Effects of heat shock, stannous chloride, and gallium nitrate on the rat inflammatory response.

Heat and a variety of other stressors cause mammalian cells and tissues to acquire cytoprotection. This transient state of altered cellular physiology is nonproliferative and antiapoptotic. In this study, male Wistar rats were stress conditioned with either stannous chloride or gallium nitrate, which have immunosuppressive effects in vivo and in vitro, or heat shock, the most intensively studied inducer of cytoprotection. The early stages of inflammation in response to topical suffusion of mesentery tissue with formyl-methionyl-leucyl-phenylalanine (FMLP) were monitored using intravital microscopy. Microvascular hemodynamics (venular diameter, red blood cell velocity [Vrbc], white blood cell [WBC] flux, and leukocyte-endothelial adhesion [LEA]) were used as indicators of inflammation, and tissue levels of inducible Hsp70, determined using immunoblot assays, provided a marker of cytoprotection. None of the experimental treatments blocked decreases in WBC flux during FMLP suffusion, an indicator of increased low-affinity interactions between leukocytes and vascular endothelium known as rolling adhesion. During FMLP suffusion LEA, an indicator of firm attachment between leukocytes and vascular endothelial cells increased in placebo and gallium nitrate-treated animals but not in heat- and stannous chloride-treated animals, an anti-inflammatory effect. Hsp70 was not detected in aortic tissue from placebo and gallium nitrate-treated animals, indicating that Hsp70-dependent cytoprotection was not present. In contrast, Hsp70 was detected in aortic tissues from heat- and stannous chloride-treated animals, indicating that these tissues were in a cytoprotected state that was also an anti-inflammatory state.

The effect of gallium nitrate on synoviocyte MMP activity.

Gallium, a group IIIa metal salt, has been demonstrated to be an effective immunosuppressive agent. Gallium has also been shown to inhibit the production of inflammatory cytokines, such as IL-1beta, produced by macrophage-like cells in vitro. To further characterize the effects of gallium on the inflammatory process, we examined the effects of gallium nitrate on matrix metalloproteinase (MMP) activity utilizing the rabbit synoviocyte cell line HIG-82. HIG-82 cells were incubated with IL-1beta and TPA, with and without increasing concentrations of gallium nitrate. Conditioned medium was collected and assayed for MMP activity using a synthetic substrate and substrate gel zymography. IL-1beta and TPA alone induced MMP activity in HIG-82 cells. A dose-dependent inhibition of IL-1beta and TPA stimulated MMP activity by gallium nitrate at increasing concentrations was observed. This study demonstrates that gallium nitrate can inhibit the activity of MMPs and may be useful as a modulator of inflammation in arthritis.

Tissue-level cytoprotection.

In vitro and ex vivo tissue models provide a useful level of biological organization for cytoprotection studies positioned between cultured cells and intact animals. We have used 2 such models, primary tissue cultures of winter flounder renal secretory epithelium and ex vivo preparations of rat intestinal tissues, the latter to access the microcirculation of exposed mesentery tissues. Herein we discuss studies indicating that differentiated functions are altered in thermotolerant or cytoprotected tissues. These functions include transepithelial transport in renal epithelium and attachment and transmigration of leukocytes across vascular endothelium in response to mediators of inflammation. Evidence pointing to inflammation as a major venue for the heat shock response in vertebrates continues to mount. One such venue is wound healing. Heat shock proteins are induced early in wound responses, and some are released into the extracellular wound fluid where they appear to function as proinflammatory cytokines. However, within responding cells in the wound, heat shock proteins contribute to the acquisition of a state of cytoprotection that protects cells from the hostile environment of the wound, an environment created to destroy pathogens and essentially sterilize the wound. We propose that the cytoprotected state is an anti-inflammatory state that contributes to limiting the inflammatory response; that is, it serves as a brake on inflammation.

The mechanism of beta-glycerophosphate action in mineralizing chick limb-bud mesenchymal cell cultures.

