Plasmodium falciparum glyoxalase II: Theorell-Chance product inhibition patterns, rate-limiting substrate binding via Arg(257)/Lys(260), and unmasking of acid-base catalysis

Glyoxalase II (GloII) is a ubiquitous thioester hydrolase catalyzing the last step of the glutathione-dependent conversion of 2-oxoaldehydes to 2-hydroxycarboxylic acids. Here, we present a detailed structure-function analysis of cGloII from the malaria parasite Plasmodium falciparum. The activity of the enzyme was salt-sensitive and pH-log k(cat) and pH-log k(cat)/K-m profiles revealed acid-base catalysis. An acidic pK(a)(app) value of approximately 6 probably reflects hydroxide formation at the metal center. The glutathione-binding site was analyzed by site-directed mutagenesis. Substitution of residue Arg(154) caused a 2.5-fold increase of K-m(app), whereas replacements of Arg(257) or Lys(260) were far more detrimental. Although the glutathione-binding site and the catalytic center are separated, six of six single mutations at the substrate-binding site decreased the k(cat)(app) value. Furthermore, product inhibition studies support a Theorell-Chance Bi Bi mechanism with glutathione as the second product. We conclude that the substrate is predominantly bound via ionic interactions with the conserved residues Arg(257) and Lys(260), and that correct substrate binding is a pH-and salt-dependent rate-limiting step for catalysis. The presented mechanistic model is presumably also valid for GloII from many other organisms. Our study could be valuable for drug development strategies and enhances the understanding of the chemistry of binuclear metallohydrolases

'Greengold' - A Late Season Avocado

'Greengold' is a high-quality, heavy-bearing, late-season avocado (Persea americana L.) developed by the Department of Horticulture, University of Hawai'i at Manoa. It meets the need for a quality, high-yielding cultivar suitable for local and export markets

Proinflammatory cytokines inhibit osteogenic differentiation from stem cells: implications for bone repair during inflammation

SummaryObjectiveThe effects of inflammation on bone development from mesenchymal stem cells (MSC) are unclear due to the difficulty in isolating MSC. The aim of this study was to develop a MSC isolation method and to determine the in vitro effects of interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) on their osteogenic differentiation.MethodsMurine MSC were isolated from the limbs of C57/Bl6 mice through collagenase digestion of bone and enriched as the Stem cell antigen (Sca-1)+ CD31− CD45− population, using lineage immunodepletion, followed by fluorescence-activated cell sorting (FACS). They were differentiated along the osteoblast linage in the presence or absence of IL-1β and TNFα. Mineralization was measured as was the expression of a number of osteogenic genes by quantitative polymerase chain reaction (PCR).ResultsWe show that osteogenic differentiation from the MSC population is suppressed by IL-1β and TNFα. In addition to suppression of bone mineralization, both cytokines inhibited the differentiation-associated increases in alkaline phosphatase (ALP) activity and the gene expression for ALP, α1(I) procollagen, runt-related transcription factor 2 (Runx2) and osterix. However, only TNFα inhibited osteonectin and osteopontin mRNA expression and only IL-1β reduced cell proliferation.ConclusionsThe convenient isolation technique enables the easy generation of sufficient MSC to permit the molecular analysis of their differentiation. We were thus able to show that the proinflammatory cytokines, IL-1β and TNFα, can compromise bone development from this primary MSC population, although with some significant differences. The potential involvement of specific inflammatory mediators needs to be taken into account if optimal bone repair and presumably that of other tissues are to be achieved with MSC

