Variation in the ovarian reserve Is linked to alterations in intrafollicular estradiol production and ovarian biomarkers of follicular differentiation and oocyte quality in cattle

The mechanisms whereby the high variation in numbers of morphologically healthy oocytes and follicles in ovaries (ovarian reserve) may have an impact onovarian function, oocyte quality, and fertility are poorly understood. The objective was to determine whether previously validated biomarkers for follicular differentiation and function, as well as oocyte quality differed between cattle with low versus a high antral follicle count (AFC). Ovaries were removed (n = 5 per group) near the beginning of the nonovulatory follicular wave, before follicles could be identified via ultrasonography as being dominant, from heifers with high versus a low AFC. The F1, F2, and F3 follicles were dissected and diameters determined. Follicular fluid and thecal, granulosal, and cumulus cells and the oocyte were isolated and subjected to biomarker analyses. Although the size and numerous biomarkers of differentiation, such as mRNAs for the gonadotropin receptors, were similar, intrafollicular concentrations of estradiol and the abundance of mRNAs for CYP19A1 in granulosal cells and ESR1, ESR2, and CTSB in cumulus cells were greater, whereas mRNAs for AMH in granulosal cells and TBC1D1 in thecal cells were lower for animals with low versus a high AFC during follicle waves. Hence, variation in the ovarian reserve may have an impact on follicular function and oocyte quality via alterations in intrafollicular estradiol production and expression of key genes involved in follicle-stimulating hormone action (AMH) and estradiol (CYP19A1) production by granulosal cells, function and survival of thecal cells (TBC1D1), responsiveness of cumulus cells to estradiol (ESR1, ESR2), and cumulus cell determinants of oocyte quality (CTSB).

A contribution to the differentiation between nectar honey and honeydew honey

Thirty genuine honey samples were analyzed for pH, acidity, water, ash, net absorbance, total polyphenols (Folin-Ciocalteau method) and glucose, fructose, melezitose and erlose (as their trimethylsilyl oximes and trimethylsilyl ethers) by capillary gas chromatography. The resulting data were used, along with palynological analysis, to characterize the samples in relation to their possible source (nectar, honeydew and mixture honeys). Some minor components (carboxylic acids and cyclitols), eluting before monosaccharides, were also determined. One of these compounds was quercitol (1,3,4/2,5-cyclohexane-pentol), a deoxyinositol which has been previously determined in Quercus sp. samples. Quercitol was present in a broad concentration range (0.01-1.50 g/100 g) in honeys whose major source was honeydew but it was never higher than 0.01 g/100 g in samples characterized as nectar honeys. Quercitol concentrations appear to be related to the presence and amount of Quercus sp. honeydew as honey source, although further research is required to confirm this. (C) 2004 Elsevier Ltd. All rights reserved.

A novel technique for differentiation of proteins in the development of acid gel structure from control and heat treated milk using confocal scanning laser microscopy

The incorporation of caseins and whey proteins into acid gels produced from unheated and heat treated skimmed milk was studied by confocal scanning laser microscopy (CSLM) using fluorescent labelled proteins. Bovine casein micelles were labelled using Alexa Fluor 594, while whey proteins were labelled using Alexa Fluor 488. Samples of the labelled protein solutions were introduced into aliquots of pasteurised skim milk, and skim milk heated to 90 degrees C for 2 min and 95 degrees C for 8 min. The milk was acidified at 40 degrees C to a final pH of 4.4 using 20 g gluconodelta-lactone/l (GDL). The formation of gels was observed with CSLM at two wavelengths (488 nm and 594 nm), and also by visual and rheological methods. In the control milk, as pH decreased distinct casein aggregates appeared, and as further pH reduction occurred, the whey proteins could be seen to coat the casein aggregates. With the heated milks, the gel structure was formed of continuous strands consisting of both casein and whey protein. The formation of the gel network was correlated with an increase in the elastic modulus for all three treatments, in relation to the severity of heat treatment. This model system allows the separate observation of the caseins and whey proteins, and the study of the interactions between the two protein fractions during the formation of the acid gel structure, on a real-time basis. The system could therefore be a valuable tool in the study of structure formation in yoghurt and other dairy protein systems.

A role for Notch signaling in human corneal epithelial cell differentiation and proliferation

