Disparate patterns of thermal adaptation between life stages in temperate vs. tropical Drosophila melanogaster.

Many terrestrial ectothermic species exhibit limited variation in upper thermal tolerance across latitude. However, these trends may not signify limited adaptive capacity to increase thermal tolerance in the face of climate change. Instead, thermal tolerance may be similar among populations because behavioural thermoregulation by mobile organisms or life stages may buffer natural selection for thermal tolerance. We compared thermal tolerance of adults and embryos among natural populations of Drosophila melanogaster from a broad range of thermal habitats around the globe to assess natural variation of thermal tolerance in mobile vs. immobile life stages. We found no variation among populations in adult thermal tolerance, but embryonic thermal tolerance was higher in tropical strains than in temperate strains. We further report that embryos live closer to their upper thermal limits than adults - that is, thermal safety margins are smaller for embryos than adults. F1 hybrid embryos from crosses between temperate and tropical populations had thermal tolerance that matched that of tropical embryos, suggesting the dominance of heat-tolerant alleles. Together, our findings suggest that thermal selection has led to divergence in embryonic thermal tolerance but that selection for divergent thermal tolerance may be limited in adults. Further, our results suggest that thermal traits should be measured across life stages to better predict adaptive limits.

Simulation and analysis of conjunctive use with MODFLOW's farm process.

The extension of MODFLOW onto the landscape with the Farm Process (MF-FMP) facilitates fully coupled simulation of the use and movement of water from precipitation, streamflow and runoff, groundwater flow, and consumption by natural and agricultural vegetation throughout the hydrologic system at all times. This allows for more complete analysis of conjunctive use water-resource systems than previously possible with MODFLOW by combining relevant aspects of the landscape with the groundwater and surface water components. This analysis is accomplished using distributed cell-by-cell supply-constrained and demand-driven components across the landscape within "water-balance subregions" comprised of one or more model cells that can represent a single farm, a group of farms, or other hydrologic or geopolitical entities. Simulation of micro-agriculture in the Pajaro Valley and macro-agriculture in the Central Valley are used to demonstrate the utility of MF-FMP. For Pajaro Valley, the simulation of an aquifer storage and recovery system and related coastal water distribution system to supplant coastal pumpage was analyzed subject to climate variations and additional supplemental sources such as local runoff. For the Central Valley, analysis of conjunctive use from different hydrologic settings of northern and southern subregions shows how and when precipitation, surface water, and groundwater are important to conjunctive use. The examples show that through MF-FMP's ability to simulate natural and anthropogenic components of the hydrologic cycle, the distribution and dynamics of supply and demand can be analyzed, understood, and managed. This analysis of conjunctive use would be difficult without embedding them in the simulation and are difficult to estimate a priori.

Methicillin-resistant Staphylococcus aureus (MRSA) detection: comparison of two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) with three selective MRSA agars (MRSA ID, MRSASelect, and CHROMagar MRSA) for use with infection-control swabs.

Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing problem. Rapid detection of MRSA-colonized patients has the potential to limit spread of the organism. We evaluated the sensitivities and specificities of MRSA detection by two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) and three selective MRSA agars (MRSA ID, MRSASelect, and CHROMagar MRSA), using 205 (101 nasal, 52 groin, and 52 axillary samples) samples from consecutive known MRSA-infected and/or -colonized patients. All detection methods had higher MRSA detection rates for nasal swabs than for axillary and groin swabs. Detection of MRSA by IDI-MRSA was the most sensitive method, independent of the site (94% for nasal samples, 80% for nonnasal samples, and 90% overall). The sensitivities of the GenoType MRSA Direct assay and the MRSA ID, MRSASelect, and CHROMagar MRSA agars with nasal swabs were 70%, 72%, 68%, and 75%, respectively. All detection methods had high specificities (95 to 99%), independent of the swab site. Extended incubation for a further 24 h with selective MRSA agars increased the detection of MRSA, with a corresponding decline in specificity secondary to a significant increase in false-positive results. There was a noticeable difference in test performance of the GenoType MRSA Direct assay in detection of MRSA (28/38 samples [74%]) compared with detection of nonmultiresistant MRSA (17/31 samples [55%]) (susceptible to two or more non-beta-lactam antibiotics). This was not observed with selective MRSA agar plates or IDI-MRSA. Although it is more expensive, in addition to rapid turnaround times of 2 to 4 h, IDI-MRSA offers greater detection of MRSA colonization, independent of the swab site, than do conventional selective agars and GenoType MRSA Direct.

