Phosphorylation of F-actin-associating G protein gamma12 subunit enhances fibroblast motility

Eleven isoforms of G protein gamma subunit have been found thus far, but the precise roles of individual gamma subunits are not known. The gamma12 subunit has two unique properties: phosphorylation by protein kinase C and association with F-actin. To elucidate the role of gamma12, we overexpressed gamma12 and other gamma subunits in NIH 3T3 cells together with the beta1 subunit. The overexpressed gamma12 as well as endogenous gamma12, but not gamma2, gamma5, and gamma7 subunits, associated with cytoskeletal components. Expression of gamma12 induced remarkable changes including cell rounding, disruption of stress fibers, and enhancement of cell migration, but expression of other gamma subunits did not induce significant changes. Deletion of the N-terminal region of gamma12 decreased the abilities of gamma12 to associate with cytoskeletal fractions, to induce cell rounding, and to increase cell motility. Replacement by alanine of Ser2 of gamma12 (Ser1 of a mature gamma12 protein), a phosphorylation site for protein kinase C, eliminated these effects of gamma12, whereas a mutant in which Ser2 was replaced with glutamic acid showed effects equivalent to wild-type gamma12. These results indicate that phosphorylation of gamma12 at Ser2 enhances the motility of cells.     

Synthesis and degradation of a 28-kDa pod storage protein in French bean (Phaseolus vulgaris) plants

Pod storage protein (PSP) accumulated in developing pods of French bean (Phaseolus vulgaris L.) plants, and increasing the PSP mRNA level by pod removal resulted in the enhancement of PSP accumulation in pods that formed later. Pod storage protein was detected in flowers, young leaves and young stem internodes in addition to pods. Accumulation of PSP and its mRNA was induced by sink-removal in an organ-specific manner. In addition, wounding induced PSP accumulation systemically in leaves. Methyl jasmonate did not induce PSP synthesis but enhanced the synthesis that was induced by wounding. In senescing pods, PSP was degraded, and degradation products with molecular masses of 20 and 17 kDa were detected in the pods. The amount of 20-kDa degradation product was greater than that of the 17 kDa product. Received: 26 May 1999 / Accepted: 24 June 1999     

The role of serum imbibition for skin grafts

Numerous studies of grafted skin suggest that full-thickness skin grafts are nourished by exudate from the recipient bed called a serum imbibition. However, whether serum imbibition by itself is sufficient for nourishment of skin grafts has not been shown definitely and directly. To clarify the role of serum imbibition, we performed a comparative study between 20 skin grafts and 20 musculocutaneous flaps. The nourishment of the cell in the skin graft is by serum imbibition. That in musculocutaneous flaps is mainly derived from blood supply. We evaluated the nourishment by means of the unique characteristics of the cell cycle. Once cells are put into a synthetic phase, they cannot reverse or stop the progress of the cell cycle. To take advantage of this characteristic of the cell cycle, prewounding methods (40 flaps were lifted once and put back to the original sites prior to the evaluation) were intended for the cells in pre-elevated skin to turn into a proliferating phase. Cells were examined by antibody against proliferating cell nuclear antigen immunohistologically, to determine whether they had turned into the proliferating phase or not. After 3 days, all flaps were reelevated; half (20 flaps) had their muscle layer and the neurovascular bundle removed to make a full-thickness skin graft. The rest (20 flaps) were only lifted. They were sutured back to the original sites. Ten skin grafts and musculocutaneous flaps each were harvested at 3 hours (1st day) and at 11 days (11th day) after the second operation. Bromodeoxyuridine, which is a thymidine analog and is taken into the cells in the synthetic phase, was introduced intraperitoneally 2 hours before the harvest. All flaps and grafts were evaluated histologically and immunohistologically. Proliferating cell nuclear antigen analysis showed that the prewounding method induced the cells of skin grafts and musculocutaneous flaps to proliferate before the implantation. Regarding the bromodeoxyuridine uptake, no significant differences could be seen between skin grafts and musculocutaneous flaps irrespective of their different nourishment. No structural changes, such as degenerative or necrotic, could be seen at the hair follicle and other glands even at the 11th day. Almost all of the layers of skin grafts survived as long as they were checked by light microscopy (hematoxylin and eosin stain). No differences could be seen between musculocutaneous flaps and skin grafts or between the 1st and 11th days in this study. We concluded that serum imbibition is sufficient for nourishment of skin grafts, just as blood supply is sufficient for nourishment of musculocutaneous flaps.     

Differential regulation of adenine nucleotide translocators by hypertonicity in the brain

To determine the gene(s) induced by hypertonicity in the brain, we performed a differential display analysis using RNA isolated from isotonic and hypertonic rat astrocytes. One cDNA rapidly up-regulated by hypertonicity was isolated, and the DNA sequence revealed that it was identical to adenine nucleotide translocator (ANT)2. ANT2 protein exchanges intramitochondrial ATP for cytoplasmic ADP. Among three ANT isoforms, only ANT2 mRNA was up-regulated markedly from 1 to 4 h after exposure to hypertonicity. Induction of the mRNA did not require de novo protein synthesis. Furthermore, ADP translocase activity in mitochondria of astrocytes was increased significantly by hypertonicity. To see the localization and regulation of ANT2 mRNA in the brain, we performed in situ hybridization of rat brain after intraperitoneal injection of a high concentration of NaCl. Although there were only weak signals in the control, intense hybridization signals were seen in hypertonic rat whole brain. Microscopic examination showed that ANT2 signals were present in the neurons, as well as glial cells. These results suggest that ANT2 may play a role in brain cells to adapt to the hypertonic environment.     

