The Pneumocystis carinii drug target S-adenosyl-L-methionine:sterol C-24 methyl transferase has a unique substrate preference

Pneumocystis is an opportunistic pathogen that can cause pneumonitis in immunodeficient people such as AIDS patients. Pneumocystis remains difficult to study in the absence of culture methods for luxuriant growth. Recombinant protein technology now makes it possible to avoid some major obstacles. The P. carinii expressed sequence tag (EST) database contains 11 entries of a sequence encoding a protein homologous to S-adenosyl-L-methionine (SAM):C-24 sterol methyl transferase (SMT), suggesting high activity of this enzyme in the organism. We sequenced the erg6 cDNA, identified the putative peptide motifs for the sterol and SAM binding sites in the deduced amino acid sequence and expressed the protein in Escherichia coli. Unlike SAM:SMT from other organisms, the P. carinii enzyme had higher affinities for lanosterol and 24-methylenelanosterol than for zymosterol, the preferred substrate in other fungi. Cycloartenol was not a productive substrate. With lanosterol and 24-methylenelanosterol as substrates, the major reaction products were 24-methylenelanosterol and pneumocysterol respectively. Thus, the P. carinii SAM:SMT catalysed the transfer of both the first and the second methyl groups to the sterol C-24 position, and the substrate preference was found to be a unique property of the P. carinii SAM:SMT. These observations, together with the absence of SAM:SMT among mammals, further support the identification of sterol C-24 alkylation reactions as excellent targets for the development of drugs specifically directed against this pathogen.     

Development and Application of a New Method for Specific and Sensitive Enumeration of Spores of Nonproteolytic Clostridium botulinum Types B,E, and F in Foods and Food Materials