Differentiating chick limb-bud mesenchymal cells plated in micromass culture form a cartilage matrix that can be mineralized in the presence of 4 mM inorganic phosphate (Pi), and 1 mM calcium. Previous studies showed that when beta-glycerophosphate (beta GP) is used in place of Pi, the mineral crystals formed are larger and differ in distribution. The present study shows that the difference in distribution is not associated with alterations in cell proliferation, protein synthesis, or with collagen, proteoglycan core protein, or alkaline phosphatase gene expression. Cultures with 2.5, 5, and 10 mM beta GP did show different levels of alkaline phosphatase activity, and in the presence of low (0.3 mM) Ca had different Pi contents (4, 6 and 9 mM, respectively), indicating that the increase in CaxP product may in part be responsible for the altered pattern of mineralization. However, cultures with beta GP in which alkaline phosphatase activity was inhibited with levamisole still had an altered mineral distribution as revealed by Fourier transform-infrared (FT-IR) microspectroscopy. The presence of a casein kinase II-like activity in the mineralizing cultures, the ability of specific inhibitors of this enzyme to block mineralization, and the known ability of beta GP to block phosphoprotein phosphatase activity suggests that altered patterns of matrix protein phosphorylation may influence mineral deposition in these cultures.

Identification of proteolytic activities in ROS 17/2.8 cell lysates which cleave peptide substrates for protein kinase C-mediated phosphorylation.

We have observed two proteolytic activities in cell lysates from the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation, and other peptides containing similar sequences. Both activities are inhibited by Pefabloc, a serine protease inhibitor, while one of the activities is inhibited by either EDTA or aprotinin. The protease inhibitors pepstatin, bestatin, E-64, leupeptin and phosphoramidon do not block either of these proteolytic activities.

Peptide bond cleavage site determination of novel proteolytic enzymes found in ROS 17/2.8 cell lysates.

We have identified proteolytic activities in the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation (PSPKC, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys). Using polyacrylamide gel electrophoresis conditions similar to those used to resolve small molecular weight proteins, the peptide bonds of PSPKC which are cleaved by the proteolytic activities present in ROS 17/2.8 cell lysates have been determined. These activities cleave the Ser-Arg, Thr-Leu, and Ser-Val peptide bonds. To date, no proteolytic activities present in osteoblast cell lysates have been described with the aforementioned peptide bond specificities, suggesting that these activities are novel. The PSPKC-cleaved peptide fragment pattern generated was similar for several different osteoblast cell lysates. Lysates generated from different rat tissues were also able to cleave PSPKC, but the peptide fragment pattern generated by ROS 17/2.8 cell lysates appeared to be unique amongst these tissues.

Gallium nitrate increases type I collagen and fibronectin mRNA and collagen protein levels in bone and fibroblast cells.

Gallium is a Group IIIa transitional element with therapeutic efficacy in the treatment of metabolic bone disorders. Previously described antiresorptive effects of gallium on osteoclasts are not sufficient to account for the full range of effects of gallium on bone structure and metabolism. We have recently shown that gallium nitrate inhibits osteocalcin gene expression and the synthesis of osteocalcin protein, an osteoblast-specific bone matrix protein that is thought to serve as a signal to trigger osteoclastic resorption. Here we present evidence for an additional mechanism by which gallium may function to augment bone mass by altering matrix protein synthesis by osteoblastic and fibroblastic cells. Rat calvarial explants exposed to gallium nitrate for 48 h showed increased incorporation of 3H-proline into hydroxyproline and collagenase digestible protein. In addition, gallium treatment increased steady-state mRNA levels for fibronectin and type I procollagen chains in primary rat calvarial osteoblast-enriched cultures, the ROS 17/2.8 osteoblastic osteosarcoma line, and nontransformed human dermal fibroblasts. These findings suggest that the exposure of mesenchymally-derived cells to gallium results in an altered pattern of matrix protein synthesis that would favor increased bone formation.

Gallium nitrate inhibits alkaline phosphatase activity in a differentiating mesenchymal cell culture.

The effect of gallium nitrate on alkaline phosphatase activity in a differentiating chick limb-bud mesenchymal cell culture was monitored in order to gain insight into the observation that rachitic rats treated with gallium nitrate failed to show the expected increase in serum alkaline phosphatase activity. Cultures maintained in media containing 15 microM gallium nitrate showed drastically decreased alkaline phosphatase activities in the absence of significant alterations in total protein synthesis and DNA content. However, addition of 15 microM gallium nitrate to cultures 18 h before assay for alkaline phosphatase activity had little effect. At the light microscopic and electron microscopic level, gallium-treated cultures differed morphologically from gallium-free cultures: with gallium present, there were fewer hypertrophic chondrocytes and cartilage nodules were flatter and further apart. Because of altered morphology, staining with an antibody against chick cartilage alkaline phosphatase appeared less extensive; however, all nodules stained equivalently relative to gallium-free controls. Histochemical staining for alkaline phosphatase activity was negative in gallium-treated cultures, demonstrating that the alkaline phosphatase protein present was not active. The defective alkaline phosphatase activity in cultures maintained in the presence of gallium was also evidenced when cultures were supplemented with the alkaline phosphatase substrate, beta-glycerophosphate (beta GP). The data presented suggest that gallium inhibits alkaline phosphatase activity in this culture system and that gallium causes alterations in the differentiation of mesenchymal cells into hypertrophic chondrocytes.

