Prevention of chemotherapy-induced osteoporosis by cyclophosphamide with a long-acting form of parathyroid hormone.

BACKGROUND: Most chemotherapeutics reduce bone mineral density (BMD) and increase risk for fractures by causing gonadal suppression, which in turn increases bone removal. Cyclophosphamide (CYP) also has a direct effect of inhibiting bone formation and removal, making the resulting bone loss particularly difficult to treat with antiresorptive therapy. AIM: We tested whether a single dose of the anabolic agent PTH linked to a collagen binding domain (PTHCBD) could prevent the effects of CYP-induced bone loss. METHODS: Mice received either buffer alone, CYP, or CYP+ PTH-CBD. BMD and alkaline phosphatase were measured every 2 weeks for a total of 8 weeks. RESULTS: After 6 weeks, mice treated with CYP showed expected reductions in BMD (increase from baseline: 7.4 ± 6.9 vs 24.35 ± 4.86% in mice without chemotherapy, p<0.05) and decrease in alkaline phosphatase levels (42.78 ± 6.06 vs 60.62 ± 6.23 IU/l in mice without chemotherapy, p<0.05), consistent with osteoporosis from impaired bone formation. Administration of a single dose of PTH-CBD (320 µg/kg ip) prior to CYP treatment improved BMD (change from baseline: 23.4 ± 5.4 vs 7.4 ± 6.9%, CYP treatment alone, p<0.05) and increased alkaline phosphatase levels (50.14 ± 4.86 vs 42.78 ± 6.06 IU/l in CYP treatment alone, p<0.05). BMD values and alkaline phosphatase levels were restored to those seen in mice not receiving chemotherapy. CONCLUSIONS: A single dose of PTHCBD prior to chemotherapy reversed CYP-induced suppression of bone formation and prevented CYP-induced bone loss in mice.

Monthly administration of a novel PTH-collagen binding domain fusion protein is anabolic in mice.

We synthesized fusion proteins of parathyroid hormone (PTH) (1-33) and the collagen binding domain of ColH (CBD) and tested them for anabolic bone activity in mice. Two fusion proteins were synthesized, linking the carboxy terminus of PTH(1-33) either directly to the amino terminal of the CBD or to the CBD through an adjacent ColH domain (PTH-PKD-CBD). Both PTH-CBD and PTH-PKD-CBD increased cAMP accumulation in cells stably transfected with the PTH/PTHrP receptor, and both peptides bound to type 1 collagen in flow-through assays. Distribution studies indicated that the PTH-CBD was concentrated in the bone and skin, tissues with abundant collagen and blood flow. Administration of 320 µg/kg PTH-CBD either weekly (for 8 weeks) or monthly (for 6 months) to 7-week-old C57BL/6J mice resulted in a sustained increase in bone mineral density (BMD) (15% for weekly studies, 13% for monthly studies; P < 0.05). PTH-PKD-CBD showed only 5% increases in BMD after weekly administration, and, as expected, neither weekly nor monthly PTH(1-34) affected BMD. PTH-CBD increased serum alkaline phosphatase levels. Importantly, there were no significant increases in serum calcium observed. Collectively, the data suggest that PTH-CBD has a sustained anabolic effect in bone with either weekly or monthly administration. This approach of targeted delivery of PTH to bone may show promise for the treatment of disorders of low bone mass, such as postmenopausal osteoporosis.

Case study of volatile organic compounds in indoor air of a house before and after repair where sick building syndrome occurred.

A housewife in her late thirties, mother of two children, had an indefinite complaint about the indoor air quality of her house. Inspectors from a public health center treating the housewife's complaint quantified formaldehyde (FA) in high concentration exceeding Japanese national guideline of FA in some rooms of the house. We also determined FA and total volatile organic compounds (TVOCs) in higher concentrations more than the national guidelines. Remodeling of the house was performed to improve the air quality as follows. Vinyl wallpaper was exchanged to plant made paper, plywood made doors were exchanged to pure wood made doors, plywood stairs were covered with plant cork and so on. After remodeling the house, we measured the concentrations of FA and TVOCs again. The concentrations of the chemicals in the indoor air decreased which approve effectiveness of the remodeling. Moreover complaints of the housewife lessened. This also proved the effectiveness of the remodeling. Four years after the inspection, we visited the house again and found that the concentration of FA in the house was still lower than that of national guideline. The housewife was evaluated in a good healthy condition by her answers to our questions related to indoor air quality, daily life, physical condition, and so on.

