A human glucagon-like peptide-1 receptor polymorphism results in reduced agonist responsiveness

Glucagon-like peptide-1 (GLP-1) and its cognate receptor play an important physiological role in maintaining blood glucose homeostasis. A GLP-1 receptor (GLP-1R) polymorphism in which threonine 149 is substituted with a methionine residue has been recently identified in a patient with type 2 diabetes but was not found in non-diabetic control subjects. We have functionally assessed the recombinant GLP-1R variant after transient expression in COS-7 and HEK 293 cells. Compared to the wild type receptor, the variant GLP-1R showed (i) similar expression levels, (ii) 60-and 5-fold reduced binding affinities, respectively, for two GLP-1R full agonists, GLP-1 and exendin-4, and (iii) markedly decreased potencies of these peptides in triggering cAMP-mediated signaling (despite conserved efficacies). In contrast to full agonists, the efficacy of the primary GLP-1 metabolite/GLP-1R partial agonist, GLP-1 (9-36) amide, was essentially abolished by the T149M substitution. By hydropathy analysis, the polymorphism localizes to transmembrane domain 1, suggesting this receptor segment as a novel determinant of agonist affinity/efficacy. These findings reveal that naturally occurring sequence variability of the GLP-1R within the human population can result in substantial loss-of-function. A genetic link between the T149M variant and increased susceptibility to type 2 diabetes remains to be established.     

Effect of potentiation and stretching on maximal force, rate of force development, and range of motion

The purpose of this investigation was to compare the effects of stretching vs. potentiation on subsequent maximal force and rate of force development capabilities of subjects in an isometric squat. Ten male collegiate athletes participated as subjects in this study. Subjects were tested during 3 separate sessions that involved joint range of motion (ROM) measurements of the lower body and isometric squat trials on a force plate to determine peak force (PF) and rate of force development (RFD) values. One testing session was preceded by 10 minutes of quiet sitting (C), 1 by a 30-minute lower-body stretching protocol (S), and 1 by 3 sets of a leg press exercise using 90% of the subjects' previously determined 1 repetition maximum (P). Three repetitions were performed for each set of the leg press, with a 3-minute rest period between each set. PF during the isometric squat was not significantly different following any of the 3 conditions (C: 100.0 +/- 0.0%, S: 101.2 +/- 6.5%, P: 98.6 +/- 6.2%). However, RFD was significantly lower in P (87.5 +/- 12.8%) compared with both C (100.0 +/- 0.0%) and S (102.6 +/- 18.5%). Significant improvement in ROM occurred only following P. It appears the potentiation protocol used in the current investigation may actually have had fatiguing effects instead of potentiating effects, but it did result in significant increases in ROM.     

Signal amplification in molecular imaging by pretargeting a multivalent, bispecific antibody

Here we describe molecular imaging of cancer using signal amplification of a radiotracer in situ by pretargeting a multivalent, bispecific antibody to carcinoembryonic antigen (CEA), which subsequently also captures a radioactive hapten-peptide. Human colon cancer xenografts as small as approximately 0.15 g were disclosed in nude mice within 1 h of giving the radiotracer, with tumor/blood ratios increased by >or=40-fold (approximately 10:1 at 1 h, approximately 100:1 at 24 h), compared to a (99m)Tc-labeled CEA-specific F(ab') used clinically for colorectal cancer detection, while also increasing tumor uptake tenfold ( approximately 20% injected dose/g) under optimal conditions. This technology could be adapted to other antibodies and imaging modalities.     

APDS: the autonomous pathogen detection system

We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system was developed to provide early warning to civilians in the event of a bioterrorism incident and can be used at high profile events for short-term, intensive monitoring or in major public buildings or transportation nodes for long-term monitoring. The APDS is completely automated, offering continuous aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and nucleic acid-based polymerase chain reaction (PCR) amplification and detection. Highly multiplexed antibody-based and duplex nucleic acid-based assays are combined to reduce false positives to a very low level, lower reagent costs, and significantly expand the detection capabilities of this biosensor. This article provides an overview of the current design and operation of the APDS. Certain sub-components of the ADPS are described in detail, including the aerosol collector, the automated sample preparation module that performs multiplexed immunoassays with confirmatory PCR, and the data monitoring and communications system. Data obtained from an APDS that operated continuously for 7 days in a major U.S. transportation hub is reported.     

