Gene and protein expressions of type I collagen are regulated tissue-specifically in rat hyaline cartilages in vivo

The present study was designed to investigate how rat hyaline cartilages at various sites in vivo express the gene and protein of type I collagen using in situ hybridization and immunohistochemistry. The gene of pro alpha 1(I) collagen was expressed by chondrocytes in articular cartilage, and the protein of type I collagen was identified in the cartilage matrix. In contrast, growth plate cartilage expressed the gene of pro alpha 1(I) collagen, but no protein of type I collagen. Neither gene nor protein of type I collagen was expressed in cartilages of trachea and nasal septum. The present study suggested that expression of type I collagen in hyaline cartilages may be regulated tissue-specifically at gene and/or protein levels.     

DNA substrate specificity of reverse gyrase from extremely thermophilic archaebacteria

It has been shown earlier that eukaryotic type I DNA topoisomerases act on duplex DNA regions, while eubacterial type I topoisomerases require single-stranded regions. The present paper demonstrates that the type I topoisomerase from extremely thermophilic archaebacteria, reverse gyrase, winds DNA by binding to single-stranded DNA regions. Thus, type I topoisomerases, both relaxing one in eubacteria and reverse gyrase in extremely thermophilic archaebacteria share a substrate specificity to melted DNA regions. The important consequence of this specificity is that the cellular DNA superhelical stress actively controlled by bacterial topoisomerases is confined to a narrow range characterized by a low stability of the double helix. Hence we suppose that bacterial topoisomerase systems control duplex stability near its minimum, for which purpose they create an appropriate negative superhelicity at moderate temperatures or a positive one at extremely high temperatures, the feedback being ensured by the aforesaid specificity of type I bacterial topoisomerases.     

Induction of Mx protein by interferon and double-stranded RNA in salmonid cells

Mx protein is one of several antiviral proteins that are induced by the type I interferons (IFN), IFNalpha and beta, in mammals. In this work induction of a 76 kDa Mx protein by double-stranded RNA (dsRNA) or type I IFN-like activity in Atlantic salmon macrophages, Atlantic salmon fibroblast cells (AS cells) and in Chinook salmon embryo cells (CHSE-214) is reported. Type I IFN-like activity was produced by the stimulation of Atlantic salmon macrophages with the synthetic dsRNA polyinosinic polycytidylic acid (poly I:C). A correlation appeared to exist between Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) induced by IFN in CHSE-214 cells. Several observations in the present work suggest that, as in mammals, the induction of Mx protein by dsRNA in fish cells primarily occurs via induction of type I IFN. First, type I IFN-like activity but not poly I:C, induced Mx protein expression in CHSE-214 cells. These cells apparently lack the ability to produce IFN in response to poly I:C. Second, the putative IFN induced maximal Mx protein expression 48 h earlier than poly I:C in AS cells. Third, the peak expression of Mx protein in macrophages induced by poly I:C occurred after 48 h whereas peak in IFN-like activity was observed by 24 h after addition of poly I:C. The present work supports the notion of using Mx protein as a molecular marker for the production of putative type I IFN in fish.     

Polymorphic forms of troponin T and troponin C and their localization in striated muscle cell types

1. 1. Immunochemical studies have shown that the major forms of troponin T present in fast skeletal, slow skeletal and cardiac muscles are different proteins.

2. 2. Similar studies indicate that the major form of troponin C present in fast skeletal muscles differs from troponin C present in slow skeletal and cardiac muscle cells. The forms of troponin C present in slow skeletal and cardiac muscles are immunochemically very similar.

3. 3. The antibodies to the polymorphic forms of troponin T and troponin C are specific for the muscle type, except in the case of the slow skeletal and cardiac muscle forms of troponin C.

4. 4. By the immunoperoxidase technique, it has been shown that the fast skeletal muscle troponin T is localized in type II cells and slow skeletal muscle troponin T in type I cells.

5. 5. Fast skeletal muscle troponin C is present in type II cells and a different troponin C, identified by its reaction with the antibody against cardiac troponin C, is present in type I cells.

6. 6. It is concluded that in normal adult skeletal muscle, fast muscle forms of troponin I, troponin T and troponin C are present together as a homocomplex in type II cells and the slow muscle forms exist as an analagous homocomplex in type I cells.

