Loss of glial glutamate transporters and induction of neuronal expression of GLT-1B in the hypoxic neonatal pig brain

The homeostasis of glutamate is critical to normal brain function; deficiencies in the regulation of extracellular glutamate are thought to be a major determinant of damage in hypoxic brains. Extracellular levels of glutamate are regulated mainly by plasmalemmal glutamate transporters. We have evaluated the distribution of the glutamate transporter GLAST and two splice variants of GLT-1 in the hypoxic neonatal pig brain using this as model of neonatal humans. In response to severe hypoxic insults, we observe a rapid loss of two glial glutamate transporters from specific brain regions, such as the CA1 region of the hippocampus, but not the dentate gyrus. The spatial distribution of loss accords with patterns of damage in these brains. Conversely, we demonstrate that hypoxia evokes the expression of a splice variant of GLT-1 in neurons. We suggest that this expression may be induced in response to elevated extracellular glutamate around these neurons, and that this splice variant may represent a useful marker for direct quantification of the extent of likely neuronal damage in hypoxic brains. © 2004 Elsevier B.V. All rights reserved.

Neonatal calyceal dilation and renal fibrosis resulting from loss of Adamts-1 in mouse kidney is due to a developmental dysgenesis

Background. A disintegrin and metalloproteinase with thrombospondin motifs 1, Adamts-1, is important for the development and function of the kidney. Mice lacking this protein present with renal lesions comprising enlarged calyces, and reduced cortex and medulla layers. Our current findings are consistent with the defect occurring due to a developmental dysgenesis. Methods. We generated Adamts-1 null mice, and further investigated their kidney phenotype in a time course study ranging from E18.5 to 12 months of age. Immunohistochemistry was used to assess the localization of type IV collagen, TGF-beta and F4/80-positive macrophages in the kidneys of Adcants-1 null mice compared to wild-type control animals. The expression of Adamts-1 mRNA was determined in metanephric kidney explants by in situ hybridization. Results. Adamts-1 null mice have a gross kidney defect. At day 18.5 of gestation, the Adcants-1 null kidney has a normal appearance but at birth when the kidney begins to function, the defect becomes evident. During development of the kidney Adamts-1 expression was specifically detected in the developing loops of Henle, as well as in the proximal and distal convoluted tubules. Expression was not detected in the ureter, ureteric bud or its derivatives as had been previously suggested. At 6 months and I year of age, the Adamts-1 null mice displayed interstitial fibrosis in the cortical and medullary regions of the kidney. At I year of age, the Adamts-1 null mice displayed mild interstitial matrix expansion associated with increased collagen type IV expression, without apparent tubular dilatation, compared to wild-type animals. Immunohistochemical analysis demonstrated TGF-beta protein localized to infiltrating macrophages and glomeruli of Adamts-1 null mice. Conclusions. Adamts-1 is required for the normal development of the kidney. The defect observed in its absence results from a dysgenic malformation affecting the medulla that becomes apparent at birth, once the kidneys start to function.

Socioeconomic causes of loss of animal genetic diversity: analysis and assessment

The proximate causes and processes involved in loss of breeds are outlined. The path-dependent effect and Swanson's dominance-effect are discussed in relation to lock-in of breed selection. These effects help to explain genetic erosion. It is shown that the extension of markets and economic globalisation have contributed significantly to the loss of breeds. The decoupling of animal husbandry from surrounding natural environmental conditions is further eroding the stock of genetic resources. Recent trends in animal husbandry raise serious sustainability issues, apart from animal welfare concerns. The extension of markets and economic globalisation have contributed significantly to the rapid loss of domestic breeds, especially livestock. (C) 2003 Elsevier Science B.V. All rights reserved.

Persistent colonization with Tannerella forsythensis and loss of attachment in adolescents

Colonization with Tannerella forsythensis may characterize the conversion of periodontally healthy sites into diseased sites. This three-year study describes the prevalence of T forsythensis and its relationship to clinical loss of attachment (LOA) in a group of adolescents considered at risk of developing early chronic periodontitis. Adolescents with (LOA+) and without (LOA-) loss of attachment were examined at baseline and 1.5 and 3 yrs subsequently. On each occasion, attachment loss was measured on selected teeth, and the presence of T. forsythensis in their subgingival plaque samples was determined by PCR. T. forsythensis prevalence in LOA+ subjects at baseline (64%) increased to 82% and 86% on subsequent examinations. In contrast, prevalence of T. forsythensis in LOA- subjects was always significantly lower (25%, 36%, and 32%, respectively). The odds of loss of attachment were 8.16 times greater in subjects infected with T. forsythensis at each examination. These results suggest that T. forsythensis is strongly associated with loss of attachment in this adolescent population.

