Curcumin-arteether combination therapy of Plasmodium berghei-infected mice prevents recrudescence through immunomodulation

Earlier studies in this laboratory have shown the potential of artemisinin-curcumin combination therapy in experimental malaria. In a parasite recrudescence model in mice infected with Plasmodium berghei (ANKA), a single dose of alpha,beta-arteether (ART) with three oral doses of curcumin prevented recrudescence, providing almost 95% protection. The parasites were completely cleared in blood with ART-alone (AE) or ART+curcumin (AC) treatments in the short-term, although the clearance was faster in the latter case involving increased ROS generation. But, parasites in liver and spleen were not cleared in AE or AC treatments, perhaps, serving as a reservoir for recrudescence. Parasitemia in blood reached up to 60% in AE-treated mice during the recrudescence phase, leading to death of animals. A transient increase of up to 2–3% parasitemia was observed in AC-treatment, leading to protection and reversal of splenomegaly. A striking increase in spleen mRNA levels for TLR2, IL-10 and IgG-subclass antibodies but a decrease in those for INFγ and IL-12 was observed in AC-treatment. There was a striking increase in IL-10 and IgG subclass antibody levels but a decrease in INFγ levels in sera leading to protection against recrudescence. AC-treatment failed to protect against recrudescence in TLR2^−/− and IL-10^−/− animals. IL-10 injection to AE-treated wild type mice and AC-treated TLR2^−/− mice was able to prolong survival. Blood from the recrudescence phase in AE-treatment, but not from AC-treatment, was able to reinfect and kill naïve animals. Sera from the recrudescence phase of AC-treated animals reacted with several parasite proteins compared to that from AE-treated animals. It is proposed that activation of TLR2-mediated innate immune response leading to enhanced IL-10 production and generation of anti-parasite antibodies contribute to protective immunity in AC-treated mice. These results indicate a potential for curcumin-based combination therapy to be tested for prevention of recrudescence in falciparum and relapse in vivax malaria.     

Unique properties of Plasmodium falciparum porphobilinogen deaminase

The hybrid pathway for heme biosynthesis in the malarial parasite proposes the involvement of parasite genome-coded enzymes of the pathway localized in different compartments such as apicoplast, mitochondria, and cytosol. However, knowledge on the functionality and localization of many of these enzymes is not available. In this study, we demonstrate that porphobilinogen deaminase encoded by the Plasmodium falciparum genome (PfPBGD) has several unique biochemical properties. Studies carried out with PfPBGD partially purified from parasite membrane fraction, as well as recombinant PfPBGD lacking N-terminal 64 amino acids expressed and purified from Escherichia coli cells (DeltaPfPBGD), indicate that both the proteins are catalytically active. Surprisingly, PfPBGD catalyzes the conversion of porphobilinogen to uroporphyrinogen III (UROGEN III), indicating that it also possesses uroporphyrinogen III synthase (UROS) activity, catalyzing the next step. This obviates the necessity to have a separate gene for UROS that has not been so far annotated in the parasite genome. Interestingly, DeltaPfP-BGD gives rise to UROGEN III even after heat treatment, although UROS from other sources is known to be heat-sensitive. Based on the analysis of active site residues, a DeltaPfPBGDL116K mutant enzyme was created and the specific activity of this recombinant mutant enzyme is 5-fold higher than DeltaPfPBGD. More interestingly, DeltaPfPBGDL116K catalyzes the formation of uroporphyrinogen I (UROGEN I) in addition to UROGEN III, indicating that with increased PBGD activity the UROS activity of PBGD may perhaps become rate-limiting, thus leading to non-enzymatic cyclization of preuroporphyrinogen to UROGEN I. PfPBGD is localized to the apicoplast and is catalytically very inefficient compared with the host red cell enzyme.     

An improved phage-display panning method to produce an HM-1 killer toxin anti-idiotypic antibody

Background  

Phage-display panning is an integral part of biomedical research. Regular panning methods are sometimes complicated by inefficient detachment of the captured phages from the antigen-coated solid supports, which prompted us to modify. Here, we produce an efficient antigen-specific single chain fragment variable (scFv) antibody by using a target-related molecule that favored selection ofrecombinant antibodies.     

Antimycobacterial activities of novel 2-(sub)-3-fluoro/nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid

Various 2-(sub)-3-fluoro/nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid derivatives were synthesized from 2-aminothiophenol by a five-step reaction, evaluated for in-vitro and in-vivo antimycobacterial activities against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB), and Mycobacterium smegmatis (MC2), and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from M. smegmatis. Among the thirty-four synthesized compounds, 2-(3-(diethylcarbamoyl)piperidin-1-yl)-)-3-fluoro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid (7l) was found to be the most active compound in vitro with MIC of 0.18 and 0.08 microM against MTB and MTR-TB, respectively. Compound 7l was found to be 2 and 570 times more potent than isoniazid against MTB and MDR-TB, respectively. In the in-vivo animal model 7l decreased the bacterial load in lung and spleen tissues with 2.78 and 3.12-log10 protections, respectively, at the dose of 50 mg/kg body weight.     

