Isolation and characterization of the papaya MADS-box E-class genes, CpMADS1 and CpMADS3, and a TM6 lineage gene CpMADS2.

Papaya (Carica papaya L.) plants are polygamous, with female, male, and hermaphroditic flowers. To understand the roles of MADS-box genes in flower development and sex determination, we cloned cDNAs of E-class genes CpMADS1 and CpMADS3 and a TM6 lineage of the B-class gene CpMADS2 from young flower buds of papaya. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses revealed that CpMADS1 and CpMADS3 were preferentially expressed in the carpel and also in petals and stamens. CpMADS2 was expressed in both petals and stamens early during floral development. Comparison of 10 papaya genotypes of 5 different sex phenotypes - hermaphrodite, male, female, progeny-all-hermaphrodite, and progeny-all-male - by Southern blot analysis of genomic DNAs with probes of the 3 genes revealed similar restriction patterns and copy number, suggesting a low relationship of the 3 CpMADS genes with sex expression of papaya plants at the genomic level.

LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2.

Neurofibromatosis type 2 (NF2) is caused by mutations in the NF2 gene that encodes a tumor-suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss-of-function is associated with increased activity of Rac and p21-activated kinases (PAKs) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates the actin severing and depolymerizing activity of cofilin. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2(ΔEx2)) exhibited increased levels of LIMK1, LIMK2 and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared with wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared with normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2(ΔEx2) MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs. The decreased viability of Nf2(ΔEx2) MSCs was not due to caspase-dependent or -independent apoptosis, but rather due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrests cells in early mitosis by decreasing aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

Drug reaction with eosinophilia and systemic symptoms syndrome associated myocarditis: a survival experience after extracorporeal membrane oxygenation support.

WHAT IS KNOWN AND OBJECTIVE: Myocarditis that develops because of the drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome is a life-threatening disease. We report a case of DRESS-associated myocarditis with cardiac failure that required extracorporeal membrane oxygenation (ECMO) for cardiovascular support. CASE SUMMARY: A 14-year-old boy experienced DRESS-associated myocarditis after anticonvulsive therapy with carbamazepine, clonazepam and phenytoin. The clinical signs included hypotension, cardiac arrhythmia and poor left ventricular (LV) performance. Laboratory investigations showed elevated levels of cardiac enzymes. Systemic corticosteroid pulse therapy for 3 days was administered for treating the DRESS syndrome. The patient required inotropic drugs including dopamine, dobutamine and milrinone because of refractory hypotension and poor LV function. He was placed on ECMO support, and intra-aortic balloon pumping was initiated because of poor response to inotropic drugs and stasis of blood flow in the ventricle on hospital day 17. Plasma exchanges for four separate times over 8 days were also performed during ECMO support on day 22. His condition stabilized 13 days after ECMO support was initiated. The patient was discharged on hospital day 50, and the seizure was controlled by the oral form clonazepam, phenobarbital, topiramate and levetiracetam. Three months later, an echocardiogram showed mild dilated cardiomyopathy. WHAT IS NEW AND CONCLUSION: Drug reaction with eosinophilia and systemic symptoms-associated fulminant myocarditis is a life-threatening disease. Traditionally, systemic corticosteroid administration, plasmapheresis, intravenous immunoglobulin infusion and ventricular assist device implantation have been used for the treatment of this disease. To our knowledge, this is the first case of DRESS-associated fulminant myocarditis treated successfully with ECMO support. However, echocardiogram should be followed regularly because dilated cardiomyopathy may be the late sequela.

Time-resolved measurements of PM2.5 carbonaceous aerosols at Gosan, Korea.

