Macromolecular organization of collagen fibres in natural and tanned basement membrane

The elastic constants and ultrastructure of natural and tanned basement membrane of the crystalline lens of the adult rat have been investigated. Sonicated and negatively stained specimens of both membranes show parallel filaments that have similar spacing of 3.5(+/- 0.1) nm and a different periodicity. In natural membrane the periodicity is 3.7(+/- 0.13) nm, whilst in tanned basement membrane the periodicity is 3.2(+/- 0.15) nm. The periodicity ratio of tanned membrane to natural membrane was 0.86 +/- 0.04, whilst the elongation ratio of tanned membrane compared with natural membrane was 0.88 +/- 0.05. In contrast to this, the thickness ratio of tanned to natural membrane was 1.098 +/- 0.045. Tanned basement membrane showed a shrinkage of 12% in length but an increase in thickness of about 10%. These data suggest, firstly, that the degree of extension of the superhelices of the filaments follows closely the degree of extension of the intact membrane and, secondly, that the coiled superhelices of tanned membrane have an angle of tilt of about 42 degrees compared with those of natural membrane, where the angle is about 50 degrees. The Young's modulus of elasticity and ultimate stress of tanned basement membrane are, respectively, eight times greater and one-third as great as natural membrane. The entropy change in basement membrane was calculated from the external work necessary to extend the tanned membrane, and was estimated to be -13.5(+/- 2.4) J K-1 mol-1. An estimate of the change in entropy from thermodynamic measurements made on a suspension of collagen tanned with glutaraldehyde was found to be -30.1(+/- 9.5) J K-1 mol-1. The two different estimates of the change in entropy of collagen following tanning suggest that in basement membrane only about 45% of the collagenous protein has an extensile helical structure.     

Calreticulin, PDI, Grp94 and BiP chaperone proteins are associated with retained COMP in pseudoachondroplasia chondrocytes.

Cartilage oligomeric matrix protein (COMP), a large pentameric glycoprotein and member of the thrombospondin (TSP) group of extracellular proteins, is found in the territorial matrix surrounding chondrocytes. More than 50 unique COMP mutations have been identified as causing two skeletal dysplasias: pseudoachondroplasia (PSACH); and multiple epiphyseal dysplasia (EDM1). Recent studies suggest that calcium-binding and calcium-induced protein folding differ between wild type and mutant proteins, and abnormal processing of the mutant COMP protein contributes to the characteristic enlarged lamellar appearing rER cisternae in PSACH and EDMI chondrocytes in vivo and in vitro. Towards the goal of delineating the pathogenesis of PSACH and EDM1, in-vivo PSACH growth plate and in-vitro PSACH chondrocytes cultured in alginate beads were examined to identify and localize the chaperone proteins participating in the processing of the retained extracellular matrix proteins in the PSACH rER. Aggrecan was localized to both the rER cisternae and matrix while COMP and type IX collagen were only found in the rER. Type II collagen was solely found in the ECM suggesting that it is processed and transported differently from other retained ECM proteins. Five chaperone proteins: BiP (Grp78); calreticulin (CRT); protein disulfide (PDI); ERp72; and Grp94, demonstrated immunoreactivity in the enlarged PSACH cisternae and the short rER channels of chondrocytes from both in-vivo and in-vitro samples. The chaperone proteins cluster around the electron dense material within the enlarged rER cisternae. CRT, PDI and GRP94 AB-gold particles appear to be closely associated with COMP. Immunoprecipitation and Western blot, and Fluorescence Resonance Energy Transfer (FRET) analyses indicate that CRT, PDI and GRP94 are in close proximity to normal and mutant COMP and BiP to mutant COMP. These results suggest that these proteins play a role in the processing and transport of wild type COMP in normal chondrocytes and in the retention of mutant COMP in PSACH chondrocytes.     

Population status,habitat dependence,and reproductive ecology of Bahama Orioles: a critically endangered synanthropic species

ABSTRACT Recent elevation of critically endangered Bahama Orioles (Icterus northropi) to species status prompted us to evaluate their population status, habitat use, and breeding ecology. From surveys, we estimated that at least 141 to 254 individuals remain globally, with 90 to 162, 24 to 44, and 27 to 48 individuals remaining on North Andros Island, Mangrove Cay, and South Andros Island, The Bahamas, respectively. Orioles were observed nesting exclusively in anthropogenic habitat (residential and agricultural land), but home ranges also included nearby pine forest and coppice (dry broadleaf forest). Most nests (40 of 46, or 87%) were in nonnative coconut palm (Cocos nucifera), with native Sabal palmetto and Thrinax morrisii, and an introduced Brassaia actinophylla also used. Trees selected by orioles for nesting were significantly taller, less likely to have shrubs underneath, further from cover, and had more palm trees nearby than randomly selected palm trees. Three of eight nests with known contents were parasitized by Shiny Cowbirds (Molothrus bonariensis). Lethal yellowing disease recently devastated coconut palms and reduced the number of orioles on North Andros, but palms on Mangrove Cay and South Andros remain healthy. The juxtaposition of anthropogenic habitat to suitable native habitats may be more important than any single factor for Bahama Orioles, especially for breeding adults and fledged young. Conservation of coppice habitat, at high risk for agricultural and residential development, is crucial for survival of this critically endangered synanthropic species.     

