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Fulltext Genome-wide meta-analysis identifies five new susceptibility loci for pancreatic cancer

Klein AP, Wolpin BM, Risch HA, Stolzenberg-Solomon RZ, Mocci E, Zhang M, et al.

Nat Commun, 2018 02 08;9(1):556.
PMID: 29422604 DOI: 10.1038/s41467-018-02942-5

In 2020, 146,063 deaths due to pancreatic cancer are estimated to occur in Europe and the United States combined. To identify common susceptibility alleles, we performed the largest pancreatic cancer GWAS to date, including 9040 patients and 12,496 controls of European ancestry from the Pancreatic Cancer Cohort Consortium (PanScan) and the Pancreatic Cancer Case-Control Consortium (PanC4). Here, we find significant evidence of a novel association at rs78417682 (7p12/TNS3, P = 4.35 × 10-8). Replication of 10 promising signals in up to 2737 patients and 4752 controls from the PANcreatic Disease ReseArch (PANDoRA) consortium yields new genome-wide significant loci: rs13303010 at 1p36.33 (NOC2L, P = 8.36 × 10-14), rs2941471 at 8q21.11 (HNF4G, P = 6.60 × 10-10), rs4795218 at 17q12 (HNF1B, P = 1.32 × 10-8), and rs1517037 at 18q21.32 (GRP, P = 3.28 × 10-8). rs78417682 is not statistically significantly associated with pancreatic cancer in PANDoRA. Expression quantitative trait locus analysis in three independent pancreatic data sets provides molecular support of NOC2L as a pancreatic cancer susceptibility gene.

Matched MeSH terms: Proteins/genetics; Repressor Proteins/genetics; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins
Fulltext Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles

Zaborowski MP, Cheah PS, Zhang X, Bushko I, Lee K, Sammarco A, et al.

Sci Rep, 2019 Nov 22;9(1):17387.
PMID: 31758005 DOI: 10.1038/s41598-019-53554-y

Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models.

Matched MeSH terms: Membrane Proteins/genetics; Membrane Proteins/metabolism*; Recombinant Proteins/genetics; Recombinant Proteins/metabolism*
Fulltext New emm (M protein gene) sequences of group A streptococci isolated from Malaysian patients

Jamal F, Pit S, Facklam R, Beall B

Emerg Infect Dis, 1999 Jan-Feb;5(1):182-3.
PMID: 10081694
Matched MeSH terms: Bacterial Outer Membrane Proteins*; Bacterial Proteins/genetics*; Carrier Proteins/genetics*
SPI1-mediated transcriptional activation of CEP55 promotes the malignant growth of triple-negative breast cancer and M2 macrophage polarization

Liu Y, Dong M, Jia Y, Yang D, Hui Y, Yang X

Pathol Res Pract, 2024 Oct;262:155544.
PMID: 39197215 DOI: 10.1016/j.prp.2024.155544

BACKGROUND: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of three receptors commonly targeted in breast cancer treatment. In this study, the research focused on investigating the role of centrosomal protein 55 (CEP55) in TNBC progression and its interaction with the transcription factor Spi-1 proto-oncogene (SPI1).
METHODS: Various techniques including qRT-PCR, western blotting, and immunohistochemistry assays were utilized to examine gene expression patterns. Functional assays such as wound-healing assay, transwell invasion assay, 5-Ethynyl-2'-deoxyuridine assay, and metabolic assays were conducted to assess the impact of CEP55 on the behaviors of TNBC cells. CD163-positive macrophages were quantified by flow cytometry. The chromatin immunoprecipitation assay and dual-luciferase reporter assay were performed to assess the association of SPI1 with CEP55. A xenograft mouse model experiment was used to analyze the impact of SPI1 on tumor development in vivo.
RESULTS: CEP55 and SPI1 expression levels were significantly upregulated in TNBC tissues and cells. The depletion of CEP55 led to decreased TNBC cell migration, invasion, proliferation, glucose metabolism, and M2 macrophage polarization, indicating its crucial role in promoting TNBC progression. Moreover, SPI1 transcriptionally activated CEP55 in TNBC cells, and its overexpression was associated with accelerated tumor growth in vivo. Further, CEP55 overexpression relieved SPI1 silencing-induced inhibitory effects on TNBC cell migration, invasion, proliferation, glucose metabolism, and M2 macrophage polarization.
CONCLUSION: SPI1-mediated transcriptional activation of CEP55 plays a key role in enhancing TNBC cell migration, invasion, proliferation, glucose metabolism, and M2 macrophage polarization. These insights provide valuable information for potential targeted therapies to combat TNBC progression by modulating the SPI1-CEP55 axis.

