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Most life forms on earth, including the cold-adapted bacteria from the polar regions, are being affected by global warming. To better understand how global warming may affect Antarctic bacteria, the heat-stress response of cold-adapted Cryobacterium sp. SO1 at the transcript level was determined in this study. A Cryobacterium sp. SO1 culture was grown at 20 °C which was its optimal growth temperature and subsequently exposed to a higher temperature of 25 °C that inhibited growth for 3 h. The transcriptome of strain SO1 was isolated, and RNA-sequencing (RNA-seq) was performed and analyzed using bioinformatics tools. The global transcriptome revealed 169 differentially expressed genes (DEGs), 108 of which were up-regulated and 61 of which were down-regulated. The strain SO1 DEGs were classified into seven categories: signal transduction, DNA modification and repair mechanisms, oxidative-stress defence, metabolism-related, cellular translational machinery, cell growth-related, and programmed cell death-related genes. Strain SO1 altered those genes to cope with the heat stress. Transposase gene up-regulation, in particular, probably promoted genome transposition in an attempt to generate genetically diverse cells in the population in the hope that some will be able to withstand the growth inhibitory temperature. Overall, the findings of this study shed some light on how Cryobacterium sp. SO1, and other Antarctic bacteria in general, will probably respond to global warming. [ABSTRACT FROM AUTHOR]

New studies point to an increasing number of identified bacteria that can nodulate and fix N2 in legumes which do not belong to the original genus of Rhizobium and the rhizobial phylogenetic lineages. This study was conducted to isolate and identify diazotrophic microsymbionts from the root nodules of Mucuna bracteata (an important cover crop for oil palm) based on nitrogenase gene (niflJ) isolation and partial 16S rDNA sequence analysis. The findings of this study indicated that the isolated microsymbionts could nodulate and promote N2-fixation activity in M. bracteata. These also contributed to enhanced plant growth in terms of leaf protein and chlorophyll content, as well as in the biomass of whole plants and nodules. Additionally, ni/Hgene fragments were successfully amplified at ~380 bp from eight of the isolates (USM accessions All, B4, B9, B12, B19, Cl, C4 and C8) using nifH4 primers, while the remaining isolates (namely, USM accessions B14, B15, B20, C2 and C9) were successfully amplified at various sizes (-550, 650, 350, 450, and 900 bp, respectively) using ni/H4 primers. The partial 16S rDNA sequencing revealed that the diazotrophic microsymbionts were not only from the traditional Alphaproteobacteria class (Brevundimonas sp.), but also from the Betaproteobacteria class (Achromobacter sp. and Burkholderia sp.) and the Gammaproteobacteria class (Stenotrophomonas sp.). Five non-rhizobial isolates were obtained and identified as Bacillus sp. from the root nodules of M. bracteata. The findings indicate the diversity of potentially-beneficial diazotrophic microsymbionts active in this emerging legume species. [ABSTRACT FROM AUTHOR]

Abstract: Epipremnum pinnatum (L.) Engl. hexane extract produced a significant growth inhibition against T-47D breast carcinoma cells and analysis of cell death mechanisms indicated that the extract elicited a non-apoptotic programmed cell death. T-47D cells exposed to the extract at EC50 concentration (72h) for 24h failed to demonstrate typical DNA fragmentation associated with apoptosis, as carried out using a modified TUNEL assay. In addition, acute exposure to the extract produced an insignificant regulation of caspase-3 and p53 mRNA expression but increased in the c-myc mRNA expression. Ultrastructural analysis using transmission electron microscope demonstrated distinct vacuolated cells, which strongly indicated a Type II non-apoptotic cell death although the changes in chromatin were also detected. The presence of non-apoptotic programmed cell death was then reconfirmed with annexin-V and propidium iodide staining. These findings suggested that up-regulation of c-myc mRNA expression may have contributed to the growth arrest and Type II non-apoptotic programmed cell death in the Epipremnum pinnatum (L.) Engl. hexane extract-treated T-47D cells. [Copyright &y& Elsevier]

