The cofactor function of the N-terminal domain of tissue factor

Tissue factor (TF) is an integral membrane protein cofactor for factor VIIa (fVIIa) that initiates the blood coagulation cascade during vascular injury. TF has two fibrinonectin type III-like domains, both of which make extensive interactions with both the light and heavy chains of fVIIa. In addition to interaction with fVIIa, the membrane proximal C-terminal domain of TF is also known to bind the natural substrates factors IX and X, thereby facilitating their assembly and recognition by fVIIa in the activation complex. Both fVIIa and TF are elongated proteins, and their complex appears to be positioned nearly perpendicular to the membrane surface. It is possible that, similar to fVIIa, the N-terminal domain of TF also contacts the natural substrates. To investigate this possibility, we substituted all 23 basic and acidic residues of the N-terminal domain of TF with Ala or Asn and expressed the mutants as soluble TF(2-219) in a novel expression/purification vector system in the periplasmic space of bacteria. Following purification to homogeneity, the cofactor properties of mutants in promoting the amidolytic and proteolytic activity of fVIIa were analyzed in appropriate kinetic assays. The amidolytic activity assays indicated that several charged residues spatially clustered at the junction of the N- and C-terminal domains of TF are required for high affinity interaction with fVIIa. On the other hand, the proteolytic activity assays revealed that none of the residues under study may be an interactive site for either factor IX or factor X. However, it was discovered the Arg(74) mutant of TF was defective in enhancing both the amidolytic and proteolytic activity of fVIIa, suggesting that this residue may be required for the allosteric activation of the protease.     

Infrared body temperature measurement of mice as an early predictor of death in experimental fungal infections

Temperatures of mice were measured using an infrared high performance non-contact thermometer, after the device had been calibrated using implantable microchips containing temperature transponders. Mice were infected with three species of Candida (isolates) and the resultant disseminated infections monitored. Mouse temperatures could be reliably measured using the infrared device and this measurement caused little distress to the mice. We were further able to demonstrate that mice rarely recovered if their body temperature dropped below 33.3 degrees C (sensitivity 68%, specificity 97%). Adoption of a 33.3 degrees C endpoint in fungal sepsis experiments measured by infrared non-contact thermometer would significantly reduce the suffering in the terminal stages of this type of infection model.     

Identification of signature and primers specific to genus <Emphasis Type="Italic">Pseudomonas</Emphasis> using mismatched patterns of 16S rDNA sequences

Background

Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus.

Results

Repeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene. Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified. The sub-sequences between the repeating patterns yielded a continuous region of 495 bases. The sub-sequences after alignment and using Shanon's entropy measure yielded a consensus pattern. A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns. Nine patterns in this stretch showed nearly 70% specificity to the target genus. These patterns were further used to obtain a signature that is highly specific to Pseudomonas. The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment.

Conclusions

The developed approach was successfully applied to genus Pseudomonas. It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

     

Breeding territory fidelity in a partial migrant, the American dipper Cinclus mexicanus

American dipper Cinclus mexicanus populations are frequently composed of resident individuals that occupy permanent territories year round and migratory individuals that overwinter with residents but migrate to breeding territories on higher elevation creeks each spring. Between 1999 and 2004 we examined how migratory strategy (resident/migratory) and sex differences influence breeding territory fidelity of American dippers occupying the Chilliwack River watershed, British Columbia, Canada. Counter to expectation we found that the migratory strategy of American dippers did not influence whether birds breeding in one year were found on their former breeding territory in the next. Migratory strategy also did not affect the probability that known surviving dippers occupied the same breeding territory in the following year. Males and females were equally likely to be found on their former territory in the following year (females 43%, males 41%) and known survivors had similar levels of breeding territory fidelity (females 74%, males 68%). However, breeding territory fidelity of males and females varied in response to different factors. Surviving female dippers were more likely to be found on their former breeding territory in the subsequent year following a successful breeding attempt than an unsuccessful breeding attempt. Prior reproductive performance did not influence whether surviving male dippers were found on their former breeding territory. Male dippers were more likely to be found on their former territory and, if they survived, have higher breeding territory fidelity when their mate also returned to that same territory. Mate retention also influenced whether females were found on their former territory in the following year but had no effect on the breeding territory fidelity of known survivors. We argue that sex‐specific dispersal decision rules in American dippers are driven by sex differences in the predictability of breeding performance between years and sex differences in how mate retention influences subsequent reproductive success.     

