Isolation and identification of a new intracellular antimicrobial peptide produced by Paenibacillus alvei AN5

A wild-type, Gram-positive, rod-shaped, endospore-forming and motile bacteria has been isolated from palm oil mill sludge in Malaysia. Molecular identification using 16S rRNA gene sequence analysis indicated that the bacteria belonged to genus Paenibacillus. With 97 % similarity to P. alvei (AUG6), the isolate was designated as P. alvei AN5. An antimicrobial compound was extracted from P. alvei AN5-pelleted cells using 95 % methanol and was then lyophilized. Precipitates were re-suspended in phosphate buffered saline (PBS), producing an antimicrobial crude extract (ACE). The ACE showed antimicrobial activity against Salmonella enteritidis ATCC 13076, Escherichia coli ATCC 29522, Bacillus cereus ATCC 14579 and Lactobacillus plantarum ATCC 8014. By using SP-Sepharose cation exchange chromatography, Sephadex G-25 gel filtration and Tricine SDS-PAGE, the ACE was purified, which produced a ~2-kDa active band. SDS-PAGE and infrared (IR) spectroscopy indicated the proteinaceous nature of the antimicrobial compound in the ACE, and liquid chromatography electrospray ionization mass spectroscopy and de novo sequencing using an automatic, Q-TOF premier system detected a peptide with the amino acid sequence F–C–K–S–L–P–L–P–L–S–V–K (1,330.7789 Da). This novel peptide was designated as AN5-2. The antimicrobial peptide exhibited stability from pH 3 to 12 and maintained its activity after being heated to 90 °C. It also remained active after incubation with denaturants (urea, SDS and EDTA).     

Epidemiological and genetic data supporting the transmission of Ancylostoma ceylanicum among human and domestic animals

Background

Currently, information on species-specific hookworm infection is unavailable in Malaysia and is restricted worldwide due to limited application of molecular diagnostic tools. Given the importance of accurate identification of hookworms, this study was conducted as part of an ongoing molecular epidemiological investigation aimed at providing the first documented data on species-specific hookworm infection, associated risk factors and the role of domestic animals as reservoirs for hookworm infections in endemic communities of Malaysia.

Methods/Findings

A total of 634 human and 105 domestic canine and feline fecal samples were randomly collected. The overall prevalence of hookworm in humans and animals determined via microscopy was 9.1% (95% CI = 7.0–11.7%) and 61.9% (95% CI = 51.2–71.2%), respectively. Multivariate analysis indicated that participants without the provision of proper latrine systems (OR = 3.5; 95% CI = 1.53–8.00; p = 0.003), walking barefooted (OR = 5.6; 95% CI = 2.91–10.73; p<0.001) and in close contact with pets or livestock (OR = 2.9; 95% CI = 1.19–7.15; p = 0.009) were more likely to be infected with hookworms. Molecular analysis revealed that while most hookworm-positive individuals were infected with Necator americanus, Ancylostoma ceylanicum constituted 12.8% of single infections and 10.6% mixed infections with N. americanus. As for cats and dogs, 52.0% were positive for A. ceylanicum, 46.0% for Ancylostoma caninum and 2.0% for Ancylostoma braziliense and all were single infections.

Conclusion

This present study provided evidence based on the combination of epidemiological, conventional diagnostic and molecular tools that A. ceylanicum infection is common and that its transmission dynamic in endemic areas in Malaysia is heightened by the close contact of human and domestic animal (i.e., dogs and cats) populations.     

cDNA sequence and molecular modeling of a nerve growth factor from Bothrops jararacussu venomous gland

The complete nucleotide sequence of a nerve growth factor precursor from Bothrops jararacussu snake (Bj-NGF) was determined by DNA sequencing of a clone from cDNA library prepared from the poly(A) + RNA of the venom gland of B. jararacussu. cDNA encoding Bj-NGF precursor contained 723 bp in length, which encoded a prepro-NGF molecule with 241 amino acid residues. The mature Bj-NGF molecule was composed of 118 amino acid residues with theoretical pI and molecular weight of 8.31 and 13,537, respectively. Its amino acid sequence showed 97%, 96%, 93%, 86%, 78%, 74%, 76%, 76% and 55% sequential similarities with NGFs from Crotalus durissus terrificus, Agkistrodon halys pallas, Daboia (Vipera) russelli russelli, Bungarus multicinctus, Naja sp., mouse, human, bovine and cat, respectively. Phylogenetic analyses based on the amino acid sequences of 15 NGFs separate the Elapidae family (Naja and Bungarus) from those Crotalidae snakes (Bothrops, Crotalus and Agkistrodon). The three-dimensional structure of mature Bj-NGF was modeled based on the crystal structure of the human NGF. The model reveals that the core of NGF, formed by a pair of beta-sheets, is highly conserved and the major mutations are both at the three beta-hairpin loops and at the reverse turn.     

