Immunolocalization of MP70 in lens fiber 16-17-nm intercellular junctions

Thin section electron microscopy reveals two different types of membrane interactions between the fiber cells of bovine lens. Monoclonal antibodies against lens membrane protein MP70 (Kistler et al., 1985, J. Cell Biol., 101:28-35) bound exclusively to the 16-17-nm intercellular junctions. MP70 localization was most dramatic in the lens outer cortex and strongly reduced deeper in the lens. In contrast, the 12-nm double membrane structures and single membranes were consistently unlabeled. In freeze-fracture replicas with adherent cortical fiber membranes, MP70 was immunolocalized in the junctional plaques which closely resemble the gap junctions in other tissues. MP70 is thus likely to be associated with intercellular communication in the lens.     

Isolation of a cDNA clone for transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids

We have isolated a cDNA clone for rat transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids. The clone was identified initially by hybrid selection of TP1 mRNA. The sequence of the 251-nucleotide cDNA includes the entire coding region for the protein, thereby confirming the identity of the clone as well as predicting two changes in the published amino acid sequence.     

cAMP-dependent protein kinase phosphorylates gap junction protein in lens cortex but not in lens nucleus

MP70 (a 70 kDa membrane protein) is a component of the gap junctions of the young fibre cells in the lens outer cortex. In the older fibres deeper in the mammalian lens (lens nucleus), MP70 is processed to MP38 by cleavage and removal of the carboxy terminal half. It is shown here that cortical MP70, and its derivative MP64, can be phosphorylated with cAMP-dependent protein kinase. In contrast, MP38 from the lens nucleus is not phosphorylated by the enzyme. Proteolytic processing and this lens region specific phosphorylation are relevant for the future development of functional assays for lens gap junctions.     

Ontogenetic transition of wound healing pattern in rat skin occurring at the fetal stage

Full-thickness excisional wounds were made in the dorsal skin of rat fetuses at day 16 and day 18 of gestation. A small patch of skin surrounding the open wound was cut out, mounted on a plastic ring and incubated in an organ culture system. In the presence of serum, the open wound in the day-16 fetal skin closed within three days of culture. During the wound-closure process, no new structures were formed in the wound space, and no conspicuous changes were noted in the histological architecture of the surrounding skin during culture, indicating that the wound closure may result from a centripetal movement of the surrounding skin only. In contrast, the size of the open wound in the day-18 fetal skin remained almost unchanged for one week, but a thin acellular network spread over the wound space within one day of culture. The predominant component of the network was cross-linked fibrin, as disclosed by scanning electron microscopy and sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting. The network served as a scaffold for the ingrowth of fibroblast-like cells. These stage-dependent differences in fetal wound healing were consistent with an in vivo study showing that the day-16 wound was covered with the surrounding skin itself, whereas the day-18 wound was covered with newly formed epidermis and invaded by inflammatory cells. The present investigation strongly indicates the prenatal occurrence of a fetal-to-adult transition in the wound-healing pattern of rat skin.     

Identification of a 70,000-D protein in lens membrane junctional domains

A 70,000-D membrane protein (MP70), which is restricted to the eye lens fibers and is present in immunologically homologous form in many vertebrate species, has been identified. By use of anti-MP70 monoclonal antibodies for immunofluorescence microscopy and electron microscopy, this polypeptide was localized in lens membrane junctional domains. Both immunofluorescence microscopy and SDS PAGE reveal an abundance of MP70 in the lens outer cortex that coincides with a high frequency of fiber gap junctions in the same region.     

Mapping nucleolar proteins with monoclonal antibodies

Using monoclonal antibodies as probes, we have characterized three antigens with respect to localization in the nucleolus, molecular weight and solubility. Two proteins, of 110,000 and 94,000 apparent molecular weight, were found associated with the ribonucleoprotein fibers. A third protein, with a molecular weight of 40,000, was accumulated at the nucleolar periphery, was present in the nucleoplasm, and may be involved in pre-ribosome maturation and transport.     

Sequential Expression of Nucleoproteins during Rat Spermiogenesis

Transition proteins and protamines are highly basic sperm-specific nuclear proteins that serve to compact the DNA during late spermiogenesis. To understand their sequential role in this function, transition protein 1 (TP1), transition protein 2 (TP2), and protamine 1 (P1) were assayed by polyacrylamide gel electrophoresis in pools of microdissected, staged seminiferous tubule segments in the rat. The results were compared with immunocytochemical analyses of squash preparations from accurately identified stages of the epithelial cycle. TP2 was the first to appear as a faint band at stages IX–XI, followed by high levels at stages XII–XIV of the cycle. TP1 showed a low expression at stage XII of the cycle and peaked at stages XIII–I, whereas protamine 1 first appeared at stage I of the cycle and remained high throughout the rest of spermiogenesis. Immunocytochemical analyses and Western blots largely confirmed these results: TP2 in steps 9–14, TP1 in steps 12–15, and P1 from late step 11 to step 19 of spermiogenesis. We propose that TP2 is the first nucleoprotein that replaces histones from the spermatid nucleus, and its appearance is associated with the onset of nuclear elongation. TP1 shows up along with the compaction of the chromatin. The two transition proteins seem to have distinct roles during transformation of the nuclei and compaction of spermatid DNA.     

Inhibition of lactate dehydrogenase in cultured SIRC cells by cigarette smoke

Summary SIRC cell monolayer cultures were exposed to whole smoke from a mid tar and nicotine level research cigarette (ASFC, 72 puffs), or from a high tar and nicotine level reference cigarette (Kentucky 2R1, 48 puffs) over a period of 65 days. The activity and distribution of lactate dehydrogenase (LDH) in the cells were investigated, and the electrophoretic characteristics of its isozymes studied. Cell morphology was examined by light microscopy and by transmission- and scanning electron microscopy.LDH activity was reduced by exposure to smoke from both cigarette types, the greater inhibitory effect being produced by that of the Kentucky cigarette. In addition, cells exposed to this high tar and nicotine smoke displayed intramitochondrial granules which were larger and more numerous than those found in cells exposed to the mid tar and nicotine smoke, or in the control cells. It is speculated that cation accumulation in the mitochondria may be involved in the observed inhibition of LDH activity.Supported by a research grant from the ASFC (Association Suisse des Fabricants de Cigarettes), Switzerland