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Fulltext Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma antarctica PI12

Ramli AN, Mahadi NM, Rabu A, Murad AM, Bakar FD, Illias RM

Microb Cell Fact, 2011;10:94.
PMID: 22050784 DOI: 10.1186/1475-2859-10-94

Cold-adapted enzymes are proteins produced by psychrophilic organisms that display a high catalytic efficiency at extremely low temperatures. Chitin consists of the insoluble homopolysaccharide β-(1, 4)-linked N-acetylglucosamine, which is the second most abundant biopolymer found in nature. Chitinases (EC 3.2.1.14) play an important role in chitin recycling in nature. Biodegradation of chitin by the action of cold-adapted chitinases offers significant advantages in industrial applications such as the treatment of chitin-rich waste at low temperatures, the biocontrol of phytopathogens in cold environments and the biocontrol of microbial spoilage of refrigerated food.

Matched MeSH terms: Fungal Proteins/metabolism; Recombinant Proteins/metabolism
Glycosylation is not necessary for recognition of the fusion glycoprotein domain of the human respiratory syncytial virus by a polyclonal antibody

Lim SH, Jahanshiri F, Jalilian FA, Rahim RA, Sekawi Z, Yusoff K

Acta Virol., 2010;54(3):181-7.
PMID: 20822310

Human respiratory syncytial virus (HRSV) is a leading pathogen causing lower respiratory tract infections in infants and young children worldwide. In line with the development of an effective vaccine against HRSV, a domain of the fusion (F) glycoprotein of HRSV was produced and its immunogenicity and antigenic properties, namely the effect of deficient glycosylation was examined. A His-tagged recombinant F (rF) protein was expressed in Escherichia coli, solubilized with 8 mol/l urea, purified by the Ni-NTA affinity chromatography and used for the raising of a polyclonal antibody in rabbits. The non-glycosylated rF protein proved to be a strong immunogen that induced a polyclonal antibody that was able to recognize also the glycosylated F1 subunit of native HRSV. The other way around, a polyclonal antibody prepared against the native HRSV was able to react with the rF protein. These results indicated that glycosylation was not necessary for the F domain aa 212-574 in order to be recognized by the specific polyclonal antibody.

Matched MeSH terms: Recombinant Fusion Proteins/metabolism; Viral Fusion Proteins/metabolism
Feeding Artemia franciscana (Kellogg) larvae with bacterial heat shock protein, protects from Vibrio campbellii infection

Sung YY, Ashame MF, Chen S, Macrae TH, Sorgeloos P, Bossier P

J Fish Dis, 2009 Aug;32(8):675-85.
PMID: 19515074 DOI: 10.1111/j.1365-2761.2009.01046.x

Among their numerous physiological effects, heat shock proteins (Hsps) are potent immunomodulators, a characteristic reflecting their potential as therapeutic agents and which led to their application in combating infection. As an example, the up-regulation of endogenous Hsp70 in the branchiopod crustacean Artemia franciscana (Kellogg) is concurrent with shielding against bacterial infection. To better understand this protective mechanism, gnotobiotic Artemia were fed with Escherichia coli treated to over-produce different prokaryotic Hsps. This was shown to increase larval resistance to experimental Vibrio campbellii exposure. Immunoprobing of Western blots showed that the enhanced resistance to V. campbellii correlated with DnaK production in E coli. A definitive role for DnaK was then demonstrated by feeding Artemia larvae with transformed bacteria over-producing only this protein, although other Hsps such as DnaJ and grpE also provided tolerance against Vibrio infection. Feeding of bacteria synthesizing selected Hsps is therefore suggested as an alternative to antibiotic use as a means of enhancing resistance of Artemia larvae to bacterial infection, which may have potential applications in aquaculture.

