Profound Effects of Aggregatibacter actinomycetemcomitans Leukotoxin Mutation on Adherence Properties Are Clarified in in vitro Experiments

Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivary-coated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previous in vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2) some caution should be used when interpreting the effect attributed to targeted gene mutations when seen in a competitive in vivo environment.     

Combined effect of 24-epibrassinolide and salicylic acid mitigates lead (Pb) toxicity by modulating various metabolites in <Emphasis Type="BoldItalic">Brassica juncea</Emphasis> L. seedlings

The present study demonstrated the combined effect of 24-epibrassinolide and salicylic acid against lead (Pb, 0.25, 0.50, and 0.75 mM) toxicity in Brassica juncea seedlings. Various parameters including water status, metal uptake, total water- and lipid-soluble antioxidants, metal chelator content (total thiols, protein-bound thiols, and non-protein-bound thiols), phenolic compounds (flavonoids, anthocyanins, and polyphenols), and organic acids were studied in 10-day-old seedlings. Dry matter content and the heavy metal tolerance index were reduced by 42.24 and 52.3%, respectively, in response to Pb treatment. Metal uptake, metal-chelating compounds, phenolic compounds, and organic acids were increased in Pb-treated seedlings as compared to control plants. The treatment of Pb-stressed seedlings with combination of EBL and SA resulted in enhancement of heavy metal tolerance index by 40.07%, water content by 1.84%, and relative water content by 23.45%. The total water- and lipid-soluble antioxidants were enhanced by 21.01 and 2.21%, respectively. In contrast, a significant decline in dry weight, metal uptake, thiol, and polyphenol contents was observed following the application of 24-epibrassinolide and salicylic acid. These observations indicate that Pb treatment has an adverse effect on B. juncea seedlings. However, co-application of 24-epibrassinolide and salicylic acid mitigates the negative effects of Pb, by lowering Pb metal uptake and enhancing the heavy metal tolerance index, water content, relative water content, antioxidative capacities, phenolic content, and organic acid levels.     

Fast Fourier infrared spectroscopy to characterize the biochemical composition in diatoms

Diatoms are photosynthetic unicellular microalgae and are nature’s hidden source of several biosynthetic metabolites with their use in biofuel, food and drug industries. They mainly contain various lipids, sterols, isoprenoids and toxins with their use in apoptotic, fertility controlling and cancer drugs. Chemical studies on diatoms are limited due to various limitations such as variation of nutrients, contaminants and change in seasonal factors in the environment. To overcome these limitations, we obtained axenic cultures of 12 fresh-water diatom strains on the 22nd day of inoculation having a dry weight of 1 mg each and performed their Fourier transform infrared (FTIR) study for the detection of functional groups responsible for their chemical moiety. The spectral mapping showed a varied level of polyunsaturated fatty acids, amides, amines, ketone bodies and esters for their applications in various pharmacological, food and biofuel industries in the exponential phase of their growth in f/2 media. The FTIR study of the 12 diatom strains showed various similarities in the form of some common peak patterns ranging from 3000 to 3600 cm^?1 for ν_O–H absorption. The symmetric stretching vibration frequency of Diadesmis confervaceae (V2) type species showed different behaviour than others in the spectral region starting from 1600 to 1700 cm^?1. The absorption between 1500 and 1575 cm^?1 reflects the presence of the –N–H group. Infrared (IR) absorptions falling between 1600 and 1700 cm^?1 reflect the presence of amide’s ν_C=O in all species. Placoneis elginensis (V8) type species showed an additional absorption band which is centred around 1735–1750 cm^?1 which perhaps reflects the presence of ester’s ν_C=O. Diadesmis confervaceae (V2), Nitzschia palea (V4), Placoneis elginensis (V8), Nitzschia palea var. debilis (V6), Nitzschia inconspicua (V10), Gomphonema parvulum (V11) and Sellaphora (V12) showed distinct structural features with important key functionalities that can make them essential drug markers in the pharmaceutical industry.     

Association of <Emphasis Type="Italic">Toll like receptor 2</Emphasis> and <Emphasis Type="Italic">9</Emphasis> gene variants with pulmonary tuberculosis: exploration in a northern Indian population

Tuberculosis (TB) is a disease of global importance. There is an increasing recognition of the role of Toll like receptors, important pattern recognition receptors of host immune system, in determining the susceptibility or resistance to TB in various populations. In an attempt to examine the importance of Toll like receptors in immune response to Mycobacterium tuberculosis infection, we explored two variants each of TLR2 and TLR9 in a population residing in Uttar Pradesh, India. Genotyping was performed to detect -196 to -174 del polymorphism and G2258A SNP (Arg753Gln, rs5743708) in TLR2 gene and -T1237C (rs5743836) and G2848A (rs352140) SNP in TLR9 gene in patients with pulmonary TB and healthy controls. The A allele of G2848A SNP in TLR9 gene was found with a marginally higher frequency among TB patients as compared to healthy controls, suggesting that A allele at position 2848 of TLR9 gene may be associated with susceptibility to TB in North Indian population [p?=?0.05, Mantel–Haenszel OR?=?1.34, 95% CI (1.0–1.82)].     

