The exceptionally broad species diversity of vascular plant genera in east Asian temperate forests, compared with their sister taxa in North America, has been attributed to the greater climatic diversity of east Asia, combined with opportunities for allopatric speciation afforded by repeated fragmentation and coalescence of populations through Late Cenozoic ice-age cycles. According to Qian and Ricklefs, these opportunities occurred in east Asia because temperate forests extended across the continental shelf to link populations in China, Korea and Japan during glacial periods, whereas higher sea levels during interglacial periods isolated these regions and warmer temperatures restricted temperate taxa to disjunct refuges. However, palaeovegetation data from east Asia show that temperate forests were considerably less extensive than today during the Last Glacial Maximum, calling into question the coalescence of tree populations required by the hypothesis of Qian and Ricklefs.

Molecular tools for tissue profiling, such as expression microarrays and real-time PCR, generally require collection of fresh frozen tissues as sources of high-quality RNA. The fragile nature of RNA prompted us to examine the effects of storage time and transport conditions with regard to RNA integrity and gene expression in nonfixed surgical human specimens. At surgery, fresh normal tonsil and colon tissue was cut into pieces and snap frozen. Additional fresh tissue pieces were (i) left at room temperature, (ii) kept on ice, (iii) in normal saline or (iv) in a commercial RNA-stabilizing buffer (RNAlater) and snap frozen after 0.5, 1, 3, 6 and 16 h. Structural RNA integrity was analysed by microchip electrophoresis. Surprisingly, RNA remained stable in both tissue types under all conditions tested for up to 6-16 h. Gene expression by real-time PCR of cfos, HIF1alpha, Bcl2, PCNA, TGFbeta1 and SMAD7 was analysed at different storage time points in tonsil tissue. Expression levels were essentially stable when samples were kept on ice, while marked regulation of single genes was observed during storage at room temperature, in normal saline and in RNAlater. Furthermore, we analysed selected tissue types from the local biobank representing 47 normal and malignant tissues transported on ice for up to 2-3 h before biobanking. RNA prepared from 45 of the 47 samples exhibited distinct ribosomal peaks indicating intact RNA. This study shows that RNA degradation is a minor problem during handling of fresh human tissue before biobanking. Our data indicate that nonfixed tissue specimens may be transported on ice for hours without any major influence on RNA quality and expression of the selected genes. However, further studies are warranted to clarify the impact of transport logistics on global gene expression.

The use of cold therapy in acute sports injuries as well as in the rehabilitation of the injured athlete has become a generally accepted treatment method. Various cooling modalities are used to apply cold to the injured area, e.g. ice packs, ice towels, ice massage, frozen gel packs, ethyl chloride and other vapocoolants, chemical reaction devices and inflatable splints using refrigerant gas. Most clinical studies report that the use of cryotherapy has a positive effect on pain reduction and on the recovery of various injuries. When the physiological processes produced by cryotherapy are examined in experimental situations, some of these reactions differ from expectations. Skin, subcutaneous, intramuscular and joint temperature changes depend on application method, initial temperature and application time. Intramuscular temperature continues to drop after the cooling modality has been removed. Results of various studies are consistent on the effects on neuromuscular and pain processes. Results of studies on cold and blood flow vary considerably, however it appears that blood flow increases with superficial cold application and decreases when cold is applied to large skin surface areas. Motor performance is affected by temperature with a critical temperature being around 18 degrees C, above and beneath which muscle performance decreases. There is also a critical temperature for the application of cold with inflammation and oedema increasing at temperatures below 15 degrees C. Precautions should be taken because prolonged application at very low temperatures could have deleterious effects.

We report here the isolation and characterization of a new member of the ice/ced-3 family of cell death genes, named ich-3. The predicted amino acid sequence of Ich-3 protein shares 54% identity with murine interleukin-1beta converting enzyme (ICE). Overexpression of ich-3 in Rat-1 and HeLa cells induces apoptosis, which can be inhibited by CrmA and Bcl-2. The mRNA and proteins of ich-3 are dramatically induced in vivo upon stimulation with lipopolysaccharide, an inducer of septic shock. The ich-3 gene product can be cleaved by cytotoxic T cells granule serine protease granzyme B, suggesting that Ich-3 may mediate apoptosis induced by granzyme B. Ich-3 does not process proIL-1beta directly but does promote proIL-1beta processing by ICE. These results suggest that Ich-3 may play a very important role in apoptosis and inflammatory responses and may be an upstream regulator of ICE.