Matched MeSH terms: Nuclear Proteins/genetics; Nuclear Proteins/metabolism; Proto-Oncogene Proteins/genetics; Proto-Oncogene Proteins/metabolism
Fulltext Whole genome sequencing and analysis reveal insights into the genetic structure, diversity and evolutionary relatedness of luxI and luxR homologs in bacteria belonging to the Sphingomonadaceae family

Gan HM, Gan HY, Ahmad NH, Aziz NA, Hudson AO, Savka MA

Front Cell Infect Microbiol, 2014;4:188.
PMID: 25621282 DOI: 10.3389/fcimb.2014.00188

Here we report the draft genomes and annotation of four N-acyl homoserine lactone (AHL)-producing members from the family Sphingomonadaceae. Comparative genomic analyses of 62 Sphingomonadaceae genomes were performed to gain insights into the distribution of the canonical luxI/R-type quorum sensing (QS) network within this family. Forty genomes contained at least one luxR homolog while the genome of Sphingobium yanoikuyae B1 contained seven Open Reading Frames (ORFs) that have significant homology to that of luxR. Thirty-three genomes contained at least one luxI homolog while the genomes of Sphingobium sp. SYK6, Sphingobium japonicum, and Sphingobium lactosutens contained four luxI. Using phylogenetic analysis, the sphingomonad LuxR homologs formed five distinct clades with two minor clades located near the plant associated bacteria (PAB) LuxR solo clade. This work for the first time shows that 13 Sphingobium and one Sphingomonas genome(s) contain three convergently oriented genes composed of two tandem luxR genes proximal to one luxI (luxR-luxR-luxI). Interestingly, luxI solos were identified in two Sphingobium species and may represent species that contribute to AHL-based QS system by contributing AHL molecules but are unable to perceive AHLs as signals. This work provides the most comprehensive description of the luxI/R circuitry and genome-based taxonomical description of the available sphingomonad genomes to date indicating that the presence of luxR solos and luxI solos are not an uncommon feature in members of the Sphingomonadaceae family.

Matched MeSH terms: Bacterial Proteins/genetics*; Bacterial Proteins/metabolism; Bacterial Proteins/chemistry; Repressor Proteins/genetics*; Repressor Proteins/metabolism; Repressor Proteins/chemistry
Review: Beta-thalassemia and molecular chaperones

Sumera A, Radhakrishnan A, Baba AA, George E

Blood Cells Mol. Dis., 2015 Apr;54(4):348-52.
PMID: 25648458 DOI: 10.1016/j.bcmd.2015.01.008

Thalassemia is known as a diverse single gene disorder, which is prevalent worldwide. The molecular chaperones are set of proteins that help in two important processes while protein synthesis and degradation include folding or unfolding and assembly or disassembly, thereby helping in cell homeostasis. This review recaps current knowledge regarding the role of molecular chaperones in thalassemia, with a focus on beta thalassemia.