Aspergillosis in German cockroach Blattella germanica (L.) (Blattoidea: Blattellidae)

Natural infection of Aspergillus flavus was observed in adults of Blattella germanica. Though the adult insects exhibited no external symptoms, they became hypoactive and later died. The dead and experimentally infected insects repeatedly yielded Aspergillus flavus in culture on Czapek's medium. Direct microscopic observation of the tissues of infected insects revealed fungal material. The blood films stained with Giemsa stain showed granulocytes (GRs) engulfing fungal hyphae. A remarkable increase in GR and plasmatocyte (PL) counts occurred in differential haemocyte counts (DHCs) of the infected insects. Two main types of immunological responses of the insect noticed were phagocytosis and encapsulation. DHC showed maximum involvement of GRs and PLs in immune mechanism. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies on the expression of 80-kDa human sperm antigen in rat testis and epididymis.

The 80-kDa human sperm antigen (HSA) has demonstrated to be a promising candidate for development of an antifertility vaccine because it is a sperm-specific, conserved, and immunogenic protein. The present study demonstrates the androgen-regulated expression of 80-kDa HSA in testis and epididymis of rat by immunohistochemistry (IHC), using its specific antibodies. Developmental expression of 80-kDa HSA was investigated on days 10, 20, 40, 60, and 90 of age in the testis and epididymis by IHC, and relative staining intensity was estimated by image analysis using BIOVIS software. On days 10 and 20, no significant staining was observed in the testis and epididymis, whereas it gradually increased from day 40 onwards. The highest staining was seen on day 90 in both testis and epididymis. Gradual increase in expression of 80-kDa HSA after day 40 suggests that it is possibly regulated by androgen. To study the androgen-regulated expression of 80-kDa, adult male rats were treated with 75 mg/kg body weight of ethylene dimethane sulfonate (EDS), which selectively destroys Leydig cells and thus induces complete androgen withdrawal. It was observed that the staining intensity decreased following EDS treatment in rat testis as well as epididymis, and it was regained after supplementation with dihydrotestosterone. Increased expression during sexual maturation at the time of testosterone surge and its regulation by antiandrogen/androgen treatment suggest androgen-dependent expression of 80-kDa HSA in rat testis and epididymis.     

In Vivo Addition of Poly(A) Tail and AU-Rich Sequences to the 3′ Terminus of the Sindbis Virus RNA Genome: a Novel 3′-End Repair Pathway

Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5′ end and polyadenylated at its 3′ end. The 3′ nontranslated region (3′NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3′CSE) and a poly(A) tail. This 3′CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693–2704, 1997). Here, we demonstrate that the 3′-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3′NTR, we generated a battery of mutant genomes with mutations in the 3′NTR and tested their ability to generate infectious virus and undergo 3′ polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3′CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3′CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3′NTR. These results indicate the ability of alphavirus RNAs to undergo 3′ repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3′ end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.     

Click and Cut: a click chemistry approach to developing oxidative DNA damaging agents

Metallodrugs provide important first-line treatment against various forms of human cancer. To overcome chemotherapeutic resistance and widen treatment possibilities, new agents with improved or alternative modes of action are highly sought after. Here, we present a click chemistry strategy for developing DNA damaging metallodrugs. The approach involves the development of a series of polyamine ligands where three primary, secondary or tertiary alkyne-amines were selected and ‘clicked’ using the copper-catalysed azide-alkyne cycloaddition reaction to a 1,3,5-azide mesitylene core to produce a family of compounds we call the ‘Tri-Click’ (TC) series. From the isolated library, one dominant ligand (TC1) emerged as a high-affinity copper(II) binding agent with potent DNA recognition and damaging properties. Using a range of in vitro biophysical and molecular techniques—including free radical scavengers, spin trapping antioxidants and base excision repair (BER) enzymes—the oxidative DNA damaging mechanism of copper-bound TC1 was elucidated. This activity was then compared to intracellular results obtained from peripheral blood mononuclear cells exposed to Cu(II)–TC1 where use of BER enzymes and fluorescently modified dNTPs enabled the characterisation and quantification of genomic DNA lesions produced by the complex. The approach can serve as a new avenue for the design of DNA damaging agents with unique activity profiles.     

Structural and functional characterization of a ubiquitin variant engineered for tight and specific binding to an alpha‐helical ubiquitin interacting motif

Ubiquitin interacting motifs (UIMs) are short α‐helices found in a number of eukaryotic proteins. UIMs interact weakly but specifically with ubiquitin conjugated to other proteins, and in so doing, mediate specific cellular signals. Here we used phage display to generate ubiquitin variants (UbVs) targeting the N‐terminal UIM of the yeast Vps27 protein. Selections yielded UbV.v27.1, which recognized the cognate UIM with high specificity relative to other yeast UIMs and bound with an affinity more than two orders of magnitude higher than that of ubiquitin. Structural and mutational studies of the UbV.v27.1‐UIM complex revealed the molecular details for the enhanced affinity and specificity of UbV.v27.1, and underscored the importance of changes at the binding interface as well as at positions that do not contact the UIM. Our study highlights the power of the phage display approach for selecting UbVs with unprecedented affinity and high selectivity for particular α‐helical UIM domains within proteomes, and it establishes a general approach for the development of inhibitors targeting interactions of this type.