Cloning and chromosomal localization of a gene encoding a novel serine/threonine kinase belonging to the subfamily of testis-specific kinases.

Using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to two highly conserved motifs within the protein kinase family of catalytic domains, we isolated a PCR fragment encoding a novel member of the testis-specific serine/threonine kinases (STK) from mouse male mixed germ cell mRNA. This PCR fragment recognized a 1020-bp transcript in male germ cells by northern blot analysis and was used to clone a full-length cDNA from a mouse mixed germ cell cDNA library. This cDNA has an open reading frame of 804 bases encoding a protein of 268 amino acids. This novel gene is almost identical to Stk22c, encoding a recently described testis-specific protein kinase, except for base-pair deletions that result in a shift in the coding region and an alteration of 22 amino acids (residues 109-131). Due to its homology with Stk22c, we have called this protein kinase gene Stk22d. Northern blot analysis revealed that this protein kinase is developmentally expressed in testicular germ cells and is not present in brain, ovary, kidney, liver, or early embryonic cells. We then cloned the human homologue of this protein kinase gene (STK22C) and found it to be expressed exclusively in the testis. Fluorescence in situ hybridization with both the human and mouse cDNA clones revealed syntenic localization on chromosomes 1p34-p35 and 4E1, respectively.

Expression and localization of caveolin-1, and the presence of membrane rafts, in mouse and Guinea pig spermatozoa.

In somatic cells, caveolin-1 plays several roles in membrane dynamics, including organization of detergent-insoluble lipid rafts, trafficking of cholesterol, and anchoring of signaling molecules. Events in sperm capacitation and fertilization require similar cellular functions, suggesting a possible role for caveolin-1 in spermatozoa. Immunoblot analysis demonstrated that caveolin-1 was indeed present in developing mouse male germ cells and both mouse and guinea pig spermatozoa. In mature spermatozoa, caveolin-1 was enriched in a Triton X-100-insoluble membrane fraction, as well as in membrane subdomains separable by means of their light buoyant densities through sucrose density gradient centrifugation. These data indicated the presence of membrane rafts enriched in caveolin-1 in spermatozoa. Indirect immunofluorescence analysis revealed caveolin-1 in the regions of the acrosome and flagellum in sperm of both species. Confocal immunofluorescence analysis of developing mouse male germ cells demonstrated partial co-localization with a marker for the acrosome. Furthermore, syntaxin-2, a protein involved in acrosomal exocytosis, was present in both raft and nonraft fractions in mature sperm. Together, these data indicated that sperm membranes possess distinct raft subdomains, and that caveolin-1 localized to regions appropriate for involvement with acrosomal biogenesis and exocytosis, as well as signaling pathways regulating such processes as capacitation and flagellar motility.

Improved high-performance liquid chromatographic procedure for the separation and quantification of hydroxytestosterone metabolites.

A reproducible, sensitive high-performance liquid chromatography (HPLC) method has been developed to quantify hydroxytestosterone metabolites. The method features a simple one-step linear gradient of methanol in water (10%-60% methanol) for separation of the testosterone metabolites on a Supelcosil LC-18 column; metabolites are detected at 247 nm. This method provides a distinct advantage over previously developed assays in that the solvent gradient does not contribute to baseline changes throughout the chromatographic run. In this way, the flat baseline markedly improves robustness of the assay and simplifies peak integration and quantification. At the same time, resolution of 15 different steroid metabolites catalyzed by the cytochrome P-450 enzymes are readily separated in rat or mouse liver microsomes. An internal standard, cortexolone, was selected for use based on its structural and spectral similarity to the hydroxytestosterone metabolites, and quantification is based on the molar response for the testosterone/cortexolone peak area ratio. The limit of detection (LOD) is 1 pmol on-column with a limit of quantitation (LOQ) of 4 pmol on-column. The intraday repeatability is approximately 3%. This simplified procedure is straightforward and should greatly facilitate the routine use of the testosterone hydroxylation assay to measure cytochrome P-450 isozyme activity.

In vitro studies on the role of titanium in aseptic loosening.

