Growth response of Casuarina equisetifolia Forst. rooted stem cuttings to Frankia in nursery and field conditions

Casuarina equisetifolia Forst. is a tree crop that provides fuel wood, land reclamation, dune stabilization, and scaffolding for construction, shelter belts, and pulp and paper production. C. equisetifolia fixes atmospheric nitrogen through a symbiotic relationship with Frankia, a soil bacterium of the actinobacteria group. The roots of C. equisetifolia produce root nodules where the bacteria fix atmospheric nitrogen, which is an essential nutrient for all plant metabolic activities. However, rooted stem cuttings of elite clones of C. equisetifolia by vegetative propagation is being planted by the farmers of Pondicherry as costeffective method. As the vegetative propagation method uses inert material (vermiculite) for rooting there is no chance for Frankia association. Therefore after planting of these stocks the farmers are applying 150 kg of di-ammonium phosphate (DAP)/acre/year. To overcome this fertilizer usage, the Frankia-inoculated rooted stem cuttings were propagated under nursery conditions and transplanted in the nutrient-deficient soils of Karaikal, Pondicherry (India), in this study. Under nursery experiments the growth and biomass of C. equisetifolia rooted stem cuttings inoculated with Frankia showed 3 times higher growth and biomass than uninoculated control. These stocks were transplanted and monitored for their growth and survival for 1 year in the nutrient-deficient farm land. The results showed that the rooted stem cuttings of C. equisetifolia significantly improved growth in height (8.8 m), stem girth (9.6 cm) and tissue nitrogen content (3.3 mg g^?1) than uninoculated controls. The soil nutrient status was also improved due to inoculation of Frankia.     

Comparative radiolabeling and distribution of a tumour-directed monoclonal antibody

The monoclonal antibody (MAb) 155H.7, raised against a synthetic β-anomer of the Thomsen-Friedenreich antigen (S-TAG), was radioiodinated using iodine monochloride, chloramine-T and Iodogen and radiolabeled with ¹¹¹In using the bromoacetamido-derivative of benzyl-EDTA. The in vitro immunoreactivity of the MAb was assessed using an ELISA with the S-TAG and the in vivo distribution of the radioiodinated and radiochelated MAb was determined in the murine mammary carcinoma TA3/Ha tumour model. Both chloramine-T and iodine monochloride radioiodination greatly reduced the immunoreactivity of the MAb compared to radioiodination using lodogen. Bifunctional chelate labeling was comparable to Iodogen in reducing the immunoreactivity of the MAb and subsequent chelation of ¹¹¹In did not further compromise the immunoreactivity of the MAb. The in vivo distribution data showed significantly different distributions of the radiolabels after injection of the radioiodinated and radiochelated MAb. The ¹³¹I-MAb showed some tumour association as compared to the distribution of an ¹²⁵I-non-specific protein and the data also indicates that there is preferential dehalogenation of the radioiodinated MAb. ¹¹¹In from the radiochelated MAb showed significantly higher uptake in the tumour than ¹³¹I from the ¹³¹I-MAb. It is suggested that the differing fates of the two radiolabels within the tumour cell is responsible for the difference in retention observed and not necessarily due to the lack of MAb uptake by the tumour. Overall, the radiochelate label for MAb 155H.7 appears to be superior to radioiodine for in vivo use.     

Expression of Human Carbonic Anhydrase in the Cyanobacterium Synechococcus PCC7942 Creates a High CO(2)-Requiring Phenotype : Evidence for a Central Role for Carboxysomes in the CO(2) Concentrating Mechanism