Optimization of media composition for Nattokinase production by Bacillus subtilis using response surface methodology

Response surface methodology and central composite rotary design (CCRD) was employed to optimize a fermentation medium for the production of Nattokinase by Bacillus subtilis at pH 7.5. The four variables involved in this study were Glucose, Peptone, CaCl(2), and MgSO(4). The statistical analysis of the results showed that, in the range studied; only peptone had a significant effect on Nattokinase production. The optimized medium containing (%) Glucose: 1, Peptone: 5.5, MgSO(4): 0.2 and CaCl(2): 0.5 resulted in 2-fold increased level of Nattokinase (3194.25U/ml) production compared to initial level (1599.09U/ml) after 10h of fermentation. Nattokinase production was checked with fibrinolytic activity.     

Phenotypic Assessment of Probiotic and Bacteriocinogenic Efficacy of Indigenous LAB Strains from Human Breast Milk

Breast milk is the combination of bioactive compounds and microflora that promote newborn’s proper growth, gut flora, and immunity. Thus, it is always considered the perfect food for newborns. Amongst their bioactives, probiotic communities—especially lactic acid bacteria (LAB)—are characterized from breast milk over the first month of parturition. In this study, seven LAB were characterized phenotypically and genotypically as Levilactobacillus brevis BDUMBT08 ({"type":"entrez-nucleotide","attrs":{"text":"MT673657","term_id":"1863149999"}}MT673657), L. gastricus BDUMBT09 ({"type":"entrez-nucleotide","attrs":{"text":"MT774596","term_id":"1873222494"}}MT774596), L. paracasei BDUMBT10 ({"type":"entrez-nucleotide","attrs":{"text":"MT775430","term_id":"1874262304"}}MT775430), L. brevis BDUMBT11 ({"type":"entrez-nucleotide","attrs":{"text":"MW785062","term_id":"2009762994"}}MW785062), L. casei BDUMBT12 ({"type":"entrez-nucleotide","attrs":{"text":"MW785063","term_id":"2009820773"}}MW785063), L. casei BDUMBT13 ({"type":"entrez-nucleotide","attrs":{"text":"MW785178","term_id":"2010296340"}}MW785178), and Brevibacillus brevis M2403 ({"type":"entrez-nucleotide","attrs":{"text":"MK371781","term_id":"1548854744"}}MK371781) from human breast milk. Their tolerance to lysozyme, acid, bile, gastric juice, pancreatic juice, and NaCl and potential for mucoadhesion, auto-aggregation, and co-aggregation with pathogens are of great prominence in forecasting their gut colonizing ability. They proved their safety aspects as they were negative for virulence determinants such as hemolysis and biofilm production. Antibiogram of LAB showed their sensitivity to more than 90% of the antibiotics tested. Amongst seven LAB, three isolates (L. brevis BDUMBT08 and BDUMBT11, and L. gatricus BDUMBT09) proved their bacteriocin producing propensity. Although the seven LAB isolates differed in their behavior, their substantial probiotic properties with safety could be taken as promising probiotics for further studies to prove their in vivo effects, such as health benefits, in humans.     

Role of heme in the synthesis of cytochrome c oxidase in Neurospora crassa

The role of heme in the synthesis of cytochrome c oxidase has been investigated in the mold Neurospora crassa. Iron-deficient cultures of the mold have low levels of cytochrome oxidase and delta-aminolevulinate dehydratase, the latter being the rate-limiting enzyme of the heme-biosynthetic pathway in this organism. Addition of iron to the iron-deficient cultures results in an immediate increase in the levels of delta-aminolevulinate dehydratase followed by an increase in the rate of heme synthesis and cytochrome oxidase levels. The rate of precursor labeling of the mitochondrial subunits of cytochrome oxidase is decreased preferentially under conditions of iron deficiency and addition of iron corrects this picture. Exogenous hemin addition which prevents iron-mediated induction of delta-aminolevulinate dehydratase also inhibits the increase in the activity of cytochrome oxidase and the enhanced precursor labeling of the mitochondrial subunits of cytochrome oxidase. Protein synthesis on mitoribosomes measured in vivo and in vitro is decreased under conditions of heme deficiency. Hemin addition in vitro to mitochondrial lysates prepared from heme-deficient mycelia restores a near normal rate of protein synthesis. It is concluded that heme is required for the optimal rate of translation on mitoribosomes.     

A Cytochemical Study on the Mycorrhizae of Spathoglottis Plicata

This paper describes the hitherto unreported aspects of orchid mycorrhizae. The host cells harbour upto 4 generations of fungal pelotons which are formed after each peloton is digested. There are two types of hyphae in a host cell, one forming the pelotons, and the other which lies close to the host cell wall and separated from the former by a callosic wall. The later, called non-pelotonic hyphae form the fresh peloton when the former is digested. Consequently, there are also cytochemical differences between these two types of hyphae. This revised version was published online in July 2006 with corrections to the Cover Date.