The highly potent botulinum neurotoxins are responsible for botulism, a severe neuroparalytic disease. Strains of nonproteolytic Clostridium botulinum form neurotoxins of types B, E, and F and are the main hazard associated with minimally heated refrigerated foods. Recent developments in quantitative microbiological risk assessment (QMRA) and food safety objectives (FSO) have made food safety more quantitative and include, as inputs, probability distributions for the contamination of food materials and foods. A new method that combines a selective enrichment culture with multiplex PCR has been developed and validated to enumerate specifically the spores of nonproteolytic C. botulinum. Key features of this new method include the following: (i) it is specific for nonproteolytic C. botulinum (and does not detect proteolytic C. botulinum), (ii) the detection limit has been determined for each food tested (using carefully structured control samples), and (iii) a low detection limit has been achieved by the use of selective enrichment and large test samples. The method has been used to enumerate spores of nonproteolytic C. botulinum in 637 samples of 19 food materials included in pasta-based minimally heated refrigerated foods and in 7 complete foods. A total of 32 samples (5 egg pastas and 27 scallops) contained spores of nonproteolytic C. botulinum type B or F. The majority of samples contained <100 spores/kg, but one sample of scallops contained 444 spores/kg. Nonproteolytic C. botulinum type E was not detected. Importantly, for QMRA and FSO, the construction of probability distributions will enable the frequency of packs containing particular levels of contamination to be determined.Food-borne botulism is a severe and deadly intoxication caused by the consumption of food containing as little as 30 to 100 ng of preformed botulinum neurotoxin (45). More than 2,500 cases of botulism were reported in Europe in 1999 and 2000, with the majority of cases in the east of the continent (44). Currently, 25 to 50 food-borne botulism cases are diagnosed annually in the United States (27). There are seven distinct botulinum neurotoxins (types A to G) and a number of subtypes (6, 26, 45). In view of the potency of the botulinum neurotoxin and the severity of botulism, four phylogenetically distinct bacteria are grouped together as the Clostridium botulinum species, solely on the basis of their ability to form botulinum neurotoxin. The divergence between these four distinct bacteria is strong enough to merit their classification as distinct species and in some cases is significantly greater than that between bacteria belonging to different genera, e.g., Bacillus subtilis and Staphylococcus aureus (7). Two of these bacteria (proteolytic C. botulinum and nonproteolytic C. botulinum) are responsible for the majority of cases of food-borne botulism. Strains of proteolytic C. botulinum produce neurotoxins of type A, B, or F, form spores of high heat resistance, and have a minimum growth temperature of approximately 12°C (39). Strains of nonproteolytic C. botulinum produce neurotoxins of type B, E, or F, form spores of moderate heat resistance, and are able to grow and form toxin at 3°C (18, 48) and are recognized as the major hazard associated with minimally heated refrigerated foods (4, 37, 43, 44, 48). These new foods meet consumer demand for high-quality, convenient foods that are low in preservatives, and sales are presently increasing by about 10% per annum in many countries (3, 47).Quantitative microbiological risk assessment (QMRA) is now established as an important microbiology food safety tool (42). Process risk models have been used to assess the safety of specific foods with respect to nonproteolytic C. botulinum and the food-borne botulism hazard (e.g., 2, 41). These process risk models benefit from high-quality information, including that on the incidence of spores of nonproteolytic C. botulinum spores in food materials. The implementation of food safety objectives (FSO) also benefits from the availability of high-quality information on the microbial contamination of foods and food materials (24). This information is most effective in the form of probability distributions rather than as average spore concentrations or other statistics.The difficulty with enumerating nonproteolytic C. botulinum in foods is that there is no effective selective culture medium available. Surveys of the extent of contamination of foods and food materials have used a nonselective enrichment followed by either testing for neurotoxin using a mouse test or enzyme-linked immunosorbent assay (ELISA) or testing for the presence of neurotoxin genes using a PCR test (3, 10, 13, 35, 38, 39). This approach, however, is not optimized for nonproteolytic C. botulinum or proteolytic C. botulinum (therefore potentially failing to recover all spores of either organism) and may also not distinguish nonproteolytic C. botulinum from proteolytic C. botulinum. Heating at 80°C for 10 min followed by incubation at 35°C (54) may be reasonably selective for proteolytic C. botulinum, but there is no similar approach for nonproteolytic C. botulinum, although incubation at 28°C (54) may offer an element of selection. It is necessary, therefore, to develop a method to enumerate spores of nonproteolytic C. botulinum in food materials that is robust and optimized, as well as sensitive and specific for this particular pathogen (and does not also detect proteolytic C. botulinum). When enumerating bacteria in foods, it is essential to demonstrate the efficiency of the method by verifying that small concentrations (in the present study, spores of nonproteolytic C. botulinum) can be detected following addition to test samples.This paper describes the development, validation, and application of a new method to enumerate spores of nonproteolytic C. botulinum in foods and in food materials. This method has been designed to generate data for the construction of probability distributions that can be used in QMRA and FSO settings. Most of the effort has been dedicated to the development and evaluation of the enrichment procedure rather than the PCR test, as the PCR test has received much attention from others (e.g., 3, 10, 16, 36, 38). A low-temperature selective-enrichment procedure is described that has been optimized specifically for nonproteolytic C. botulinum over proteolytic C. botulinum and other bacteria. In order to detect low concentrations of spores, large quantities (200 g) of food materials and foods have been tested. Specific detection of neurotoxin genes is achieved by the use of an established multiplex PCR (36), with an internal amplification control now included (25). By the use of a set of control samples inoculated with defined concentrations of spores of nonproteolytic C. botulinum, the detection limit has been estimated for each food material and food tested. The method has been used in an extensive survey of raw materials intended for use in pasta ready meals, as well as the final meals themselves. The implications for risk assessment and risk management of chilled foods are discussed.     

The role of resource dispersion in promoting the co‐occurrence of dominant and subordinate ant species

Dominant competitors govern resource use in many communities, leading to predictions of local exclusion and lower species diversity where dominant species are abundant. However, subordinate and dominant species frequently co‐occur. One mechanism that could facilitate resource sharing and co‐occurrence of dominant and subordinate competitors is fine‐scale resource dispersion. Here, we distributed 6 g of a food resource into 1, 2, 8, 32 or 64 units in small 0.40 m² areas centred on nests of the dominant ant Monomorium sydneyense. We tested three hypotheses. First, we hypothesized that the species richness and abundance of foraging ants would increase with increasing resource dispersion. Accordingly, species richness doubled and total ant abundance was two orders of magnitude higher in high resource dispersion treatments. Secondly, we hypothesized that increasing resource dispersion would reduce competitive interactions such as resource turnover events and lower the probability of food resources being occupied. Substantial support for this hypothesis was observed. Finally, we tested the hypothesis that the foraging time of each species would be proportional to the relative abundance of each species solely in high resource dispersion treatments. Expected and observed foraging times were statistically similar for only the dominant ant M. sydneyense. The subdominant Pheidole rugosula increased its foraging time much more than was expected, while two subordinate ants showed no relationship between observed and expected times. Thus, while increasing resource dispersion significantly increased overall species richness, this increase in co‐occurrence did not correlate with a significant increase in foraging time for the two subordinate species. Rather, changes in resource dispersion appeared to benefit only the subdominant species. Inter‐site variation appeared more important for other subordinate species indetermining co‐occurrence and foraging time. Multiple mechanisms facilitate co‐occurrence and resource sharing in this community, and probably in most other communities.     

