Morphological and Molecular Identification of Spirometra Tapeworms (Cestoda: Diphyllobothriidae) from Carnivorous Mammals in the Serengeti and Selous Ecosystems of Tanzania

Spirometra tapeworms (Cestoda: Diphyllobothriidae) collected from carnivorous mammals in Tanzania were identified by the DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and internal transcribed spacer 1 (ITS1), and by morphological characteristics. A total of 15 adult worms were collected from stool samples and carcasses of Panthera leo, Panthera pardus, and Crocuta crocuta in the Serengeti and Selous ecosystems of Tanzania. Three Spirometra species: S. theileri, S. ranarum and S. erinaceieuropaei were identified based on morphological features. Partial cox1 sequences (400 bp) of 10 specimens were revealed. Eight specimens showed 99.5% similarity with Spirometra theileri ({"type":"entrez-nucleotide","attrs":{"text":"MK955901","term_id":"1796114604","term_text":"MK955901"}}MK955901), 1 specimen showed 99.5% similarity with the Korean S. erinaceieuropaei and 1 specimen had 99.5% similarity with Myanmar S. ranarum. Sequence homology estimates for the ITS1 region of S. theileri were 89.8% with S. erinaceieuropaei, 82.5% with S. decipiens, and 78.3% with S. ranarum; and 94.4% homology was observed between S. decipiens and S. ranarum. Phylogenetic analyses were performed with 4 species of Spirometra and 2 species of Dibothriocephalus (=Diphyllobothrium). By both ML and BI methods, cox1 and ITS1 gave well supported, congruent trees topology of S. erinaceieuropaei and S. theileri with S. decipiens and S. ranarum forming a clade. The Dibothriocephalus species were sisters of each other and collectively forming successive outgroups. Our findings confirmed that 3 Spirometra species (S. theileri, S. ranarum, and S. erinaceieuropaei) are distributed in the Serengeti and Selous ecosystems of Tanzania.     

Selective allele loss and interference between cauliflower mosaic virus DNAs

Summary Some cauliflower mosaic virus (CaMV) alleles are selectively lost during growth of the virus in mixedly infected turnip plants. Viral DNA from plants co-inoculated with DNA of the cabbage S isolate and infectious cabbage S DNA with an extra EcoRI restriciion site lacked the extra site. The EcoRI allele was also lost in most plants co-inoculated with a non-infectious mutant of cabbage S DNA while little selective allele loss was observed with two other non-infectious mutant DNAs. Plants co-inoculated with DNAs of closely-related isolates (CM4-184 and W) contained both parental viral DNAs and some DNAs with characteristics of both parents. Interference, scored as a reduced frequency of infection or a delay in symptom appearance relative to plants inoculated with wild-type DNA, occurred when plants were inoculated with wild-type and mutant DNAs covalently attached to one another in partial dimer plasmid DNAs. Similarities in the conditions leading to selective allele loss and those leading to interference suggest that both may have been due to active gene conversion between CaMV DNA molecules.     

Disruption of phospholipid asymmetry in erythrocyte vesicles deficient in spectrin

The transbilayer distribution of phospholipids in right-side-out and inside-out vesicles derived from human erythrocytes was studied by phospholipase A2 digestion assays and by staining with the fluorescent dye merocyanine 540. In both types of vesicles, the normal asymmetric distribution of phospholipids characteristic of intact cells was disrupted. Because both types of vesicles are deficient in spectrin, the major protein of the cytoskeletal network which normally underlies the membrane, these results support the contention that spectrin is involved in the maintenance of phospholipid asymmetry.     

Sequence analysis of the 5′-flanking region of the gene encoding human N-acetylglucosaminyltransferase III

We have isolated and characterized the immediate (1651 bp) 5′-flanking region of the gene (GnT-III) encoding N-acetylglucosaminyltransferase III (GnT-III) from a human placental genomic library. Analysis of promoter elements shows a similarity to the 5′-flanking region of murine 1,4-galactosyltransferase. The sequence lacks obvious TATA elements and CCAAT boxes; however, putative regulatory sites, including 2 potential cAMP-response regulatory elements (CRE), 11 insulin-response element consensus sequences (IRE), 7 potential AP-2-binding sites, 2 SP1 consensus sequences (GC boxes) and 2 sequences similar to the half-palindromic glucocorticoid-responsive element (GRE), are present.     