Active human carbonic anhydrase II (HCAII) protein was expressed in the cyanobacterium Synechococcus PCC7942 by means of transformation with the bidirectional expression vector, pCA. This expression was driven by the bacterial Tac promoter and was regulated by the IacIQ repressor protein, which was expressed from the same plasmid. Expression levels reached values of around 0.3% of total cell protein and this protein appeared to be entirely soluble in nature and located within the cytosol of the cell. The expression of this protein has dramatic effects on the photosynthetic physiology of the cell. Induction of expression of carbonic anhydrase (CA) activity in both high dissolved inorganic carbon (C_i) and low C_i grown cells leads the creation of a high C_i requiring phenotype causing: (a) a dramatic increase in the K_0.5 (C_i) for photosynthesis, (b) a loss of the ability to accumulate internal C_i, and (c) a decrease in the lag between the initial C_i accumulation following illumination and the efflux of CO₂ from the cells. In addition, the effects of the expressed CA can largely be reversed by the carbonic anhydrase inhibitor ethoxyzolamide. As a result of the above findings, it is concluded that the CO₂ concentrating mechanism in Synechococcus PCC7942 is largely dependent on (a) the absence of CA activity from the cytosol, and (b) the specific localization of CA activity in the carboxysome. A theoretical model of photosynthesis and C_i accumulation is developed in which the carboxysome plays a central role as both the site of CO₂ generation from HCO_3⁻ and a resistance barrier to CO₂ efflux from the cell. There is good qualitative agreement between this model and the measured physiological effects of expressed cytosolic CA in Synechococcus cells.     

Simultaneous production of citric acid and invertase by Yarrowia lipolytica SUC+ transformants

Simultaneous production of citric acid (CA) and invertase by Yarrowia lipolytica A-101-B56-5 (SUC⁺ clone) growing from sucrose, mixture of glucose and fructose, glucose or glycerol was investigated. Among the tested substrates the highest concentration of CA was reached from glycerol (57.15 g/L) with high yield (Y_CA/S = 0.6 g/g). When sucrose was used, comparable amount of CA was secreted (45 g/L) with slightly higher yield (Y_CA/S = 0.643 g/g). In all cultures amount of isocitrate (ICA) was below 2% of total citrates. Considering invertase production, the best carbon source appeared to be sucrose (72 380 U/L). The highest yield of CA and invertase biosynthesis calculated for 1 g of biomass was obtained for cells growing from glycerol (9.9 g/g and 4325 U/g, respectively). Concentrates of extra- and intracellular invertase of the highest activity were obtained from sucrose as substrate (0.5 and 1.8 × 10⁶ U/L, respectively).     

Taq DNA polymerase slippage mutation rates measured by PCR and quasi-likelihood analysis: (CA/GT)n and (A/T)n microsatellites

During microsatellite polymerase chain reaction (PCR), insertion–deletion mutations produce stutter products differing from the original template by multiples of the repeat unit length. We analyzed the PCR slippage products of (CA)_n and (A)_n tracts cloned in a pUC18 vector. Repeat numbers varied from two to 14 (CA)_n and four to 12 (A)_n. Data was generated on approximately 10 single molecules for each clone type using two rounds of nested PCR. The size and peak areas of the products were obtained by capillary electrophoresis. A quasi- likelihood approach to the analysis of the data estimated the mutation rate/repeat/PCR cycle. The rate for (CA)_n tracts was 3.6 × 10^–3 with contractions 14 times greater than expansions. For (A)_n tracts the rate was 1.5 × 10^–2 and contractions outnumbered expansions by 5-fold. The threshold for detecting ‘stutter’ products was computed to be four repeats for (CA)_n and eight repeats for (A)_n or ~8 bp in both cases. A comparison was made between the computationally and experimentally derived threshold values. The threshold and expansion to contraction ratios are explained on the basis of the active site structure of Taq DNA polymerase and models of the energetics of slippage events, respectively.     

