Characterization of gp120 and its single-chain derivatives, gp120-CD4D12 and gp120-M9: implications for targeting the CD4i epitope in human immunodeficiency virus vaccine design

Single-chain derivatives of JRFL gp120 linked to the first two domains of human CD4 (gp120-CD4D12) or to the CD4 miniprotein analog CD4M9 (gp120-M9), have been constructed. Biacore studies revealed that gp120-CD4D12 and gp120-M9 bound to antibody 17b with dissociation constants of 0.8 and 25 nM, respectively, at pH 7.0, while gp120 alone did not bind. The binding of gp120-CD4D12 to 17b is not affected by the addition of excess soluble CD4D12, while the binding of gp120-M9 is enhanced. This finding indicates that the M9 component of the single chain interacts relatively weakly with gp120 and can be displaced by soluble CD4D12. Immunogenicity studies of gp120, gp120-CD4D12, and gp120-M9 were carried out with guinea pigs. All three molecules were highly immunogenic. The resulting antisera were examined for neutralizing activities against various human immunodeficiency virus type 1 isolates. Broadly neutralizing activity was observed only with sera generated against gp120-CD4D12. These antisera were depleted of anti-CD4D12 antibodies by being passed over a column containing immobilized CD4D12. The depleted sera showed a loss of broadly neutralizing activity. Sera that were affinity purified over a column containing immobilized gp120-M9 also lacked such neutralizing activity. This finding suggests that the broadly neutralizing response observed is exclusively due to anti-CD4 antibodies. Competition experiments showed that only antisera generated against gp120-CD4D12 competed with the CD4i antibody 17b and that this activity was not affected by depletion of anti-CD4 antibodies. The data indicate that although antibodies targeting the CD4i epitope were generated by the gp120-CD4D12 immunogen, these antibodies were nonneutralizing.     

Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents

Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS deficiency whereas a reverse trend was observed with apicidin. Together, these finding provide a new strategy for differential mitochondrial targeting in cancer therapy.Cancer cells favor glycolysis over oxidative phosphorylation (OXPHOS) to meet their energy demand,¹ suggesting that they have adapted to survive and proliferate in the absence of fully functional mitochondria. Research in the last two decades demonstrates that, in addition to generation of energy, mitochondria including cancer cell mitochondria regulate multiple cellular signaling pathways encompassing cell death, proliferation, cellular redox balance, and metabolism.^{2, 3} As cancer cells possess defects in these pathways that provide an opportunity to target this organelle for therapeutic purposes. Subsequently, several agents have been developed that target cancer cell mitochondria to induce apoptosis, a cell death pathway, and eradicate cancer cells.^{4, 5} Cancer cell mitochondria harbor several proapoptotic proteins including cytochrome c, which is released from mitochondria in response to anticancer agents and activates caspases to execute apoptosis.^{5, 6} Thus, anticancer agents that induce cytochrome c release from mitochondria will be beneficial for induction of apoptosis in cancer cells. Indeed, several such agents have been developed, which include inhibitors targeting prosurvival Bcl-2 family members including Bcl-2, Bcl-xL, and Mcl-1.^{7, 8, 9} Unfortunately, cancer cells have developed multiple mechanisms to resist or overcome cytochrome c release and evade apoptosis.Although underlying mechanisms of cancer cell resistance to apoptosis are still undefined, the OXPHOS defect is known to be one of the key reasons for the attenuation of apoptosis in cancer cells.^{10, 11} Multiple lines of evidence support the notion that cancer cell survival and proliferation commonly associate with an OXPHOS defect in cancer.^{1, 12} Active OXPHOS is an efficient form of respiration but also regulates apoptosis through the OXPHOS complexes. The OXPHOS system consists of five multimeric protein complexes (I, II, III, IV, and V). The components of these complexes (except complex-II) are encoded by both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA).^{12, 13} Thus mutations, deletions, and translocations in either mtDNA or nDNA can potentially result in OXPHOS deficiency. MtDNA mutations associate with inhibition of apoptosis, induction of angiogenesis, invasion and metastasis of various types of cancer.^{3, 12, 14} Thus, mtDNA could potentially be an important target to restore cell death in cancer and attenuate cancer growth. Therefore, there is an urgent need to investigate the role of OXPHOS in the molecular mechanisms underlying cancer cell death.We investigated the effects of several anticancer agents of different classes including DNA-damaging agents (etoposide and doxorubicin), protein kinase inhibitors (staurosporine and sorafenib), mitotic inhibitor (taxol), ER stressor/inhibitor of Ca²⁺-ATPases (thapsigargin), and histone deacetylase (HDAC) inhibitor (apicidin) on mtDNA. We also determined the impact of OXPHOS defects on apoptosis induction by these agents. Although most anticancer agents induced caspase activation and apoptosis, the mtDNA level was elevated maximally by etoposide and it was not modulated by a caspase inhibitor but reduced by an autophagy inhibitor. Induction of mtDNA is associated with increased reactive oxygen species (ROS) production and elevated mitochondrial mass. Pharmacologic inhibition of OXPHOS complexes reduced the etoposide-induced elevation in mtDNA, suggesting the involvement of these complexes in etoposide-induced apoptosis. Together, we define the impact of mtDNA and OXPHOS function on mitochondrial apoptosis, which has significance in restoring cancer cell apoptosis for therapeutic purposes.     