The ability of cytolytic cells to cause apoptosis in target cells is in part due to the action of the serine proteinase granzyme B. We demonstrate that granzyme B is inhibited, with an association rate constant of 2.9 x 10(5) M-1 s-1, by the cowpox viral serpin cytokine response modifier A (CrmA). Previously we have shown CrmA to be an inhibitor of the cysteine proteinase interleukin-1 beta-converting enzyme (ICE). Thus the mechanism of CrmA involves the unusual ability to efficiently inhibit proteinases from two distinct catalytic classes, in this case serine and cysteine proteinases. Granzyme B and ICE are both used to combat viral infection, and we propose that cowpox virus uses CrmA to evade the contribution of these two proteinases. Thus, through CrmA, the virus may influence two of the pathways normally used to kill virus-infected cells: acting on endogenous proteinases such as ICE and on exogenous proteinases delivered by cytotoxic lymphocytes to infected cells.

The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca(2+)-bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFPice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the "anchored clathrate" mechanism of AFP action.

Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitin-interacting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.

The parasites of the order kinetoplastidae including Leishmania spp. emerge from most ancient phylogenic branches of unicellular eukaryotic lineages. In their life cycle, topoisomerase I plays a significant role in carrying out vital cellular processes. Camptothecin (CPT), an inhibitor of DNA topoisomerase I, induces programmed cell death (PCD) both in the amastigotes and promastigotes form of L. donovani parasites. CPT-induced cellular dysfunction in L. donovani promastigotes is characterized by several cytoplasmic and nuclear features of apoptosis. CPT inhibits cellular respiration that results in mitochondrial hyperpolarization taking place by oligomycin-sensitive F0-F1 ATPase-like protein in leishmanial cells. During the early phase of activation, there is an increase in reactive oxygen species (ROS) inside cells, which causes subsequent elevation in the level of lipid peroxidation and decrease in reducing equivalents like GSH. Endogenous ROS formation and lipid peroxidation cause eventual loss of mitochondrial membrane potential. Furthermore, cytochrome c is released into the cytosol in a manner independent of involvement of CED3/CPP32 group of proteases and unlike mammalian cells it is insensitive to cyclosporin A. These events are followed by activation of both CED3/CPP32 and ICE group of proteases in PCD of Leishmania. Taken together, our study indicates that different biochemical events leading to apoptosis in leishmanial cells provide information that could be exploited to develop newer potential therapeutic targets.

Engagement of CD95 or tumor necrosis factor 1 receptor (TNFR-1) by ligand or agonist antibodies is capable of activating the cell death program, the effector arm of which is composed of mammalian interleukin-1beta converting enzyme (ICE)-like cysteine proteases (designated caspases) that are related to the Caenorhabditis elegans death gene, CED-3. Caspases, unlike other mammalian cysteine proteases, cleave their substrates following aspartate residues. Furthermore, proteases belonging to this family exist as zymogens that in turn require cleavage at internal aspartate residues to generate the two-subunit active enzyme. As such, family members are capable of activating each other. Remarkably, both CD95 and TNFR-1 death receptors initiate apoptosis by recruiting a novel ICE/CED-3 family member, designated FLICE/MACH, to the receptor signaling complex. Therefore, FLICE/MACH represents the apical triggering protease in the cascade. Consistent with this, recombinant FLICE was found capable of proteolytically activating downstream caspases. Furthermore, CrmA, a pox virus-encoded serpin that inhibits Fas and tumor necrosis factor-induced cell death attenuates the ability of FLICE to activate downstream caspases.

