决明胰蛋白酶抑制剂1活性相关残基的定点突变与抑制活性分析

决明胰蛋白酶抑制剂1（CoTI1）属于Kunitz胰蛋白酶抑制剂家族成员,通过序列比对预测Arg86、Leu84和Thr88等3个氨基酸残基可能是CoTI1发挥抑制作用的关键残基。通过定点突变的方法将Arg86、Leu84与Thr88残基分别突变为Asp残基,并考察各突变体对胰蛋白酶及棉铃虫等鳞翅目害虫消化酶的抑制作用。与CoTI1相比,CoTI1^R86D、CoTI1^T88D与CoTI1^L84D突变体对胰蛋白酶的抑制活性分别下降了93%、64%与59%;对棉铃虫、甜菜夜蛾、斜纹夜蛾等3种鳞翅目害虫消化酶的平均抑制活性分别下降了88.7%、57%与60.7%。以上结果表明Arg86、Leu84与Thr88是CoTI1发挥抑制作用的关键残基,这为CoTI1的抑制分子机制及抗虫研究提供了重要的理论依据。     

Molecular cloning and characterization of <Emphasis Type="Italic">α</Emphasis>-amylase/subtilisin inhibitor from rhizome of <Emphasis Type="Italic">Ligusticum chuanxiong</Emphasis>

Objectives

To clone and characterize a novel bi-functional α-amylase/subtilisin inhibitor (LASI) from the rhizome of Ligusticum chuanxiong, a traditional Chinese medicine.

Results

The LASI showed strong homology with members of the Kunitz trypsin inhibitor family. Its putative amino acid sequence has a 40 % identity with that of the α-amylase/subtilisin inhibitor from rice. LASI gene without signal peptide was expressed in E. coli Rosetta. After purification, the recombinant LASI protein was inhibitory against not only α-amylase from porcine pancreas, Helicoverpa armigera, Spodoptera litura and Plutella xylostella, but also subtilisin A, but not against trypsin or chymotrypsin. In addition, the expression level of LASI in rhizome was higher than that in leaf and LASI expression was enhanced by salt, chilling and drought treatment.

Conclusions

This is the first member of the Kunitz-protease inhibitor family identified in traditional Chinese medicine and it might be involved in the plant defense responses against lepidopterous pests, microorganisms and abiotic stresses.

     

Biodegradation optimization and metabolite elucidation of Reactive Red 120 by four different <Emphasis Type="Italic">Aspergillus</Emphasis> species isolated from soil contaminated with industrial effluent

Azo dyes are recalcitrant owing to their xenobiotic nature and exhibit high resistance to degradation processes. In the present study, different Aspergillus species (A. flavus, A. fumigatus, A. niger, and A. terreus) isolated from soil samples contaminated with industrial effluent, collected from Jeddah, Saudi Arabia, were analyzed for azo dye, Reactive Red 120 (RR120) biodegradation. The physicochemical parameters such as carbon (sucrose) and nitrogen (ammonium sulfate) sources, pH, and temperature affecting the biodegradation of RR120 were optimized using central composite design–response surface methodology (CCD-RSM). The maximum RR120 degradation was found to be 84% (predicted) at the optimum level of sucrose (11.73 g/L), ammonium sulfate (1.26 g/L), pH (5.71), and temperature (28.26 °C). Further, the validation results confirmed that the predicted values are in good agreement with the experimental results for RR120 degradation by A. flavus (86%), A. fumigatus (84%), A. niger (85%), and A. terreus (86%). The metabolic product of RR120 after biodegradation by different Aspergillus species was identified as sodium 2-aminobenzenesulfonate. The present study suggests that Aspergillus species are good candidates for azo dye-loaded effluent treatment.     

Inactivation of proteolytic enzymes by cestodes

Inhibitor activity of cestodes from intestines of different hosts (sea birds, salt-water fish, and freshwater fish) was investigated. Alcataenia larina, Arctotaenia tetrabothrioides, Tetrabothrius erostris, T. minor, Wardium cirrosa, Bothriocephalus scorpii, Eubothrium rugosum, and Triaenophorus nodulosus were able to inhibit the activity of the commercial trypsin and activity of proteinase homogenates of the intestinal mucosa of the hosts. It was suggested that the inhibitor produced by the cestodes is specific for trypsin and protects them from the digestive enzymes of the host.     