Matched MeSH terms: HSP70 Heat-Shock Proteins/metabolism; Escherichia coli Proteins/metabolism
Cloning and expression of kiss2 in the zebrafish and medaka

Kitahashi T, Ogawa S, Parhar IS

Endocrinology, 2009 Feb;150(2):821-31.
PMID: 18927220 DOI: 10.1210/en.2008-0940

Newly discovered kisspeptin (metastin), encoded by the Kiss1/KISS1 gene, is considered as a major gatekeeper of puberty through the regulation of GnRH. In the present study, we cloned a novel kisspeptin gene (kiss2) in the zebrafish Danio rerio and the medaka Oryzias latipes, which encodes a sequence of 125 and 115 amino acids, respectively, and its core sequence (FNLNPFGLRF, F-F form) is different from the previously characterized kiss1 (YNLNSFGLRY, Y-Y form). Our in silico data mining shows kiss1 and kiss2 are highly conserved across nonmammalian vertebrate species, and we have identified two putative kisspeptins in the platypus and three forms in Xenopus. In the brain of zebrafish and medaka, in situ hybridization and laser capture microdissection coupled with real-time PCR showed kiss1 mRNA expression in the ventromedial habenula and the periventricular hypothalamic nucleus. The kiss2 mRNA expression was observed in the posterior tuberal nucleus and the periventricular hypothalamic nucleus. Quantitative real-time PCR analysis during zebrafish development showed a significant increase in zebrafish kiss1, kiss2 (P < 0.002), gnrh2, and gnrh3 (P < 0.001) mRNA levels at the start of the pubertal phase and remained high in adulthood. In sexually mature female zebrafish, Kiss2 but not Kiss1 administration significantly increased FSH-beta (2.7-fold, P < 0.05) and LH-beta (8-fold, P < 0.01) mRNA levels in the pituitary. These results suggest that the habenular Kiss1 and the hypothalamic Kiss2 are potential regulators of reproduction including puberty and that Kiss2 is the predominant regulator of gonadotropin synthesis in fish.

Matched MeSH terms: Fish Proteins/metabolism; Zebrafish Proteins/metabolism
In-vitro digestibility and amino acid composition of soy protein isolate cross-linked with microbial transglutaminase followed by heating with ribose

Gan CY, Cheng LH, Azahari B, Easa AM

Int J Food Sci Nutr, 2009;60 Suppl 7:99-108.
PMID: 19194813 DOI: 10.1080/09637480802635090

Cross-linked soy protein isolate (SPI) gels were produced via single-treatment of SPI with microbial transglutaminase (MTG) for 5 h or 24 h, or with ribose for 2 h, or via combined-treatments of SPI with MTG followed by heating with ribose. Assessment of gel strength and solubility concluded that measures which increased protein cross-links resulted in improved gel strength; however, in most cases the digestibility and amino acid content of the gels were reduced. The combined treated gel of SPI/MTG for 24 h/ribose was more easily digested by digestive enzymes and retained higher amounts of amino acids compared with the control Maillard gels of SPI with ribose. MTG consumed lysine and glutamine and reduced the availability of amino acids for the Maillard reaction with ribose. MTG was able to preserve the nutritional value of SPI against the destructive effect of the Maillard reaction and cross-links.

Matched MeSH terms: Vegetable Proteins/metabolism*; Soybean Proteins/metabolism*
Minichromosome maintenance protein 2 is a reliable proliferative marker in breast carcinoma

Reena RM, Mastura M, Siti-Aishah MA, Munirah MA, Norlia A, Naqiyah I, et al.

Ann Diagn Pathol, 2008 Oct;12(5):340-3.
PMID: 18774496 DOI: 10.1016/j.anndiagpath.2008.04.001

This is a study aimed to examine the distribution pattern of a specific minichromosome maintenance protein 2 (MCM2) in benign and malignant breast tissue. We also aim to correlate the frequency of expression of MCM2 with the degree of tumor differentiation. We used immunohistochemistry to examine the distribution and expression pattern of MCM2 on formalin-fixed paraffin-embedded tissue sections of benign (n = 30) and malignant breast tissue (n = 70) (IDC 56, DCIS 4, ILC 2, nonductal 4, mixed type 4). We quantified MCM2 expression by calculating a labeling index, which represents the percentage of epithelial nuclei that stained positively. Immunoreactivity was heterogenous in all the 70 malignant cases examined. Epithelial cells in cycle are most frequent at the tumor periphery. Labeling index of MCM2 was greatest in grade 3 (poorly differentiated) and lowest in grade 1 tumors (well differentiated). Minichromosome maintenance protein 2 expression in breast cancer showed a positive association with histologic grade (P < .05). In all the benign breast tissue examined, no proliferating compartments could be characterized. Minichromosome maintenance protein 2 is a useful proliferative marker of breast carcinoma. The frequency of expression of MCM2 showed an inverse correlation with the degree of tumor differentiation.