Genetic modification is critically enabling for studies addressing specification and maintenance of cell fate; however, methods for engineering modifications are inefficient. We demonstrate a rapid and efficient recombination system in which an inducible, floxed cre allele replaces itself with an incoming transgene. We target this inducible cassette exchange (ICE) allele to the (HPRT) locus and demonstrate recombination in murine embryonic stem cells (ESCs) and primary cells from derivative ICE mice. Using lentivectors, we demonstrate recombination at a randomly integrated ICE locus in human ESCs. To illustrate the utility of this system, we insert the myogenic regulator, Myf5, into the ICE locus in each platform. This enables efficient directed differentiation of mouse and human ESCs into skeletal muscle and conditional myogenic transdetermination of primary cells cultured in vitro. This versatile tool is thus well suited to gain-of-function studies probing gene function in the specification and reprogramming of cell fate.

In freeze-fracture (FF) preparations of ADH-stimulated toad urinary bladder, characteristic intramembrane particle (IMP) aggregates are seen on the protoplasmic (P) face of the luminal membrane of granular cells while complementary parallel grooves are found on the exoplasmic (E) face. These IMP aggregates specifically correlate with ADH-induced changes in water permeability. Tubular cytoplasmic structures whose membranes contain IMP aggregates which look identical to the IMP aggregates in the luminal membrane have also been described in granular cells from unstimulated and ADH-stimulated bladders. The diameter of these cytoplasmic structures (0.11 +/- 0.004 micrometers) corresponds to that of tubular invaginations of the luminal membrane seen in thin sections of ADH-treated bladders (0.13 +/- 0.005 micrometers). Continuity between the membranes of these cytoplasmic structures (which are not granules) and the luminal membrane has been directly observed in favorable cross-fractures. In FF preparations of the luminal membrane, these apparent fusion events are seen as round, ice-filled invaginations (0.13 +/- 0.01 micrometer Diam), of which about half have the characteristic ADH-associated aggregates near the point of membrane fusion. They are less numerous than, but linearly related to, the number of aggregates counted in the same preparations (n = 78, r = 0.71, P less than 0.01). These observations suggest that the IMP aggregates seen in luminal membrane after ADH stimulation are transferred preformed by fusion of cytoplasmic with luminal membrane.

Potassium (K+) deprivation-induced apoptosis of cerebellar granule neurons requires new mRNA and protein synthesis. Using a fluorogenic substrate for interleukin-1beta converting enzyme (ICE), we show that K+ deprivation of cerebellar granule neurons induces cycloheximide-sensitive ICE-like protease activity. A peptide inhibitor of ICE-like protease activity, Ac-YVAD-chloromethylketone (Ac-YVAD-CMK), prevents K+ deprivation-induced apoptosis. Further, reactive oxygen species (ROS) are essential mediators of K+ deprivation-induced apoptosis of cerebellar granule neurons because neuronal death is also blocked by superoxide dismutase, N-acetyl-L-cysteine, and free radical spin traps. Using fluorescent assays, we show that ROS production after K+ deprivation is blocked by actinomycin D, cycloheximide, and Ac-YVAD-CMK, suggesting that ROS act downstream of gene transcription, mRNA translation, and ICE activation. Taken together, we show that new mRNA and protein synthesis, activation of ICE-like proteases, and ROS production are sequential events in K+ deprivation-induced apoptosis of cerebellar granule neurons.

BACKGROUND

The proportion of paratyphoid fever cases to typhoid fever cases may change due to urbanization and increased dependency on food purchased from street vendors. For containment of paratyphoid a different strategy may be needed than for typhoid, because risk factors for disease may not coincide and current typhoid vaccines do not protect against paratyphoid fever.

OBJECTIVE

To determine risk factors for typhoid and paratyphoid fever in an endemic area.

METHODS

Community-based case-control study conducted from June 2001 to February 2003 in hospitals and outpatient health centers in Jatinegara district, Jakarta, Indonesia. Enrolled participants were 1019 consecutive patients with fever lasting 3 or more days, from which 69 blood culture-confirmed typhoid cases, 24 confirmed paratyphoid cases, and 289 control patients with fever but without Salmonella bacteremia were interviewed, plus 378 randomly selected community controls.

METHODS

Blood culture-confirmed typhoid or paratyphoid fever; risk factors for both diseases.

