Detection and characterization of new genotypes of Myzus antirrhinii (Hemiptera: Aphididae) in Japan, with evidence for their production of sexual morphs.

Two forms of Myzus persicae (Sulzer), F-15 and R306N', resembling M. antirrhinii (Macchiati) in esterase profile have been found in Japan. To determine the genetic relationship of F-15 and R306N' with M. antirrhinii, clonal lineages of F-15, R306N' and those of 14 other forms of M. persicae collected in Japan were analysed for 17 polymorphic microsatellite DNA loci. Two microsatellite multi-locus (MS) genotypes of M. persicae and two MS genotypes, Ma3 and Ma4, of M. antirrhinii from Australia were used as references. The 16 Japanese forms were represented by different MS genotypes. F-15 and R306N' had, respectively, 44.1% and 35.3% of their alleles at the 17 loci in common with Ma3 and Ma4, and some of the common alleles were peculiar in size. F-15 and R306N' shared 22.6% and 29.6%, respectively, of their alleles with the other 16 MS genotypes of M. persicae. The corresponding values for Ma3 and Ma4 were both 27.4%. F-15 and R306N' were grouped reliably with the two Australian MS genotypes of M. antirrhinii both by neighbour-joining method based on shared allele distance and by a Bayesian method with Markov Chain Monte Carlo algorithm. These results suggest that F-15 and R306N' are genetically closely related to M. antirrhinii and isolated from the gene pool of M. persicae despite their ability to produce sexual morphs. It is therefore proposed that F-15 and R306N' should be classified as M. antirrhinii.

Two amino acid substitutions in acetylcholinesterase associated with pirimicarb and organophosphorous insecticide resistance in the cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae).

The complete coding sequences of two acetylcholinesterase (AChE) genes, Ace1 (orthologous to Drosophila Ace) and Ace2 (paralogous to Ace), from the cotton aphid (Aphis gossypii) were identified and sequences from carbamate resistant and susceptible strains compared. No change in the amino acid sequences was found in Ace1, while two amino acid substitutions, Ser431Phe and Ala302Ser, were detected between resistant and susceptible strains in Ace2. The position of Ser431Phe corresponds to one of fourteen aromatic residues lining the active site gorge and is located in the acyl pocket. Ala302Ser is located at one of the three residues which form the oxyanion hole in the active site of AChE. The Ser431Phe and Ala302Ser substitutions may play a role in pirimicarb and organophosphate resistance, respectively.

An inwardly rectifying K+ channel in bovine parotid acinar cells: possible involvement of Kir2.1.

Using electrophysiological and molecular techniques, we investigated the molecular nature of an inwardly rectifying K+ channel in bovine parotid acinar (BPA) cells and examined its role in setting resting membrane potential. In whole-cell recordings from freshly isolated BPA cells, a predominant current was a K+ current rectified strongly in the inward direction. An inward conductance of the inwardly rectifying K+ (Kir) current was proportional to [K+]o(0.57). The selectivity sequence based on permeability ratios was K+ (1.00) > Rb+ (0.63) >> Li+ (0.04) = Na+ (0.02) and the sequence based on conductance ratios was K+ (1.00) >> Rb+ (0.03) = Li+ (0.03) = Na+ (0.02). The current was blocked by extracellular Ba2+ and Cs+ in a voltage- and a concentration-dependent manner, with a Kd at 0 mV of 11.6 microM and 121 mM, respectively. Cell-attached patch measurements identified 27 pS K+ channels as being the most likely to mediate whole-cell Kir currents. Addition of Ba2+ (100 microM) to the bathing solution reversibly depolarized the resting membrane potential in intact unstimulated cells. RT-PCR of RNA from bovine parotid cells revealed transcripts of bovine Kir2.1 (bKir2.1). HEK293 cells stably expressing bKir2.1 cloned from bovine parotid exhibited whole-cell and single channel Kir currents, of which electrophysiological characteristics were quantitatively similar to those of native Kir currents. Immunohistochemical studies showed a bKir2.1 immunoreactivity in BPA cells. Collectively, these results suggest that Kir2.1 may mediate native Kir currents responsible for setting resting membrane potential in BPA cells and might be, at least in part, involved in spontaneous secretion in ruminant parotid glands.