Enhanced Expression of Rabies Virus Surface G-Protein in Escherichia coli using SUMO Fusion

Fusion systems are known to increase the expression of difficult to express recombinant proteins in soluble form to facilitate their purification. Rabies glycoprotein was also tough to express at sufficient level in soluble form in both E. coli and plant. The present work was aimed to over-express and purify this membrane protein from soluble extract of E. coli. Fusion of Small Ubiqutin like Modifier (SUMO) with rabies glycoprotein increased ~1.5 fold higher expression and ~3.0 fold solubility in comparison to non-fused in E. coli. The SUMO fusion also simplified the purification process. Previously engineered rabies glycoprotein gene in tobacco plants provides complete protection to mice, but the expression was very low for purification. Our finding demonstrated that the SUMO-fusion was useful for enhancing expression and solubility of the membrane protein and again proves to be a good alternative technology for applications in biomedical and pharmaceutical research.     

3-Formylchromone interacts with cysteine 38 in p65 protein and with cysteine 179 in IκBα kinase, leading to down-regulation of nuclear factor-κB (NF-κB)-regulated gene products and sensitization of tumor cells

3-Formylchromone (3-FC) has been associated with anticancer potential through a mechanism yet to be elucidated. Because of the critical role of NF-κB in tumorigenesis, we investigated the effect of this agent on the NF-κB activation pathway. Whether activated by inflammatory agents (such as TNF-α and endotoxin) or tumor promoters (such as phorbol ester and okadaic acid), 3-FC suppressed NF-κB activation. It also inhibited constitutive NF-κB expressed by most tumor cells. This activity correlated with sequential inhibition of IκBα kinase (IKK) activation, IκBα phosphorylation, IκBα degradation, p65 phosphorylation, p65 nuclear translocation, and reporter gene expression. We found that 3-FC inhibited the direct binding of p65 to DNA, and this binding was reversed by a reducing agent, thus suggesting a role for the cysteine residue. Furthermore, mutation of Cys38 to Ser in p65 abolished this effect of the chromone. This result was confirmed by a docking study. 3-FC also inhibited IKK activation directly, and the reducing agent reversed this inhibition. Furthermore, mutation of Cys179 to Ala in IKK abolished the effect of the chromone. Suppression of NF-κB activation led to inhibition of anti-apoptotic (Bcl-2, Bcl-xL, survivin, and cIAP-1), proliferative (cyclin D1 and COX-2), invasive (MMP-9 and ICAM-1), and angiogenic (VEGF) gene products and sensitization of tumor cells to cytokines. Thus, this study shows that modification of cysteine residues in IKK and p65 by 3-FC leads to inhibition of the NF-κB activation pathway, suppression of anti-apoptotic gene products, and potentiation of apoptosis in tumor cells.     

