The characterization of the first anti-mouse Muc6 antibody shows an increased expression of the mucin in pancreatic tissue of Cftr-knockout mice

Gel-forming mucins are large high-molecular weight secreted O-glycoproteins responsible for the gel-properties of the mucus blanket. Five orthologous gel-forming mucins have been cloned in human and mouse. Among them, the mucin MUC6 has been less studied, particularly in rodents and no anti rodent-Muc6 antibody has been reported yet. In order to further study Muc6 in mice, our aims were to obtain a specific Muc6 antibody, to validate it and to test it in Cftr deficient mice. A polyclonal serum named CP4 was isolated from a rabbit immunized by a mouse Muc6 peptide. In Western blot experiments, the antibody detected a high-molecular weight molecule secreted by the gastric tissue. Using immunohistochemistry, we showed that the antibody reacted strongly with deep glands of duodenum and ileum and mucous neck cells of gastric body. CP4 also recognized Muc6 protein secreted at the surface of the stomach and renal collecting tubules. The centroacinar cells of pancreatic tissue also reacted with the antibody. Cftr−/− mice showed a higher expression of Muc6 at both protein and RNA levels compared with their control Cftr+/+ littermates suggesting that as in the human disease, Muc6 may contribute to the formation of materials that block pancreatic acini and ducts in mouse models of cystic fibrosis. The rabbit anti-mouse Muc6 polyclonal antibody seems highly specific to the mouse mucin and will be useful to study pancreatic pathology in cystic fibrosis.     

Genetic analysis of resistance to Puccinia striiformis f.sp. tritici in cv. Aflak at adult plant stage

In order to investigate heritability and gene action for yellow rust resistance in wheat, a resistance yellow rust cultivar Aflak was crossed to susceptible cultivar Avocet‘s’. Parents, F₁, F₂ and F₃ generations were cultured according to randomised complete block design with two replications in the research station of Gharakhil, Iran. Parents and other generations were inoculated with 70E0A+ race. Traits including severity and infection type were recorded and then coefficient of infection was calculated. For this trait, generations mean and variance analysis were performed and results showed that there were significant differences among generations for coefficient of infection. Results showed that in addition to additive and dominance effects, at least one kind of epistasis interaction (additive × additive) control this trait. Although additive and dominance effects control this trait, but with attention to generations variance analysis, the results showed that additive variance had important role to control this trait.     

MUC6 mucin expression inhibits tumor cell invasion

The MUC6 mucin has a critical protective function in the normal stomach, pancreas and duodenum and is aberrantly expressed during the progression of some gastrointestinal cancers. Our aim was to determine whether MUC6 contributes to the etiology or progression of pancreatic cancer and elucidate the molecular basis of its involvement. Expression of MUC6 glycoprotein was examined in pancreatic cancer tissues by immunofluorescence and loss of MUC6 was observed. Next, to determine whether MUC6 inhibits tumor growth and metastasis by altering cell adhesion and invasion, recombinant MUC6 cDNA and separate MUC6 N-terminal and C-terminal domains were transfected into pancreatic, colorectal and breast cancer cell lines. The recombinant N- and C-terminal proteins were each seen to oligomerize under non-reducing conditions. Overexpression of both domains of the MUC6 glycoprotein significantly inhibited cell adhesion to matrix proteins (collagen I, collagen IV, fibronectin and laminin) in LS 180 but not in PANC-1 cells. Moreover, the N- and C-terminal domains of MUC6 inhibited invasion of both LS 180 and PANC-1 cells by 40% and 70%, respectively, in comparison with controls. These results suggest that MUC6 may inhibit invasion of tumor cells through the basement membrane of the pancreatic duct and slow the development of infiltrating carcinoma.     

Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary

Background  

The aging of reproductive organs is not only a major social issue, but of special interest in aging research. A long-standing view of 'immortal germ line versus mortal soma' poses an important question of whether the reproductive tissues age in similar ways to the somatic tissues. As a first step to understand this phenomenon, we examine global changes in gene expression patterns by DNA microarrays in ovaries and testes of C57BL/6 mice at 1, 6, 16, and 24 months of age. In addition, we compared a group of mice on ad libitum (AL) feeding with a group on lifespan-extending 40% calorie restriction (CR).     

Tumor suppression by collagen XV is independent of the restin domain

Non-fibrillar collagen XV is a chondroitin sulfate modified glycoprotein that is associated with the basement membrane zone in many tissues. Its precise functions remain to be fully elucidated though it clearly plays a critical role in the structural integrity of the extracellular matrix. Loss of collagen XV from the basement membrane zone precedes invasion of a number of tumor types and we previously showed that collagen XV functions as a dose-dependent suppressor of tumorigenicity in cervical carcinoma cells. The carboxyl terminus of another non-fibrillar collagen (XVIII) is cleaved to produce endostatin, which has anti-angiogenic effects and thus may act as a tumor suppressor in vivo. Since collagen XV has structural similarity with collagen XVIII, its C-terminal restin domain could confer tumor suppressive functions on the molecule, though our previous data did not support this. We now show that expression of collagen XV enhances the adhesion of cervical carcinoma cells to collagen I in vitro as does the N-terminus and collagenous regions of collagen XV, but not the restin domain. Destruction of a cysteine residue in the collagenous region that is critical for intermolecular interactions of collagen XV abolished the enhanced adhesion to collagen I. Finally, we demonstrate that unlike full length collagen XV, expression of the restin domain alone does not suppress tumorigenicity of cervical carcinoma cells in vivo; hence, this process is dependent on functions and interactions of other parts of the protein.     

