Growth-related changes in histology and immunolocalization of steroid hormone receptors in gonads of the immature male green turtle (Chelonia mydas)

Studies on the population dynamics of sea turtles require histological evaluation of the ontogenetic development and the activity of the gonads for reproduction. To investigate the growth-related changes of gonads in the immature male green turtle (Chelonia mydas), the histological changes of testes and epididymides and the localization of the androgen receptor, estrogen receptor alpha, estrogen receptor beta, and progesterone receptor were examined. The testes were categorized histologically into six developmental stages, and a scarce relationship between straight carapace length and gonadal development was confirmed based on the histological analysis. Several kinds of steroid hormone receptors were examined to show distributions in both testes and epididymides, for which their immunoreactivities were enhanced according to the developmental stage of the testes. These results suggest that straight carapace length is not an adequate indicator of maturity determination, whereas histological and immunohistochemical evaluations are useful in identifying the growth stages of green turtles, owing to the higher sensitivity to steroid hormones that appear during growth.     

CYCLIC NUCLEOTIDE-GATED ION CHANNEL 2 modulates auxin homeostasis and signaling

Cyclic nucleotide-gated ion channels (CNGCs) have been firmly established as Ca²⁺-conducting ion channels that regulate a wide variety of physiological responses in plants. CNGC2 has been implicated in plant immunity and Ca²⁺ signaling due to the autoimmune phenotypes exhibited by null mutants of CNGC2 in Arabidopsis thaliana. However, cngc2 mutants display additional phenotypes that are unique among autoimmune mutants, suggesting that CNGC2 has functions beyond defense and generates distinct Ca²⁺ signals in response to different triggers. In this study, we found that cngc2 mutants showed reduced gravitropism, consistent with a defect in auxin signaling. This was mirrored in the diminished auxin response detected by the auxin reporters DR5::GUS and DII-VENUS and in a strongly impaired auxin-induced Ca²⁺ response. Moreover, the cngc2 mutant exhibits higher levels of the endogenous auxin indole-3-acetic acid, indicating that excess auxin in the cngc2 mutant causes its pleiotropic phenotypes. These auxin signaling defects and the autoimmunity syndrome of the cngc2 mutant could be suppressed by loss-of-function mutations in the auxin biosynthesis gene YUCCA6 (YUC6), as determined by identification of the cngc2 suppressor mutant repressor of cngc2 (rdd1) as an allele of YUC6. A loss-of-function mutation in the upstream auxin biosynthesis gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1, WEAK ETHYLENE INSENSITIVE8) also suppressed the cngc2 phenotypes, further supporting the tight relationship between CNGC2 and the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS–YUCCA -dependent auxin biosynthesis pathway. Taking these results together, we propose that the Ca²⁺ signal generated by CNGC2 is a part of the negative feedback regulation of auxin homeostasis in which CNGC2 balances cellular auxin perception by influencing auxin biosynthesis.

One-sentence summary: The immunity-related Ca²⁺ channel CYCLIC NUCLEOTIDE-GATED CHANNEL 2 modulates auxin homeostasis and balances cellular auxin perception by influencing auxin biosynthesis.     

Outbreak of central nervous system disease associated with hand,foot, and mouth disease in Japan during the summer of 2000: detection and molecular epidemiology of enterovirus 71

Few outbreaks of the serious enterovirus 71 (EV71) infections, which affect the central nervous system (CNS), had been reported in Japan before 2000. During June through August 2000, a patient died of pulmonary edema caused by brainstem encephalitis accompanied by EV71-induced hand, foot, and mouth disease (HFMD), and many patients complicated by serious CNS disease, including paralysis, were hospitalized in a restricted area in Hyogo Prefecture, Japan (K-area). During the same period, endemics of HFMD were reported in other areas in Hyogo Prefecture, where EV71 was isolated from HFMD patients, but few patients developed aseptic meningitis. The isolations of EV71 from K-area patients were difficult with the use of Vero cells, so the strains were isolated by use of GL37 cells; Vero cells, however, could isolate EV71 strains from other areas in Hyogo Prefecture. We sequenced VP4 coding regions of these EV71 isolates and found that the isolates from K-area had the same sequence, which, except for one isolate, was different from the sequences of EV71 strains isolated from other areas of Hyogo Prefecture. Although these results were not enough to state that EV71 from K-area was a virulent strain, it seemed reasonable to conclude that serious CNS diseases in K-area were caused by EV71 because it was the only infectious agent detected in the inpatients of K-area.     

cDNA cloning and predicted primary structure of scallop sarcoplasmic reticulum Ca-ATPase

Sarcoplasmic reticulum (SR) Ca²⁺-ATPase of the scallop cross-striated adductor muscle was purified with deoxycholate and digested with lysyl endopeptidase for sequencing of the digested fragments. Overlapping cDNA clones of the ATPase were isolated by screening the cDNA library with an RT-PCR product as a hybridization probe, which encodes the partial amino acid sequence of the ATPase. The predicted amino acid sequence of the ATPase contained all the partial sequences determined with the proteolytic fragments and consisted of the 993 residues with 70% overall sequence similarity to those of the SR ATPases from rabbit fast-twitch and slow-twitch muscles. An outline of the structure of the scallop ATPase molecule is predicted to mainly consist of ten transmembrane and five ‘stalk’ domains with two large cytoplasmic regions as observed with the rabbit ATPase molecules. The sequence relationship between scallop and other sarco/endoplasmic reticulum-type Ca²⁺-ATPases is discussed.     

Enzymes related to catecholamine biosynthesis in Tetrahymena pyriformis. Presence of GTP cyclohydrolase I.