RESULTS

In 1019 fever patients we identified 88 (9%) Salmonella typhi and 26 (3%) Salmonella paratyphi A infections. Paratyphoid fever among cases was independently associated with consumption of food from street vendors (comparison with community controls: odds ratio [OR], 3.34; 95% confidence interval [CI], 1.41-7.91; with fever controls: OR, 5.17; 95% CI, 2.12-12.60) and flooding (comparison with community controls: OR, 4.52; 95% CI, 1.90-10.73; with fever controls: OR, 3.25; 95% CI, 1.31-8.02). By contrast, independent risk factors for typhoid fever using the community control group were mostly related to the household, ie, to recent typhoid fever in the household (OR, 2.38; 95% CI, 1.03-5.48); no use of soap for handwashing (OR, 1.91; 95% CI, 1.06-3.46); sharing food from the same plate (OR, 1.93; 95% CI, 1.10-3.37), and no toilet in the household (OR, 2.20; 95% CI, 1.06-4.55). Also, typhoid fever was associated with young age in years (OR, 0.96; 95% CI, 0.94-0.98). In comparison with fever controls, risk factors for typhoid fever were use of ice cubes (OR, 2.27; 95% CI, 1.31-3.93) and female sex (OR, 1.79; 95% CI, 1.04-3.06). Fecal contamination of drinking water was not associated with typhoid or paratyphoid fever. We did not detect fecal carriers among food handlers in the households.

CONCLUSIONS

In Jakarta, typhoid and paratyphoid fever are associated with distinct routes of transmission, with the risk factors for disease either mainly within the household (typhoid) or outside the household (paratyphoid).

Cysteine proteases related to mammalian interleukin-1 beta-converting enzyme (ICE) and the nematode cell death abnormal ced-3 gene product have been implicated in the effector mechanism of apoptotic cell death. Two novel members of this new family of ICE/CED-3-related proteases, designated ICErel-II and ICErel-III, were cloned from human monocytic cells. Both were highly homologous to human ICE (52% identical) and CED-3 (25% identical) and both contained the absolutely conserved pentapeptide sequence Gln-Ala-Cys-Arg-Asp containing the catalytic cysteine residue. Other structural motifs that were comparable with ICE suggest that ICErel-II and ICErel-III are also synthesized as larger proenzymes which are proteolytically processed to form heterodimeric active enzymes. Pro-interleukin-1 beta processing activity could not be detected in cells transfected with ICErel-II or ICErel-III, but pro-domain-less truncated forms of ICErel-II and ICErel-III were capable of effectively inducing fibroblast apoptosis. ICErel-II and ICErel-III may, therefore, participate in proteolytic events culminating in the apoptotic death of human cells.

BACKGROUND

This large outbreak of foodborne disease highlights the challenge of investigating outbreaks caused by intentional contamination and demonstrates the vulnerability of self-service foods to intentional contamination.

OBJECTIVE

To investigate a large community outbreak of Salmonella Typhimurium infections.

METHODS

Epidemiologic investigation of patients with Salmonella gastroenteritis and possible exposures in The Dalles, Oregon. Cohort and case-control investigations were conducted among groups of restaurant patrons and employees to identify exposures associated with illness.

METHODS

A community in Oregon. Outbreak period was September and October 1984.

METHODS

A total of 751 persons with Salmonella gastroenteritis associated with eating or working at area restaurants. Most patients were identified through passive surveillance; active surveillance was conducted for selected groups. A case was defined either by clinical criteria or by a stool culture yielding S Typhimurium.