Abnormal formation of sarcoplasmic reticulum networks and triads during early development of skeletal muscle cells in mitsugumin29-deficient mice.

Recently, we detected a novel membrane protein, mitsugumin29 (MG29), in the triads in rabbit skeletal muscle cells and suggested important roles for this membrane protein in the formation of the sarcoplasmic reticulum (SR) networks and triads in muscle cells. In the present study, we examined the development of skeletal muscle cells in MG29-deficient mice to try to determine the roles played by MG29 in the formation of the SR networks and triads. Ultrastructural observations revealed some morphological abnormalities in these mice, such as incomplete formation of the SR networks, an irregular running of the transverse tubule and a partial defect in the triads at the A-I junctional region. These ultrastructural abnormalities occurred during early myogenesis and were preserved until the adult stage. The possible roles for MG29 in the formation of SR networks and triads in skeletal muscle cells are discussed in the light of these observations.

Deficiency of triad junction and contraction in mutant skeletal muscle lacking junctophilin type 1.

In skeletal muscle excitation-contraction (E-C) coupling, the depolarization signal is converted from the intracellular Ca2+ store into Ca2+ release by functional coupling between the cell surface voltage sensor and the Ca2+ release channel on the sarcoplasmic reticulum (SR). The signal conversion occurs in the junctional membrane complex known as the triad junction, where the invaginated plasma membrane called the transverse-tubule (T-tubule) is pinched from both sides by SR membranes. Previous studies have suggested that junctophilins (JPs) contribute to the formation of the junctional membrane complexes by spanning the intracellular store membrane and interacting with the plasma membrane (PM) in excitable cells. Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle. To examine the physiological role of JP-1 in skeletal muscle, we generated mutant mice lacking JP-1. The JP-1 knockout mice showed no milk suckling and died shortly after birth. Ultrastructural analysis demonstrated that triad junctions were reduced in number, and that the SR was often structurally abnormal in the skeletal muscles of the mutant mice. The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+. Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E-C coupling in skeletal muscle.

Japanese red-bellied newts in Space--AstroNewt experiment on Space Shuttle IML-2 and Space Flyer Unit.

Biological effects of gravity was examined in embryonic development of Japanese red bellied newt. Two space newt missions were conducted in 1994 and 1995. The Second International Microgravity Laboratory was flown in 1994 as one of the SpaceLab missions. Space Flyer Unit, a Japanese space platform, was delivered to the earth orbit by the third launch of the H-II rocket and retrieved by Space Shuttle in 1996. Female newts were induced to lay eggs in orbit at these two space missions. Eggs were successfully obtained on both missions, and exposed to space environment from its early developmental stages. Morphology of the embryos was found not deviated from those developed on ground, as long as in the images taken in orbit or the examined specimen retrieved to ground. On the other hand, pathological changes were discovered in several organs of the adult newts that returned alive from their space flight.

Junctophilins: a novel family of junctional membrane complex proteins.

Junctional complexes between the plasma membrane (PM) and endoplasmic/sarcoplasmic reticulum (ER/ SR) are a common feature of all excitable cell types and mediate cross-talk between cell surface and intracellular ion channels. We have identified the junctophilins (JPs), a novel conserved family of proteins that are components of the junctional complexes. JPs are composed of a carboxy-terminal hydrophobic segment spanning the ER/SR membrane and a remaining cytoplasmic domain that shows specific affinity for the PM. In mouse, there are at least three JP subtypes: JP-1, -2, and -3. JP-2 is abundantly expressed in the heart, and mutant mice lacking JP-2 exhibited embryonic lethality. Cardiac myocytes from the mutant mice showed deficiency of the junctional membrane complexes and abnormal Ca2+ transients. Our results suggest that JPs are important components of junctional membrane complexes.

In vitro organogenesis of pancreas in Xenopus laevis dorsal lips treated with retinoic acid.