Phased A-tracts bind to the alpha subunit of RNA polymerase with increased affinity at low temperature.

Previously we showed that the expression of a Clostridium perfringens phospholipase C gene (plc) is activated by promoter upstream phased A-tracts in a low temperature-dependent manner. In this paper we characterize the interaction between the alpha subunit of C. perfringens RNA polymerase and the phased A-tracts. Hydroxyl radical footprinting and fluorescence polarization assaying revealed that the alpha subunit binds to the minor grooves of the phased A-tracts through its C-terminal domain with increased affinity at low temperature. The result provides a molecular mechanism underlying the activation of the plc promoter by the phased A-tracts.

Clostridial hydrolytic enzymes degrading extracellular components.

Bacteria belonging to the genus Clostridium, both glycolytic and proteolytic, and both pathogenic and non-pathogenic, produce a battery of hydrolytic enzymes to obtain nutrients from various biopolymers. The clostridial hydrolytic enzymes are diverse, and are used or are potentially useful for fundamental and applied research purposes. Among them, enzymes degrading the major components in the extracellular matrix or on the cell surface in vertebrates are herein reviewed with special emphasis on recent knowledge gained through molecular biology of clostridial collagenases, sialidases and hyaluronidases. This paper also reviews some literature on the biotechnological approach to the designing of new molecular tools and drug delivery systems involving clostridial hydrolytic enzymes.

Cleavage of a C-terminal peptide is essential for heptamerization of Clostridium perfringens epsilon-toxin in the synaptosomal membrane.

Activation of Clostridium perfringens epsilon-protoxin by tryptic digestion is accompanied by removal of the 13 N-terminal and 22 C-terminal amino acid residues. In this study, we examined the toxicity of four constructs: an epsilon-protoxin derivative (PD), in which a factor Xa cleavage site was generated at the C-terminal trypsin-sensitive site; PD without the 13 N-terminal residues (DeltaN-PD); PD without the 23 C-terminal residues (DeltaC-PD); and PD without either the N- or C-terminal residues (DeltaNC-PD). A mouse lethality test showed that DeltaN-PD was inactive, as is PD, whereas DeltaC-PD and DeltaNC-PD were equally active. DeltaC-PD and DeltaNC-PD, but not the other constructs formed a large SDS-resistant complex in rat synaptosomal membranes as demonstrated by SDS-polyacrylamide gel electrophoresis. When DeltaNC-PD and DeltaC-PD, both labeled with (32)P and mixed in various ratios, were incubated with membranes, eight distinct high molecular weight bands corresponding to six heteropolymers and two homopolymers were detected on a SDS-polyacrylamide gel, indicating the active toxin forms a heptameric complex. These results indicate that C-terminal processing is responsible for activation of the toxin and that it is essential for its heptamerization within the membrane.

Substrate recognition by the collagen-binding domain of Clostridium histolyticum class I collagenase.

Clostridium histolyticum type I collagenase (ColG) has a segmental structure, S1+S2+S3a+S3b. S3a and S3b bound to insoluble collagen, but S2 did not, thus indicating that S3 forms a collagen-binding domain (CBD). Because S3a+S3b showed the most efficient binding to substrate, cooperative binding by both domains was suggested for the enzyme. Monomeric (S3b) and tandem (S3a+S3b) CBDs bound to atelocollagen, which contains only the collagenous region. However, they did not bind to telopeptides immobilized on Sepharose beads. These results suggested that the binding site(s) for the CBD is(are) present in the collagenous region. The CBD bound to immobilized collagenous peptides, (Pro-Hyp-Gly)(n) and (Pro-Pro-Gly)(n), only when n is large enough to allow the peptides to have a triple-helical conformation. They did not bind to various peptides with similar amino acid sequences or to gelatin, which lacks a triple-helical conformation. The CBD did not bind to immobilized Glc-Gal disaccharide, which is attached to the side chains of hydroxylysine residues in the collagenous region. These observations suggested that the CBD specifically recognizes the triple-helical conformation made by three polypeptide chains in the collagenous region.

Collagen-binding domain of a Clostridium histolyticum collagenase exhibits a broad substrate spectrum both in vitro and in vivo.

