Intracellular interaction of collagen-specific stress protein HSP47 with newly synthesized procollagen.

Heat shock protein 47 (HSP47), a collagen-specific stress protein, has been postulated to be a collagen-specific molecular chaperone localized in the ER. We previously demonstrated that HSP47 transiently associated with newly synthesized procollagen in the ER (Nakai, A., M. Satoh, K. Hirayoshi, and K. Nagata. 1992. J. Cell Biol. 117:903-914). In the present work, we examined the location where HSP47 binds to and dissociates from newly synthesized procollagen within the cells, and whether HSP47 associates with nascent single procollagen polypeptide chains and/or with mature triple-helix procollagen. This was accomplished by biochemical coprecipitation with anti-HSP47 and anticollagen antibodies, combined with pulse-label and chase experiments in the presence or absence of various inhibitors for protein secretion, as well as by confocal laser microscopic observation of the cells double stained with both antibodies. We further examined whether the RDEL (Arg-Asp-Glu-Leu) sequence at the COOH terminus of HSP47 can act as an ER-retention signal, as the KDEL sequence does. When the secretion of procollagen was inhibited by the presence of alpha, alpha'-dipyridyl, an iron chelator that inhibits procollagen triple-helix formation, or by the presence of brefeldin A. which inhibits protein transport between the ER and the Golgi apparatus, procollagen was found to be bound to HSP47 during the chase period in the intermediate compartment. In contrast, the dissociation of procollagen chains from HSP47 was not inhibited when procollagen secretion was inhibited by monensin or bafilomycin A1, both of which are known to be inhibitors of post-cis-Golgi transport. These findings suggest that HSP47 and procollagen dissociated between the post-ER and the cis-Golgi compartments. HSP47 was shown to bind to nascent, single-polypeptide chains of newly synthesized procollagen, as well as to the mature triple-helix form of procollagen. HSP47 with the RDEL sequence deleted was secreted out of the cells, which suggests that the RDEL sequence actually acts as an ER-retention signal, as the KDEL sequence does. This secreted HSP47 did not acquire endoglycosidase H resistance. The biological significance of the interaction between HSP47 and procollagen in the central secretory pathway, as well as possible mechanisms for this pathway, will be discussed.

Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis.

Triple helix formation of procollagen after the assembly of three alpha-chains at the C-propeptide is a prerequisite for refined structures such as fibers and meshworks. Hsp47 is an ER-resident stress inducible glycoprotein that specifically and transiently binds to newly synthesized procollagens. However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo. Here, we describe the establishment of Hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of alpha1(I) collagen and fibril structures in mesenchymal tissues. The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes. The homozygous mice did not survive beyond 11.5 days postcoitus (dpc), and displayed abnormally orientated epithelial tissues and ruptured blood vessels. When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47+/+ and Hsp47+/- cells were resistant, but those of Hsp47-/- cells were sensitive. These results indicate for the first time that type I collagen is unable to form a rigid triple-helical structure without the assistance of molecular chaperone Hsp47, and that mice require Hsp47 for normal development.

Alternative 5' splice site selection induced by heat shock.

The mouse HSP47 gene consists of six exons separated by five introns. Three HSP47 cDNAs differing only in their 5' noncoding regions have been reported. One of these alternatively spliced mRNAs was detected only after heat shock, which caused an alternative 5' splice donor site selection. Other stress inducers, including an amino acid analog and sodium arsenite, had no effect on the alternative splicing. The alternatively spliced mRNA, which was 169 nucleotides longer in the 5' noncoding region compared to mRNA transcribed in non-heat shock conditions, was efficiently translated under heat shock conditions. This novel finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression.

Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids.

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.

Unique characteristics of community-onset healthcare- associated bloodstream infections: a multi-centre prospective surveillance study of bloodstream infections in Japan.