Matched MeSH terms: Blood Proteins/genetics*; Blood Proteins/metabolism; Blood Proteins/chemistry; HSP70 Heat-Shock Proteins/genetics*; HSP70 Heat-Shock Proteins/metabolism; HSP70 Heat-Shock Proteins/chemistry
Strain measurements and fracture resistance of endodontically treated premolars restored with all-ceramic restorations

Seow LL, Toh CG, Wilson NH

J Dent, 2015 Jan;43(1):126-32.
PMID: 25448436 DOI: 10.1016/j.jdent.2014.10.001

OBJECTIVES: The aim of this study was to investigate the recovery of cuspal stiffness and fracture resistance in endodontically treated maxillary premolars restored with bonded ceramic inlays and onlays of various designs.
METHODS: Seventy intact premolars were selected for this study; six cavity designs were investigated: (i) mesio-occlusal-distal (MOD) inlay (I), (ii) MOD inlay with palatal cusp coverage (IPC), (iii) MOD onlay (O), (iv) MOD inlay with pulp chamber extension (IPE), (v) MOD inlay with palatal cusp coverage and pulp chamber extension (IPCPE), and (vi) MOD onlay with pulp chamber extension (OPE). Intact teeth acted as control. Strain gauges were attached to the buccal and palatal surfaces of the teeth to measure cuspal stiffness under static loading. All specimens were eventually subjected to compressive load to failure. Cuspal stiffness and fracture resistance data were analyzed using ANOVA and Tukey test.
RESULTS: The I and IPE restorations restored cuspal stiffness to 75% of the sound tooth value. The O and OPE restored teeth had stiffness values greater than that of a sound tooth. The I, IPC, O, IPE, IPCPE and OPE restored teeth demonstrated fracture strength values of 938N±113 N (s.d.), 1073N±176 N and 1317N±219 N, 893N±129 N, 1062N±153 N and 1347N±191 N respectively.
CONCLUSIONS: Within the limitations of this study, it was concluded that the all-ceramic onlay or inlay with palatal cusp coverage provided best biomechanical advantage in restoring an endodontically treated maxillary premolar tooth.
CLINICAL SIGNIFICANCE: The onlay approach which is more conservative compared to full coverage restoration is considered an appropriate approach to the restoration of endodontically treated maxillary premolars. The addition of a pulpal extension to the all-ceramic restorations, apart from being technically challenging, was not found to offer any biomechanical advantage to the restored teeth.
KEYWORDS: Endodontically treated teeth; Fracture strengths; Inlay; Onlay; Pulp chamber extension; Strains

Matched MeSH terms: Nerve Tissue Proteins; RNA-Binding Proteins
Fulltext Sumoylation controls the timing of Tup1-mediated transcriptional deactivation

Ng CH, Akhter A, Yurko N, Burgener JM, Rosonina E, Manley JL

Nat Commun, 2015 Mar 13;6:6610.
PMID: 25766875 DOI: 10.1038/ncomms7610

The small ubiquitin-like modifier (SUMO) is implicated in various cellular activities, including transcriptional regulation. We previously showed that the yeast activator Gcn4 becomes sumoylated during activation, facilitating its eventual promoter eviction and transcriptional shut off. Here we show that the corepressor Tup1 is sumoylated, at two specific lysines, under various stress conditions. Mutation of these sites has no effect on Tup1 recruitment or RNAP II promoter occupancy immediately following induction. However, Tup1 levels subsequently decrease, while RNAP II and transcription increase in Tup1 mutant cells. Consistent with this, a Tup1 mutant displaying increased sumoylation led to reduced transcription. We also show that coordinated sumoylation of Gcn4 and Tup1 enhances Gcn4 promoter eviction and that multiple Tup1-interacting proteins become sumoylated after stress. Together, our studies provide evidence that coordinated sumoylation of Gcn4, Tup1 and likely other factors dampens activated transcription by stabilizing Tup1 binding and stimulating Gcn4 and RNAP II removal.

