Morphogenetic activity of extracellular matrices on cultured podocytes. Laminin accelerates podocyte process formation in vitro.

Morphogenetic effects of various extracellular matrix proteins on the renal podocyte were investigated using the conditionally immortalized podocyte cell line. Podocytes were plated on glass coverslips and coated with the following matrix proteins: laminin-10/11, laminin-1, fibronectin, collagen type IV, collagen type I. Three hours after plating, podocytes on laminins developed prominent processes, while those on other matrix proteins started to elongate processes after two days. Vinculin-immunolabeling showed that podocytes plated on laminins possessed thin rod-shaped focal contacts, whereas those on fibronectin showed large dot-shaped focal contacts. Inhibition of serine/threonine protein kinases induced podocyte process formation in an extracellular matrix-independent manner. The present study reveals the significance of laminin on podocyte morphogenesis in vitro, and shows that different extracellular matrix proteins trigger different intracellular signals governing podocyte morphogenesis. Taken together with our previous studies, podocyte process formation is thought to be regulated by protein Ser/Thr phosphorylation.

Herpes simplex virus in the vestibular ganglion and the geniculate ganglion-role of loose myelin.

This study presents the first direct evidence for herpes simplex virus type 1 (HSV-1) infection in the neurons of the vestibular ganglion. Although many investigators have reported electron microscopic evidence of HSV-1 infection in sensory ganglia, HSV-1 infection in the vestibular ganglion has not been described. Vestibular ganglion neurons have a unique structure, with a loose myelin sheath instead of the satellite cell sheath that is seen in other ganglia. This loose myelin is slightly different from compact myelin which is known as too tight for HSV-1 to penetrate. The role of loose myelin in terms of HSV-1 infection is completely unknown. Therefore, in an attempt to evaluate the role of loose myelin in HSV-1 infection, we looked for HSV-1 particles, or any effects mediated by HSV-1, in the vestibular ganglion as compared with the geniculate ganglion. At the light microscopic level, some neurons with vacuolar changes were observed, mainly in the distal portion of the vestibular ganglion where the communicating branch from the geniculate ganglion enters. At the electron microscopic level, vacuoles, dilated rough endoplasmic reticulum and Golgi vesicles occupied by virus were observed in both ganglia neurons. In contrast, viral infections in Schwann and satellite cells were observed only in the geniculate ganglion, but not in the vestibular ganglion. These results suggest that loose myelin is an important barrier to HSV-1 infection, and it must play an important role in the prevention of viral spread from infected neurons to other cells.

[Regulation of the microtubule-dependent process formation. A review based on a comparison between the neuron and the renal glomerular podocyte].

Microtubular cytoskeletons play a crucial role in the morphogenesis of process-bearing cells, such as the neuron and the renal glomerular podocyte. Microtubules are bundled and stabilized by various microtubule-associated proteins, providing a mechanical basis to maintain the deviated morphology of cell processes. To support the process morphology, microtubules are also associated with other cytoskeletal elements such as actin and intermediate filaments. The microtubular polarity is uniformly plus-end-distal in neuronal axons, whereas in dendrites as well as in podocytes, the polarity is revealed to be non-uniform (i.e., both plus-end-distal and minus-end-distal microtubules are present in cell processes). Recently, this non-uniformity is reported to be established by a microtubule-dependent motor protein. Motor proteins are capable to drive the intracellular transport of cytoskeletal elements in addition to that of membrane vesicles. It is still an open question whether cytoskeletal elements are transported along cell processes as subunits or as polymers.

[Morphological transformation of sensory ganglion neurons and satellite cells].

Sensory ganglion neurons in higher vertebrates are unique in that they are pseudounipolar with a single stem process that divides at some distance from the cell body into central and peripheral processes. In the early stages of development, these neurons are bipolar but later they became pseudounipolar. This developmental process of sensory ganglion neurons with satellite cells was examined by scanning electron microscopy following removal of connective tissue. This pseudo-unipolarization began earlier but proceeded at a slower rate in chick than in rat embryos. This difference may due to the difference found in the extent and intimacy of satellite cell investments in these two animals, which was due to the fact that sensory neurons undergo pseudo-unipolarization only in the presence of satellite cells in vitro. The neuronal perikaryal projections were observed by scanning electron microscopy after removal of connective tissue and satellite cells. Morphometric analysis reveal that perikaryal projections were more numerous on the surface of mature pseudounipolar neurons than on the surface of premature bipolar neurons, and that the number of projections increased as the neuronal cell bodies grew larger. This may support the hypothesis that perikaryal projections are structural devices for increasing the neuron-satellite interface and for improving the efficiency of metabolic exchange between these two cell types. These results suggest that satellite cells play an important role in neuronal maturation.