<Emphasis Type=Italic>Ex situ</Emphasis> conservation programmes in European zoological gardens: Can we afford to lose them?

The role of ex situ activities for the conservation of biodiversity, and of zoos and aquaria in particular, is open to continuing debate. The present note highlights the conservation breeding potential of zoological gardens and aquaria in the European union, but it also recognises the lack of a convincing scientific and legal framework that encourages ex situ activities for ‘exotic’ species. If ex situ programmes are considered essential for global biodiversity conservation, the EU should not limit itself to regulating zoos through the zoo directive, but should actively promote and support their ex situ conservation activities.     

Radial and axial variations of bacteria within the cecum and proximal colon of guinea pigs revealed by PCR-DGGE

We investigated the local variability of bacterial species within the lumen of the cecum and proximal colon of guinea pigs by using PCR-denaturing gradient gel electrophoresis (DGGE). The DGGE banding profiles revealed radial and axial variation of the bacteria. This variation of bacteria within the large intestinal lumen might be of physiological importance for the degradation of dietary fiber and interaction between bacteria and the mucosal immune system.     

Protein kinase C isoenzymes in mouse Harderian gland. Differential expression of the α- and ε-isoforms during pregnancy

Protein kinase C (PKC) is known to be involved in the regulation of exocytosis in different cell lines and tissues. Experiments were designed to determine whether the Harderian gland of CD-1 mouse produces PKC isoenzymes and whether the expression of the isoforms changes during pregnancy. The presence of the isoenzymes was assessed by immunoblotting experiments using extract of total Harderian gland and polyclonal antisera specific for nine different PKC isoforms. Antisera giving a positive staining on Western blots were subsequently used for immunohistochemical investigation using a secondary antibody conjugated to alkaline phosphatase. Immunoblotting experiments revealed that the Harderian gland from female mouse expresses PKC isoforms-,-,- and-. These isoforms were also detected in the Harderian gland from 13-day pregnant mouse; however, striking quantitative changes were seen concerning the - and -isoforms. The 80-kDa native form of PKC- almost doubled in the pregnant mouse in comparison with normal female mouse whereas the amount of 50-kDa catalytic domain did not change. Protein kinase C- appeared as a 92- to 93-kDa form and a 67-kDa form. While the 92- to 93-kDa protein was expressed to a similar extent in both types of mouse, the 67-kDa form was more abundant inthe Harderian gland from normal female mouse. These data were corroborated by immunohistochemical experiments and showing a diffuse and granular staining of the adenomeres. These observations demonstrate for the first time (to our knowledge) that the mouse Harderian gland produces several PKC isoenzymes that could be involved in the regulation of exocytosis and/or other functions. Moreover, the expression of the - and -isoforms could be regulated by sexual hormones, as suggested by the differential abundance of these two proteins in the gland of pregnant mouse compared with normal female mouse.     

Synthesis of two different molecular forms of ribulose-bisphosphate carboxylase/oxygenase in Rhodopseudomonas blastica grown in batch culture

The expression of the two different molecular forms (form I and form II) of ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) in Rhodopseudomonas blastica during growth in batch on pyruvate–malate medium was investigated. During the early exponential phase of growth, only form I enzyme was synthesized. At the mid-exponential phase of growth, both forms were expressed, although form I enzyme was predominant. At the late exponential phase, form I and form II enzymes were synthesized but form II enzyme predominated. It is concluded that form I and form II RuBisCO enzymes in R. blastica are differentially expressed and this may be mediated by the level of CO2 in the growth medium.     