Matched MeSH terms: DNA-Binding Proteins/metabolism; Nuclear Proteins/metabolism*; Repressor Proteins/metabolism*; Saccharomyces cerevisiae Proteins/metabolism*
Mechanisms of action of 17βH-neriifolin on its anticancer effect in SKOV-3 ovarian cancer cell line

Mutalip SS, Yunos NM, Abdul-Rahman PS, Jauri MH, Osman A, Adenan MI

Anticancer Res, 2014 Aug;34(8):4141-51.
PMID: 25075041

AIM: Abnormalities in apoptotic signalling pathways often occur in cancer cells and limit the successful chemotherapy outcomes in cancers. Therefore, there is an urgent need to discover new anticancer agents with novel mechanisms of action to overcome the resistance effect in chemotherapy.
MATERIALS AND METHODS: In the present study, the anticancer effects and the mechanisms of action of 17βH-neriifolin (cardiac glycoside) were evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and a proteomic approach in treated and non-treated SKOV-3 ovarian cancer cells.
RESULTS: 17βH-neriifolin was found to be active with IC50 values of 0.01 ± 0.001 in SKOV-3 ovarian cancer cell line, as evaluated by the sulforhodamine B (SRB) assay. RESULTS from TUNEL assay indicated that 17βH-neriifolin caused apoptosis in SKOV-3 cells in a dose-dependent manner. Based on differential analysis of treated and non-treated SKOV-3 two-dimensional electrophoresis (2-DE) profiles, four proteins, namely vimentin (VIM), pyruvate kinase, muscle (PKM), heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) and transgelin (TAGLN1) were identified to be involved in apoptosis. Other proteins including piggybac transposable element derived 5 (PGBD5), DENN/MADD domain containing 2D (DENND2D) and formin-like 1(FMNL) have also been identified to be associated in SKOV-3 cell death induced by 17βH-neriifolin.
CONCLUSION: These findings may provide new insights on the potential of 17βH-neriifolin's mechanism of action in killing ovarian cancer cells.

Matched MeSH terms: Carrier Proteins/physiology; Membrane Proteins/physiology; Microfilament Proteins/physiology; Muscle Proteins/physiology
Unscrambling the effect of C-terminal tail deletion on the stability of a cold-adapted, organic solvent stable lipase from Staphylococcus epidermidis AT2

Kamarudin NH, Rahman RN, Ali MS, Leow TC, Basri M, Salleh AB

Mol Biotechnol, 2014 Aug;56(8):747-57.
PMID: 24771007 DOI: 10.1007/s12033-014-9753-1

Terminal moieties of most proteins are long known to be disordered and flexible. To unravel the functional role of these regions on the structural stability and biochemical properties of AT2 lipase, four C-terminal end residues, (Ile-Thr-Arg-Lys) which formed a flexible, short tail-like random-coil segment were targeted for mutation. Swapping of the tail-like region had resulted in an improved crystallizability and anti-aggregation property along with a slight shift of the thermostability profile. The lipolytic activity of mutant (M386) retained by 43 % compared to its wild-type with 18 % of the remaining activity at 45 °C. In silico analysis conducted at 25 and 45 °C was found to be in accordance to the experimental findings in which the RMSD values of M386 were more stable throughout the total trajectory in comparison to its wild-type. Terminal moieties were also observed to exhibit large movement and flexibility as denoted by high RMSF values at both dynamics. Variation in organic solvent stability property was detected in M386 where the lipolytic activity was stimulated in the presence of 25 % (v/v) of DMSO, isopropanol, and diethyl ether. This may be worth due to changes in the surface charge residues at the mutation point which probably involve in protein-solvent interaction.