This study was designed to define the role titanium debris plays in aseptic loosening. Macrophages exposed to commercially pure titanium (1-3 microns) exhibit the same mediator profile as those exposed to polymethylmethacrylate. This response consists of increased release of tumor necrosis factor but not prostaglandin E 2 or interleukin-1. Osteoblasts increase production of prostaglandin E 2 when exposed to media from titanium stimulated macrophages but not interleukin-6 or granulocyte macrophage colony stimulating factor. Media from macrophages exposed to titanium did not lead to bone resorption, as measured by calcium 45 release, in organ culture. The cellular response to titanium is characterized by release of tumor necrosis factor from macrophages and prostaglandin E 2 from osteoblasts exposed to the macrophage conditioned medium. A comparison of the results of this study with those of others involving exposure of macrophages in tissue culture suggests that titanium may not be as inflammatory as other particles in the aseptically loose joint.

Evaluation in rats of the dose-response relationship among colonic mucosal growth, colonic fermentation, and dietary fiber.

The dose-response relationship among dietary fiber, colonic fermentation, fecal weight, and mucosal growth were evaluated in this study. The morphometric parameter of total mucosal volume was used to assess diet-induced differences in colonic mucosal growth. Dietary fibers with a wide range of fermentability and that have previously been shown to inhibit the development of colonic neoplasia in rats were used. Sprague-Dawley rats were fed Purina Rodent Chow, AIN-76a fiber-free diet, or an AIN-76a diet supplemented with three different dietary fibers, (cellulose, guar gum, or wheat bran) at 2, 5, 10, or 15% of the diet. Diets were fed for 28 days. Total colonic mucosal volume was determined using stereologic principles and computerized image analysis; 48-hr fecal weight was measured; and the concentration of short-chain fatty acids (SCFA) in colonic contents was determined at study termination. Each type of fiber induced a dose-dependent increase in total mucosal volume of the colon and fecal weight. Mucosal volume and fecal weight were closely correlated (R2 > 0.95). Total mucosal volume was not correlated with the concentration of total SCFA or butyrate in the colon. These results indicate that diet-induced change in colonic mucosal growth, as measured by total mucosal volume, is positively correlated with fecal weight and not related to alterations in colonic fermentation. Enhanced colonic mucosal growth occurs in rats fed dietary fibers that have previously been shown to inhibit the development of genotoxin-induced colonic neoplasia in rats.

The interactions of diet and colonic microflora in regulating colonic mucosal growth.

The colonic mucosa can adapt its growth to alterations in diet. Metabolites from colonic microflora are frequently implicated as the primary factor in mediating the colonic mucosal response to diet; however, there is also evidence indicating that diet may have a direct effect in mediating this response. The aim of this study was to determine the role of diet, microflora, and microflora metabolites in altering the growth of the colonic mucosa. Two 28-day feeding studies were conducted using Sprague-Dawley rats. The first study compared the growth of the colonic mucosa in germ-free and conventional rats fed 6 different diets. The second study compared the growth of the colonic mucosa to the concentration of bacterial-derived short-chain fatty acids (SCFs), bile acids, and ammonia. The diets that were fed consisted of (1) AIN-76a diet without dietary fiber; (2) standard AIN-76a diet, which contained 5% cellulose; (3) AIN-76a diet with 5% guar gum; (4) a "Western" human diet with 20% fat and 10% cellulose; (5) AIN-76a diet formulated to mimic Diet 4 in fat content but with 2.5% cellulose; and (6) Purina Rodent Chow. Quantitative volumetric and stereologic analysis was used to assess changes in total colonic mucosal volume as a measure of mucosal growth. In germ-free rats, Diets 2-4 and 6 induced a significant increase (18-38%) in mucosal volume compared to Diet 1. In conventional animals, only Diets 4 and 6 induced a significant increase (up to 63%) in mucosal volume compared to Diet 1. Relative to the germ-free animals, only conventional animals on Diets 4 and 6 had an increase in mucosal volume. The increases in mucosal volume in Diets 4 and 6 were not consistently associated with increased SCFAs, ammonia, or bile acids. There was a wide range in the colonic concentrations of SCFAs (2-fold), ammonia (6-fold), and bile acids (10-fold). The presence of colonic microflora in and of itself does not lead to enhanced colonic mucosal growth. Rather, there are unique interactions between specific types of diet and microflora that lead to a growth-promoting effect. This effect could not be explained by alterations in the concentration of SCFAs, ammonia, or bile acids in colonic contents.

