First fossil evidence of samaras of Ventilago Gaertn. (Rhamnaceae) from India and its implications

The Ventilago Gaertn. (Rhamnaceae) is widely distributed in pantropical areas of Africa, Asia, and Australia. However, fossil records of this taxon are sparse, which limits understanding of the evolution and biogeographic history of the genus. In the present study, we report and describe two new fossil species of Ventilago, V. siwalika sp. nov. from the Miocene sediments of Himachal Pradesh, western Himalaya, and V. pliocenica sp. nov. from the Pliocene sediments of Jharkhand, eastern India based on single-winged samaras. Ventilago pliocenica is characterized by a prominent midvein, obtuse to sub-round apex with mucronate tip, longitudinal secondary veins extending the full length of the fruit, and reticulate nature of higher-order veins, the presence of equatorial rim, the hypanthium, and short pedicel. On the other hand, V. siwalika is characterized by a prominent midvein, obtuse to sub-round apex with mucronate tip, longitudinal secondary veins extending the full length of the fruit, and reticulate nature of higher-order veins. Our discovery represents the first unambiguous fossil record of single-winged samara of Ventilago from India and provides valuable insights into the evolution of this genus. In this paper, we also review its biogeographic history and add new information to understand its hypothetical migration route. Present and earlier records of Ventilago also suggest that this genus was a common forest element during Neogene (Miocene time) in Asia.     

Mechanism of Ds1 excision from the genome of maize streak virus

Summary We have previously shown that the maize transposable element Ds1 introduced into maize plants by agroinfection can be excised from the genome of geminivirus maize streak virus (MSV). Excision depended strictly on the presence of an active Ac element in the plants. In this study, the excision products or footprints left in the MSV genome after Ds1 excision were extensively characterized and the effects of flanking sequences on Ds1 excision were analysed. Most types of footprints obtained were comparable to those described for Ds1 excision in the maize genome, and could be explained by the models proposed for excision of plant transposable elements. In two revertants, however, some terminal sequences of the Ds1 element were found to have been left behind at the excision site. The finding of this novel type of Ds1 footprint indicated that gene conversion events occurred during and/or after Ds1 excision from the MSV genome. A partial deletion of one copy of the 8 by duplications flanking the Ds1 element had no effect on the frequency or on the types of footprints of Ds1 excision from the MSV genome. Thus, the duplicated 8 by sequences flanking the transposable element are not involved in Ds1 excision. These results, as well as a statistical analysis of the modifications of the bases flanking the Ds1 element after excision, are discussed in terms of excision models.     

Human-Associated Methicillin-Resistant Staphylococcus aureus from a Subtropical Recreational Marine Beach

Reports of Staphylococcus aureus including methicillin-resistant S. aureus (MRSA) detected in marine environments have occurred since the early 1990s. This investigation sought to isolate and characterize S. aureus from marine waters and sand at a subtropical recreational beach, with and without bathers present, in order to investigate possible sources and to identify the risks to bathers of exposure to these organisms. During 40 days over 17 months, 1,001 water and 36 intertidal sand samples were collected by either bathers or investigators at a subtropical recreational beach. Methicillin-sensitive S. aureus (MSSA) and MRSA were isolated and identified using selective growth media and an organism-specific molecular marker. Antimicrobial susceptibility, staphylococcal cassette chromosome mec (SCCmec) type, pulsed-field gel electrophoresis (PFGE) pattern, multi-locus sequence type (MLST), and staphylococcal protein A (spa) type were characterized for all MRSA. S. aureus was isolated from 248 (37 %) bather nearby water samples at a concentration range of <2–780 colony forming units per ml, 102 (31 %) ambient water samples at a concentration range of <2–260 colony forming units per ml, and 9 (25 %) sand samples. Within the sand environment, S. aureus was isolated more often from above the intertidal zone than from intermittently wet or inundated sand. A total of 1334 MSSA were isolated from 37 sampling days and 22 MRSA were isolated from ten sampling days. Seventeen of the 22 MRSA were identified by PFGE as the community-associated MRSA USA300. MRSA isolates were all SCCmec type IVa, encompassed five spa types (t008, t064, t622, t688, and t723), two MLST types (ST8 and ST5), and 21 of 22 isolates carried the genes for Panton–Valentine leukocidin. There was a correlation (r?=?0.45; p?=?0.05) between the daily average number of bathers and S. aureus in the water; however, no association between exposure to S. aureus in these waters and reported illness was found. This report supports the concept that humans are a potential direct source for S. aureus in marine waters.     

