Electron microscopic observation and single-stranded DNA binding activity of the Mcm4,6,7 complex

Mcm2-7 proteins that play an essential role in eukaryotic DNA replication contain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. Our group has reported that a DNA helicase activity is associated with a 600 kDa human Mcm4, 6 and 7 complex. The structure of the Mcm4,6,7 complex was visualized by electron microscopy after negative staining with uranyl acetate. The complex contained toroidal forms with a central channel and also contained structures with a slit. Gel-shift analysis indicated that the level of affinity of the Mcm4,6,7 complex for single-stranded DNA was comparable to that of SV40 T antigen, although the Mcm4,6,7 complex required longer single-stranded DNA for the binding than did SV40 T antigen. The nucleoprotein complexes of Mcm4,6,7 and single-stranded DNA were visualized as beads in a queue or beads on string-like structures. The formation of these nucleoprotein complexes was erased by Mcm2 that is a potential inhibitor of the Mcm4,6,7 helicase. We also found that the DNA helicase activity of Mcm4,6,7 complex was inhibited by the binding of Mcm3,5 complex. These results support the notion that the Mcm4,6,7 complex functions as a DNA helicase and the formation of 600 kDa complex is essential for the activity.     

Photosynthesis-Dependent Volume Regulation in Guard Cell Protoplasts from Vicia faba L.

A rapid and convenient procedure was developed for isolatingguard cell protoplasts (GCPs) from epidermal strips of Viciafaba L. The mean rates of O₂ uptake in the dark and evolutionin light of the isolated GCPs were 200 and 290 µmol O₂mg^–1 Chl h^–1, respectively, showing net O₂ evolutionin light. Photosynthetic O₂ evolution was suppressed completelyby 5 µM DCMU. Addition of 5 µM DCMU to the incubationmedium after 30 min of light exposure also suppressed the light-inducedswelling of GCP, indicating possible participation of PS IIin volume regulation in GCP. ⁴Present address: Division of Environmental Biology, The NationalInstitute for Environmental Studies, Yatabe machi, Tsukuba,Ibaraki 305, Japan. (Received December 17, 1983; Accepted March 21, 1984)     

Macromolecular structure of reassembled neurofilaments as revealed by the quick-freeze deep-etch mica method: difference between NF-M and NF-H subunits in their ability to form cross-bridges.

Neurofilament (NF) structure and ability to form cross-bridges were examined by quick-freeze deep-etch mica and low-angle rotary-shadow electron microscopy in NFs purified from bovine spinal cord and reassembled in various combinations of NF subunits. When NFs were reassembled from triplet proteins, NF-L, NF-M and NF-H, they were oriented randomly and often fragmented, but their elongated filaments (12-15 nm wide) and the cross-bridges (4-5 nm wide) connecting them were similar in appearance to those of isolated bovine NFs or in vivo rat NFs. Projections extended from the wall of the core filament in almost the same pattern as the cross-bridges and were the same in width and interval (minimum interval, 20-25 nm) as the cross-bridges. Projections were more conspicuous when core filaments were separated by 60 to 80 nm or more, while cross-bridges were more conspicuous when core filaments were close to each other. Projections or cross-bridges extended bilaterally at intervals of 20 to 25 nm where core filaments expanded and formed a network between filaments which were far from one another. When NFs were reconstructed from NF-L alone, only core filaments appeared, the same width as the filaments of triplet NFs. The core filaments were occasionally in almost direct contact with each other, with no projection or cross-bridge. When NFs were reassembled from NF-M alone or NF-L + NF-M, although NF-M core filaments were shorter and slightly thinner than NF-L + NF-M core filaments, both had projections, and both had cross-bridges, but cross-bridges were less evident. Cross-bridges were almost the same in width as those of triplet NFs, but significantly shorter and much less frequent although the minimum interval was the same, and core filaments were not attached to each other. In contrast, when NFs were reconstituted from NF-H alone or NF-L + NF-H, both had conspicuous projections and cross-bridges, similar to those of triplet NFs. Thus, when NFs contained NF-H, they formed frequent cross-bridges and long projections with extensive peripheral branching. When NFs contained NF-M but no NF-H, they tended to form cross-bridges, and to form projections that were shorter and straighter and without peripheral branching. That is, there appears to be a significant difference between NF-M and NF-H in ability to form cross-bridges and thus in interaction with adjacent NFs.     

Photosynthetic Properties of Guard Cell Protoplasts from Vicia faba L.