Pharmacologic inhibition of particulate-induced bone resorption.

In this study, a rat calvaria/macrophage co-culture model was used to study the effects of various agents upon bone resorption induced by macrophage exposure to bone cement particles. The experimental group consisted of calvaria bone disks set in tissue culture medium on stainless-steel platforms into wells with macrophages adherent to the bottom which are exposed to the particles. Tumor necrosis factor alpha (TNF-alpha), prostaglandin E2 (PGE2), and calcium 45 (Ca45 were released in significant amounts in this system. Interleukin 1 alpha (IL-1 alpha) was not detected. Indomethacin inhibited the production of PGE2, but did not affect TNF release or inhibit the release of Ca45. Anti-TNF antibody neutralized the presence of TNF to undetectable levels, but did not affect PGE2 release or inhibit Ca45 release. The addition of calcitonin did not inhibit Ca45 release by calvaria. In contrast, the addition of disodium pamidronate, a member of the bisphosphonate family, was effective in inhibiting the release of Ca45 even after 96 h of incubation. In prior studies, incubation of calvaria in conditioned medium from macrophages exposed to cement particles led to resorption through a mechanism which is dependent upon TNF production by macrophages, and PGE2 production by cells in bone. In this two-way system, in which macrophages and cells in bone are allowed to interact, this dependency was no longer evident. Pamidronate was the only agent tested which suppressed the increase in bone resorption associated with macrophage exposure to bone cement particles to levels which were not significantly different from unexposed calvaria. By delaying or preventing bone resorption associated with macrophage exposure to bone cement particles, bisphosphonates may have a clinical role in cemented joint arthroplasty by decreasing the rate or incidence of aseptic loosening and prolonging implant longevity.

Role of tumor necrosis factor alpha in particulate-induced bone resorption.

The purpose of this study was to determine the role of tumor necrosis factor alpha in bone resorption secondary to mediator release from macrophages exposed to cement particles. The J774 mouse macrophage cell line was exposed to polymethylmethacrylate particles for 24 hours and the resulting conditioned medium was analyzed for prostaglandin E2, tumor necrosis factor alpha, interleukin-1 alpha and beta, and the ability to stimulate release of prostaglandin E2 and 45Ca from radiolabeled mouse calvaria. Macrophage exposure to polymethylmethacrylate particles led to a 9-fold increase in release of tumor necrosis factor alpha (p < 0.01), but did not lead to a significant increase in release of prostaglandin E2, interleukin-1 alpha, or interleukin-1 beta when compared to unexposed cells. Exposure of the macrophages to polymethylmethacrylate particles over a time course from 30 minutes to 96 hours led to an increase in the release of tumor necrosis factor alpha that was initially detected at 30 minutes and was maximum at 48 hours. Incubation of the macrophage-polymethylmethacrylate conditioned medium with rat calvaria significantly increased the release of 45Ca and prostaglandin E2 from the bone. To study the role of release of tumor necrosis factor alpha in bone resorption, the macrophage-polymethylmethacrylate conditioned medium was then preincubated with anti-tumor necrosis factor alpha antibody prior to exposure of the conditioned medium to the calvaria. This preincubation was successful in significantly inhibiting 45Ca release by calvaria (p <0.01) to levels that were not significantly different from the levels of release by unexposed calvaria. Tumor necrosis factor alpha appears to play a critical role in initiating particulate-induced bone resorption. Exposure of macrophages to polymethylmethacrylate particles leads to a significant release of tumor necrosis factor alpha in a time-dependent fashion. This macrophage-polymethylmethacrylate conditioned medium stimulated release of prostaglandin E2 and bone resorption in bone organ culture. The addition of anti-tumor necrosis factor alpha antibody to this in vitro system inhibited the bone resorption stimulated by the macrophage-polymethylmethacrylate conditioned medium and partially suppressed the production of prostaglandin E2. The sequence of events in this model for particulate-induced bone resorption appears to be initiated by the production of tumor necrosis factor alpha by the macrophage, followed by production of prostaglandin E2 by cells in bone, and then by bone resorption.

