Human mitogen-activated protein kinase kinase 7 (MKK7) is a highly conserved c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activated by environmental stresses and physiological stimuli

We report the cloning of a novel human activator of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 7 (MKK7). The mRNA for MKK7 is widely expressed in humans and mice and encodes a 47-kDa protein (419 amino acids), as determined by immunoblotting endogenous MKK7 with an antibody raised against its N terminus. The kinase domain of MKK7 is closely related to a Drosophila JNK kinase dHep (69% identity) and to a newly identified ortholog from Caenorhabditis elegans (54% identity), and was more distantly related to MKK4, MKK3, and MKK6. MKK7 phosphorylated and activated JNK1 but failed to activate p38 MAPK in co-expression studies. In hematopoietic cells, endogenous MKK7 was activated by treatment with the growth factor interleukin-3 (but not interleukin-4), or by ligation of CD40, the B-cell antigen receptor, or the receptor for the Fc fragment of immunoglobulin. MKK7 was also activated when cells were exposed to heat, UV irradiation, anisomycin, hyperosmolarity or the pro-inflammatory cytokine tumor necrosis factor-alpha. Co-expression of constitutively active mutants of RAS, RAC, or CDC42 in HeLa epithelial cells or of RAC or CDC42 in Ba/F3 factor-dependent hematopoietic cells also activated MKK7, suggesting that MKK7 will be involved in many physiological pathways.     

Effects of coenzyme Q on plasma aldosterone concentrations in dogs (author's transl)

The renin-angiotensin system, potassium and adrenocorticotropin (ACTH) are well known as control mechanism for aldosterone secretion. However, the precise mechanism of these factors for aldosterone secretion remain still unclear. Several interesting evidences related to the effects of Coenzyme Q on the secretion and biosynthesis of aldosterone have been demonstrated. Biochemical action of Coenzyme Q is generally accepted as a component of the electron transfer process of respiration in mitochondria. Fabre et al demonstrated that significant reduction of plasma aldosterone concentration in adrenal venous by the Coenzyme Q infusion. Weinstein et al observed that urine sodium excretion decreased after infusion of Coenzyme Q into renal artery. Kumagai et al suggested that Coenzyme Q inhibited the activity of 18-hydroxylase in the adrenal cortex. The present study was designed to evaluate the effects of Coenzyme Q on the secretion of aldosterone. Method: 24 cases of male among el and beagle dogs were subdivided into 4 groups. 1st group were administered intravenous infusion of Coenzyme Q, 2nd group were orally administered Coenzyme Q for 7 weeks, 3rd group were administered simultaneous infusion of Coenzyme Q and angiotensin II and 4th group were administered furosemide orally under the condition of continuous Coenzyme Q administration. Then, plasma concentrations of aldosterone, 11-OHCS and angiotensin I were determined during the time course. Results and Discussions: Plasma aldosterone concentration was significantly increased after intravenous infusion of angiotensin II and decreased 45 minutes after the beginning of infusion. However, the concentration still remained higher than control level. By the simultaneous infusion of Coenzyme Q with angiotensin II, the decreased concentration again increased significantly. It seems that above mentioned results suggest possibility that Coenzyme Q potentiate the action of angiotensin II on aldosterone secretion. By the intravenous infusion of Coenzyme Q, plasma aldosterone concentration increased significantly and concentrations of plasma 11-OHCS and angiotensin I did not affected. This result suggests that Coenzyme Q may stimulate aldosterone secretion from adrenal cortex without increase of ACTH and renin-angiotensin. 4 hours after the oral administration of Coenzyme Q, plasma aldosterone concentration was increased significantly. Na/K in 24 hours' excreta was decreased by Coenzyme Q administration. The decrease of Na/K in excreta may be reflection of the increase of aldosterone secretion. Although, plasma aldosterone concentration increased for the short duration by the Coenzyme Q, it decreased gradually and returned to the control level after 7 days under the condition of continuous oral administration. By the oral administration of furosemide under prolonged Coenzyme Q administration plasma aldosterone concentration increased significantly and remained higher than that of control.     