Matched MeSH terms: Nuclear Proteins/metabolism*; Cell Cycle Proteins/metabolism*
A genome level survey of Burkholderia pseudomallei immunome expressed during human infection

Su YC, Wan KL, Mohamed R, Nathan S

Microbes Infect., 2008 Oct;10(12-13):1335-45.
PMID: 18761419 DOI: 10.1016/j.micinf.2008.07.034

Burkholderia pseudomallei is the etiological agent of melioidosis, a severe infectious disease of humans and animals. The role of the bacterium's proteins expressed in vivo during human melioidosis continues to remain an enigma. This study's aim was to identify B. pseudomallei target proteins that elicit the humoral immune response in infected humans. A small insert genomic expression library was constructed and immunoscreened to identify peptides that reacted exclusively with melioidosis patients' sera. Sero-positive clones expressing immunogenic peptides were sequenced and annotated, and shown to represent 109 proteins involved in bacterial cell envelope biogenesis, cell motility and secretion, transcription, amino acid, ion and protein metabolism, energy production, DNA repair and unknown hypothetical proteins. Western blot analysis of three randomly selected full-length immunogenic polypeptides with patients' sera verified the findings of the immunome screening. The patients' humoral immune response to the 109 proteins suggests the induction or significant upregulation of these proteins in vivo during human infection and thus may play a role in the pathogenesis of B. pseudomallei. Identification of B. pseudomallei immunogens has shed new light on the elucidation of the bacterium's pathogenesis mechanism and disease severity. These immunogens can be further evaluated as prophylactic and serodiagnostic candidates as well as drug targets.

Matched MeSH terms: Bacterial Proteins/metabolism*; Recombinant Proteins/metabolism
Characterization of hbzE-encoded gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867

Yeo CC, Tan CL, Gao X, Zhao B, Poh CL

Res. Microbiol., 2007 Sep;158(7):608-16.
PMID: 17720458

Pseudomonas alcaligenes NCIMB 9867 (strain P25X) is known to synthesize two isofunctional gentisate 1,2-dioxygenases (GDO; EC 1.13.11.4) as well as other enzymes involved in the degradation of xylenols and cresols via the gentisate pathway. The hbzE gene encoding what is possibly the strictly inducible gentisate 1,2-dioxygenase II (GDO-II) was cloned, overexpressed and purified as a hexahistidine fusion protein from Escherichia coli. Active recombinant GDO-II had an estimated molecular mass of 150kDa and is likely a tetrameric protein with a subunit mass of approximately 40kDa, similar to the previously characterized gentisate 1,2-dioxygenase I (GDO-I) encoded by xlnE. However, GDO-II was unable to utilize gentisate that is substituted at the carbon-4 position, unlike GDO-I which had broader substrate specificity. GDO-II also possessed different kinetic characteristics when compared to GDO-I. The hbzE-encoded GDO-II shared higher sequence identities (53%) with GDOs from Ralstonia sp. U2 and Polaromonas naphthalenivorans CJ2, compared with only 35% identity with the xlnE-encoded GDO-I. The hbzE gene was found to be part of a cluster of nine genes including the putative regulatory gene designated hbzR, which encodes an LysR-type regulator and is divergently transcribed from the other genes of the hbzHIJKLFED cluster.

Matched MeSH terms: Bacterial Proteins/metabolism; Recombinant Proteins/metabolism
Purification of long helical capsid of newcastle disease virus from Escherichia coli using anion exchange chromatography

Yap CF, Tan WS, Sieo CC, Tey BT

Biotechnol Prog, 2013 Mar-Apr;29(2):564-7.
PMID: 23364925 DOI: 10.1002/btpr.1697

NP(Δc375) is a truncated version of the nucleocapsid protein of Newcastle disease virus (NDV) which self-assembles into a long helical structure. A packed bed anion exchange chromatography (PB-AEC), SepFastTM Supor Q pre-packed column, was used to purify NP(Δc375) from clarified feedstock. This PB-AEC column adsorbed 76.2% of NP(Δc375) from the clarified feedstock. About 67.5% of the adsorbed NP(Δc375) was successfully eluted from the column by applying 50 mM Tris-HCl elution buffer supplemented with 0.5 M NaCl at pH 7. Thus, a recovery yield of 51.4% with a purity of 76.7% which corresponds to a purification factor of 6.5 was achieved in this PB-AEC operation. Electron microscopic analysis revealed that the helical structure of the NP(Δc375) purified by SepFast(TM) Supor Q pre-packed column was as long as 490 nm and 22-24 nm in diameter. The antigenicity of the purified NP(Δc375) was confirmed by enzyme-linked immunosorbent assay.

