The association between lumbar paraspinal muscle functional cross-sectional area on MRI and regional volumetric bone mineral density measured by quantitative computed tomography.

Osteosarcopenia is a common condition among elderly and postmenopausal female patients. Site-specific bone mineral density is more predictive of bone-related complications. Few studies have investigated muscle-bone associations. Our results demonstrated that in women, significant positive associations between paraspinal muscles FCSA and vBMD exist at different lumbosacral levels. These regional differences should be considered when interpreting bone-muscle associations in the lumbar spine. INTRODUCTION: There is increasing evidence between bone and muscle volume associations. Previous studies have demonstrated comorbidity between osteoporosis and sarcopenia. Recent studies showed that sarcopenic subjects had a fourfold higher risk of concomitant osteoporosis compared to non-sarcopenic individuals. Although site-specific bone mineral density (BMD) assessments were reported to be more predictive of bone-related complications after spinal fusions than BMD assessments in general, there are few studies that have investigated level-specific bone-muscle interactions. The aim of this study is to investigate the associations between muscle functional cross-sectional area (FCSA) on magnetic resonance imaging (MRI) and site-specific quantitative computed tomography (QCT) volumetric bone mineral density (vBMD) in the lumbosacral region among spine surgery patients. METHODS: We retrospectively reviewed a prospective institutional database of posterior lumbar fusion patients. Patients with available MRI undergoing posterior lumbar fusion were included. Muscle measurements and FCSA were conducted and calculated utilizing a manual segmentation and custom-written program at the superior endplate of the L3-L5 vertebrae level. vBMD measurements were performed and calculated utilizing a QCT pro software at L1-L2 levels and bilateral sacral ala. We stratified by sex for all analyses. RESULTS: A total of 105 patients (mean age 61.5 years and 52.4% females) were included. We found that female patients had statistically significant lower muscle FCSA than male patients. After adjusting for age and body mass index (BMI), there were statistically significant positive associations between L1-L2 and S1 vBMD with L3 psoas FCSA as well as sacral ala vBMD with L3 posterior paraspinal and L5 psoas FCSA. These associations were not found in males. CONCLUSIONS: Our results demonstrated that in women, significant positive associations between the psoas and posterior paraspinal muscle FCSA and vBMD exist in different lumbosacral levels, which are independent of age and BMI. These regional differences should be considered when interpreting bone and muscle associations in the lumbar spine.

Postoperative decrease of regional volumetric bone mineral density measured by quantitative computed tomography after lumbar fusion surgery in adjacent vertebrae.

We investigated the effect of posterior lumbar fusion surgery on the regional volumetric bone mineral density (vBMD) measured by quantitative computed tomography. Surgery negatively affected the regional vBMD in adjacent levels. Interbody fusion was independently associated with vBMD decline and preoperative epidural steroid injections (ESIs) were associated with less postoperative vBMD decline. INTRODUCTION: Few studies investigate postoperative BMD changes after lumbar fusion surgery utilizing quantitative computed tomography (QCT). Additionally, it remains unclear what preoperative and operative factors contribute to postoperative BMD changes. The purpose of this study is to investigate the effect of lumbar fusion surgery on regional volumetric bone mineral density (vBMD) in adjacent vertebrae and to identify potential modifiers for postoperative BMD change. METHODS: The data of patients undergoing posterior lumbar fusion with available pre- and postoperative CTs were reviewed. The postoperative changes in vBMD in the vertebrae one or two levels above the upper instrumented vertebra (UIV+1, UIV+2) and one level below the lower instrumented vertebra (LIV+1) were analyzed. As potential contributing factors, history of ESI, and the presence of interbody fusion, as well as various demographic/surgical factors, were included. RESULTS: A total of 90 patients were included in the study analysis. Mean age (±SD) was 62.1 ± 11.7. Volumetric BMD (±SD) in UIV+1 was 115.4 ± 36.9 mg/cm3 preoperatively. The percent vBMD change in UIV+1 was - 10.5 ± 12.9% (p < 0.001). UIV+2 and LIV+1 vBMD changes showed similar trends. After adjusting with the interval between surgery and the secondary CT, non-Caucasian race, ESI, and interbody fusion were independent contributors to postoperative BMD change in UIV+1. CONCLUSIONS: Posterior lumbar fusion surgery negatively affected the regional vBMDs in adjacent levels. Interbody fusion was independently associated with vBMD decline. Preoperative ESIs were associated with less postoperative vBMD decline, which was most likely a result of a preoperative decrease in vBMD due to ESIs.

Role of increased circulating and renal adrenomedullin in rats with malignant hypertension.