PURPOSE. To identify the role of Notch signaling in the human corneal epithelium. METHODS. Localization of Notch1, Notch2, Delta1, and Jagged1 in the human corneal epithelium was observed with the use of indirect immunofluorescence microscopy. Gene and protein expression of Notch receptors and ligands in human corneal epithelial cells was determined by RT-PCR and Western blot analysis, respectively. The effects of Notch inhibition (by {gamma}-secretase inhibition) and activation (by recombinant Jagged1) on epithelial cell proliferation (Ki67) and differentiation (CK3) were analyzed after Western blotting and immunocytochemistry. RESULTS. Immunofluorescent labeling localized Notch1 and Notch2 to suprabasal epithelial cell layers, whereas Delta1 and Jagged1 were observed throughout the corneal epithelium. Notch1, Notch2, Delta1, and Jagged1 genes and proteins were expressed in human corneal epithelial cells. {gamma}-Secretase inhibition resulted in decreased Notch1 and Notch2 expression, with an accompanying decrease in Ki67 and increased CK3 expression. The activation of Notch by Jagged1 resulted in the upregulation of active forms of Notch1 and 2 proteins (P < 0.05), with a concurrent increase in Ki67 (P < 0.05) and a decrease in CK3 (P < 0.05) expression. Interestingly, {gamma}-secretase inhibition in a three-dimensional, stratified corneal epithelium equivalent had no effect on Ki67 or CK3 expression. In contrast, Jagged1 activation resulted in decreased CK3 expression (P < 0.05), though neither Notch activation nor inhibition affected cell proliferation in the 3D tissue equivalent. CONCLUSIONS. Notch family members and ligands are expressed in the human corneal epithelium and appear to play pivotal roles in corneal epithelial cell differentiation.

Genetic differentiation between hosts and locations in populations of latent Botrytis cinerea in southern England

This study tested the hypothesis that aggressive, localized infections and asymptomatic systemic infections were caused by distinct specialized groups of Botrytis cinerea, using microsatellite genotypes at nine loci of 243 isolates of B. cinerea obtained from four hosts (strawberry (Fragaria ´ananassa), blackberry (Rubus fruticosus agg.), dandelion, (Taraxacum of®- cinale agg.) and primrose (Primula vulgaris)) in three regions in southern England (in the vicinities of Brighton, Reading and Bath). The populations were extremely variable, with up to 20 alleles per locus and high genic diversity. Each host in each region had a population of B. cinerea with distinctive genetic features, and there were also consistent host and regional distinctions. The B. cinerea population from strawberry was distinguished from that on other hosts, including blackberry, most notably by a common 154-bp amplicon at locus 5 (present in 35 of 77 samples) that was rare in isolates from other hosts (9¤166), and by the rarity (3¤77) of a 112-bp allele at locus 7 that was common (58¤166) in isolates from other hosts. There was signi®cant linkage disequilibrium overall within the B. cinerea populations on blackberry and strawberry, but with quite different patterns of association among isolates from the two hosts. No evidence was found for differentiation between populations of B. cinerea from systemically infected hosts and those from locally infected fruits.

TRIM32 regulates skeletal muscle stem cell differentiation and is necessary for normal adult muscle regeneration

Limb girdle muscular dystrophy type 2H (LGMD2H) is an inherited autosomal recessive disease of skeletal muscle caused by a mutation in the TRIM32 gene. Currently its pathogenesis is entirely unclear. Typically the regeneration process of adult skeletal muscle during growth or following injury is controlled by a tissue specific stem cell population termed satellite cells. Given that TRIM32 regulates the fate of mammalian neural progenitor cells through controlling their differentiation, we asked whether TRIM32 could also be essential for the regulation of myogenic stem cells. Here we demonstrate for the first time that TRIM32 is expressed in the skeletal muscle stem cell lineage of adult mice, and that in the absence of TRIM32, myogenic differentiation is disrupted. Moreover, we show that the ubiquitin ligase TRIM32 controls this process through the regulation of c-Myc, a similar mechanism to that previously observed in neural progenitors. Importantly we show that loss of TRIM32 function induces a LGMD2H-like phenotype and strongly affects muscle regeneration in vivo. Our studies implicate that the loss of TRIM32 results in dysfunctional muscle stem cells which could contribute to the development of LGMD2H.

Expression of the collagen receptor glycoprotein VI during megakaryocyte differentiation

This study examined the expression of the platelet collagen receptor glycoprotein VI (GPVI) in megakaryocyte cell lines and primary megakaryocytes by reverse transcriptase-polymerase chain reaction and by flow cytometry and ligand blotting using the snake venom toxin convulxin. Expression of GPVI is increased in the megakaryoblastic cell lines HEL and CMK on differentiation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), along with the Fc receptor gamma-chain (FcR gamma-chain). The increase in GPVI expression is associated with marked potentiation of tyrosine phosphorylation and Ca(++) elevation in response to convulxin. Syk, linker for activated T cells, and phospholipase C gamma 2 (PLC gamma 2) are among the proteins tyrosine phosphorylated on convulxin stimulation in PMA-differentiated HEL cells. Studies on primary murine megakaryocytes grown in vitro confirmed that GPVI is up-regulated in parallel with functional activation, assessed by measurement of [Ca(++)](i), during differentiation. The results demonstrate that expression of GPVI is up-regulated along with the FcR gamma-chain during differentiation of megakaryocytes. (Blood. 2000;96:2740-2745)