Matched MeSH terms: Recombinant Proteins/metabolism; Capsid Proteins/metabolism
Cryptocaryon irritans recombinant proteins as potential antigens for sero-surveillance of cryptocaryonosis

Lokanathan Y, Mohd-Adnan A, Kua BC, Nathan S

J Fish Dis, 2016 Sep;39(9):1069-83.
PMID: 27086498 DOI: 10.1111/jfd.12474

Cryptocaryonosis is a major problem for mariculture, and the absence of suitable sero-surveillance tools for the detection of cryptocaryonosis makes it difficult to screen Cryptocaryon irritans-infected fish, particularly asymptomatic fish. In this study, we proposed a serum-based assay using selected C. irritans proteins to screen infected and asymptomatic fish. Eight highly expressed genes were chosen from an earlier study on C. irritans expressed sequence tags and ciliate glutamine codons were converted to universal glutamine codons. The chemically synthesized C. irritans genes were then expressed in an Escherichia coli expression host under optimized conditions. Five C. irritans proteins were successfully expressed in E. coli and purified by affinity chromatography. These proteins were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to screen sera from experimentally immunized fish and naturally infected fish. Sera from both categories of fish reacted equally well with the expressed C. irritans recombinant proteins as well as with sonicated theronts. This study demonstrated the utility of producing ciliate recombinant proteins in a heterologous expression host. An ELISA was successfully developed to diagnose infected and asymptomatic fish using the recombinant proteins as antigens.

Matched MeSH terms: Recombinant Proteins/metabolism; Protozoan Proteins/metabolism
Fulltext Unmasking Heavily O-Glycosylated Serum Proteins Using Perchloric Acid: Identification of Serum Proteoglycan 4 and Protease C1 Inhibitor as Molecular Indicators for Screening of Breast Cancer

Lee CS, Taib NA, Ashrafzadeh A, Fadzli F, Harun F, Rahmat K, et al.

PLoS One, 2016;11(2):e0149551.
PMID: 26890881 DOI: 10.1371/journal.pone.0149551

Heavily glycosylated mucin glycopeptides such as CA 27.29 and CA 15-3 are currently being used as biomarkers for detection and monitoring of breast cancer. However, they are not well detected at the early stages of the cancer. In the present study, perchloric acid (PCA) was used to enhance detection of mucin-type O-glycosylated proteins in the serum in an attempt to identify new biomarkers for early stage breast cancer. Sensitivity and specificity of an earlier developed sandwich enzyme-linked lectin assay were significantly improved with the use of serum PCA isolates. When a pilot case-control study was performed using the serum PCA isolates of normal participants (n = 105) and patients with stage 0 (n = 31) and stage I (n = 48) breast cancer, higher levels of total O-glycosylated proteins in sera of both groups of early stage breast cancer patients compared to the normal control women were demonstrated. Further analysis by gel-based proteomics detected significant inverse altered abundance of proteoglycan 4 and plasma protease C1 inhibitor in both the early stages of breast cancer patients compared to the controls. Our data suggests that the ratio of serum proteoglycan 4 to protease C1 inhibitor may be used for screening of early breast cancer although this requires further validation in clinically representative populations.

Matched MeSH terms: Blood Proteins/metabolism*; Complement C1 Inactivator Proteins/metabolism*
Fulltext Does Short-Term Dietary Omega-3 Fatty Acid Supplementation Influence Brain Hippocampus Gene Expression of Zinc Transporter-3?

Sopian NF, Ajat M, Shafie NI, Noor MH, Ebrahimi M, Rajion MA, et al.