RESULTS

The outbreak occurred in 2 waves, September 9 through 18 and September 19 through October 10. Most cases were associated with 10 restaurants, and epidemiologic studies of customers at 4 restaurants and of employees at all 10 restaurants implicated eating from salad bars as the major risk factor for infection. Eight (80%) of 10 affected restaurants compared with only 3 (11%) of the 28 other restaurants in The Dalles operated salad bars (relative risk, 7.5; 95% confidence interval, 2.4-22.7; P<.001). The implicated food items on the salad bars differed from one restaurant to another. The investigation did not identify any water supply, food item, supplier, or distributor common to all affected restaurants, nor were employees exposed to any single common source. In some instances, infected employees may have contributed to the spread of illness by inadvertently contaminating foods. However, no evidence was found linking ill employees to initiation of the outbreak. Errors in food rotation and inadequate refrigeration on ice-chilled salad bars may have facilitated growth of the S Typhimurium but could not have caused the outbreak. A subsequent criminal investigation revealed that members of a religious commune had deliberately contaminated the salad bars. An S Typhimurium strain found in a laboratory at the commune was indistinguishable from the outbreak strain.

CONCLUSIONS

This outbreak of salmonellosis was caused by intentional contamination of restaurant salad bars by members of a religious commune.

BACKGROUND

Standard nonoperative therapy for acute muscle strains usually involves short-term rest, ice, and nonsteroidal anti-inflammatory medications, but there is no clear consensus on how to accelerate recovery.

OBJECTIVE

Local delivery of platelet-rich plasma to injured muscles hastens recovery of function.

METHODS

Controlled laboratory study.

METHODS

In vivo, the tibialis anterior muscles of anesthetized Sprague-Dawley rats were injured by a single (large strain) lengthening contraction or multiple (small strain) lengthening contractions, both of which resulted in a significant injury. The tibialis anterior either was injected with platelet-rich plasma, was injected with platelet-poor plasma as a sham treatment, or received no treatment.

RESULTS

Both injury protocols yielded a similar loss of force. The platelet-rich plasma only had a beneficial effect at 1 time point after the single contraction injury protocol. However, platelet-rich plasma had a beneficial effect at 2 time points after the multiple contraction injury protocol and resulted in a faster recovery time to full contractile function. The sham injections had no effect compared with no treatment.

CONCLUSIONS

Local delivery of platelet-rich plasma can shorten recovery time after a muscle strain injury in a small-animal model. Recovery of muscle from the high-repetition protocol has already been shown to require myogenesis, whereas recovery from a single strain does not. This difference in mechanism of recovery may explain why platelet-rich plasma was more effective in the high-repetition protocol, because platelet-rich plasma is rich in growth factors that can stimulate myogenesis.

CONCLUSIONS

Because autologous blood products are safe, platelet-rich plasma may be a useful product in clinical treatment of muscle injuries.

Reliable data are scanty on the incidence of chronic diseases and life expectancy (LE) of highly trained athletes. We therefore studied Finnish male world class athletes to estimate the LE of athletes. Finnish team members in the Olympic games, World or European championships or intercountry competitions during 1920-1965 in track and field athletics, cross-country skiing, soccer, ice hockey, basketball, boxing, wrestling, weight lifting, and shooting were included (N = 2613 men). The reference cohort, 1712 men, was selected from the Finnish Defence Forces conscription register matched on age and area of residence. All referents were classified completely healthy at the time of induction to military service. The stratified Kaplan-Meier product limit method and the Cox proportional hazards model were used to estimate the life expectancies and the mortality odds ratios (OR) and their confidence limits. The mean LE adjusted for occupational group, marital status, and the age at entry to the cohort (and its 95% confidence limits) was in endurance sports (long distance running and cross-country skiing) 75.6 (73.6, 77.5) yr; in team games (soccer, ice hockey, basketball, as well as jumpers and short-distance runners from track and field (73.9 (72.7, 75.1) yr; in power sports (boxing, wrestling, weight lifting, and throwers from field athletics) 71.5 (70.4, 72.2) yr; and in the reference group 69.9 (69.0, 70.9) yr. The increased mean life expectancies were mainly explained by decreased cardiovascular mortality (endurance sports mortality odds ratio OR = 0.49 (95% CL 0.26, 0.93), team sports OR = 0.61 (0.41, 0.92) compared with referents). For maximum life span no differences between the groups were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