Matched MeSH terms: Bacterial Proteins/genetics*; Bacterial Proteins/metabolism*; Bacterial Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
Fulltext Molybdenum reduction to molybdenum blue in Serratia sp. Strain DRY5 is catalyzed by a novel molybdenum-reducing enzyme

Shukor MY, Halmi MI, Rahman MF, Shamaan NA, Syed MA

Biomed Res Int, 2014;2014:853084.
PMID: 24724104 DOI: 10.1155/2014/853084

The first purification of the Mo-reducing enzyme from Serratia sp. strain DRY5 that is responsible for molybdenum reduction to molybdenum blue in the bacterium is reported. The monomeric enzyme has an apparent molecular weight of 105 kDalton. The isoelectric point of this enzyme was 7.55. The enzyme has an optimum pH of 6.0 and maximum activity between 25 and 35°C. The Mo-reducing enzyme was extremely sensitive to temperatures above 50°C (between 54 and 70°C). A plot of initial rates against substrate concentrations at 15 mM 12-MP registered a V max for NADH at 12.0 nmole Mo blue/min/mg protein. The apparent K m for NADH was 0.79 mM. At 5 mM NADH, the apparent V max and apparent K m values for 12-MP of 12.05 nmole/min/mg protein and 3.87 mM, respectively, were obtained. The catalytic efficiency (k cat/K m ) of the Mo-reducing enzyme was 5.47 M(-1) s(-1). The purification of this enzyme could probably help to solve the phenomenon of molybdenum reduction to molybdenum blue first reported in 1896 and would be useful for the understanding of the underlying mechanism in molybdenum bioremediation involving bioreduction.

Matched MeSH terms: Bacterial Proteins/metabolism*
Statistical optimization of green fluorescent protein production from Escherichia coli BL21(DE3)

Chew FN, Tan WS, Boo HC, Tey BT

Prep Biochem Biotechnol, 2012;42(6):535-50.
PMID: 23030465 DOI: 10.1080/10826068.2012.660903

An optimized cultivation condition is needed to maximize the functional green fluorescent protein (GFP) production. Six process variables (agitation rate, temperature, initial medium pH, concentration of inducer, time of induction, and inoculum density) were screened using the fractional factorial design. Three variables (agitation rate, temperature, and time of induction) exerted significant effects on functional GFP production in E. coli shake flask cultivation and were optimized subsequently using the Box-Behnken design. An agitation rate of 206 rpm at 31°C and induction of the protein expression when the cell density (OD(600nm)) reaches 1.04 could enhance the yield of functional GFP production from 0.025 g/L to 0.241 g/L, which is about ninefold higher than the unoptimized conditions. Unoptimized cultivation conditions resulted in protein aggregation and hence reduced the quantity of functional GFP. The model and regression equation based on the shake flask cultivation could be applied to a 2-L bioreactor for maximum functional GFP production.

Matched MeSH terms: Recombinant Proteins/isolation & purification; Recombinant Proteins/standards; Recombinant Proteins/chemistry; Green Fluorescent Proteins/isolation & purification*; Green Fluorescent Proteins/standards*; Green Fluorescent Proteins/chemistry
Bacteriocin release protein-mediated secretory expression of recombinant chalcone synthase in Escherichia coli

Zakaria II, Rahman RN, Salleh AB, Basri M

Appl Biochem Biotechnol, 2011 Sep;165(2):737-47.
PMID: 21633820 DOI: 10.1007/s12010-011-9292-1

Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD(600) induction time of 0.5 was found to be the best condition for chalcone synthase secretion.

Matched MeSH terms: Plant Proteins/genetics; Plant Proteins/metabolism*; Plant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism*; Recombinant Proteins/chemistry
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/genetics*; Fungal Proteins/metabolism; Fungal Proteins/chemistry*; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
Physicochemical properties and sensory analysis of duck meatballs containing duck meat surimi-like material during frozen storage

Nurkhoeriyati T, Huda N, Ahmad R

J Food Sci, 2012 Jan;77(1):S91-8.
PMID: 22260136 DOI: 10.1111/j.1750-3841.2011.02519.x