Phytoremediation applications in natural condition and in mesocosm: The uptake of cadmium by Lemna minuta Kunth,a non-native species in Italian watercourses

Metal pollution in water and soil is an environmental and public health issue. Cadmium (Cd) is included in the list of priority hazardous substances in the European Water Framework Directive. Phytoremediation system is a cost-effective, plant-based approach that takes advantage of the ability of plants to concentrate elements and compounds from the environment and to metabolize various molecules in their tissues. We studied the presence and the importance of an invasive species, such as Lemna minuta, in the environment and the effects of Cd pollution on this species. Growth, removal, and tolerance were evaluated for different Cd concentrations and different times of plant exposure. Overall, the results show that L. minuta has a good capacity of growth, metal bioconcentration, and tolerance up to 3 days of exposure at 0.5 and 1.5 mg L^?1 of Cd. In particular, L. minuta was able to accumulate Cd up to 3771 mg kg^?1 on dry mass basis. We can conclude that L. minuta possesses a great capability of Cd absorption and accumulation, thus supporting a potential use of this species in designing a metal bioremediation system in phytoremediation field.     

The Effects of One Amino Acid Substitutions at the C-Terminal Region of Thermostable L2 Lipase by Computational and Experimental Approach

The substitutions of the amino acid at the predetermined critical point at the C-terminal of L2 lipase may increase its thermostability and enzymatic activity, or even otherwise speed up the unfolding of the protein structure. The C-terminal of most proteins is often flexible and disordered. However, some protein functions are directly related to flexibility and play significant role in enzyme reaction. The critical point for mutation of L2 lipase structure was predicted at the position 385 of the L2 sequence, and the best three mutants were determined based on I-Mutant2.0 software. The best three mutants were S385E, S385I and S385V. The effects of the substitution of the amino acids at the critical point were analysed with molecular dynamics simulation by using Yet Another Scientific Artificial Reality Application software. The predicted mutant L2 lipases were found to have lower root mean square deviation value as compared to L2 lipase. It was indicated that all the three mutants had higher compactness in the structure, consequently enhanced the stability. Root mean square fluctuation analysis showed that the flexibility of L2 lipase was reduced by mutations. Purified S385E lipase had an optimum temperature of 80 °C in Tris–HCl pH 8. The highest enzymatic activity of purified S385E lipase was obtained at 80 °C temperature in Tris–HCl pH 8, while for L2 lipase it was at 70 °C in Glycine–NaOH pH 9. The thermal stability of S385V lipase was enhanced as compared to other protein since that the melting point (T _m) value was at 85.96 °C. S385I lipase was more thermostable compared to recombinant L2 lipase and other mutants at temperature 60 °C within 16 h preincubation.     

Effect of propeptide amino acid substitution in γ‐carboxylation,activity and expression of recombinant human coagulation factor IX

The production of recombinant vitamin K dependent (VKD) proteins for therapeutic purposes is an important challenge in the pharmaceutical industry. These proteins are primarily synthesized as precursor molecules and contain pre–propeptide sequences. The propeptide is connected to γ‐carboxylase enzyme through the γ‐carboxylase recognition site for the direct γ‐carboxylation of VKD proteins that has a significant impact on their biological activity. Propeptides have different attitudes toward γ‐carboxylase and certain amino acids in propeptide sequences are responsible for the differences in γ‐carboxylase affinity. By aiming to replace amino acids in hFIX propeptide domain based on the prothrombin propeptide, pMT‐hFIX‐M14 expression cassette, containing cDNA of hFIX with substituted ?14 residues (Asp to Ala) was made. After transfection of Drosophila S2 cells, expression of the active hFIX was analyzed by performing ELISA and coagulation test. A 1.4‐fold increase in the mutant recombinant hFIX expression level was observed in comparison with that of a native recombinant hFIX. The enhanced hFIX activity and specific activity of the hFIXD‐14A (2.2 and 1.6 times, respectively) were further confirmed by comparing coagulation activity levels of substituted and native hFIX. Enrichment for functional, fully γ‐carboxylated hFIX species via barium citrate adsorption demonstrated 2‐fold enhanced recovery in the S2‐expressing hFIXD‐14A relative to that expressed native hFIX. These results show that changing ?14 residues leads to a decrease in the binding affinity to substrate, increase in γ‐carboxylation and activity of recombinant hFIX. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:515–520, 2018