To evaluate the number and time of the migration(s) that colonized the New World we analyzed all available sequences of the first hypervariable segment of the human mitochondrial DNA control region, including 544 Native Americans. Sequence and population trees showed that the Amerind, Na-Dene, and Eskimo are significantly closer among themselves than anyone is to Asian populations, with the exception of the Siberian Chukchi, that in some analyses are closer to Na-Dene and Eskimo. Nucleotide diversity analyses based on haplogroup A sequences suggest that Native Americans and Chukchi originated from a single migration to Beringia, probably from east Central Asia, that occurred approximately 30,000 or approximately 43,000 years ago, depending on which substitution rate is used, with 95% confidence intervals between approximately 22,000 and approximately 55,000 years ago. These results support a model for the peopling of the Americas in which Beringia played a central role, where the population that originated the Native Americans settled and expanded. Some time after the colonization of Beringia they crossed the Alberta ice-free corridor and peopled the rest of the American continent. The collapse of this ice-free corridor during a few thousand years 14,000-20,000 years ago isolated the people south of the ice-sheets, who gave rise to the Amerind, from those still in Beringia; the latter originated the Na-Dene, Eskimo, and probably the Siberian Chukchi.

The pancreatic beta-cell-specific expression of the insulin gene is mediated, at least in part, by the interaction of unique trans-acting beta-cell factors with a cis-acting DNA element found within the insulin enhancer (5'-GC CATCTG-3'; referred to as the insulin control element [ICE]) present in the rat insulin II gene between positions -100 and -91. This sequence element contains the consensus binding site for a group of DNA-binding transcription factors called basic helix-loop-helix proteins (B-HLH). As a consequence of the similarity of the ICE with the DNA sequence motif associated with the cis-acting elements of the B-HLH class of binding proteins (CANNTG), the ability of this class of proteins to regulate cell-type-specific expression of the insulin gene was addressed. Cotransfection experiments indicated that overexpression of Id, a negative regulator of B-HLH protein function, inhibits ICE-mediated activity. Antibody to the E12/E47 B-HLH proteins attenuated the formation, in vitro, of a previously described (J. Whelan, S. R. Cordle, E. Henderson, P. A. Weil, and R. Stein, Mol. Cell. Biol. 10:1564-1572, 1990) beta-cell-specific activator factor(s)-ICE DNA complex. Both of these B-HLH proteins (E12 and E47) bound efficiently and specifically to the ICE sequences. The role of B-HLH proteins in mediating pancreatic beta-cell-specific transcription of the insulin gene is discussed.

BACKGROUND

This paper reviews epidemiological information about methamphetamine production and use in North America.

METHODS

Information is drawn from a range of sources, including, but not limited to, historical accounts, peer-reviewed papers, population surveys and large national databases.

RESULTS

Methamphetamine and amphetamine use in North America is characterised by geographic variations, with different types of the drug, different routes of administration and different types of users at various times. Unlike some other drug use patterns in North America, the nature of methamphetamine use in Canada, Mexico and the United States has been linked closely in terms of production and supply of the drug. According to their national household surveys, the annual prevalence for 'speed' use in Canada was 0.8% in 2004, 0.3% for 'anfetaminas' and 0.1% for 'metanfetaminas' in Mexico in 2002, and 1.4% for 'stimulants' in the United States in 2006.

CONCLUSIONS

Although the data sources in the three North American countries are not consistent in methodology, terminology or frequency of reporting, all show similar trends. The type of stimulant most used has shifted from non-medical use of pharmaceutical amphetamine to use of powder methamphetamine and then to use of 'ice'. The indicators show the problem is greatest in the western parts of the countries and is moving eastward, but the decreased availability of pseudoephedrine may have a significant impact on the nature of the epidemic in the future. Nevertheless, use of methamphetamine poses a number of risks for users and specialised treatment resources for these various populations are needed.

