Due to the recent development of NMR spectroscopy, the three-dimensional structures of proteins up to 10 Kd in aqueous solution can be determined in atomic levels. In the present article, we show the intuitive description on the theoretical background of structural determination by NMR, where we take erabutoxin b as an example and compare the structures in aqueous solution and in the crystalline state. Then, we show the three-dimensional structure of mouse epidermal growth factor and human transforming growth factor a determined by NMR. On the basis of the results, we discuss the molecular mechanism of recognition between the epidermal growth factor and its receptor.

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Localization of junctions between inner enamel-secretory ameloblasts was examined by immunofluorescence microscopy using antibodies against adherens junction proteins, radixin, vinculin, and A-CAM. All antibodies used stained the boundary between the ameloblasts exclusively in the plane where F-actin was abundant. This suggests that the adherens junctions in the ameloblasts are involved in cell-to-cell movement with actin-based micro filament bundles.

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An electrode sensitive to tetraphenyl phosphonium (TPP⁺) was applied to monitor the changes in the membrane potential of the ciliate Blepharismajaponicum during the process of cell elongation. Uptake of TPP⁺ by Blepharisma cells was observed when they were incubated in a mediumcontaining 2 x 10^-6M TPP⁺. Subsequent light stimulation (3, 000 lux) led to an efflux of accumulated TPP⁺ from the cells, indicating that depolarization of the plasma membraneoccurred with the concomitant cell elongation. TPP⁺ efflux was always observed whenever cell elongation was induced in response to various stimuli: valinomycin (10^-6M), K⁺ (30 mM), Co²⁺ (5 mM), and Ca²⁺ (5 mM). Furthermore, elongation was also evoked by electrical stimulation. These observations indicate that membrane depolarization is involved in the process of cell elongation in Blepharisma.

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To study the effects of surface materials of cells on the behavior of other neighboring cells in a crowded culture, con fluent sheets of rat 3Y1 fibroblasts were fixed and then 3Y1 cells were seeded on to them. Among con fluent sheets un fixed, fixed with formalin and fixed with ethanol and an empty plastic dish surface, the substrate activity to permit cell adhesion was compared. After con fluent 3Y1 cells (mainly composed of cells with a G1-DNA content) were reseeded with fresh medium on to these substrates, the capacity to initiate DNA synthesis per attached cell was also compared. The substrate activity of the ethanol-fixed cell sheet to permit cell adhesion was as high as that of the empty dish surface, whereas that of the un fixed cell sheet and that of the formalin- fixed cell sheet were low. When the ethanol-fixed cell sheet and the empty dish surface were coated with the ethanol extract of the un fixed cell sheet, the substrate activity diminished, indicating that during the fixation process with ethanol an adhesion-inhibitory factor (s) was removed. The capacity to initiate DNA synthesis of each cell that had completed adhesion and spreading on the cell sheets un fixed, fixed with formalin, and fixed with ethanol was lower compared to the cell that had adhered to the empty dish surface. We conclude that factors over the 3Y1 cell surface inhibit the overlapping cell adhesion and the proliferation of cells contacting each other, resulting in the ordered cell configuration in the con fluent culture.

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Horseradish peroxidase (HRP, 10 mg/100 g body weight) was intravenously injected into rats in order to investigate the nature of the compartmens involved in the transcellular transport of the protein through hepatocytes into bile. Double cytochemistry for HRP and the marker enzymesfor cytoplasmic orgattelles was used. HRP was shown to be taken up by hepatocytes via vesicles at the sinusoidal surface, some of which were positive for 5'-nucleotiddse activity. HRP was then found in the smooth-surfaced vesicles and tubules which were negative in 5'-nucleotidase, glucose 6-phosphatase, thiamine pyrophosphatase and acid phosphatase activity, suggesting that the tubular structures are neither the endoplasmic reticulum, the Golgi apparatus nor lysosomes. Biochemical studies revealed that the lead procedures used for the double cytochemistry did not inhibit the peroxidatic activity of HRP, and conversely that HRP did not interfere with the marker enzyme activity. Such cytochemical observations seemed to be supported by the observation that administration of monensin (3.5 mg/100 g) and chloroquine (5 mg/100 g), which markedly aletered the structure of the Golgi apparatus and lysosomes, respectively, slightly altered the biliary excretion of HRP but not to a significant extent.

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A nonphotosynthetic, white-wild cell line of sycamore (Acer pseudoplatanus L.) contains amyloplasts as the only kind of plastic!, whereas a photosynthetically competent green variant cell line contains only chloroplasts. Transcripts of both nuclear and plastid genes for photosynthetic components in the white cells were not detectable in contrast to those in the green cells. To investigate the limiting step (s) behind these diminished levels of transcripts, we have performed in vivo pulse-chase labeling of RNAin both cell types. These studies indicated that the rates of incorporation of [3H]uridine and nucleotide pool sizes were indistinguishable between the two cell lines. Transcripts of certain nuclear (rbcS, cab, psbO) and plastid (rbcL) genes in the white cell were not detectable. Weinfer from these data that transcriptional regulation entails an important role in controlling photosynthetic RNAlevels. Related analyses exploiting plastid run-on transcription have provided supporting evidence that the transcription of the amyloplast genomein the white cell is greatly suppressed in contrast to that of the chloroplast genomein the green cell. The results support a model of selective suppression of photosynthesis genes in nonphotosynthetic higher plant cells, and indicate that gene expression in such a system is primarily controlled at the transcriptional level.