Elicitation of simian immunodeficiency virus-specific cytotoxic T lymphocytes in mucosal compartments of rhesus monkeys by systemic vaccination

Since most human immunodeficiency virus (HIV) infections are initiated following mucosal exposure to the virus, the anatomic containment or abortion of an HIV infection is likely to require vaccine-elicited cellular immune responses in those mucosal sites. Studying vaccine-elicited mucosal immune responses has been problematic because of the difficulties associated with sampling T lymphocytes from those anatomic compartments. In the present study, we demonstrate that mucosal cytotoxic T lymphocytes (CTL) specific for simian immunodeficiency virus (SIV) and simian HIV can be reproducibly sampled from intestinal mucosal tissue of rhesus monkeys obtained under endoscopic guidance. These lymphocytes recognize peptide-major histocompatibility complex class I complexes and express gamma interferon on exposure to peptide antigen. Interestingly, systemic immunization of monkeys with plasmid DNA immunogens followed by live recombinant attenuated poxviruses or adenoviruses with genes deleted elicits high-frequency SIV-specific CTL responses in these mucosal tissues. These studies therefore suggest that systemic delivery of potent HIV immunogens may suffice to elicit substantial mucosal CTL responses.     

Prior vaccination increases the epitopic breadth of the cytotoxic T-lymphocyte response that evolves in rhesus monkeys following a simian-human immunodeficiency virus infection

Although recent evidence has confirmed the importance of cytotoxic T-lymphocyte (CTL) responses in controlling human immunodeficiency virus type 1 and simian immunodeficiency virus replication, the relevance of the epitopic breadth of those CTL responses remains unexplored. In the present study, we sought to determine whether vaccination can expand CTL populations which recognize a repertoire of viral epitopes that is greater than is typically generated in the course of a viral infection. We demonstrate that potent secondary CTL responses to subdominant epitopes are rapidly generated following a pathogenic simian-human immunodeficiency virus challenge of rhesus monkeys vaccinated with plasmid DNA or recombinant modified vaccinia virus Ankara vaccines. These data indicate that prior vaccination can increase the breadth of the CTL response that evolves after an AIDS virus infection.     

TIMP-1 Inhibits Apoptosis in Lung Adenocarcinoma Cells via Interaction with Bcl-2

Tissue inhibitors of metalloproteinases (TIMPs) are multifaceted molecules that exhibit properties beyond their classical proteinase inhibitory function. Although TIMP-1 is a known inhibitor of apoptosis in mammalian cells, the mechanisms by which it exerts its effects are not well-established. Our earlier studies using H2009 lung adenocarcinoma cells, implanted in the CNS, showed that TIMP-1 overexpressing H2009 cells (HB-1), resulted in more aggressive tumor kinetics and increased vasculature. The present study was undertaken to elucidate the role of TIMP-1 in the context of apoptosis, using the same lung cancer cell lines. Overexpressing TIMP-1 in a lung adenocarcinoma cell line H2009 resulted in an approximately 3-fold increased expression of Bcl-2, with a marked reduction in apoptosis upon staurosporine treatment. This was an MMP-independent function as a clone expressing TIMP-1 mutant T2G, lacking MMP inhibition activity, inhibited apoptosis as strongly as TIMP1 overexpressing clones, as determined by inhibition of PARP cleavage. Immunoprecipitation of Bcl-2 from cell lysates also co-immunoprecipitated TIMP-1, indicative of an interaction between these two proteins. This interaction was specific for TIMP-1 as TIMP-2 was not present in the Bcl-2 pull-down. Additionally, we show a co-dependency of TIMP-1 and Bcl-2 RNA and protein levels, such that abrogating Bcl-2 causes a downregulation of TIMP-1 but not TIMP-2. Finally, we demonstrate that TIMP-1 dependent inhibition of apoptosis occurs through p90RSK, with phosphorylation of the pro-apoptotic protein BAD at serine 112, ultimately reducing Bax levels and increasing mitochondrial permeability. Together, these studies define TIMP-1 as an important cancer biomarker and demonstrate the potential TIMP-1 as a crucial therapeutic target.     

