Biomedical subjects

F L Huang

Publications and source records attributed to F L Huang.

At least 19 recordsLinked to original sources

PubMed2006

Cobrotoxin-containing analgesic compound to treat chronic moderate to severe cancer pain: results from a randomized, double-blind, cross-over study and from an open-label study.

Cobrotoxin produces intense analgesia but it has an onset of response of 1-3 h which hampers its clinical use in cancer pain. Recently, a compound analgesic formulation combining cobrotoxin, tramadol hydrochloride and ibuprofen (Compound Keluoqu, CKLQ) has become available in China. The aim of this study was to evaluate the clinical efficacy of CKLQ for moderate to severe cancer pain. A consecutive series of patients with chronic moderate to severe cancer pain was enrolled into two multicenter trials. Of the 230 eligible patients, 119 were assigned to a randomized, double-blind, cross-over study, while 111 entered an open-label study. They were all of Han-China nationality and had a mean age of 52.0 and 55.4 years and a mean body weight of 55.6 and 52.9 kg, respectively. A total of 11 patients discontinued the study, 6 (54.5%) because of insufficient pain relief and 5 due to the occurrence of adverse events. In the cross-over study, 59 patients were randomized to receive a CKLQ package with 2 CKLQ tablets (each containing 0.16 mg cobrotoxin, 25 mg tramadol hydrochloride and 50 mg ibuprofen) and 2 placebo capsules, a placebo package with 2 placebo tablets and 2 placebo capsules, and an active control package with 2 tramadol hydrochloride capsules (each containing 50 mg tramadol hydrochloride) and 2 placebo tablets (arm A), and 60 to receive a tramadol hydrochloride package, a placebo package and a CKLQ package (arm B), sequentially and only once. Patients in the open-label study only received CKLQ and were given the option to continue for up to 7 days as long as they had satisfactory pain relief. Pain response was classified as CR, PR and NC. CR was defined as 100% pain relief, with a pain score of 0 on a 0-10 VAS. PR was defined as decreased to mild pain, with a pain score of no more than 4 on a 0-10 VAS. NC was defined as pain that either remained unchanged or that was reduced from severe to moderate at baseline, with a VAS pain score of more than 4 after treatment. One hundred and eight patients completed the cross-over study with all the three drug units. The overall rate of pain relief was 93/111 (83.7%) for CKLQ, 75/110 (68.2%) for tramadol hydrochloride (P=0.011) and 39/111 (35.1%) for placebo (P<0.001). The mean duration of pain relief with CKLQ was significantly longer than that of the other two agents (P<0.001). Of the 35 patients who did not respond to tramadol hydrochloride, 27 (77.1%) responded to CKLQ, while of the 18 who did not respond to CKLQ, 8 (55.6%) achieved satisfactory pain control with tramadol hydrochloride. In the open-label study, the overall relief rate of a single-dose of CKLQ was 99/111 (89.2%). A reduction in the percentage of complete relief, an increase in that of PR and a significant decrease in duration of relief were observed after continuous treatment with at least 10 doses of CKLQ. The frequency of adverse events for CKLQ was similar to that of tramadol hydrochloride. The results of the randomized, double-blind, cross-over study and the open-label study of CKLQ in cancer patients with chronic moderate to severe cancer pain suggest that the CKLQ may be valuable for the treatment of chronic moderate to severe cancer pain. However, the tolerance of CKLQ remains to be further defined.

Adult↗

PubMed2004

Cardiac complications of enterovirus rhombencephalitis.

