Mutations in Lama1 Disrupt Retinal Vascular Development and Inner Limiting Membrane Formation

The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin α1, a basement membrane protein. Despite normal localization of laminin α1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial two-hybrid system. Therefore, this mutation could affect interactions between laminin α1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1^tm1.1Olf, was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.     

The developmental competence of bovine nuclear transfer embryos derived from cow versus heifer cytoplasts

Due to its economic importance, the production of cattle by nuclear transfer has been a primary research focus for many researchers during the past few years. While many groups have successfully produced cattle by nuclear transfer, and progress in this area continues, nuclear transfer remains a very inefficient technology. This study evaluates the effect of the oocyte source (cow and heifer) on the developmental competence of nuclear transfer embryos. In order for nuclear transfer to be successful, a differentiated donor cell must be reprogrammed and restored to a totipotent state. This reprogramming is probably accomplished by factors within the oocyte cytoplasm. This study indicates that oocytes derived from cows have a greater capacity to reprogram donor cell DNA following nuclear transfer as compared to heifer oocytes based on in vitro development to the 2-cell stage and to the compacted morula/blastocyst stages. Nuclear transfer embryos derived from cow oocytes resulted in significantly higher rates of pregnancy establishment than embryos derived from heifer oocytes and resulted in higher pregnancy retention at 90 and 180 days and a greater number of term deliveries. Following delivery more calves derived from cow oocytes tended to be healthy and normal than those derived from heifer oocytes. The differences in developmental efficiency between nuclear transfer embryos derived from cow and heifer cytoplasts demonstrate that subtle differences in oocyte biology can have significant effects on subsequent development of nuclear transfer embryos.     

PROBER: oligonucleotide FISH probe design software

PROBER is an oligonucleotide primer design software application that designs multiple primer pairs for generating PCR probes useful for fluorescence in situ hybridization (FISH). PROBER generates Tiling Oligonucleotide Probes (TOPs) by masking repetitive genomic sequences and delineating essentially unique regions that can be amplified to yield small (100-2000 bp) DNA probes that in aggregate will generate a single, strong fluorescent signal for regions as small as a single gene. TOPs are an alternative to bacterial artificial chromosomes (BACs) that are commonly used for FISH but may be unstable, unavailable, chimeric, or non-specific to small (10-100 kb) genomic regions. PROBER can be applied to any genomic locus, with the limitation that the locus must contain at least 10 kb of essentially unique blocks. To test the software, we designed a number of probes for genomic amplifications and hemizygous deletions that were initially detected by Representational Oligonucleotide Microarray Analysis of breast cancer tumors. AVAILABILITY: http://prober.cshl.edu     

Text similarity: an alternative way to search MEDLINE

MOTIVATION: The most widely used literature search techniques, such as those offered by NCBI's PubMed system, require significant effort on the part of the searcher, and inexperienced searchers do not use these systems as effectively as experienced users. Improved literature search engines can save researchers time and effort by making it easier to locate the most important and relevant literature. RESULTS: We have created and optimized a new, hybrid search system for Medline that takes natural text as input and then delivers results with high precision and recall. The combination of a fast, low-sensitivity weighted keyword-based first pass algorithm to cast a wide net to gather an initial set of literature, followed by a unique sentence-alignment based similarity algorithm to rank order those results was developed that is sensitive, fast and easy to use. Several text similarity search algorithms, both standard and novel, were implemented and tested in order to determine which obtained the best results in information retrieval exercises. AVAILABILITY: Literature searching algorithms are implemented in a system called eTBLAST, freely accessible over the web at http://invention.swmed.edu. A variety of other derivative systems and visualization tools provides the user with an enhanced experience and additional capabilities. CONTACT: Harold.Garner@UTSouthwestern.edu.     

CRTAP is required for prolyl 3- hydroxylation and mutations cause recessive osteogenesis imperfecta

Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.     

