following keywords: genes, non-syndromic cleft lip-palate, and genetics of clefts lip-palates, until January 2018. Results: Various genes are identified in different population and country, with the study using case parent’s trio. The aim of this study contributes to review relative gene which has been identify in non-syndromic cleft lip and palate, and to help to have a better understanding of the inheritance pattern of this pathology and the prevention of genetic disease. Conclusion: Although three major genes have been confirmed, the genetic research is necessary to provide an understanding of the pathophysiology of the clefts lip-palates.
{"title":"Updating Genetics Polymorphisms of Non-Syndromic Clefts Lip-Palates","authors":"A. Rafik, S. Nadifi","doi":"10.4236/AJMB.2018.83015","DOIUrl":"https://doi.org/10.4236/AJMB.2018.83015","url":null,"abstract":"Introduction: Non-Syndromic Clefts Lip-Palates (NSCLP/CP) are most common congenital malformation in the world, with very important psychic and social impact. Formation of NSCLP/CP arises from the interaction of environmental and genetic factors. This paper provides a review of recent progress in defining the genetic causes of NSCLP. Methods: A literature review was conducted on the Medline data by searching for the following keywords: genes, non-syndromic cleft lip-palate, and genetics of clefts lip-palates, until January 2018. Results: Various genes are identified in different population and country, with the study using case parent’s trio. The aim of this study contributes to review relative gene which has been identify in non-syndromic cleft lip and palate, and to help to have a better understanding of the inheritance pattern of this pathology and the prevention of genetic disease. Conclusion: Although three major genes have been confirmed, the genetic research is necessary to provide an understanding of the pathophysiology of the clefts lip-palates.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":"08 1","pages":"178-185"},"PeriodicalIF":0.0,"publicationDate":"2018-07-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44156126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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