Int J Mol Sci, 2015;16(7):15800-10.
PMID: 26184176 DOI: 10.3390/ijms160715800

Dietary omega-3 fatty acids have been recognized to improve brain cognitive function. Deficiency leads to dysfunctional zinc metabolism associated with learning and memory impairment. The objective of this study is to explore the effect of short-term dietary omega-3 fatty acids on hippocampus gene expression at the molecular level in relation to spatial recognition memory in mice. A total of 24 male BALB/c mice were randomly divided into four groups and fed a standard pellet as a control group (CTL, n = 6), standard pellet added with 10% (w/w) fish oil (FO, n = 6), 10% (w/w) soybean oil (SO, n = 6) and 10% (w/w) butter (BT, n = 6). After 3 weeks on the treatment diets, spatial-recognition memory was tested on a Y-maze. The hippocampus gene expression was determined using a real-time PCR. The results showed that 3 weeks of dietary omega-3 fatty acid supplementation improved cognitive performance along with the up-regulation of α-synuclein, calmodulin and transthyretin genes expression. In addition, dietary omega-3 fatty acid deficiency increased the level of ZnT3 gene and subsequently reduced cognitive performance in mice. These results indicate that the increased the ZnT3 levels caused by the deficiency of omega-3 fatty acids produced an abnormal zinc metabolism that in turn impaired the brain cognitive performance in mice.

Matched MeSH terms: Carrier Proteins/metabolism*; Membrane Proteins/metabolism*
Fulltext Prevention of cell-surface attachment and reduction of penicillin-binding protein 2a (PBP2a) level in methicillin-resistant Staphylococcus aureus biofilms by Acalypha wilkesiana

Santiago C, Lim KH, Loh HS, Ting KN

BMC Complement Altern Med, 2015;15:79.
PMID: 25880167 DOI: 10.1186/s12906-015-0615-6

Formation of biofilm is known to enhance the virulence of methicillin-resistance Staphylococcus aureus (MRSA), which is associated with persistent infections in hospital settings. The biofilm layer essentially forms a protective barrier encapsulating the bacterial colony and thus reduces the effectiveness of chemotherapeutics. We have isolated 9EA-FC-B bioactive fraction from Acalypha wilkesiana Müll. Arg. that reverses ampicillin resistant in MRSA through inhibition of the antibiotic resistant protein, penicillin-binding protein 2a (PBP2a). In this study, we aimed to investigate the effects of 9EA-FC-B on MRSA biofilm forming capacity.

Matched MeSH terms: Bacterial Proteins/metabolism*; Penicillin-Binding Proteins/metabolism
Identification of a 48kDa tubulin or tubulin-like C6/36 mosquito cells protein that binds dengue virus 2 using mass spectrometry

Chee HY, AbuBakar S

Biochem Biophys Res Commun, 2004 Jul 16;320(1):11-7.
PMID: 15207695

Binding of dengue virus 2 (DENV-2) to C6/36 mosquito cells protein was investigated. A 48 kDa DENV-2-binding C6/36 cells protein (D2BP) was detected in a virus overlay protein-binding assay. The binding occurred only to the C6/36 cells cytosolic protein fraction and it was inhibited by free D2BP. D2BP was shown to bind to DENV-2 E in the far-Western-binding studies and using mass spectrometry (MS) and MS/MS, peptide masses of the D2BP that matched to beta-tubulin and alpha-tubulin chains were identified. These findings suggest that DENV-2 through DENV-2 E binds directly to a 48 kDa tubulin or tubulin-like protein of C6/36 mosquito cells.

Matched MeSH terms: Viral Envelope Proteins/metabolism; Insect Proteins/metabolism
Biochemical Characterization of the Cytochrome P450 CYP107CB2 from Bacillus lehensis G1

Ang SS, Salleh AB, Chor LT, Normi YM, Tejo BA, Rahman MBA, et al.

