• Volume 37, Issue 6

December 2012,   pages  921-1121

• Clipboard: Endocannabinoid signalling is required for estrogen-dependent modulation of inhibitory transmission

• Clipboard: Etroplus suratensis (Bloch), the State Fish of Kerala

• Early modern natural history: Contributions from the Americas and India

• What history tells us XXIX. Transfers from plant biology: From cross protection to RNA interference and DNA vaccination

• Crowding, molecular volume and plasticity: An assessment involving crystallography, NMR and simulations

The discrepancy between the X-ray and NMR structures of Mycobacterium tuberculosis peptidyl-tRNA hydrolase in relation to the functionally important plasticity of the molecule led to molecular dynamics simulations. The X-ray and the NMR studies along with the simulations indicated an inverse correlation between crowding and molecular volume. A detailed comparison of proteins for which X-ray and the NMR structures appears to confirm this correlation. In consonance with the reported results of the investigations in cellular compartments and aqueous solution, the comparison indicates that the crowding results in compaction of the molecule as well as change in its shape, which could specifically involve regions of the molecule important in function. Crowding could thus influence the action of proteins through modulation of the functionally important plasticity of the molecule.

• Efficient multi-site-directed mutagenesis directly from genomic template

In this article, the traditional multi-site-directed mutagenesis method based on overlap extension PCR was improved specifically for complicated templates, such as genomic sequence or complementary DNA. This method was effectively applied for multi-site-directed mutagenesis directly from mouse genomic DNA, as well as for combination, deletion or insertion of DNA fragments.

• Transcription of gD and gI genes in BHV1-infected cells

Glycoprotein D (gD) and glycoprotein I (gI) genes of bovine herpes virus 1 (BHV1) are contiguous genes with 141 bp region between the two open reading frames (ORFs). Expression of gD and gI from a bicistronic construct containing complete gD and gI gene has been reported either through internal ribosome entry site (IRES)-like element or through the scanning and leakage model (Mukhopadhyay 2000). We here show by computational and experimental means that gD is expressed solely as bicistronic transcript comprising gD and gI coding region in BHV1-infected cells. gI ORF was also shown to express separately. An IRES-like element was also predicted by IRES predicting software in the middle of the gD coding region; within that region a putative promoter was also identified by promoterscan. The intergenic region between the two ORF showed extensive secondary structure which brings the stop codon of gD very close to start codon of gI gene. gD gene transcript in BHV1-infected cells was solely bicistronic. gI transcript was also present in the BHV1-infected cells but in low copy number. The results indicate that gI is probably transcribed from its own transcript in BHV1-infected cells.

• Cx43, ZO-1, alpha-catenin and beta-catenin in cataractous lens epithelial cells

Specimens of the anterior lens capsule with an attached monolayer of lens epithelial cells (LECs) were obtained from patients (𝑛=52) undergoing cataract surgery. Specimens were divided into three groups based on the type of cataract: nuclear cataract, cortical cataract and posterior subcapsular cataract (PSC). Clear lenses (𝑛=11) obtained from donor eyes were used as controls. Expression was studied by immunofluorescence, real-time PCR and Western blot. Statistical analysis was done using the student’s 𝑡-test. Immunofluorescence results showed punctate localization of Cx43 at the cell boundaries in controls, nuclear cataract and PSC groups. In the cortical cataract group, cytoplasmic pools of Cx43 without any localization at the cell boundaries were observed. Real-time PCR results showed significant up-regulation of Cx43 in nuclear and cortical cataract groups. Western blot results revealed significant increase in protein levels of Cx43 and significant decrease of ZO-1 in all three cataract groups. Protein levels of alpha-catenin were decreased significantly in nuclear and cortical cataract group. There was no significant change in expression of beta-catenin in the cataractous groups. Our findings suggest that ZO-1 and alpha-catenin are important for gap junctions containing Cx43 in the LECs. Alterations in cell junction proteins may play a role during formation of different types of cataract.