Evidence for new Neanderthal teeth in Tabun Cave (Israel) by the application of self-organizing maps (SOMs)

Morphological and metrical study suggested that seven human teeth from Tabun Cave, Israel were part of the upper dentition of a single, probably Neanderthal, individual renumbered as Tabun BC7. An enamel fragment gave ESR age estimates of 82+/-14 ka (early U-uptake) and 92+/-18 ka (linear uptake) and an age estimate of 90(+30)(-16) ka using U-series disequilibrium. Although metrical analyses suggested Neanderthal affinities, definitive assessment was difficult as the values often fell into the ranges of both Neanderthal and Levantine early modern human samples. Therefore, two further classification analyses were conducted (neural networks using self-organizing maps and homogeneity analysis). Both identify Tabun BC7 as a Neanderthal. Neural networks are a promising tool for paleoanthropological studies as they can provide reliable classifications even with incomplete data.     

Were neandertal and modern human cranial differences produced by natural selection or genetic drift?

Most evolutionary explanations for cranial differences between Neandertals and modern humans emphasize adaptation by natural selection. Features of the crania of Neandertals could be adaptations to the glacial climate of Pleistocene Europe or to the high mechanical strains produced by habitually using the front teeth as tools, while those of modern humans could be adaptations for articulate speech production. A few researchers have proposed non-adaptive explanations. These stress that isolation between Neandertal and modern human populations would have lead to cranial diversification by genetic drift (chance changes in the frequencies of alleles at genetic loci contributing to variation in cranial morphology). Here we use a variety of statistical tests founded on explicit predictions from quantitative- and population-genetic theory to show that genetic drift can explain cranial differences between Neandertals and modern humans. These tests are based on thirty-seven standard cranial measurements from a sample of 2524 modern humans from 30 populations and 20 Neandertal fossils. As a further test, we compare our results for modern human cranial measurements with those for a genetic dataset consisting of 377 microsatellites typed for a sample of 1056 modern humans from 52 populations. We conclude that rather than requiring special adaptive accounts, Neandertal and modern human crania may simply represent two outcomes from a vast space of random evolutionary possibilities.     

The 'human revolution' in lowland tropical Southeast Asia: the antiquity and behavior of anatomically modern humans at Niah Cave (Sarawak, Borneo)

Recent research in Europe, Africa, and Southeast Asia suggests that we can no longer assume a direct and exclusive link between anatomically modern humans and behavioral modernity (the 'human revolution'), and assume that the presence of either one implies the presence of the other: discussions of the emergence of cultural complexity have to proceed with greater scrutiny of the evidence on a site-by-site basis to establish secure associations between the archaeology present there and the hominins who created it. This paper presents one such case study: Niah Cave in Sarawak on the island of Borneo, famous for the discovery in 1958 in the West Mouth of the Great Cave of a modern human skull, the 'Deep Skull,' controversially associated with radiocarbon dates of ca. 40,000 years before the present. A new chronostratigraphy has been developed through a re-investigation of the lithostratigraphy left by the earlier excavations, AMS-dating using three different comparative pre-treatments including ABOX of charcoal, and U-series using the Diffusion-Absorption model applied to fragments of bones from the Deep Skull itself. Stratigraphic reasons for earlier uncertainties about the antiquity of the skull are examined, and it is shown not to be an 'intrusive' artifact. It was probably excavated from fluvial-pond-desiccation deposits that accumulated episodically in a shallow basin immediately behind the cave entrance lip, in a climate that ranged from times of comparative aridity with complete desiccation, to episodes of greater surface wetness, changes attributed to regional climatic fluctuations. Vegetation outside the cave varied significantly over time, including wet lowland forest, montane forest, savannah, and grassland. The new dates and the lithostratigraphy relate the Deep Skull to evidence of episodes of human activity that range in date from ca. 46,000 to ca. 34,000 years ago. Initial investigations of sediment scorching, pollen, palynomorphs, phytoliths, plant macrofossils, and starch grains recovered from existing exposures, and of vertebrates from the current and the earlier excavations, suggest that human foraging during these times was marked by habitat-tailored hunting technologies, the collection and processing of toxic plants for consumption, and, perhaps, the use of fire at some forest-edges. The Niah evidence demonstrates the sophisticated nature of the subsistence behavior developed by modern humans to exploit the tropical environments that they encountered in Southeast Asia, including rainforest.     