Protein J, 2018 04;37(2):180-193.
PMID: 29508210 DOI: 10.1007/s10930-018-9764-z

The bioconversion of vitamin D3 catalyzed by cytochrome P450 (CYP) requires 25-hydroxylation and subsequent 1α-hydroxylation to produce the hormonal activated 1α,25-dihydroxyvitamin D3. Vitamin D3 25-hydroxylase catalyses the first step in the vitamin D3 biosynthetic pathway, essential in the de novo activation of vitamin D3. A CYP known as CYP107CB2 has been identified as a novel vitamin D hydroxylase in Bacillus lehensis G1. In order to deepen the understanding of this bacterial origin CYP107CB2, its detailed biological functions as well as biochemical characteristics were defined. CYP107CB2 was characterized through the absorption spectral analysis and accordingly, the enzyme was assayed for vitamin D3 hydroxylation activity. CYP-ligand characterization and catalysis optimization were conducted to increase the turnover of hydroxylated products in an NADPH-regenerating system. Results revealed that the over-expressed CYP107CB2 protein was dominantly cytosolic and the purified fraction showed a protein band at approximately 62 kDa on SDS-PAGE, indicative of CYP107CB2. Spectral analysis indicated that CYP107CB2 protein was properly folded and it was in the active form to catalyze vitamin D3 reaction at C25. HPLC and MS analysis from a reconstituted enzymatic reaction confirmed the hydroxylated products were 25-hydroxyitamin D3 and 1α,25-dihydroxyvitamin D3 when the substrates vitamin D3 and 1α-hydroxyvitamin D3 were used. Biochemical characterization shows that CYP107CB2 performed hydroxylation activity at 25 °C in pH 8 and successfully increased the production of 1α,25-dihydroxyvitamin D3 up to four fold. These findings show that CYP107CB2 has a biologically relevant vitamin D3 25-hydroxylase activity and further suggest the contribution of CYP family to the metabolism of vitamin D3.

Matched MeSH terms: Bacterial Proteins/metabolism; Recombinant Proteins/metabolism
Cold-adapted RTX lipase from antarctic Pseudomonas sp. strain AMS8: isolation, molecular modeling and heterologous expression

Ali MS, Ganasen M, Rahman RN, Chor AL, Salleh AB, Basri M

Protein J, 2013 Apr;32(4):317-25.
PMID: 23645400 DOI: 10.1007/s10930-013-9488-z

A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S(207), D(255) and H(313), based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 °C and retained almost 50 % of its activity at 10 °C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 °C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.

Matched MeSH terms: Bacterial Proteins/metabolism; Recombinant Proteins/metabolism
Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis

Kahar UM, Ng CL, Chan KG, Goh KM

Appl Microbiol Biotechnol, 2016 Jul;100(14):6291-307.
PMID: 27000839 DOI: 10.1007/s00253-016-7451-6

Type I pullulanases are enzymes that specifically hydrolyse α-1,6 linkages in polysaccharides. This study reports the analyses of a novel type I pullulanase (PulASK) from Anoxybacillus sp. SK3-4. Purified PulASK (molecular mass of 80 kDa) was stable at pH 5.0-6.0 and was most active at pH 6.0. The optimum temperature for PulASK was 60 °C, and the enzyme was reasonably stable at this temperature. Pullulan was the preferred substrate for PulASK, with 89.90 % adsorbance efficiency (various other starches, 56.26-72.93 % efficiency). Similar to other type I pullulanases, maltotriose was formed on digestion of pullulan by PulASK. PulASK also reacted with β-limit dextrin, a sugar rich in short branches, and formed maltotriose, maltotetraose and maltopentaose. Nevertheless, PulASK was found to preferably debranch long branches at α-1,6 glycosidic bonds of starch, producing amylose, linear or branched oligosaccharides, but was nonreactive against short branches; thus, no reducing sugars were detected. This is surprising as all currently known type I pullulanases produce reducing sugars (predominantly maltotriose) on digesting starch. The closest homologue of PulASK (95 % identity) is a type I pullulanase from Anoxybacillus sp. LM14-2 (Pul-LM14-2), which is capable of forming reducing sugars from starch. With rational design, amino acids 362-370 of PulASK were replaced with the corresponding sequence of Pul-LM14-2. The mutant enzyme formed reducing sugars on digesting starch. Thus, we identified a novel motif involved in substrate specificity in type I pullulanases. Our characterization may pave the way for the industrial application of this unique enzyme.

Matched MeSH terms: Bacterial Proteins/metabolism*; Recombinant Proteins/metabolism
Fulltext Nicotine-prevented learning and memory impairment in REM sleep-deprived rat is modulated by DREAM protein in the hippocampus