Photoprotective effect of kinetin on pigment content and photochemical activities of wheat chloroplasts agingin vitro

The effects of kinetin (Kn) on pigment content and electron transport activities (ETA) in wheat leavesin vivo and chloroplastsin vitro aging in light was investigated. Excised wheat leaves were infiltrated with Kn for 3 h under irradiation. The treatment increased zeaxanthin (Zx) content by 40% and also increased chlorophyll (Chia, Chib) and major carotenoid (Car) contents in the leaves (per fresh mass unit). Chloroplasts isolated from Kn treated leaves, when incubated in light for 4 h showed relatively lower pigment loss and slower loss of ETA compared to the chloroplasts of untreated leaves. These observations suggest photoprotective action of Kn. The photoprotection was more prominent when Kn was applied directly to the irradiated chloroplastsin vitro. Moreover, chloroplasts agingin vitro under irradiation without Kn treatment lost pigments and ETA. Within 3 h of irradiation, both whole chain (H₂O to methylviologen) electron transport as well as photosystem (PS) 2 activity were completely lost. However, in the chloroplasts treated with Kn, the loss of pigments was slow and even after 4 h of irradiation the chloroplasts retained 15 % of PS 2 and 9 % of whole chain ETA. In the untreated chloroplasts, the loss of Zx after 4 h of irradiation was 49 % whereas in Kn treated samples its level was 1.3 times higher than that of control. Since a higher level of Zx was maintained in Kn treated chloroplasts, photoprotective action of Kn is possibly mediated through Zx. One of us (NKC) thanks Sambalpur University for study leave and Department of Biological Sciences, Mankato State University, Mankato for labortory facilities.     

Rapid screening of an SH2-containing gene using PCR amplification method

Summary To isolate a novel gene that contains an SH2 domain, we devised a rapid and nonradioactive cDNA library screening method using polymerase chain reaction (PCR). For PCR amplification, we designed degenerate oligonucleotide primers from the multialigned DNA sequences of SH2 domains. This method offers an inexpensive and efficient approach for the isolation of clones of interest from cDNA libraries.     

植物乳杆菌谷氨酸脱羧酶催化pH范围的理性改造及高效转化生产g-氨基丁酸

γ-氨基丁酸可由谷氨酸脱羧酶(glutamate decarboxylase, GAD)催化谷氨酸一步合成,反应体系成分简单、环境友好。然而,绝大多数GAD酶催化pH偏酸性且反应范围狭小,需要加入无机盐维持最适催化环境,增加了生产附加成分。此外,随着产物γ-氨基丁酸的生成,溶液pH会逐渐上升,不利于GAD酶的持续转化。本研究首先从实验室保藏的一株高产γ-氨基丁酸的植物乳杆菌(Lactobacillus plantarum)中克隆得到谷氨酸脱羧酶LpGAD,基于酶蛋白表面电荷修饰,选择9个位点进行定点突变及组合突变,酶学性质表征结果显示三突变体LpGAD^{S24R/D88R/Y309K}在催化pH区间内酶活力整体提高,尤其拓宽了在偏中性pH 6.0下的酶活,为野生酶的1.68倍。接下来,通过分子动力学模拟解析了酶活提高的机理。此外,将Lpgad与Lpgad^{S24R/D88R/Y309K}突变基因分别在谷氨酸棒杆菌(Corynebacterium glutamicum) E01中过表达,通过优化确定了摇瓶最适转化条件为反应温度40 ℃,菌体量OD₆₀₀=20,底物L-谷氨酸100.0 g/L,5-磷酸吡哆醛添加量为100 μmol/L。5 L发酵罐中,不调节pH,通过分批投料底物L-谷氨酸,γ-氨基丁酸产量高达402.8 g/L,较对照菌株提高了1.63倍。本研究成功拓宽了LpGAD的pH催化范围及酶活,提高了γ氨基丁酸的转化效率,为实现其规模化工业生产奠定了基础。