Large-Scale Structure at z~2.5

We have made a statistically complete, unbiased survey of C IV systems toward a region of high QSO density near the South Galactic Pole using 25 lines of sight spanning $1.5<z<2.8$. Such a survey makes an excellent probe of large-scale structure at early epochs. We find evidence for structure on the $15-35h^{-1}$ proper Mpc scale ($H_0 \equiv 100$ km $s^{-1}$ Mpc${-1}$) as determined by the two point C IV - C IV absorber correlation function, and reject the null hypothesis that C IV systems are distributed randomly on such scales at the $\sim 3.5\sigma$ level. The structure likely reflects the distance between two groups of absorbers subtending $\sim~ 13 \times 5 \times 21h^{-3}$ and $\sim 7 \times 1 \times 15h^{-3}$ Mpc$^3$ at $z\sim 2.3$ and $z \sim 2.5$ respectively. There is also a marginal trend for the association of high rest equivalent width C IV absorbers and QSOs at similar redshifts but along different lines of sight. The total number of C IV systems detected is consistent with that which would be expected based on a survey using many widely separated lines of sight. Using the same data, we also find 11 Mg II absorbers in a complete survey toward 24 lines of sight; there is no evidence for Mg II - Mg II or Mg II - QSO clustering, though the sample size is likely still small to detect such structure if it exists.Comment: 56 pages including 32 of figures, in gzip-ed uuencoded postscript format, 1 long table not included, aastex4 package. Accepted for publication in ApJ Supplement

Antimicrobial and brine shrimp toxicity of some plants used in traditional medicine in Bukoba District, north-western Tanzania

Herbal medicines constitute a potentially important resource for new and safe drugs for the management of microbial infections and other diseases. In this study, dichloromethane, ethylacetate and ethanol extracts of Canarium schweinfurthii Engl., Dissotis brazzae Cong., Iboza urticifolia (Bak) E.A.Bruce, Isoglosa lacteal Lindau, Strombosia Scheffleri Engl., and Whitfieldia elongate T. Anders were tested for antimicrobial activity and brine shrimp toxicity. The objective was to validate claims that they are used to treat bacterial infections, diarrhoea and heal wounds among the Haya tribe of north-western Tanzania. At least one extract of each plant showed antibacterial activity. Dichloromethane extracts were the most active while ethanol extracts were the least active. Extracts of Whitfieldia elongate and Isoglossa lacteal were the most and least active with MICs in the range 0.08-0.62 mg/ml and 15.6-62.5 mg/ml, respectively. The dichloromethane extract of Whitfieldia elongate exhibited strong antifungal activity against Cryptococcus neoformans. Against brine shrimp larvae, the extracts from the six plants exhibited a low to very low toxicity with LC50 values ranging from 15.35-374.0µg/ml. However, ethanol extracts of Dissotis brazzae and Strombosia scheffleri had LC50 values of >1000µg/ml. The seemingly innocuous nature and relatively good antibacterial activity against skin infections and gastrointestinal pathogenic bacteria support the traditional uses of the plants and deserve more detailed studies

Proteomic profiling of urine for the detection of colon cancer.

BACKGROUND: Colorectal cancer is the second most common cause of cancer related death in the developed world. To date, no blood or stool biomarkers with both high sensitivity and specificity for potentially curable early stage disease have been validated for clinical use. SELDI and MALDI profiling are being used increasingly to search for biomarkers in both blood and urine. Both techniques provide information predominantly on the low molecular weight proteome (<15 kDa). There have been several reports that colorectal cancer is associated with changes in the serum proteome that are detectable by SELDI and we hypothesised that proteomic changes would also be detectable in urine. RESULTS: We collected urine from 67 patients with colorectal cancer and 72 non-cancer control subjects, diluted to a constant protein concentration and generated MALDI and SELDI spectra. The intensities of 19 peaks differed significantly between cancer and non-cancer patients by both t-tests and after adjusting for confounders using multiple linear regressions. Logistic regression classifiers based on peak intensities identified colorectal cancer with up to 78% sensitivity at 87% specificity. We identified and independently quantified 3 of the discriminatory peaks using synthetic stable isotope peptides (an 1885 Da fragment of fibrinogen and hepcidin-20) or ELISA (beta2-microglobulin). CONCLUSION: Changes in the urine proteome may aid in the early detection of colorectal cancer

Blood parasites in endangered wildlife - Trypanosomes discovered during a survey of haemoprotozoa from the Tasmanian devil