The substrate spectrum of the tandem collagen-binding domain (CBD) of Clostridium histolyticumclass I collagenase (ColG) was examined both in vitro and in vivo. CBD bound to insoluble type I, II, III and IV collagens in vitro, and to skin, aorta, tendon, kidney, trachea and corneal tissues containing various types of collagen fibrils or sheets. CBD bound to all kinds of collagen fibrils regardless of their diameters and also bound to sheet-forming collagen in the glomerular basal lamina or Descemet's membrane of the cornea. This wide substrate spectrum expands possible applications of the drug delivery system we proposed previously (PNAS 95:7018-7023, 1998). Therapeutic agents fused with CBD will bind not only to subcutaneous tissues, but also to other tissues containing non-type I collagen.

Construction and virulence testing of a collagenase mutant of Clostridium perfringens.

Clostridium perfringens produces several extracellular toxins and enzymes, including an extracellular collagenase or kappa toxin that is encoded by the colA gene. To determine if the ability to produce collagenase was a significant virulence factor in cases of gas gangrene or clostridial myonecrosis that are caused by C. perfringens, a chromosomal colA mutant was constructed by homologous recombination and subsequently virulence tested in the mouse myonecrosis model. The results clearly indicate that loss of the ability to produce collagenase does not alter the ability of the mutant to establish a virulent infection. By contrast, infection with a mutant unable to produce alpha-toxin led to a marked decrease in virulence. These results indicate that collagenase is not a major determinant of virulence in C. perfringens -mediated clostridial myonecrosis.

The hydA gene encoding the H(2)-evolving hydrogenase of Clostridium perfringens: molecular characterization and expression of the gene.

A putative hydrogenase (hydA) gene of Clostridium perfringens encodes a protein with strong identity to Clostridium pasteurianum hydrogenase I. Disruption of the hydA gene abolished H(2) productivity, confirming its function. A putative butyrate kinase gene (buk) is adjacent to the hydA gene. When cultures were grown in medium with glucose, 1.8-kb hydA and 2.1-kb buk transcripts and a 3. 9-kb transcript hybridized with both hydA and buk-probe were detectable in all the exponential growth phases. In medium without glucose, these transcripts were decreased rapidly after the mid-exponential phase. These results suggest that the transcription of these two genes is probably regulated by a similar mechanism in response to glucose availability.

A Clostridium perfringens hem gene cluster contains a cysG(B) homologue that is involved in cobalamin biosynthesis.

The hem gene cluster, which consists of hemA, cysG(B), hemC, hemD, hemB, and hemL genes, and encodes enzymes involved in the biosynthetic pathway from glutamyl-tRNA to uroporphyrinogen III, has been identified by the cloning and sequencing of two overlapping DNA fragments from Clostridium perfringens NCTC8237. The deduced amino acid sequence of the N-terminal region of C. perfringens HemD is homologous to those reported for the C-terminal region of Salmonella typhimurium CysG and Clostridium josui HemD. C. perfringens CysG(B) is a predicted 220-residue protein which shows homology to the N-terminal region of S. typhimurium CysG. Disruption of the cysG(B) gene in C. perfringens strain 13 by homologous recombination reduced cobalamin (vitamin B12) levels by a factor of 200. When grown in vitamin B12-deficient medium, the mutant strain showed a four-fold increase in its doubling time compared with that of the wild-type strain, and this effect was counteracted by supplementing the medium with vitamin B12. These results suggest that C. perfringens CysG(B) is involved in the chelation of cobalt to precorrin II as suggested for the CysG(B) domain of S. typhimurium CysG, enabling the synthesis of cobalamin.

Promoter upstream bent DNA activates the transcription of the Clostridium perfringens phospholipase C gene in a low temperature-dependent manner.

The phospholipase C gene (plc) of Clostridium perfringens possesses three phased A-tracts forming bent DNA upstream of the promoter. An in vitro transcription assay involving C.perfringens RNA polymerase (RNAP) showed that the phased A-tracts have a stimulatory effect on the plc promoter, and that the effect is proportional to the number of A-tracts, and more prominent at lower temperature. A gel retardation assay and hydroxyl radical footprinting revealed that the phased A-tracts facilitate the formation of the RNAP-plc promoter complex through extension of the contact region. The upstream (UP) element of the Escherichia coli rrnB P1 promoter stimulated the downstream promoter activity temperature independently, differing from the phased A-tracts. When the UP element was placed upstream of the plc promoter, low temperature-dependent stimulation was observed, although this effect was less prominent than that of the phased A-tracts. These results suggest that both the phased A-tracts and UP element cause low temperature-dependent activation of the plc promoter through a similar mechanism, and that the more efficient low temperature-dependent activation by the phased A-tracts may be due to an increase in the bending angle at a lower temperature.