     

Localization of precursor proteins and mRNA of type I and III collagens in usual interstitial pneumonia and sarcoidosis

Summary The aim of this study was to assess and compare the accumulation and distribution of newly synthesized type I and III collagens in usual interstitial pneumonia (UIP) and pulmonary sarcoidosis. Lung biopsies from 10 patients with UIP and 13 patients with sarcoidosis were investigated by immunohistochemical technique and mRNA in situ hybridization. The antibodies for the aminoterminal propeptide of type I procollagen and the aminoterminal propeptide of type III procollagen (PINP and PIIINP, respectively) were used. When compared to healthy lung, levels of type I pN- and type III pN-collagens were increased in both of these disorders. Type I procollagen was mostly present as intracellular spots in newly formed fibrosis in UIP while type III pN-collagen was expressed extracellularly underneath metaplastic alveolar epithelium. Type I procollagen was present intracellularly within and around the granulomas of sarcoidosis, whereas type III pN-collagen was expressed extracellularly, mainly around the granulomas. mRNAs of both collagens colocalized with the precursor proteins. We conclude that the expression of precursor proteins and mRNA of type I and type III collagens is increased in UIP and sarcoidosis, reflecting mainly active synthesis of these collagens in different areas of the lung.     

Cloning and sequencing of the cDNA encoding an isoform of microtubule-associated protein tau containing four tandem repeats: differential expression of tau protein mRNAs in human brain.

We have isolated cDNA clones encoding a 383-amino acid isoform of the human microtubule-associated protein tau. It differs from previously determined tau sequences by the presence of an additional repeat of 31 amino acids, giving four, rather than three, tandem repeats in its carboxy-terminal half. The extra repeat is encoded by a separate exon. Probes derived from cDNA clones encoding the three (type I) and four repeat (type II) tau protein isoforms detected mRNAs for both forms in all adult human brain areas examined. However, in foetal brain only type I mRNA was found. Type I and type II mRNAs were present in pyramidal cells in cerebral cortex. In the hippocampal formation, type I mRNA was found in pyramidal and granule cells; type II mRNA was detected in most, though not all, pyramidal cells but not in granule cells. These observations indicate that tau protein mRNAs are expressed in a stage- and cell-specific manner. Tau protein is found in the protease-resistant core of the paired helical filament, the major constituent of the neurofibrillary tangle in Alzheimer's disease. Taken in conjunction with previous findings, the present results indicate that both the three and four repeat-containing tau protein isoforms are present in the core of the paired helical filament.     

Developmental modulation of embryonic cardiac myocyte adhesion to cardiac collagens in vitro.

The goal of this investigation is to identify molecules that mediate embryonic cardiac myocyte adhesion during chick cardiac morphogenesis. The assay used employs culturing embryonic myocytes on substrata containing embryonic heart proteins separated by molecular weight. This assay shows that embryonic myocytes from 10- to 14-day-old embryos will bind to 140,000 and 128,000 Da proteins present in embryonic hearts and do not require Mg2+ or Ca2+ for adhesion. Myocytes from embryos younger than 10 days or older than 14 days display little or no binding. Embryonic heart fibroblasts collected at these same ages do not bind to these proteins. The 140- and 128-kDa proteins were found to copurify in extraction procedures for procollagens. Amino acid analysis shows that both proteins contain high glycine and hydroxyproline, indicating that they are collagens. However, glycine and imino acid levels are low relative to other known collagens, indicating a nonhelical domain present in each molecule and most closely resembled levels present in procollagens. Immunoblots show that antisera to chick collagen type I recognizes the 128-kDa protein while anti-collagen type III recognizes the 140-kDa protein. Monoclonal antibodies to the amino terminal propeptide of collagen type I recognize the 128-kDa protein in immunoblotting procedures. Embryonic chick myocytes bind to 140/128 kDa proteins present in extracts of sympathetic trunk, although they do not bind to 140/128 kDa proteins in embryonic tendon. The findings thereby indicate that forms of type III and type I collagens in embryonic heart support direct adhesion of embryonic myocytes for a restricted period of cardiac myogenesis and that these proteins differ from collagen types I and III present in other tissues and from fully processed collagen types I and III.     