The multiligand-binding protein gC1qR, putative C1q receptor, is a mitochondrial protein

A protein of 33 kDa (p33) that tightly binds to the globular domains of the first complement component, C1q, is thought to serve as the major C1q receptor (gC1qR) on B cells, neutrophils, and mast cells. However, the cellular routing and the subcellular localization of p33/gC1qR are unknown. We have performed confocal laser-scanning microscopy and found that p33/gC1qR is present in intracellular compartments, where it colocalizes with the mitochondrial marker protein, pyruvate dehydrogenase. No surface staining for p33/gC1qR on endothelial EA.hy926 cells was observed. A fusion protein of the p33/gC1qR presequence with green fluorescent protein translocated to the mitochondria of transfected COS-7 cells. Concomitantly, a 6-kDa portion of the fusion protein was proteolytically removed. The 33 amino-terminal residues of the presequence proved sufficient to direct reporter constructs to mitochondria. Association of p33/gC1qR with mitoplasts indicated that the mature protein of 209 residues resides in the matrix and/or the inner membrane of mitochondria. Immunocytochemistry of fetal mice tissues revealed a ubiquitous expression of p33/gC1qR, most prominently in tissues that are rich in mitochondria. Thus, the candidate complement receptor p33/gC1qR of intact cells cannot interact with plasma C1q due to mutually exclusive localizations of the components. The functional role of p33/gC1qR needs to be reconsidered.     

Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p involved in mRNA processing and transport

RanGAP1 is the GTPase activator for the nuclear Ras-related regulatory protein Ran, converting it to the putatively inactive GDP-bound state. Here, we report the amino acid sequence of RanGAP1, derived from cDNA and peptide sequences. We found it to be homologous to murine Fug1, implicated in early embryonic development, and to Rna1p from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Mutations of budding yeast RNA1 are known to result in defects in RNA processing and nucleocytoplasmic mRNA transport. Concurrently, we have isolated Rna1p as the major RanGAP activity from Sc. pombe. Both this protein and recombinant Rna1p were found to stimulate RanGTPase activity to an extent almost identical to that of human RanGAP1, indicating the functional significance of the sequence homology. The Ran-specific guanine nucleotide exchange factor RCC1 and its yeast homologues are restricted to the nucleus, while Rna1p is reported to be localized to the cytoplasm. We suggest a model in which both activities, nuclear GDP-to-GTP exchange on Ran and cytoplasmic hydrolysis of Ran-bound GTP, are essential for shuttling of Ran between the two cellular compartments. Thus, a defect in either of the two antagonistic regulators of Ran would result in a shutdown of Ran-dependent transport processes, in agreement with the almost identical phenotypes described for such defects in budding yeast.     

Increase of 5-HT7 (serotonin-7) and 5-HT1A (serotonin-1A) receptor mRNA expression in rat hippocampus after adrenalectomy

Kinematic and electromyographic (EMG) analysis of a target-directed, maximal velocity movement was used to investigate the effects of high-force eccentric exercise on the neuromuscular control of elbow flexion. Ten non-weight-trained females [19.6 (1.6) years old] performed 50 maximal velocity elbow flexion movements from 0 to 1.58 rad (90 degrees), as rapidly as possible in response to a light stimulus, while kinematic and triphasic EMG parameters were measured. This was done three times pre-exercise, immediately and 1, 2, 3, 4, and 5 days following the 50 maximal eccentric elbow flexion actions. The eccentric exercise caused lengthening of kinematic parameters including total movement time and time to peak velocity. The EMG elements of the biceps brachii (b.) motor time, time to peak EMG, biceps b. burst duration, and the latency period between biceps b. and triceps b. bursts were lengthened post-exercise. These changes persisted for up to 5 days post-exercise. The exercise also caused a large increase in serum creatine kinase (CK) activity. It was concluded that high-force eccentric exercise in this population caused prolonged changes in neuromuscular control that were a function of exercise-induced disruption of the skeletal muscle. Compensation in the central motor program was such that the components of the triphasic EMG pattern were systematically lengthened.     

Identification of a new nuclear gene (CEM1) encoding a protein homologous to a beta-keto-acyl synthase which is essential for mitochondrial respiration in Saccharomyces cerevisiae

We have analysed a new gene, CEM1, from Saccharomyces cerevisiae. Inactivation of this gene leads to a respiratory-deficient phenotype. The deduced protein sequence shows strong similarities with beta-keto-acyl synthases or condensing enzymes. Typically, enzymes of this class are involved in the synthesis of fatty acids or similar molecules. An analysis of the mitochondrial lipids and fatty acids shows no major difference between the wild type and deleted strains, implying that the CEM1 gene product is not involved in the synthesis of the bulk fatty acids. Thus it is possible that the CEM1 protein is involved in the synthesis of a specialized molecule, probably related to a fatty acid, which is essential for mitochondrial respiration.     

