Haploinsufficiency for one COL3A1 allele of type III procollagen results in a phenotype similar to the vascular form of Ehlers-Danlos syndrome, Ehlers-Danlos syndrome type IV

Mutations in the COL3A1 gene that encodes the chains of type III procollagen result in the vascular form of Ehlers-Danlos syndrome (EDS), EDS type IV, if they alter the sequence in the triple-helical domain. Although other fibrillar collagen-gene mutations that lead to allele instability or failure to incorporate proalpha-chains into trimers-and that thus reduce the amount of mature molecules produced-result in clinically apparent phenotypes, no such mutations have been identified in COL3A1. Furthermore, mice heterozygous for Col3a1 "null" alleles have no identified phenotype. We have now found three frameshift mutations (1832delAA, 413delC, and 555delT) that lead to premature termination codons (PTCs) in exons 27, 6, and 9, respectively, and to allele-product instability. The mRNA from each mutant allele was transcribed efficiently but rapidly degraded, presumably by the mechanisms of nonsense-mediated decay. In a fourth patient, we identified a point mutation, in the final exon, that resulted in a PTC (4294C-->T [Arg1432Ter]). In this last instance, the mRNA was stable but led to synthesis of a truncated protein that was not incorporated into mature type III procollagen molecules. In all probands, the presenting feature was vascular aneurysm or rupture. Thus, in contrast to mutations in genes that encode the dominant protein of a tissue (e.g., COL1A1 and COL2A1), in which "null" mutations result in phenotypes milder than those caused by mutations that alter protein sequence, the phenotypes produced by these mutations in COL3A1 overlap with those of the vascular form of EDS. This suggests that the major effect of many of these dominant mutations in the "minor" collagen genes may be expressed through protein deficiency rather than through incorporation of structurally altered molecules into fibrils.     

Effect of high-fat diet on the expression of proteins in muscle, adipose tissues, and liver of C57BL/6 mice

In the present study, the effect of a high fat diet on the expression of proteins in insulin target tissues was analyzed using a proteomic approach. Gastrocnemius muscle, white and brown adipose tissue, and liver were taken from C57BL/6 mice either fed on a high-fat or a chow diet. Expression levels of approximately 10 000 polypeptides for all the four tissues were assessed by two-dimensional gel electrophoresis (2-DE). Computer-assisted image analysis allowed the detection of 50 significantly (p < 0.05) differentially expressed proteins between obese and lean mice. Interestingly, more than half of these proteins were detected in the brown adipose tissue. The differentially expressed proteins were identified by tandem mass spectrometry. Several stress and redox proteins were modulated in response to the high-fat diet. A key glycolytic enzyme was found to be downregulated in adipose tissues and muscle, suggesting that at elevated plasma fatty acid concentrations, fatty acids compete with glucose as an oxidative fuel source. Furthermore, in brown adipose tissue there were significant changes in mitochondrial enzymes involved in the Krebs tricarboxylic acid (TCA) cycle and in the respiratory chain in response to the high-fat diet. The brown adipose tissue is an energy-dissipating tissue. Our data suggest that the high-fat diet treated mice were increasing energy expenditure to defend against weight gain.     

The role of cadherin,beta-catenin,and AP-1 in retinoid-regulated carcinoma cell differentiation and proliferation

Vitamin A derivatives (retinoids) are potent regulators of cell proliferation and differentiation. Retinoids inhibit the function of the oncogenic AP-1 and beta-catenin/TCF pathways and also stabilize components of the adherens junction, a tumor suppressor complex. When treated with retinoic acid (RA), the breast cancer cell line, SKBR3, undergoes differentiation and reduction in cell proliferation. The present work demonstrates that in SKBR3 cells, which exhibit high AP-1 activity, RA-regulation of cadherin expression and function, but not changes in AP-1 (or beta-catenin/TCF) signaling, is responsible for the epithelial differentiation. However, cadherin function and recruitment of beta-catenin to the membrane is not required for RA to regulate DNA synthesis in these cells. RA also reduces the activity of an AP-1 and TCF-sensitive cyclin D1 reporter in SKBR3 cells in a manner that is independent of the TCF site. In contrast, in SW480 cells, which have high levels of beta-catenin/TCF signaling, the activity and retinoid responsiveness of the cyclin D1 promoter was markedly inhibited by mutation of the TCF site. These data indicate that the remarkably broad effects of RA on the growth and differentiation of many different epithelial cancers may well be explained by the ability of RA to differentially regulate the activity of RAR/RXR, AP-1, and beta-catenin/TCF pathways.     