Gene expression in the hippocampus of inbred alcohol-preferring and -nonpreferring rats

The hippocampus is sensitive to the effects of ethanol and appears to have a role in the development of alcohol tolerance. The objective of this study was to test the hypothesis that there are innate differences in gene expression in the hippocampus of inbred alcohol-preferring (iP) and -nonpreferring (iNP) rats that may contribute to differences in sensitivity to ethanol and/or in the development of tolerance. Affymetrix microarrays were used to measure gene expression in the hippocampus of alcohol-naive male iP and iNP rats in two experiments (n=4 and 6 per strain in the two experiments). Combining data from the two experiments, there were 137 probesets representing 129 genes that significantly differed (P < or = 0.01); 62 probesets differed at P < or = 0.001. Among the 36% of the genes that were expressed more in the iP than iNP rat at this level of significance, many were involved in cell growth and adhesion, cellular stress reduction and anti-oxidation, protein trafficking, regulation of gene expression, synaptic function and metabolism. Among the 64% of the genes that had lower expression in the hippocampus of iP than iNP rats were genes involved in metabolic pathways, cellular signaling systems, protein trafficking, cell death and neurotransmission. Overall, the data indicate that there are significant innate differences in gene expression in the hippocampus between iP and iNP rats, some of which might contribute to the differences observed in the development of alcohol tolerance between the selectively bred P and NP lines.     

Flavobacterium johnsoniae gliding motility genes identified by mariner mutagenesis

Cells of Flavobacterium johnsoniae glide rapidly over surfaces. The mechanism of F. johnsoniae gliding motility is not known. Eight gld genes required for gliding motility have been described. Disruption of any of these genes results in complete loss of gliding motility, deficiency in chitin utilization, and resistance to bacteriophages that infect wild-type cells. Two modified mariner transposons, HimarEm1 and HimarEm2, were constructed to allow the identification of additional motility genes. HimarEm1 and HimarEm2 each transposed in F. johnsoniae, and nonmotile mutants were identified and analyzed. Four novel motility genes, gldK, gldL, gldM, and gldN, were identified. GldK is similar in sequence to the lipoprotein GldJ, which is required for gliding. GldL, GldM, and GldN are not similar in sequence to proteins of known function. Cells with mutations in gldK, gldL, gldM, and gldN were defective in motility and chitin utilization and were resistant to bacteriophages that infect wild-type cells. Introduction of gldA, gldB, gldD, gldFG, gldH, gldI, and gldJ and the region spanning gldK, gldL, gldM, and gldN individually into 50 spontaneous and chemically induced nonmotile mutants restored motility to each of them, suggesting that few additional F. johnsoniae gld genes remain to be identified.     

Nucleotide dissociation from NBD1 promotes solute transport by MRP1

MRP1 transports glutathione-S-conjugated solutes in an ATP-dependent manner by utilizing its two NBDs to bind and hydrolyze ATP. We have found that ATP binding to NBD1 plays a regulatory role whereas ATP hydrolysis at NBD2 plays a dominant role in ATP-dependent LTC4 transport. However, whether ATP hydrolysis at NBD1 is required for the transport was not clear. We now report that ATP hydrolysis at NBD1 may not be essential for transport, but that the dissociation of the NBD1-bound nucleotide facilitates ATP-dependent LTC4 transport. These conclusions are supported by the following results. The substitution of the putative catalytic E1455 with a non-acidic residue in NBD2 greatly decreases the ATPase activity of NBD2 and the ATP-dependent LTC4 transport, indicating that E1455 participates in ATP hydrolysis. The mutation of the corresponding D793 residue in NBD1 to a different acidic residue has little effect on ATP-dependent LTC4 transport. The replacement of D793 with a non-acidic residue, such as D793L or D793N, increases the rate of ATP-dependent LTC4 transport. Along with their higher transport activities, their Michaelis constant Kms (ATP) are also higher than that of wild-type. Coincident with their higher Kms (ATP), their Kds derived from ATP binding are also higher than that of wild-type, implying that the rate of dissociation of the bound nucleotide from the mutated NBD1 is faster than that of wild-type. Therefore, regardless of whether the bound ATP at NBD1 is hydrolyzed or not, the release of the bound nucleotide from NBD1 may bring the molecule back to its original conformation and facilitate the protein to start a new cycle of ATP-dependent solute transport.     

