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The murine multiple drug resistance (mdr) gene, mdr1a, encodes a 170-kDa transmembrane protein that is expressed in many tissues including intestinal epithelial cells, a subset of lymphoid cells and hematopoietic cells. We report that mdr1a knockout (mdr1a-/-) mice are susceptible to developing a severe, spontaneous intestinal inflammation when maintained under specific pathogen-free animal facility conditions. The intestinal inflammation seen in mdr1a-/- mice has a pathology similar to that of human inflammatory bowel disease (IBD) and is defined by dysregulated epithelial cell growth and leukocytic infiltration into the lamina propria of the large intestine. Treating mdr1a-/- mice with oral antibiotics can both prevent the development of disease and resolve active inflammation. Lymphoid cells isolated from mice with active colitis are functionally reactive to intestinal bacterial Ags, providing evidence that there is enhanced immunologic responsiveness to the normal bacterial flora during IBD. This study is the first description of spontaneous colitis in a gene knockout mouse with an apparently intact immune system. This novel model of spontaneous colitis may provide new insight into the pathogenesis of IBD, the nature of dysregulated immune reactivity to intestinal bacterial Ags, and the potential functional role of mdr genes expressed in the cells and tissues of the colonic microenvironment.

The hemidesmosome is a complex junction containing many proteins. The keratin cytoskeleton attaches to its cytoplasmic plaque, while its transmembrane elements interact with components of the extracellular matrix. Hemidesmosome assembly involves recruitment of alpha 6 beta 4 integrin heterodimers, as well as cytoskeletal elements and cytoskeleton-associated proteins to the cell surface. In our cell culture models, these phenomena appear to be triggered by laminin-5 in the extracellular matrix. Cell interaction with laminin-5 apparently induces both phosphorylation and dephosphorylation of subunits of alpha 6 beta 4 integrin. There is emerging evidence that such events are necessary for subsequent cytoskeleton anchorage to the hemidesmosome cytoplasmic plaque. Once assembled, the hemidesmosome plays an essential role in maintaining firm epithelial adhesion to the basement membrane, with hemidesmosome disruption being a hallmark of certain devastating blistering diseases. However, the hemidesmosome is more than just a stable anchor, as it may also be the site of signal transduction, mediated by its alpha 6 beta 4 integrin component. This review discusses our current knowledge of the structure and assembly of the hemidesmosome.

In a 5-year prospective study, computed tomographic (CT) morphometry of the lumbosacral vertebral canal was performed on 42 large-breed dogs (21 controls and 21 dogs with lumbosacral stenosis). Dogs were allotted to 4 groups. Group 1 (n = 13) consisted of cadaver specimens obtained from dogs that died or were euthanatized for reasons unrelated to the spine; group 2 (n = 8) consisted of live dogs with no history of clinical signs related to the spine and with normal neurologic examination findings; group 3 (n = 10) consisted of dogs with surgically confirmed lumbosacral stenosis; and group 4 (n = 11) consisted of dogs with suspected lumbosacral stenosis that were managed conservatively. The CT scans were performed, using 5-mm contiguous slices obtained perpendicular to the vertebral canal, from the midbody of the 5th lumbar vertebra through the caudal endplate of the sacrum (L5-S3). Lumbosacral lordosis was minimized in all dogs by positioning them in dorsal recumbency with the hind limbs flexed. A tuberculin syringe calibration phantom was placed within the scanning field of view, parallel to the axis of the spine. In each dog, 11 CT slice locations within the lumbosacral spine were evaluated. At each slice location, sagittal plane diameter, dorsal plane diameter, and transverse area of the vertebral canal, vertebral body, and calibration phantom were measured, using the CT computer's software programs for distance and area calculation. Window/level settings were constant, and all measurements were made by the same operator (JCJ). Accuracy of calibration phantom CT measurements was 100% for sagittal and dorsal plane diameter and was 85% for transverse area.(ABSTRACT TRUNCATED AT 250 WORDS)

We have analyzed gene-specific and strand-specific DNA damage and repair in the dihydrofolate reductase gene in hamster cells. Cells were UV-irradiated or treated with two types of chemotherapeutics, alkylating agents or cisplatin. UV-induced pyrimidine dimers were detected using a previously published technique in which the T4 endonuclease V enzyme is used to create nicks at the lesion sites. 6-4 photoproducts were detected in a similar assay using ABC excinuclease after prior reversal of the pyrimidine dimers with photolyase. Adducts formed by the alkylating agents nitrogen mustard and dimethyl sulfate were quantitated by generating strand breaks at basic sites after neutral depurination. Cisplatin-induced intrastrand adducts were detected with ABC excinuclease, and cisplatin interstrand cross-links were detected using a denaturation-reannealing reaction before electrophoresis. In accord with previous reports by other investigators, we find distinct strand specificity of the repair of pyrimidine dimers after UV; the transcribed strand was much more efficiently repaired than the nontranscribed strand. In contrast, there was little or no strand bias in the repair of the 6-4 photoproducts. For alkylating agents, a slight bias toward repair in the transcribed strand was found after treatment with nitrogen mustard, but there appeared to be no bias in the repair after treatment with dimethyl sulfate. Cisplatin interstrand cross-links are repaired with equal efficiency from the two strands, but the more common cisplatin-induced lesion, the intrastrand adduct, is preferentially repaired from the transcribed strand. In conclusion, there is strand bias in the repair of pyrimidine dimers and cisplatin intrastrand adducts, but the strand specificity of repair may not be a general feature for all DNA lesions, as we found little or no strand bias in the repair of other lesions studied.