PCR-based assays for the fish pathogen Aeromonas salmonicida. I. Evaluation of three PCR primer sets for detection and identification

In an effort to develop a rapid diagnostic test for the fish pathogen Aeromonas salmonicida, the performance of 2 polymerase chain reaction (PCR) primer sets (AP and PAAS) targeting the fish pathogen A. salmonicida and 1 PCR primer set (MIY) targeting A. salmonicida subsp. salmonicida were evaluated. Initially, the PCR assays were used to screen purified DNA extracted from 308 A. salmonicida isolates. The AP and PAAS PCR tests were demonstrated to be 100% specific for the species A. salmonicida and did not cross-react with any of the non-target organisms (bacterial species other than A. salmonicida) used in this study. The combined sensitivity of the AP and PAAS tests was 99.4% and offered the best coverage in terms of identifying the target organism. The MIY PCR appeared to be 100% sensitive and specific for A. salmonicida subsp. salmonicida. Studies with tissues, spiked with known quantities of bacteria, were conducted to determine the lower detection limit of the PCR tests, and then the ability of these PCR tests to detect A. salmonicida in experimentally infected salmonids was assessed.     

Identification of a key residue in the conformational stability of acyl carrier protein

Conformational flexibility of acyl carrier protein (ACP) is important for its ability to interact with multiple enzymes in bacterial fatty acid metabolism. We have recently shown that, unlike the prototypical ACP from Escherichia coli, the more acidic Vibrio harveyi ACP is largely unfolded at physiological pH. Mutations D18K, A75H and A75H/D18K were made in recombinant V. harveyi ACP (rACP) to determine the importance of basic residues Lys-18 and His-75 in maintaining the native conformation of E. coli ACP. Both D18K and A75H ACPs were fatty acylated by acyl-ACP synthetase, showing that neither mutation grossly alters tertiary structure. Circular dichroism (CD) indicated that rACP refolded upon addition of MgCl(2) at 100-fold lower concentrations (<1 mM) than KCl, suggesting that divalent cations stabilize rACP by interaction at specific sites. Surprisingly, mutants A75H and A75H/D18K exhibited native-like conformation in the absence of MgCl(2), while the D18K mutant was comparable to rACP. Moreover, the alpha-helical content of A75H, A75H/D18K and E. coli ACPs was more sensitive than that of rACP or D18K ACP to modification by the histidine-selective reagent diethylpyrocarbonate. Together, these results suggest that the partial positive charge of His-75 may be important in maintaining the conformational stability of E. coli ACP at a neutral pH.     

Neurotrophin receptors and nerve growth factor are differentially expressed in adjacent nonneuronal cells of normal and injured tooth pulp

High-affinity tyrosine kinase A (trkA) neurotrophin receptors on neurons and nonneuronal cells elicit differentiation or survival functions in response to nerve growth factor (NGF), whereas the low-affinity neurotrophin (p75) receptor modulates trkA activity or can independently cause apoptosis or NFkappaB-mediated survival functions. We examined dental tissues for the presence of trkA-like immunoreactivity (trkA-IR), to determine which nonneuronal cell types express it in normal compared with inflamed teeth and how the trkA-positive cells relate to those expressing the p75 receptor and/or NGF. Normal and injured rat molars (dentin cavity for 4 h, 16-24 h, 3 days, 16 days, or 5 weeks) were immunoreacted using the ABC detection system for two anti-trkA antibodies (sTA, Santa Cruz Biotechnology; rTA, L. Reichardt) and antibodies against p75 and NGF, all of which also stained pulpal nerve fibers. We report that, when using the sTA antibody (recognizing the intracellular carboxy terminal), nonneuronal trkA-IR was found in odontoblasts of normal teeth and also in invading polymorphonuclear leukocytes (PMNs) and reparative odontoblasts after injury. When using rTA (recognizing the extracellular domain of the receptor), nonneuronal trkA-IR was only found in odontoblasts. Odontoblasts also had NGF-IR but did not label for NGF mRNA. The lack of odontoblast NGF mRNA suggests that NGF is passed from fibroblasts to the adjacent odontoblasts, where it is picked up by receptor-mediated mechanisms for regulation of odontoblast function. Tooth injury disrupts this system such that trkA-IR decreases in injured odontoblasts, p75 decreases in fibroblasts, and NGF is upregulated by fibroblasts and accumulates in the injured pulp and surviving odontoblasts. Pulpal NGF may contribute to chemoattraction for the invading leukocytes or their sTA-IR may have been induced in response to pulpal NGF. Thus, tooth pulp has a different distribution of nonneuronal NGF and its paracrine receptors during inflammation compared with normal conditions.     