The physicochemical properties and sensory analysis of duck meatballs containing duck meat surimi-like material during frozen storage were evaluated. Properties of meatballs containing duck surimi-like material prepared by acid solubilization (ACDS), alkaline solubilization (ALDS), and conventional processing (CDS) as well as duck mince (as the control, CON) were compared. ACDS had significantly higher (P < 0.05) moisture and protein content and lower fat content compared with CON. The thiobarbituric acid-reactive substances (TBARS) value of all samples increased as the storage time increased up to week 8 (P < 0.05), but thereafter it decreased in most of the samples. ACDS and ALDS had significantly higher TBARS values (P < 0.05), and these values remained higher than those of the other samples throughout the frozen storage period. Addition of surimi-like material to the meatballs had significant effects (P < 0.05) on springiness, gumminess, and chewiness values of all samples. Ingredients and frozen storage affected most sensory attributes in samples significantly (P<0.05). No significant increase in growth of organisms occurred during 12-wk frozen storage The results indicate that acid-alkaline solubilization methods improve both physicochemical and sensory properties of duck meatballs containing duck surimi-like material. Thus, these techniques should be applicable to product development of duck surimi-like material.

Matched MeSH terms: Muscle Proteins/analysis; Muscle Proteins/isolation & purification; Muscle Proteins/chemistry*; Avian Proteins/analysis; Avian Proteins/isolation & purification; Avian Proteins/chemistry*
Fulltext Klebsiella pneumoniae yggG gene product: a zinc-dependent metalloprotease

Kuan CS, Wong MT, Choi SB, Chang CC, Yee YH, Wahab HA, et al.

Int J Mol Sci, 2011;12(7):4441-55.
PMID: 21845088 DOI: 10.3390/ijms12074441

Klebsiella pneumoniae causes neonatal sepsis and nosocomial infections. One of the strains, K. pneumoniae MGH 78578, shows high level of resistance to multiple microbial agents. In this study, domain family, amino acid sequence and topology analyses were performed on one of its hypothetical protein, YggG (KPN_03358). Structural bioinformatics approaches were used to predict the structure and functionality of YggG protein. The open reading frame (ORF) of yggG, which was a putative metalloprotease gene, was also cloned, expressed and characterized. The ORF was PCR amplified from K. pneumoniae MGH 78578 genomic DNA and cloned into a pET14-b vector for heterologous expression in Escherichia coli. The purified YggG protein was subsequently assayed for casein hydrolysis under different conditions. This protein was classified as peptidase M48 family and subclan gluzincin. It was predicted to contain one transmembrane domain by TMpred. Optimal protein expression was achieved by induction with 0.6 mM isopropyl thiogalactoside (IPTG) at 25 °C for six hours. YggG was purified as soluble protein and confirmed to be proteolytically active under the presence of 1.25 mM zinc acetate and showed optimum activity at 37 °C and pH 7.4. We confirmed for the first time that the yggG gene product is a zinc-dependent metalloprotease.

Matched MeSH terms: Bacterial Proteins/genetics; Bacterial Proteins/metabolism*; Bacterial Proteins/chemistry; Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/chemistry
Fluorescent quantitation method for differentiating the nativity of green fluorescent protein

Chew FN, Tan WS, Tey BT

J Biosci Bioeng, 2011 Feb;111(2):246-8.
PMID: 21036662 DOI: 10.1016/j.jbiosc.2010.10.004

A gel imaging method was employed to quantitate the GFP that had been subjected to denaturation and degradation treatments. This method is able to differentiate the nativity of GFP by relating the observed changes in the position of fluorescent bands which is unable to be detected using the spectrofluorometric method.

Matched MeSH terms: Green Fluorescent Proteins/analysis*
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/genetics*; Fish Proteins/isolation & purification*; Fish Proteins/metabolism; Zebrafish Proteins/genetics; Zebrafish Proteins/isolation & purification; 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/isolation & purification; Vegetable Proteins/metabolism*; Vegetable Proteins/chemistry*; Soybean Proteins/isolation & purification; Soybean Proteins/metabolism*; Soybean Proteins/chemistry*
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/genetics; Bacterial Proteins/immunology*; Bacterial Proteins/metabolism*; Recombinant Proteins/genetics; Recombinant Proteins/immunology; Recombinant Proteins/metabolism

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