A thermostable cysteine protease precursor from a tropical plant contains an unusual C-terminal propeptide: cDNA cloning, sequence comparison and molecular modeling studies

We report here the cloning and characterization of the entire cDNA of a papain-like cysteine protease from a tropical flowering plant. The 1098-bp ORF of the cDNA codify a protease precursor having a signal peptide of 19 amino acids, a cathepsin-L like N-terminal proregion of 114 amino acids, a mature enzyme part of 208 amino acids and a C-terminal proregion of 24 amino acids. The derived amino acid sequence of the mature part tallies with the thermostable cysteine protease Ervatamin-C--as was aimed at. The C-terminal proregion of the protease has altogether a different sequence pattern not observed in other members of the family and it contains a negatively charged helical zone. The three-dimensional model of the precursor, based on the homology modeling and X-ray structure, shows that the extended peptide stretch region of the N-terminal propeptide, covering the interdomain cleft, contains protruding side chains of positively charged residues. This study also indicates that the negatively charged zone of C-terminal propeptide may interact with the positively charged zone of the N-terminal propeptide in a cooperative manner in the maturation process of this enzyme.     

Maintaining Specimen Integrity for G6PD Screening by Cytofluorometric Assays

Cytochemical staining remains an efficient way of identifying females who are heterozygous for the X chromosome-linked glucose-6-phosphate dehydrogenase (G6PD) gene. G6PD is highly polymorphic with certain alleles resulting in low intracellular G6PD activity in red blood cells. Low intracellular G6PD activity is associated with a risk of severe hemolysis when exposed to an oxidative stress such as fava beans, certain drugs and infections. Heterozygous females express the enzyme from both X-chromosome alleles resulting in two red blood cell populations each with G6PD enzyme characteristics representative of each allele; for example, normal and deficient. Cytochemical staining is the only way to determine the relative representation of each allele in red blood cells, a feature that is critical when assessing the risk for severe hemolysis when exposed to an oxidant such as the anti-malarial drug primaquine. This letter discusses red blood cell integrity with respect to the cytofluorometric assays for G6PD activity. An approach to making this test more robust is suggested. The approach makes this test more reliable and extends its use to a broader range of blood specimens.     

C-Terminal extension of a plant cysteine protease modulates proteolytic activity through a partial inhibitory mechanism

The amino acid sequence of ervatamin-C, a thermostable cysteine protease from a tropical plant, revealed an additional 24-amino-acid extension at its C-terminus (CT). The role of this extension peptide in zymogen activation, catalytic activity, folding and stability of the protease is reported. For this study, we expressed two recombinant forms of the protease in Escherichia coli, one retaining the CT-extension and the other with it truncated. The enzyme with the extension shows autocatalytic zymogen activation at a higher pH of 8.0, whereas deletion of the extension results in a more active form of the enzyme. This CT-extension was not found to be cleaved during autocatalysis or by limited proteolysis by different external proteases. Molecular modeling and simulation studies revealed that the CT-extension blocks some of the substrate-binding unprimed subsites including the specificity-determining subsite (S2) of the enzyme and thereby partially occludes accessibility of the substrates to the active site, which also corroborates the experimental observations. The CT-extension in the model structure shows tight packing with the catalytic domain of the enzyme, mediated by strong hydrophobic and H-bond interactions, thus restricting accessibility of its cleavage sites to the protease itself or to the external proteases. Kinetic stability analyses (T(50) and t(1/2) ) and refolding experiments show similar thermal stability and refolding efficiency for both forms. These data suggest that the CT-extension has an inhibitory role in the proteolytic activity of ervatamin-C but does not have a major role either in stabilizing the enzyme or in its folding mechanism.