The adhesion complex of the corneal epithelium consists of the hemidesmosome and its associated structures, such as anchoring filaments, lamina densa of the basement membrane, and anchoring fibrils. It contributes to the adhesion of the corneal epithelium to Bowman's layer. To understand the adhesion complex better, an electron microscopic and immunofluorescence analysis was done of the reformation of the adhesion complex in small (1 mm) keratectomy wounds in the guinea pig cornea. In these wounds, the epithelium, hemidesmosomes, basal lamina, anchoring fibrils, and anterior stroma were removed. The wound bed was epithelialized completely by 24 hr after wounding. Immunofluorescence analyses involved the use of antibodies against plaque components of the hemidesmosome, an antibody against laminin, and an antibody against the collagen VII component of anchoring fibrils. At 18 hr after wounding, there was no morphologic evidence of hemidesmosomes at the epithelial-stromal interface. At 24 hr, hemidesmosomes were observed, with or without subjacent lamina densa. Furthermore, plaque components were detected by immunofluorescence in those cells in contact with the wound bed. In contrast, no type VII collagen was detected. On day 7, collagen VII, laminin, and bullous pemphigoid autoantibody markers colocalized along the wound bed as determined by immunofluorescence. However, at the ultrastructural level, even though the lamina densa of the basal lamina was observed primarily where hemidesmosomes were present, it remained incomplete. In this study, the precise temporal sequence in which components are incorporated into the assembling adhesion complex was described during wound healing. Furthermore, the possibility that the hemidesmosomal plaque nucleates the formation of the underlying basal lamina was discussed.

We have used three methods to study the formation and repair of intrastrand adducts and interstrand cross-links in the DNA of Chinese hamster ovary cells induced by the anticancer drug cis-diamminedichloroplatinum II (cisplatin). Using atomic absorption spectroscopy, we found that 21% of the total genomic cisplatin adducts were removed at 8 h and 42% at 24 h. We used ABC excinuclease digestion, coupled with out previously reported methodology to quantify DNA in specific genomic regions. These adducts were removed faster in the transcribed dihydrofolate reductase and c-myc genes compared to a noncoding fragment, a region containing the little or nontranscribed c-fos oncogene, and to the overall genome. Interstrand cross-links in specific sequences were quantified by Southern hybridization of denatured-renatured DNA separated on a neutral gel. We found that cross-links were removed more efficiently from the gene regions than intrastrand adducts and, at high levels of cross-linking, removal was similar from transcribed and from nontranscribed regions.

According to the concept of keratin pairing defined by tissue coexpression, a 55-kD/64-kD keratin pair is a marker of "corneal-type" differentiation. Intermediate filament (IF)-enriched preparations from guinea pig and bovine corneal epithelium were analyzed, and a rabbit antiserum was generated against a 55-kD polypeptide enriched in these preparations. This antiserum generated a typical IF-like pattern in cultured bovine corneal epithelial cells. Immunofluorescence microscopic analysis of frozen sections of guinea pig and bovine tissue revealed that the 55-kD antiserum labeled corneal and limbal epithelium. In addition, the antiserum stained a subpopulation of peripheral limbal cells that were distributed in both basal and suprabasal layers of the epithelium. The monoclonal antibody AE5 was used to investigate the distribution of the 64-kD polypeptide in guinea pig and bovine tissue. Immunoblotting analysis revealed that AE5 antibodies recognized a 64-kD polypeptide in guinea pig cornea, but recognized a 66-kD polypeptide in bovine cornea. Immunofluorescence microscopic analysis of guinea pig tissue revealed that AE5 antibodies labeled suprabasal layers of corneal epithelium, in suprabasal layers of limbal epithelium, and in groups of cells in the peripheral limbal epithelium. We discuss the possibility that the ocular epithelial cells recognized by either the 55-kD or the 64-kD antibodies in the peripheral limbus may play a role in the reepithelialization of the cornea after wounding.

Les moustiques femellesAedes aegypti L., alourdies par des poids places sur leur thorax ou leur abdomen, peuvent encore se repaitre normalement de sang humain. Sur le thorax, elles tolerent 2 fois le poids de leur corps et 3 a 5 fois sur l'abdomen. Le fait d'etre attachees ne restreint pas leur capacite d'absorber du sang. En expose le moyen de tenir des moustiques en laisse.