The human proto-oncogene bcl-2 and its Caenorhabditis elegans homologue ced-9 inhibit programmed cell death. In contrast, members of the human interleukin-1beta converting enzyme (ICE) family of cysteine proteases and their C. elegans homologue CED-3 promote the death program. Genetic experiments in C. elegans have shown that ced-9 is formally a negative regulator of ced-3 function, but neither those studies nor others have determined whether CED-9 or Bcl-2 proteins act biochemically upstream or downstream of CED-3/ICE proteases. CPP32, like all known members of the CED-3/ICE family, is synthesized as a proenzyme that is subsequently processed into an active protease with specificity for cleavage at Asp-X peptide bonds. In this report, we demonstrate that the CPP32 proenzyme is proteolytically processed and activated in Jurkat cells induced to die by Fas ligation. CPP32 activation is blocked by cell-permeable inhibitors of aspartate-directed, cysteine proteases, suggesting that pro-CPP32 is cleaved by active CPP32 or by other ICE family members. Heterologous expression of Bcl-2 in Jurkat cells prevents Fas-induced cell death as well as proteolytic processing and activation of CPP32. Thus, Bcl-2 acts at or upstream of the CPP32 activation step to inhibit apoptosis induced by Fas stimulation.

The ability to control extracellular ice formation during freezing is critical to the survival of freezing-tolerant plants. Antifreeze proteins, which are proteins that have the ability to retard ice crystal growth, were recently identified as the most abundant apoplastic proteins in cold-acclimated winter rye (Secale cereale L.) leaves. In the experiments reported here, amino-terminal sequence comparisons, immuno-cross-reactions, and enzyme activity assays all indicated that these antifreeze proteins are similar to members of three classes of pathogenesis-related proteins, namely, endochitinases, endo-beta-1,3-glucanases, and thaumatin-like proteins. Apoplastic endochitinases and endo-beta-1,3-glucanases that were induced by pathogens in freezing-sensitive tobacco did not exhibit antifreeze activity. Our findings suggest that subtle structural differences may have evolved in the pathogenesis-related proteins that accumulate at cold temperatures in winter rye to confer upon these proteins the ability to bind to ice.

Postoperative changes in eight dietary variables were compared at 6-mo intervals over 24 mo in 53 horizontal-gastroplasty (HGP) and 51 Roux-en-Y gastric-bypass (RYGB) patients; the variables included 1) calorie intake; percent intake of 2) protein, 3) carbohydrate, and 4) fat; 5) sweets and high-calorie beverages (SWS) and 6) milk and ice cream (MIC) as percent of calories; and 7) high-calorie liquids (HCL) and 8) nonliquid sweets (NLS) as percentage of dietary sugar. Weight and calorie intake were significantly less after RYGB than after HGP after 6 mo (p less than or equal to 0.01). Protein intake was significantly increased at all intervals after RYGB and at 6 and 12 mo after HGP (p less than 0.05). After RYGB, intakes of SWS, MIC, and HCL were significantly decreased at all intervals (p less than 0.05). SWS and MIC consumption was also significantly less after RYGB than after HGP (p less than or equal to 0.05). Decreased SWS and MIC consumption in RYGB patients suggests that food-preference differences are partially responsible for the lower calorie intake and greater weight loss after RYGB than after HGP.

The advent of complete mitochondrial DNA (mtDNA) sequence data has ushered in a new phase of human evolutionary studies. Even quite limited volumes of complete mtDNA sequence data can now be used to identify the critical polymorphisms that define sub-clades within an mtDNA haplogroup, providing a springboard for large-scale high-resolution screening of human mtDNAs. This strategy has in the past been applied to mtDNA haplogroup V, which represents <5% of European mtDNAs. Here we adopted a similar approach to haplogroup H, by far the most common European haplogroup, which at lower resolution displayed a rather uninformative frequency distribution within Europe. Using polymorphism information derived from the growing complete mtDNA sequence database, we sequenced 1580 base pairs of targeted coding-region segments of the mtDNA genome in 649 individuals harboring mtDNA haplogroup H from populations throughout Europe, the Caucasus, and the Near East. The enhanced genealogical resolution clearly shows that sub-clades of haplogroup H have highly distinctive geographical distributions. The patterns of frequency and diversity suggest that haplogroup H entered Europe from the Near East approximately 20,000-25,000 years ago, around the time of the Last Glacial Maximum (LGM), and some sub-clades re-expanded from an Iberian refugium when the glaciers retreated approximately 15,000 years ago. This shows that a large fraction of the maternal ancestry of modern Europeans traces back to the expansion of hunter-gatherer populations at the end of the last Ice Age.