Structural interactions in chondroitin 4-sulfate mediated adherence of Plasmodium falciparum infected erythrocytes in human placenta during pregnancy-associated malaria

Infection with Plasmodium falciparum during pregnancy results in the adherence of infected red blood cells (IRBCs) in placenta, causing pregnancy-associated malaria with severe health complications in mothers and fetuses. The chondroitin 4-sulfate (C4S) chains of very low sulfated chondroitin sulfate proteoglycans (CSPGs) in placenta mediate the IRBC adherence. While it is known that partially sulfated but not fully sulfated C4S effectively binds IRBCs, structural interactions involved remain unclear and are incompletely understood. In this study, structurally defined C4S oligosaccharides of varying sulfate contents and sizes were evaluated for their ability to inhibit the binding of IRBCs from different P. falciparum strains to CSPG purified from placenta. The results clearly show that, with all parasite strains studied, dodecasaccharide is the minimal chain length required for the efficient adherence of IRBCs to CSPG and two 4-sulfated disaccharides within this minimal structural motif are sufficient for maximal binding. Together, these data demonstrate for the first time that the C4S structural requirement for IRBC adherence is parasite strain-independent. We also show that the carboxyl group on nonreducing end glucuronic acid in dodecasaccharide motif is important for IRBC binding. Thus, in oligosaccharides containing terminal 4,5-unsaturated glucuronic acid, the nonreducing end disaccharide moiety does not interact with IRBCs due to the altered spatial orientation of carboxyl group. In such C4S oligosaccharides, 14-mer but not 12-mer constitutes the minimal motif for inhibition of IRBC binding to placental CSPG. These data have important implications for the development and evaluation of therapeutics and vaccine for placental malaria.     

Circadian rhythm of airways caliber and its autonomic modulation

ABSTRACT

The autonomic nervous system (ANS) is one of the effector pathways for circadian variation of many physiological parameters. Autonomic tone and airways caliber have been reported to exhibit circadian variation in separate studies. A simultaneous investigation of heart rate variability (HRV) and airway caliber might ascertain how airway caliber is modulated by autonomic tone. This study was planned to identify the variations in airway caliber and autonomic function tone during a 24-hour span. A total of 56 healthy male subjects with almost similar daily routines were studied. Time domain, frequency domain and nonlinear analysis of R-R interval from 5 min electrocardiogram (ECG) was done seven times during the daytime wake span at 3-hour intervals starting at 05:00 h in the morning until 23:00 h in the night. Simultaneously peak expiratory flow rate (PEFR) was determined using a mini Wright’s peak flow meter. Rhythmometric analysis was done for PEFR and HRV parameters. Significant circadian variation in low frequency (LF) and high frequency (HF) variance was identified in this group of healthy subjects. The circadian rhythm of LF variance was characterized by a gradual increase and corresponding reciprocal change in HF variance from morning until night. The LF/HF ratio and SD2/SD1 ratio reflecting sympatho-vagal balance showed low to high values from morning to evening. The acrophase of the PEFR temporal pattern is similar to that of LF power and almost opposite in phase to that of HF power. PEFR is positively correlated with LF power. The circadian rhythm of airway caliber co-varies with cardiac autonomic tone. It appears that the temporal pattern of cardiac autonomic tone precedes in time that of airways caliber, thereby suggesting the latter operates under the modulatory effect of the 24-hour pattern in sympatho-vagal balance.     

Stage-specific induction of systemic acquired resistance by fruit extracts of Azadirachta indica

In the present study, the efficacy of aqueous fruit extract of Azadirachta indica A. Juss. (Neem) on induction of systemic acquired resistance (SAR) in tomato against Pseudomonas syringae pv. tomato through enhancement in the activity of polyphenol oxidase (PPO) at different ages was studied. Plants at 6, 8, 10 and 12?weeks of age were selected. A single leaf at the third node from the base of each plant was treated either singly or with different combinations of the pathogen and Neem fruit extract. Samples were collected from the non-treated leaves at an interval of 24?h up to five?days for enzyme assay and after two?weeks for disease development from both treated and untreated plants. The results demonstrate that the neem fruit extract could induce additional PPO isoforms both locally as well as systemically. The PPO activity was observed to be elevated in both the treated and non-treated leaves leading to induction of SAR. The induction of SAR enhances with the increase in the age of the plant.     