BACKGROUND: Epidemics of enterovirus 71 infection have caused the death of many children throughout the world. Rhombencephalitis, brain stem encephalitis, and heart failure were present in all of the fatal cases. However, no evidence of myocarditis was noted in the heart specimens, and the mechanism of heart failure remains unknown. AIMS: To characterise the presentation of cardiac complications in children with enterovirus rhombencephalitis and discuss its pathogenesis. METHODS: Ninety one consecutive patients with enterovirus rhombencephalitis underwent echocardiography. Of these, 17 patients (nine male, eight female; median age 14 months, range 4-57 months) with left ventricular dysfunction were studied. RESULTS: Tachycardia was noted in all patients and systemic hypertension in 12. Muscle-brain fraction of creatine kinase was >5% in 14 patients. Plasma norepinephrine and epinephrine levels were significantly raised in the three patients in whom these were analysed. Electrocardiographic abnormalities were noted in eight patients. Pulmonary oedema was complicated in 15 patients. The initial ejection fraction of the left ventricle was 22-58% (mean 37%, SD 11%). All patients deteriorated to hypotensive shock within 12 hours and 13 died. Heart specimens from seven patients showed no evidence of myocarditis, but significant coagulative myocytolysis, myofibrillar degeneration, and cardiomyocyte apoptosis were observed. CONCLUSIONS: Acute heart failure was noted in 19% of patients with enterovirus rhombencephalitis, which had a fatality rate of 77%. It was not caused by myocarditis but possibly by neurogenic cardiac damage.

Blood Pressure↗

PubMed2002

Age-related deficits in long-term potentiation are insensitive to hydrogen peroxide: coincidence with enhanced autophosphorylation of Ca2+/calmodulin-dependent protein kinase II.

Reactive oxygen species (ROS) can have deleterious effects for both normal aging and Alzheimer's disease (AD). We examined the hypothesis that synapses undergoing long-term potentiation (LTP) are preferentially at risk for ROS-mediated oxidative stress during aging. We observed age-dependent deficits in LTP induced by a high-frequency stimulation (HFS) protocol in the CA1 region of hippocampus from C57BL/6 mice. There was a significant difference between LTP measured over 60 min in young (1-2 months) and old (23-26 months) mice. In oxidative stress studies, exogenous H(2)O(2) (580 micro M) significantly inhibited LTP in young mice; a similar dose of H(2)O(2) failed to inhibit LTP in slices from adult (2-4 months) or from old mice. The results show that there are significant deficits in LTP in aging mice, but such deficits are insensitive to H(2)O(2). Western immunoblotting studies in young mice show that the relative levels of autophosphorylated alpha-Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are unchanged in hippocampal CA1 treated with H(2)O(2) relative to untreated controls. However with aging, there is a significant enhancement in the levels of autophosphorylated CaMKII in H(2)O(2)-treated CA1 of older mice. Phosphorylation of RC3/neurogranin (Ng) by protein kinase C (PKC) is decreased in CA1 in response to H(2)O(2) treatment, irrespective of age. We propose that, during aging, enhanced local release of H(2)O(2) from mitochondria may induce a compensatory "ceiling" effect at synapses, so that the levels of autophosphorylated alpha CaMKII are aberrantly saturated, leading to alterations in synaptic plasticity.

Aging↗

PubMed2001

Localization of A-type K+ channel subunit Kv4.2 in rat brain.

Kv4.2, a voltage-gated K+ (Kv) channel subunit, has been suggested to be the key component of the subthreshold A-type K+ currents (I(SA)s) recorded from the specific subcellular compartments of certain CNS neurons. To correlate Kv4.2 localization with the I(SA)s detected, immunohistochemistry will be useful. Although the Kv4.2 immunostaining pattern in the hippocampus and cerebellum has been reported, the Kv4.2 antibody used was not specific. Furthermore, Kv4.2 localization in other brain regions remains unclear. In this report, we first demonstrated the specificity of a new Kv4.2 antibody, and then used it to examine Kv4.2 localization throughout adult rat brain by immunohistochemistry. At the cellular level, Kv4.2 was found in neurons but not glias. At the subcellular level, Kv4.2 was localized in the somatodendritic compartment of most neurons examined. Nevertheless, our preliminary data indicated that Kv4.2 might be also present in the axon/terminal compartment. At the functional level, our data indicates that Kv4.2 localization and I(SA) correlate quite well in some CNS neurons, supporting that Kv4.2 is the key component of some I(SA)s recorded in vivo.