In order to better understand the characteristics of atmospheric carbonaceous aerosol at a background site in Northeast Asia, semicontinuous organic carbon (OC) and elemental carbon (EC), and time-resolved water-soluble organic carbon (WSOC) were measured by a Sunset OC/ EC and a PILS-TOC (particle-into-liquid sampler coupled with an online total organic carbon) analyzer, respectively, at the Gosan supersite on Jeju Island, Korea, in the summer (May 28-June 17) and fall (August 24-September 30) of 2009. Hourly average OC concentration varied in the range of approximately 0.87-28.38 microgC m-3, with a mean of 4.07+/- 2.60 microgC m-3, while the hourly average EC concentration ranged approximately from 0.04 to 8.19 .microgC m-3, with a mean of 1.35 +/- 0.71 microgC m-3, from May 28 to June 17, 2009. During the fall season, OC varied in the approximate range 0.9-9.6 microgC m-3, with a mean of 2.30 +/-0.80 microgC m-3, whereas EC ranged approximately from 0.01 to 5.40 microgC m-3, with a mean of 0.66 +/- 0.38 microgC m-3. Average contributions of EC to TC and WSOC to OC were 26.0% +/- 9.7% and 20.6% +/-7.4%, and 37.6% +/- 23.5% and 57.2% +/- 22.2% during summer and fall seasons, respectively. As expected, clear diurnal variation of WSOC/OC was found in summer, varying from 0.22 during the nighttime up to 0.72 during the daytime, mainly due to the photo-oxidation process. In order to investigate the effect of air mass pathway on the characteristics of carbonaceous aerosol, 5-day back-trajectory analysis was conducted using the HYSPLIT model. The air mass pathways were classified into four types: Continental (CC), Marine (M), East Sea (ES) and Korean Peninsula (KP). The highest OC/EC ratio of 3.63 was observed when air mass originated from the Continental area (CC). The lowest OC/EC ratio of 0.79 was measured when air mass originated from the Marine area (M). A high OC concentration was occasionally observed at Gosan due to local biomass burning activities. The contribution of secondary OC to total OC varied approximately between 8.4% and 32.2% and depended on air mass type.

Furano-1,2-naphthoquinone inhibits EGFR signaling associated with G2/M cell cycle arrest and apoptosis in A549 cells.

Furano-1,2-naphthoquinone (FNQ), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits an anti-carcinogenic effect. FNQ exerted anti-proliferative activity with the G(2)/M cell cycle arrest and apoptosis in A549 cells. FNQ-induced G(2)/M arrest was correlated with a marked decrease in the expression levels of cyclin A and cyclin B, and their activating partner cyclin-dependent kinases (Cdk) 1 and 2 with concomitant induction of p53, p21, and p27. FNQ-induced apoptosis was accompanied with Bax up-regulation and the down-regulation of Bcl-2, X-linked inhibitor of apoptosis (XIAP), and survivin, resulting in cytochrome c release and sequential activation of caspase-9 and caspase-3. Western blot analysis revealed that FNQ suppressed EGFR phosphorylation and JAK2, STAT3, and STAT5 activation, but increased in activation of p38 MAPK and c-Jun NH2-terminal kinase (JNK) stress signal. The combined treatment of FNQ with AG1478 (a specific EGFR inhibitor) significantly enhanced the G(2)/M arrest and apoptosis, and also led to up-regulation in Bax, p53, p21, p27, release of mitochondrial cytochrome c, and down-regulation of Bcl-2, XIAP, survivin, cyclin A, cyclin B, Cdk1, and Cdk2 in A549 cells. These findings suggest that FNQ-mediated cytotoxicity of A549 cell related with the G(2)/M cell cycle arrest and apoptosis via inactivation of EGFR-mediated signaling pathway.

[A pocket pRb mutation induces the increase in its affinity to E2F4 coupled with activation of muscle differentiation].