Yrb2p is a nuclear protein that interacts with Prp20p, a yeast Rcc1 homologue

A conserved family of Ran binding proteins (RBPs) has been defined by their ability to bind to the Ran GTPase and the presence of a common region of approximately 100 amino acids (the Ran binding domain). The yeast Saccharomyces cerevisiae genome predicts only three proteins with canonical Ran binding domains. Mutation of one of these, YRB1, results in defects in transport of macromolecules across the nuclear envelope (Schlenstedt, G., Wong, D. H., Koepp, D. M., and Silver, P. A. (1995) EMBO J. 14, 5367-5378). The second one, encoded by YRB2, is a 327-amino acid protein with a Ran binding domain at its C terminus and an internal cluster of FXFG and FG repeats conserved in nucleoporins. Yrb2p is located inside the nucleus, and this localization relies on the N terminus. Results of both genetic and biochemical analyses show interactions of Yrb2p with the Ran nucleotide exchanger Prp20p/Rcc1. Yrb2p binding to Gsp1p (yeast Ran) as well as to a novel 150-kDa GTP-binding protein is also detected. The Ran binding domain of Yrb2p is essential for function and for its association with Prp20p and the GTP-binding proteins. Taken together, we suggest that Yrb2p may play a role in the Ran GTPase cycle distinct from nuclear transport.     

The effects of coenzyme Q10 treatment on maternally inherited diabetes mellitus and deafness, and mitochondrial DNA 3243 (A to G) mutation

Little is known about cisplatin ototoxicity in pediatric patients. Measurement of otoacoustic emissions is a rapid, reproducible, objective method of evaluating hearing. We examined whether transient-evoked otoacoustic emissions in pediatric patients exposed to cisplatin in the past correlated with audiographic findings. Twelve patients were entered into the study (mean age at treatment 7.8 years, mean cumulative dose 442.5 mg/mm2, mean 7.1 doses). Hearing at 3000 Hz was preserved in 82.6% of patients. In the higher frequencies significant sensorineural hearing loss was noted: 43.5% at 4 kHz; 81.0% at 6 kHz; and 90.5% at 8 kHz. Transient-evoked otoacoustic emissions were measurable in 11 of 12 patients. Middle ear disease accounted for abnormal otoacoustic emission seen in three patients (1 with effusion, 2 with significant negative middle ear pressure). When the middle ear was normal, a statistically significant correlation was seen between the transient-evoked otoacoustic emissions reproducibility and pure-tone threshold (correlation coefficient = -0.69, p = 0.008). Increased hearing loss was also associated with young age at first dose of cisplatin (p = 0.044), high number of chemotherapy cycles (p = 0.042), and high cumulative dose (p = 0.042).     

Cat carotid body chemosensory discharge (in vitro) is insensitive to charybdotoxin

Charybdotoxin (ChTX), a venom protein, suppresses Ca2+-activated K+ (K+(Ca)) currents in the glomus cell of neonatal rat carotid body. If it works similarly for cat carotid body chemoreceptors, charybdotoxin is expected to stimulate the chemosensory discharge during normoxia, and particularly hypoxia and hypercapnia. We studied the effects of charybdotoxin (20-40 nM) in vitro (perfused/superfused) on the cat carotid chemosensory discharge, and simultaneously tissue PO2 (PtiO2), as a measure of positive control. ChTX (20 nM) only increased PtiO2 and decreased carotid chemosensory discharge during hypoxia, indicating vasodilation. We conclude that K+(Ca) channels do not appear to play a significant role in chemotransduction in the cat carotid body.     