A choice behavior for morphine reveals experience-dependent drug preference and underlying neural substrates in developing larval zebrafish.

Transparent larval zebrafish offer the opportunity to unravel genetic and neuronal networks underlying behavior in a developing system. In this study, we developed a choice chamber paradigm to measure reward-associated behavior in larval zebrafish. In the chamber where larval zebrafish have a choice of spending their time in either a water- or morphine-containing compartment, larvae that have previously experienced morphine spend significantly more time in the compartment containing morphine. This behavior can be attentuated by pre-treatment with antagonists of the opioid receptor or the dopamine receptor, and furthermore, is impaired in the too few mutant, which has a genetic deficiency in the production of specific groups of dopaminergic and serotonergic neurons in the ventral forebrain. These results uncover a choice behavior for an addictive substance in larval zebrafish that is mediated through central opioid and monoaminergic neurotransmitter systems.

Applications of supercritical fluid extraction and chromatography in forensic science.

Supercritical fluid technology is a rapidly expanding analytical technique. Here we give a brief insight into the background of supercritical fluid technology and how supercritical fluid extraction and supercritical fluid chromatography work in analysis. The applications of these two techniques in forensic science are known to be important. The main area of forensic use of supercritical fluid technology is in the sample preparation and separation of drugs of abuse particularly opiates, cannabinoids, cocaine and sedatives. Supercritical fluid technology can be used for both time-of-death-related drug analysis and for obtaining information relating to long term drug abuse. We also give a review of the use of supercritical fluids in two other major forensic areas, fingerprinting and the extraction and separation of explosives from both bombing events and gunshot residues. Overall we show that supercritical fluid technology is fast becoming a major part of forensic investigations and that it is an invaluable analysis technique.

Stress infrared telethermography is useful in the diagnosis of complex regional pain syndrome, type I (formerly reflex sympathetic dystrophy).

OBJECTIVE: To assess the sensitivity, specificity, and predictive value (PV) of stress infrared telethermography (IRT) in the complex regional pain syndrome, type I (CRPS-I). METHODS: One hundred eighty-five consecutive patients (47 men, 138 women) with 205 pairs of chronically painful limbs (upper, lower, or both) were examined by pain specialists in neurology, physiatry, and anesthesia, who then reached a consensus diagnosis. A clinical diagnosis of CRPS-I required at least two of the following observations: burning pain, vasomotor changes, diaphoresis, trophic changes, allodynia. Patients with only one criterion were classified as possible CRPS-I; those with none were judged not to have CRPS-I. Patients and 24 asymptomatic control subjects underwent stress IRT, which was considered positive for CRPS-I if it showed three of the following: quantitative thermal emission of > or = 1.00 degree C, abnormal distal thermal gradient patterns, presence of a "thermal marker," and abnormal response to functional cold water autonomic stress testing. RESULTS: By clinical criteria, CRPS-I was diagnosed in 73 pairs of limbs; not CRPS-I was diagnosed in 70; and 62 pairs had possible CRPS-I. Excluding possible CRPS-I cases, there were 5 false-negative stress IRTs (sensitivity 93%) and 7 false-positive results (specificity 89%). Based on estimated 50% prior probability for our population, the positive PV is 90% and the negative PV 94%. None of the control subjects exhibited thermographic evidence of CRPS-I. CONCLUSION: Stress IRT is a sensitive and specific indicator of CRPS-I.