Animals↗

PubMed2001

Neurogranin null mutant mice display performance deficits on spatial learning tasks with anxiety related components.

Neurogranin/RC3 is a protein that binds calmodulin and serves as a substrate for protein kinase C. Neuronally distributed in the hippocampus and forebrain, neurogranin is highly expressed in dendritic spines of hippocampal pyramidal cells, implicating this protein in long-term potentiation and in learning and memory processes. Null mutation of the neurogranin gene Ng generated viable knockout mice for analysis of the behavioral phenotype resulting from the absence of neurogranin protein. Ng -/- mice were normal on measures of general health, neurological reflexes, sensory abilities, and motor functions, as compared to wild type littermate controls. On the Morris water task, Ng -/- mice failed to reach acquisition criterion on the hidden platform test and did not show selective search on the probe trial. In the Barnes circular maze, another test for spatial navigation learning, Ng -/- mice showed impairments on some components of transfer, but normal performance on time spent around the target hole. Abnormal and idiosyncratic behaviors were detected, that appeared to represent an anxiogenic phenotype in Ng -/- mice, as measured in the light<-->dark exploration test and the open field center time parameter. These findings of apparent deficits in spatial learning and anxiety-like tendencies in Ng -/- support a role for neurogranin in the hippocampally-mediated interaction between stress and performance.

Animals↗

PubMed2000

Genomic organization and the promoter region of the round-spotted pufferfish (Tetraodon fluviatilis) CDC37 gene.

The CDC37 gene was isolated from a round-spotted pufferfish genomic library and characterized. This gene is composed of nine exons spanning 3.5 kb. Exon 1 contains the 5'-untranslated region and exon 2 contains the putative translation initiation site. By 5'-RACE (rapid amplication of cDNA ends) and sequence analysis, we deduced the promoter region for the CDC37 gene and found that it does not contain typical TATA or CCAAT box. The 1.8 kb DNA fragment upstream of the putative transcription initiation site contains numerous potential binding sites for transcription factors including CREB, E2A, Ets-1, GATA, NF-IL6 and PEA3. When this DNA fragment was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme four times more efficiently than the promoterless pCAT-Basic did. In addition, the CDC37 gene is linked to the TYK2 gene in a tail-to-head manner with a small intergenic region of 292 bp.

5' Untranslated Regions↗

PubMed2000

Glutathiolation of proteins by glutathione disulfide S-oxide derived from S-nitrosoglutathione. Modifications of rat brain neurogranin/RC3 and neuromodulin/GAP-43.

S-Nitrosoglutathione (GSNO) undergoes spontaneous degradation that generates several nitrogen-containing compounds and oxidized glutathione derivatives. We identified glutathione sulfonic acid, glutathione disulfide S-oxide (GS(O)SG), glutathione disulfide S-dioxide, and GSSG as the major decomposition products of GSNO. Each of these compounds and GSNO were tested for their efficacies to modify rat brain neurogranin/RC3 (Ng) and neuromodulin/GAP-43 (Nm). Among them, GS(O)SG was found to be the most potent in causing glutathiolation of both proteins; four glutathiones were incorporated into the four Cys residues of Ng, and two were incorporated into the two Cys residues of Nm. Ng and Nm are two in vivo substrates of protein kinase C; their phosphorylations by protein kinase C attenuate the binding affinities of both proteins for calmodulin. When compared with their respective unmodified forms, the glutathiolated Ng was a poorer substrate and glutathiolated Nm a better substrate for protein kinase C. Glutathiolation of these two proteins caused no change in their binding affinities for calmodulin. Treatment of [(35)S]cysteine-labeled rat brain slices with xanthine/xanthine oxidase or a combination of xanthine/xanthine oxidase with sodium nitroprusside resulted in an increase in cellular level of GS(O)SG. These treatments, as well as those by other oxidants, all resulted in an increase in thiolation of proteins; among them, thiolation of Ng was positively identified by immunoprecipitation. These results show that GS(O)SG is one of the most potent glutathiolating agents generated upon oxidative stress.