Glycosylation at Asn-184 inhibits the conversion of single-chain to two-chain tissue-type plasminogen activator by plasmin

Tissue-type plasminogen activator (tPA) is a glycosylated serine protease which is an effective thrombolytic agent. Native single-chain tPA (sc-tPA) is converted to two-chain tPA (tc-tPA) by plasmin, the product of the reaction of plasminogen with tPA. Native sc-tPA occurs as two glycoforms. Type I sc-tPA is fully glycosylated, while type II lacks glycosylation at Asn-184. The rates at which type I and type II human melanoma sc-tPA were converted to type I and type II tc-tPA by plasmin were determined by two different methods. In each case, the second-order rate constant (kcat/Km) for type II sc-tPA (approximately 8 microM-1 s-1) was about twice that for type I sc-tPA (approximately 4 microM-1 s-1). These results indicate that glycosylation at Asn-184 hinders the conversion of sc-tPA to tc-tPA and suggest that under physiological conditions type I sc-tPA may persist in the single-chain form longer than type II sc-tPA. Previous studies have shown that type I tc-tPA has a lower activity than type II tc-tPA and that sc-tPA has a lower activity and susceptibility to inhibition when compared to tc-tPA. The present work provides further evidence that tPA glycosylation serves to modulate activity. The two major glycoforms may represent more persistent but slow acting (type I) and less persistent but faster acting (type II) variants of tPA.     

Type I IGF receptors of Leydig cells are upregulated by human chorionic gonadotropin

The effects of human chorionic gonadotropin (hCG) on type I insulin-like growth factor (IGF) receptors of purified Leydig cells were investigated. Sprague-Dawley rats (50 day-old) were treated with a single injection of hCG 10 units intraperitoneally, type I IGF receptors were then determined daily for 4 days. HCG caused a rapid increase in type I IGF receptors within 24 h, which returned to basal by 72 h. There was no significant change in binding affinity. Our present study indicates that type I IGF receptors of Leydig cells are up regulated by hCG, and this may be one mechanism by which hCG and IGF-I interact to enhance Leydig cell steroidogenesis.     

Extracellular-signal regulated kinase signaling pathway mediates downregulation of type I procollagen gene expression by FGF-2, PDGF-BB, and okadaic acid in osteoblastic cells

Although basic fibroblast growth factor (FGF-2) had been shown to inhibit type I collagen gene expression in osteoblast, its inhibitory mechanism is unknown. In the present study, we investigated the underlying mechanisms by which growth factors downregulate type I collagen gene expression. Treatment of mouse osteoblastic MC3T3-E1 cells with okadaic acid (40 ng/ml), an inhibitor of phosphoserine/threonine-specific protein phosphatase and activator of ERK1/2, for 24 h and 48 h completely inhibited steady-state mRNA levels of type I collagen. FGF-2 (30 ng/ml), platelet-derived growth factor-BB (PDGF-BB), 30 ng/ml, and serum, which activate ERK mitogen-activated protein kinase (MAPK) pathway also inhibited collagen type I gene expression, suggesting that the activation of ERK pathway mediates inhibition of type I collagen mRNA. This observation was further confirmed by experiments using inhibitors of the ERK pathway (i.e., PD and U0126), which increased type I collagen mRNA in MC3T3-E1 cells, indicating that the inhibition of ERK pathway upregulates type I collagen gene expression. Low serum (0.3%) markedly increased type I collagen mRNA. MEK inhibitor PD inhibited c-fos induction by FGF-2 and PDGF-BB, suggesting that c-fos is the downstream target of ERK pathway. Our data have clearly demonstrated for the first time that the ERK MAPK pathway play an important role in the regulation of type I collagen gene expression in osteoblastic cells. Results also showed that one of the mechanisms by which FGF-2 and PDGF-BB downregulate type I collagen gene expression in the osteoblast is through the activation of ERK signaling pathway.     