Uptake of amino acids by the parasitic, flagellated protist Trichomonas vaginalis.

HPLC techniques have been applied to study amino acid uptake and release by Trichomonas vaginalis under a variety of conditions. Studies on the growth of T. vaginalis in complex media and the survival of the parasite in simple media, with and without amino acids and/or maltose, have shown that the growth or survival of T. vaginalis is better in the presence of maltose than when it is absent, and that greater amounts of amino acids are consumed by T. vaginalis in the absence of maltose. The results are consistent with several amino acids, notably arginine, threonine, leucine and methionine, being used by T. vaginalis as energy substrates. T. vaginalis released alanine and glycine into the culture media, the excretion being greater in the presence of maltose. These studies have provided new data on the uptake and release of amino acids by T. vaginalis and pave the way for detailed analysis of key enzymes and the regulation of the pathways involved.

Developing the role of vice president of patient care services: pulling the clinical team together.

Making the transition from the traditional Nurse Executive role to Vice President of Patient Care Services can improve the quality of patient care while encouraging interdepartmental cooperation and streamlining the organization. This author who has experience coordinating two executive restructuring projects provides suggestions to ease the transition.

Nongenotoxic effects of polycyclic aromatic hydrocarbons and their oxygenation by-products on the intercellular communication of rat liver epithelial cells.

Since polycyclic aromatic hydrocarbons (PAHs) are known to have epigenetic effects, we evaluated the effect of the parent chemical and the ozonated products on in vitro cell to cell communication bioassays which measures a nongenotoxic event. The scrape loading/dye transfer (SL/DT) technique was used to determine the effect of the following PAHs on gap-junction intercellular communication (GJIC): fluorene, 1-methyl-fluorene, fluoranthene, anthracene, 9-methyl-anthracene, phenanthrene, pyrene, benzo(a)pyrene, and benzo(e)pyrene. The methylated PAHs were more inhibitory to GJIC than the unmethylated counterparts. Fluoranthene, which has an additional ring added to fluorene, was more effective in inhibiting GJIC than fluorene. The three-ringed PAHs were also more inhibitory than the four- and five-ringed PAHs. A time-course study of fluoranthene and of pyrene resulted in maximal inhibition occurring within 30 min of incubation with the cells. The cells recovered from the inhibition within 1 hr after fluoranthene and pyrene were removed from the cell culture medium. Pyene, vbenzo(a)pyrene, fluorene, and fluoranthene were ozonated until the parent compound was completely eliminated as determined by reverse-phase high-pressure liquid chromatography (RP-HPLC). An increased level of inhibition of GJIC was observed for the ozonated mixtures of by-products of pyrene, fluoranthene, and benzo(a)pyrene, but not for fluorene, as monitored with the SL/DT technique. The products of the ozonated pyrene mixture were fractionated and collected by RP-HPLC. Each fraction was found to be inhibitory to GJIC as monitored by fluorescence recovery after photobleaching. In conclusion, current treatment technologies, such as ozonation or biologically based oxidations and methylations, do not necessarily eliminate toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and molecular cloning of four cysteine proteinase genes from the pathogenic protozoon Trichomonas vaginalis.