Dorsal lips of Xenopus laevis may differentiate into pancreas after treatment with retinoic acid in vitro. The dorsal lip region is fated to be dorsal mesoderm and anterior endoderm. Dorsal lip cells isolated from stage 10 early gastrula differentiate into tissues such as notochord, muscle and pharynx. However, in the present study, dorsal lips treated with 10(-4) M retinoic acid for 3 h differentiated into pancreas-like structures accompanied by notochord and thick endodermal epithelium. Sections of the explants showed that some cells gathered and formed an acinus-like structure as observed under microscopes. In addition to the morphological changes, expressions of the pancreas-specific molecular markers, XIHbox8 and insulin, were induced in retinoic acid-treated dorsal lip explants. Therefore, it is suggested that retinoic acid may induce the dorsal lip cells to differentiate into a functional pancreas. However, continuous treatment with retinoic acid did not induce pancreas differentiation at any concentration. Dorsal lips treated with retinoic acid within 5 h after isolation differentiated into pancreas-like cells, while those treated after 15 h or more did not. The present study provided a suitable test system for analyzing pancreas differentiation in early vertebrate development.

Occurrence of immunoreactive Activin/Inhibin beta(B) in thyrotropes and gonadotropes in the bullfrog pituitary: possible Paracrine/Autocrine effects of activin B on gonadotropin secretion.

Occurrence of immunoreactive activin/inhibin beta(B) in the bullfrog (Rana catesbeiana) pituitary was investigated immunocytochemically by use of antibody against Xenopus activin/inhibin beta(B) subunit. Thyrotropes were demonstrated to contain activin/inhibin beta(B)-immunoreactive substances. Moreover, immunoelectron microscopy revealed that in the secretory granules of thyrotropes and, to a lesser extent, in those of gonadotropes, activin/inhibin beta(B)-immunoreactive substances were present. Based on this observation, we investigated the effect of activin B on the release of gonadotropins from dispersed anterior pituitary cells of the bullfrog. Activin B stimulated the release of not only follicle-stimulating hormone (FSH) but also luteinizing hormone (LH) dose dependently. Under the culture conditions used in this experiment, inhibin B, as well as follistatin, did not affect the basal levels of LH and FSH, but they suppressed the activin-induced release of these hormones. This is the first study on the effect of activin on pituitary hormone secretion in lower tetrapods.

Molecular cloning and characterization of a microsatellite locus found in an RAPD marker of a spider mite, Panonychus citri (Acari: Tetranychidae).

Genetic markers were searched using PCR with 40 kinds of decanucleotide primers to investigate DNA polymorphism in Panonychus citri. A region consisting of a variable number of CT tandem repeats (microsatellite) was found in a fragment amplified with the OPB10 primer. The microsatellite differed in size by ca. 100bp among several P. citri populations screened and was derived from at least seven alleles. This region was characteristic of P. mori and P. osmanthi, but was lacking in P. ulmi. The flanking regions were highly conserved among these species.

In vitro pancreas formation from Xenopus ectoderm treated with activin and retinoic acid.

In the present study, isolated presumptive ectoderm from Xenopus blastula was treated with activin and retinoic acid to induce differentiation into pancreas. The presumptive ectoderm region of the blastula consists of undifferentiated cells and is fated to become epidermis and neural tissue in normal development. When the region is isolated and cultured in vitro, it develops into atypical epidermis. Isolated presumptive ectoderm was treated with activin and retinoic acid. The ectoderm frequently differentiated into pancreas-like structures accompanied by an intestinal epithelium-like structure. Sections of the explants viewed using light and electron microscopy showed some cells clustered and forming an acinus-like structure, including secretory granules. The pancreas-specific molecular markers insulin and XIHbox8 were also expressed in the treated explants. The pancreatic hormones, insulin and glucagon, were detected in the explants using immunohistochemistry. Therefore, sequential treatment with activin and retinoic acid can induce presumptive ectoderm to differentiate into a morphological and functional pancreas in vitro. When ectoderm was immediately treated with retinoic acid after treatment with activin, well-differentiated pronephric tubules were seen in a few of the differentiated pancreases. Treatment with retinoic acid 3-5 h after activin treatment induced frequent pancreatic differentiation. When the time lag was longer than 15h, the explants developed into axial mesoderm and pharynx. The present study provides an effective system for analyzing pancreas differentiation in vertebrate development.

Interspecific diversity of mitochondrial COI sequences in Japanese Panonychus species (Acari: Tetranychidae).