Abd Rashid N, Hapidin H, Abdullah H, Ismail Z, Long I

Brain Behav, 2017 06;7(6):e00704.
PMID: 28638710 DOI: 10.1002/brb3.704

INTRODUCTION: REM sleep deprivation is associated with impairment in learning and memory, and nicotine treatment has been shown to attenuate this effect. Recent studies have demonstrated the importance of DREAM protein in learning and memory processes. This study investigates the association of DREAM protein in REM sleep-deprived rats hippocampus upon nicotine treatment.
METHODS: Male Sprague Dawley rats were subjected to normal condition, REM sleep deprivation and control wide platform condition for 72 hr. During this procedure, saline or nicotine (1 mg/kg) was given subcutaneously twice a day. Then, Morris water maze (MWM) test was used to assess learning and memory performance of the rats. The rats were sacrificed and the brain was harvested for immunohistochemistry and Western blot analysis.
RESULTS: MWM test found that REM sleep deprivation significantly impaired learning and memory performance without defect in locomotor function associated with a significant increase in hippocampus DREAM protein expression in CA1, CA2, CA3, and DG regions and the mean relative level of DREAM protein compared to other experimental groups. Treatment with acute nicotine significantly prevented these effects and decreased expression of DREAM protein in all the hippocampus regions but only slightly reduce the mean relative level of DREAM protein.
CONCLUSION: This study suggests that changes in DREAM protein expression in CA1, CA2, CA3, and DG regions of rat's hippocampus and mean relative level of DREAM protein may involve in the mechanism of nicotine treatment-prevented REM sleep deprivation-induced learning and memory impairment in rats.

Matched MeSH terms: Repressor Proteins/metabolism*; Kv Channel-Interacting Proteins/metabolism*
Fulltext Molecular characterization of the viral structural protein genes in the first outbreak of dengue virus type 2 in Hunan Province, inland China in 2018

Guan J, He Z, Qin M, Deng X, Chen J, Duan S, et al.

BMC Infect Dis, 2021 Feb 10;21(1):166.
PMID: 33568111 DOI: 10.1186/s12879-021-05823-3

BACKGROUND: An unexpected dengue outbreak occurred in Hunan Province in 2018. This was the first dengue outbreak in this area of inland China, and 172 cases were reported.
METHODS: To verify the causative agent of this outbreak and characterise the viral genes, the genes encoding the structural proteins C/prM/E of viruses isolated from local residents were sequenced followed by mutation and phylogenetic analysis. Recombination, selection pressure, potential secondary structure and three-dimensional structure analyses were also performed.
RESULTS: Phylogenetic analysis revealed that all epidemic strains were of the cosmopolitan DENV-2 genotype and were most closely related to the Zhejiang strain (MH010629, 2017) and then the Malaysia strain (KJ806803, 2013). Compared with the sequence of DENV-2SS, 151 base substitutions were found in the sequences of 89 isolates; these substitutions resulted in 20 non-synonymous mutations, of which 17 mutations existed in all samples (two in the capsid protein, six in the prM/M proteins, and nine in the envelope proteins). Moreover, amino acid substitutions at the 602nd (E322:Q → H) and 670th (E390: N → S) amino acids may have enhanced the virulence of the epidemic strains. One new DNA binding site and five new protein binding sites were observed. Two polynucleotide binding sites and seven protein binding sites were lost in the epidemic strains compared with DENV-2SS. Meanwhile, five changes were found in helical regions. Minor changes were observed in helical transmembrane and disordered regions. The 429th amino acid of the E protein switched from a histamine (positively charged) to an asparagine (neutral) in all 89 isolated strains. No recombination events or positive selection pressure sites were observed. To our knowledge, this study is the first to analyse the genetic characteristics of epidemic strains in the first dengue outbreak in Hunan Province in inland China.
CONCLUSIONS: The causative agent is likely to come from Zhejiang Province, a neighbouring province where dengue fever broke out in 2017. This study may help clarify the intrinsic geographical relatedness of DENV-2 and contribute to further research on pathogenicity and vaccine development.

Matched MeSH terms: Viral Nonstructural Proteins/metabolism; Capsid Proteins/metabolism
Cloning, extracellular expression and characterization of a predominant beta-CGTase from Bacillus sp. G1 in E. coli

Ong RM, Goh KM, Mahadi NM, Hassan O, Rahman RN, Illias RM

J Ind Microbiol Biotechnol, 2008 Dec;35(12):1705-14.
PMID: 18726621 DOI: 10.1007/s10295-008-0462-2

The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60 degrees C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% beta-cyclodextrin (CD) and 10% gamma-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of beta-CD.

Matched MeSH terms: Bacterial Proteins/metabolism*; Recombinant Proteins/metabolism*

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