BACKGROUND: Analysis of bloodstream infections (BSIs) is valuable for their diagnosis, treatment and prevention. However, limited data are available in Japan. AIM: To investigate the characteristics of patients with bacteraemia in Japan. METHODS: This study was conducted in five hospitals from October 2012 to September 2013. Clinical, demographic, microbiological and outcome data for all blood-culture-positive cases were analysed. FINDINGS: In total, 3206 cases of BSI were analysed: 551 community-onset healthcare-associated (CHA)-BSIs, 1891 hospital-acquired (HA)-BSIs and 764 community-acquired (CA)-BSIs. The seven- and 30-day mortality rates were higher in patients with CHA- and HA-BSIs than in patients with CA-BSIs. The odds ratios (ORs) for seven-day mortality were 2.56 [95% confidence interval (CI) 1.48-4.41] and 2.63 (95% CI 1.64-4.19) for CHA- and HA-BSIs, respectively. The ORs for 30-day mortality were 2.41 (95% CI 1.63-3.57) and 3.31 (95% CI 2.39-4.59) for CHA- and HA-BSIs, respectively. There were 499 cases (15.2%) of central-line-associated BSI and 163 cases (5.0%) of peripheral-line-associated BSI. Major pathogens included coagulase-negative staphylococci (N = 736, 23.0%), Escherichia coli (N = 581, 18.1%), Staphylococcus aureus (N = 294, 9.2%) and Klebsiella pneumoniae (N = 263, 8.2%). E. coli exhibited a higher 30-day mortality rate among patients with HA-BSIs (22.3%) compared with patients with CHA-BSIs (12.3%) and CA-BSIs (3.4%). K. pneumoniae exhibited higher 30-day mortality rates in patients with HA-BSIs (22.0%) and CHA-BSIs (22.7%) compared with patients with CA-BSIs (7.8%). CONCLUSION: CHA- and HA-BSIs had higher mortality rates than CA-BSIs. The prognoses of E. coli- and K. pneumonia-related BSIs differed according to the category of bacteraemia.

The human genome has only one functional hsp47 gene (CBP2) and a pseudogene (pshsp47).

Among all the species investigated to date, only in humans is hsp47 reported to exist as two separate genes. Here we examined whether hsp47 forms a gene family, and if so, how many genes constitute the family. Cloning and sequencing of human hsp47 cDNA revealed that only one gene, identical to CBP2, was transcribed. No transcript corresponding to colligin, which was reported to be a human homologue of hsp47, was found. Genomic southern hybridization using the exon III fragment of mouse hsp47 as a probe, however, showed two bands for several restriction enzyme digests. We cloned and sequenced the gene corresponding to the extra band and found that a pseudogene (pshsp47) existed in the human genome. We have mapped this pseudogene to chromosome 9p12-p13 by fluorescent in situ hybridization (FISH) using a 3.5kb genomic fragment containing the entire pshsp47 sequence as a probe. These results suggested that functional hsp47 exists as CBP2, not as colligin, and a highly conserved pseudogene is present in the human genome.

Structure of the gene encoding the mouse 47-kDa heat-shock protein (HSP47).

HSP47, a 47-kDa heat-shock protein (HSP), is a member of a group of HSPs with the unique characteristics of collagen binding as well as transformation sensitivity. The protein belongs to the serpin (serine protease inhibitor) superfamily as determined from its amino acid sequence homology. We have isolated and characterized the mouse HSP47 including about 1 kb of the 5'-flanking region. This gene spans about 7.8 kb, consisting of six exons separated by five introns. This exon-intron structure is different from other serpin family proteins. Southern blot analysis revealed the existence of a single copy of HSP47. The promoter region contains a TATA box, four Sp1-binding sites and one AP-1-binding site. A complete heat-shock element (HSE) was found between nucleotides (nt) -61 and -79. Furthermore, the heat inducibility was reproduced by transfecting mouse BALB/3T3 cells with a plasmid carrying cat under the control of the HSE-containing fragment (nt -197 and +38) of HSP47. Computer analysis of the promoter region did not show marked homology to other vertebrate promoters.

