Disease-associated glycosylated molecular variants of human C-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and Indian visceral leishmaniasis

Human C-reactive protein (CRP), as a mediator of innate immunity, removed damaged cells by activating the classical complement pathway. Previous studies have successfully demonstrated that CRPs are differentially induced as glycosylated molecular variants in certain pathological conditions. Affinity-purified CRPs from two most prevalent diseases in India viz. tuberculosis (TB) and visceral leishmaniasis (VL) have differential glycosylation in their sugar composition and linkages. As anemia is a common manifestation in TB and VL, we assessed the contributory role of glycosylated CRPs to influence hemolysis via CRP-complement-pathway as compared to healthy control subjects. Accordingly, the specific binding of glycosylated CRPs with erythrocytes was established by flow-cytometry and ELISA. Significantly, deglycosylated CRPs showed a 7–8-fold reduced binding with erythrocytes confirming the role of glycosylated moieties. Scatchard analysis revealed striking differences in the apparent binding constants (10⁴–10⁵ M⁻¹) and number of binding sites (10⁶–10⁷sites/erythrocyte) for CRP on patients’ erythrocytes as compared to normal. Western blotting along with immunoprecipitation analysis revealed the presence of distinct molecular determinants on TB and VL erythrocytes specific to disease-associated CRP. Increased fragility, hydrophobicity and decreased rigidity of diseased-erythrocytes upon binding with glycosylated CRP suggested membrane damage. Finally, the erythrocyte-CRP binding was shown to activate the CRP-complement-cascade causing hemolysis, even at physiological concentration of CRP (10 μg/ml). Thus, it may be postulated that CRP have a protective role towards the clearance of damaged-erythrocytes in these two diseases.     

Effect of Ethanol, Methanol and Acetone on Rooting Etiolated Cuttings of Vigna radiata in Presence of Sucrose and Auxin

Root formation on etiolated hypocotyl cuttings of Vigna radiataL. is greatly enhanced in the presence of ethanol, methanoland acctone. Low concentrations of solvents plus sucrose slightlypromoted the formation of roots. Vigna radiata, alcohol, rooting, auxin     

Frequent alterations of SLIT2–ROBO1–CDC42 signalling pathway in breast cancer: clinicopathological correlation

The aim of the study was to understand the role of SLIT2–ROBO1/2–CDC42 signalling pathways in development of breast cancer (BC). Primary BC samples (n=150), comprising of almost equal proportion of four subtypes were tested for molecular alterations of SLIT2, ROBO1, ROBO2 and CDC42, the key regulator genes of this pathway. Deletion and methylation frequencies of the candidate genes were seen in the following order: deletion, SLIT2 (38.6%) >ROBO1 (30%) >ROBO2 (7.3%); methylation, SLIT2 (63.3%) >ROBO1 (26.6%) >ROBO2 (9.3%). Majority (80%, 120/150) of the tumours showed alterations (deletion/methylation) in at least one of the candidate genes. Overall, alterations of the candidate genes were as follows: SLIT2, 75.3% (101/150); ROBO1, 45.3% (68/150); ROBO2, 15.3% (23/150). Significantly, higher alteration of SLIT2 locus was observed in triple negative breast cancer (TNBC) over HER2 subtype (P=0.0014). Similar trend is also seen in overall alterations of SLIT2 and/or ROBO1, in TNBC than HER2 subtype (P=0.0012); of SLIT2 and/or ROBO2 in TNBC than luminal A (P=0.014) and HER2 subtype (P=0.048). Immunohistochemical analysis of SLIT2, ROBO1/2 showed reduced expression, concordant with their molecular alterations. Also, high expression of total CDC42 (49/52; 94.2%) and reduced expression of phospho Serine-71 CDC42 (41/52; 78.8%) was observed. Coalterations of SLIT2 and/or ROBO1, SLIT2 and/or ROBO2 had significant association with reduced expression of phospho Serine-71 CDC42 (P=0.0012–0.0038). Alterations of SLIT2 and/or ROBO1, reduced expression of phospho Serine-71 CDC42 predicted poor survival of BC patients. Results indicate the importance of SLIT2–ROBO1–CDC42 signalling pathway in predicting tumour progression.     