The parasitic protozoon Trichomonas vaginalis produces multiple forms of cysteine proteinase (CP). The molecular basis for this has now been examined by cloning DNA fragments encoding CPs. Using generic degenerate oligonucleotide primers based on two well-conserved regions within the central region of all eukaryotic CPs, several polymerase chain reaction fragments were isolated from T. vaginalis genomic DNA and shown to encode different CPs. One fragment with a well-represented sequence was used as a general probe to screen a T. vaginalis cDNA library at moderate stringency and five different cDNA clones were isolated. Preliminary sequencing showed that they encoded similar but distinct CPs. In the process of confirming the 5' end of one of these cDNA clones using RACE-PCR (rapid amplification of cDNA 5' ends-polymerase chain reaction), an additional sequence encoding a different CP was identified. The corresponding clone (TvCP3) and the three longest clones from the library screen (TvCP1, TvCP2 and TvCP4) were characterized further. TvCP1 and TvCP2 were full-length and TvCP3 and TvCP4 were apparently slightly less than full-length. Comparison of the predicted amino acid sequences of the four clones showed that TvCP1 and TvCP4 are related (72% identity). TvCP2 is closer to TvCP1 (60%) and TvCP4 (65%) than is TvCP3, which has 53%, 59% and 56% identity to TvCP1, TvCP2 and TvCP4, respectively. Comparison with the sequences of other known CPs indicated that the T. vaginalis gene products all belong to the cathepsin L/cathepsin H/papain branch of the papain superfamily. The TvCP1, TvCP2 and TvCP4 sequences are related (38-45% identity) to those of CP2 of Dictyostelium discoideum, human cathepsin L, three CPs from lobster and CPs from black gram, oilseed rape and rice (oryzains alpha and beta). TvCP3 shows less identity to the other eukaryotic CPs but is most similar to D. discoideum CP2 (38%). The four predicted amino acid sequences share some features distinct from the majority of CPs, which suggests they might have had a common evolutionary origin. The most striking feature of sequences TvCP1, TvCP2 and TvCP3 is the apparent lack of a pre-sequence (signal sequence) for TvCP1 and very short pre-sequences for TvCP2 and TvCP3. Southern analysis indicated that the organization of the genes corresponding to the TvCP cDNAs differs. The TvCP1, TvCP2 and TvCP3 genes are single-copy, whereas the TvCP4 gene appeared to be multiple-copy.(ABSTRACT TRUNCATED AT 400 WORDS)

Hydrolysis of L-leucine methyl ester by Leishmania mexicana mexicana amastigote cysteine proteinases.

The main cysteine proteinases of the amastigote form of Leishmania mexicana mexicana were partially purified by gel filtration and ion exchange chromatography. The latter procedure resulted in the separation of some individual cysteine proteinases, as demonstrated by gelatin-sodium dodecyl sulphatepolyacrylamide gel electrophoresis. Fractions containing the partially purified proteinases rapidly hydrolysed L-leucine methyl ester to leucine. The activity towards this compound co-eluted with and resembled the parasite's cysteine proteinase activity. The results suggest that amastigotes of L.m.mexicana are susceptible to L-leucine methyl ester because this compound is rapidly hydrolysed by cysteine proteinases that occur in abundance in the megasomes of this stage.

Purification and characterization of methionine gamma-lyase from Trichomonas vaginalis.

Methionine gamma-lyase (EC 4.4.1.11) was purified to homogeneity from the anaerobic protozoan parasite Trichomonas vaginalis by a series of f.p.l.c. procedures. The enzyme catalyses alpha gamma- and alpha beta-elimination reactions of a number of derivatives of methionine and cysteine. It also catalyses gamma-replacement reactions of the thiomethyl group of methionine, homocysteine and ethionine to yield the corresponding S-substituted homocysteine derivative. The enzyme is pyridoxal 5'-phosphate-dependent, has a native molecular mass of approx. 160 kDa and consists of four apparently identical subunits of molecular mass 43-45 kDa. The absorption spectrum of the enzyme is typical of those obtained for other pyridoxal 5'-phosphate-dependent enzymes, and the holoenzyme can be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of the cofactor. The enzyme activity is significantly affected by carbonyl and thiol reagents, is competitively inhibited by a number of substrate analogues and is completely inactivated by the suicide inhibitor DL-propargylglycine. The T. vaginalis enzyme is similar, in terms of activity and properties, to the enzymes found in a number of species of bacteria that metabolize methionine under anaerobic conditions. It is suggested that methionine catabolism may be of particular importance to the survival of T. vaginalis under microaerophilic conditions in its host.

Analysis of the proteinases of Trypanosoma brucei.