We first identified GTP cyclohydrolase I activity (EC 3.5.4.16) in the ciliated protozoa, Tetrahymena pyriformis. The V_max value of the enzyme in the cellular extract of T. pyriformis was 255 pmol mg⁻¹ protein h⁻¹. Michaelis–Menten kinetics indicated a positive cooperative binding of GTP to the enzyme. The GTP concentration producing half-maximal velocity was 0.8 mM. By high-performance liquid chromatography (HPLC) with fluorescence detection, a major peak corresponding to -monapterin (2-amino-4-hydroxy-6-[(1′R,2′R)-1′,2′,3′-trihydroxypropyl]pteridine, -threo-neopterin) and minor peaks of -erythro-neopterin and -erythro-biopterin were found to be present in the cellular extract of Tetrahymena. Thus, it is strongly suggested that Tetrahymena converts GTP into unconjugated pteridine derivatives. In this study, dopamine was detected as the major catecholamine, while neither epinephrine nor norepinephrine was identified. Indeed, this protozoa was shown to possess the activity of a dopamine synthesizing enzyme, aromatic -amino acid decarboxylase. On the other hand, activities of tyrosine hydroxylase or tyrosinase which converts tyrosine into dopa, the substrate of aromatic -amino acid decarboxylase, could not be detected in this protozoa. Furthermore, neither dopamine β-hydroxylase activity nor phenylethanolamine N-methyltransferase activity could be identified by the HPLC methods.     

Noise of secretagogue-induced inward currents dependent on extracellular calcium in rat mast cells

We analyzed the noise of the inward currents induced by stimulation of rat peritoneal mast cells with compound 48/80 (48/80), a secretagogue, and examined the role of extracellular Ca2+ in generation of the large noise. In the presence of 2 mM Ca2+ in the external solution, the power density spectra of the 48/80-induced inward currents in most cells were fitted with the sum of two Lorentzian functions. The cut-off frequencies (fc) at -50 mV for the low and high frequency components were 16.3 +/- 7.3 (n = 10) and 180 +/- 95 (n = 9) Hz. Involvement of a cation-selective channel in the large noise was identified in some cells, but the single channel current amplitude estimated from parameters of the noise varied among cells (0.20-2.47 pA at -50 mV), thereby indicating that the currents were mediated by more than two classes of channel. The low frequency component of the 48/80-induced currents was suppressed by lowering the extracellular Ca2+ concentration to 1 microM with the addition of EGTA, without appreciable changes in the high frequency component. When the extracellular Ca2+ was reduced to 1 microM by EGTA 1 min prior to stimulation, 48/80 induced little or no currents in most cells and small currents in some cells. The power density spectra of the small currents were fitted mainly by a single Lorentzian curve with an fc of 150 +/- 5.8 Hz (n = 3). Re-admission of 1.3 mM Ca2+ produced a low frequency part of current noise with an fc of 18.8 (n = 2) Hz.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apoptosis signal-regulating kinase 1 is involved not only in apoptosis but also in non-apoptotic cardiomyocyte death

The molecular basis of myocardial cell death in the ischemia-reperfused heart still remains to be clarified. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in stress-induced apoptosis. We studied ASK1(-/-) mice to examine the role of ASK1 in ischemia-reperfusion injury. In the wild-type heart, ischemia-reperfusion resulted in necrotic injury, whereas infarct size was drastically reduced in the ASK1(-/-) heart. The necrotic injury was not accompanied with any evidence of apoptosis such as an increase in TUNEL-positive cells, DNA fragmentation or the activation of caspase-3. ASK1(-/-) cardiomyocytes were more resistant to H(2)O(2)- or Ca(2+)-induced apoptotic and non-apoptotic cell death compared with wild-type cells. These data suggest that ASK1 is involved in necrosis as well as apoptosis and that ASK1-dependent necrosis is likely to contribute to myocardial cell death in the ischemia-reperfused heart.     

Angiotensin II suppresses growth arrest specific homeobox (Gax) expression via redox-sensitive mitogen-activated protein kinase (MAPK)

Oxidative stress is known to be involved in growth control of vascular smooth muscle cells (VSMCs). We and others have demonstrated that angiotensin II (Ang II) has an important role in vascular remodeling. Several reports suggested that VSMC growth induced by Ang II was elicited by oxidative stress. Gax, growth arrest-specific homeobox is a homeobox gene expressed in the cardiovascular system. Over expression of Gax is demonstrated to inhibit VSMC growth. We previously reported that Ang II down-regulated Gax expression. To address the regulatory mechanism of Gax, we investigated the significance of oxidative stress in Ang II-induced suppression of Gax expression. We further examined the involvement of mitogen-activated protein kinases (MAPKs), which is crucial for cell growth and has shown to be activated by oxidative stress, on the regulation of Gax expression by Ang II. Ang II markedly augmented intracellular H2O2 production which was decreased by pretreatment with N-acetylcystein (NAC), an anti-oxidant. Ang II and H2O2 decreased Gax expression dose-dependently and these effects were blocked by administration of both NAC and pyrrolidine dithiocarbamate (PDTC), another anti-oxidant. Ang II and H2O2 induced marked activation of extracellular signal-responsive kinase1/2 (ERK1/2), which was blocked by NAC. Ang II and H2O2 also activated p38MAPK, and they were blocked by pre-treatment with NAC. However, the level of activated p38MAPK was quite low in comparison with ERK1/2. Ang II- or H2O2 -induced Gax down-regulation was significantly inhibited by PD98059, an ERK1/2 inhibitor but not SB203580, a p38MAPK inhibitor. The present results demonstrated the significance of regulation of Gax expression by redox-sensitive ERK1/2 activation.