Thylakoid Membrane Development and Differentiation: Assembly of the Chlorophyll a-b Light-Harvesting Complex and Evidence for the Origin of Mr=19, 17.5 and 13.4 kDa Proteins

Pea plants were grown under intermittent illumination (ImL)conditions. The low dosage of light given to ImL plastids limitedthe rate of chlorophyll (Chl) a and Chl b biosynthesis and,therefore, it retarded the rate of photosynthetic unit formationand thylakoid membrane development. Depending on the developmentalstage of the photosynthetic unit, ImL plastids had variableChl a/Chl b ratios (2.7 <Chl a/Chlb<20) and showed distinctintermediates in the assembly of the chlorophyll a–b light-harvestingcomplex (LHC) of photosystem-II (PSII). The results are consistentwith a step-wise increment in the PSII antenna size involvingthree distinct forms of the PSII unit: (i) a PSII-core formwith about 37 Chl a molecules; (ii) a PSIL_ß form containingthe PSII-core and the LHC-II-inner antenna with a total of about130 Chl (a + b) molecules, and (iii) the mature PSII_a form containingPSII_ß and the LHC-II-peripheral antenna with a totalof 210–300 Chl (a + b) molecules. The thylakoid membranecontained polypeptide subunits b, c and d (the Lhcb1, 2 and3 gene products, respectively) when only the LHC-II-inner waspresent. Polypeptide subunit a, (the apoprotein of the chlorophyll-proteinknown as CP29), along with increased amounts of b and c appearedlater in the development of thylakoids, concomitant with theassembly of the LHC-II-peripheral. The results suggest thatpolypeptide subunit d has priority of assembly over subunita. It is implied that, of all LHC-II constituent proteins, subunitd is most proximal to the PSII-core complex and that it servesas a linker in the transfer of excitation energy from the bulkLHC-II (subunits b and c) to the PSII-core. The work also addressesthe origin of low-molecular-weight proteins (M_r = 19, 17.5 and13.4 kDa) which co-isolate with intact developing plastids andwhose abundance decreases during plastid development. Aminoacid compositional and immunoblot analyses show a nuclear histoneorigin for these low-molecular-weight proteins and suggest co-isolationof histone-containing nuclear vesicles along with intact developingplastids. ¹Present address: Plant Physiology Research Group, The Universityof Calgary, Department of Biological Sciences, 2500 UniversityDrive N.W., Calgary, Alberta CANADA T2N 1N4.     

Biochemical analysis of pleiotropy in Dictyostelium.

The patterns of accumulation of five developmentally regulated enzymes were determined in four aggregateless mutant strains of Dictyostelium. Previous analyses of these strains had predicted that they should not accumulate these markers. The results are consistent with the concept that a linear dependent pathway controls the temporal expression of development in this system.     

Independence of 1,25-dihydroxyvitamin D3-mediated calcium transport from de novo RNA and protein synthesis

The administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to rachitic chicks produces an increase in (a) RNA and protein synthesis, (b) calcium binding protein (CaBP) concentration, and (c) alkaline phosphatase activity in the duodenum. These events occur concomitantly with enhanced calcium transport. We inhibited RNA and protein synthesis in richitic chicks and measured the subsequent response to 1,25(OH)2D3. Actinomycin D, injected prior to and following 1,25(OH)2D3 administration, inhibited intestinal RNA polymerase activity, blocked the rise in serum calcium, reduced the amount of CaBP, and increased alkaline phosphatase activity. Cycloheximide injected in similar fashion, inhibited the 1,25(OH)2D3-mediated increase in intestinal protein synthesis, serum calcium, CaBP, and alkaline phosphatase activity. Neither inhibitor blocked the ability of 1,25(OH)2D3 to stimulate calcium transport as measured in isolated duodenal loops in vivo. The ability of either inhibitor to block 1,25(OH)2D3-mediated calcium transport despite inhibition of CaBP production and alkaline phosphatase activity (by cycloheximide) indicates that de novo RNA and protein synthesis, and in particular CaBP and alkaline phosphatase, are not required for the 1,25(OH)2D3 stimulation of calcium transport.     

Unilateral Lateral Entorhinal Inactivation Impairs Memory Expression in Trace Eyeblink Conditioning

Memory in trace eyeblink conditioning is mediated by an inter-connected network that involves the hippocampus (HPC), several neocortical regions, and the cerebellum. This network reorganizes after learning as the center of the network shifts from the HPC to the medial prefrontal cortex (mPFC). Despite the network reorganization, the lateral entorhinal cortex (LEC) plays a stable role in expressing recently acquired HPC-dependent memory as well as remotely acquired mPFC-dependent memory. Entorhinal involvement in recent memory expression may be attributed to its previously proposed interactions with the HPC. In contrast, it remains unknown how the LEC participates in memory expression after the network disengages from the HPC. The present study tested the possibility that the LEC and mPFC functionally interact during remote memory expression by examining the impact of pharmacological inactivation of the LEC in one hemisphere and the mPFC in the contralateral hemisphere on memory expression in rats. Memory expression one day and one month after learning was significantly impaired after LEC-mPFC inactivation; however, the degree of impairment was comparable to that after unilateral LEC inactivation. Unilateral mPFC inactivation had no effect on recent or remote memory expression. These results suggest that the integrity of the LEC in both hemispheres is necessary for memory expression. Functional interactions between the LEC and mPFC should therefore be tested with an alternative design.     

Solid-phase synthesis of naphthylamidines as factor VIIa/tissue factor inhibitors

Reductive amination followed by acylation of polymer-linked formyl aryl amidines generate combinatorial libraries of aryl amidines 8-13. Potent small molecule naphthylamidine inhibitors 12 (Ki<100 nM) of FVIIa/TF have been discovered and their activity against other serine proteases in the coagulation cascade is reported.