A method comprising enzyme separation by SDS-PAGE and subsequent use of peptidyl aminomethylcoumarins as substrates has been used to study proteinases of the protozoan parasite Trypanosoma brucei. The application of this method has allowed investigation of the substrate specificities of individual proteinases in cell lysates without the need for enzyme purification. The results show that T. brucei contains a group of cysteine proteinases, probably four in number, with substrate and inhibitor specificities similar to those of cathepsin L. A second group of proteinases, larger enzymes with significantly different substrate specificities and sensitivity to inhibitors, was also detected. Peptidyl diazomethanes inhibited the cysteine proteinases and also parasite growth, offering promise that peculiarities in the substrate specificity of trypanosomal cysteine proteinases could be exploited by compounds of this type.

Abnormal chromosome complement resulting from a familial inversion of chromosome 2.

It has been suggested that pericentric inversions of chromosome 2 increase the risk for spontaneous abortion but do not increase the risk for unbalanced recombinant offspring. We report our experience of a familial pericentric inversion of chromosome 2 resulting in two unbalanced recombinant offspring. Both subjects have 46,XX,rec(2),dup q,inv(2)(p25q35).

Proteinase activity in rumen ciliate protozoa.

Azocasein-degrading proteinase activity was detected in all rumen ciliate protozoa that were examined from four entodiniomorphid and two holotrich genera. All of the activities were optimal in the range pH 4.0-5.0 and were inhibited by cysteine proteinase inhibitors, notably leupeptin. The inhibition profiles and extent of inhibition observed with the different groups of inhibitors were organism-specific. Gelatin-SDS-polyacrylamide gel electrophoresis of protozoal lysates revealed multiple forms of the proteinases in the species examined. The number of enzymes detected, their molecular masses, the level of activity and inhibitor susceptibility was genus-dependent. The proteinase profiles of the two holotrich species differed and inter-species differences were also apparent among species of the genus Entodinium. The characteristics and molecular size distribution of rumen bacterial proteinases were different to the protozoal proteinases. Low levels of proteinase activity, of apparently bacterial origin, were detected by gelatin-SDS-PAGE analysis of cell-free rumen liquor.

The release of hydrolases from Trichomonas vaginalis and Tritrichomonas foetus.

Trichomonas vaginalis and Tritrichomonas foetus were found to release large amounts of beta-N-acetylglucosaminidase (EC 3.2.1.30), alpha-mannosidase (EC 3.2.1.24), beta-glucosidase (EC 3.2.1.21), acid phosphatase (EC 3.1.3.2) and proteinases during axenic growth in vitro. The enzymes were released continually throughout the growth phase, with the extracellular activity being of the same order as that within the cells. There was differential release of proteinases from Trichomonas vaginalis. The subcellular localization of the hydrolases was determined by differential and isopycnic centrifugation. The intracellular enzymes were shown to be mostly located within particle populations. Centrifugation on Percoll gradients allowed the separation of sub-populations of the particles in T. vaginalis; two distinct sub-populations were apparent with equilibrium densities in 20% (v/v) Percoll of 1.035 and 1.050 g cm-3 respectively. The higher density particles were rich in the hydrolases released most abundantly, suggesting a possible link between enzyme release and these organelles. Distinct subpopulations of hydrolase-containing particles were not detected in Tritrichomonas foetus. The results demonstrate that hydrolytic enzyme release represents a major activity during trichomonad growth.

Multiple cysteine proteinase forms during the life cycle of Dictyostelium discoideum revealed by electrophoretic analysis.