• Comparative analysis of fecal microflora of healthy full-term Indian infants born with different methods of delivery (vaginal vs cesarean): Acinetobacter sp. prevalence in vaginally born infants

In this study fecal microflora of human infants born through vaginal delivery (VB) and through cesarean section (CB) were investigated using culture-independent 16S rDNA cloning and sequencing approach. The results obtained clearly revealed that fecal microbiota of VB infants distinctly differ from those in their counterpart CB infants. The intestinal microbiota of infants delivered by cesarean section appears to be more diverse, in terms of bacteria species, than the microbiota of vaginally delivered infants. The most abundant bacterial species present in VB infants were Acinetobacter sp., Bifidobacterium sp. and Staphylococcus sp. However, CB infant’s fecal microbiota was dominated with Citrobacter sp., Escherichia coli and Clostridium difficile. The intestinal microbiota of cesarean section delivered infants in this study was also characterized by an absence of Bifidobacteria species. An interesting finding of our study was recovery of large number of Acinetobacter sp. consisting of Acinetobacter pittii (former Acinetobacter genomic species 3), Acinetobacter junii and Acinetobacter baumannii in the VB infants clone library. Among these, Acinetobacter baumannii is a known nosocomial pathogen and Acinetobacter pittii (genomic species 3) is recently recognized as clinically important taxa within the Acinetobacter calcoaceticusAcinetobacter baumannii (ACB) complex. Although none of the infants had shown any sign of clinical symptoms of disease, this observation warrants a closer look.

• Sialyl Lewis x expression in cervical scrapes of premalignant lesions

Sialylated oligosaccharides of glycoproteins and glycolipids have been implicated in tumour progression and metastases. Altered expression of glycosidic antigens has been reported in cervical cancer. In cervix premalignant lesions, an increased expression of sialic acid has been reported. In the present study we determined the expression profiles of the glycosidic antigens Tn, sialyl Tn (sTn), Lewis a (Lea), sialyl Lewis a (sLea), Lewis x (Lex) and sialyl Lewis x (sLex) in cervical scrapes with cytological diagnoses of normal, low-grade squamous intraepithelial lesions (LGSIL) and high-grade squamous intraepithelial lesions (HGSIL). Cervical scrapings were collected to detect tumour antigens expressions by flow cytometry using monoclonal antibodies. Cytometry analysis of Tn, sTn, Lea and Lex did not reveal differences at the expression level among groups. The number of positive cells to sLea antigen increased in the HGSIL group with respect to the normal group (𝑝=0.0495). The number of positive cells to sLex antigen in the samples increased with respect to the grade of squamous intraepithelial lesion (SIL) (𝑝 &lt; 0.001, Mann–Whitney U test). The intensity of expression of this antigen increased in the HGSIL samples with respect to normal samples (𝑝 &lt; 0.0068). sLex antigen could be a candidate to be used as biomarker for the early diagnosis of cervical cancer.

• Swainsonine promotes apoptosis in human oesophageal squamous cell carcinoma cells in vitro and in vivo through activation of mitochondrial pathway

Swainsonine, a natural indolizidine alkaloid, has been reported to have antitumour effects, and can induce apoptosis in human gastric and lung cancer cells. In the present study, we evaluated the antitumour effects of swainsonine on several oesophageal squamous cell carcinoma cells and investigated relative molecular mechanisms. Swainsonine treatment inhibited the growth of Eca-109, TE-1 and TE-10 cells in a concentration-dependent manner as measured by MTT assay. Morphological observation, DNA laddering detection and flow cytometry analysis demonstrated that swainsonine treatment induced Eca-109 cell apoptosis in vitro. Further results showed that swainsonine treatment up-regulated Bax, down-regulated Bcl-2 expression, triggered Bax translocation to mitochondria, destructed mitochondria integrity and activated mitochondria-mediated apoptotic pathway, followed by the release of cytochrome c, which in turn activated caspase-9 and caspase-3, promoted the cleavage of PARP, resulting in Eca-109 cell apoptosis. Moreover, swainsonine treatment inhibited Bcl-2 expression, promoted Bax translocation, cytochrome c release and caspase-3 activation in xenograft tumour cells, resulting in a significant decrease of tumour volume and tumour weight in the swainsonine-treated xenograft mice groups compared with that in the control group. Taken together, this study demonstrated that swainsonine inhibited Eca-109 cells growth through activation of mitochondria-mediated caspase-dependent pathway.

• Effects of diet-induced hypercholesterolemia on amyloid accumulation in ovariectomized mice

A central hypothesis in the study of Alzheimer’s disease (AD) is the accumulation and aggregation of 𝛽-amyloid peptide (A𝛽). Recent epidemiological studies suggest that patients with elevated cholesterol and decreased estrogen levels are more susceptible to AD through A𝛽 accumulation. To test the above hypothesis, we used ovariectomized with diet-induced hypercholesterolemia (OVX) and hypercholesterolemia (HCL) diet alone mouse models. HPLC analysis reveals the presence of beta amyloid in the OVX and HCL mice brain. Congo red staining analysis revealed the extent of amyloid deposition in OVX and hypercholesterolemia mice brain. Overall, A𝛽 levels were higher in OVX mice than in HCL. Secondly, estrogen receptors 𝛼 (ER𝛼) were assessed by immunohistochemistry and this suggested that there was a decreased expression of ER 𝛼 in OVX animals when compared to hypercholesterolemic animals. A𝛽 was quantified by Western blot and ELISA analysis. Overall, Aβ levels were higher in OVX mice than in HCL mice. Our experimental results suggested that OVX animals were more susceptible to AD with significant increase in A𝛽 peptide.