<Emphasis Type="Italic">CDKN2A</Emphasis> and <Emphasis Type="Italic">MC1R</Emphasis> variants found in Cypriot patients diagnosed with cutaneous melanoma

The prevalence of genetic variants associated to cutaneous melanoma (CM) has never been determined within Cypriot melanomas. This study evaluates the frequency of variants in cyclin-dependent kinase inhibitor 2A (CDKN2A) and melanocortin-1 receptor (MC1R) in 32 patients diagnosed with CM. Other characteristics and risk factors were also assessed. CDKN2A p.Ala148Thr was detected in three of 32 patients, while the control group revealed no variations within CDKN2A. MC1R screening in 32 patients revealed the following variations: p.Val60Leu in 11 patients, p.Arg142His in four patients, p.Thr314Thr in one patient, p.Arg160Trp in one patient, p.Val92Met/p.Thr314Thr in one patient and p.Val92Met/p.Arg142His/p.Thr314Thr in one patient. The control group revealed only p.Val60Leu (in 10 of 45 individuals), which is frequently found in general populations. Two unrelated patients carried CDKN2A p.Ala148Thr in combination with MC1R p.Arg142His, suggesting digenic inheritance that may provide evidence of different gene variants acting synergistically to contribute for CM development. This study confirms the presence of CDKN2A and MC1R variants among Cypriot melanomas and supports existing evidence of a role for these variants in susceptibility to melanoma.     

Reliability of the search for 19 common mutations in the <Emphasis Type="Italic">CFTR</Emphasis> gene in Russian cystic fibrosis patients and the calculated frequency of the disease in Russian Federation

A study of Russian cystic fibrosis (CF) patient DNA was conducted to assess the incidence frequency of 19 mutations, namely CFTRdele2,3(21kb), F508del, I507del, 1677delTA, 2143delT, 2184insA, 394delTT, 3821delT, L138ins, 604insA, 3944delGT, G542X, W1282X, N1303K, R334W, and 3849 + 10kbC > T, S1196X, 621 + 1g > t, and E92K of the CFTR gene. We also sought to determine the estimated CF frequency in Russian Federation. In addition, we determined the total information content of the approach for 19 common mutations registration in the CFTR gene, 84.6%, and the allelic frequencies of the examined mutations: three mutations were observed with a frequency exceeding 5% (F508del, 53.98%, E92K, 6.47%, CFTRdele2,3(21kb), 5.35%); other mutations were observed with frequencies ranging from 0.13 to 3.0%. The CF population carrier frequency was 1 in 38 subjects, while the predicted CF frequency was 1 in 5776 newborns.     

Potential Anticoagulant Activity of Trypsin Inhibitor Purified from an Isolated Marine Bacterium <Emphasis Type="Italic">Oceanimonas</Emphasis> Sp. BPMS22 and its Kinetics

Protease inhibitors control major biological protease activities to maintain physiological homeostasis. Marine bacteria isolated from oligotrophic conditions could be taxonomically distinct, metabolically unique, and offers a wide variety of biochemicals. In the present investigation, marine sediments were screened for the potential bacteria that can produce trypsin inhibitors. A moderate halotolerant novel marine bacterial strain of Oceanimonas sp. BPMS22 was isolated, identified, and characterized. The effect of various process parameters like salt concentration, temperature, and pH was studied on the growth of the bacteria and production of trypsin inhibitor. Further, the trypsin inhibitor was purified to near homogeneity using anion exchange, size exclusion, and affinity chromatography. The purified trypsin inhibitor was found to competitively inhibit trypsin activity with an inhibition coefficient, K_i, of 3.44?±?0.13 μM and second-order association rate constant, k_ass, of 1.08?×?10³ M^?1 S^?1. The proteinaceous trypsin inhibitor had a molecular weight of approximately 30 kDa. The purified trypsin inhibitor showed anticoagulant activity on the human blood samples.     