Co-ordination of proliferation and differentiation in cells committed to muscle fate requires the interaction of the retinoblastoma gene product (pRb) with transcription factors of the E2F family. pRb has different affinities for distinct E2Fs, however, the mechanism involved in pRb-E2Fs interaction has not been completely investigated. We have found that pRb carrying a small deletion at the end of the T antigen binding region (deltaS/N), and unable to interact with large T antigen, could induce acute cell cycle block, stable prolongation of the cell cycle in G0/G1 and G2/M phases and suppression of the growth of tumor cells. The deltaS/N showed increased affinity for E2F4, bound hyperphosphorylated forms of E2F4 and induced its nuclear compartmentalization. The ability of deltaS/N to form complexes with E2F4 on DNA was associated with an increase in formation of "free" E2F4 and transsuppression of the specific reporter through preferential binding to E2F4 but not t o E2F1. Stable expression of deltaS/N in multi-potent fibroblasts promoted early muscle commitment. The results obtained suggest that a mutation in the T antigen binding region may increase in affinity of the pRb for E2F4 combined with activation of muscle differentiation.

[Drosophila melanogaster gene Merlin interacts with the clathrin adaptor protein gene lap].

The protein Merlin is involved in the regulation of cell proliferation and differentiation in the eyes and wings of Drosophila and is a homolog of the human protein encoded by the Neurofibromatosis 2 (NF2) gene whose mutations cause auricular nerve tumors. Recent studies show that Merlin and Expanded cooperatively regulate the recycling of membrane receptors, such as the epidermal growth factor receptor (EGFR). By performing a search for potential genetic interactions between Merlin (Mer) and the genes important for vesicular trafficking, we found that ectopic expression in the wing pouch of the clathrin adapter protein Lap involved in clathrin-mediated receptor endocytosis resulted in the formation of extra vein materials. On the one hand, coexpression of wild-type Merlin and lap in the wing pouch restored normal venation, while overexpression of a dominant-negative mutant Mer(DBB) together with lap enhanced ectopic vein formation. Using various constructs with Merlin truncated copies, we showed the C-terminal portion of the Merlin protein to be responsible for the Merlin-lap genetic interaction. Furthermore, we showed that the Merlin and Lap proteins colocalized at the cortex of the wing imaginal disc cells.

[Role of the porcupine gene in the development of the wing imaginal disk of Drosophila melanogaster].

A search for the genes interacting with the Merlin tumor suppressor gene revealed a Merlin-porcupine interaction during wing morphogenesis. Ectopic expression of the porcupine gene in the wing imaginal disk reduced the adult wing, while addition of an UAS construct with a full-length or truncated copy of the Merlin gene partly restored the wing phenotype. The highest restoration level was observed upon adding the fragments coding for the C end of the Merlin protein. In addition, the porcupine gene was shown to mediate the wingless gene autoregulation, which occurs at two ontogenetic stages, segmentation during embryo development and determination of the wg expression band at the boundary between the dorsal and ventral compartments of the wing imaginal disk.

First Report of Papaya leaf curl virus Infecting Papaya Plants in Taiwan.

Papaya leaf curl disease was first reported in India in 1939 (1). Caused by begomovirus, Papaya leaf curl virus (PaLCV) (2), this disease was discovered in the papaya orchards of southern Taiwan in 2002. Infected papaya developed symptoms such as downward curling of leaves, twisted petioles, vein enation, and stunting. Diseased plants produced small and distorted fruits that tend to fall prematurely. Typical twin virion was observed in the diseased papaya cells by electron microscopy. In addition, our whitefly-transmission test demonstrated that the sweet potato whitefly (Bemisia tabaci) could transmit this virus. For further molecular identification, two opposing primers were selected for the polymerase chain reaction (PCR) detection of PaLCV from the published nucleotide sequences of PaLCV (Genbank Accession No. NC004147) (3). The primer pair, composed of the forward primer 5' -GCT AGA AAT TAT GTC GAA GCG-3' and the reverse primer 5'-TCA ACT ACA ACC TGA GGA AAG C-3', was designed to amplify a PaLCV-specific 1,031-bp fragment containing 774 bp of the coat protein gene open reading frame (CP-ORF) using PCR. Five diseased papaya samples with typical leaf-curl symptoms tested positive in the PCR-based assay with this specific primer pair, whereas five healthy papaya samples tested negative. However, the sequencing results of the PCR product from five PaLCV-infected papayas indicated the CP-ORF of PaLCV in Taiwan (PaLCV-Tw) was somewhat different from PaLCV in India (PaLCV-Id). The DNA sequences (Genbank Accession No. AY183472) of CP-ORF of PaLCV-Tw were 80% identical to those of PaLCV-Id, and their translated amino acid sequences were 77% identical. This indicates that PaLCV-Tw and PaLCV-Id are two different species or strains. References: (1) K. M. Thomas and C. S. Krishnaswamy. Curr. Sci. 8:316,1939. (2) S. Saxena et al. Plant Dis. 82:126, 1998. (3) S. Saxena et al. Biochem. Mol. Biol. Int. 45:101, 1998.