Mechanisms of cellular recruitment in aseptic loosening of prosthetic joint implants.

The association of macrophages engaged in polymethylmethacrylate (PMMA) particle phagocytosis with pockets of inflammatory cells is a pathognomonic feature of the aseptically loose interface not present at the well-fixed interface. The mechanism by which the presence of PMMA particles leads to cellular recruitment, bone resorption, and ultimate loosening is poorly understood. Granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), cytokines released by osteoblasts, stimulate the recruitment of macrophages into sites of inflammation. We show that exposure of macrophages to PMMA particles stimulated release of tumor necrosis factor (TNF), but no increase in prostaglandin E2 (PGE-2) or interleukin 1. Incubation of osteoblasts with conditioned medium from macrophages exposed to PMMA particles led to release of GM-CSF, IL-6, and PGE-2. Incubation of the PMMA/macrophage medium with antibodies to TNF prior to osteoblast exposure inhibited release of GM-CSF, IL-6, and PGE-2 by the osteoblasts. Our data demonstrate that exposure of macrophages to PMMA particles leads to the release of TNF which then stimulates osteoblasts to produce GM-CSF, IL-6 and PGE-2. Based upon the results of this study , we propose that the process of cellular recruitment in aseptic loosening is initiated when the mechanical failure of the cement mantle leads to the production of PMMA particles. These particles are phagocytized by macrophages leading to the production of TNF. TNF stimulates surrounding osteoblasts to produce GM-CSF, IL-6, and PGE-2 which leads to recruitment of macrophages and osteoclasts into the area of the bone-cement interface. The recruitment of these cells potentiates this process leading to bone resorption and ultimately, clinical loosening of prosthetic joint implants.

Mediator interactions in macrophage/particulate bone resorption.

The purpose of this study was to examine the mechanism by which mediators released from macrophages exposed to cement particles may interact with cells in bone to ultimately lead to bone resorption. Macrophages were exposed to cement particles, and then this conditioned medium was exposed to rat calvarial bones in vitro. The macrophage conditioned medium contained increased levels of tumor necrosis factor, but not interleukin 1 or prostaglandin E2. Exposure of this medium to the calvaria led to release of prostaglandin E2 by the calvaria, but not tumor necrosis factor or interleukin 1. Bone resorption was assessed by measuring the release of calcium 45 from the newborn rat calvarial bones. At 48, 72, and 96 h of incubation, the macrophage/cement particle-conditioned medium led to the release of both prostaglandin E2 and calcium 45 from the calvaria. To determine whether the release of calcium 45 was dependent on prostaglandin E2 production by the cells in bone, the calvaria were incubated with 600 ng/ml of indomethacin in addition to the macrophage-conditioned medium. The addition of indomethacin was effective in inhibiting both prostaglandin E2 and calcium 45 release from the calvaria, even after 96 h of exposure to the macrophage-conditioned medium. This study suggests that tumor necrosis factor produced in association with macrophage/cement particle osteolysis arises from macrophages and not cells in bone, and that prostaglandin E2 originates from cells in bone and not from macrophages. Interleukin 1 was not found to be produced by macrophages or bone, and appears to have a lesser role.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophage inflammatory proteins 1 and 2: expression by rat alveolar macrophages, fibroblasts, and epithelial cells and in rat lung after mineral dust exposure.