Cloning and expression analysis of zygote arrest 1 (<Emphasis Type="Italic">Zar1</Emphasis>) in New Zealand white rabbits

Zygote arrest 1 (Zar1) is an oocyte-specific maternal-effect gene. Previous studies indicate that Zar1 plays important role in early embryo development, but little is known about its function in rabbit. The objectives of this study were to clone the New Zealand white rabbit Zar1 gene and to investigate its expression in various organs in groups of animals with different reproductive traits. We obtained a 709-bp Zar1 cDNA fragment consisting of an 8-bp exon 1, 161-bp exon 2, 75-bp exon 3, 271-bp exon 4 and 194-bp 3 ^' sequences. The rabbit Zar1 nucleotide sequence showed per cent identities of 91, 88, 88, 87, 86, 87, 76 and 82% with Zar1 orthologues in human, cattle, sheep, pig, mouse, rat, zebrafish and Xenopus laevis, respectively, indicating a high homology with other species and evolutionary conservation. Quantitative real-time polymerase chain reaction analyses revealed nonoocyte-specific Zar1 expression, with expression in spleen, lung, ovary, uterus, heart, liver and kidney. The expression level was highest in the lung. This study may lay the theoretical foundation for the study of ZAR1’s biological function.     

The homologous identification of the stem rust resistance genes <Emphasis Type="Italic">Rpg5</Emphasis>, <Emphasis Type="Italic">Adf3</Emphasis> and <Emphasis Type="Italic">RGA1</Emphasis> in the relatives of barley

The barley genes Rpg5, RGA1 and Adf3, which provide a strong resistance to many pathotypes of stem rust, were cloned a few years ago, but it was still unclear whether their homologues were represented in wheat and in related species. The paper describes the results of a bioinformatic research to determine the homologues of Rpg5, RGA1 and Adf3 in the genomes of Triticum aestivum and several wild grasses, which breeders usually use as sources of stem rust resistance, and which are available in the genome databases. It was found that the Th. elongatum sequence Q9FEC6 and T. aestivum sequence Q43655 were the highly identical homologues of the Adf3 sequence. T. urartu M8A999 sequence and T. aestivum W5FCU1 sequence were found to be the closest homologues of Rpg5 complete protein sequence, but the identity of their kinase domains was not as clear as that of the other domains. The separate Rpg5 kinase part analysis did not provide the strong evidences that its orthologs were present in our corn species. T. urartu M7ZZX9 sequence and T. aestivum W5FFP0 and W5FI33 sequences were shown to be the homologues of RGA1. The analysis of the predicted active sites allowed finding out the difference between sequences of Rpg5, RGA1, Adf3 protein and their homologues.     

Toxicity,membrane binding and uptake of the <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> agglutinin (SSA) in different insect cell lines

The fungal lectin purified from Sclerotinia sclerotiorum, further referred to as Sclerotinia sclerotiorum agglutinin or SSA, possesses insecticidal activity against important pest insects such as pea aphids (Acyrthosiphon pisum). This paper aims at a better understanding of its activity at cellular level. Therefore, different insect cell lines were treated with SSA. These cell lines were derived from different tissues and represent the three major orders of insects important in agriculture: CF-203 (midgut Choristoneura fumiferana, Lepidoptera), GUTAW1 (midgut, Helicoverpa zea, Lepidoptera), High5 cells (ovary, Trichoplusia ni, Lepidoptera), Sf9 (ovary cells from Spodoptera frugiperda, Lepidoptera), S2 (hemocyte, Drosophila melanogaster, Diptera), and TcA (whole body, Tribolium castaneum, Coleoptera). Although the sensitivity to SSA differs between the cell lines, SSA clearly showed toxicity in all six cell lines with median effect concentrations (EC₅₀) ranging between 9 and 42 μg/ml. An in-depth analysis of the mechanism of uptake in the cells revealed superior amounts of FITC-SSA at the membrane of CF-203 cells compared to Sf9 cells, while a similar small amount of SSA was internalized in both cell lines. Pre-incubation with the clathrin-mediated endocytosis inhibitor phenylarsine oxide inhibited the internalization of SSA into the CF-203 and Sf9 cells with a respective reduction of 6- and 1.7-fold. The data are discussed in relation to the importance of cellular uptake mechanism for SSA binding and cytotoxicity.     