Animals↗

PubMed2000

Involvement of neurogranin in the modulation of calcium/calmodulin-dependent protein kinase II, synaptic plasticity, and spatial learning: a study with knockout mice.

Neurogranin/RC3 is a neural-specific Ca(2+)-sensitive calmodulin (CaM)-binding protein whose CaM-binding affinity is modulated by phosphorylation and oxidation. Here we show that deletion of the Ng gene in mice did not result in obvious developmental or neuroanatomical abnormalities but caused an impairment of spatial learning and changes in hippocampal short- and long-term plasticity (paired-pulse depression, synaptic fatigue, long-term potentiation induction). These deficits were accompanied by a decreased basal level of the activated Ca(2+)/CaM-dependent kinase II (CaMKII) ( approximately 60% of wild type). Furthermore, hippocampal slices of the mutant mice displayed a reduced ability to generate activated CaMKII after stimulation of protein phosphorylation and oxidation by treatments with okadaic acid and sodium nitroprusside, respectively. These results indicate a central role of Ng in the regulation of CaMKII activity with decisive influences on synaptic plasticity and spatial learning.

Animals↗

PubMed2000

Calcium-sensitive interaction between calmodulin and modified forms of rat brain neurogranin/RC3.

Neurogranin (NG) binding of calmodulin (CaM) at its IQ domain is sensitive to Ca(2+) concentration and to modifications by protein kinase C (PKC) and oxidants. The PKC phosphorylation site of NG is within the IQ domain whereas the four oxidant-sensitive Cys residues are outside this region. These Cys residues were oxidized forming two pairs of intramolecular disulfides, and could also be glutathiolated by S-nitrosoglutathione resulting in the incorporation of four glutathiones per NG. Circular dichroism (CD) showed that modification of NG by phosphorylation, oxidation forming intramolecular disulfides, or glutathiolation did not affect the alpha-helical content of this protein. Mutation of the four Cys residues [Cys(-)-NG] to Gly and Ser did not affect the alpha-helical content either. Interaction of CaM with the reduced (red)-, glutathiolated (GS)-, or Cys(-)-NG in the Ca(2+)-free solution resulted in an increase in the alpha-helicity determined by their CD spectra, but relatively little change was seen with the oxidized NG (ox-NG) or phosphorylated NG (PO(4)-NG). The binding affinities between the various modified forms of NG and CaM were determined by CD spectrometry and sedimentation equilibrium: their affinities were Cys(-)-NG > red-NG, GS-NG > ox-NG > PO(4)-NG. Unlike Cys(-)-, red-, and GS-NG, neither ox- nor PO(4)-NG bound to a CaM-affinity column. Thus, both oxidation of NG to form intramolecular disulfides and phosphorylation of NG by PKC are effective in modulating the intracellular level of CaM. These results indicate that modification of NG to form intramolecular disulfides outside the IQ domain provides an alternative mechanism for regulation of its binding affinity to CaM.

Animals↗

PubMed2000

Phosphorylation of HMG-I by protein kinase C attenuates its binding affinity to the promoter regions of protein kinase C gamma and neurogranin/RC3 genes.

A 20-kDa DNA-binding protein that binds the AT-rich sequences within the promoters of the brain-specific protein kinase C (PKC) gamma and neurogranin/RC3 genes has been characterized as chromosomal nonhistone high-mobility-group protein (HMG)-I. This protein is a substrate of PKC alpha, beta, gamma, and delta but is poorly phosphorylated by PKC epsilon and zeta. Two major (Ser44 and Ser64) and four minor phosphorylation sites have been identified. The extents of phosphorylation of Ser44 and Ser64 were 1:1, whereas those of the four minor sites all together were <30% of the major one. These PKC phosphorylation sites are distinct from those phosphorylated by cdc2 kinase, which phosphorylates Thr53 and Thr78. Phosphorylation of HMG-I by PKC resulted in a reduction of DNA-binding affinity by 28-fold as compared with 12-fold caused by the phosphorylation with cdc2 kinase. HMG-I could be additively phosphorylated by cdc2 kinase and PKC, and the resulting doubly phosphorylated protein exhibited a >100-fold reduction in binding affinity. The two cdc2 kinase phosphorylation sites of HMG-I are adjacent to the N terminus of two of the three predicted DNA-binding domains. In comparison, one of the major PKC phosphorylation sites, Ser64, is adjacent to the C terminus of the second DNA-binding domain, whereas Ser44 is located within the spanning region between the first and second DNA-binding domains. The current results suggest that phosphorylation of the mammalian HMG-I by PKC alone or in combination with cdc2 kinase provides an effective mechanism for the regulation of HMG-I function.