Macrophage inflammatory proteins 1 alpha and 2 (MIP-1 alpha, MIP-2) are members of a growing family of cytokines thought to play a role in host defense. MIP-1 alpha and MIP-2 were previously identified in the mouse and shown to stimulate inflammatory cell recruitment. To better understand the potential role of MIP-1 alpha and MIP-2 in lung defense, we investigated the ability of rat lung cells to express mRNA for and/or secrete MIP-1 alpha and MIP-2 proteins in vitro and characterized expression of these cytokines in rat lung after in vivo exposure to silica (SiO2) or titanium dioxide (TiO2). In response to lipopolysaccharide, rat alveolar macrophages expressed increased levels of MIP-1 alpha and MIP-2 mRNA and secreted proteins (identified by N-terminal sequencing) homologous to mouse MIP-1 alpha and MIP-2. Rat alveolar macrophage MIP-1 alpha and MIP-2 mRNA expression was also increased by tumor necrosis factor-alpha (TNF) and adherence to plastic. Studies with a rat fibroblast and epithelial cell line demonstrated that MIP-2, but not MIP-1 alpha, expression can be detected in these cells after stimulation with TNF. Intratracheal instillation studies with SiO2 and TiO2 showed that inflammatory doses of these dusts increase MIP-1 alpha and MIP-2 mRNA expression in whole lung and that increased gene expression preceded the accumulation of inflammatory cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Matrix-assisted laser desorption mass spectrometry of proteins isolated by capillary zone electrophoresis.

A simple method for the off-line coupling of laser desorption mass spectrometry (LDMS) and capillary zone electrophoresis (CZE) is described. Representative mass spectra of subpicomole quantities of proteins isolated from CZE are presented and discussed. The current detection limit for bovine alpha-lactalbumin is 100 fmols injected onto the CZE column. Horse heart myoglobin was demonstrated to be stable in CHES/KCl, a CZE buffer, for at least 1 month, suggesting that some isolates can be safely stored for long time periods prior to LDMS analysis. Protein stability in 0.1% aqueous trifluoroacetic acid (TFA), a common solvent for LDMS, must also be considered. In the special case of porcine pepsinogen, significant (greater than 50%) degradation was observed within 5 min in TFA. In favorable cases, mass measurement accuracies of +/- 0.02% were obtained for protein isolates. Factors limiting mass measurement accuracy are presented. Finally, the possibility of identifying protein isolates, by combining N-terminal sequencing, molecular mass measurements, and selective peptide "mapping" procedures, is discussed.

Bovine aortic endothelial cell transglutaminase. Enzyme characterization and regulation of activity.

Bovine aortic endothelial cells contain Ca2+-dependent tissue-type transglutaminase. Its activity in these cells was high, with apparent Km and Vmax. values with respect to putrescine of 0.203 mM and 18.5 nmol/min per mg of protein, and its activity was inhibited by the three competitive inhibitors dansylcadaverine, spermine and methylamine. The molecular mass of endothelial cell transglutaminase estimated by gel filtration chromatography was 88 kDa and it was immunoprecipitated by rabbit monospecific antiserum raised against rat liver transglutaminase. Its enzymic activity rose when the cell cultures reached confluence, and was further increased when their proliferation was arrested (synchronized at G0/G1 phase). Most of the enzymic activity was found in the 15,000 g soluble fraction, with only 4-22% of the activity found in the particulate fraction, depending on the state of cell proliferation. Examination of these cellular fractions by SDS/polyacrylamide-gel electrophoresis and immunoblotting revealed that at confluence endothelial cells have accumulated transglutaminase antigen in their 15,000 g particulate fraction. A series of experiments demonstrated the existence of a latent transglutaminase form in non-proliferating cells, and suggested that this might involve the formation of an inhibitory complex. Treatment of cell lysates and the 15,000 g particulate fraction with high salt concentration showed a significant increase in transglutaminase activity. Mixing experiments using the 100,000 g particulate fraction or purified rat liver transglutaminase on one hand and the cytosolic fraction on the other showed dose-dependent inhibition of the transglutaminase activity of the latter. It is concluded that endothelial cells contain a particulate fraction-residing inhibitor of transglutaminase which interacts via ionic interaction with the enzyme.