Estradiol-17beta stimulates the renewal of spermatogonial stem cells in males.

In this work, we examined the functions of the female hormone "estrogen" on spermatogenesis of the Japanese eel (Anguilla japonica). Estradiol-17beta (E(2)), a natural estrogen in vertebrates, was present in the serum and its receptor was expressed in the testis during the whole process of spermatogenesis. Spermatogonial stem cell renewal was promoted by E(2) implantation but was suppressed by tamoxifen (an antagonist of estrogen). In vitro, 10 pg/ml of E(2) was sufficient to induce spermatogonial stem cell division in cultured testicular tissue, therefore confirming the in vivo observations. These results clearly show that estrogen is an indispensable "male hormone" in the early spermatogenetic cycle.     

Oxidative phosphorylation in Micrococcus denitrificans. II. The properties of pyridine nucleotide transhydrogenase

A pyridine nucleotide transhydrogenase activity, supported by ATP or by succinate oxidation, was demonstrated in phosphorylating membrane fragments from Micrococcus denitrificans. The ATP-supported reaction was inhibited by various energy-transfer inhibitors and uncouplers or by treatment with high concentrations of LiCl. P_i and arsenate showed a stimulatory effect on the ATP-supported activity; half-maximal stimulation was attained by about 80 μM phosphate.

The transhydrogenase reaction dependent on succinate oxidation was not appreciably inhibited by energy-transfer inhibitors, although oleate and pentachlorophenol were almost equally effective in both reactions. P_i did not stimulate the succinate-supported activity.

From the effects of thyroxine and its derivatives on the energy-dependent and independent reductions of NAD⁺ by NADPH, the involvement of the same transhydrogenase enzyme in both reactions was suggested.

These and other results indicated that the energy-transfer system of M. denitrificans was very similar to, though not identical with, that of mammalian mitochondria.     

MICROSATELLITE MARKERS REVEAL POPULATION GENETIC STRUCTURE OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM TAMARENSE (DINOPHYCEAE) IN JAPANESE COASTAL WATERS1

This is the first report to explore the fine‐scale diversity, population genetic structure, and biogeography of a typical planktonic microbe in Japanese and Korean coastal waters and also to try to detect the impact of natural and human‐assisted dispersals on the genetic structure and gene flow in a toxic dinoflagellate species. Here we present the genetic analysis of Alexandrium tamarense (Lebour) Balech populations from 10 sites along the Japanese and Korean coasts. We used nine microsatellite loci, which varied widely in number of alleles and gene diversity across populations. The analysis revealed that Nei's genetic distance correlated significantly with geographic distance in pair‐wise comparisons, and that there was genetic differentiation in about half of 45 pair‐wise populations. These results clearly indicate genetic isolation among populations according to geographic distance and restricted gene flow via natural dispersal through tidal currents among the populations. On the other hand, high P‐values in Fisher's combined test were detected in five pair‐wise populations, suggesting similar genetic structure and a close genetic relationship between the populations. These findings suggest that the genetic structure of Japanese A. tamarense populations has been disturbed, possibly by human‐assisted dispersal, which has resulted in gene flow between geographically separated populations.     

Anatomy of the vessel network within and between tree rings of Fraxinus lanuginosa (Oleaceae)

The three-dimensional (3-D) arrangement of vessels and the vessel-to-vessel connections in the secondary xylem of the stem of the ring-porous hardwood tree Fraxinus lanuginosa were studied in series of thick transverse sections with epifluorescence microscope and confocal laser scanning microscope. Vessels were traced in sequential sections, and vessel networks were reconstructed in two segments of wood with dimensions of 2 × 1.4 × 21.2 mm(3) and 2 × 1.4 × 5.8 mm(3) (tangential × radial × axial). The arrangement of vessels and intervessel pits were visualized by scanning electron microscopy in low-density polyethylene microcasts and on exposed tangential faces of growth-ring boundaries. The vessels deviated from the stem axis in the tangential direction and, to a lesser extent, in the radial direction. Some neighboring vessels were twisted around each other. Vessels that appeared solitary in single sections were found to be sequentially contiguous with a number of other vessels, forming networks that extended in the tangential direction and across growth-ring boundaries. In the 21.2-mm wood block, all earlywood vessels at the growth-ring boundary made contact with latewood vessels in the previous tree ring. Within a growth ring however, only a single contact was observed between individual earlywood and latewood vessels. Densely arranged intervessel pits were characteristic in the regions where adjacent vessels made contact with each other. Such bordered pits were abundant in the tangential walls of vessel elements adjacent to growth-ring boundaries. Therefore, bordered pits appear to provide the pathway for the radial transport of water via the vessel network across growth-ring borders. Fiber-tracheids, observed as terminal cells in the tree rings, might also contribute to the apoplastic transfer of water across ring borders.