Identification of ArgBP1, an Arg protein tyrosine kinase binding protein that is the human homologue of a CNS-specific Xenopus gene

Cardiac fibroblasts constitute greater than 90% of the non-myocyte cells in the heart. Previously, it was established that cardiac fibroblasts are predisposed to transformation into a phenotype with muscle-specific features and that transforming growth factor-beta 1 (TGF-beta 1) is a specific inducer of this event. In this study the hypothesis that TGF-beta 1-induced phenotypic modulation of cardiac fibroblasts is associated with their altered proliferative capacity is tested. Therefore the effects of TGF-beta 1 on DNA synthesis in cardiac fibroblasts under normal conditions of cell culture and in response to a potent mitogen, basic fibroblasts growth factor (bFGF) were determined. The results showed that TGF-beta 1 at 15 ng/ml (a concentration that induces fibroblast "transformation") had a regulatory effect on proliferative capacity of cardiac fibroblasts which varied as the function of cell density in culture. In subconfluent and confluent cultures, pre-treatment of cardiac fibroblasts with TGF-beta 1 for 24 h resulted in a dramatic shift in the bFGF-induced stimulation of DNA synthesis. TGF-beta 1-induced inhibition of DNA synthesis in cardiac fibroblasts coincided with their phenotypic modulation as evidenced by the expression of sarcomeric actin mRNA and morphological changes. Cross-linking studies with [125I]-labeled TGF-beta 1 showed the presence of conventional types I, II and III TGF-beta 1 receptor complexes on cardiac fibroblasts and their binding to TGF-beta 1 under the experimental conditions. In summary, these data indicate that the proliferative capacity of cardiac fibroblasts is controlled by TGF-beta 1. They further suggest that the TGF-beta 1-induced phenotypic modulation of cardiac fibroblasts may be extended to include their altered proliferative capacity.     

Mammalian homologue of the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth is a protein kinase C zeta-interacting protein

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C zeta (PKCzeta) as a bait, we have cloned a gene coding for a novel PKCzeta-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCzeta and weakly with that of PKCepsilon. In the COS-7 cells coexpressing FEZ1 and PKCzeta, FEZ1 was present mainly in the plasma membrane, associating with PKCzeta and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCzeta. When the constitutively active mutant of PKCzeta was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCzeta activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCzeta stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCzeta.     

A homologue of Saccharomyces cerevisiae Dpm1p is not sufficient for synthesis of dolichol-phosphate-mannose in mammalian cells

Dolichol-phosphate-mannose (Dol-P-Man) serves as a donor of mannosyl residues in major eukaryotic glycoconjugates. It donates four mannosyl residues in the N-linked oligosaccharide precursor and all three mannosyl residues in the core of the glycosylphosphatidylinositol anchor. In yeasts it also donates one mannose to the O-linked oligosaccharide. The yeast DPM1 gene encodes a Dol-P-Man synthase that is a transmembrane protein expressed in the endoplasmic reticulum. We cloned human and mouse homologues of DPM1, termed hDPM1 and mDPM1, respectively, both of which encode proteins of 260 amino acids, having 30% amino acid identity with yeast Dpm1 protein but lacking a hydrophobic transmembrane domain, which exists in the yeast synthase. Human and mouse DPM1 cDNA restored Dol-P-Man synthesis in mouse Thy-1-deficient mutant class E cells. Mouse class E mutant cells had an inactivating mutation in the mDPM1 gene, indicating that mDPM1 is the gene for class E mutant. In contrast, hDPM1 and mDPM1 cDNA did not complement another Dol-P-Man synthesis mutant, hamster Lec15 cells, whereas yeast DPM1 restored both mutants. Therefore, in contrast to yeast, mammalian cells require hDPM1/mDPM1 protein and a product of another gene that is defective in Lec15 mutant cells for synthesis of Dol-P-Man.     

Cloning of the human mitochondrial 51 kDa subunit (NDUFV1) reveals a 100% antisense homology of its 3''UTR with the 5''UTR of the gamma-interferon inducible protein (IP-30) precursor: is this a link between mitochondrial myopathy and inflammation?