Canine cryptorchism and subsequent testicular neoplasia: case-control study with epidemiologic update

A retrospective study of 2,912 cryptorchid dogs identified 14 breeds with significantly high risk. Among six distinct closely interrelated breed groups (e.g., toy, miniature, and standard poodles), the risk in the smaller breed was always greater than that in the larger relative, suggesting that genetically influenced maldescent could be, in part, related to physical size or the rate of growth of the involved structures. Testicular tumors were diagnosed in 5.7% of the cryptorchid dogs; half had only Sertoli cell tumors, one-third had only seminomas. The relative risk for Sertoli cell tumor or seminoma was not directly related to a familial risk for cryptorchism. Using the health experience of a control population composed of male dogs with anal sac disease (N = 4,184), there is an estimated relative risk of 9.2 in cryptorchid dogs to develop a testis tumor (95% confidence interval, 5.9-14.3) and 4.2 in dogs with inguinal hernia (95% confidence interval, 1.8-9.5). Considering that the anatomical development of the genital tract, testis descent, and tunic relationships in dog are very similar to that in man, and that the associations of cryptorchism and inguinal hernia with testis neoplasms are also similar, the dog should be an excellent model system to further investigate the causes of human cryptorchism.     

Identification of two mosquitocidal Bacillus cereus strains showing different host ranges

Mosquitocidal bacteria, M413 and C32 have been isolated from sediment samples collected from woodland and ditch, respectively. Gas chromatographic analysis of fatty acids methyl esters (GC-FAME) and 16S rRNA gene sequence alignment results showed these isolates belong to Bacillus cereus. The SDS-PAGE analysis of sporulated cultures of both isolates showed two major bands very similar in size. Interestingly, however, M413 is mainly toxic to 4th instars of Ochlerotatus taeniorhynchus whereas C32 is to those of Culex quinquefasciatus.     

The USDA Barley Core Collection: Genetic Diversity,Population Structure,and Potential for Genome-Wide Association Studies

New sources of genetic diversity must be incorporated into plant breeding programs if they are to continue increasing grain yield and quality, and tolerance to abiotic and biotic stresses. Germplasm collections provide a source of genetic and phenotypic diversity, but characterization of these resources is required to increase their utility for breeding programs. We used a barley SNP iSelect platform with 7,842 SNPs to genotype 2,417 barley accessions sampled from the USDA National Small Grains Collection of 33,176 accessions. Most of the accessions in this core collection are categorized as landraces or cultivars/breeding lines and were obtained from more than 100 countries. Both STRUCTURE and principal component analysis identified five major subpopulations within the core collection, mainly differentiated by geographical origin and spike row number (an inflorescence architecture trait). Different patterns of linkage disequilibrium (LD) were found across the barley genome and many regions of high LD contained traits involved in domestication and breeding selection. The genotype data were used to define ‘mini-core’ sets of accessions capturing the majority of the allelic diversity present in the core collection. These ‘mini-core’ sets can be used for evaluating traits that are difficult or expensive to score. Genome-wide association studies (GWAS) of ‘hull cover’, ‘spike row number’, and ‘heading date’ demonstrate the utility of the core collection for locating genetic factors determining important phenotypes. The GWAS results were referenced to a new barley consensus map containing 5,665 SNPs. Our results demonstrate that GWAS and high-density SNP genotyping are effective tools for plant breeders interested in accessing genetic diversity in large germplasm collections.     

Measurement of cell proliferation by heavy water labeling

DNA replication occurs almost exclusively during S-phase of the cell cycle and represents a simple biochemical metric of cell division. Previous methods for measuring cell proliferation rates have important limitations. Here, we describe experimental protocols for measuring cell proliferation and death rates based on the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells. Label incorporation is measured by gas chromatography/mass spectrometry. Modifications of the basic protocol permit analysis of small cell samples (down to 2,000 cells). The theoretical basis and operational requirements for effective use of these methods to measure proliferation and death rates of cells in vivo are described. These methods are safe for use in humans, have technical and interpretation advantages over alternative techniques and can be used on small numbers of cells. The protocols enable definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans.     

Cold-stage scanning electron microscope measurement of ice morphology in apple tissue as a function of freezing rate

A cold-stage SEM was used to document the morphology of ice in apple tissue and a sucrose solution frozen at rates ranging from 450 to 0.03 degrees K/min. Freezing rates of 3-mm-thick apple discs were measured with a differential thermocouple technique, which gave measurement of the growth velocity and the temperature gradient through the solidified specimen as well as the cooling rate during solidification. Cold-stage SEM micrographs were used to measure the dendritic spacing of the ice structures, and these data were found to correlate linearly with the square root of the cooling rate during solidification as would be predicted by a theoretical analysis of mass transfer in the formation of dendrites. Comparison of freeze-substituted and freeze-dried apple-tissue micrographs with those from a cold-stage SEM showed that the cold-stage SEM technique was the only one which correctly represented ice morphology in apple tissue.