Physiological Studies with Heavy Water. II. Germination and Growth Inhibition of Barley by D2O

Seed germination and barley seedling growth in various D₂O concentrations have been studied. It was observed that the emergence of root and shoot was delayed, there being greater delay in shoot than in root emergence. A complete block was observed in germination in pure D₂O and the germination rate was slowed down significantly in lower concentrations. An initial germination delay by different D₂O concentrations seemed to cause a subsequent retardation in the growth measured as shoot and root length. A comparison of root and shoot length with their respective dry weights suggested that the growth by cell division/elongation might have been affected more than the transport of food materials from the endosperm to the embryo. Analysis of the total sugars of the endosperm and the embryo at 8 hour intervals showed that while the hydrolysis of starch to sugars was progressively decreased by increasing D₂O concentrations, the transport rate from endosperm to embryo showed a sharp inhibition in 50% D₂O and above. This indicated that the inhibition in the transport of materials, besides less hydrolysis of reserve food materials, may also be a causal factor of germination and growth inhibition in D₂O.     

Role of C-reactive protein in complement-mediated hemolysis in Malaria

Human C-reactive protein (CRP) is a clinically important classical acute phase protein. Although CRP has been reported to bind with many nucleated cells, the direct binding of CRP to erythrocytes in diseases remains largely unexplored. The main focus of the present study was to investigate the binding of disease-specific CRP to erythrocytes of same patients. Distinct molecular variant of disease-specific CRP was affinity purified from sera of malaria patients (CRP_Mal). This CRP showed strong binding with malaria erythrocytes (RBC_Mal) as confirmed by flow cytometric analysis (FACS), enzyme-linked immunosorbent assays (ELISA), and radio binding assays. Calcium and phosphoryl choline (PC) were found to be essential for this interaction. A 2.3-fold increased binding of induced CRP to RBC_Mal as compared to normal erythrocytes (RBC_N) confirmed disease-specificity. Preincubation of RBC_Mal with unconjugated CRP showed 3–5 fold inhibition. The association constant of CRP and RBC_Mal was 4.7 × 10⁶ cpm/μg with the corresponding number of receptors/cell being 4.3 × 10⁵. The effector function of CRP_Mal has been demonstrated by its potency to activate the complement pathway. An optimal dose of 10 μg/ml of CRP induced three-fold higher hemolysis of patient erythrocytes as compared to RBC_N. These studies provide direct evidence for an important phagocytic functional interaction of this acute-phase protein by triggering the CRP-complement pathway after the binding of CRP_Mal with RBC_Mal. Hemolysis as triggered by this pathway may be one of the causative factors of anemia, a common clinical manifestation of this disease.     

Ascorbic Acid and Heterocyst Development in the Blue–Green Alga Amabaema ambigua

The nitrogen–fixing blue–green alga Anabaena ambigua was grown in a medium which contained either ammonium chloride as nitrogen source or molecular nitrogen. In the latter case the alga produced heterocysts. The material was analysed for ascorbic acid, dehydro-ascorbic acid and diketogulonic acid. The amount of a,scorbic acid was found always to he higher in the alga grown with molecular nitrogen. When the alga grown with combined nitrogen was transferred to the medium lacking it, there was an increase in the ascorbic acid content. Conversely, when material cultured on the nitrogen–free medium was suspended in the medium containing ammonium chloride, there was a decrease in the cellular ascorbic acid. Esogenously added ascorbic acid, up to 0.5 mg per ml, increased the heterocyst frequency to nearly three times that of the control. D–isoascorbic acid, an analogue of ascorbic acid, also showed an enhancement of heterocyst production. Algal extracts were fractionated by poiyacrylamide electro–phoresis, and the presence of ascorbic acid oxidase was detected on the gels. Two bands, with Rf values 0.34 and 1.0, were found to give positive test: for the enzyme. The total enzyme activity was 16.7 % higher in cells grown with molecular nitrogen than in those grown with combined nitrogen. The exact location of the enzyme in the alga ist not known although the heterocysts were earlier shown to contain ascorbic acid. Cytochemical tests, however, indicated strong per–oxidase activity in the heterocysts.