Gallium nitrate regulates rat osteoblast expression of osteocalcin protein and mRNA levels.

Gallium nitrate, a group IIIa metal salt, has been found to be clinically effective for the treatment of accelerated bone resorption in cancer-related hypercalcemia and Paget's disease. Here we report the effects of gallium nitrate on osteocalcin mRNA and protein levels on the rat osteoblast-like cell line ROS 17/2.8. Gallium nitrate reduced both constitutive and vitamin D3-stimulated osteocalcin protein levels in culture medium by one-half and osteocalcin mRNA levels to one-third to one-tenth of control. Gallium nitrate also inhibited vitamin D3 stimulation of osteocalcin and osteopontin mRNA levels but did not affect constitutive osteopontin mRNA levels. Among several different metals examined, gallium was unique in its ability to reduce osteocalcin mRNA levels without decreasing levels of other mRNAs synthesized by ROS 17/2.8 cells. The effects of gallium nitrate on osteocalcin mRNA and protein synthesis mimic those seen when ROS 17/2.8 cells are exposed to transforming growth factor beta 1 (TGF beta 1); however, TGF-beta 1 was not detected in gallium nitrate-treated ROS 17/2.8 cell media. Use of the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that gallium nitrate did not alter the stability of osteocalcin mRNA. Transient transfection assays using the rat osteocalcin promoter linked to the bacterial reporter gene chloramphenicol acetyltransferase indicated that gallium nitrate blocked reporter gene expression stimulated by the osteocalcin promoter. This is the first reported effect of gallium nitrate on isolated osteoblast cells.

Prostaglandin E1 inhibits collagenase gene expression in rabbit synoviocytes and human fibroblasts.

Cartilage breakdown, as seen in inflammatory and degenerative joint diseases, can be mediated by proteolytic enzymes, such as the metalloproteinase collagenase, the only enzyme able to digest collagen at neutral pH. In vitro collagenase gene expression can be stimulated by the phorbol ester tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. We have investigated the effect of prostaglandin E1 (PGE1) on 12-O-tetradecanoyl-phorbol-13-acetate-stimulated collagenase mRNA levels in the rabbit synoviocyte cell line HIG-82. PGE1, but not PGE2 or PGF2 alpha, was able to selectively reduce collagenase mRNA levels in a dose-dependent fashion. PGE1 markedly increased intracellular levels of cAMP, while PGE2 and PGF2 alpha had little or no effect on cAMP production in the HIG-82 synoviocytes. Agents known to increase intracellular cAMP levels, such as the adenyl cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), mimicked the effect of PGE1, on collagenase mRNA levels. PGE1, forskolin, and IBMX also decreased collagenase mRNA levels in human skin fibroblasts, demonstrating that this observation was not unique to the HIG-82 cell line. Transient transfection experiments carried out in HIG-82 cells using a 1.2-kilobase portion of the 5'-flanking region of the human collagenase gene linked to the reporter gene luciferase demonstrated that PGE1, forskolin, and IBMX exert their inhibitory effect on the promoter region of the collagenase gene.

Tumor necrosis factor induces phosphorylation of a 28-kDa mRNA cap-binding protein in human cervical carcinoma cells.