Selective loss of NMDA receptor NR1 subunit isoforms in Alzheimer's disease

Previous work had shown that the ratio of NMDA receptor NR1 subunit mRNA transcripts containing an N-terminal splice cassette to those that do not is markedly lower in regions of the Alzheimer's disease (AD) brain that are susceptible to pathological damage, compared with spared regions in the same cases or homotropic regions in controls. To elucidate the origins of this difference in proportionate expression, we measured the absolute levels of each of the eight NR1 transcripts by quantitative internally standardized RT-PCR assay. Expression of transcripts with the cassette was strongly attenuated in susceptible regions of Alzheimer's brain, whereas expression of non-cassette transcripts differed little from that in controls. The expression of other NR1 splice variants was not associated with pathology relevant to disease status, although some combinations of splice cassettes were well maintained in AD cases. The population profile of NR1 transcripts in occipital cortex differed from the profiles in other brain regions studied. Western analysis confirmed that the expression of protein isoforms containing the N-terminal peptide was very low in susceptible areas of the Alzheimer's brain. Cells that express NR1 subunits with the N-terminal cassette may be selectively vulnerable to toxicity in AD.

Loss of PKR Activity in Chronic Lymphocytic Leukemia

There are a number of observations that suggest the dsRNA-activated protein kinase, PKR, may play an active role in formation and maintenance of leukemia, including nonrandom chromosomal deletions in acute leukemia as well as truncations and deletions of the PKR gene in some leukemia cell lines. However, there is little direct evidence from patient material that this is so. Here we show that full-length PKR is present but not active in 21 of 28 patient samples from B-cell chronic lymphocytic leukemia (B-CLL). PKR from these patients was unable to auto-activate or phosphorylate substrates but was able to bind dsRNA. Furthermore, the lack of PKR activation was not due to differing levels of the PKR activator, PACT nor of the PKR inhibitor, p58(IPK). We compared PKR status with clinical parameters and disease staging. No differences were found between the 2 groups in terms of staging (modified Rai or Binet), age, CD38 status, p53 status, 11q23 deletion status or CEP12 deletion status. However, there was a significant correlation between deletion in 13q14.3 and lack of PKR activity. We show that B-CLL cells appear to contain a soluble inhibitor of PKR, as lysates from cells lacking PKR activity were able to inhibit exogenous PKR in mixing experiments. Finally, we show suppression of PKR activity was still present following ultrafilitration through a 10,000 Da cutoff filter but was lost upon extraction with phenol/chloroform or by high salt washing. This data suggests loss of PKR activity may contribute to the formation and/or maintenance of CLL. (C) 2004 Wiley-Liss, Inc.

Functional analysis of the a-defensin disulfide array in mouse cryptdin-4

The alpha-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines alpha-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell alpha-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and proCrp4 molecules to degradation by MMP-7. Although native Crp4 and the alpha-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining alpha-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.

Failure of a medulloblastoma-derived mutant of SUFU to suppress WNT signaling

Germline mutations of APC in patients with Turcot syndrome (colon cancer and medulloblastoma), was well as somatic mutations of APC, beta-catenin, and Axin in sporadic medulloblastomas (MBs) have shown the importance of WNT signaling in the pathogenesis of MB. A subset of children with MB have germline mutations of SUFU, a known inhibitor of Hedgehog signal transduction. A recent report suggested that murine Sufu can bind beta-catenin, export it from the nucleus, and thereby repress beta-catenin/T-cell factor (Tcf)-mediated transcription. We show that an MB-derived mutant of SUFU has lost the ability to decrease nuclear levels of beta-catenin, and cannot inhibit beta-catenin/Tcf-mediated transcription as compared to wild type SUFU. Our results suggest that loss of function of SUFU results in overactivity of both the Sonic Hedgehog, and the WNT signaling pathways, leading to excessive proliferation and failure to differentiate resulting in MB.