Presence and growth of naturalized Escherichia coli in temperate soils from Lake Superior watersheds

The presence of Escherichia coli in water is used as an indicator of fecal contamination, but recent reports indicate that soil populations can also be detected in tropical, subtropical, and some temperate environments. In this study, we report that viable E. coli populations were repeatedly isolated from northern temperate soils in three Lake Superior watersheds from October 2003 to October 2004. Seasonal variation in the population density of soilborne E. coli was observed; the greatest cell densities, up to 3 x 10(3) CFU/g soil, were found in the summer to fall (June to October), and the lowest numbers, < or =1 CFU/g soil, occurred during the winter to spring months (February to May). Horizontal, fluorophore-enhanced repetitive extragenic palindromic PCR (HFERP) DNA fingerprint analyses indicated that identical soilborne E. coli genotypes, those with > or =92% similarity values, overwintered in frozen soil and were present over time. Soilborne E. coli strains had HFERP DNA fingerprints that were unique to specific soils and locations, suggesting that these E. coli strains became naturalized, autochthonous members of the soil microbial community. In laboratory studies, naturalized E. coli strains had the ability to grow and replicate to high cell densities, up to 4.2 x 10(5) CFU/g soil, in nonsterile soils when incubated at 30 or 37 degrees C and survived longer than 1 month when soil temperatures were < or =25 degrees C. To our knowledge, this is the first report of the growth of naturalized E. coli in nonsterile, nonamended soils. The presence of significant populations of naturalized populations of E. coli in temperate soils may confound the use of this bacterium as an indicator of fecal contamination.     

Golgin-160 is required for the Golgi membrane sorting of the insulin-responsive glucose transporter GLUT4 in adipocytes

The peripheral Golgi protein golgin-160 is induced during 3T3L1 adipogenesis and is primarily localized to the Golgi cisternae distinct from the trans-Golgi network (TGN) in a general distribution similar to p115. Small interfering RNA (siRNA)-mediated reduction in golgin-160 protein resulted in an increase accumulation of the insulin-responsive amino peptidase (IRAP) and the insulin-regulated glucose transporter (GLUT4) at the plasma membrane concomitant with enhanced glucose uptake in the basal state. The redistribution of GLUT4 was rescued by expression of a siRNA-resistant golgin-160 cDNA. The basal state accumulation of plasma membrane GLUT4 occurred due to an increased rate of exocytosis without any significant effect on the rate of endocytosis. This GLUT4 trafficking to the plasma membrane in the absence of golgin-160 was independent of TGN/Golgi sorting, because it was no longer inhibited by the expression of a dominant-interfering Golgi-localized, gamma-ear-containing ARF-binding protein mutant and displayed reduced binding to the lectin wheat germ agglutinin. Moreover, expression of the amino terminal head domain (amino acids 1-393) had no significant effect on the distribution or insulin-regulated trafficking of GLUT4 or IRAP. In contrast, expression of carboxyl alpha helical region (393-1498) inhibited insulin-stimulated GLUT4 and IRAP translocation, but it had no effect on the sorting of constitutive membrane trafficking proteins, the transferrin receptor, or vesicular stomatitis virus G protein. Together, these data demonstrate that golgin-160 plays an important role in directing insulin-regulated trafficking proteins toward the insulin-responsive compartment in adipocytes.     

Recovery from an activity-induced metabolic acidosis in the American alligator, Alligator mississippiensis