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Transcriptional regulation has been shownto operate as a selective control mechanismof expression of photosynthetic genes in the nonphotosynthetic plastids, amyloplasts, of a white-wild cell line of sycamore (Acer pseudoplatanus L.). To elaborate the mechanisms governing the transcriptional regulation at the molecular level, we have examined the template activity of the amyloplast DNA compared to the chloroplast DNA by using the in vitro run-off transcription assay system with extracts of the two plastid types. The results of these assays clearly indicate that most of the amyloplast DNA regions do not serve as a template for the in vitro transcription regardless of the plastid extracts; this is in contrast to the chloroplast DNA which serves as an active template. It is highly likely that the template activity of amyloplast DNA per se is the modulating element of transcriptional regulation. Parallel experiments determining the DNA base content by HPLC analysis have shown that a variety of methylated bases, especially 5-methylcytosine, are localized in the DNAregions containing suppressed genes of the amyloplast genome. In sharp contrast, methylated bases were undetectable in the expressed gene regions of amyloplast and whole chloroplast genomes. The overall findings strongly support the notion that DNAmethylation is involved in the selective suppression of photosynthetic genes in the nonphotosynthetic plastids of cultured sycamore cells.

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Infection of the human lymphocyte CEMcell line with the HIV-1 (human immunodeficiency virus type-1, LAV-1 strain) results in cell death. A fluoroquinolone antibiotic, ofloxacin, protected the infected cells from HIV-1-mediated cytolysis. Other fluoroquinolones, e.g. ciprofloxacin, norfloxacin, and enoxacin, also protected the infected cells from HIV-1-mediated cytolysis. The d-isomer of ofloxacin (DR-3354) was about 50- fold less effective than the 1-isomer (DR-3355). Almost none of the rescued cells had detectable HIV-antigens and they could be maintained for long periods in vitro without drugs.

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The biosynthesis and intracellular localization of nonspecific lipid transfer protein (nsLTP) in control human subjects and in patients with peroxisome-deficient disorders were investigated. The molecular mass of human nsLTP was indistinguishable from that of rat nsLTP (13 kDa) by an immunoblot analysis. Intracellular localization was identical with that of catalase, a marker enzyme of peroxisomal matrix, by a double immunofluorescence study. The nsLTP was deficient in liver tissues or fibroblasts from patients with peroxisomedeficient disorders such as Zellweger syndrome and neonatal adrenoleukodystrophy (ALD). Pulse-chase experiments showed that nsLTP was synthesized as a large precursor in both the control and Zellweger fibroblasts. However, the processing to the 13 kDa mature protein was disturbed and the degradation was rapid in Zellweger fibroblasts. After somatic cell fusion using Zellweger fibroblasts from different genetic groups, the processing was normalized. These results suggest that the biosynthesis and localization of human nsLTP are similar to those of rat nsLTP and that the defect of nsLTP in peroxisome-deficient disorders is a phenomenon secondary to an abnormal transport mechanism of peroxisomal proteins. The defect of nsLTP may play an important role in metabolic disturbances in bile acid synthesis and steroidogenesis in peroxisome-deficient disorders.

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Cortical granule breakdown in sea urchin eggs has been investigated with a video microscope system using Nomarski differential interference contrast optics, when induced by fertilization, microinjecting inositol 1, 4, 5-trisphosphate (IP₃) or Ca-EGTA buffer solution into the egg, or per fusing a medium containing 1 mM Ca²⁺ to isolated cortices. The cortical granule increased up to 1.2 times in diameter and broke down within 40 msec. These values were almost constant among the three methods used to induce cortical granule breakdown. Upon fertilization, the cortical granule breakdown propagated over the egg surface at a speed of 3.3 μm/sec in Clypeaster japonicus eggs, which indicates that cortical granule breakdown propagated through the 3.3-μm-wide egg surface within 1 sec. In such a small area of the egg surface, however, it took much more than 1 sec for all cortical granules to break down because the maximal rate of breakdown was 7.6%/sec; that is, it took 9 sec and 18 sec for 50%and 90%, respectively, of cortical granules to break down. Moreover, the rate did not simply decrease with time, and a shoulder was found during the reducing phase, which suggests that cortical granules are divided into fast and slow breakdown groups according to the responsiveness to the breakdown stimulus. The cortical granule breakdown induced by microinjecting the Ca-EGTA buffer and IP₃ solutions propagated at 68 μm/sec and 35 μm/sec, respectively. The stimulus for cortical granule breakdown is discussed concerning the transient intracellular Ca²⁺ increase.

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When macrophages derived from bone marrow were incubated with lignin derivative at 10 jug/ml, they formed multinucleated giant cells containing more than sixty nuclei. Cine-microscopic observation revealed that the formation of the giant cells was due to cell fusion. This phenomenon was specific for primary-cultured macrophages, and was not observed with either a macrophage cell line or established cell lines derived from other tissues.

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