• Identification and characterization of a gene encoding a putative lysophosphatidyl acyltransferase from Arachis hypogaea

Lysophosphatidyl acyltransferase (LPAT) is the important enzyme responsible for the acylation of lysophosphatidic acid (LPA), leading to the generation of phosphatidic acid (PA) in plant. Its encoding gene is an essential candidate for oil crops to improve oil composition and increase seed oil content through genetic engineering. In this study, a full-length AhLPAT4 gene was isolated via cDNA library screening and rapid amplification of cDNA ends (RACE); our data demonstrated that AhLPAT4 had 1631 nucleotides, encoding a putative 43.8 kDa protein with 383 amino acid residues. The deduced protein included a conserved acyltransferase domain and four motifs (I–IV) with putative LPA and acyl-CoA catalytic and binding sites. Bioinformatic analysis indicated that AhLPAT4 contained four transmembrane domains (TMDs), localized to the endoplasmic reticulum (ER) membrane; detailed analysis indicated that motif I and motifs II–III in AhLPAT4 were separated by the third TMD, which located on cytosolic and ER luminal side respectively, and hydrophobic residues on the surface of AhLPAT4 protein fold to form a hydrophobic tunnel to accommodate the acyl chain. Subcellular localization analysis confirmed that AhLPAT4 was a cytoplasm protein. Phylogenetic analysis revealed that AhLPAT4 had a high homology (63.7–78.3%) with putative LPAT4 proteins from Glycine max, Arabidopsis thaliana and Ricinus communis. AhLPAT4 was ubiquitously expressed in diverse tissues except in flower, which is almost undetectable. The expression analysis in different developmental stages in peanut seeds indicated that AhLPAT4 did not coincide with oil accumulation.

• COCHLEATA controls leaf size and secondary inflorescence architecture via negative regulation of UNIFOLIATA (LEAFY ortholog) gene in garden pea Pisum sativum

UNIFOLIATA [(UNI) or UNIFOLIATA-TENDRILLED ACACIA (UNI-TAC)] expression is known to be negatively regulated by COCHLEATA (COCH) in the differentiating stipules and flowers of Pisum sativum. In this study, additional roles of UNI and COCH in P. sativum were investigated. Comparative phenotyping revealed pleiotropic differences between COCH (UNI-TAC and uni-tac) and coch (UNI-TAC and uni-tac) genotypes of common genetic background. Secondary inflorescences were bracteole-less and bracteolated in COCH and coch genotypes, respectively. In comparison to the leaves and corresponding sub-organs and tissues produced on COCH plants, coch plants produced leaves of 1.5-fold higher biomass, 1.5-fold broader petioles and leaflets that were 1.8-fold larger in span and 1.2-fold dorso-ventrally thicker. coch leaflets possessed epidermal cells 1.3-fold larger in number and size, 1.4-fold larger spongy parenchyma cells and primary vascular bundles with 1.2-fold larger diameter . The transcript levels of UNI were at least 2-fold higher in coch leaves and secondary inflorescences than the corresponding COCH organs. It was concluded that COCH negatively regulated UNI in the differentiating leaves and secondary inflorescences and thereby controlled their sizes and/or structures. It was also surmised that COCH and UNI (LFY homolog) occur together widely in stipulate flowering plants.

• Characteristic differences in metabolite profile in male and female plants of dioecious Piper betle L.

Piper betle is a dioecious pan-Asiatic plant having cultural and medicinal uses. It belongs to the family Piperaceae and is a native of the tropics although it is also cultivated in subtropical areas. Flowering in P. betle occurs only in tropical regions. Due to lack of inductive floral cycles the plant remains in its vegetative state in the subtropics. Therefore, due to lack of flowering, gender distinction cannot be made the in the subtropics. Gender distinction in P. betle in vegetative state can be made using Direct Analysis in Real Time Mass Spectroscopy (DARTMS), a robust high-throughput method. DARTMS analysis of leaf samples of two male and six female plants showed characteristic differences in the spectra between male and female plants. Semi-quantitative differences in some of the identified peaks in male and female landraces showed gender-based differences in metabolites. Cluster analysis using the peaks at m/z 151, 193, 235 and 252 showed two distinct clusters of male and female landraces. It appears that male and female plants besides having flowers of different sexes also have characteristic differences in the metabolites representing two metabolic types.