Metabolism of 2'-carboxyarabinitol in leaves

Results presented here indicate that 2′-carboxyarabinitol (CA) is the in vivo precursor and product of 2′-carboxyarabinitol 1-phosphate (CA1P) metabolism in leaves. When [2-¹⁴C]CA was fed in the light to leaves of five species known to be highly active in CA1P metabolism (Phaseolus vulgaris, Lycopersicon esculentum, Helianthus annuus, Petunia hybrida, and Beta vulgaris), [¹⁴C]CA1P was formed in the dark. Reillumination of a Phaseolus leaf caused this [¹⁴C]CA1P to be rapidly metabolized to [¹⁴C]CA (t_½ = 1 min). The epimer 2′-carboxyribitol could not substitute for CA in the dark synthesis of CA1P, and CA in the anionic form was a better substrate than CA in the lactone form. In leaves of Phaseolus vulgaris, the active CA pool size used in the dark synthesis of CA1P is between about 70 and 110 nanomoles per milligram of chlorophyll. The photosynthetic electron transport inhibitor diuron did not affect the dark synthesis of [¹⁴C]CA1P, but did greatly reduce the rate of its subsequent light degradation (t_½ = approximately 10 min). Dark synthesis of [¹⁴C]CA1P was inhibited by dithiothreitol and NaF. From the present data, we suggest that CA1P and CA participate in a metabolic substrate cycle in vivo.     

Identification of potential tumour-associated carbonic anhydrase isozyme IX inhibitors: atom-based 3D-QSAR modelling,pharmacophore-based virtual screening and molecular docking studies

Abstract

Tumour hypoxia results in dramatic changes in the gene expression, proliferation and survival of tumour cells. The tumour cells shift towards anaerobic glycolysis which results in change of pH in their microenvironment. In response to this stress, over expression of carbonic anhydrase IX (CA IX) genes is observed in many solid tumours. So, selective inhibition of CA IX can be a promising target for anti-cancer drugs. In this work in silico tools like atom-based 3D-QSAR modelling, pharmacophore-based virtual screening and molecular docking were used to identify potential CA IX inhibitors. Based on the training set used in the QSAR model, twenty pharmacophore models were generated. Out of these, HHHR_1, AHHR_1, DHHHR_1, AHHHR_1 model was used to screen a database of 1,50,000 compounds retrieved from ZINC 15 database. R² and Q² was 0.9864 and 0.8799, respectively, for the developed QSAR model. 163 compounds showed a phase screen score above 2.4 in which ZINC02260669 was the highest ranked (screen score, 2.852058) compound in all the four models. Built QSAR model was used to predict the activity of all these 163 compounds and ZINC72370966 showed the highest predicted activity with pKi value of 7.649. These compounds were docked against CA IX (human) protein (PDB ID 5FL6) and molecular docking results showed favourable binding interactions for the best ten identified hits. This work gives design insights and some potential scaffolds which can be developed as CA IX inhibitors.

Communicated by Ramaswamy H. Sarma     

Ovarian sequestration of [14C]-inulin by an insect: An index of vitellogenesis

[¹⁴C]-Inulin injected into the blood of female milkweed bugs (Oncopeltus fasciatus) undergoing vitellogenesis is sequestered in the eggs. Determination of ovarian radiocarbon uptake gives a reproducible index of the progression of vitellogenesis that permits quantitative measurements prior to oviposition or under conditions where vitellogenesis is incomplete. This assay allows intra- and interspecific comparisons of the rates of juvenile hormone biosynthesis by corpora allata (CA) when these endocrine glands are transplanted into milkweed bug females, whose CA have been chemically destroyed with precocene. The method has the potential of distinguishing between nervous and hormonal regulation of the CA.     

Feeding corn grain steeped in citric acid modulates rumen fermentation and inflammatory responses in dairy goats

