A functional RNAi screen identifies hexokinase 1 as a modifier of type II apoptosis

Tumor necrosis factor alpha (TNF-α) signals through NF-κB, JNK, and caspase modules to drive physiological responses that range from inflammation to apoptosis. The balance between the individual modules determines the nature of the response, and deregulated TNF signaling has been implicated in numerous pathological conditions. We used a quantitative high-throughput RNA interference assay to probe the entire complement of human kinases and phosphatases for gene products that tilt the balance of TNF signal transduction in favor of cell death or cell viability. Of all gene products tested, loss of hexokinase 1 resulted in the greatest elevations in TNF-dependent death. In secondary assays, we demonstrated that hexokinase 1 does not alter TNF-dependent activation of NF-κB or JNK modules. Instead, hexokinase 1 modifies the induction of caspase-driven cell death. Specifically, we showed that hexokinase 1 inhibits the formation of active, pro-apoptotic caspases in response to extrinsic inducers of apoptosis. These data are the first loss-of-function reports to examine the involvement of hexokinase 1 in the transduction of cell death signals and indicate that hexokinases are critical determinants of the viability of cells in response to extrinsic apoptotic cues.     

Anaesthetic considerations of adults with Morquio's syndrome - a case report

Background

Base deficit (BD) is commonly used in the operating room (OR) as an endpoint of resuscitation. BD is used as a surrogate marker for the accumulation of lactic acid(Lac). However, the BD can be affected by large amounts of saline.

Methods

We conducted a survey of anesthesiologists regarding the use of BD. We also studied the reliability of BD to determine the presence of hyperlactatemia (HL). Patients undergoing general anesthesia were eligible for enrollment if they were receiving an arterial line as part of their routine care. If an arterial blood gas was drawn by the operative team as part of the routine care, the remainder of the unused blood was also used to measure Lac.

Results

Survey: 73 staff anesthesiologists were surveyed. Over 70% of respondents used BD as an endpoint of resuscitation. Base Deficit Study: 35 patients were enrolled resulting in 88 arterial blood gases with corresponding Lac. Mean age was 61.4 ± 14.3 years, 43% were male. Mean pH was 7.39 ± 0.05, the mean bicarbonate was 23.0 ± 2.3 meq/L, the mean BD 1.34 ± 2.3, and the mean Lac was 1.58 ± 0.71 mmol/L. Mean ASA risk score was 3.16 ± 0.71. ROC area under the curve for base deficit to detect HL was 0.58.

Conclusion

BD can often mislead the clinician as to the actual Lac. Lac can now be measured in the OR in real time. Therefore, if clinicians in the operative setting want to know the Lac, it should be measured directly.     

Phosphorylation of the β1 Integrin Cytoplasmic Domain: Toward an Understanding of Function and Mechanism

As F9 stem cells differentiate into parietal endoderm they form focal adhesion sites. There is a concomitant decrease in the level of phosphorylation of S785 in the cytoplasmic domain of the β1 integrin subunit. Previous transfection studies demonstrate that site-specific mutations at this residue, mimicking different phosphorylation states, can alter the subcellular localization of the subunit in differentiating F9 cells. We now extend these observations in an attempt to substantiate the function of β1 phosphorylation and determine how the phosphorylation levels are regulated. We show that treatment of parietal endoderm with okadaic acid induces an increase in β1 phosphorylation and selective loss of β1 from focal adhesion sites. Using a PCR approach, we identify two phosphatases expressed in parietal endoderm, including PP2A. Using a crosslinking approach, where antibodies are added to live cells, we show that the catalytic subunit of PP2A co-immunoprecipitates with β1. Immunocytochemistry shows PP2A colocalizing to focal adhesion sites with β1. In addition integrin-linked kinase (ILK) co-immunoprecipitates with β1 in parietal endoderm and localizes to focal adhesion sites. Okadaic acid treatment significantly decreases the level of ILK associated with β1. A possible role for regulated β1 phosphorylation in cell migration is discussed.     