Involvement of L-arginine/nitric oxide pathway at the paraventricular nucleus of hypothalamus in central neural regulation of penile erection in the rat.

The objective of this study is to investigate whether the L-arginine/nitric oxide pathway is involved in the neurotransmission of paraventricular nucleus of hypothalamus (PVN) activation-induced penile erection in the rat. Male adult Sprague-Dawley rats anesthetized with pentobarbital were used. The femoral artery was cannulated to measure systemic and mean arterial pressure (SAP and MAP), and heart rate (HR). A 26-gauge needle was inserted into corpus cavernosum to measure the intracavernous pressure (ICP) simultaneously with SAP, MAP and HR on a polygraph. Four groups of study were arranged: (1) stereotaxically delivery of L-arginine (500 nmol/500 nl) into PVN; (2) administration of a mixture (1 microl) containing N(G)-Nitro-L-arginine methyl ester (L-NAME) 500 nmol and L-arginine 500 nmol into PVN; (3) microinjection of saline 500 nl into PVN as a vehicle control; and (4) intracavernous injection of L-arginine (100 nmol/50 microl). The ICP, SAP, MAP and HR were monitored for at least 2 h after each administration of the experimental agents. Upon administration of L-arginine into PVN, there was a significant increase of ICP from resting 9.6+/-2.5 mmHg to peaked at 64.4+/-9.8 mmHg after a latency of 3016.0+/-1749.7 s and with a duration of 27.6+/-15.8 min. There was no change of resting ICP after administration of the mixture of L-NAME and L-arginine into PVN. Application of saline to PVN and intracavernous injection of L-arginine failed to increase ICP. Based on elicitation of penile erection upon administration of L-arginine into PVN, and elimination of this L-arginine induced penile erection by co-administration of L-NAME with L-arginine, the results of this study suggest that L-arginine/nitric oxide pathway may be involved in the neurotransmission of PVN activation-induced penile erection in the rat.

The correlation between pretreatment serum hormone levels and treatment outcome for patients with prostatic cancer and bony metastasis.

OBJECTIVE: To evaluate whether pretreatment serum hormone levels are a prognostic factor for prostatic cancer with bony metastasis under hormonal treatment. PATIENTS AND METHODS: Between 1980 and 1994, 96 patients with prostate cancer and bony metastasis were included for an evaluation by a retrospective review of their charts. All 96 had received hormonal treatment after a diagnosis of metastatic prostatic carcinoma. Serum testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were assessed before treatment. The patients were divided into two groups according to their response during the follow-up. Group 1 (good response) had no change or resolution of metastatic lesion(s) on the bone scan and a declining prostate-specific antigen (PSA) level. Group 2 had increased PSA or progression of metastatic lesion(s) on the bone scan. Tumours were graded as low (2-4), intermediate (5-7) and high (8-10) using the Gleason score. RESULTS: There were 43 patients in group 1 and 53 in group 2; the overall mean (sd) age was 72.5 (6.8) years and the follow-up 29.5 (0.5) months. The respective mean (sd) levels of testosterone, LH, FSH and prolactin before treatment were 4.6 (1.6) ng/mL, 20.2 (13.3) mIU/mL, 19.6 (18.6) mIU/mL and 20.7 (12.1) ng/mL in group 1, and 2.6 (1.0) ng/mL, 27.3 (11.0) mIU/mL, 27.1 (9.8) mIU/mL and 41.3 (28.4) ng/mL in group 2. The level of testosterone was significantly higher in group 1 than in group 2, while LH, FSH and prolactin were significantly lower in group 1 than in group 2. When stratified by tumour grade, patients in group 1 still had significantly higher pretreatment testosterone and lower LH, FSH and prolactin than those in group 2. CONCLUSION: Higher testosterone and lower LH, FSH and prolactin levels were good prognostic factors for patients with metastatic prostatic cancer under hormonal treatment, irrespective of tumour grading.