Animals↗

PubMed2000

Congenital muscular dystrophy.

Congenital muscular dystrophy (CMD) is a rare heterogeneous disease found in the oriental population, especially the occidental type of CMD. We report a case of a one-year-old infant who presented with early onset hypotonia, muscular weakness, delayed motor development and normal intelligence. A muscle biopsy revealed dystrophic muscle fibers. A high creatine kinase (CK) level, mostly of the MM type, was also noted. Further study of brain images showed hyperintense lesions in the white matter area. The patient showed the clinical and laboratory findings characteristic of CMD, more likely to be of the occidental type. Further genetic or histopathologic studies, especially merosin investigation, are suggested for improved classification and prognosis prediction.

Humans↗

PubMed1999

N-methyl-D-aspartate induces neurogranin/RC3 oxidation in rat brain slices.

Neurogranin/RC3 (Ng), a postsynaptic neuronal protein kinase C (PKC) substrate, binds calmodulin (CaM) at low level of Ca2+. In vitro, rat brain Ng can be oxidized by nitric oxide (NO) donors and by oxidants to form an intramolecular disulfide bond with resulting downward mobility shift on nonreducing SDS-polyacrylamide gel electrophoresis. The oxidized Ng, as compared with the reduced form, is a poorer substrate of PKC but like the PKC-phosphorylated Ng has a lower affinity for CaM than the reduced form. To investigate the physiological relevance of Ng oxidation, we tested the effects of neurotransmitter, N-methyl-D-aspartate (NMDA), NO donors, and other oxidants such as hydrogen peroxide and oxidized glutathione on the oxidation of this protein in rat brain slices. Western blot analysis showed that the NMDA-induced oxidation of Ng was rapid and transient, it reached maximum within 3-5 min and declined to base line in 30 min. The response was dose-dependent (EC50 approximately 100 microM) and could be blocked by NMDA-receptor antagonist 2-amino-5-phosphonovaleric acid and by NO synthase inhibitor NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine. Ng was oxidized by NO donors, sodium nitroprusside, S-nitroso-N-acetylpenicillamine, and S-nitrosoglutathione, and H2O2 at concentrations less than 0.5 mM. Oxidation of Ng in brain slices induced by sodium nitroprusside could be reversed by dithiothreitol, ascorbic acid, or reduced glutathione. Reversible oxidation and reduction of Ng were also observed in rat brain extracts, in which oxidation was enhanced by Ca2+ and the oxidized Ng could be reduced by NADPH or reduced glutathione. These results suggest that redox of Ng is involved in the NMDA-mediated signaling pathway and that there are enzymes catalyzing the oxidation and reduction of Ng in the brain. We speculate that the redox state of Ng, similar to the state of phosphorylation of this protein, may regulate the level of CaM, which in turn modulates the activities of CaM-dependent enzymes in the neurons.

2-Amino-5-phosphonovalerate↗

PubMed1999

Purification, characterization, and molecular cloning of carp hyosophorin.