Procollagen binds to both prolyl 4-hydroxylase/protein disulfide isomerase and HSP47 within the endoplasmic reticulum in the absence of ascorbate.

In cells, only properly folded procollagen trimers are secreted from the endoplasmic reticulum (ER), while improperly folded abnormal procollagens are retained within the ER. Ascorbic acid is a co-factor in procollagen hydroxylation, which in turn is required for trimer formation. We examined chaperone proteins which bound to procollagen in the absence of ascorbic acid, a model which mimics the human disease scurvy at the cellular level. We found that both prolyl 4-hydroxylase (P4-H)/protein disulfide isomerase (PDI) and HSP47 bound to procollagen in the absence of ascorbic acid. However, the binding of PDI to procollagen decreased when HSP47 was co-transfected, suggesting that HSP47 and PDI compete for binding to procollagen. These data indicate that P4-H/PDI and HSP47 have cooperative but distinct chaperone functions during procollagen biosynthesis.

Quercetin, a bioflavonoid, inhibits the increase of human multidrug resistance gene (MDR1) expression caused by arsenite.

Expression of the MDR1 gene, which encodes P-glycoprotein, is increased under some stress conditions. We have reported that quercetin, a bioflavonoid, inhibits the expression of heat-shock proteins. We have identified the effects of quercetin on the MDR1 gene expression in the human hepatocarcinoma cells line, HepG2. The increase of P-glycoprotein synthesis and MDR1 mRNA accumulation caused by exposure to arsenite were inhibited by quercetin. The CAT assay suggested that quercetin suppressed the transcriptional activation of the MDR1 gene after exposure to arsenite. Although many drugs that prevent the P-glycoprotein function have been reported, this is the first report to describe the inhibition of MDR1 expression by a reagent.

N-myc suppression and cell cycle arrest at G1 phase by prostaglandins.

Effects of cyclopentenone prostaglandins, delta 12-prostaglandin (PG) J2 and PGA2 on the expression of N-myc in relation to the effects on cell cycle progression were investigated using human neuroblastoma cell line GOTO. Both PGs suppressed N-myc expression within several hours prior to inducing G1 arrest. The N-myc suppression with delta 12-PGJ2 was continued but with PGA2 it was gradually released, followed by the release of G1 arrest. These results suggest that delta 12-PGJ2 and PGA2 inhibit cell cycle progression in strong association with N-myc suppression and delta 12-PGJ2 is more potent and has a longer effect than PGA2.

The effect of quercetin on cell cycle progression and growth of human gastric cancer cells.

Quercetin, a flavonoid, is found in many plants, including edible fruits and vegetables. We examined the effects on cell growth of human malignant cells derived from the gastrointestinal tract and on cell cycle progression. Quercetin markedly inhibited the growth of human gastric cancer cells and the IC50 value was 32-55 microM. DNA synthesis was suppressed to 14% of the control level by the treatment with 70 microM quercetin for 2 days. Furthermore, quercetin blocked cell progression from the G1 to the S phase.

Heat shock protein (HSP) 47 and collagen are upregulated during neointimal formation in the balloon-injured rat carotid artery.

Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is thought to be essential for the proper processing and secretion of procollagen molecules. We investigated the time course and localization of HSP47 and collagen expression after balloon catheter angioplasty in the rat carotid artery, based on the premise that accumulation of extracellular matrix components is a main feature of intimal hyperplasia in humans and in laboratory animals. Low levels of HSP47 expression were evident in uninjured carotid arteries. Northern blot analysis revealed that HSP47 mRNA expression was markedly stimulated 1--3 days after the induced injury and a high level was maintained for 7 days, followed by a gradual decline for up to 21 days after the injury. These changes in HSP47 expression paralleled changes in alpha 1(I) collagen expression. Immunohistochemical staining revealed colocalization of HSP47 and collagen in smooth muscle cells (SMCs) of the media and intima. In situ hybridization analysis showed that activated SMCs, which proliferated and migrated into the intima, expressed high levels of HSP47. In cultured human aortic SMCs, similar upregulation of HSP47 and alpha1(I) collagen by TGF-beta was noted. These results show that SMCs activated after balloon injury express high levels of HSP47 and collagen during cell proliferation and migration, hence an overproduction of collagen and development of intimal thickening. Thus, HSP47 plays a role in the formation and progression of neointima after angioplasty.

