Genetic Regulation of Melanocyte Differentiation

Melanocytes originate from the neural crest in vertebrates and migrate to the body surface where they differentiate into functional cells. Genes involved in melanocyte differentiation can be classified into two groups. One of them consists of the functional genes that control proteins specific to the function of the melanocyte. As the representative gene of this category, albino (c) locus in the mouse is considered to control tyrosinase, the key enzyme in melanogenesis. cDNA for mouse tyrosinase has been cloned and sequenced. The cDNA can be used to detect tyrosinase mRNA synthesized during melanocyte differentiation. On the other hand, genes such as brown (b) or pink-eyed dilution (p) have been assumed to control melanosome proteins. The other category consists of genes that regulate the expression of these functional genes directly or indirectly. In the mouse, so-called white-spotting genes and genes of the agouti series are considered to fall into this category. Based on the fact that mutations at the white-spotting loci result in the absence of melanocytes in a particular area of skin, it is assumed that some of these loci control the factors that promote either differentiation or migration of melanoblasts and are candidates for the classic regulator genes Genes at the agouti (a) locus in the mouse determine the type of melanin synthesized in hair follicle melanocytes, that is eumelanin or pheomelanin. An interesting feature of this locus is that the site of gene action is not within the melanocytes but in the cells surrounding them. The results of our study indicate that the gene product of the a-locus interacts with α-MSH at the α-MSH receptor site, regulates the cellular cAMP level via a signal transduction system and, in turn, determines the type of melanin synthesized in the cells.     

Immunofluorescent and immunochemical evidence for the expression of cytovillin in the microvilli of a wide range of cultured human cells

We have previously purified a Mr 75,000 protein, cytovillin, from cultured human choriocarcinoma cells (JEG-3) and shown that this protein was specifically confined to the microvillus membrane of these cells. I have now studied the expression and the subcellular distribution of cytovillin in eighteen normal and transformed human cell lines and strains by using immunoblotting and indirect immunofluorescence microscopy. In all cell types, cytovillin was highly enriched in cell surface protrusions. When cell types were ranked according to their staining intensity, choriocarcinoma was highest, then amniotic epithelial cells, other choriocarcinoma cells and tumor cells, and finally fibroblastoid cells. The latter only gave faint diffuse fluorescence on the plasma membrane and, occasionally, on the microvilli. However, detergent extracts of all cell types could be shown to contain cytovillin by the use of immunoblotting techniques. Metabolic pulse-chase labelling experiments with JEG-3 cells demonstrated synthesis of cytovillin as a single-chain polypeptide. No precursor forms or specific proteolytic cleavage products could be seen either by immunoblotting or immunoprecipitation. The protein was found to be very stable with a biologic half-life of about 25 hours. The pI determined by isoelectric focusing was 6.1. These results were consistent with cytovillin being an integral component of the microvilli and other surface extensions of all human cell types examined.     

Centrifugation of 2-cell mouse ova: cytoplasm stratification and recovery

Summary Two-cell mouse ova, which were centrifuged for l h at 70 000–90 000xg, showed a precise stratification of the cytoplasm and an elongation of the nucleus. The ova were fixed at different times and observed by light and electron microscopy using cytochemical methods and detergent extractions. Within 40 min after centrifugation the normal-looking morphology was recovered except for the persisting lipid caps at the centripetal poles of the blastomeres. Cleavage, compaction and blastulation were not prevented by centrifugation. Treatments with colcemid or cytochalasin D delayed but did not impair recovery. These results suggest that a resilient cytoskeletal structure may be involved in this kind of embryonic regulation.     

Contribution of midbody channels to embryogenesis in the mouse

Summary We have examined the persistence of midbody channels during the second, third, and fourth cleavage cycles of the mouse using immunofluorescence to map the distribution of midbody microtubule bundles in intact embryos. Electron microscopy showed these bundles to be a characteristic feature of midbodies throughout the interphase period. In recently-divided embryos at each cleavage stage the number of midbodies was half the number of blastomeres, and declined towards zero as the next cleavage approached. This indicated to us that the only midbodies present in each stage were those which had arisen in the immediately-preceding division. Of those blastomeres which were in mitosis at the time of fixation, less than 4% were connected via a midbody to another blastomere, demonstrating that persistence of midbodies beyond a single cleavage cycle is a rare event. We conclude that midbody channels in our embryos are likely to connect only pairs of sister blastomeres because midbodies do not persist through multiple cleavage cycles. Midbody channels cannot, therefore, be regarded as providing extensive cell coupling in advance of the onset of gap junctional communication.     

The X-ray induced G2 arrest in mouse eggs: a maternal effect involving a lack of polypeptide phosphorylation

Summary In some strains of mice, eggs when X irradiated during the pronuclear stage, undergo a mitotic block in the G₂ phase of the first cell cycle and cleave when the second division takes place in controls. The importance of this effect varies considerably with the strain and depends exclusively on the maternal genotype. In previous work, two-dimensional electrophoresis showed that eggs blocked at the one-cell stage after irradiation, undergo the same modifications in polypeptide synthesis as two-cell controls of the same age, except at the time of normal first mitosis, where three polypeptide sets of 30, 35 and 45 kDa appear only in cleaving controls. In the present study, we have found phosphorylations in dividing controls, on polypeptides of 30, 35 and 45 kDa. These phosphorylations are not seen in blocked irradiated eggs.     