Thirteen out of the 15 known Cryobacterium spp. were from extremely cold environments. However, the fundamental question on their cold adaptation strategies to survive in the cold has not been addressed adequately. Hence, this work was conducted to determine the Cryobacterium sp. SO1 cold adaptation strategies. Cryobacterium sp. SO1 that grew optimally at 20 °C was exposed to a sub-optimal temperature of 10 °C. Its mRNA was extracted, sequenced, and analyzed. Strain SO1 global transcriptional profiles revealed a total of 182 differential expressed genes. Four hydrolases, a clp protease, and novel YraN family endonuclease that were related to the programmed cell death pathway were upregulated, indicating that the temperature drop was probably lethal to some cells. Three highly upregulated transcriptional regulators were likely to be the key components to regulate genome-wide expression to adapt to the cold. Meanwhile, the oligo-ribonuclease and a Clp protease gene were upregulated probably to remove accumulated misfolded mRNA and proteins, respectively. The SerB gene was upregulated probably to provide more L-serine residue for the biosynthesis of cold-adapted proteins. Interestingly, most of the stress protein genes in the genome, such as the reactive oxygen species (ROS)-scavenging enzymes were not upregulated. Instead, strain SO1 upregulated the six ribosomal genes which were the target of oxidative nucleobase damage caused by the ROS. This mechanism was probably to ensure that the protein biosynthesis machinery was not affected. Overall, strain SO1 had all the necessary genes and well-coordinated mechanisms to adapt to the sub-optimal growth temperature. [ABSTRACT FROM AUTHOR]

The coding region of guinea pig peroxisome proliferator activated receptor gamma1 (gpPPARγ1) cDNA was successfully cloned from adipose tissue by reverse transcription polymerase chain reaction (RT-PCR) using the designated primers based on the conserved regions of the other mammalian PPARγ1 sequence. From RT-PCR, a combination of three cDNA fragments that comprised of the full length coding region PPARγ1 cDNA gene were amplified, with the size of 498, 550 and 557 bp, respectively. All three fragments were then successfully assembled by utilising the internal restriction sites present at the overlapping regions to give rise to the full-length coding region of gpPPARγ1 with the size of 1428 bp and consisting of 475 amino acids. Guinea pig PPARγ1 is highly conserved with those of other species at protein and nucleotide levels. Gene expression studies showed that gpPPARγ mRNA was predominantly expressed in adipose tissue followed by lung and spleen. However, at the protein level, PPARγ was also found to be expressed in skeletal muscle. [Copyright &y& Elsevier]

Paenibacillus durus strain ATCC 35681T is a Gram‐positive diazotroph that displayed capability of fixing nitrogen even in the presence of nitrate or ammonium. However, the nitrogen fixation activity was detected only at day 1 of growth when cultured in liquid nitrogen‐enriched medium. The transcripts of all the nifH homologues were present throughout the 9‐day study. When grown in nitrogen‐depleted medium, nitrogenase activities occurred from day 1 until day 6 and the nifH transcripts were also present during the course of the study albeit at different levels. In both studies, the absence of nitrogen fixation activity regardless of the presence of the nifH transcripts raised the possibility of a post‐transcriptional or post‐translational regulation of the system. A putative SigA box sequence was found upstream of the transcription start site of nifB1, the first gene in the major nitrogen fixation cluster. The upstream region of nifB2 showed a promoter recognizable by SigE, a sigma factor normally involved in sporulation. [ABSTRACT FROM AUTHOR]