Cereal grains treated with organic acids were proved to increase ruminal resistant starch and can relieve the risk of ruminal acidosis. However, previous study mainly focussed on acid-treated barley, the effects of organic acid-treated corn is still unknown. The objectives of this study were to evaluate whether feeding ground corn steeped in citric acid (CA) would affect ruminal pH and fermentation patterns, milk production and innate immunity responses in dairy goats. Eight ruminally cannulated Saanen dairy goats were used in a crossover designed experiment. Each experimental period was 21 day long including 14 days for adaption to new diet and 7 days for sampling and data collection. The goats were fed high-grain diet contained 30% hay and 70% corn-based concentrate. The corn was steeped either in water (control) or in 0.5% (wt/vol) CA solution for 48 h. Goats fed CA diet showed improved ruminal pH status with greater mean and minimum ruminal pH, and shorter (P<0.05) duration of ruminal pH<5.6 and less area of ruminal pH<5.6, 5.8 and 6.0. Concentration of total volatile fatty acid and molar proportion of propionate were less but the molar proportion of acetate was greater (P<0.05) in goats fed the CA diet than the control diet. Concentration of ruminal lipopolysaccharide (LPS) was lower (P<0.05) and that of lactic acid also tended (P<0.10) to be lower in goats fed CA than the control. Although dry matter intake, actual milk yield, yield and content of milk protein and lactose were not affected, the milk fat content and 4% fat-corrected milk tended (P<0.10) to be greater in goats fed CA diet. For the inflammatory responses, peripheral LPS did not differ, whereas the concentration of LPS binding protein and serum amyloid A tended (P<0.10) to be less in goats fed CA diet. Similarly, goats fed CA diet had less (P<0.05) concentration of haptoglobin and tumour necrosis factor. These results indicated that feeding ground corn treated with CA effectively improved ruminal pH status, thus alleviated the risk of ruminal acidosis, reduced inflammatory response, and tend to improve milk yield and milk fat test.     

The relationship between phosphorylation status of focal adhesion kinases,molecular subtypes,tumour microenvironment and survival in patients with primary operable ductal breast cancer

BackgroundDespite advances in therapies to treat breast cancer, over 100,000 patients die in the UK of this disease per year, highlighting the need to develop effect predictive and prognostic markers for patients with primary operable ductal breast cancer. Therefore, the aim of the present study was to examine the relationship between membranous, cytoplasmic and nuclear expression of focal adhesion kinase (phosphorylated at Y 397, Y 861 and Y 925), molecular subtypes, tumour microenvironment and survival in patients with primary operable ductal breast cancer.MethodsFour hundred and seventy-four patients presenting between 1995 and 1998 with primary operable ductal breast cancer were included in this study. Using tissue microarrays expression of membranous, cytoplasmic and nuclear tumour cell phosphorylation of FAK at Y³⁹⁷, Y⁸⁶¹ and Y⁹²⁵ was assessed, and associations with clinicopathological characteristics, tumour microenvironment and cancer-specific survival (CSS) were examined.ResultsNo significant association was observed for ph-FAK Y⁸⁶¹ with survival at all sites. However, high expression of membranous ph-FAK Y³⁹⁷ was associated with increased tumour grade (P < .001), molecular subtypes (P < .001), increased tumour necrosis (P < .001), high Klintrup–Mäkinen grade (P < .001), increased CD138+ plasma cells (P = .031), endocrine therapy (P = .001) and poor cancer specific survival (P = .040). Similarly, high expression of nuclear ph-FAK Y³⁹⁷ was associated with decreased age (P = .042), increased CD138+ plasma cells (P = .001) and poor cancer specific survival (P = .003). Furthermore, high expression of cytoplasmic ph-FAK Y⁹²⁵ was associated with decreased tumour grade (P < .001), less involved lymph node (P = .020), molecular subtypes (P < .001), decreased tumour necrosis (P < .001), low Klintrup–Mäkinen grade (P < .001), decreased CD4+ T-cells (P = .006), decreased CD138+ plasma cells (P = .034), endocrine therapy (P < .001), chemotherapy (P = .048), and improved cancer specific survival (P = .044). On multivariate analysis, high expression of nuclear ph-FAK Y³⁹⁷ was independently associated with reduced cancer specific survival (P = .017).ConclusionThe results of the present study show that membranous and nuclear ph-FAK Y³⁹⁷ and cytoplasmic ph-FAK Y⁹²⁵ were associated with prognosis in patients with primary operable ductal breast cancer. In addition, high expression of nuclear ph-FAK Y³⁹⁷ was an independent prognostic factor in patients with primary operable ductal breast cancer and could be incorporated into clinical practice.     