Expression of cell-cycle regulator CDK2-associating protein 1 (p12CDK2AP1) in transgenic mice induces testicular and ovarian atrophy in vivo

The novel cell-cycle regulator p12(CDK2AP1) (p12) gene encodes a cyclin-dependent kinase 2 (CDK2) partner that participates in cell-cycle regulation, apoptosis, and proliferation. CDK2 has been implicated in maintenance of gonadal homeostasis, as knockout mice display reproductive abnormalities. To investigate the role of p12 in homeostasis of gonadal tissues in vivo, we generated a transgenic mouse model driven by the human keratin 14 promoter, reported to target transgene expression to gonadal tissues and also stratified epithelia. Overexpression of the transgene was associated with a gonadal atrophy phenotype in mice of both sexes, yet fertility was not impaired. Histological evaluation of testes showed seminiferous tubule degeneration and decreased tubule diameter. Female transgenic mice had small ovaries, with a higher number of atretic follicles/mm(2) as compared to control nontransgenic mice. Also observed was increased germ cell apoptosis in both sexes (TUNEL). These results suggest that overexpression of p12 leads to testicular and ovarian abnormalities, a phenotype closely related to that of cdk2-/- mice. In combination, these observations suggest that the p12/CDK2 signaling pathways are carefully orchestrated to maintain proper gonadal tissue homeostasis. We suggest that the mechanisms of this regulation may be through p12-mediated altered expression of gonadal-specific genes and apoptotic pathways.     

Inhibition of stretch-activated channels during eccentric muscle contraction attenuates p70S6K activation.

Eccentric contractions (EC) are known to result in muscle hypertrophy, potentially through activation of the Akt-mammalian target of rapamycin-p70 S6 kinase (p70S6K) signaling pathway. Previous work has also demonstrated that EC result in the opening of stretch-activated channels (SAC), and inhibition of these channels resulted in an attenuation of EC-induced muscle hypertrophy. The purpose of this study was to test the hypothesis that a known intracellular pathway directly associated with muscle hypertrophy is coupled to the opening of SAC. Specifically, we measured the activation of the Akt, GSK-3beta, p70S6K, and ribosomal protein S6 following a single bout of EC in the rat tibialis anterior (TA) muscle. The TA muscles performed four sets of six repetitions of EC. In vivo blockade of SAC was performed by a continuous oral treatment with streptomycin in the drinking water (4 g/l) or by intravenous infusion of 80 micromol/kg gadolinium (Gd3+). EC increased the degree of Akt and p70S6K phosphorylation in the TA muscle, whereas in animals in which SAC had been inhibited, there was a reduced capacity for EC to induce Akt or p70S6K phosphorylation. Accompanying this reduced activation of Akt and p70S6K was a failure to phosphorylate GSK-3beta or S6 when SAC were inhibited. The results from these data indicate the necessity of functional SAC for the complete activation of Akt and p70S6K pathway in response to EC.     

Experimental evidence that source genetic variation drives pathogen emergence

A pathogen can readily mutate to infect new host types, but this does not guarantee successful establishment in the new habitat. What factors, then, dictate emergence success? One possibility is that the pathogen population cannot sustain itself on the new host type (i.e. host is a sink), but migration from a source population allows adaptive sustainability and eventual emergence by delivering beneficial mutations sampled from the source''s standing genetic variation. This idea is relevant regardless of whether the sink host is truly novel (host shift) or whether the sink is an existing or related, similar host population thriving under conditions unfavourable to pathogen persistence (range expansion). We predicted that sink adaptation should occur faster under range expansion than during a host shift owing to the effects of source genetic variation on pathogen adaptability in the sink. Under range expansion, source migration should benefit emergence in the sink because selection acting on source and sink populations is likely to be congruent. By contrast, during host shifts, source migration is likely to disrupt emergence in the sink owing to uncorrelated selection or performance tradeoffs across host types. We tested this hypothesis by evolving bacteriophage populations on novel host bacteria under sink conditions, while manipulating emergence via host shift versus range expansion. Controls examined sink adaptation when unevolved founding genotypes served as migrants. As predicted, adaptability was fastest under range expansion, and controls did not adapt. Large, similar and similarly timed increases in fitness were observed in the host-shift populations, despite declines in mean fitness of immigrants through time. These results suggest that source populations are the origin of mutations that drive adaptive emergence at the edge of a pathogen''s ecological or geographical range.