Carp hyosophorin (HSP) is purified from oocytes. It is a highly glycosylated protein (10% protein and 90% carbohydrate) of high molecular weight (>100 kDa) and is localized in the cortical granules of oocytes. During cortical reaction carp HSP is exocytosed into the perivitelline space and is rapidly cleaved to the low-molecular-weight forms of 20 to 30 kDa. The major part of carp HSP cDNA is composed of tandem repeats, the repetitive domain. A repeat is 36 base pairs (bp) in length, which encodes 12 amino acid residues. The sequences of repeats vary within a given cDNA and among different cDNAs. The predominant sequences of repeats are DDGSGSNATTTQ. In addition, the length of the repetitive domain is highly variable among different genes and cDNAs, and ranges from 170 to 1,010 bp. Transcription of carp HSP is restricted in oocytes and starts very early during oogenesis. Carp HSP is highly species-specific. The RNA of goldfish ovary shows no positive signals when probed by carp HSP cDNA.

Amino Acid Sequence↗

PubMed1999

Purification, characterization, and molecular cloning of an outer layer protein of carp fertilization envelope.

An outer layer protein of carp fertilization envelope (FE), FEO-1, was purified from carp oocytes. The cDNAs encoding FEO-1 were cloned. The mature protein of FEO-1 is 21 kDa in molecular weight and contains 177 amino acid residues whose sequence has 58% identity to the outer layer protein of chick vitelline membrane. In situ hybridization and immunocytochemistry show that FEO-1 is expressed in oocytes and liver. In oocytes, FEO-1 is stored in the cortical granules. During cortical reaction, it is exocytosed to the perivitelline space and then gradually added to the outer layer of FE (FE(o)). FEO-1 first appears as discrete deposits along FE(o), then merges to form a continuous layer. The thickness of FE(o) increases as cortical reaction proceeds. In addition to FEO-1, FE(o) contains cystatin, fibroin-like substance (FLS), and cathepsin-like substance (CLS) as well. They are stored in the cortical granules and are exocytosed to FE(o) simultaneously with FEO-1 during cortical reaction. In FE(o), FEO-1 is present in monomer form and can be completely extracted by sodium dodecyl sulfate (SDS)-mercaptoethanol (MSH). On the other hand, the cystatin, FLS, and CLS present in FE(o)are cross-linked together. They are partially extracted by SDS-MSH but can be completely extracted by guanidium thiocyanate-lauroylsarcosine.

Amino Acid Sequence↗

PubMed1999

Hypoxia/ischemia induces dephosphorylation of rat brain neuromodulin/GAP-43 in vivo.

The in vivo state of phosphorylation and the modification of two Cys residues of neuromodulin/ GAP-43 (Nm) were analyzed by electrospray ionization-mass spectrometry (ES-MS). The protein was purified from rat brain with homogenization buffer containing 1% Nonidet P-40, protease inhibitors, protein phosphatase inhibitors, and sulfhydryl reagent, 4-vinylpyridine. Nm was purified by HPLC and ion-exchange chromatography, and the various fractions were identified by ES-MS as unphosphorylated and mono-, di-, tri-, and tetraphosphorylated species. All of these Nm species contained 2 mol of added 4-vinylpyridine per mol of Nm, suggesting that the two Cys residues are in the reduced form in the brain. In vivo, the majority of Nm is in the phosphorylated form (approximately 80%), of which the levels of the mono- and diphospho forms are higher than those of the tri- and tetraphospho species. Four in vivo phosphorylation sites, Ser41, Thr95, Ser142, and Thr172, were identified by amino acid sequencing and tandem ES-MS of the peptides derived from Lys-C endoproteinase digestion. Among these sites, only Ser41 is a known target of PKC, whereas the kinases responsible for the phosphorylation of the other three novel sites are unknown. Hypoxia/ischemia caused a preferential dephosphorylation of Ser41 and Thr172, whereas Thr95 is the least susceptible to dephosphorylation.

Amino Acid Sequence↗

PubMed1998

Overexpression of protein kinase C alpha enhances lipopolysaccharide-induced nitric oxide formation in vascular smooth muscle cells.