• Rates and pattern of ovule abortion vis-à-vis in situ pollen germination in some populations of Trifolium fragiferum L.

The present study is based on four populations of Trifolium fragiferum L. of the family Fabaceae growing at four different sites in Jammu region, India. The species, which grows as a common weed in the area of study, follows an annual life cycle of about 3½month in the subtropical climates of Jammu region. While all of these populations were recorded in full bloom during February and March, they displayed a temporal scatter. Detailed studies revealed these population types to be morphologically similar but distinct in the many aspects studied. An interesting phenomenon noted for the plants of this species was in situ pollen germination, which was recorded in about 28.8% of the flowers studied. The species under investigation also showed an appreciable amount of ovule abortion. The ovule abortion in pistils was found to be non-random, with the peduncular ovule aborting at a higher rate than the stylar one. The rates and patterns of ovule abortion were studied vis-à-vis in situ pollen germination and were compared between different populations. Interesting results were obtained, indicative of the fact that precocious pollen germination does affect the ovule abortion in one way or other.

• Aluminium localization and toxicity symptoms related to root growth inhibition in rice (Oryza sativa L.) seedlings

We correlated root growth inhibition with aluminium (Al3+) localization and toxicity symptoms in rice roots using seedlings of two genotypes (tolerant and sensitive) that were exposed to different AlCl3 concentrations. Al3+ localization was evaluated by hematoxylin in primary roots and by morin in cross-sections of the root tips. Neutral invertase enzyme activity and callose (1$\to$3, 𝛽-D-glucan) accumulation were observed and compared with Al3+ accumulation sites. Root growth was inhibited by Al3+ in a concentration-specific manner and proportional to the increase of hematoxylin staining, being more pronounced in the sensitive genotype. Morin staining showed the presence of Al3+ deep within the roots of the sensitive genotype, indicating that the metal was able to penetrate beyond the first few cell layers. In the tolerant genotype, Al3+ penetration was restricted to the first two cell layers. Ruptures in exodermis and epidermis layers by lateral root protrusions in both genotypes allowed Al3+ to enter into the roots. More intense activity of invertase in roots of the tolerant genotype was also observed, which could be related to greater root growth of this cultivar when submitted to Al3+ stress. Moreover, Al3+-induced callose accumulation was a late response occurring in the same areas where Al3+ was present.

• What in silico molecular docking can do for the `bench-working biologists'

Required by an increasing amount of scientists, the in silico docking field is in full expansion, new algorithms and methods appearing at an exponential rate. The sheer range of available programs is overwhelming for the bench-working biologist, which is often discouraging by the lack of a graphical user interface, good user manual or literature to validate a given program. This mini-review attempts to present the docking problem and available solutions from a non-bioinformatician point of view and makes a selection of the available servers and programs. These tools are evaluated from several points of view, as numbers of citations, ease of usage and computer requirements. Finally, the capabilities and limitations as well as specific applications of in silico docking techniques are presented.

• The hnRNP A1 homolog Hrp36 is essential for normal development, female fecundity, omega speckle formation and stress tolerance in Drosophila melanogaster

Hrp36/Hrb87F is one of the most abundant and well-characterized hnRNP A homolog in Drosophila and is shown to have roles in regulation of alternative splicing, heterochromatin formation, neurodegeneration, etc. Yet, hrp36 null individuals were reported to be viable and without any apparent phenotype, presumably because of overlapping functions provided by Hrp38 and related proteins. Here we show that loss of both copies of hrp36 gene slows down development with significant reduction in adult life span, decreased female fecundity and high sensitivity to starvation and thermal stresses. In the absence of Hrp36, the nucleoplasmic omega speckles are nearly completely disrupted. The levels of nuclear matrix protein Megator and the chromatin remodeller ISWI are significantly elevated in principal cells of larval Malpighian tubules, which also display additional endoreplication cycles and good polytene chromosomes. We suggest that besides the non-coding hsr𝜔-n transcripts, the Hrp36 protein is also a core constituent of omega speckles. The heat-shock-induced association of other hnRNPs at the hsr𝜔 locus is affected in hrp36 null cells, which may be one of the reasons for their high sensitivity to cell stress. Therefore, in spite of the functional redundancy provided by Hrp38, Hrp36 is essential for normal development and for survival under conditions of stress.

• Acknowledgements

• # Journal of Biosciences

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July 2019

• # Editorial Note on Continuous Article Publication

Posted on July 25, 2019