• Volume 33, Issue 1

March 2008,   pages  1-155

• Clipboard: Selecting cells to make cerebral cortex

• Commentary: Sex determination: Are two mechanisms better than one?

• Biological time is fractal: Early events reverberate over a life time

• What history tells us XII. Boris Ephrussi's continuing efforts to create a enetics of differentiation"

• A gene-trap strategy identifies quiescence-induced genes in synchronized myoblasts

Cellular quiescence is characterized not only by reduced mitotic and metabolic activity but also by altered gene expression. Growing evidence suggests that quiescence is not merely a basal state but is regulated by active mechanisms. To understand the molecular programme that governs reversible cell cycle exit, we focused on quiescence-related gene expression in a culture model of myogenic cell arrest and activation. Here we report the identification of quiescence-induced genes using a gene-trap strategy. Using a retroviral vector, we generated a library of gene traps in C2C12 myoblasts that were screened for arrest-induced insertions by live cell sorting (FACS-gal). Several independent genetrap lines revealed arrest-dependent induction of 𝛽gal activity, confirming the efficacy of the FACS screen. The locus of integration was identified in 15 lines. In three lines, insertion occurred in genes previously implicated in the control of quiescence, i.e. EMSY – a BRCA2-interacting protein, p8/com1– a p300HAT-binding protein and MLL5 – a SET domain protein. Our results demonstrate that expression of chromatin modulatory genes is induced in G0, providing support to the notion that this reversibly arrested state is actively regulated.

• Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis

Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis. In the present investigation, we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis. The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver. Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis. Platelet aggregation was also measured in the presence of the eNOS inhibitor, diphenylene iodinium chloride (DIC). The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects. During the course of platelet aggregation, concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples, whereas the elevation was not significant in platelets of patients with cirrhosis. A parallel increase was observed in the levels of NO and NOS activity. In the presence of the eNOS inhibitor, platelet aggregation was enhanced and accompanied by an elevated calcium level. The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS. Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis. The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance, and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.

• Apoptosis induced by (di-isopropyloxyphoryl-Trp)2-Lys-OCH3 in K562 and HeLa cells

According to the method used in our laboratory, our group synthesized (DIPP-Trp)2-Lys-OCH3. It inhibited the proliferation of K562 and HeLa cells in a dose- and time-dependent manner with an IC50 of 15.12 and 42.23 𝜇M, respectively. (DIPP-Trp)2-Lys-OCH3 induced a dose-dependent increase of the G2/M cell population in K562 cells, and S cell population in HeLa cells; the sub-G0 population increased dramatically in both cell lines as seen by PI staining experiments using a FACS Calibur Flow cytometer (BeckmanCoulter, USA). Phosphatidylserine could significantly translocate to the surface of the membrane in (DIPP-Trp)2-Lys-OCH3-treated K562 and HeLa cells. The increase of an early apoptotic population was observed in a dose-dependent manner by both annexin-FITC and PI staining. It was concluded that (DIPP-Trp)2-Lys-OCH3 not only induced cells to enter into apoptosis, but also affected the progress of the cell cycle. It may have arrested the K562 and HeLa cells in the G2/M, S phases, respectively. The apoptotic pathway was pulsed at this point, resulting in the treated cells entering into programmed cell death. (DIPP-Trp)2-Lys-OCH3 is a potential anticancer drug that intervenes in the signalling pathway.

• Effect of tetrahydrocurcumin on insulin receptor status in type 2 diabetic rats: studies on insulin binding to erythrocytes

Curcumin is the most active component of turmeric. It is believed that curcumin is a potent antioxidant and anti-inflammatory agent. Tetrahydrocurcumin (THC) is one of the major metabolites of curcumin, and exhibits many of the same physiological and pharmacological activities as curcumin and, in some systems, may exert greater antioxidant activity than curcumin. Using circulating erythrocytes as the cellular mode, the insulin-binding effect of THC and curcumin was investigated. Streptozotocin (STZ)–nicotinamide-induced male Wistar rats were used as the experimental models. THC (80 mg/kg body weight) was administered orally for 45 days. The effect of THC on blood glucose, plasma insulin and insulin binding to its receptor on the cell membrane of erythrocytes were studied. Mean specific binding of insulin was significantly lowered in diabetic rats with a decrease in plasma insulin. This was due to a significant decrease in mean insulin receptors. Erythrocytes from diabetic rats showed a decreased ability for insulin–receptor binding when compared with THC-treated diabetic rats. Scatchard analysis demonstrated that the decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with erythrocytes of diabetic rats having less insulin receptor sites per cell than THC-treated rats. High affinity (Kd1), low affinity (Kd2) and kinetic analyses revealed an increase in the average receptor affinity of erythrocytes from THC-treated rats compared with those of diabetic rats. These results suggest that acute alteration of the insulin receptor on the membranes of erythrocytes occurred in diabetic rats. Treatment with THC significantly improved specific insulin binding to the receptors, with receptor numbers and affinity binding reaching near-normal levels. Our study suggests the mechanism by which THC increases the number of total cellular insulin binding sites resulting in a significant increase in plasma insulin. The effect of THC is more prominent than that of curcumin.

