Tzong-Hsiung Hseu

The major capsid protein, VP1, of human JC virus expressed in Escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells.

The full-length major capsid protein, VP1, of the human polyomavirus JC virus was cloned and expressed in Escherichia coli. VP1 protein expressed in E. coli self-assembled into capsid-like particles and caused haemagglutination of human O-type red blood cells. Caesium chloride density-gradient centrifugation analysis revealed that the capsid-like particles consisted of virion-like pseudovirion and empty capsid-like pseudocapsid populations. The morphology of pseudo-virion and pseudocapsid particles was observed under the electron microscope. The pseudovirions contained DNA and RNA molecules but the pseudocapsids did not contain any nucleic acid, as analysed by DNA extraction. DNA-binding activity of VP1 was also demonstrated by the South-Western probing method in vitro. Furthermore, pseudocapsids were able to deliver exogenous DNA into human foetal kidney epithelial cells. These results indicate that recombinant JC virus VP1 is able to self-assemble into capsid-like particles and to package DNA in the absence of the minor capsid proteins, VP2 and VP3. This prokaryotic assembly system may facilitate the investigation of maturation mechanism(s) of polyomaviruses. Furthermore, capsid-like particles of JC virus VP1 generated in E. coli potentially could be used as a human gene transfer vector.

Structure and genomic organization of porcine RACK1 gene.

The cDNA encoding porcine RACK1 protein was isolated from porcine spleen cDNA library. The deduced protein sequence of porcine RACK1 cDNA shows that it contains 317 amino acid residues, and shares nearly 100% identity with its vertebrate counterparts. Noticeably, the RACK1 protein was differentially expressed in various porcine tissues. High expression of RACK1 protein was observed in the tissues including thymus, pituitary, spleen and liver, whereas there was no detectable expression in muscle. The genomic DNA of porcine RACK1 with approximate 7.5 kb was constructed by both polymerase chain reaction amplification and genomic library screening. It consists of eight exons intervened by seven introns, and most of the intron/exon splice sites conform to the GT/AG rule. The promoter region contains functional serum response element, YY1-like binding site and AP1 site, which is supported by the finding that the expression of RACK1 gene in cultured porcine ST cells has a serum response as well as a TPA response.

Molecular cloning and sequence analysis of the cellobiohydrolase I gene from Trichoderma koningii G-39.

The cellobiohydrolase I gene,cbh1, has been cloned from an enhanced cellulase-producing strain,Trichoderma koningii G-39. Sequence comparisons show thatT. koningii cbhl is identical to that ofT. reesei with the exception of 6 bp-two causing silent substitutions in the coding region, three differing in one of the introns, and one in 5′-noncoding region. Thus, it should encode an identical CBHI to that ofT. reesei despite the differences in morphological characters of the two species. Analysis of approximately 1.4 kb of the 5′ flanking region shows a number of surprisingly interesting putative regulatory features. There are no unusual features within about 600 bp upstream of the translation start ATG. However, prior to the 600-bp region, there are seven CAAT sequences, a number of direct and inverted repeats, and two C/T-rich regions. Also, there are five consensus 5′-(G/C)PyGGGG-3′ sequences that have been identified to be carbon catabolite repressor binding sites ofAspergillus nidulans CREA andSaccharomyces cerevisiae MIG1 repressors. The structural organization arround these consensus sequence regions is similar to those ofA. nidulas alcR andalcA promoters. While the production of large amounts of CBHI byT. koningii upon induction apparently correlates with the large number of CAAT boxes in the 5′ upstream untranslated region ofcbh1, the presence of five CREA/MIG1 repressor-binding consensus sequences in the region suggests the wide-domain carbon catabolite repression regulatory system that controls theA. nidulans ethanol regulon, and yeast GAL genes transcription might also be operative and responsible for regulation ofT. koningii cbh1 transcription.

Synthesis and structure-activity relationship of 6-arylureido-3-pyrrol-2-ylmethylideneindolin-2-one derivatives as potent receptor tyrosine kinase inhibitors.

A series of new ureidoindolin-2-one derivatives were synthesized and evaluated as inhibitors of receptor tyrosine kinases. Investigation of structure-activity relationships at positions 5, 6, and 7 of the oxindole skeleton led to the identification of 6-ureido-substituted 3-pyrrolemethylidene-2-oxindole derivatives that potently inhibited both the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) families of receptor tyrosine kinases. Several derivatives showed potency against the PDGFR inhibiting both its enzymatic and cellular functions in the single-digit nanomolar range. Among them, compound 35 was a potent inhibitor against tyrosine kinases, including VEGFR and PDGFR families, as well as Aurora kinases. Inhibitor 36 (non-substituted on the pyrrole or phenyl ring) had a moderate pharmacokinetic profile and completely inhibited tumor growth initiated with the myeloid leukemia cell line, MV4-11, in a subcutaneous xenograft model in BALB/c nude mice.

Laser raman studies on the conformation of Pro-Leu-Gly-NH2

Laser Raman spectra of Pro-Leu-Gly-NH2, the factor that inhibits release of pituitary melanotropin, have been obtained in the solid state, in dimethylsuloxide and in aqueous solution. The amide I frequencies were observed at 1650 and 1687 cm-1 in the solid state and at 1669 cm-1 in dimethylsulfoxide. The conformation of this tripeptide has been proposed by 1H-NMR studies in [2H]-dimethylsulfoxide and revealed by X-ray analysis to be type II beta-turn. These observed amide I frequencies thus are characteristic of type II beta-turn conformation. The relatively lower amide I frequency, 1645 cm-1, observed in 2H2O indicates that the conformation of the peptide in aqueous solution could be different from those in solid state and in dimethylsulfoxide. pH changes hae no significant effect on aqueous solution spectra, except for the shape of the amide III band. The maximum of the amide III band shifted from 1259 cm-1 at pH 2.0 to 1242 cm-1 at pH 11.7. The amide III peaks in the solid state were at 1234 and 1268 cm-1.