Expression of A chain and B chain of beta-bungarotoxin from taiwan banded krait: the functional implication of the interchain disulfide bond between A chain and B chain.

beta-Bungarotoxin (beta-Bgt), the main presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of two dissimilar polypeptide chains, the A chain and B chain, cross-linked by an interchain disulfide bond. The A and B chain cDNAs were subcloned into expression vectors pT7-7 and pET20b(+), respectively, and transformed into Escherichia coli strain BL21(DE3). The expressed protein was isolated from the inclusion bodies of E. coli and subjected to refolding into its folded structure. The yields of the refolded A and B chains increased markedly by at least 100-fold after substituting Ser for Cys1S of A chain and Cys55 of B chain, which formed an interchain disulfide bond. Either the A(C15) chain or B(C55S) chain alone or in combination cannot exhibit the phospholipase A2 activity or synaptosome binding activity of beta-Bgt. Nevertheless, the results of competitive enzyme-linked immunoassay, CD spectra, and fluorescence measurement revealed that the A(C15S) chain and B(C55S) chain possessed a native-like structure like the subunits of native beta-Bgt. Moreover, the interfacial interaction between the A and B chains explored by glutaraldehyde cross-linking revealed the essential aspects of the intact interchain disulfide bond in this interaction. This suggests that the formation of the interchain disulfide bond should not be a crucial step for the formation of folded A and B chains in the venom glands, and that the integrity of the interchain disulfide linkage favors the subunit interaction that consequently fulfills the functional mechanism of beta-Bgt.

High failure rate using allograft fascia lata in pubovaginal sling surgery for female stress urinary incontinence.

OBJECTIVES: To present our unfavorable experiences using allograft fascia lata. Allograft fascia lata is an attractive sling material providing less pain, a shorter operation time, and a reported effectiveness equal to autologous fascia. METHODS: A total of 18 women (mean age 51.7 years, range 37 to 76) underwent pubovaginal sling surgery for stress urinary incontinence between March 1999 and July 1999 and were enrolled in this study. Solvent dehydrated gamma-irradiated human fascia lata with a size of 7 x 2 cm was used as the sling. The results were collected with a questionnaire survey. RESULTS: All patients were followed up for a mean of 9.2 months (range 6.9 to 11.6). Thirteen patients considered the surgery successful or to have provided improvement, with a mean of 82.5% (range 50% to 100%) subjective improvement. Five patients (27.8%) had significant failure with full recurrence of incontinence within 3 to 6 months. CONCLUSIONS: Solvent dehydrated gamma-irradiated allograft fascia is not reliable in pubovaginal sling surgery. The high failure rates within a short period prohibit its use in the operative management of stress urinary incontinence.

Glutaraldehyde cross-linking alters the environment around Trp(29) of cobrotoxin and the pathway for regaining its fine structure during refolding.