Community and hospital acquired methicillin resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> efficiently retain the <Emphasis Type="Italic">Van A</Emphasis> determinant

Dissemination of vancomycin resistance from hospital to community strains is a serious threat to public health. Our study aimed to provide evidence for transmission of Van A type resistance from the hospital to the community. Wild-type community and hospital associated methicillin resistant Staphylococcus aureus strains were studied in vitro and in a model that mimicked a natural environment to ascertain their ability to acquire and maintain the vancomycin resistance determinant (Van A gene) from vancomycin resistant Enterococcus faecalis. Fitness was assessed and the cost of Van A acquisition and retention was estimated. In vitro mating experiments were carried out using a filter mating technique and a model of a natural water body environment. Transfer of resistance was carried out in two different conditions: restricted and favorable. Transconjugants were confirmed by E test and PCR using specific primer sets. Growth kinetics and fitness measurements were done by spectrometric analysis. Using the in vitro filter mating technique, high transfer frequencies that ranged from 0.7 × 10^–3(0.0006) to 3.1 × 10^–4(0.00011) were recorded, with the highest transfer frequencies for CA MRSA (thermosensitively homogenous) (0.7 × 10^–3), and 1.2 × 10^–4 to 2.4 × 10^–6 in the model. HA MRSA (homogenous) showed a greater capacity (3.6 × 10^–4) to receive the Van A gene, while CA MRSA showed a reduced ability to maintain the gene after serial subcultures. CA and HA thermosensitively heterogeneous MRSA transconjugants exhibited higher growth rates. The present study provides evidence for the enhanced ability of CA and HA MRSA clones to acquire and maintain Van A type resistance.     

Evidence for selective sulfhydryl reactivity in cytochalasin A--mediated bacterial inhibitions.

Cytochalasin A (CA) at 5 × 10^?5$\text{M}$ strongly inhibits glucose transport in Arthrobacter sialophilis. This effect and other bacteriostatic and metabolic inhibitions of gram-positive bacteria are not caused by the closely related congeners cytochalasin B or D. Inhibitions by CA are nullified by prior drug incubation with sulfhydryl compounds. It was also found that the characterized adduct of CA with β-mercaptoethanol is devoid of biological activity. N-ethylmaleimide, p-chloromercuribenzoate and ethacrynic acid (a known, liposoluble, sulfhydryl reactant) were each shown at 5 × 10^?5$\text{M}$ to be relatively ineffective in inhibiting D-glucose transport in $\text{A. sialophilus}$. These observations suggest that CA reacts at the molecular biological level in a site-specific manner.     

The effect of juvenile hormone on protein synthesis in the transparent accessory gland of male Rhodnius prolixus

Feeding stimulates the accumulation of protein by the transparent accessory reproductive glands (TARG), allatectomy abolishes the increase, and severing the nervous connections between the corpus allatum (CA) and the brain raises the level of accumulation after feeding. TARG removed from allatectomised females exhibit a markedly decreased level of incorporation of [³H]-leucine into protein in vitro, and topical application of juvenile hormone (JH) to allatectomised males restores the ability of the glands to incorporate [³H]-leucine in vitro. Severing the nerves between the CA and the brain increases the rate of incorporation in vitro, but in both operated and normal males, the rate of incorporation decreases with time after feeding. JH applied topically to intact animals during the falling phase of incorporation, or applied directly to TARG in vitro from such animals, stimulates incorporation. These data indicate that JH from the CA controls protein synthesis in the TARG and that the CA in the male of Rhodnius may be subject to different controls than in the female.     