Expression and localization of collagen-binding stress protein Hsp47 in mouse embryo development: comparison with types I and II collagen.

Heat shock protein (Hsp)47 is a collagen-binding stress protein localized in the endoplasmic reticulum and is thought to have chaperone-like functions that are specific to procollagen biosynthesis. In previous papers, we reported that the expression of Hsp47 is closely correlated with that of various types of collagen in various cell lines and also in the progression of experimental liver fibrosis. In the present study, the expression of Hsp47 was examined during the development of mouse embryos by immunostaining with an anti-Hsp47 antiserum. The spatio-temporal correlation of the expression of Hsp47 with those of types I and II collagen was also examined using specific antisera. Hsp47 expression during embryogenesis was observed mainly in mesoderm and in tissues that are derived from mesoderm, such as connective tissue, cartilage, bone, notochord and somites. Hsp47 was also detected in tissues derived from the neural crest mesenchyme. In the central nervous system, Hsp47 was detected in some restricted regions where cells proliferate, such as the ventral area of the neural tube and choroid plexus. Immunostaining for types I and II collagen revealed the spatial and temporal correlations of the expression of these proteins with that of Hsp47. These results suggest the biological importance of Hsp47 as a collagen-specific molecular chaperone in the mouse developmental program.

HSP47, a collagen-specific molecular chaperone, delays the secretion of type III procollagen transfected in human embryonic kidney cell line 293: a possible role for HSP47 in collagen modification.

HSP47 is a stress protein (heat shock protein) which resides in the endoplasmic reticulum, and is postulated to function as a collagen-specific molecular chaperone. To elucidate the role of HSP47 in procollagen biosynthesis, we have established human embryonic kidney 293 cell lines, which were stably transfected with alpha1(III) procollagen chains with or without HSP47. 293 cells do not produce any extracellular matrix proteins including collagens, and the level of HSP47 expression is almost undetectable in this cell line. Recombinant type III procollagens in 293 cells form trypsin-resistant homotrimers, which are secreted into the medium as trimers in the presence or absence of recombinant mouse HSP47. The secretion of procollagen III was delayed in 293 cells stably transfected with proalpha1(III) collagen chains [293+proalpha1(III) cells] in comparison with human rhabdomyosarcoma cell line RD, which normally produces type III procollagens. In this study, we examined the rate of type III procollagen secretion in detail. In cells cotransfected with mouse HSP47 [293+proalpha1(III)+HSP47 cells], the rate of type III procollagen secretion was slower than in 293+proalpha1(III) cells. The binding of HSP47 with proalpha1(III) collagen chains was confirmed by immunoprecipitation using the chemical cross-linker, DSP. The electrophoretic mobility of proalpha1(III) collagen chains in 293+proalpha1(III) cells was slightly slower than that in RD cells, whereas the recombinant proalpha1(III) chains of 293+proalpha1(III)+HSP47 cells showed almost the same electrophoretic mobility as those of RD cells. The melting temperature (Tm) of type III procollagen in 293+proalpha1(III)+HSP47 cells was almost the same as that in RD cells, and the Tm in 293+proalpha1(III) cells was slightly higher than that in RD cells. These data suggest that the recombinant proalpha1(III) collagen chain is overmodified in 293+proalpha1(III) cells, but not in 293+proalpha1(III)+HSP47 cells.

Occasional infection of hepatitis C virus occurring in haemodialysis units identified by serial monitoring of the virus infection.