Purification of bovine S100A12 from recombinant Escherichia coli.

S100A12, a member of the S100 family of EF-hand calcium-binding proteins, was purified from Escherichia coli cells expressing the corresponding cDNA. The procedure involved washing induced E. coli cells with EDTA-containing hypotonic solution, ion-exchange chromatography, and HPLC. Recombinant S100A12 was purified to homogeneity with the final yield around 6.7 mg per 20 ml of culture. The purified protein was identical to native S100A12 in the N-terminal amino acid sequence, lysylendopeptidase peptide mapping, mass spectrum, and Ca2+-dependent binding affinity to amlexanox, an antiallergy drug. However, the N-terminal methionine residue of the purified protein was not cleaved off as in the native protein. The method used in the present study permits the purification of recombinant S100A12 in large quantities and may also be applicable to preparation of other S100 family proteins.

Identification of metal ligands in the Clostridium histolyticum ColH collagenase.

A Clostridium histolyticum 116-kDa collagenase has an H415EXXH motif but not the third zinc ligand, as found in already characterized zinc metalloproteinases. To identify its catalytic site, we mutated the codons corresponding to the three conserved residues in the motif to other amino acid residues. The mutation affecting His415 or His419 abolished catalytic activity and zinc binding, while that affecting Glu416 did the former but not the latter. These results suggest that the motif forms the catalytic site. We also mutated the codons corresponding to other amino acid residues that are likely zinc ligands. The mutation affecting Glu447 decreased markedly both the enzymatic activity and the zinc content, while that affecting Glu446 or Glu451 had smaller effects on activity and zinc binding. These mutations caused a decrease in kcat but no significant change in Km. These results are consistent with the hypothesis that Glu447 is the third zinc ligand. The spacing of the three zinc ligands is the same in all known clostridial collagenases but not in other known gluzincins, indicating that they form a new gluzincin subfamily. The effects of mutations affecting Glu446 and Glu451 suggest that the two residues are also involved in catalysis, possibly through an interaction with the two zinc-binding histidine residues.

Three distinct anti-allergic drugs, amlexanox, cromolyn and tranilast, bind to S100A12 and S100A13 of the S100 protein family.

To investigate the roles of calcium-binding proteins in degranulation, we used three anti-allergic drugs, amlexanox, cromolyn and tranilast, which inhibit IgE-mediated degranulation of mast cells, as molecular probes in affinity chromatography. All of these drugs, which have different structures but similar function, scarcely bound to calmodulin in bovine lung extract, but bound to the same kinds of calcium-binding proteins, such as the 10-kDa proteins isolated in this study, calcyphosine and annexins I-V. The 10-kDa proteins obtained on three drug-coupled resins and on phenyl-Sepharose were analysed by reversed-phase HPLC. It was found that two characteristic 10-kDa proteins, one polar and one less polar, were bound with all three drugs, although S100A2 (S100L), of the S100 family, was bound with phenyl-Sepharose. The cDNA and deduced amino acid sequence proved our major polar protein to be identical with the calcium-binding protein in bovine amniotic fluid (CAAF1, S100A12). The cDNA and deduced amino acid sequence of the less-polar protein shared 95% homology with human and mouse S100A13. In addition, it was demonstrated that the native S100A12 and recombinant S100A12 and S100A13 bind to immobilized amlexanox. On the basis of these findings, we speculate that the three anti-allergic drugs might inhibit degranulation by binding with S100A12 and S100A13.

Gene duplication and multiplicity of collagenases in Clostridium histolyticum.

Clostridium histolyticum collagenase contains a number of different active components. Previously we have shown that colH encodes a 116-kDa collagenase (ColH) and a 98-kDa gelatinase. We purified a different 116-kDa collagenase (ColG) from the culture supernatant and sequenced its gene (colG). We also identified four other gelatinases (105, 82, 78, and 67 kDa) and determined their N-terminal amino acid sequences, all of which coincided with that of either ColG or ColH. Hybridization experiments showed that each gene is present in a single copy and each gene is transcribed into a single mRNA. These results suggest that all the gelatinases are produced from the respective full-length collagenase by the proteolytic removal of C-terminal fragments. The substrate specificities of the enzymes suggest that colG and colH encode class I and class II enzymes, respectively. Analysis of their DNA locations by pulsed-field gel electrophoresis and nucleotide sequencing of their surrounding regions revealed that the two genes are located in different sites on the chromosome. C. histolyticum colG is more similar to C. perfringens colA than to colH in terms of domain structure. Both colG and colA have a homologous gene, mscL, at their 3' ends. These results suggest that gene duplication and segment duplication have occurred in an ancestor cell common to C. histolyticum and C. perfringens and that further divergence of the parent gene produced colG and colA.