Inducible nitric oxide synthase-deficient mice show exacerbated inflammatory process and high production of both Th1 and Th2 cytokines during paracoccidioidomycosis

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by fungus Paracoccidioides brasiliensis. To analyze the influence of inducible nitric oxide synthase (iNOS) in this disease, iNOS-deficient (iNOS^−/−) and wild-type (WT) mice were infected intravenously with P. brasiliensis 18 isolate. We found that, unlike WT mice, iNOS^−/− mice did not control fungal proliferation, and began to succumb to infection by day 50 after inoculation of yeast cells. Typical inflammatory granulomas were found in WT mice, while, iNOS^−/− mice presented incipient granulomas with intense inflammatory process and necrosis. Additionally, splenocytes from iNOS^−/− mice did not produce nitric oxide, however, their proliferative response to Con-A was impaired, just like infected WT mice. Moreover, infected iNOS^−/− mice presented a mixed pattern of immune response, releasing high levels of both Th1 (IL-12, IFN-γ and TNF-α) and Th2 (IL-4 and IL-10) cytokines. These data suggest that the enzyme iNOS is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, by influencing cytokines production, and by appeasing the development of a high inflammatory response and consequently formation of necrosis. However, iNOS-derived nitric oxide seems not being the unique factor responsible for immunosuppression observed in infections caused by P. brasiliensis.     

Mechanism of radiation-induced reactions in aqueous solution of coumarin-3-carboxylic acid: Effects of concentration,gas and additive on fluorescent product yield

Abstract

The radiation-induced reactions of a water-soluble coumarin derivative, coumarin-3-carboxyl acid (C3CA), have been investigated in aqueous solutions by pulse radiolysis with a 35 MeV electron beam, final product analysis following ⁶⁰Co γ-irradiations and deterministic model simulations. Pulse radiolysis revealed that C3CA reacted with both hydroxyl radicals (^?OH) and hydrated electrons (e^? _aq) with near diffusion-controlled rate constants of 6.8 × 10⁹ and 2.1 × 10¹⁰ M^?1 s^?1, respectively. The reactivity of C3CA towards O₂^{? ?} was not confirmed by pulse radiolysis. Production of the fluorescent molecule, 7-hydroxy-coumarin-3-carboxylic acid (7OH-C3CA), was confirmed by final product analysis with a fluorescence spectrometer coupled to a high performance liquid chromatography (HPLC) system. Production yields of 7OH-C3CA following ⁶⁰Co γ-irradiations depended on the irradiation conditions and ranged from 0.025 to 0.18 (100 eV) ^?1. Yield varied with saturating gas, additive and C3CA concentration, implying the presence of at least two pathways capable of providing 7OH-C3CA as a stable product following the scavenging reaction of C3CA with ^?OH, including a peroxidation/elimination sequence and a disproportionation pathway. A reaction mechanism for the two pathways was proposed and incorporated into a deterministic simulation, showing that the mechanism can explain experimentally measured 7OH-C3CA yields with a constant conversion factor of 4.7% from ^?OH scavenging to 7OH-C3CA production, unless t-BuOH was added.     

Hepatocyte transplantation in bile salt export pump‐deficient mice: selective growth advantage of donor hepatocytes under bile acid stress

The bile salt export pump (Bsep) mediates the hepatic excretion of bile acids, and its deficiency causes progressive familial intrahepatic cholestasis. The current study aimed to induce bile acid stress in Bsep^−/− mice and to test the efficacy of hepatocyte transplantation in this disease model. We fed Bsep^−/− and wild-type mice cholic acid (CA) or ursodeoxycholic acid (UDCA). Both CA and UDCA caused cholestasis and apoptosis in the Bsep^−/− mouse liver. Wild-type mice had minimal liver injury and apoptosis when fed CA or UDCA, yet had increased proliferative activity. On the basis of the differential cytotoxicity of bile acids on the livers of wild-type and Bsep^−/− mice, we transplanted wild-type hepatocytes into the liver of Bsep^−/− mice fed CA or CA + UDCA. After 1–6 weeks, the donor cell repopulation and canalicular Bsep distribution were documented. An improved repopulation efficiency in the CA + UDCA-supplemented group was found at 2 weeks (4.76 ± 5.93% vs. 1.32 ± 1.48%, P = 0.0026) and at 4–6 weeks (12.09 ± 14.67% vs. 1.55 ± 1.28%, P < 0.001) compared with the CA-supplemented group. Normal-appearing hepatocytes with prominent nuclear staining for FXR were noted in the repopulated donor nodules. After hepatocyte transplantation, biliary total bile acids increased from 24% to 82% of the wild-type levels, among which trihydroxylated bile acids increased from 41% to 79% in the Bsep^−/− mice. We conclude that bile acid stress triggers differential injury responses in the Bsep^−/− and wild-type hepatocytes. This strategy changed the balance of the donor–recipient growth capacities and was critical for successful donor repopulation.     