Initial Studies on the Regulation of Toluene Degradation by Pseudomonas Putida F1

Pseudomonas putida Fl oxidizes toluene through cis-toluene dihydrodiol to 3-methylcatechol. The latter compound is the substrate for “meta” fission of the aromatic nucleus. Kinetic and induction experiments indicate that the genes encoding enzymes for these reactions are part of an operon, designated the tod operon, that is coordinately induced and regulated. Strains unable to utilize toluene as a growth substrate were isolated at high frequencies by using screening procedures that utilize the redox dye, 2,3,5-triphenyl-2H-tetrazolium chloride. Biochemical characterization of strains with mutations in the structural genes of the tod operon showed that toluene induces the first four enzymes in toluene degradation by P. putida Fl. The isolation and characterization of pleiotropicnegative mutants together with mutants altered in terms of their expression of tod genes suggests that the tod operon may be under the control of a positive regulatory element.     

黑河林区驼鹿冬季食性研究

1987—1988年在黑龙江省黑河林区,应用粪便显微组织学分析技术,结合野外啃食调查,对驼鹿冬季食物组成、食物选择性和利用率进行了研究。结果表明,冬季驼鹿共采食31种(属)植物,其中柳、榛、桦、红松、杨和紫椴是主要的冬季食物(19.9％、18.0％、16.7％、14.9％、7.3％和6.7％)。驼鹿对杨、柳、红松和紫椴有正选择性,对榛、桦和毛赤杨有负选择性。选择性的强弱顺序为:杨>柳>红松>紫椴>榛>桦>毛赤杨。驼鹿对柳的选用率最高(32.1％),对桦的利用率最低(12.1％)。     

Heterogeneous distribution of the precursor of type I and type III collagen and fibronectin in the rough endoplasmic reticulum of palatal mesenchymal cells of the mouse embryo cultured in ascorbate-depleted medium

Summary In order to examine the intracellular distribution of precursors of type I and type III collagen and fibronectin in the palatal mesenchymal (MEPM) cells of the mouse embryo cultured under ascorbate-deficient conditions, immuno-electron-microscopic studies were carried out by use of affinity purified antibodies for these proteins. MEPM cells were obtained from the palatal shelves of 14-day-old mouse fetuses and cultured for 3–7 days in medium, either with or without 50 ng/dish/day ascorbic acid. Results obtained were as follows: (1) Although the rough endoplasmic reticulum (rER) of MEPM cells cultured for 5 days in ascorbate-supplemented medium was flattened, that in cells cultured in ascorbate-deficient medium had a distended or vesicular appearance. (2) Vesicular or distended rER showed heterogeneous staining for both type I and type III collagen, namely, some parts of rER showed positive staining for both types of collagen, while others showed negative staining. (3) Both type I and type III collagen showed codistribution in the same vesicular rER. (4) Vesicular rER showed negative or very faint labelling for fibronectin. These results may suggest regional differences in the function of rER.     

Cellular differences in the regeneration of murine skeletal muscle: a quantitative histological study in SJL/J and BALB/c mice

Summary Skeletal muscle regeneration in SJL/J and BALB/c mice subjected to identical crush injuries is markedly different: in SJL/J mice myotubes almost completely replace damaged myofibres, whereas BALB/c mice develop fibrotic scar tissue and few myotubes. To determine the cellular changes which contribute to these differential responses to injury, samples of crushed tibialis anterior muscles taken from SJL/J and BALB/c mice between 1 and 10 days after injury were analysed by light and electron microscopy, and by autoradiography. Longitudinal muscle sections revealed about a 2-fold greater total mononuclear cell density in SJL/J than BALB/c mice at 2 to 3 days after injury. Electron micrographs identified a similar proportion of cell types at 3 days after injury. Autoradiographic studies showed that the proportions of replicating mononuclear cells in both strains were similar: therefore greater absolute numbers of cells (including muscle precursors and macrophages) were proliferating in SJL/J muscle. Removal of necrotic muscle debris in SJL/J mice was rapid and extensive, and by 6 to 8 days multinucleated myotubes occupied a large part of the lesion. By contrast, phagocytosis was less effective in BALB/c mice, myotube formation was minimal, and fibrotic tissue conspicuous. These data indicate that the increased mononuclear cell density, more efficient removal of necrotic muscle, together with a greater capacity for myotube formation in SJL/J mice, contribute to the more successful muscle regeneration seen after injury.     

Occurrence of unusual heterogeneous lipid-containing granule-storing cells in the main excretory duct epithelium of the male mouse submandibular gland

Summary The fine structure of the main excretory duct epithelium of the male mouse submandibular glands was investigated by scanning and transmission electron microscopy. Three principal cell-types were observed: type I and II, and basal cells. This epithelium was characterized by the presence of intercellular canaliculi. Type-I cells were the most numerous. They had an abundance of mitochondria, well-developed Golgi apparatus, a few electron-lucent lipid-containing granules and poorly developed basal infoldings. These cells were also characterized by many glycogen granules throughout the cytoplasm and abundant smooth endoplasmic reticulum in the apical cytoplasm. Type-II cells were the second most numerous. Their most characteristic feature was the presence of abundant heterogeneous lipid-containing granules having acid phosphatase activity at the periphery. They were concentrated in the infra- and supranuclear cytoplasm. The granules may be derived from mitochondrial transformation and seem to be a special kind of secondary autolysosome. Type-II cells also contained abundant mitochondria throughout the cytoplasm, much smooth endoplasmic reticulum in the apical cytoplasm, a well developed Golgi apparatus adjacent to the heterogeneous lipid-containing granules and no basal infoldings. Basal cells were situated adjacent to the basal lamina. They had a large nucleus and the cytoplasm was filled with glycogen granules.