Genetic diversity and population structure of <Emphasis Type="Italic">Litsea glutinosa</Emphasis> (Lour.) in Central India

Litsea glutinosa (Lour.), one of the most dwindling forest species in central India, is represented by highly fragmented populations that have been drastically reduced for the last 40 years, promulgating government ban on its extraction. For the first time with the help of ISSR markers, we investigated genetic variation and population structure of L. glutinosa in central Indian states. A total of 84 genotypes from 10 populations covering the entire potential pockets of the species in central India were collected. The percentage of polymorphic loci ranged from 44.79% (Rewa) to 94.79% (Marvahi) with a mean value of 70.10%. The sampled populations harbored high level of genetic diversity (mean h?=?0.294 and I?=?0.424) that was partitioned more within populations (73%) than between populations (27%). Bayesian structure analysis revealed the existence of four admixed genetic pools in L. glutinosa. The unsustainable extraction rather than genetic factor seems to be responsible for population fragmentation and dwindling status of this species. The dioecious nature of the species advocates an in-situ conservation to be the most suited approach for which Chhindwara, Jagdalpur, Balaghat and Jabalpur populations are appropriate.     

Plasma concentrations of luteinizing hormone and progesterone during superovulation of dairy cows using follicle stimulating hormone or pregnant mare serum gonadotrophin

Eighteen lactating Holstein cows were randomly divided into three groups of equal size. Six cows were not superovulated; the remaining cows were superovulated using either FSH-P or PMSG beginning on Day 12 of the estrous cycle (day of ovulation = Day 0). Animals treated with FSH-P were injected intramuscularly (i.m.) with 4 mg FSH-P every 12 h for 5 d. PMSG was administered i.m. as a single injection of 2350 IU. Cloprostenol (PG, 500 ug) was injected i.m. 56 and 72 h after commencement of treatment and at the same time in the cycle of controls. All cows were inseminated 56, 68 and 80 h after the first PG injection. Blood samples (5 ml) were collected daily and every 15 min for a period of 9 h on Days -1, 0, 2, 8 and 10, with continuous blood sampling at 15-min intervals during Days 3 to 6. Ovulation rate was 27.7 +/- 8.22 in animals treated with PMSG, and 8.0 +/- 3.2 embryos per donor were recovered. In the FSH group, ovulation rate was 8.3 +/- 1.48 and 3.0 +/- 1.1 embryos per donor were recovered. Progesterone concentrations were similar in all three groups until the onset of the LH surge, when progesterone concentrations were greater (P<0.05) in animals of the PMSG group. After the preovulatory LH surge, concentrations of progesterone started increasing earlier (44 h) in cows treated with PMSG, followed by FSH-treated cows (76 h) and controls (99 h). The LH surge occurred earlier (P<0.05) in PMSG-treated cows (37 h after first PG treatment), than in animals treated with FSH-P (52 h) or controls (82 h). In animals treated with FSH-P, the magnitude of the preovulatory LH surge (24.2 +/- 1.02 ng/ml) was higher (P<0.05) than in the other two groups (PMSG = 17.1 +/- 2.04 ng/ml; control, 16.7 +/- 1.24 ng/ml). Superovulation with FSH-P or PMSG did not affect either mean basal LH concentration, frequency or amplitude of LH pulses during Days -1, 0, 2, 3, presurge periods, or Days 8 and 10 post-treatment. At ovariectomy, 8 d post-estrus, more follicles > 10 mm diam. were observed in the ovaries after treatment with PMSG (8.5 +/- 5.66) than after treatment with FSH-P (0.7 +/- 0.42) (P<0.05). Maximum concentrations of PMSG were measured 24 h after administration. Following this peak, PMSG levels declined with two slopes, with half-lives of 36 h and 370 h.     

Mapping QTLs for 15 morpho-metric traits in Arabidopsis thaliana using Col-0 × Don-0 population

Genome wide quantitative trait loci (QTL) mapping was conducted in Arabidopsis thaliana using F2 mapping population (Col-0 × Don-0) and SNPs markers. A total of five linkage groups were obtained with number of SNPs varying from 45 to 59 per linkage group. The composite interval mapping detected a total of 36 QTLs for 15 traits and the number of QTLs ranged from one (root length, root dry biomass, cauline leaf width, number of internodes and internode distance) to seven (for bolting days). The range of phenotypic variance explained (PVE) and logarithm of the odds ratio of these 36 QTLs was found be 0.19–38.17% and 3.0–6.26 respectively. Further, the epistatic interaction detected one main effect QTL and four epistatic QTLs. Five major QTLs viz. Qbd.nbri.4.3, Qfd.nbri.4.2, Qrdm.nbri.5.1, Qncl.nbri.2.2, Qtd.nbri.4.1 with PVE > 15.0% might be useful for fine mapping to identify genes associated with respective traits, and also for development of specialized population through marker assisted selection. The identification of additive and dominant effect QTLs and desirable alleles of each of above mentioned traits would also be important for future research.     