Sequences of a part of the mitochondrial cytochrome oxidase subunit I (COI) gene were analyzed in four Japanese Panonychus species to determine their phylogenetic relationships. Neighbor-Joining and maximum likelihood analysis resulted in a high bootstrap support of the relationships within the genus Panonychus. In contrast with a previous study based on ribosomal DNA data, the COI phylogeny suggested that P. mori was more distantly related to P. citri than to P. ulmi. This study shows for the first time that P. osmanthi is closely related to P. citri. Intraspecific variation analysis shows that the genetic distance between two local populations of P. mori is higher than between P. citri and P. osmanthi.

Abnormal features in skeletal muscle from mice lacking mitsugumin29.

Physiological roles of the members of the synaptophysin family, carrying four transmembrane segments and being basically distributed on intracellular membranes including synaptic vesicles, have not been established yet. Recently, mitsugumin29 (MG29) was identified as a novel member of the synaptophysin family from skeletal muscle. MG29 is expressed in the junctional membrane complex between the cell surface transverse (T) tubule and the sarcoplasmic reticulum (SR), called the triad junction, where the depolarization signal is converted to Ca(2+) release from the SR. In this study, we examined biological functions of MG29 by generating knockout mice. The MG29-deficient mice exhibited normal health and reproduction but were slightly reduced in body weight. Ultrastructural abnormalities of the membranes around the triad junction were detected in skeletal muscle from the mutant mice, i.e., swollen T tubules, irregular SR structures, and partial misformation of triad junctions. In the mutant muscle, apparently normal tetanus tension was observed, whereas twitch tension was significantly reduced. Moreover, the mutant muscle showed faster decrease of twitch tension under Ca(2+)-free conditions. The morphological and functional abnormalities of the mutant muscle seem to be related to each other and indicate that MG29 is essential for both refinement of the membrane structures and effective excitation-contraction coupling in the skeletal muscle triad junction. Our results further imply a role of MG29 as a synaptophysin family member in the accurate formation of junctional complexes between the cell surface and intracellular membranes.

Immunolocalization of mitsugumin29 in developing skeletal muscle and effects of the protein expressed in amphibian embryonic cells.

The temporal appearance and subcellular distribution of mitsugumin29 (MG29), a 29-kDa transmembrane protein isolated from the triad junction in skeletal muscle, were examined by immunohistochemistry during the development of rabbit skeletal muscle. MG29 appeared in the sarcoplasmic reticulum (SR) in muscle cells at fetal day 15 before the onset of transverse tubule (T tubule) formation. In muscle cells at fetal day 27, in which T tubule and triad formation is ongoing, both SR and triad were labeled for MG29. In muscle cells at newborn 1 day, the labeling of the SR had become weak and the triads were well developed and clearly labeled for MG29. Specific and clear labeling for MG29 was restricted to the triads in adult skeletal muscle cells. When MG29 was expressed in amphibian embryonic cells by injection of the cRNA, a large quantity of tubular smooth-surfaced endoplasmic reticulum (sER) was formed in the cytoplasm. The tubular sER was 20-40 nm in diameter and appeared straight or reticular in shape. The tubular sER was formed by the fusion of coated vesicles [budded off from the rough-surfaced endoplasmic reticulum (rER)] and vacuoles of rER origin. The present results suggest that MG29 may play important roles both in the formation of the SR and the construction of the triads during the early development of skeletal muscle cells.

Degradation of yolk platelets in the early amphibian embryo is regulated by fusion with late endosomes.

The eggs of many animal species contain a large store of yolk platelets, lipid droplets and glycogen granules; these are consumed during early embryogenesis. However, the mechanisms by which degradation of these stored materials occurs during early embryogenesis are not clearly understood. The mechanisms underlying yolk degradation in amphibian (newt) embryos were investigated. Electron microscopy using an anion marker, cationic ferritin, revealed that yolk platelets were degraded after fusion with late endosomes containing primary lysosomes. Electron microscopy and the results of experiments using a number of reagents with selective effects on intracellular transport suggested that yolk degradation activity in early amphibian embryos may be regulated at the point of fusion between late endosomes and yolk platelets.