The impact of emerging infectious diseases is increasingly recognised as a major threat to wildlife. Wild populations of the endangered Tasmanian devil, Sarcophilus harrisii, are experiencing devastating losses from a novel transmissible cancer, devil facial tumour disease (DFTD); however, despite the rapid decline of this species, there is currently no information on the presence of haemoprotozoan parasites. In the present study, 95 Tasmanian devil blood samples were collected from four populations in Tasmania, Australia, which underwent molecular screening to detect four major groups of haemoprotozoa: (i) trypanosomes, (ii) piroplasms, (iii) Hepatozoon, and (iv) haemosporidia. Sequence results revealed Trypanosoma infections in 32/95 individuals. Trypanosoma copemani was identified in 10 Tasmanian devils from three sites and a second Trypanosoma sp. was identified in 22 individuals that were grouped within the poorly described T. cyclops clade. A single blood sample was positive for Babesia sp., which most closely matched Babesia lohae. No other blood protozoan parasite DNA was detected. This study provides the first insight into haemoprotozoa from the Tasmanian devil and the first identification of Trypanosoma and Babesia in this carnivorous marsupial

Structural identification and biological activity of 7-methyl-10,11-ethylenedioxy-20(S)-camptothecin, a photodegradant of lurtotecan

An additional chromatographic peak was observed in plasma samples of patients receiving NX 211, a liposomal formulation of the topoisomerase I inhibitor lurtotecan. We have isolated and purified this product by sequential solid-phase extractions, and we report its structure and cytotoxicity relative to lurtotecan and related agents. Nuclear magnetic resonance data indicate that cleavage of the piperazino moiety occurred at the N-C bond of the B-ring, yielding 7-methyl-10,11-ethylenedioxy-20(S)-camptothecin (MEC). Tests of the growth inhibition potential of MEC in seven human tumor cell lines showed that the compound was approximately 2-18-fold more cytotoxic than lurtotecan, topotecan, and 7-ethyl-10-hydroxy-20(S)-camptothecin (SN-38). Subsequently, we found that MEC was the product of rapid photolysis of lurtotecan, with the rate of degradation inversely proportional to NX 211 concentrations, and greatly depends on light intensity. Furthermore, MEC concentrations were found to increase significantly in plasma samples exposed to laboratory light but not in blood. MEC was not produced from NX 211 in the presence of human liver microsomes, suggesting that it is not a product of cytochrome P-450 metabolism. Using a validated analytical method, trace levels of MEC were quantitated in blood samples of two patients. These observations confirm that the precautions for protection from light currently specified for preparation and administration of NX 211 dose solutions are critical. Procedures to minimize formation of MEC, by the use of amber vials for NX 211 and by preparation of dilutions immediately before clinical use in a fashion completely protected from light, are now being routinely implemented

Neutron Stars and Nuclei in the Modified Relativistic Hartree Approximation

We have examined the properties of neutron-rich matter and finite nuclei in the modified relativistic Hartree approximation for several values of the renormalization scale, $\mu$, around the standard choice of $\mu$ equal to the nucleon mass $M$. Observed neutron star masses do not effectively constrain the value of $\mu$. However for finite nuclei the value $\mu/M=0.79$, suggested by nuclear matter data, provides a good account of the bulk properties with a sigma mass of about 600 MeV. This value of $\mu/M$ renders the effective three and four body scalar self-couplings to be zero at 60\% of equilibrium nuclear matter density, rather than in the vacuum. We have also found that the matter part of the exchange diagram has little impact on the bulk properties of neutron stars.Comment: 33 pages, Latex, 8 figures (available from authors by fax), Minnesota preprint NUC-MINN-93/7-

Evidence for Shape Co-existence at medium spin in 76Rb

Four previously known rotational bands in 76Rb have been extended to moderate spins using the Gammasphere and Microball gamma ray and charged particle detector arrays and the 40Ca(40Ca,3pn) reaction at a beam energy of 165 MeV. The properties of two of the negative-parity bands can only readily be interpreted in terms of the highly successful Cranked Nilsson-Strutinsky model calculations if they have the same configuration in terms of the number of g9/2 particles, but they result from different nuclear shapes (one near-oblate and the other near-prolate). These data appear to constitute a unique example of shape co-existing structures at medium spins.Comment: Accepted for publication in Physics Letters