• Human papillomavirus genotyping by multiplex pyrosequencing in cervical cancer patients from India

Cervical cancer is a leading cause of cancer-related deaths among women in India. Human papillomavirus (HPV) infection is the causative agent of cervical cancer; and infection with the high-risk genotypes, predominantly HPV16 and 18, is the biggest risk factor. Vaccines targeting HPV16 and 18 have been found to confer protection in large-scale clinical trials. HPV genotyping has traditionally been carried out to screen the population “at risk” using indirect methods based on polymerase chain reaction (PCR) using consensus primers combined with various DNA hybridization techniques, and often followed by the sequencing of candidate products. Recently, a high-throughput and direct method based on DNA sequencing has been described for HPV genotyping using multiplex pyrosequencing. We present a pilot study on HPV genotyping of cervical cancer and non-malignant cervical samples using multiplex pyrosequencing. Using genomic DNA from cell lines, cervical biopsies, surgical tissues or formalin-fixed, paraffin-embedded tissue samples, we could successfully resolve 6 different HPV types out of the 7 tested, with their prevalence found to be in agreement with earlier reports. We also resolved coinfections with two different HPV types in several samples. An HPV16 genotype with a specific and recurrent sequence variation was observed in 8 cancer samples and one non-malignant sample. We find this technique eminently suited for high-throughput applications, which can be easily extended to large sample cohorts to determine a robust benchmark for HPV genotypes prevalent in India.

• Defence transcriptome profiling of Zingiber zerumbet (L.) Smith by mRNA differential display

Soft rot is a serious disease in ginger (Zingiber officinale Roscoe), imposing a considerable economic loss annually in all ginger-producing countries. In this study, mRNA differential display was employed to identify genes whose expression was altered in a soft rot-resistant accession of Zingiber zerumbet (L.) Smith, a wild relative of ginger, in response to Pythium aphanidermatum (Edson) Fitzp., which is the principal causative agent of soft-rot disease in ginger. Analysis using 68 primer combinations identified 70 differentially expressed transcript-derived fragments (TDFs), of which 34 TDFs were selected for further analysis following reverse northern screening. Cloning and sequence characterization of the 34 TDFs yielded a total of 54 distinct clones. Functional categorization of these clones revealed seven categories, of which the defence/stress/signalling group was the largest, with clones homologous to genes known to be actively involved in various pathogenesis-related functions in other plant species. The significance of these genes in relation to the resistance response in Z. zerumbet is discussed. This study has provided a pool of candidate genes for detailed molecular dissection of the defence mechanisms in Z. zerumbet and for accessing wild genetic resources for the transgenic improvement of ginger.

• Cloning and expression of antiviral/ribosome-inactivating protein from Bougainvillea xbuttiana

A full-length cDNA encoding ribosome-inactivating/antiviral protein (RIP/AVP) from the leaves of Bougainvillea xbuttiana was isolated. The cDNA consisted of 1364 nucleotides with an open reading frame (ORF) of 960 nucleotides encoding a 35.49 kDa protein of 319 amino acids. The deduced amino acid sequence has a putative active domain conserved in RIPs/AVPs and shows a varying phylogenetic relationship to the RIPs from other plant species. The deduced protein has been designated BBAP1 (Bougainvillea xbuttiana antiviral protein1). The ORF was cloned into an expression vector and expressed in E. coli as a fusion protein of ∼78 kDa. The cleaved and purified recombinant BBAP1 exhibited ribosome-inhibiting rRNA 𝑁-glycosidase activity, and imparted a high level of resistance against the tobacco mosaic virus (TMV).

• Molecular cloning and characterization of a gene encoding RING zinc finger ankyrin protein from drought-tolerant Artemisia desertorum

A RING zinc finger ankyrin protein gene, designated AdZFP1, was isolated from drought-tolerant Artemisia desertorum Spreng by mRNA differential display and RACE. Its cDNA was 1723 bp and encoded a putative protein of 445 amino acids with a predicted molecular mass of 47.9 kDa and an isoelectric point (pI) of 7.49. A typical C3HC4-type RING finger domain was found at the C-terminal region of the AdZFP1 protein, and several groups of ankyrin repeats were found at the N-terminal region. Alignments of amino acid sequence showed that AdZFP1 was 66% identical to the Arabidopsis thaliana putative RING zinc finger ankyrin protein AAN31869. Transcriptional analysis showed that AdZFP1 was inducible under drought stress in root, stem and leaf of the plant. Semi-quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the transcript of AdZFP1 was strongly induced by exogenous abscisic acid (ABA) and also by salinity, cold and heat to some extent. Overexpression of the AdZFP1 gene in transgenic tobacco enhanced their tolerance to drought stress.