Modeling plague persistence in host-vector communities in California

Plague is an enzootic disease in the western United States, even though long-term persistent infections do not seem to occur. Enzootic persistence may occur as a function of dynamic interactions between flea vectors and transiently infected hosts, but the specific levels of vector competence, host competence, and transmission and recovery rates that would promote persistence and emergence among wild hosts and vectors are not known. We developed a mathematical model of enzootic plague in the western United States and implemented the model with the following objectives: 1) to use matrix manipulation within a classic susceptible-->infective-->resistant-->susceptible (SIRS) model framework to describe transmission of the plague bacterium Yersinia pestis among rodents and fleas in California, 2) to perform sensitivity analysis with model parameters and variables to indicate which values tended to dominate model output, and 3) to determine whether enzootic maintenance would be predicted with realistic parameter values obtained from the literature for Y. pestis in California rodents and fleas. The model PlagueSIRS was implemented in discrete time as a computer simulation incorporating environmental stochasticity and seasonality, by using matrix functions in the computer language R, allowing any number of rodent and flea species to interact through parasitism and disease transmission. Sensitivity analysis indicated that the model was sensitive to flea attack rate, host recovery rate, and rodent host carrying capacity but relatively insensitive to changes in the duration of latent infection in the flea, host and vector competence, flea recovery from infection, and host mortality attributable to plague. Realistic parameters and variable values did allow for the model to predict enzootic plague in some combinations, specifically when rodent species that were susceptible to infection but resistant to morbidity were parasitized by multiple poorly competent flea species, including some that were present year-round. This model could be extended to similar vectorborne disease systems and could be used iteratively with data collection in sylvatic plague studies to better understand plague persistence and emergence in nature.     

Campylobacter insulaenigrae isolates from northern elephant seals (Mirounga angustirostris) in California

There are only two reports in the literature demonstrating the presence of Campylobacter spp. in marine mammals. One report describes the isolation of a new species, Campylobacter insulaenigrae sp. nov., from three harbor seals (Phoca vitulina) and a harbor porpoise (Phocoena phocoena) in Scotland, and the other describes the isolation of Campylobacter jejuni, Campylobacter lari, and an unknown Campylobacter species from northern elephant seals (Mirounga angustirostris) in California. In this study, 72 presumptive C. lari and unknown Campylobacter species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42 degrees C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were C. insulaenigrae, with one isolate being similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus. Phylogenetic analysis identified two C. insulaenigrae clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all Campylobacter species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of Campylobacter isolates from marine mammals.     

Epistatic interactions of three loci regulate flowering time under short and long daylengths in a backcross population of rice

The short-day plant rice varies greatly in photoperiod sensitivity (PS) for flowering. The hybrid F₁ from a cross between the day-neutral pure line EM93-1 and the weedy rice accession SS18-2 had stronger PS than SS18-2. Some BC₁ (EM93-1/F₁) segregates were even more sensitive to photoperiod than the F₁, as indicated by later flowering or no flowering after 250 days under a 14-h long daylength. A genome-wide scan identified the quantitative trait loci Se _7.1, Se _7.2 and Se ₈ for PS from the BC₁ population, with all alleles that inhibit flowering derived from SS18-2. These three loci regulate the time of flowering under long daylength through their main effects, and di- and trigenic epistases. Under a 10-h short daylength, the regulation is through Se _7.1 and Se ₈ main effects and digenic epistases involving all three loci. The short daylength not only nullified the main effect of Se _7.2, but also changed its epistatic effects from inhibiting flowering under long daylength to promoting flowering. The epistases indicate that genes underlying the three PS loci work in the same pathway for the control of flowering. Many non-flowered BC₁s were the trigenic heterozygote; this suggests that the three PS loci are also involved in genetic control of critical daylength.     

Campylobacter insulaenigrae Isolates from Northern Elephant Seals (Mirounga angustirostris) in California

There are only two reports in the literature demonstrating the presence of Campylobacter spp. in marine mammals. One report describes the isolation of a new species, Campylobacter insulaenigrae sp. nov., from three harbor seals (Phoca vitulina) and a harbor porpoise (Phocoena phocoena) in Scotland, and the other describes the isolation of Campylobacter jejuni, Campylobacter lari, and an unknown Campylobacter species from northern elephant seals (Mirounga angustirostris) in California. In this study, 72 presumptive C. lari and unknown Campylobacter species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42°C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were C. insulaenigrae, with one isolate being similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus. Phylogenetic analysis identified two C. insulaenigrae clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all Campylobacter species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of Campylobacter isolates from marine mammals.     

C-8 Modifications of 3-alkyl-1,8-dibenzylxanthines as inhibitors of human cytosolic phosphoenolpyruvate carboxykinase

New modifications on the C-8 4-aminobenzyl unit of the previously reported 3-alkyl-1,8-dibenzylxanthine inhibitors of cPEPCK are presented. The most active compound reported here is the 5-chloro-1,3-dimethyl-1H-pyrazole-4-sulfonic acid amide derivative 2 with an IC(50) of 0.29+/-0.08 microM. An X-ray analysis of a heteroaromatic sulfonamide is presented showing a new pi-pi interaction.