Cobrotoxin, purified from the venom of Naja naja atra (Taiwan cobra), was subjected to modification with glutaraldehyde in order to prepare intra- and intermolecule cross-linked derivatives. Monomeric and dimeric derivatives were separated from polymeric derivatives by gel filtration. The results of amino acid analysis and sequence determination revealed that only Lys residues were selectively modified by glutaraldehyde. Glutaraldehyde cross-linking was accompanied by a change in the gross conformation of cobrotoxin as revealed by circular dichroism spectra of the modified derivatives. Compared with cobrotoxin, Trp(29) of monomeric and dimeric derivatives was in an apolar microenvironment. This was in agreement with acrylamide quenching studies showing that the spatial position of the Trp indole ring became buried in the interior of the molecule after glutaraldehyde cross-linking. Moreover, the Trp of modified derivatives was less accessible for iodide than that observed with cobrotoxin. Notably, disulfide reduction could not completely unfold the structure of glutaraldehyde-modified derivatives as evidenced by the results of acrylamide quenching studies and enzyme-linked immunoassay. Study of the characteristic changes in Trp fluorescence after the initiation of refolding suggested that the fine structure around Trp(29) of cobrotoxin and glutaraldehyde-modified derivatives was formed differently. These results suggest that glutaraldehyde cross-linking leads to a change in the microenvironment of cobrotoxin Trp(29) and alters the pathway of its fine structure formation during the refolding of cobrotoxin.

Probing the structural diversities of long alpha-neurotoxins by fluorescence quenching studies.

Trp fluorescence of Ophiophagus hannah neurotoxins (Oh-4, Oh-6A, Oh-7, and Oh-8) and Bungarus multicinctus (alpha-bungarotoxin was quenched by acrylamide and iodide. Acrylamide quenching studies indicated that the degree of exposure of Trp residues in the neurotoxins followed the order Oh-8 > Oh-7 > Oh-6A > Oh-4 > alpha-bungarotoxin, as did the accessibility for iodide. These results reveal that the exposed degree of Trp residues and the microenvironment surrounding Trp residues in the neurotoxins differ, even though their Trp residues and positively charged residues are located at the same or homologous positions. In contrast to unfolded Oh-4, Oh-6A, Oh-7, and alpha-bungarotoxin, unfolding of Oh-8 by reduction and S-carboxymethylation caused a notable decrease in the susceptibility of their Trp residues for iodide. These observations support the view that the side chains of Trp residues and positively charged residues in their native structure do not point toward the same spatial positions. Computer models of the neurotoxins are in good agreement with this proposition. These results elucidate why the conserved Trp residues and cationic groups do not always play the same roles in the biological activities of the neurotoxins.

Oxytocinergic neurotransmission at the hippocampus in the central neural regulation of penile erection in the rat.

OBJECTIVES: To investigate whether there is an oxytocinergic neurotransmission at the hippocampus involved in the central regulation of penile erection in the rat. METHODS: Male adult Sprague-Dawley (n = 27) rats anesthetized with pentobarbital were used. A 26-gauge needle was inserted into the corpus cavernosum to measure the intracavernous pressure (ICP) and the systemic and mean arterial pressure and heart rate simultaneously. The following studies were performed: stereotaxic delivery of oxytocin acetate (3 pmol/100 nL) into the hippocampus; microinjection of a mixture of [d(CH(2))(5)-Tyr(Me)(2)-Orn(8)]-vasotocin (3 pmol/100 nL) and oxytocin (3 pmol/100 nL) into the hippocampus; injection of saline into the hippocampus; and intracavernous injection of oxytocin (3 pmol/100 microL). The ICP and hemodynamic parameters were monitored after each administration of the experimental agents. RESULTS: After administration of oxytocin into the hippocampus, a significant increase in the ICP occurred from resting (8.8 +/- 1.7 mm Hg) to a peak at 49.6 +/- 12.5 mm Hg and persisted for 18.6 +/- 9.4 minutes after an onset latency of 500.0 +/- 389.7 seconds. However, no change in the ICP occurred after administration of [d(CH(2))(5)-Tyr(Me)(2)-Orn(8)]-vasotocin plus oxytocin into the hippocampus. In addition, no elevation of ICP occurred after administration of saline to the hippocampus or after intracavernous injection of oxytocin. CONCLUSIONS: The results demonstrate that administration of oxytocin into the hippocampus induces penile erection in the rat. However, concomitant administration of oxytocin and its antagonist was ineffective in eliciting penile erection. These observations suggest that oxytocinergic neurotransmission at the hippocampus may be involved in the central neural regulation of the penile erection in the rat.