To examine whether hepatitis C virus (HCV) infection still occurs in haemodialysis units even after a decrease in the number of blood transfusions and in those contaminated with HCV, we tested anti-HCV antibodies and HCV RNA in 142 patients from Japanese haemodialysis units, and examined the serial prevalence of anti-HCV antibodies in 86 patients from 1992 to 1997. A high prevalence of HCV infection was observed: 34 (23.9%) and 38 (26.8%) of the 142 patients were positive for serum anti-HCV antibodies and HCV RNA, respectively. These positive rates were related to the duration of haemodialysis. We found that five patients treated in the same unit seroconverted from 1993 to 1995. Four of the five patients had been treated at the same shift and were affected at the same time. Phylogenetic analysis of the hypervariable region 1 (HVR1) sequence of HCV from serum of these patients showed that three of the four patients' sequences were phylogenetically clustered and that two of the three were closely related. Thus, an occasional transmission of HCV occurred in the haemodialysis unit. The universal precautions by staff such as carefully changing gloves may be important in inhibiting spread of HCV because no instances of infection have been seen since the instigation of educational measures for unit staff.

Effect of thiazinotrienomycin B, an ansamycin antibiotic, on the function of epidermal growth factor receptor in human stomach tumor cells.

Thiazinotrienomycin B (TT-B), an ansamycin isolated from fermentation broths of Streptomyces sp. MJ672-m3, inhibited the growth in vitro of human stomach tumor SC-6 cells over 10 times more strongly than the growth of other human tumor cells, such as HeLa (cervix), T24 (bladder) and LX-1 (lung). The extent of growth inhibition by TT-B of SC-6, but not of LX-1 nor T24, was lowered in a competitive manner by raising serum concentrations in the culture medium. TT-B inhibited the cell cycle progression of SC-6 at an early stage of the progression from G0/G1 to S. The inhibition was again competitive with serum concentrations in the culture medium. No direct inhibition of DNA synthesis was observed at the concentration range which caused the cell cycle arrest. TT-B and anti-epidermal growth factor receptor (anti-EGFR) were antagonistic to each other in inhibiting the cell cycle progression of SC-6 from G0/G1 to S, suggesting that the two compounds share the same target, EGFR. The kinase activity of EGFR was little inhibited by TT-B in a cell-free system.

New triene-ansamycins, thiazinotrienomycins F and G and a diene-ansamycin, benzoxazomycin.

New triene-ansamycins designated thiazinotrienomycins F (TT-F) and G (TT-G) and a new diene-ansamycin, benzoxazomycin, were isolated from a culture broth of Streptomyces sp. MJ672-m3 and their structures were elucidated by spectroscopic analyses. The Mean Graphs of TT-G suggests that the tumor growth inhibitory activities are almost as strong as TT-B, in respect of GI50 and TGI against several human cancer cell lines.

Hydroxymycotrienins A and B, new ansamycin group antibiotics.

New ansamycins designated hydroxymycotrienins A and B were isolated from culture broths of Bacillus sp. BMJ958-62F4. The two antibiotics inhibited more strongly the growth of human cervical cancer cell lines of human papilloma virus (HPV) positive than that of HPV negative cell lines. The structures, some biological and biochemical properties are reported.

Thiazinotrienomycins, new ansamycin group antibiotics.

New triene-ansamycins designated thiazinotrienomycins A, B, C, D and E were isolated from culture broth of Streptomyces sp. MJ672-m3 for their activities against cervical cancer cell lines. The structures and some biological and biochemical properties of the antibiotics were determined.

Melleolides K, L and M, new melleolides from Armillariella mellea.

Three new sesquiterpenoid aromatic esters designated melleolides K (1), L (2) and M (3) were isolated from the cultured mycelia of Armillariella mellea (Vahl. ex Fr.) Karst. Structures of these compounds were determined on the basis of various NMR spectral data, chemical transformations and X-ray analysis. Compounds 1, 2 and 3 showed antimicrobial activities.