Guard cell protoplasts were isolated enzymatically from theepidermis of Vicia faba L. and their photosynthetic activitieswere investigated. Time courses of light-induced changes inthe chlorophyll a fluorescence intensity of these protoplastsshowed essentially the same induction kinetics as found formesophyll protoplasts of Vicia. The transient change in thefluorescence intensity was affected by DCMU, an inhibitor ofphotosystem II; by phenylmercuric acetate, an inhibitor of ferredoxinand ferredoxin NADP reductase; and by methyl viologen, an acceptorof photosystem I. Low temperature (77 K) emission spectra ofthe protoplasts had peaks at 684 and 735 nm and a shoulder near695 nm. A high O₂ uptake (175 µmol mg^–1 Chl hr^–1)was observed in guard cell protoplasts kept in darkness, whichwas inhibited by 2 mM KCN or NaN₃ by about 60%. On illumination,this O₂ uptake was partially or completely suppressed, but itssuppression was removed by DCMU, which indicates that oxygenwas evolved (150 µmol mg^–1 Chl hr^–1) photosynthetically.We concluded that both photosystems I and II function in guardcell chloroplasts and that these protoplasts have high respiratoryactivity. (Received January 30, 1982; Accepted May 15, 1982)     

Fine structure of the ependyma and intercellular junctions in the area postrema of the rat

Summary Ependymal cells and their junctional complexes in the area postrema of the rat were studied in detail by tracer experiments using horseradish peroxidase (HRP) and colloidal lanthanum and by freeze-etch techniques, in addition to routine electron microscopy. The ependyma of the area postrema is characterized as flattened cells possessing very few cilia, a moderate amount of microvilli, a well-developed Golgi apparatus and rough endoplasmic reticulum. Numerous vesicles or tubular formations with internal dense content were found to accumulate in the basal processes of ependymal cells; the basal process makes contact with the perivascular basal lamina. It is suggested that the dense material in the tubulovesicular formations is synthesized within the ependymal cell and discharged into the perivascular space. The apical junctions between adjacent ependymal cells display very close apposition, with a gap of 2–3 nm, but no fusion of adjacent plasma membranes; they thus represent a transitional form between the zonulae adhaerentes present in the ordinary mural ependyma and the zonulae occludentes in the choroidal epithelium. A direct intercommunication between the ventricular cerebrospinal fluid (CSF) and the blood vascular system indicates that a region exists lacking a blood-ventricular CSF barrier.     

Expression of an inwardly rectifying K+ channel, Kir4.1, in satellite cells of rat cochlear ganglia

Satellite cellsare glial cells wrapped around somata of sensory and autonomic ganglionneurons. Neither their functional roles nor electrical properties havebeen fully clarified so far. Using immunohistochemistry, we found thatinwardly rectifying K⁺ channelsubunit Kir4.1 (also called Kir1.2 orK_AB-2) was expressed prominently in the satellite cells of cochlear ganglia. The Kir4.1 immunoreactivity was localized specifically at the myelin sheaths ofsatellite cells wrapping the somata of the ganglion neurons. Developmental expression of Kir4.1 in satellite cells paralleled development of the action potential in the auditory nerve. These results suggest that this channel in satellite cells may be responsible for the regulation of K⁺ extrudedfrom the ganglion neurons during excitation.

     

Closer association of mitochondria with lipid droplets in hepatocytes and activation of Kupffer cells in resveratrol-treated senescence-accelerated mice

Resveratrol has been extensively investigated because of its beneficial effects in delaying age-related diseases, thus extending the lifespan, possibly by mimicking calorie restriction. For this study, cell biological techniques were used to examine how resveratrol influenced hepatocytes in a senescence-accelerated mouse P10 (SAMP10), treated from 35 to 55 weeks of age, with special emphasis on the relationship between mitochondria and lipid droplets. Survival ratio, body weight and food intake of SAMP10 did not differ significantly between the control and resveratrol-treated groups. Compared with the control, the treated livers were altered significantly, as follows. Lipid droplets were reduced and mitochondria were increased in number in hepatocytes. Phosphorylation of acetyl-CoA carboxylase and the expression of both the mitochondrial ATP synthase β subunit and Mn superoxide dismutase (SOD2) were increased. Mitochondria, expressing more SOD2, were more tightly associated with lipid droplets, suggesting the enhancement of lipolysis through the activation of mitochondrial functions. Cathepsin D expression was less in hepatocytes but enhanced in Kupffer cells, which were increased in number and size with more numerous lysosome-related profiles. Together, resveratrol may activate mitochondria resulting in consuming lipids, and may also activate Kupffer cells by which a beneficial milieu for hepatocytes may be created. Both might be related to improvement in the functioning of the liver, which is the organ that is central to metabolic regulation.