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.

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.

Decay of female sexual behavior under parthenogenesis.

A laboratory strain of Drosophila mercatorum has existed for 20 years without males and therefore without natural selection operating to maintain the genetic basis of female mating behavior. The females of this strain have recently experienced a genetic impairment of mating capacity. This observation exemplifies the mode of evolution of vestigial characters and supports Muller's theory that random mutation will tend to destroy the genetic basis of a character from which selection has been removed.

[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.

Differential roles of two tandem E2F sites in repression of the human p107 promoter by retinoblastoma and p107 proteins.

Although many lines of evidence indicate that the cellular protein p107 is closely related to the retinoblastoma protein, the exact function of the p107 gene and its regulation are presently not known. To investigate the molecular mechanism controlling expression of the human p107 gene, a 5' flanking sequence of this gene was isolated and shown to promote high-level expression of a luciferase reporter gene in cycling human 293 and Saos-2 cells. Sequencing and transcription mapping analyses showed that the human p107 promoter is TATA-less and contains a tandem, direct repeat of E2F-binding sites, with the 3' copy overlapping the major transcription initiation site. Deletion analysis of the p107 promoter showed that a promoter DNA fragment containing only the two E2F sites together with the leader sequence could direct relatively efficient expression in 293 cells. Site-directed mutagenesis of these E2F sites revealed that although both sites were important for p107 promoter activity, mutation on the proximal, initiation site copy of the E2F site showed a stronger effect. The human p107 promoter could be repressed by the retinoblastoma protein and its own gene product. Interestingly, the repression was found to be mediated through the 5' copy of the E2F site. These studies demonstrate for the first time differential roles of two tandem E2F sites in promoter regulation.

p53 dependence of topoisomerase I recruitment in vivo.

DNA damage is attended by rapid recruitment of endogenous type I topoisomerase (topo I) into covalent cleavage complexes with genomic DNA in vivo. In contrast, endogenous topoisomerase II alpha and beta are not stimulated by DNA damage. We show that topo I and p53 are able to associate at arrested topo I-genomic DNA covalent complexes in vivo, suggesting that p53 directly stimulates topo I activity and damage to the genome of the afflicted cell. Moreover, cells that express wild-type p53 are most proficient at recruiting topo I after DNA damage; however, the p53 dependence is conditional because topo I recruitment after DNA damage can be restored if p53 mutant cells (containing a single mutant allele) are artificially held in G1. In contrast, p53 null mutants do not recruit topo I after DNA damage under any conditions (although camptothecin-dependent topo I/DNA complexes readily form in the nulls). These results show that topo I activation after DNA damage depends on the p53 status of the cell. It also depends upon the cell cycle in a way that is very different from that observed with DNA replication-dependent, camptothecin-mediated DNA breaks. The data suggest a model where p53 activates topo I, which inflicts additional genomic damage after the initial UV damage events. Topoisomerases therefore contribute to the p53 commitment to apoptosis, and topo I might assist in elimination of DNA-damaged cells as part of the cellular proofreading function inherent in the p53 pathway.

Repressed expression of the HER-2/c-erbB-2 proto-oncogene by the adenovirus E1a gene products.

The E3 region of adenovirus induces down-regulation of epidermal growth factor receptor (EGFR) through endocytosis. Here we report that an EGFR-related protein, the HER-2/c-erbB-2 gene product, p185, is also down-regulated by adenovirus, but via a different mechanism. We found that the adenovirus E1a gene is responsible for the repression of HER-2/c-erbB-2 at the RNA level.

The N-terminal amino group essential for the biological activity of notexin from Notechis scutatus scutatus venom.