Seasonal variations of blood haptoglobin level of brown bears in Japan.

Haptoglobin (Hp), a hemoglobin-binding protein, is known as an acute phase protein increasing in blood during inflammation in most mammals. On the basis of our previous studies on purification and characterization of bear Hp (Comp. Biochem. Physiol. 110B, 785-789, 1995), in this study, we developed an immunoassay method to measure serum Hp level in bear, and measured the concentration of Hp in blood samples collected from 84 reared and 25 wild brown bears in Hokkaido, Japan. The mean serum Hp concentration was 0.94 +/- 0.25 mg/ml in wild bears, which is nearly equal to those reported in other species. In reared bears, the Hp concentration was apparently higher (3.82 +/- 0.29 mg/ml), although total protein and albumin concentrations were nearly equal in the two groups. A significant seasonal variation of serum Hp, low in spring and high in autumn and winter, was found in reared bears. Possible factors participating in the seasonal variation were discussed with special references to hibernation.

Haptoglobin in Carnivora: a unique molecular structure in bear, cat and dog haptoglobins.

Haptoglobin (Hp), a hemoglobin-binding protein in plasma, consists of alpha and beta subunits and has a tetra-chain arrangement (beta-alpha-alpha-beta) connected by disulfide bridges in most mammals so far examined. Dog Hp has been reported to be unique compared with other Hps in respect that (1) the two alpha beta units are joined by a non-covalent interaction rather than a disulfide bridge and (2) the alpha chain has an oligosaccharide-binding sequence (Asn-X-Ser/Thr) and is glycosylated. To determine whether the unique structures of dog Hp are common in the Carnivora, we purified Hps from sera of bear and cat, and analyzed their subunit structure and partial amino acid sequences. The analyses by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and non-reducing conditions, revealed that bear and cat Hps have similar subunit arrangements to dog Hp, suggesting the absence of a disulfide bridge between two alpha chains. This was confirmed by amino acid sequence analysis of the alpha chains: that is, Cys15 participating in the inter-alpha chain disulfide bridge was replaced by Val in bear or Leu in cat and dog. Thus, the unique subunit arrangement of Hp reported in dog may be common in the Carnivora. In contrast to dog Hp, however, alpha chains of bear and cat Hps were found not to have the typical oligosaccharide binding sequence on their alpha chains and were not glycosylated.

Isolation and primary culture of bovine hepatocytes: albumin synthesis and adrenergic activation of glycogenolysis.

We describe a technique for isolation and primary culture of bovine hepatocytes, and their metabolic characterization. Hepatocytes were isolated from the caudate lobe of bovine liver by perfusion with 0.25 mM ethylene-glycol tetraacetic acid and 0.05% collagenase. The viability and yield of the cells were 70-92% and 0.1-3.6 x 10(7) cells/g liver, respectively. When the isolated hepatocytes were cultured in Williams' medium E, they began to spread in 3 hr and formed monolayers in 24 hr. These monolayers were retained for at least 6 days. To monitor the metabolic activities specific to liver, synthesis and secretion of albumin were measured by labeling with [35S]-methionine and immunoprecipitation. This activity was low in isolated hepatocytes, but increased after culturing 1-3 days, and decreased again after 6 days. Glycogenolytic activity was also assessed by measuring glucose release to the medium by stimulation with epinephrine. The glycogenolytic response to epinephrine was also enhanced by culturing the hepatocytes 1-3 days, but was decreased after 6 days. Since the isolated bovine hepatocytes retained the liver-specific activities of albumin synthesis and glycogenolysis for several days in culture, these cells are useful for cellular and molecular studies on the functions of bovine liver.