Fine mapping of the tomato I-3 gene for fusarium wilt resistance and elimination of a co-segregating resistance gene analogue as a candidate for I-3

The I-3 gene from the wild tomato species Lycopersicon pennellii confers resistance to race 3 of the devastating vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici. As an initial step in a positional cloning strategy for the isolation of I-3, we converted restriction fragment length polymorphism and conserved orthologue set markers, known genes and a resistance gene analogue (RGA) mapping to the I-3 region into PCR-based sequence characterised amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers. Additional PCR-based markers in the I-3 region were generated using the randomly amplified DNA fingerprinting (RAF) technique. SCAR, CAPS and RAF markers were used for high-resolution mapping around the I-3 locus. The I-3 gene was localised to a 0.3-cM region containing a RAF marker, eO6, and an RGA, RGA332. RGA332 was cloned and found to correspond to a putative pseudogene with at least two loss-of-function mutations. The predicted pseudogene belongs to the Toll interleukin-1 receptor-nucleotide-binding site-leucine-rich-repeat sub-class of plant disease resistance genes. Despite the presence of two RGA332 homologues in L. esculentum, DNA gel blot and PCR analysis suggests that no other homologues are present in lines carrying I-3 that could be alternative candidates for the gene.

Identification of functional sequences in the pregenomic RNA promoter of the Banana streak virus Cavendish strain (BSV-Cav)

The promoter regions of plant pararetroviruses direct transcription of the full-length viral genome into a pregenomic RNA that is an intermediate in the replication of the virus. It serves as template for reverse transcription and as polycistronic mRNA for translation to viral proteins. We have identified functional promoter elements in the intergenic region of the Cavendish isolate of Banana streak virus (BSV-Cav), a member of the genus Badnavirus. Potential binding sites for plant transcription factors were found both upstream and downstream of the transcription start site by homology search in the PLACE database of plant cis-acting elements. The functionality of these putative cis-acting elements was tested by constructing loss-of-function and regain-of-function mutant promoters whose activity was quantified in embryogenic sugarcane suspension cells. Four regions that are important for activity of the BSV-Cav promoter were identified: the region containing an as-l-like element, the region around-141 and down to -77, containing several putative transcription factor binding sites, the region including the CAAT-box, and the leader region. The results could help explain the high BSV-Cav promoter activity that was observed previously in transgenic sugarcane plants and give more insight into the plant cell-mediated replication of the viral genome in banana streak disease. (C) 2004 Elsevier B.V. All rights reserved.

Using the canonical modelling approach to simplify the simulation of function in functional-structural plant models

Functional-structural plant models that include detailed mechanistic representation of underlying physiological processes can be expensive to construct and the resulting models can also be extremely complicated. On the other hand, purely empirical models are not able to simulate plant adaptability and response to different conditions. In this paper, we present an intermediate approach to modelling plant function that can simulate plant response without requiring detailed knowledge of underlying physiology. Plant function is modelled using a 'canonical' modelling approach, which uses compartment models with flux functions of a standard mathematical form, while plant structure is modelled using L-systems. Two modelling examples are used to demonstrate that canonical modelling can be used in conjunction with L-systems to create functional-structural plant models where function is represented either in an accurate and descriptive way, or in a more mechanistic and explanatory way. We conclude that canonical modelling provides a useful, flexible and relatively simple approach to modelling plant function at an intermediate level of abstraction.

Activation of the cAMP pathway by variant human MC1R alleles expressed in HEK and in melanoma cells

alpha-Melanocyte-stimulating hormone (alpha-MSH) activates the melanocortin-1 receptor (MC1R) on melanocytes to promote a switch from red/yellow pheomelanin synthesis to darker eumelanins via positive coupling to adenylate cyclase. The human MC1R locus is highly polymorphic with the specific variants associated with red hair and fair skin (RHC phenotype) postulated to be loss-of-function receptors. We have examined the ability of MC1R variants to activate the cAMP pathway in stably transfected REK293 cells. The RHC associated variants, Arg151Cys, Arg160Trp and Asp294His, demonstrated agonist-mediated increases in cAMP and phosphorylation of cAMP-responsive element-binding protein (CREB). Whereas the Asp294His variant showed severely impaired functional responses, the Arg151Cys and Arg160Trp variants retained considerable signaling capacity. Melanoma cells homozygous for either the Arg151Cys variant or consensus sequence both elicited CREB phosphorylation in response to alpha-MSH in the presence of IBMX. The common RHC alleles, Arg151Cys, Arg160Trp and Asp294His, are neither complete loss-of-function receptors nor are they functionally equivalent. (c) 2005 Elsevier Inc. All rights reserved.