Quantifying torpor in small mammals non-invasively using infrared thermocouples

Measurements of torpor use are pivotal for many research areas concerning the thermal biology of endotherms. Here, I used infrared thermocouples to non-invasively examine torpor patterns in the small marsupial fat-tailed dunnart (Sminthopsis crassicaudata). Sensors were installed inside the nesting chambers to continuously monitor fur temperature in undisturbed animals. Firstly, to verify the measurements, fur temperature was monitored simultaneously with body temperature using internal radio transmitters (n=6). Secondly, I conducted a food restriction study to demonstrate the reliability of the method within a physiological experiment (n=8). Based on the correspondence of simultaneously measured fur and body temperature during torpor bouts, I was able to confirm that infrared thermocouples provide reliable temporal information on torpor patterns. Furthermore, torpor use was successfully monitored over a 20-day food restriction study. The method can easily be adapted to suit other small mammal or bird species and presents a useful, inexpensive approach for examining torpor patterns remotely and non-invasively in the laboratory.     

Role of hindbrain in inner ear morphogenesis: analysis of Noggin knockout mice

Signaling from rhombomeres 5 and 6 of the hindbrain is thought to be important for inner ear patterning. In Noggin −/− embryos, the gross anatomy of the inner ear is distorted and malformed, with cochlear duct outgrowth and coiling most affected. We attributed these defects to a caudal shift of the rhombomeres caused by the shortened body axis and the kink in the neural tube. To test the hypothesis that a caudal shift of the rhombomeres affects inner ear development, we surgically generated chicken embryos in which rhombomeres 5 and 6 were similarly shifted relative to the position of the inner ears, as in Noggin mutants. All chicken embryos with shifted rhombomeres showed defects in cochlear duct formation indicating that signaling from rhombomeres 5 and 6 is important for cochlear duct patterning in both chicken and mice. In addition, the size of the otic capsule is increased in Noggin −/− mutants, which most likely is due to unopposed BMP signaling for chondrogenesis in the peri-otic mesenchyme.     

Specificity and reactive loop length requirements for crmA inhibition of serine proteases

The viral serpin, crmA, is distinguished by its small size and ability to inhibit both serine and cysteine proteases utilizing a reactive loop shorter than most other serpins. Here, we characterize the mechanism of crmA inhibition of serine proteases and probe the reactive loop length requirements for inhibition with two crmA reactive loop variants. P1 Arg crmA inhibited the trypsin-like proteases, thrombin, and factor Xa, with moderate efficiencies (approximately 10(2)-10(4) M(-1)sec(-1)), near equimolar inhibition stoichiometries, and formation of SDS-stable complexes which were resistant to dissociation (k(diss) approximately 10(-7) sec(-1)), consistent with a serpin-type inhibition mechanism. Trypsin was not inhibited, but efficiently cleaved the variant crmA as a substrate (k(cat)/K(M) of approximately 10(6) M(-1) sec(-1)). N-terminal sequencing confirmed that the P1 Arg-P1'Cys bond was the site of cleavage. Altering the placement of the Arg in a double mutant P1 Gly-P1'Arg crmA resulted in minimal ability to inhibit any of the trypsin family proteases. This variant was cleaved by the proteases approximately 10-fold less efficiently than P1 Arg crmA. Surprisingly, pancreatic elastase was rapidly inhibited by wild-type and P1 Arg crmAs (10(5)-10(6) M(-1)sec(-1)), although with elevated inhibition stoichiometries and higher rates of complex dissociation. N-terminal sequencing showed that elastase attacked the P1'Cys-P2'Ala bond, indicating that crmA can inhibit proteases using a reactive loop length similar to that used by other serpins, but with variations in this inhibition arising from different effective P2 residues. These results indicate that crmA inhibits serine proteases by the established serpin conformational trapping mechanism, but is unusual in inhibiting through either of two adjacent reactive sites.     

A novel mode of action for a microbial-derived immunotoxin: the cytolethal distending toxin subunit B exhibits phosphatidylinositol 3,4,5-triphosphate phosphatase activity

The Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) is a potent immunotoxin that induces G(2) arrest in human lymphocytes. We now show that the CdtB subunit exhibits phosphatidylinositol (PI)-3,4,5-triphosphate phosphatase activity. Breakdown product analysis indicates that CdtB hydrolyzes PI-3,4,5-P(3) to PI-3,4-P(2) and therefore functions in a manner similar to phosphatidylinositol 5-phosphatases. Conserved amino acids critical to catalysis in this family of enzymes were mutated in the cdtB gene. The mutant proteins exhibit reduced phosphatase activity along with decreased ability to induce G(2) arrest. Consistent with this activity, Cdt induces time-dependent reduction of PI-3,4,5-P(3) in Jurkat cells. Lymphoid cells with defects in SHIP1 and/or ptase and tensin homolog deleted on chromosome 10 (PTEN) (such as Jurkat, CEM, Molt) and, concomitantly, elevated PI-3,4,5-P(3) levels were more sensitive to the toxin than HUT78 cells which contain functional levels of both enzymes and low levels of PI-3,4,5-P(3). Finally, reduction of Jurkat cell PI-3,4,5-P(3) synthesis using the PI3K inhibitors, wortmannin and LY290004, protects cells from toxin-induced cell cycle arrest. Collectively, these studies show that the CdtB not only exhibits PI-3,4,5-P(3) phosphatase activity, but also that toxicity in lymphocytes is related to this activity.     

Soluble M1 protein of Streptococcus pyogenes triggers potent T cell activation

Streptococcus pyogenes of the M1 serotype is commonly associated with large outbreaks of invasive streptococcal infections and development of streptococcal toxic shock syndrome (STSS). The pathogenesis behind these infections is believed to involve bacterial superantigens that induce potent inflammatory responses, but the reason why strains of the M1 serotype are over-represented in STSS is still not understood. In the present investigation, we show that a highly purified soluble form of the M1 protein from S. pyogenes , which lacks the membrane-spanning region, is a potent inducer of T cell proliferation and release of Th1 type cytokines. M1 protein-evoked T cell proliferation was HLA class II-dependent but not MHC-restricted, did not require intracellular processing and was Vβ-restricted. Extensive mass spectrometry studies indicated that there were no other detectable proteins in the preparation. Taken together, our data demonstrate that soluble M1 protein is a novel streptococcal superantigen, which likely contributes to the excessive T cell activation and hyperinflammatory response seen in severe invasive streptococcal infections.     

MULTIVARIATE PHENOTYPIC DIFFERENTIATION AMONG BOTTLENECK LINES OF THE HOUSEFLY

Multivariate phenotypic differentiation in eight morphometric traits was examined in bottleneck lines of the housefly initiated with one, four, or 16 pairs of flies from a natural outbred population. Differentiation was assessed using a Mahalanobis' distance metric in units of additive genetic variance and covariance estimated from the ancestral population (i.e., generalized genetic distance). This distance metric was partitioned into contributions of size and shape to total distance. Bottleneck lines of all sizes diverged significantly from the ancestral line, but the direction of these shifts differed among the lines of different initial founding size. Those populations founded with single pairs diverged from the ancestral line mostly in shape; the 16-pair lines differentiated almost entirely in size, and the four-pair lines were intermediate in the relative contribution of shape to differentiation from the control. Bottlenecks serve to alter the genetic relationships among traits within the derived populations and in doing so could promote speciation by permitting differentiation of the populations along evolutionary trajectories less accessible to the base population.