Tumor necrosis factor alpha (TNF-alpha) stimulated the phosphorylation of a 28-kDa protein (p28) in the ME-180 line of human cervical carcinoma cells. The effect of TNF-alpha on the phosphorylation state of p28 was rapid (4-fold increase within 15 min) and persistent, remaining above the basal level for at least 2 hr. The specific binding of 125I-labeled TNF-alpha to cell-surface binding sites, the stimulation of p28 phosphorylation by TNF-alpha, and the inhibition of cell proliferation by TNF-alpha occurred with nearly identical dose-response relationships. Two-dimensional SDS/PAGE resolved p28 into two isoforms having pI values of 6.2 and 6.1. A phosphorylated cap-binding protein was substantially enriched from lysates of control or TNF-alpha-treated ME-180 cells by affinity chromatography with 7-methylguanosine 5'-triphosphate-Sepharose. The phosphoprotein recovered from this procedure was the substrate for TNF-alpha-promoted phosphorylation, p28. Thus, TNF-alpha stimulates the phosphorylation of this mRNA cap-binding protein, which may be involved in the transduction of TNF-alpha-receptor binding into cellular responses.

Selective release from cultured mammalian cells of heat-shock (stress) proteins that resemble glia-axon transfer proteins.

Cultured rat embryo cells were stimulated to rapidly release a small group of proteins that included several heat-shock proteins (hsp110, hsp71, hscp73) and nonmuscle actin. The extracellular proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. Heat-shocked cells released the same set of proteins as control cells with the addition of the stress-inducible hsp110 and hsp71. Release of these proteins was not blocked by either monensin or colchicine, inhibitors of the common secretory pathway. A small amount of the glucose-regulated protein grp78 was externalized by this pathway. The extracellular accumulation of these proteins was inhibited after they were synthesized in the presence of the lysine analogue aminoethyl cysteine. It is likely that the analogue-substituted proteins were misfolded and could not be released from cells, supporting our conclusion that a selective release mechanism is involved. Remarkably, actin and the squid heat-shock proteins homologous to rat hsp71 and hsp110 are also among a select group of proteins transferred from glial cells to the squid giant axon, where they have been implicated in neuronal stress responses (Tytell et al.: Brain Res., 363:161-164, 1986). Based in part on the similarities between these two sets of proteins, we hypothesized that these proteins were released from labile cortical regions of animal cells in response to perturbations of homeostasis in cells as evolutionarily distinct as cultured rat embryo cells and squid glial cells.

The human thyrotropin beta-subunit gene differs in 5' structure from murine TSH-beta genes.

The gene encoding the beta-subunit of human thyrotropin (hTSH-beta) was isolated, and its nucleotide sequence was determined. The gene is 4.3 kb in length, consists of three exons and two introns, and is present as a single copy as determined by Southern blot analysis of total genomic DNA. The protein coding portion of the gene, which includes exons 2 and 3, was isolated from a human genomic phage library, while exon 1, which encodes only 5' untranslated mRNA sequence, was isolated from a plasmid library of size-selected genomic DNA fragments. Here we describe the isolation of the 5' untranslated exon of the hTSH-beta subunit and 5'-flanking region. The structure of the hTSH-beta gene is very similar to the previously characterized TSH-beta genes from mouse and rat. The genes from all three species have two distinct promoter regions, but while both promoters are utilized by the murine TSH-beta genes, the human TSH-beta gene apparently utilizes only the proximal promoter for transcription initiation. A striking difference in hTSH-beta gene structure compared to the murine genes is that exon 1 of the human gene is 36 nucleotides. An analysis of the mouse, rat, and human exon 1 and 5'-flanking region shows a high percentage of sequence homology, with the exception of a 9-nucleotide insertion 13 bases 3' from the proximal TATA box found in the human gene but not found in the other two species. We propose that this insertion results in the additional length of human exon 1 compared to the mouse and rat genes. By isolating the promoter region of the hTSH-beta gene, we can begin to identify specific sequences involved in the regulation of hTSH gene expression.

Two thyroid hormone regulated genes, the beta-subunits of nerve growth factor (NGFB) and thyroid stimulating hormone (TSHB), are located less than 310 kb apart in both human and mouse genomes.

Two thyroid hormone regulated genes, the beta-subunits of nerve growth factor (NGFB) and thyroid stimulating hormone (TSHB), have been assigned to mouse chromosome 3 and human chromosome 1p22. We have used the techniques of linkage analysis and pulsed field gel electrophoresis to determine the proximity of these two antithetically regulated genes in this conserved linkage group. Four novel restriction fragment length polymorphisms were identified at the human TSHB gene. Two-point linkage analysis between TSHB and NGFB in 46 families, including the Centre d'Etude du Polymorphisme Humain (CEPH) reference panel, demonstrated no recombination (theta = 0.00, Z = 42.8). Analysis of this region by pulsed field gel electrophoresis showed that the genes for TSHB and NGFB are located less than 310 kb apart in man and 220 kb in the mouse.