Notexin from Notechis scutatus scutatus snake venom was modified with trinitrobenzenesulfonic acid, and the major trinitrophenylated (TNP) derivative was separated by high-performance liquid chromatography. Modification resulted in the incorporation of only one TNP group on the N-terminal alpha-amino group. The TNP derivative showed a precipitous decrease in enzymatic activity and lethal toxicity, whereas the antigenicity remained unchanged. However, trinitrophenylation did not significantly affect the secondary structure of the toxin molecule as revealed by the CD spectra. The results, that the modification reaction was accelerated by the Ca2+ and that the TNP derivative retains its affinity for Ca2+, indicate that the N-terminal alpha-amino group did not participate in the Ca2(+)-binding. The TNP derivative could be regenerated with hydrazine hydrochloride. The biological activities of the regenerated notexin are almost the same as those of native notexin. These results suggest that the N-terminal alpha-amino group is essential for the phospholipase A2 activity and lethal toxicity of notexin, and that incorporation of the TNP group on the N-terminal alpha-amino group might give rise to a distortion of the active conformation of notexin.

cDNA sequence analysis and expression of cardiotoxin V and a new cardiotoxin VII from Naja naja atra (Taiwan cobra).

The cDNAs encoding cardiotoxin V and a new cardiotoxin VII were constructed from the cellular RNA isolated from the venom glands of Naja naja atra by reverse transcription-polymerase chain reaction. Although 95% nucleotide sequence homology was observed with the two cardiotoxins, there were nine amino-acid substitutions between cardiotoxin V and cardiotoxin VII. The cardiotoxins were subcloned into the expression vector pET 20b(+) and transformed into BL21(DE3) E. coli strain. The expressed protein was isolated from the inclusion bodies of E. coli, and purified by reverse-phase high-performance liquid chromatography. The purified recombinant cardiotoxin showed immunoreactivity with anti-cardiotoxin III antibodies as revealed by immunoblot analysis.

Identification of functional involvement of tryptophan residues in phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom.

Phospholipase A2 (PLA2) from Naja naja atra snake venom was subjected to Trp modification with 2-nitrophenylsulfenyl chloride (NPS-Cl), and six derivatives were separated by HPLC. The results of amino-acid analysis and sequence determination revealed that Trp-18, Trp-19 and Trp-61 were modified by NPS-Cl. The order of accessibilities of the three Trp residues for NPS-Cl was Trp-18 > Trp-19 > Trp-61. Sulfenylation of Trp-18 caused a 92% drop in enzymatic activity. Modification of Trp-19 and Trp-61 resulted in a decrease in enzymatic activity of PLA2 by 45.5% and 51%, respectively. The enzyme modified on both Trp-18 and Trp-19 or on both Trp-18 and Trp-61 retained little PLA2 activity. It is evident that Trp-18 plays a more crucial function in PLA2 than Trp-19 and Trp-61. Sulfenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca2+ binding and antigenicity of sulfenylated PLA2 was unaffected. These observations, together with the fact that Trp-18 is involved in the substrate binding of PLA2, suggest that incorporation of a bulky NPS group on Trp-18 might give rise to a direct distortion of the interaction between substrate and the enzyme molecule. Alternatively, modification of Trp-19 and Trp-61 might indirectly affect the interfacial binding of PLA2 with its substrate.

Specific frequency properties of renal and superior mesenteric arterial beds in rats.

In order to investigate the effects of arterial beds on pressure waveforms in arteries, the pressure waves observed in the rat tail artery were resolved into their Fourier moduli before and during ligation of the left renal artery and the superior mesenteric artery. Consistently different patterns of waveform changes in the tail artery were seen on occlusion of these vessels. Ligation of the renal artery reduced, and of the superior mesenteric artery increased, the pressure harmonics over most of the spectra. These results imply that to study the changes in the pressure contours as the observation point is moved downstream, one may have to account separately for the contributions of individual arterial beds. This is relevant in considering the degree to which it is appropriate for data to be amalgamated in models of the systemic arterial bed.

The murine erythrocyte protein-4.2-encoding gene: similarities and differences in structure and expression from its human counterpart.

We have isolated a complete cDNA encoding for the mouse erythrocyte protein 4.2 (P4.2). The entire P4.2 cDNA consists of 3465 nt with an open reading frame (ORF) of 691 amino acids. Northern blot analysis of mouse reticulocyte or spleen RNA using the P4.2 cDNA as a probe, detected a 3.5-kb message. The size of the mouse P4.2 cDNA or message that we obtained, appears to be different from those reported recently. Despite the similarity to the human P4.2 cDNAs, the mouse cDNA has a longer 3' untranslated region. A genomic clone covering the first exon and flanking sequences of the mouse P4.2 gene was isolated. Sequencing results from the first exon-intron junction region and polymerase chain reaction (PCR) experiments revealed that the mouse reticulocyte P4.2 RNA does not exhibit alternative splicing in the region identified in the human P4.2 RNA.