An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate

Immunohistochemical localization of type I and type II collagens was examined in the rat mandibular condylar cartilage (as the secondary cartilage) and compared with that in the tibial growth plate (as the primary cartilage) using plastic embedded tissues. In the condylar cartilage, type I collagen was present not only in the extracellular matrix (ECM) of the fibrous, proliferative, and transitional cell layers, but also in the ECM of the maturative and hypertrophic cell layers. Type II collagen was present in the ECM of the maturative and hypertrophic cell layers. In the growth plate, type II collagen was present in the ECM of whole cartilaginous layers; type I collagen was not present in the cartilage but in the perichondrium and the bone matrices. These results indicate that differences exist in the components of the ECM between the primary and secondary cartilages. It is suggested that these two tissues differ in the developmental processes and/or in the reactions to their own local functional needs.     

Chemistry of the collagen cross-links. Nature of the cross-links in the polymorphic forms of dermal collagen during development.

Both the type I and type III collagens present in embryonic dermis are stabilized by the intermolecular cross-link, hydroxylysino-5-oxonorleucine, derived from hydroxylysine-aldehyde, although the type I collagen possesses a significant proportion of dehydrohydroxylysinonorleucine. However, concurrent with the change in the proportion of the two types of collagen during postnatal development there is a change-over with both type I and III collagens to the labile cross-link, dehydrohydroxylysinonorleucine, derived from lysine aldehyde. The results indicate that the change in the nature of the cross-link with development is determined primarily by the change in the extent of hydroxylation of the lysine residues in the terminal non-helical regions rather than being due to the change in the type of collagen.     

Modification of pyruvate kinase isozymes in prolonged primary cultures of adult rat hepatocytes.

Pyruvate kinase isozymic changes were studied in the adult hepatocyte cultures, by electrophoretic, kinetic and immunological methods. We were able to maintain parenchymal cells from normal adult rat liver in non-proliferating monolayer cultures up to 10 days. Hepatocytes appeared to contain a dominant PK I type up to 4-5 days of culture. After day 5, PK III type was regularly present with PK I and after 7 days PK III type was always the only isozyme detected in culture. It must be pointed out that, by the Ouchterlony method and sometimes by electrophoresis, concentrated extracts from freshly isolated hepatocytes or starting hepatocyte cultures did also contain Pyruvate kinase PK III type. These results suggest that Pyruvate kinase III is present but partly repressed in the adult parenchymal cells and becomes derepressed in culture.     

An insilico approach to structural elucidation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Arabidopsis thaliana: hints for herbicide design

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS), the first enzyme of the shikimate pathway, is responsible for the synthesis of aromatic amino acids in microorganisms and plants. The pathway has been of increasing interest in the recent past as the enzymes are being targeted for antimicrobial drug and herbicide design. In the present work the three dimensional structure of the type II DAHPS present in Arabidopsis thaliana (At-DAHPS) is described and compared with type I DAHPS. The structure shows that the enzyme belongs to the (β/α)(8) TIM barrel family and that most of the active site residues are conserved in the type I DAHPS enzymes. Although the overall structures of the type I and type II enzymes are similar, there are differences in the extra barrel elements which may explain the different modes of enzyme regulation. At the N-terminus of At-DAHPS, there are three non-core helices, α0a (Ala72-Lys83), α0b (Ala94-Ala106) and α0c (Ala113-Val128), but no β(0), in contrast to the microbial type II DAHPS. Also, the (I/L)GAR motif in the type I DAHPS is substituted with xGxR in the case of type II DAHPS. Also, a motif NK(/I)PGR(/K) is present in the sequences of type II DAHPS including At-DAHPS. The elucidation of the active site architecture of At-DAHPS may provide a structural framework useful for the design of specific inhibitors towards herbicide development.     

Developmental modulation of embryonic cardiac myocyte adhesion to cardiac collagens in vitro