Sennoside B inhibits PDGF receptor signaling and cell proliferation induced by PDGF-BB in human osteosarcoma cells.

AIMS To address the possibility that sennoside B inhibition of cell proliferation is mediated via interference with platelet-derived growth factor (PDGF) signaling. MAIN METHODS Human osteosarcoma MG63 cells were treated with PDGF in the presence or absence of sennoside B. Activation of the PDGF signaling pathway was monitored using western immunoblotting with specific antibodies against the PDGF receptor, phosphotyrosine and components of the downstream signaling cascade. Activation of cell metabolism and proliferation was assessed by chromogenic reduction of MTT. KEY FINDINGS Sennoside B was found to inhibit PDGF-BB-induced phosphorylation of the PDGF receptor (PDGFR) in human MG63 osteosarcoma cells. Downstream signaling was also affected; pre-incubation of PDGF-BB with sennoside B inhibited the phosphorylation of pathway components including Ak strain transforming protein (AKT), signal transducer and activator of transcription 5 (STAT-5) and extracellular signal-regulated kinase 1/2 (ERK1/2). Further, we found that sennoside B can bind directly to the extracellular domains of both PDGF-BB and the PDGF-beta receptor (PDGFR-beta). The effect was specific for sennoside B; other similar compounds including aloe-emodin, rhein and the meso isomer (sennoside A) failed to inhibit PDGFR activation or downstream signaling. Sennoside B also inhibited PDGF-BB stimulation of MG63 cell proliferation. SIGNIFICANCE These results indicate that sennoside B can inhibit PDGF-stimulated cell proliferation by binding to PDGF-BB and its receptor and by down-regulating the PDGFR-beta signaling pathway. Sennoside B is therefore of potential utility in the treatment of proliferative diseases in which PDGF signaling plays a central role.

Induction of cellulase by cello-oligosaccharides in Trichoderma koningii G-39

Abstract A fraction of cello-oligosaccharides with a high degree of polymerization (Gx, degree of polymerization greater than 10) was found to be a potent cellulase inducer of Trichoderma koningii G-39. It was prepared by partial sulfuric acid hydrolysis of cellulose. A comparison of the induction potential and efficiency of Gx and sophorose indicated that the latent period of induction was longer and the maximal enzyme production lower for Gx than for sophorose. The optimal concentrations are 1% with Gx and 0.1% (w/v) with sophorose with a corresponding latent period and a maximum carboxymethylcellulase (CMCase) activity, which accumulated in the culture filtrate, of about 8 h and 5.4 units for Gx and 4 h and 7.2 units (in mg glucose equivalent per ml culture filtrate) for sophorose. The apparent differences between the two compounds are attributable to the interplay of catabolite repression in the inducing effect of Gx which is subjected to cellulase degradation with glucose as the major end products. HPLC sugar analysis of the culture filtrate incubated with Gx suggests that the inducing potential and efficiency of cello-oligosaccharides depend on its degree of polymerization. The protein patterns as revealed by SDS-polyacrylamide gel analysis of the culture filtrates from incubation with Avicel, phosphoric acid-swollen Avicel, Gx or sophorose showed no inducerspecific protein bands except variation in intensity and induction time. A dot hybridization analysis of poly(A) RNA from washed mycelium incubated with Gx or sophorose using a 650 basepair cDNA fragment of cellobiohydrolase I gene as the probe indicates that the inductive formation of cellulase by both inducers in this strain is operative at the transcriptional level.

Specificity of the acid protease from Monascus kaoliang towards the B-chain of oxidized insulin.

The proteolytic specificity of the acid protease from Monascus kaoliang has been investigated using the B-chain of performic acid-oxidized insulin as peptide substrate. Six splittings were detected after 1 h digestion and 12 splittings were found after 12 h incubation at 37 degrees C, pH 4.8. The bonds most susceptible to the acton of M. kaoliang acid protease were Phe(24)-Phe(25), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). Among the acid proteases compared, the specificity of M. kaoliang acid protease on the B-chain of oxidized insulin is more closely related to that of penicillopepsin with which it has ten cleavage sites in common. N-Acetyl-L-phenylalanyl-L-3,5-diiodotyrosine, a synthetic substrate for pepsin, was resistant to the hydrolysis of M. kaoliang acid protease.

Novel azulene-based derivatives as potent multi-receptor tyrosine kinase inhibitors.

A series of azulene-based derivatives were synthesized as potent inhibitors for receptor tyrosine kinases such as FMS-like tyrosine kinase 3 (FLT-3). Systematic side chain modification of prototype 1a was carried out through SAR studies. Analogue 22 was identified from this series and found to be one of the most potent FLT-3 inhibitors, with good pharmaceutical properties, superior efficacy, and tolerability in a tumor xenograft model.

Cell growth restoration and high level protein expression by the promoter of hexose transporter, HXT7, from Saccharomyces cerevisiae

The promoters of high-affinity hexose transporter, HXT6 and HXT7, are sufficient for complementary expression of invertase to restore the growth of Saccharomyces cerevisiae in raffinose medium. The HXT7 promoter produced higher invertase activity at 139- and 30-fold of the basal activity in strains GN 3C.2 and W303-1, respectively. In addition, the HXT7 promoter expressed 3- to 9-fold more of enhanced green fluorescent protein than that of the constitutive ADH1 in three different S. cerevisiae strains, even during short-term incubation in glucose medium. Therefore, HXT7 promoter could be used for heterologous protein expression in S. cerevisiae.