Oxidative damage to proteins and decrease of antioxidant capacity in patients with varicocele.

To examine oxidative damage to blood proteins in the spermatic vein and seminal plasma antioxidant capacity of patients with varicocele, 30 young male patients with varicocele (group 1), 25 young male patients with subclinical varicocele (group 2), and 15 normal young males without varicocele (group 3) were recruited in this study. Varicocele and subclinical varicocele were confirmed by physical examination and Doppler ultrasonography. Blood samples were drawn from peripheral and spermatic veins before varicocelectomy. Plasma protein carbonyls were measured by a spectrophotometric assay after reacting with 2,4-dinitrophenylhydrazine. Protein thiols and ascorbic acid of seminal plasma were measured by spectrophotometric methods. We found that plasma protein carbonyls in the spermatic veins were significantly higher than those of corresponding peripheral veins in all 30 patients in group 1 and 12 patients in group 2 receiving varicocelectomy. Protein carbonyls in the spermatic veins of patients with varicocele (3.72 +/- 0.56 nmole/mg protein) and patients with subclinical varicocele (3.50 +/- 0.30 nmole/mg protein) were found to be higher than those of the control (2.35 +/- 0.33 nmole/mg protein). Protein thiols were 0.97 +/- 0.96, 1.50 +/- 0.89, and 3.49 +/- 0.81 nmole/ml, and ascorbic acid levels were 1.87 +/- 0.42, 2.13 +/- 0.24, and 2.38 +/- 0.07 mg/dl, in seminal plasma of the patients in groups 1, 2, and 3, respectively. Seminal plasma protein thiols and ascorbic acid levels in group 1 were significantly lower than those in groups 2 and 3, respectively. These results indicate that oxidative stress in the patients with varicocele and subclinical varicocele was higher than that of the control. We suggest that plasma protein carbonyls, and protein thiols and ascorbic acid of seminal plasma are useful markers for the assessment of oxidative stress in patients with varicocele and subclinical varicocele.

Refolding of Taiwan cobra neurotoxin: intramolecular cross-link affects its refolding reaction.

In order to explore the effect of intramolecular cross-linking in the folding reaction of cobrotoxin from Naja naja atra (Taiwan cobra) venom, the toxin molecule was modified with glutaraldehyde (GA). The monomeric GA-modified cobrotoxin (mGA-cobrotoxin) was separated from the dimeric and trimeric derivatives using gel filtration. The results of electrophoretic and chromatographic analyses revealed that mGA-cobrotoxin comprised two modified derivatives, which contained modified Lys residues at positions 26 and 27 and at positions 26, 27, and 47, respectively. Moreover, an intramolecular cross-linking of loops II and III by Lys residues was noted with the monomeric derivative containing three modified Lys residues. In sharp contrast to cobrotoxin observations, the folding rate of mGA-cobrotoxin decreased in the presence of GSH/ GSSG, but notably increased in the absence of thiol compounds. Particularly, the accelerated effect of GSH/GSSG on the refolding reaction was affected by the presence of the intramolecular cross-link. Comparative analyses on cobrotoxin and mGA-cobrotoxin CD spectra revealed that modification with the GA reagent caused a change in the gross conformation of cobrotoxin. Fluorescence measurement revealed that the stability of the microenvironment around the single Trp-29 in mGA-cobrotoxin and unfolded mGA-cobrotoxin was appreciably higher than in cobrotoxin and unfolded toxin. Moreover, the ordered structure formation around Trp-29 in refolded mGA-cobrotoxin was faster than in refolded cobrotoxin as evidenced by fluorescence quenching studies. Taken together, these results suggest that the structural flexibility of unfolded cobrotoxin should be favorable for the thiol catalyst to exert its action in the refolding reaction after modification with GA.