The goal of this investigation is to identify molecules that mediate embryonic cardiac myocyte adhesion during chick cardiac morphogenesis. The assay used employs culturing embryonic myocytes on substrata containing embryonic heart proteins separated by molecular weight. This assay shows that embryonic myocytes from 10- to 14-day-old embryos will bind to 140,000 and 128,000 Da proteins present in embryonic hearts and do not require Mg²⁺ or Ca²⁺ for adhesion. Myocytes from embryos younger than 10 days or older than 14 days display little or no binding. Embryonic heart flbroblasts collected at these same ages do not bind to these proteins. The 140- and 128-kDa proteins were found to copurify in extraction procedures for procollagens. Amino acid analysis shows that both proteins contain high glycine and hydroxyproline, indicating that they are collagens. However, glycine and imino acid levels are low relative to other known collagens, indicating a nonhelical domain present in each molecule and most closely resembled levels present in procollagens. Immunoblots show that antisera to chick collagen type I recognizes the 128-kDa protein while anti-collagen type III recognizes the 140-kDa protein. Monoclonal antibodies to the amino terminal propeptide of collagen type I recognize the 128-kDa protein in immunoblotting procedures. Embryonic chick myocytes bind to 140/128 kDa proteins present in extracts of sympathetic trunk, although they do not bind to 140/128 kDa proteins in embryonic tendon. The findings thereby indicate that forms of type III and type I collagens in embryonic heart support direct adhesion of embryonic myocytes for a restricted period of cardiac myogenesis and that these proteins differ from collagen types I and III present in other tissues and from fully processed collagen types I and III.     

Biochemical and kinetic analysis of the RNase active sites of the integrase/tyrosine family site-specific DNA recombinases

In this study, we have used multiple strategies to characterize the mechanisms of the type I and type II RNA cleavage activities harbored by the Flp (pronounced here as "flip") site-specific DNA recombinase (Flp-RNase I and II, respectively). Reactions using half-sites pre-bound by step-arrest mutants of Flp agree with a "shared active site" being responsible for the type I reaction (as is the case with normal DNA recombination). In a "pre-cleaved" type I substrate containing a 3'-phosphotyrosyl bond, the Flp-RNase I activity can be elicited by either wild type Flp or by Flp(Y343F). Kinetic analyses of the type I reaction are consistent with the above observations and support the notion that the DNA recombinase and type I RNase active sites are identical. The type II RNase activity is expressed by Flp(Y343F) in a half-site substrate and is unaffected by the catalytic constitution of a Flp monomer present on a partner half-site. Reaction conditions that proscribe the assembly of a DNA bound Flp dimer have no effect on Flp-RNase II. These biochemical results, together with kinetic data, are consistent with the reaction being performed from a "non-shared active site" contained within a single Flp monomer. The Flp-related recombinase Cre, which utilizes a non-shared recombination active site, exhibits the type I RNA cleavage reaction. So far, we have failed to detect the type II RNase activity in Cre. Despite their differences in active site assembly, Cre functionally mimics Flp in being able to provide two functional active sites from a trimer of Cre bound to a three-armed (Y-shaped) substrate.     

An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate

Summary Immunohistochemical localization of type I and type II collagens was examined in the rat mandibular condylar cartilage (as the secondary cartilage) and compared with that in the tibial growth plate (as the primary cartilage) using plastic embedded tissues. In the condylar cartilage, type I collagen was present not only in the extracellular matrix (ECM) of the fibrous, proliferative, and transitional cell layers, but also in the ECM of the maturative and hypertrophic cell layers. Type II collagen was present in the ECM of the maturative and hypertrophic cell layers. In the growth plate, type II collagen was present in the ECM of whole cartilaginous layers; type I collagen was not present in the cartilage but in the perichondrium and the bone matrices. These results indicate that differences exist in the components of the ECM between the primary and secondary cartilages. It is suggested that these two tissues differ in the developmental processes and/or in the reactions to their own local functional needs.     

The effect of food restriction and low protein diet upon collagen type I and III ratio in rat skin

It has been demonstrated that the content of the collagen type I is more affected by both chronic low protein diet feeding and chronic food deprivation (50% food intake) than the content of collagen type III. By introducing these dietary regimes the proportion of collagen type I to collagen type III ratio drops from 2.1 to 1.3 indicating the higher proportion of collagen type III in the skin at the end of the experiment (after 18 months of chronic feeding). It was also observed that the total concentration of hydroxyproline (hyp) in the skin decreases considerably in both food restricted animals and those fed a low protein diet. It is suggested that, under the present experimental conditions, the balance between collagen break-down and synthesis is shifted and, furthermore, that this shift is different for collagen type I and III and results in an altered ratio of these two collagen species in the skin. Refeeding of animals leads to a higher than normal collagen type I to III ratio indicating thus a relatively higher proportion of collagen type I in this tissue.