Physical mapping of an adult plant stripe rust resistance gene from Triticum monococcum

Stripe rust (Puccinia striiformis f. sp. tritici) is one of the major devastating disease which causes large reduction in wheat yield. T. monococcum is an attractive diploid species for gene discovery in wheat with smaller genome size of 5700 Mb compared to 17,300 Mb of bread wheat. An adult plant stripe rust resistance QTL QYrtm.pau-2A was mapped on chromosome 2A flanked by two SSR markers Xwmc170 and Xwmc407. In the present study, two gene based markers Pau_Ta2AL_Gene45 and Pau_Ta2AL_Gene54 developed from 2A specific ESTs were found to map close to QYrtmpau-2A to narrow down the region for candidate gene identification. Utilizing sequence information of these two markers, four BAC clones were identified from the Minimum Tiling Path of 2AL assembly and were sequenced. SSR markers were designed from these BAC sequences and mapped to chromosome 2A. A 50 Mb region of wheat chromomse 2A was identified to harbor stripe rust resistance gene of T. monococcum. Gene based markers identified in the present investigation can be used for marker assisted introgression of QYrtm.pau-2A from T. monococcum to cultivated wheat.     

Biotransformation of linear alkylbenzene sulfonate (LAS) by Phanerochaete chrysosporium: oxidation of alkyl side-chain

The white rot fungus Phanerochaete chrysosporium, which generally mineralizes substituted aromatics to CO₂, transformed linear alkylbenzene sulfonate (LAS) surfactants mainly at their alkyl side chain. Degradation of LAS was evidenced by a zone of clearing on LAS-containing agar plates and colorimetric analysis of liquid cultures. Disappearance of LAS was virtually complete within 10 days in low nitrogen (2.4 mM N), high nitrogen (24 mM N) and malt extract (ME) liquid media. After 5 days of incubation in ME medium, transformation of LAS was complete at concentrations4 mg l^-1, but decreased at higher concentrations. The LAS degradation was not dependent on lignin peroxidases (LiPs) and manganese-dependent peroxidases (MnPs). Mineralization of¹⁴C-ring-LAS to ¹⁴CO₂ by P. chrysosporium was <1% regardless of the culture conditions used. Thin layer chromatography and mass spectral analyses indicated that P. chrysosporium transformed LAS to sulfophenyl carboxylates (SPCs) through oxidative shortening of the alkyl side-chains. While LAS disappearance in the cultures was not dependent on LiPs and MnPs, transformation of the parent LAS moieties to SPCs was more extensive in low N medium that favors expression of these enzymes. The SPCs produced in LN cultures were shorter in chain-length than those produced in ME cultures. Also there was a notable shift in the relative abundance of odd and even chain length metabolites compared to the starting LAS particularly in the low N cultures suggesting the possible involvement of processes other than or in addition to-oxidation in the chain-shortening process.     

Blood parameters of two hill stream cobitids in relation to their habitat

The blood of two fresh water cobitids — Botia lohachata, an exclusive water breathing form and Lepidocephalus guntea a dual breather, — showed a comparatively higher range of Hb (16.0–19.0 g%), Hct (50.0–61.1%) and number of RBC (2.71–6.7 millions/mm³) than many other water and air breathing fishes. Significant sexual difference exists in these characteristics (P > 0.05).The impact of life in oxygen depleted water, also inhabited by L. guntea as a result of its air breathing habit, is well reflected in its greater RBC size (11.86 × 8.66 µm) and their larger surface area (83.96 µm²) compared to that of Botia (53.16 µm²). The smaller size (9.92 × 6.45 µm) and consequently greater number of erythrocytes (4.67 millions/mm³) in Botia, are related to its active mode of life in the swiftly flowing water of hilly rivers. Though the higher nucleo-cytoplasmic ratio of 1 : 5.2 in Botia and 1 : 6.9 in Lepidocephalus suggest a slower red cell metabolism, the greater number of erythrocytes seems to have compensated for their active mode of life.