Norepinephrine (NE) induces a sigmoidal dose-response curve for perfusion pressure and a bell-shaped curve for oxygen consumption (VO2) in the constant-flow perfused hindlimb of Wistar rats. These effects are now described in spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats (WKY). In SHR, the pressure curve was shifted left- and upward whereas the VO2 curve was shifted left- but downward, when compared with WKY. In the presence of 10 microM propranolol, prazosin (2.5 nM) shifted the pressure and VO2 curves much more than yohimbine (0.1 microM) to the right in both strains and its effects were greater in SHR, suggesting that these effects were mediated largely by alpha-1 receptors, particularly in SHR. In the presence of propranolol plus yohimbine, the pressure curve was markedly shifted to the right by both the selective alpha-1A-antagonist 5-methylurapidil (3.3 nM), and by the alpha-1D antagonist BMY 7378 (0.1 microM) or SK&F 105854 (2 microM) in SHR but not in WKY. With respect to the VO2 curve, 5-methylurapidil attenuated the descending limb without affecting the ascending limb. Similar effects were also obtained with another alpha-1A antagonist 1 nM KMD-3213 in both SHR and WKY. In contrast, BMY and SK&F markedly inhibited the ascending limb of the VO2 curve. These results indicate that both alpha-1A- and alpha-1D subtypes are functionally up-regulated in SHR muscle vascular bed where the ascending limb of VO2 is predominantly mediated by the alpha-1D at a much lower concentration for NE than the descending limb which is predominantly mediated by the alpha-1A subtype.     

The human Musashi homolog 1 (MSI1) gene encoding the homologue of Musashi/Nrp-1, a neural RNA-binding protein putatively expressed in CNS stem cells and neural progenitor cells

In 1992 Congress mandated the Department of Veterans Affairs to provide treatment to veterans traumatized by sexual assault experienced during active military duty. A 1995 survey of how VA medical centers had responded to this mandate indicated that 51 percent of 136 centers had established a sexual trauma treatment team. Teams treated a mean+/-SD of 5.5+/-10 patients a week, and newly referred veterans waited a mean of 3.3+/-4 days for evaluation. Teams varied in the discipline mix of providers, training, organizational structure, services offered, and caseload. Medical centers without dedicated treatment teams offered nonspecialized services to sexually traumatized veterans or offered community referrals for sexual trauma treatment services.     

One-step synthesis and structural quantitative analysis of La_(2/3)Sr_(1/3)MnO_3/La_(1.4)Sr_(1.6)Mn_2O_7 composites

La2/3Sr1/3MnO3/La1.4Sr1.6Mn2O7 composites with arbitrary weight percentage were prepared using a one-step solid-state reaction method. The experimental results demonstrated that addition of K2CO3 during preparation favored the formation of the composites even though the K+ ions were volatilized under the high temperatures of sintering. Full quantitative analysis with the Rietveld method showed that the content of La1.4Sr1.6Mn2O7 phase decreased and the fraction of the La2/3Sr1/3MnO3 phase increased as the a...     

A1 is a constitutive and inducible Bcl-2 homologue in mature human neutrophils

Digestion of blood within the mosquito midgut is mediated primarily by a series of proteases, and several previous studies have described protease activity within homogenates of the midgut of the malaria vector Anopheles stephensi. We have expanded on these previous data by resolving protease isoforms from the midgut as well as the hemolymph of adult An. stephensi mosquitoes via gel electrophoresis and zymography. Using this procedure, we have been able to identify multiple isozymes of trypsin, chymotrypsin, and aminopeptidase. We were able to detect an increase in the intensity of some of these protease bands plus the appearance of new bands 24 hr after mosquitoes had taken a blood meal. Furthermore, we detected 2 endogenous trypsin isozymes within the hemolymph. There was no upregulation of these hemolymph isozymes after a blood meal, thus suggesting that they may not be involved in digestion of the blood meal by the mosquito.     

Yeast cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication

Sister chromatid cohesion is crucial for chromosome segregation during mitosis. Loss of cohesion very possibly triggers sister separation at the metaphase --> anaphase transition. This process depends on the destruction of anaphase inhibitory proteins like Pds1p (Cut2p), which is thought to liberate a sister-separating protein Esp1p (Cut1p). By looking for mutants that separate sister centromeres in the presence of Pds1p, this and a previous study have identified six proteins essential for establishing or maintaining sister chromatid cohesion. Four of these proteins, Scc1p, Scc3p, Smc1p, and Smc3p, are subunits of a 'Cohesin' complex that binds chromosomes from late G1 until the onset of anaphase. The fifth protein, Scc2p, is not a stoichiometric Cohesin subunit but it is required for Cohesin's association with chromosomes. The sixth protein, Eco1p(Ctf7p), is not a Cohesin subunit. It is necessary for the establishment of cohesion during DNA replication but not for its maintenance during G2 and M phases.