Effects of body size and resource availability on dispersal in a native and a non-native estuarine snail

I manipulated snail densities of two coexisting species of salt marsh snail, Cerithidea californica Haldeman (native) and Batillaria attramentaria Sowerby (non-indigenous) to investigate how resource levels set by intraspecific competition may influence dispersal rates. I used two distinct size classes of the snails (mature and immature) to determine if the effects of competition on dispersal differed between developmental stages of a consumer. Dispersal attempts were measured within enclosure pens by counting snails climbing the sides of the enclosure. The influence of snail density per se and resource levels (which were set by snail densities) on dispersal rates were separated by comparing responses of snails to density before and after resources became depleted. For large snails of both species, dispersal increased as resource levels decreased, supporting the hypothesis that competition influences dispersal rates. Small snails of both species, in contrast, always dispersed at relatively higher rates than larger individuals, but were not influenced by variation in resource levels. This result corroborates other studies that have shown reduced competition in these species at smaller size, and suggests that another mechanism, such as genetically coded behavior to disperse when young, influences their behavior. Previous experiments demonstrated Batillaria's superior resource conversion efficiency; therefore, I had hypothesized that for any given resource level, Cerithidea would disperse more, because it was more affected by resource availability. Adult Batillaria, however, responded more sensitively to resource levels (i.e., dispersed more at any given resource level) than Cerithidea. This counter-intuitive result illustrates the potential importance of genetic limitations on behavioral responses available to a species. Constraints on behavioral responses may have been accentuated since Batillaria is a non-indigenous species whose evolved behavioral responses are not necessarily adapted to its present, non-native environment.     

Activation of phospholipase D by PKC and GTPgammaS in human neuroblastoma cells overexpressing MARCKS

Regulation of phospholipase D (PLD) activity participating in signal transduction involves complex interactions with small G-proteins (ARF, Rho) and protein kinase C isoforms (PKCalpha). In SK-N-MC human neuroblastoma cells, phorbol ester (TPA) activation of PLD was enhanced by overexpressing myristoylated alanine-rich C kinase substrate (MARCKS). To study MARCKS interactions with PLD, we investigated PLD isoform expression and activation by TPA and GTPgammaS in intact and digitonin-permeabilized clones transfected with MARCKS (M22). PLD2 was in both cytosol and membrane fractions while PLD1 was primarily membrane-associated in both vector control and M22 cells; location or quantities were unaltered by TPA treatment. TPA-stimulated PLD activity was higher in both intact and digitonin-permeabilized M22 cells than in vector controls. In contrast, GTPgammaS-stimulated PLD activity was independent of MARCKS expression but was additive with MARCKS-PKC-dependent activation in permeabilized cells. Combinations of PKC inhibition and down-regulation in intact and permeabilized (with GTPgammaS present) cells indicated that a PKC-mediated phosphorylation event was necessary in intact cells without access to GTPgammaS, stimulation of PLD mediated by GTPgammaS was independent of PKC, and PLD activation by PKC in permeabilized cells was kinase-independent. Western blot analysis showed that MARCKS, PKCalpha, PLD1 and PLD2 were present in a detergent-insoluble fraction (DIF); GTPgammaS increased recovery of PLD2 in DIF. Disruption of cholesterol-rich DIFs with digitonin, cyclodextrin or filipin potentiated activation of PLD by TPA. Our studies suggest that activation of PLD by PKC requires MARCKS and can involve both phosphorylation-independent and -dependent processes. As PLD activation by GTPgammaS is PKC-MARCKS-independent, MARCKS may provide a fine tuning component in conjunction with G-protein-mediated mechanisms for regulation of PLD.     

The Nova Scotia (type D) form of Niemann-Pick disease is caused by a G3097-->T transversion in NPC1.

Niemann-Pick type D (NPD) disease is a progressive neurodegenerative disorder characterized by the accumulation of tissue cholesterol and sphingomyelin. This disorder is relatively common in southwestern Nova Scotia, because of a founder effect. Our previous studies, using classic linkage analysis of this large extended kindred, defined the critical gene region to a 13-cM chromosome segment between D18S40 and D18S66. A recently isolated gene from this region, NPC1, is mutated in the majority of patients with Niemann-Pick type C disease. We have identified a point mutation within this gene (G3097-->T; Gly992-->Trp) that shows complete linkage disequilibrium with NPD, confirming that NPD is an allelic variant of NPC1.     

Genetic components of variation in Nemophila menziesii undergoing inbreeding: morphology and flowering time.

The standard approaches to estimation of quantitative genetic parameters and prediction of response to selection on quantitative traits are based on theory derived for populations undergoing random mating. Many studies demonstrate, however, that mating systems in natural populations often involve inbreeding in various degrees (i.e. , self matings and matings between relatives). Here we apply theory developed for estimating quantitative genetic parameters for partially inbreeding populations to a population of Nemophila menziesii recently obtained from nature and experimentally inbred. Two measures of overall plant size and two of floral size expressed highly significant inbreeding depression. Of three dominance components of phenotypic variance that are defined under partial inbreeding, one was found to contribute significantly to phenotypic variance in flower size and flowering time, while the remaining two components contributed only negligibly to variation in each of the five traits considered. Computer simulations investigating selection response under the more complete genetic model for populations undergoing mixed mating indicate that, for parameter values estimated in this study, selection response can be substantially slowed relative to predictions for a random mating population. Moreover, inbreeding depression alone does not generally account for the reduction in selection response.