Proteinases of the cellular slime mould Dictyostelium discoideum have been analysed using electrophoresis on polyacrylamide gels containing gelatin (gelatin/PAGE). Multiple proteinase forms were apparent in vegetative myxamoebae, but the presence of individual enzyme forms depended on the manner in which the cells were grown. Axenic cells had a characteristic A-pattern of proteinases consisting of six bands, the most active enzymes having apparent Mr values of 51,000 and 45,000 (these have been named ddCP51 and ddCP45, respectively). Some of the proteinases were also present in the medium, the major extracellular form was ddCP42, a 42,000-Mr enzyme. Cells grown in association with bacteria had a distinct B-pattern with three main enzymes that had apparent Mr values of 48,000, 43,000 and 38,000. All of the A- and B-pattern proteinases were most active at acid pH in the presence of dithiothreitol and were inhibited by various agents such as trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), leupeptin and chymostatin, which inactivate cysteine proteinases. One of the enzymes, ddCP30, was identified as cysteine proteinase B which had been purified and characterized previously [North, M.J. & Whyte, A. (1984) J. Gen. Microbiol. 130, 123-134]. During starvation of axenic cells in shaken suspensions some of the vegetative proteinases disappeared, ddCP42 was released from the cells and one new enzyme with an apparent Mr of 48,000 appeared. Addition of cyclic AMP had little effect on these changes. When the axenically grown myxamoebae underwent development on filters, similar changes in band pattern were observed and the aggregation stage was characterized by the presence of three cysteine proteinase bands (apparent Mr values of 48,000, 45,000 and 43,000). Proteinases, especially ddCP42, were released from the cells and could be collected from the buffer-saturated pads which supported the filters. The results demonstrate that cysteine proteinases are present throughout growth and development of D. discoideum and that the forms present are subject to nutritional and developmental regulation.

The use of a highly sensitive electrophoretic method to compare the proteinases of trichomonads.

A highly sensitive electrophoretic method involving gelatin-containing polyacrylamide gels has been used to analyse trichomonad proteinases. Multiple forms, optimally active at pH 5-6, were present in all four species examined, but the species could be distinguished from one another by both quantitative and qualitative differences. The intestinal parasites, Trichomitus batrachorum and Pentatrichomonas hominis, had lower specific activities than the urogenital parasites, Trichomonas vaginalis and Tritrichomonas foetus, and, in the case of P. hominis, there were fewer enzyme forms. The high activity proteinases of Tritrichomonas foetus had low apparent molecular weights (less than 25 kDa), while the predominant enzymes of Trichomonas vaginalis were of high apparent molecular weight (68-110 kDa). Distinct differences were also observed between the proteinase patterns of various isolates of T. vaginalis. All of the enzymes were stimulated by dithiothreitol, suggesting that they were cysteine proteinases. This was confirmed for the T. vaginalis and Tritrichomonas foetus proteinases from their inhibition by antipain, leupeptin, TLCK and iodoacetic acid. The method allows the detection of proteinases in samples of Trichomonas vaginalis containing as few as 10(4) cells or as little as 1 microgram protein. It was also possible to detect proteinase activity released into the medium. For both T. vaginalis and Tritrichomonas foetus, the extracellular enzymes present during early log phase were qualitatively different from the intracellular proteinases, although the latter were present in samples of media obtained from later cultures (cell densities greater than 1 X 10(5) parasites ml-1). The results show the potential of this technique for detecting proteinases in trichomonad samples in studies aimed at determining proteinase function in pathogenesis and host-parasite relationships.

A randomized study of cyclosporine with and without prednisone in renal allograft recipients. Canadian Transplant Group.

Sixty-nine patients receiving Cs after cadaveric or LRD renal transplants were randomly allocated to receive prednisone or no prednisone beginning on the day of transplant. There were 36 in the prednisone group and 33 in the group assigned to no prednisone. Of these latter, only seven (21%) never received prednisone and an additional four had one short course for rejection episodes (11%). Of the remaining 22 who were placed on continuous steroids, only 12 met rejection criteria and either some or all of the remainder probably had Cs nephrotoxicity. The patient and graft survival were better but not statistically so in the no-prednisone group (97% v 89%) and (88% v 78%), and the number of infections was only half that of the prednisone-treated group (22% v 42%). A policy of withholding steroids except for rejection episodes does not prejudice graft or patient survival in Cs-treated patients.