Vision of the body and the differentiation of perceived body side in touch

Although tactile representations of the two body sides are initially segregated into opposite hemispheres of the brain, behavioural interactions between body sides exist and can be revealed under conditions of tactile double simultaneous stimulation (DSS) at the hands. Here we examined to what extent vision can affect body side segregation in touch. To this aim, we changed hand-related visual input while participants performed a go/no-go task to detect a tactile stimulus delivered to one target finger (e.g., right index), stimulated alone or with a concurrent non-target finger either on the same hand (e.g., right middle finger) or on the other hand (e.g., left index finger = homologous; left middle finger = non-homologous). Across experiments, the two hands were visible or occluded from view (Experiment 1), images of the two hands were either merged using a morphing technique (Experiment 2), or were shown in a compatible vs incompatible position with respect to the actual posture (Experiment 3). Overall, the results showed reliable interference effects of DSS, as compared to target-only stimulation. This interference varied as a function of which non-target finger was stimulated, and emerged both within and between hands. These results imply that the competition between tactile events is not clearly segregated across body sides. Crucially, non-informative vision of the hand affected overall tactile performance only when a visual/proprioceptive conflict was present, while neither congruent nor morphed hand vision affected tactile DSS interference. This suggests that DSS operates at a tactile processing stage in which interactions between body sides can occur regardless of the available visual input from the body.

Restriction fragment length polymorphism of polymerase chain reaction products applied to the differentiation of poultry campylobacters for epidemiological investigations

A technique for subtyping Camplobacter jejuni isolates has been developed by using the restriction fragment length polymorphism (Rnp) of polymerase chain reaction (PCR) products of the fluA and flaB genes. The technique was validated by using strains representing 28 serotypes of C jejuni and it may also be applied to C coli. From these strains 12 distinct RFLP profiles were observed but there was no direct relationship between the RFLP profile and the serotype. One hundred and thirty-five campylobacter isolates from 15 geographically distinct broiler flocks were investigated. All the isolates could be subtyped by using the RFLP method. Isolates from most of the flocks had a single RFLP profile despite data indicating that several serotypes were involved. Although it is possible that further restriction analysis may have demonstrated profile variations in these strains, it is more likely that antigenic variation can occur within genotypically related campylobacters. As a result, serotyping may give conflicting information for veterinary epidemiological purposes. This RFLP typing scheme appears to provide a suitable tool for the investigation of the sources and routes of transmission of campylobacters in chickens.

The development of a ligase mediated PCR with potential for the differentiation of serovars within Leptospira interrogans

A ligase mediated polymerase chain reaction (LMPCR) was developed to amplify between the repetitive element, IS1533, of Leptospira and adjacent chromosomally located BglII restriction endonuclease enzyme sites. To do this, complimentary oligonucleotide linkers designed to anneal together with an overhanging BglII end were ligated to BglII digested DNA from 35 leptospiral reference strains and field isolates, This ligated DNA was used as template for PCR with oligonucleotide primers specific for the linker and for the repetitive element IS1533. The resultant amplicon profile hybridised a 102 hp region derived from the terminus of IS1533 thus confirming that amplicons generated by LMPCR contained part of IS1533. The number of fragments generated containing IS1533 was significantly fewer than that generated by RFLP but the LMPCR method has the potential to use far less template DNA and be quicker than standard RFLP. Obvious and reproducible interserovar differences were demonstrated by LMPCR whereas for 20 of 21 L. hardjo-bovis isolates tested no intraserovar differences were observed. Of those serovars known to possess IS1533 homologues and tested here by LMPCR, each produced a unique amplicon profile which hybridised the IS1533 terminus probe. The limited heterogeneity amongst hardjo-bovis isolates is discussed as is the potential contribution of this method to diagnosis, differentiation and the phylogenetics of the Leptospires.

Tax principles, product differentiation and the nature of competition

We analyze the choice between the origin and destination principles of taxation when there is product differentiation and Bertrand competition. If taxes are redistributed to consumers and demand is linear the origin principle dominates the destination principle whatever the degree of product differentiation and extent of economic integration. With nonlinear demand the origin principle dominates if there is sufficient economic integration. When the social value assigned to tax revenue is higher than the private value, the destination principle dominates for intermediate values of product differentiation and economic integration. The same results are also shown to hold with Cournot competition.

Differentiation of neotropical ecosystems by modern soil phytolith assemblages and its implications for palaeoenvironmental and archaeological reconstructions

The interpretation of Neotropical fossil phytolith assemblages for palaeoenvironmental and archaeological reconstructions relies on the development of appropriate modern analogues. We analyzed modern phytolith assemblages from the soils of ten distinctive tropical vegetation communities in eastern lowland Bolivia, ranging from terra firme humid evergreen forest to seasonally-inundated savannah. Results show that broad ecosystems – evergreen tropical forest, semi-deciduous dry tropical forest, and savannah – can be clearly differentiated by examination of their phytolith spectra and the application of Principal Component Analysis (PCA). Differences in phytolith assemblages between particular vegetation communities within each of these ecosystems are more subtle, but can still be identified. Comparison of phytolith assemblages with pollen rain data and stable carbon isotope analyses from the same vegetation plots show that these proxies are not only complementary, but significantly improve taxonomic and ecosystem resolution, and therefore our ability to interpret palaeoenvironmental and archaeological records. Our data underline the utility of phytolith analyses for reconstructing Amazon Holocene vegetation histories and pre-Columbian land use, particularly the high spatial resolution possible with terrestrial soil-based phytolith studies.