TIE-2/VEGF-R2 SAR and in vitro activity of C3-acyl dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazole analogs

Orally bioavailable, dual inhibitors of TIE-2/VEGF-R2 were identified by elaborating the C3/N13 SAR around a fused pyrrolodihydroindazolocarbazole scaffold. Analogs bearing a C3-thiophencarbonyl group were evaluated in enzymatic and cellular biochemical assays; two orally bioavailable analogs were further profiled in functional assays and found to inhibit microvessel growth in rat aortic explant cultures and inhibit Ang-1-stimulated chemotaxis of HUVECs.     

Natural Variation in Maize Aphid Resistance Is Associated with 2,4-Dihydroxy-7-Methoxy-1,4-Benzoxazin-3-One Glucoside Methyltransferase Activity

Plants differ greatly in their susceptibility to insect herbivory, suggesting both local adaptation and resistance tradeoffs. We used maize (Zea mays) recombinant inbred lines to map a quantitative trait locus (QTL) for the maize leaf aphid (Rhopalosiphum maidis) susceptibility to maize Chromosome 1. Phytochemical analysis revealed that the same locus was also associated with high levels of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) and low levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). In vitro enzyme assays with candidate genes from the region of the QTL identified three O-methyltransferases (Bx10a-c) that convert DIMBOA-Glc to HDMBOA-Glc. Variation in HDMBOA-Glc production was attributed to a natural CACTA family transposon insertion that inactivates Bx10c in maize lines with low HDMBOA-Glc accumulation. When tested with a population of 26 diverse maize inbred lines, R. maidis produced more progeny on those with high HDMBOA-Glc and low DIMBOA-Glc. Although HDMBOA-Glc was more toxic to R. maidis than DIMBOA-Glc in vitro, BX10c activity and the resulting decline of DIMBOA-Glc upon methylation to HDMBOA-Glc were associated with reduced callose deposition as an aphid defense response in vivo. Thus, a natural transposon insertion appears to mediate an ecologically relevant trade-off between the direct toxicity and defense-inducing properties of maize benzoxazinoids.     

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.