Our previous studies showed that lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis in cardiovascular tissues is attenuated by protein kinase C (PKC) inhibitors. In the current study, we identify a specific PKC isotype involved in the LPS signal transduction pathway that leads to NO formation in rat vascular smooth muscle cells (VSMC). VSMC were transfected with a mammalian expression vector containing a full length PKCalpha cDNA insert, and a stable transfectant overexpressing PKCalpha was obtained as evidenced by increased expression of PKCalpha mRNA and protein. In response to 100 ng/ml LPS stimulation, the PKCalpha transfectants showed a 1.8-fold increase in PKC activity at 30 min and a twofold increase in NO production over 24 hr compared with cells transfected with control plasmids. The LPS-stimulated increase in NO synthesis in PKCalpha transfectants was blocked by the specific PKCalpha inhibitor Gö 6976. After 6 hr LPS treatment, PKCalpha-transfected and control cells showed equivalent increases in mRNA and protein for the inducible NO synthase. NO synthase activity of the cell extracts assayed in the presence of excess substrate and cofactors was not significantly different between PKCalpha-transfected and control cells after LPS stimulation. However, mRNA levels for GTP cyclohydrolase I, a key enzyme in (6R)-tetrahydro-L-biopterin synthesis, and cationic amino acid transporter-2, involved in L-arginine transport, was enhanced in cells overexpressing PKCalpha compared with control cells. These results suggest that PKCalpha plays an important role in LPS-induced NO formation and that a significant portion of this effect may be by means of enhanced substrate availability to the inducible nitric oxide synthase enzyme.

Amino Acid Transport Systems, Basic↗

PubMed1998

Identification of cystatin as a component of carp chorion.

An ovary-specific cystatin is immunocytochemically demonstrated to be localized in the chorions, cortical granules, and yolk granules of carp oocytes, as well as in the follicle cells surrounding oocytes. During cortical reaction, cystatin is exocytosed from cortical granules into the perivitelline space. In situ hybridization confirms that cystatin is synthesized by oocytes and follicle cells. Western blotting reveals that chorion cystatin appears in multiple bands of high molecular weight (from 65 kDa to larger than 200 kDa). No cystatin monomer of 14 kDa is found. These results indicate that chorion cystatin is conjugated with other chorion components. Two forms of conjugates are found. In one form, cystatin, ZP2, fibroin-like substance (FLS), and cathepsin-like substance (CLS) are conjugated, which is extracted by sodium dodecyl sulfate. In the other form, cystatin, FLS, and CLS are conjugated, which is extracted by guanidine thiocyanate (GTC). Most chorion cystatin of oocytes and ovulated eggs is solubilized by GTC, while a large amount of cystatin remains in the fertilization envelope of cortical reacted eggs after extraction by GTC.

Animals↗

PubMed1997

Purification and cloning of carp nephrosin, a secreted zinc endopeptidase of the astacin family.

We have purified a secreted proteinase of 23 kDa from carp head kidney by sequential column chromatography on a Reactive Blue 72-agarose dye affinity column and an FPLC Mono-P column. The secretion of this proteinase from carp head kidney can be stimulated by high concentrations of potassium. Since the carp proteinase is present mainly in the head kidney, kidney, and spleen (all of which are lymphohematopoietic organs), it is named nephrosin. The carp nephrosin is most sensitive to metal chelators, but not to inhibitors specific for other classes of proteinases. A cDNA clone has been isolated from a carp head kidney cDNA library by immunoscreening with a polyclonal antiserum raised against purified nephrosin. The cloned cDNA is 1086 base pairs in length and has an open reading frame encoding a protein of 273 amino acids, including a 19-amino acid signal peptide and 56-amino acid propeptide. The deduced amino acid sequence shows moderate levels of identity to medaka HCE1 (52.5%), medaka LCE (50.7%), crayfish astacin (33.2%), murine meprin-alpha (34%), and murine meprin-beta (33.5%), all members of the astacin family of zinc endopeptidases. Nephrosin is the first member of the astacin family found in lymphohematopoietic tissues.

Adrenal Cortex↗