Culture Optimization Strategy for 1-Deoxynojirimycin-producing <Emphasis Type="Italic">Bacillus methylotrophicus</Emphasis> K26 Isolated from Korean Fermented Soybean Paste, <Emphasis Type="Italic">Doenjang</Emphasis>

1-Deoxynojirimycin (1-DNJ) is an α-glucosidase inhibitor that is used for the treatment of type 2 diabetes. In this study, we isolated Bacillus methylotrophicus K26 with α-glucosidase inhibition (AGI) activity from Korean fermented soybean paste (Doenjang) and confirmed that the genome harbored the DNJ biosynthesis genes including gabT1, yktc1, and gutB1 by PCR screening, while 1-DNJ production was confirmed by ultra-performance liquid chromatography–quadrupole time-of-flight–mass spectrometry. To increase 1-DNJ production by B. methylotrophicus K26, culture conditions were optimized with one-factor-ata- time (OFAT) and response surface methodology (RSM) approaches. Screen of 11 carbon and 9 nitrogen sources by the OFAT method identified sucrose and yeast extract as optimal culture components. Sucrose concentration (X₁), yeast extract concentration (X₂), and culture temperature (X₃) were selected as independent variables for central composite design. The coefficient of determination (R²) for the model was 0.927, and the probability value of the regression model was highly significant. RSM predicted the optimal conditions for 1-DNJ production by B. methylotrophicus K26 as sucrose and yeast extract concentrations of 4.61% and 7.03%, respectively, at a temperature of 34°C. Under these conditions, AGI activity was experimentally measured as 89.3%, which was close to the predicted value of 91.9%.     

Connection between proliferation rate and temozolomide sensitivity of primary glioblastoma cell culture and expression of <Emphasis Type="Italic">YB-1</Emphasis> and <Emphasis Type="Italic">LRP/MVP</Emphasis>

Glioblastomas (GBL) are the most common and aggressive brain tumors. They are distinguished by high resistance to radiation and chemotherapy. To find novel approaches for GBL classification, we obtained 16 primary GBL cell cultures and tested them with real-time PCR for mRNA expression of several genes (YB-1, MGMT, MELK, MVP, MDR1, BCRP) involved in controlling cell proliferation and drug resistance. The primary GBL cultures differed in terms of proliferation rate, wherein a group of GBL cell cultures with low proliferation rate demonstrated higher resistance to temozolomide. We found that GBL primary cell cultures characterized by high proliferation rate and lower resistance to temozolomide expressed higher mRNA level of the YB-1 and MDR1 genes, whereas upregulated expression of MVP/LRP mRNA was a marker in the group of GBL with low proliferation rate and high resistance. A moderate correlation between expression of YB-1 and MELK as well as YB-1 and MDR1 was found. In the case of YB-1 and MGMT expression, no correlation was found. A significant negative correlation was revealed between mRNA expression of MVP/LRP and MELK, MDR1, and BCRP. No correlation in expression of YB-1 and MVP/LRP genes was observed. It seems that mRNA expression of YB-1 and MVP/LRP may serve as a marker for GBL cell cultures belonging to distinct groups, each of which is characterized by a unique pattern of gene activity.     

Case report of whole genome sequencing in the XY female: identification of a novel <Emphasis Type="Italic">SRY</Emphasis> mutation and revision of a misdiagnosis of androgen insensitivity syndrome

Background

The 46,XY female is characterised by a male karyotype and female phenotype arising due to any interruption in the sexual development pathways in utero. The cause is usually genetic and various genes are implicated.

Case presentation

Herein we describe a 46,XY woman who was first diagnosed with androgen insensitivity syndrome (testicular feminisation) at 18 years; however, this was later questioned due to the presence of intact Müllerian structures. The clinical phenotype suggested several susceptibility genes including SRY, DHH, NR5A1, NR0B1, AR, AMH, and AMHR2. To study candidate genes simultaneously, we performed whole genome sequencing. This revealed a novel and likely pathogenic missense variant (p.Arg130Pro, c.389G>C) in SRY, one of the major genes implicated in complete gonadal dysgenesis, hence securing this condition over androgen insensitivity syndrome as the cause of the patient’s disorder of sexual development.

Conclusion

This case highlights the emerging clinical utility of whole genome sequencing as a tool in differentiating disorders of sexual development.

     

Molecular phylogeny of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the yeast genus <Emphasis Type="Italic">Saccharomyces</Emphasis>

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum), 94–98.3% for S. kudriavzevii, and 96.8–100% for S. paradoxus/S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking.