In vitro assembly and structure of trichocyte keratin intermediate filaments: a novel role for stabilization by disulfide bonding

Intermediate filaments (IF) have been recognized as ubiquitous components of the cytoskeletons of eukaryotic cells for 25 yr. Historically, the first IF proteins to be characterized were those from wool in the 1960s, when they were defined as low sulfur keratins derived from "microfibrils." These proteins are now known as the type Ia/type IIa trichocyte keratins that constitute keratin IF of several hardened epithelial cell types. However, to date, of the entire class of >40 IF proteins, the trichocyte keratins remain the only ones for which efficient in vitro assembly remains unavailable. In this paper, we describe the assembly of expressed mouse type Ia and type IIa trichocyte keratins into IF in high yield. In cross-linking experiments, we document that the alignments of molecules within reduced trichocyte IF are the same as in type Ib/IIb cytokeratins. However, when oxidized in vitro, several intermolecular disulfide bonds form and the molecular alignments rearrange into the pattern shown earlier by x-ray diffraction analyses of intact wool. We suggest the realignments occur because the disulfide bonds confer substantially increased stability to trichocyte keratin IF. Our data suggest a novel role for disulfide bond cross linking in stabilization of these IF and the tissues containing them.     

Syncoilin isoform organization and differential expression in murine striated muscle

Syncoilin is a 64 kDa intermediate filament (IF) protein expressed in myocytes at the sarcolemma, perinucleus, myotendenous and neuromuscular junctions. Here we present a revised domain projection and structural analysis for the original isoform (sync-1) and introduce two novel syncoilin isoforms (sync-2 and sync-3) generated by exon splicing. On the basis of consensus identity we propose that syncoilin be reclassified as a type III IF protein. All three syncoilin isoforms lack a L1 domain, a significant departure from standard IF rod domain projections that is likely to impact significantly on their biological function. Our analyses indicate that syncoilin is unlikely to form classical intermediate filament structures by itself, and that the significant difference in C-terminal structure between the three isoforms indicates that they may play divergent roles in myocytes. We show that despite lacking an apparent structural role in striated muscle, syncoilin isoforms are differentially and strongly upregulated in response to cardiotoxin induced regeneration and denervation induced atrophy in the C57BL/6 mouse, possibly suggesting an atypical role for syncoilin in muscle.     

Chromatid repulsion associated with Roberts/SC phocomelia syndrome is reduced in malignant cells and not expressed in interspecies somatic-cell hybrids.

Different cell types from a female patient with Roberts/SC phocomelia syndrome were evaluated quantitatively for the presence of repulsion of heterochromatin and satellite regions of mitotic chromosomes. Whereas EBV-transformed lymphoblasts from an established cell line revealed these phenomena at frequencies equal to those in PHA-stimulated lymphocytes and cultured skin fibroblasts, aneuploid cells from a metastatic melanoma displayed them at 50% lower frequency. Cocultivation of the patient's fibroblasts with either an immortal Chinese hamster cell line or with a human male fibroblast strain carrying a t(4;6)(p14;q21) translocation showed that the phenomenon was not corrected or induced by a diffusible factor or by cell-to-cell contact. In each experiment, only the patient's metaphase spreads revealed chromatid repulsion. In fusion hybrids between the patient's fibroblasts and an established Chinese hamster cell line, the human chromosomes behaved perfectly normally, suggesting that the gene product which is missing or mutant in Roberts/SC phocomelia syndrome is supplied by the Chinese hamster genome.     

Toxicity and DNA damage induced by 1-nitropyrene and its derivatives in Chinese hamster lung fibroblasts

1-Nitropyrene and its chemically synthesised derivatives were investigated for their cytotoxicity and ability to induce DNA-strand breaks in Chinese hamster lung fibroblasts. Both 1-nitrosopyrene (0.25-60 micrograms/ml) and 1-aminopyrene (0.25-25 micrograms/ml) were cytotoxic, and induced the formation of DNA lesions, which were measured as DNA single-strand breaks after sedimentation in alkaline sucrose-density gradients. Higher doses of 1-aminopyrene (25-60 micrograms/ml) inhibited the formation of DNA single-strand breaks. 1-Nitropyrene was not toxic (0.25-60 micrograms/ml) and induced low levels of detectable DNA strand breaks, whilst N-acetyl-1-aminopyrene was inactive. The post-mitochondrial supernatant fraction of Aroclor-induced rat-liver containing 4 mM NADPH (S9 mix) did not promote the activation of 1-nitropyrene. In fact DNA strand breaks induced by either 1-nitropyrene or 1-nitrosopyrene was abolished in the presence of S9 mix. The 1-nitropyrene reduced intermediate, N-hydroxy-1-aminopyrene was synthesised by the reduction of 1-nitrosopyrene with ascorbic acid. In the presence of ascorbic acid, 1-nitrosopyrene caused a 5-fold increase in the number of DNA single-strand breaks when compared to cells treated with 1-nitrosopyrene alone. The results are discussed in terms of the metabolic activation of 1-nitropyrene and 1-aminopyrene in Chinese hamster lung cells.     

Immunohistochemical localisation of cyclooxygenase in the human uterus

The distribution in the human uterus of the enzyme cyclooxygenase, involved in prostaglandin synthesis, was studied using the peroxidase-antiperoxidase immunohistochemical technique. Specific staining was observed only during the luteal phase of the menstrual cycle. It was found that cyclooxygenase was localised in the surface and glandular epithelium of the endometrium but could not be demonstrated in the endometrial stroma, myometrium or blood vessels. The location of specific staining for cyclooxygenase and its variation during the menstrual cycle was the same regardless of the amount of menstrual blood loss. Tissue samples obtained from early pregnant and post-menopausal patients failed to exhibit any specific staining.