The 73 kilodalton heat shock cognate protein purified from rat brain contains nonesterified palmitic and stearic acids.

A protein related to the 71 kilodalton inducible rat heat shock protein was purified to electrophoretic homogeneity in milligram amounts from brain tissue of nonheat-stressed rats. The protein has been designated as a stress cognate protein based on previous studies and data presented herein that this protein cross-reacted with a monoclonal antibody originally raised against the Drosophila 70 kilodalton heat shock protein. The purified protein had an apparent molecular mass of 73 kilodaltons when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and an apparent mass of 150 kilodaltons as determined by nondissociative gel chromatography, suggesting that the purified protein is a homodimer. The purified protein had isoelectric points of 5.0 under nondissociative conditions and 5.6 when exposed to protein denaturants, suggesting loss of bound anionic molecules and/or net exposure of basic residues upon denaturation. Chloroform/methanol extraction of the purified protein and subsequent analyses by thin layer and gas-liquid chromatography resulted in the identification of palmitic and stearic acids noncovalently bound to the protein. Approximately four molecules of fatty acids were bound per dimer with palmitic and stearic acids present in a one-to-one ratio. The purified protein did not bind exogenously added radioactive palmitate, indicating that the fatty acid-binding sites of the cognate protein were fully occupied and that the associated fatty acids were too tightly bound to exchange readily. The possible significance of the fatty acids associated with the 73 kilodalton stress cognate protein is discussed.

Purification and initial characterization of the 71-kilodalton rat heat-shock protein and its cognate as fatty acid binding proteins.

The major rat heat-shock (stress) protein and its cognate were purified to electrophoretic homogeneity from livers of heat-shocked rats. Both proteins exhibited similar behavior on a variety of column chromatography matrices but were separable by preparative isoelectric focusing under nondenaturing conditions by virtue of a 0.2 pH unit difference in isoelectric point. Both purified proteins had similar physical properties, suggesting the possibility that they may have similar biological functions as well. Both proteins were homodimers under nondissociative conditions (Mr 150 000) with isoelectric points of 5.0 (cognate) and 5.2 (major stress protein). After denaturation, both proteins had an increase in isoelectric point of 0.6 pH unit, and the resulting polypeptide chains had apparent molecular weights of 73 000 (cognate) and 71 000 (major stress protein). Similarities in the electrophoretic properties of these two proteins and serum albumin, which also undergoes a large basic shift in isoelectric point due to loss of fatty acids and conformational changes accompanying denaturation, prompted us to search for lipids associated with the purified 71-kilodalton stress protein and its cognate. Thin-layer chromatography of chloroform/methanol extracts of these two proteins revealed nonesterified fatty acids bound to both proteins. Palmitic acid, stearic acid, and a small amount of myristic acid were identified by gas chromatography/mass spectroscopy. Both proteins contained approximately four molecules of fatty acid per dimer with palmitate and stearate present in a one to one molar ratio. Possible roles of the major stress protein and its cognate as fatty acid associated proteins in cellular responses to stress are discussed.

[Systematization of the musculo-tendinous architecture of the human biceps (musculus biceps brachii)]. / Systématisation de l'architecture musculo-tendineuse du biceps humain (musculus biceps brachii).

The structure of the brachial biceps is studied based upon the bilateral dissections of six subjects. The object of this work consists of determining the distribution of the group and muscular fibers in relation to the two proximal insertions and the two distal insertions. It is possible to describe the subgroups within each structure. The objective of each of these subgroups is considered within the framework of the coordination between the two muscular formation and the double insertions on the structure of the forearm. It is probable that this muscular formation corresponds to two different movements of the forearm during the locomotory progression. This work can be utilised in the sphere of the comparative biomechanical observation. Also electromyographic and the histochemical characteristics of the muscular group.

Development of five-hour radiometric serum antibacterial assay for gram-positive cocci.

A preliminary report on a 5-hr radiometric serum antibacterial assay (ABA) for Gram-positive cocci is presented. The method agreed within +/- one twofold dilution with static ABA endpoints in 24/26 (92%) of the assays and with cidal ABA endpoints in 23/26 (88%) of the assays performed.