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.

Serum myoglobin in Duchenne muscular dystrophy carrier detection: a comparison with creatine kinase and hemopexin using logistic discrimination.

Logistic discrimination was used to assess the effectiveness of serum myoglobin (Mb), creatine kinase (CK), and hemopexin (H) measurements in identifying Duchenne muscular dystrophy (DMD) carriers. Subjects included 36 obligate carriers, 46 age-matched control women, 30 mothers of isolated cases, and 14 DMD patients. The percentages of obligate carriers with logistic carrier probabilities exceeding the upper normal 95th centile (or 97.5th centile) were: CK alone, 63% (50%); Mb alone, 65% (62%); CK and Mb, 66% (62%); CK and H, 78% (65%); CK H and Mb, 72% (65%). In this study, Mb identified more carriers than CK at the 97.5% level, but there was no advantage in using Mb measurements with CK. CK provided slightly better overall separation of the control and carrier groups than Mb. CK, Mb, and H in combination provided significantly better separation than CK and H, or CK alone.

The structural loop II of cobrotoxin is the main binding region for nAChR and epitope in the region is conformation-dependent.

Modification of positively charged residues, Lys and Arg, in cobrotoxin revealed that Lys-27, Lys-47, Arg-28, Arg-30, Arg-33, and Arg-36 of cobrotoxin were essential for the lethality and binding activity to nicotinic acetylcholine receptor (nAChR). The antigenicity of cobrotoxin was drastically diminished when Lys-47, Arg-28, Arg-30, Arg-33, and Arg-36 were modified, while that of the Lys-27-modified derivative was not significantly changed. The CD spectra of cobrotoxin displayed similar patterns after modification of Lys-27, Lys-47, and Arg-28. These findings suggest that Lys-27, Lys-47, and Arg-28 residues may be related to the direct binding to nAChR, and that there is no involvement of Lys-27 in the antigenic determinants of cobrotoxin. Extending the modification to Arg-30, Arg-33, and Arg-36 caused progressive conformational changes of cobrotoxin and resulted in decreased binding activity to antibody and nAChR. This indicates that Arg-30, Arg-33, and Arg-36 may be of structural importance for maintaining the active conformation of cobrotoxin. These results, together with the facts that Tyr-25, Tyr-35, and Trp-29 of cobrotoxin are important in nAChR binding activity, but Trp-29 and Tyr-35 residues are not essential for the antigenicity, suggest that the structural loop II of cobrotoxin is the main binding region for nAChR and the epitope in that region is conformation-dependent.

Identification of arg-30 as the essential residue for the enzymatic activity of Taiwan cobra phospholipase A2.

Taiwan cobra (Naja naja atra) phospholipase A2 (PLA2) was inactivated by arginine-specific reagents, phenylglyoxal and 1, 2-cyclohexanedione. Kinetic analyses of the modification reaction revealed that the inactivation of PLA2 followed pseudo-first-order kinetics and the loss of activity was correlated with the incorporation of one molecule of modification reagent per PLA2 molecule. This was confirmed by the results of amino acid composition determination, that showed that a marked decrease in enzymatic activity was associated with the modification of one arginine residue. Tryptic cleavage of the modified protein and microsequencing revealed that Arg-30 was the functionally essential residue. The incorporation of a modifier into the PLA2 did not significantly affect the secondary structure of the enzyme, as revealed by the CD spectrum, and Ca2+-binding of the modified PLA2 was unaffected. Nevertheless, the nonpolarity of the active site of PLA2 markedly decreased with the arginine modification, as evidenced by the decreases in the enhancement of Trp and 8-anilinonaphthalene sulfonate fluorescence. These results, together with those of X-ray crystallographic analysis of N. naja atra PLA2 [Scott et al. (1990) Science 250, 1541-1546], demonstrate that Arg-30 is one of the residues involved in the interfacial binding of a PLA2 molecule with its substrate.