Ice recrystallization during thawing post-cryopreservation results in extensive cellular damage and ultimately leads to cell death and reduced cell viability. Antifreeze proteins (AFPs) are a group of proteins that allow organisms to survive in subzero environments. These proteins have thermal hysteresis and ice recrystallization inhibitory activities. In this present study, we demonstrated the efficiency of a recombinant antifreeze protein from the Antarctic yeast, Glaciozyma antarctica, as a recrystallization inhibitor (RI) of ice growth and assessed its application as a cryopreservative of the fungal cutinase enzyme against freeze-thaw cycles. Recombinant Afp1 from G. antarctica, a psychrophilic yeast, has been produced in a methylotrophic yeast, Pichia pastoris, system that results in the expression of a hyper-glycoprotein (~55 kDa). Recombinant Afp1 exhibits antifreeze functions: thermal hysteresis (TH) and recrystallization inhibition where the highest TH values recorded for ~0.5°C at 10 mg/mL. The cryoprotective effects of Afp1 on purified recombinant cutinase showed that Afp1 can retain enzymatic activity up to ~20% when subjected to several cycles of freeze thawing. These findings indicate that Afp1 might act as a cryoprotective agent and thus, has great potential in biotechnology applications. [ABSTRACT FROM AUTHOR]

Psychrophilic and psychrotrophic bacteria play important roles in nutrient cycling in cold environments. These bacteria are suitable as model organisms for studying cold-adaptation, and sources of cold-active enzymes and metabolites for industrial applications. Here, we report the genome sequences of two Cryobacterium sp. strains SO1 and SO2. Genes coding major proteins related to cold- or thermal-stress adaptations and those with industrial applications found in their genomes are described. [ABSTRACT FROM AUTHOR]

To survive in extremely cold environments, psychrophilic microorganisms produce exopolysaccharides (EPS), which are carbohydrate polymers that constitute a substantial component of the extracellular polymers surrounding cells of these microorganisms. EPS can interfere with RNA extraction and decrease the purity of the RNA extracted from EPS producing microorganisms. In this work, six commercial RNA extraction kits and two published protocols for RNA extraction were evaluated for total RNA extraction from the psychrophilic yeast, Glaciozyma antarctica. All the protocols were optimised to obtain the highest quality of total RNA. The results show that all of the tested commercial kits and the tested conventional methods yielded RNA from G. antarctica, albeit with varying quality. The protocol that utilises TRIzol® reagent was the most effective method for isolating total RNA from G. antarctica of which this protocol resulted in the highest RNA yield and purity compared to other methods. This method of RNA extraction produced RNA of sufficient quality for reverse transcriptase PCR (RT-PCR) to detect the expression of the G. antarctica delta 9-fatty acid desaturase gene as well as for the construction of a G. antarctica cDNA library. [ABSTRACT FROM AUTHOR]

The cytotoxic activities of several diterpenoid constituents of the medicinal plant, “hempedu bumi,” or Andrographis paniculata Nees (Acanthaceae), were evaluated. The seven diterpenoid compounds used were andrographolide, 14-deoxyandrographolide, andrographiside, deoxyandrographiside, 14-deoxy-12-methoxyandrographolide, neoandrographolide, and 14-deoxy-11,12-didehydroandrographolide. The activities of these compounds were evaluated with various human tumor cell lines such as Caov-3 (human ovarian carcinoma cell line), T-47D (human breast carcinoma cell line), Hs-578T (human breast carcinoma cell line), Hep G2 (human hepatocarcinoma cell line), and NCI–H23 (human lung adenocarcinoma cell line). Cell survival was measured using the MTS assay after 72 h of incubation. Andrographolide, neoandrographolide, andrographiside, deoxyandrographiside, and 14-deoxy-12-methoxyandrographolide appeared to be noncytotoxic against all the cell lines. Only 14-deoxyandrographolide and 14-deoxy-11,12-didehydroandrographolide exhibited cytotoxic activities (based on EC 50 values), but this was limited to the T-47D cell line (EC 50 values of 2.8 µg/ml and 1.5 µg/ml, respectively). As one of the principle diterpenoids of Andrographis paniculata , 14-deoxy-11,12-didehydroandrographolide appeared to be the most potent when compared with the rest of the compounds examined. The effects of 14-deoxy-11,12-didehydroandrographolide on T-47D cells were further confirmed to be nonapoptotic, non-necrotic, but programmed in nature, as demonstrated using a DNA fragmentation detection assay, Trypan blue exclusion assay, and annexin V–propidium iodide staining. [ABSTRACT FROM AUTHOR]