The metabolic acidosis resulting from an intense exercise bout is large in crocodilians. Here we studied recovery from this pH perturbation in the American alligator. Metabolic rate, minute ventilation, arterial pH and gases, and strong ion concentration were measured for 10 h after exhaustion to elucidate the mechanisms and time course of recovery. Exhaustion resulted in a significant increase in lactate, metabolic rate, and ventilation, and a decrease in arterial PCO2), pH and bicarbonate. By 15 min after exhaustion, oxygen consumption returned to rest though carbon dioxide excretion remained elevated for 30 min. Arterial PO2), [Na+], and [K+], increased following exhaustion and recovered by 30 min post-exercise. Minute ventilation, tidal volume, [Cl-], and respiratory exchange ratio returned to resting values by 1 h. The air convection requirement for oxygen was elevated between 15 and 60 min of recovery. Breathing frequency and pH returned to resting values by 2 h of recovery. Lactate levels remained elevated until 6 h post-exercise. Arterial PCO2) and [HCO3-] were depressed until 8 h post-exercise. Compensation during recovery of acid-base balance was achieved by altering ventilation: following the initial metabolic acidosis and titration of bicarbonate, a relative hyperventilation prevented a further decrease in pH.     

REV1 and polymerase ζ facilitate homologous recombination repair

REV1 and DNA Polymerase ζ (REV3 and REV7) play important roles in translesion DNA synthesis (TLS) in which DNA replication bypasses blocking lesions. REV1 and Polζ have also been implicated in promoting repair of DNA double-stranded breaks (DSBs). However, the mechanism by which these two TLS polymerases increase tolerance to DSBs is poorly understood. Here we demonstrate that full-length human REV1, REV3 and REV7 interact in vivo (as determined by co-immunoprecipitation studies) and together, promote homologous recombination repair. Cells lacking REV3 were hypersensitive to agents that cause DSBs including the PARP inhibitor, olaparib. REV1, REV3 or REV7-depleted cells displayed increased chromosomal aberrations, residual DSBs and sites of HR repair following exposure to ionizing radiation. Notably, cells depleted of DNA polymerase η (Polη) or the E3 ubiquitin ligase RAD18 were proficient in DSB repair following exposure to IR indicating that Polη-dependent lesion bypass or RAD18-dependent monoubiquitination of PCNA are not necessary to promote REV1 and Polζ-dependent DNA repair. Thus, the REV1/Polζ complex maintains genomic stability by directly participating in DSB repair in addition to the canonical TLS pathway.     

Susceptibility to infection and pathogenicity of White Spot Disease (WSD) in non-model crustacean host taxa from temperate regions

Despite almost two decades since its discovery, White Spot Disease (WSD) caused by White Spot Syndrome Virus (WSSV) is still considered the most significant known pathogen impacting the sustainability and growth of the global penaeid shrimp farming industry. Although most commonly associated with penaeid shrimp farmed in tropical regions, the virus is also able to infect, cause disease and kill a wide range of other decapod crustacean hosts from temperate regions, including lobsters, crabs, crayfish and shrimp. For this reason, WSSV has recently been listed in European Community Council Directive 2006/88. Using principles laid down by the European Food Safety Authority (EFSA) we applied an array of diagnostic approaches to provide a definitive statement on the susceptibility to White Spot Syndrome Virus (WSSV) infection in seven ecologically or economically important crustacean species from Europe. We chose four marine species: Cancer pagurus, Homarus gammarus, Nephrops norvegicus and Carcinus maenas; one estuarine species, Eriocheir sinensis and two freshwater species, Austropotamobius pallipes and Pacifastacus leniusculus. Exposure trials based upon natural (feeding) and artificial (intra-muscular injection) routes of exposure to WSSV revealed universal susceptibility to WSSV infection in these hosts. However, the relative degree of susceptibility (measured by progression of infection to disease, and mortality) varied significantly between host species. In some instances (Type 1 hosts), pathogenesis mimicked that observed in penaeid shrimp hosts whereas in other examples (Types 2 and 3 hosts), infection did not readily progress to disease, even though hosts were considered as infected and susceptible according to accepted principles. Results arising from challenge studies are discussed in relation to the potential risk posed to non-target hosts by the inadvertent introduction of WSSV to European waters via trade. Furthermore, we highlight the potential for susceptible but relatively resistant hosts to serve as models to investigate natural mitigation strategies against WSSV in these hosts. We speculate that these non-model hosts may offer a unique insight into viral handling in crustaceans.