Association of IL-6, TNF-α and IL-10 gene polymorphisms with type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is a metabolic pro-inflammatory disorder characterized by chronic hyperglycemia and increased levels of circulating cytokines suggesting a causal role of inflammation in its etiology. Polymorphism of cytokine genes including interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were studied in T2DM patients as well as in normal healthy controls. Genomic DNA was isolated from both T2DM patients and controls followed by quantification and genotyping by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) using suitable primers. The genotypic, allelic and carriage rate frequency distribution in patients and controls were analyzed by SPSS (version 15.0). Odd ratios with 95 % confidence interval was determined to describe the strength of association by logistic regression model. Double and triple combinations of genotypes were analyzed by χ² test. Gene–gene interaction and linkage disequilibrium tests were performed using SHEsis software. Individually, IL-6, TNF-α and IL-10 did not show any association. In double combination, IL-6 ?597 GA and TNF-α ?308 GG genotypes increased the risk up to 21 times and in triple combination IL-6 ?597 AA, TNF-α ?308 GG and IL-10 ?592 CA increased the risk of T2DM up to 314 times. In gene–gene interaction allele ‘A’ of all studied polymorphisms increased the risk of T2DM up to 1.41 times. Our results suggest that individuals having a haplotype combination of AA, GG and CA for IL-6, TNF-α and IL-10 gene polymorphisms will have higher susceptibility and be at greater risk of developing T2DM.     

Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120

Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO₃ ^? dehydration and the subsequent fixation of CO₂. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein–protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k _cat of 2.0 × 10⁴ s^?1 and k _cat/K _m of 4.1 × 10⁶ M^?1 s^?1 at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25–35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO₂ analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes.     

In vitro evaluation of antibacterial and immunomodulatory activities of Pelargonium reniforme,Pelargonium sidoides and the related herbal drug preparation EPs® 7630

The importance of Pelargonium species, most notably Pelargonium reniforme and Pelargonium sidoides, in traditional medicine in the Southern African region is well documented. Nowadays, a modern aqueous-ethanolic formulation of the roots of P. sidoides (EPs^® 7630) is successfully employed for the treatment of ear, nose and throat disorders as well as respiratory tract infections. To provide a scientific basis of its present utilization in phytomedicine, EPs^® 7630, extracts and isolated constituents of the titled Pelargoniums with emphasis on P. sidoides were evaluated for antibacterial activity and for their effects on nonspecific immune functions. The samples exhibited merely moderate direct antibacterial capabilities against a spectrum of Gram-positive and Gram-negative bacteria. Functional bioassays including an in vitro model for intracellular diseases, a fibroblast-lysis assay (tumour necrosis factor (TNF) activity), a fibroblast-virus protection assay (IFN activity) and a biochemical assay for nitric oxides revealed significant immunomodulatory properties. Gene expression experiments (iNOS, IFN-α, IFN-γ, TNF-α, Interleukin (IL)-1, IL-10, IL12, IL-18) not only confirmed functional data, they also clearly showed differences in the response of infected macrophages when compared to that of noninfected cells. ELISA confirmed the protein production of TNF-α, IL-1α and IL-12, while FACS analyses reaffirmed the cytokines IL-1α and IL-12 at the singular cell level. The current data provide convincing support for the improvement of immune functions at various levels, hence, validating the medicinal uses of EPs^® 7630. Despite considerable efforts, the remedial effects cannot yet be related to a chemically defined principle.