Microsatellite evolution in congeneric mammals: domestic and bighorn sheep

We compared genotypes at eight (AC)n microsatellite loci in domestic sheep (Ovis aries) and wild Rocky Mountain bighorn sheep (O. canadensis). The domestic sheep had greater genetic variation, higher allele-size variances, and larger allele sizes than the wild sheep. Accumulating evidence from higher taxonomic comparisons shows that these parameters are biased if microsatellite loci are selected in one taxon and used in another. Our results demonstrate similar biases between congeneric species. We compared standard measures of genetic variation, differentiation, and distance within and between species (H, D, FST) to newer measures based on allele-size variance (SW, SB, RST). The size-based distances better detected species-level divergence, but standard measures better distinguished allopatric populations. Empirical calibration of these measures at the subspecies level is needed to establish their useful ranges.      

Distribution of flourescently labeled α-actinin in living and fixed fibroblasts

The distribution of flourescently labeled α-actinin after microinjection into fibroblasts has been determined in both living and fixed cells. We have found that the distribution of the injected tetramethylrhodamine isthiocyanate-labeled protein (TMRITC-α-actinin) in living cells, which is in ruffling membranes, actin microfilament bundles, and polygonal microfilament networks (Feramisco, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:3967-3971), was virtually unaffected by the fixation (3.5 percent formaldehyde) and extraction (absolute acetone) used for the preparation of the cells for immunoflourescence. Also, these patterns were found to coincide with the α-actinin revealed by immunoflourescence. Also, these patterns were found to coincide with the α-actinin revealed by immunoflourescence. These findings offer, for the first time, evidence indicating the validity of the immunoflourescence technique in the localization of α-actinin in cultured cells. With the combination of the injection procedure and the immunoflourescence localization of endogenous structural proteins, it was determined that nearly all of the actin stress fibers were decorated in a periodic manner with the injected α-actinin. Endogenous tropomyosin in the injected cells was found to be distributed with a periodic pattern along the stress fibers that was antiperiodic to the pattern observed for the microinjected α-actinin. The tropomyosin antibody stained the polygonal microfilament networks and was excluded from the foci, whereas the microinjected α-actinin was incorporated into the foci of the networks. Thus, the microinjected fluorescent derivative of α-actinin appears to be incorporated into the functional pools of α-actinin within the living cell and to be utilized by the cell with fidelity.     

Distribution of an endogenous lectin in the developing chick optic tectum

We determined the cellular localization of an endogenous lectin at various times during the development of a well-characterized region of chick brain, the optic tectum. This lectin is a carbohydrate-binding protein that interacts with lactose and other saccharides, undergoes striking changes in specific activity with development, and has previously been purified by affinity chromatography from extracts of embryonic chick brain and muscle. Cellular localization in the tectum was done by indirect immunofluoresecent staining, using immunoglobulin G derived from an antiserum raised against pure lectin. No lectin was detectable in the optic tectum examined at 5 days of embryonic development. From approximately 7 days of development, neuronal cell bodies and fibers were labeled by the antibody; and extracts of tectum contained hemagglutination activity that could be inhibited by lactose or by the antiserum. Lectin remained present in many tectal neuronal layers after hatching; but in 2-month-old chicks it was sparse or absent in most of the tectum except for prominent labeling of fibers in the stratum album centrale. The initial appearance of lectin in the optic tectum was not dependent on innervation by optic nerve fibers since bilateral enucleation during embryogenesis did not affect it. Lectin was detectable on the surface of embryonic optic tectal neurons dissociated with a buffer containing EDTA.     

Capacity of biological soil crusts colonized by the lichen <Emphasis Type="Italic">Diploschistes</Emphasis> to metabolize simple phenols

Background and aims

Soil compaction strongly affects water uptake by roots. The aim of the work was to examine soil—plant interactions with focus on the impact of distribution of compacted soil layers on growth and water uptake by wheat roots.

Methods

The growth-chamber experiment was conducted on wheat growth in soil with compacted soil layers. The system for maintaining constant soil water potential and measurement of daily water uptake from variously compacted soil layers was used.

Results

Layered soil compaction differentiated vertical root distribution to higher extent for root length than root mass. The propagation rate of a water extraction front was the highest through layers of moderately compacted soil. The root water uptake rate was on average 67 % higher from moderately than heavily compacted soil layers. Correlations between water uptake and the length of thick roots were increasing with increasing level of soil compaction.

Conclusions

The study shows that root amount, water uptake, propagation of water extraction and shoot growth strongly depend on the existence of compacted layers within soil profile. The negative effects of heavily compacted subsoil layer on water uptake were partly compensated by increased uptake from looser top soil layers and significant contribution of thicker roots in water uptake.