• In vitro production of azadirachtin from cell suspension cultures of Azadirachta indica

The present study aimed to elucidate the effect of nutritional alteration on biomass content and azadirachtin production in cell suspensions of the elite neem variety crida-8. Variations in total nitrogen availability in the medium in terms of different ratios of nitrate:ammonium showed that the ratio 4:1 revealed a profound effect, leading to a 1.5-fold increase in the total extracellular azadirachtin production (5.59 mg/l) over the standard MS medium. Reduction in sucrose (15 mg/l) in the medium exhibited a reduction in biomass and absence of azadirachtin, whereas total phosphate reduction raised intracellular azadirachtin production (6.98 mg/l). An altered medium with a nitrate:ammonium ratio of 4:1 coupled with complete elimination of phosphate enhanced biomass by 36% (59.36 g/l).

• Contribution of pitcher fragrance and fluid viscosity to high prey diversity in a Nepenthes carnivorous plant from Borneo

Mechanisms that improve prey richness in carnivorous plants may involve three crucial phases of trapping: attraction, capture and retention. Nepenthes rafflesiana var. typica is an insectivorous pitcher plant that is widespread in northern Borneo. It exhibits ontogenetic pitcher dimorphism with the upper pitchers trapping more flying prey than the lower pitchers. While this difference in prey composition has been ascribed to differences in attraction, the contribution of capture and retention has been overlooked. This study focused on distinguishing between the prey trapping mechanisms, and assessing their relative contribution to prey diversity. Arthropod richness and diversity of both visitors and prey in the two types of pitchers were analysed to quantify the relative contribution of attraction to prey trapping. Rate of insect visits to the different pitcher parts and the presence or absence of a sweet fragrance was recorded to clarify the origin and mechanism of attraction. The mechanism of retention was studied by insect bioassays and measurements of fluid viscosity. Nepenthes rafflesiana was found to trap a broader prey spectrum than that previously described for any Nepenthes species, with the upper pitchers attracting and trapping a greater quantity and diversity of prey items than the lower pitchers. Capture efficiency was low compared with attraction or retention efficiency. Fragrance of the peristome, or nectar rim, accounted mainly for the observed non-specific, better prey attraction by the upper pitchers, while the retentive properties of the viscous fluid in these upper pitchers arguably explains the species richness of their flying prey. The pitchers of N. rafflesiana are therefore more than simple pitfall traps and the digestive fluid plays an important yet unsuspected role in the ecological success of the species.

• Mycobacteria and innate cells: critical encounter for immunogenicity

Protective immunity against mycobacterial infections such as Mycobacterium tuberculosis is mediated by interactions between specific T cells and activated macrophages. To date, many aspects of mycobacterial immunity have shown that innate cells are the key elements that substantially influence the subsequent adaptive host response. During the early phases of infection, phagocytic cells and innate lymphocyte subsets play a pivotal role. Here we summarize the findings of recent investigations on macrophages, dendritic cells and 𝛾𝛿 T lymphocytes in the response to mycobacteria.

• A songbird forebrain area potentially involved in auditory discrimination and memory formation

Songbirds rely on auditory processing of natural communication signals for a number of social behaviors, including mate selection, individual recognition and the rare behavior of vocal learning – the ability to learn vocalizations through imitation of an adult model, rather than by instinct. Like mammals, songbirds possess a set of interconnected ascending and descending auditory brain pathways that process acoustic information and that are presumably involved in the perceptual processing of vocal communication signals. Most auditory areas studied to date are located in the caudomedial forebrain of the songbird and include the thalamo-recipient field L (subfields L1, L2 and L3), the caudomedial and caudolateral mesopallium (CMM and CLM, respectively) and the caudomedial nidopallium (NCM). This review focuses on NCM, an auditory area previously proposed to be analogous to parts of the primary auditory cortex in mammals. Stimulation of songbirds with auditory stimuli drives vigorous electrophysiological responses and the expression of several activity-regulated genes in NCM. Interestingly, NCM neurons are tuned to species-specific songs and undergo some forms of experience-dependent plasticity in-vivo. These activity-dependent changes may underlie long-term modifications in the functional performance of NCM and constitute a potential neural substrate for auditory discrimination. We end this review by discussing evidence that suggests that NCM may be a site of auditory memory formation and/or storage.

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