HPC-1/syntaxin 1A suppresses exocytosis of PC12 cells.

The membrane protein syntaxin (originally named HPC-1) is involved in vesicle trafficking and required for neurotransmitter release at nerve terminals. The presence of syntaxin on target membranes is hypothesized to confer specificity to targeting and fusion via interactions with complementary vesicle-associated proteins. To elucidate the function of syntaxin 1A in exocytosis, HPC-1/syntaxin 1A-reduced PC12h cells (PC12h/Deltasyx) that were stably transfected with a plasmid for antisense syntaxin 1A expression were constructed. Depolarizing stimulation of PC12h/Deltasyx enhanced dopamine release, compared with PC12h. There was a strong inverse correlation between syntaxin 1A protein expression and enhancement of dopamine release. Reduction of syntaxin 1A had no effect on increase of the cytoplasmic free Ca2+ concentration by depolarized stimulation. Moreover, PC12h/Deltasyx clones similarly enhanced of exocytosis by native secretagogues. These results indicate that syntaxin 1A has more than one function in exocytosis.

Morphological abnormalities of adrenal gland and hypertrophy of liver in mutant mice lacking ryanodine receptors.

Ryanodine receptors (RyRs), which form Ca2+ channels in the membrane of the endoplasmic reticulum, consist of three subtypes (RyR1, RyR2, and RyR3). The RyRs release Ca2+ from the endoplasmic reticulum into the cytoplasm and thus play an important role, especially in the contraction of skeletal and cardiac muscle cells. The genes of these RyRs are also expressed in many non-muscle tissues, but the role played by RyRs in non-muscle cells is not fully understood. In the present study, we examined the morphological changes in such cells caused by a deficiency of RyRs genes using three mutant mice lacking RyR1, RyR3, or both RyR1 and RyR3. The results showed morphological abnormalities in the adrenal cortical cells in all three mutant mice. In addition, an excessive accumulation of glycogen granules in hepatic cells, and a hypertrophy of the liver were both present in those mutant mice lacking both RyR1 and RyR3. We discuss the relationship between the morphological abnormalities of the adrenal cortex and liver induced by a deficiency of RyRs, and the possible causes of these abnormalities.

Effect of calcium on development of amiloride-blockable Na+ transport in axolotl in vitro.

The axolotl, Ambystoma mexicanum, which has no specific calcium-containing sieve layer in the dermis, provides useful material for the study of the effect of Ca2+ on the development of amiloride-blockable active Na+ transport across the skin of amphibians. We raised axolotls in thyroid hormone or aldosterone or cultured the skin with corticoid plus one of several Ca2+ concentrations and found that 1) although the short-circuit current (SCC) was increased by both aldosterone and 3,3',5-triiodo-L-thyronine in vivo, only corticoid was necessary for such an increase in vitro; 2) the development of the SCC in vitro was both corticoid and Ca2+ dependent, because the SCC was well developed with over 100 microM Ca2+ but not with under 10 microM Ca2+ in the presence of corticoid, nor even with 300 microM Ca2+ without corticoid; and 3) Ca2+, but not corticoid, was necessary for the formation of cell-to-cell junctions, because the resistance of the skin was well developed with 300 microM Ca2+ without corticoid.

Structure and expression of mitsugumin29 gene.

Recently mitsugumin29 unique to the triad junction in skeletal muscle was identified as a novel member of the synaptophysin family; the members of this family have four transmembrane segments and are distributed on intracellular vesicles. In this study, we isolated and analyzed mouse mitsugumin29 cDNA and genomic DNA containing the gene. The mitsugumin29 gene mapped to the mouse chromosome 3 F3-H2 is closely related to the synaptophysin gene in exon-intron organization, which indicates their intimate relationship in molecular evolution. RNA blot hybridization and immunoblot analysis revealed that mitsugumin29 is expressed abundantly in skeletal muscle and at lower levels in the kidney. Immunofluorescence microscopy demonstrated that mitsugumin29 exists specifically in cytoplasmic regions of the proximal and distal tubule cells in the kidney. The results obtained may suggest that mitsugumin29 is involved in the formation of specialized endoplasmic reticulum systems in skeletal muscle and renal tubule cells.