Tinkering with heparan sulfate sulfation to steer development

Heparan sulfate (HS) proteoglycans, at the cell surface and extracellular matrix, facilitate ligand-receptor interactions crucial to many physiological processes. The distinct sulfation patterns of HS sugar chains presented by their protein core are key to HS proteoglycan activity. Tight regulation of several Golgi complex enzyme families is crucial to produce complex tissue-specific HS sequences. Several in vivo models deficient in HS biosynthesis enzymes demonstrate that developmental abnormalities result from modified HS structure. This review will discuss the plasticity of sulfation requirements on HS for activating protein ligands, which might reflect a flexible HS biosynthetic mechanism. In addition, the latest discovery of HS acting enzymes, the Sulfs, responsible for extracellular tweaking of HS sulfation levels subsequent to biosynthesis will be considered.     

Enhanced cadmium-induced testicular necrosis and renal proximal tubule damage caused by gene-dose increase in a Slc39a8-transgenic mouse line

Resistance to cadmium (Cd)-induced testicular necrosis is an autosomal recessive trait defined as the Cdm locus. Using positional cloning, we previously identified the Slc39a8 (encoding an apical-surface ZIP8 transporter protein) as the gene most likely responsible for the phenotype. In situ hybridization revealed that endothelial cells of the testis vasculature express high ZIP8 levels in two sensitive inbred mouse strains and negligible amounts in two resistant strains. In the present study, we isolated a 168.7-kb bacterial artificial chromosome (BAC), carrying only the Slc39a8 gene, from a Cd-sensitive 129/SvJ BAC library and generated BAC-transgenic mice. The BTZIP8-3 line, having three copies of the 129/SvJ Slc39a8 gene inserted into the Cd-resistant C57BL/6J genome (having its normal two copies of the Slc39a8 gene), showed tissue-specific ZIP8 mRNA expression similar to wild-type mice, mainly in lung, testis, and kidney. The 2.5-fold greater expression paralleled the fact that the BTZIP8-3 line has five copies, whereas wild-type mice have two copies, of the Slc39a8 gene. The ZIP8 mRNA and protein localized especially to endothelial cells of the testis vasculature in BTZIP8-3 mice. Cd treatment reversed Cd resistance (seen in nontransgenic littermates) to Cd sensitivity in BTZIP8-3 mice; reversal of the testicular necrosis phenotype confirms that Slc39a8 is unequivocally the Cdm locus. ZIP8 also localized specifically to the apical surface of proximal tubule cells in the BTZIP8-3 kidney. Cd treatment caused acute renal failure and signs of proximal tubular damage in the BTZIP8-3 but not nontransgenic littermates. BTZIP8-3 mice should be a useful model for studying Cd-induced disease in kidney. kidney; testis; ZIP8; bacterial artificial chromosome     

Chemotherapy-induced diarrhea is associated with changes in the luminal environment in the DA rat

The microflora of the gastrointestinal tract (GIT) are a complex ecosystem, performing a number of beneficial functions. Irinotecan causes both early and late diarrhea, the latter possibly caused, in part, by changes in the microflora of the GIT. Female DA rats were given atropine subcutaneously, prior to a single 200 mg/kg intraperitoneal dose of irinotecan. Animals were monitored for diarrhea and killed at 30 and 60 mins, 2, 6, 12, 24, 48, and 72 hrs after chemotherapy administration. Control rats received no treatment. Fecal samples and stomach, jejunum, and colon samples were collected and stored at -70 degrees C until required. Standard microbiological culture techniques were used to grow and isolate the flora. Biochemical tests were used to identify the bacteria. The level of growth was noted for relative comparison between time points and graded accordingly. Early diarrhea was observed in the rats from 2-6 hrs after treatment, after which time the diarrhea resolved. Late onset diarrhea was apparent 72 hrs after treatment. Changes were seen in the flora of the stomach, jejunum, colon and feces. The majority of microflora changes were seen 6, 12, and 24 hrs after treatment, with a relative increase or decrease in the presence of bacteria in comparison with control rats. In some rats bacteria were not observed at all time points, and different bacteria not seen in control animals were identified in rats treated with irinotecan. These changes were observed up to 72 hrs after treatment. In conclusion, irinotecan treatment causes changes in the flora of the stomach, jejunum, colon, and feces of rats and is associated with the development of diarrhea. These changes in flora may have systemic effects and in particular may contribute to the development of chemotherapy-induced mucositis.