Collagen-binding growth factors: production and characterization of functional fusion proteins having a collagen-binding domain.

The autocrine/paracrine peptide signaling molecules such as growth factors have many promising biologic activities for clinical applications. However, one cannot expect specific therapeutic effects of the factors administered by ordinary drug delivery systems as they have limited target specificity and short half-lives in vivo. To overcome the difficulties in using growth factors as therapeutic agents, we have produced fusion proteins consisting of growth factor moieties and a collagen-binding domain (CBD) derived from Clostridium histolyticum collagenase. The fusion proteins carrying the epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) at the N terminal of CBD (CBEGF/CBFGF) tightly bound to insoluble collagen and stimulated the growth of BALB/c 3T3 fibroblasts as much as the unfused counterparts. CBEGF, when injected subcutaneously into nude mice, remained at the sites of injection for up to 10 days, whereas EGF was not detectable 24 h after injection. Although CBEGF did not exert a growth-promoting effect in vivo, CBFGF, but not bFGF, strongly stimulated the DNA synthesis in stromal cells at 5 days and 7 days after injection. These results indicate that CBD may be used as an anchoring unit to produce fusion proteins nondiffusible and long-lasting in vivo.

A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase.

The Clostridium histolyticum 116-kDa collagenase consists of four segments, S1, S2a, S2b, and S3. A 98-kDa gelatinase, which can degrade denatured but not native collagen, lacks the C-terminal fragment containing a part of S2b and S3. In this paper we have investigated the function of the C-terminal segments using recombinant proteins. Full-length collagenase degraded both native type I collagen and a synthetic substrate, Pz-peptide, while an 88-kDa protein containing only S1 and S2a (S1S2a) degraded only Pz-peptide. Unlike the full-length enzyme, S1S2a did not bind to insoluble type I collagen. To determine the molecular determinant of collagen binding activity, various C-terminal regions were fused to the C terminus of glutathione S-transferase. S3 as well as S2bS3 conferred collagen binding. However, a glutathione S-transferase fusion protein with a region shorter than S3 exhibited reduced collagen binding activity. S3 liberated from the fusion protein also showed collagen binding activity, but not S2aS2b or S2b. S1 had 100% of the Pz-peptidase activity but only 5% of the collagenolytic activity of the full-length collagenase. These results indicate that S1 and S3 are the catalytic and binding domains, respectively, and that S2a and S2b form an interdomain structure.

Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N- and C-terminal peptides.

The effect of lambda-toxin, a thermolysin-like metalloprotease of Clostridium perfringens, on the inactive epsilon-prototoxin produced by the same organism was examined. When the purified epsilon-prototoxin was incubated with the purified lambda-toxin at 37 C for 2 hr, the 32.5-kDa epsilon-prototoxin was processed into a 30.5-kDa polypeptide, as determined by SDS-polyacrylamide gel electrophoresis. A mouse lethality test showed that the treatment activated the prototoxin: the 50% lethal doses (LD50) of the prototoxin with and without lambda-toxin treatment were 110 and 70,000 ng/kg of body weight, respectively. The lethal activity of the prototoxin activated by lambda-toxin was comparable to that with trypsin plus chymotrypsin and higher than that with trypsin alone: LD50 of the prototoxin treated with trypsin and trypsin plus chymotrypsin were 320 and 65 ng/kg of body weight, respectively. The epsilon-toxin gene was cloned and sequenced. Determination of the N-terminal amino acid sequence of each activated epsilon-prototoxin revealed that lambda-toxin cleaved between the 10th and 11th amino acid residues from the N-terminus of the prototoxin, while trypsin and trypsin plus chymotrypsin cleaved between the 13th and 14th amino acid residues. The molecular weight of each activated epsilon-prototoxin was also determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The C-terminus deduced from the molecular weight is located at the 23rd or 30th amino acid residue from the C-terminus of the prototoxin, suggesting that removal of not only N-terminal but also C-terminal peptide is responsible for activation of the prototoxin.