A pH-stable laccase from alkali-tolerant γ-proteobacterium JB: Purification, characterization and indigo carmine degradation

γ-Proteobacterium JB, an alkali-tolerant soil isolate, produced laccase constitutively in unbuffered medium. The enzyme was purified to homogeneity by ammonium sulphate precipitation, DEAE-sepharose anion exchange chromatography and preparatory polyacrylamide gel electrophoresis. The purified enzyme was a monomeric polypeptide (MW 120 kDa) and absorbed at 590 nm indicating the presence of Type I Cu²⁺-centre. It worked optimally at 55 °C and showed different pH optima for different substrates. The enzyme was highly stable in the pH range 4–10 even after 60 days at 4 °C. K_m and V_max values for syringaldazine, catechol, pyrogallol, p-phenylenediamine, l-methyl DOPA and guaiacol substrates were determined. Inhibitors, viz. azide, diethyldithiocarbamate, thioglycollate and cysteine-hydrochloride all inhibited laccase non-competitively using guaiacol as substrate at pH 6.5. The enzyme degraded indigo carmine (pH 9, 55 °C) to anthranilic acid via isatin as determined spectrophotometrically and by HPLC analysis. Degradation was enhanced in the presence of syringaldehyde (571%), vanillin (156%) and p-hydroxybenzoic acid (91.6%) but not HOBT.     

Functional divergence and comparative in‐silico study of Cas4 proteins of DUF83 class

Clustered Regularly Interspaced Short Palindromic Repeats‐ C RISPR a ssociated (CRISPR‐Cas) systems present in genomes of bacteria and archaea have been the focus of many research studies recently. The Cas4 proteins of these systems are thought to be responsible for the adaptation step in the CRISPR mechanism. Cas4 proteins exhibit low sequence similarity among themselves and are currently classified into 2 main classes: DUF83 and DUF911. The characteristic features of Cas4 proteins belonging to DUF83 class have been elucidated by determining the structures of Cas4 protein from Sulfolobus solfataricus and Pyrobaculum calidifontis. Although, both Cas4 proteins characterized structurally are of same DUF83 class, these 2 proteins do exhibit significant biochemical and functional differences. The aim of the present study was to explore the structural and evolutionary features responsible for these differences. Our study predicts residues which might be responsible for such differences. Functional divergence analysis was used to predict sites exhibiting type I divergence, where certain amino acids are conserved in 1 clade whereas the same site is highly variable in the other clade. Our intra‐molecular interaction analysis reinforces the influence of such divergence sites on the other functionally important amino acids. In general, this study identifies some of the divergence hotspots that could be the focus of future experimental studies for better understanding of Cas4 enzymatic activity in CRISPR mechanism.     

Biochemical and histological changes during in vitro rooting of microcuttings of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.) Wettst.

The present study was conducted to investigate the biochemical changes vis-à-vis histological changes during adventitious rooting of microcuttings of Bacopa monnieri (L.) Wettst. The rooting in these microcuttings was induced on basal MS medium and medium supplemented with different concentrations of indole-3-acetic acid and indole-3-butyric acid (IBA). Presence of lower auxin concentration (1.0 µM) in the medium enhanced rooting and significantly improved number of roots per shoot but maximum root length was observed on basal MS medium. Histological studies were conducted to identify different phases of rooting in these microcuttings. The root meristemoids with distinct polarity become visible after 3 days and mark the beginning of in vitro root initiation phase. It was followed by primordia elongation, root emergence and visible rooting on the 5th day of culture on medium supplemented with auxins. Biochemical studies were also conducted from basal portions of microcuttings cultured on MS medium supplemented with 1.0 µM IBA and control (basal MS medium) from 0 to 7 days. Total carbohydrate content was lower during initial periods (up to day 1) and was found to increase during root initiation and primordia development, which reflects high energy demands for active cell divisions. A significantly higher level of phenols was recorded in microcuttings on medium supplemented with IBA. Polyphenol oxidase, peroxidase (POX), ascorbate peroxidase activities were also found to vary during different phases of rhizogenesis. Early phases were also marked with the lower activities of POX and IAAO. This study revealed significant role of enzymes, sugars and phenols during different phases of rooting.     

Supply-Side Analysis of Growth of Bacillus subtilis on Glucose-Citrate Medium: Feasible Network Alternatives and Yield Optimality

Our prior work revealed that compared to the case for glucose metabolism, increased carbon yield and nil acid formation result when Bacillus subtilis grows on glucose medium containing citrate. To scrutinize further how citrate addition may alter metabolic flux regulation and the degree that the observed carbon yield corresponds to the maximal value, experimental (by least-squares analysis) and optimal (by linear programming) fluxes and yields were contrasted. Networks with differing reaction routes, directionality constraints, and transhydrogenase activities were examined. To attain an elevated carbon yield, citrate-glucose utilization need not alleviate any stoichiometric constraints that can sometimes interfere with the attainment of network objectives. Rather, the high carbon yield and nil acid formation attained may be linked to restriction of glycolytic capacity, particularly at the level of pyruvate kinase, which is consistent with a hypothesized effect of coupled metal-citrate uptake. Allowing for malic enzyme activity, hexose monophosphate pathway cycling, and transhydrogenase activity may also lead to the flux distributions underlying the high carbon yield observed. Finally, the observed carbon yield corresponded well to the maximum yield provided by all the network alternatives examined. Collectively, these results suggest that (i) the observed carbon yield is essentially equal to the maximal values associated with plausible networks and (ii), as suggested by others, nonoptimal flux regulation may contribute significantly to apparent cellular maintenance requirements.