Although it has been reported that the circulating adrenomedullin (AM) level is elevated in hypertension and renal failure, the pathophysiological significance of circulating and intrarenal AM in malignant hypertension remains unknown. We investigated the circulating and intrarenal AM system in rats with malignant hypertension by measuring the plasma level, renal tissue level, and mRNA abundance of AM and the mRNA abundance of AM receptor. We also investigated the effects of intravenously infused calcitonin gene-related peptide (CGRP)-(8-37), an antagonist of AM, on the hemodynamics and renal tubular function. We studied the following four groups: control Wistar-Kyoto rats (WKY), control spontaneously hypertensive rats (C-SHR), salt-loaded SHR (S-SHR), and DOCA-salt SHR (D-SHR). After 3 wk of DOCA treatment, D-SHR developed malignant hypertension. D-SHR were characterized by higher blood pressure, kidney weight, urinary protein excretion and blood urea nitrogen, and lower creatinine clearance compared with the other three groups. The plasma AM level and urinary excretion of AM were markedly higher in D-SHR than in the other three groups. In the kidney, the tissue AM level and the expression of AM mRNA in the renal medulla were significantly increased in D-SHR compared with the other three groups, whereas there were no significant differences in these levels in the renal cortex among the four groups. In the renal AM receptor system, the expression of the gene for receptor activity modifying protein 3 was significantly increased in the renal medulla in D-SHR compared with the other three groups. An immunohistochemical study revealed that AM immunostaining in renal collecting duct cells and distal tubules was more intense in D-SHR than in the other three groups. After CGRP-(8-37) infusion, blood pressure increased significantly and urinary sodium excretion and urine flow decreased significantly only in D-SHR. These results suggest that the increased circulating AM and renal AM and the increased expression of the mRNA for AM and its receptor may at least partly compensate for the malignant hypertensive state in certain forms of malignant hypertension via the hypotensive, natriuretic, and diuretic actions of AM.

Phenotypic changes of adrenomedullin receptor components, RAMP2, and CRLR mRNA expression in cultured rat vascular smooth muscle cells.

Adrenomedullin is known to inhibit cell proliferation in cultured rat vascular smooth muscle cells, through a cAMP-dependent process. The calcitonin receptor-like receptor could function as an adrenomedullin receptor when co-expressed with receptor activity-modifying protein 2. To determine whether vascular adrenomedullin receptor components, the calcitonin receptor-like receptor and the receptor activity-modifying protein 2, phenotypically change during in vitro culture conditions, we examined the expression of adrenomedullin receptor components, adrenomedullin-induced cAMP production, and the inhibition of cell proliferation in culture rat vascular smooth muscle cells during serial passages. The results demonstrated that the receptor activity-modifying protein 2 and calcitonin receptor-like receptor mRNAs increased in a passage-dependent manner in rat vascular smooth muscle cells. Furthermore, the responses of both the elevation of cAMP and the inhibition of cell proliferation became larger in vascular smooth muscle cells with an increasing number of passages. The results suggest that the increase in functional AM receptor during phenotypic change may in part contribute to the development of vascular lesions, such as in atherosclerosis.

Alterations of intrarenal adrenomedullin and its receptor system in heart failure rats.

Calcitonin receptor-like receptor/receptor activity-modifying protein 2 (CRLR/RAMP2) and CRLR/RAMP3 complexes have been reported to be specific adrenomedullin (AM) receptors. In the present study, we evaluated the pathophysiological significance of renal AM and its receptor system in aortocaval shunt (ACS) rats. Renal AM levels were measured serially during 5 weeks after the operation. Renal gene expressions of AM, CRLR, RAMP2, and RAMP3 were measured at 2 weeks (decompensated phase) and 5 weeks (compensated phase) after the operation. Immunohistochemical localizations of renal AM were also evaluated. Furthermore, the relations between urinary sodium excretion (UNaV) and renal AM levels were evaluated. Renal AM levels were higher in ACS than in control animals only at 1, 2, and 3 weeks after the operation. At 2 weeks after the operation, renal AM mRNA expression was also higher in ACS than in control animals. CRLR, RAMP2, and RAMP3 mRNAs were expressed in the kidney, but there were no differences between the 2 groups. Immunohistochemistry revealed the positive AM immunostaining within the renal tubular cells, and it was more intense in ACS than in control animals. There were significant correlations between UNaV and renal AM levels. At 5 weeks after the operation, there were no differences in mRNA levels of AM, CRLR, RAMP2, and RAMP3 between the 2 groups. There was a significant correlation between UNaV and medullary AM levels. The present findings suggest that increased renal AM levels in decompensated heart failure, presumably due to increased AM production in renal tubules, in part, are involved in the regulation of sodium excretion.

Decreased gene expression of adrenomedullin receptor in mouse lungs during sepsis.

Plasma concentrations of adrenomedullin (AM) are markedly increased during sepsis, but the role of AM has not been clarified. Coexpression of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 2 or 3 have been reported to form the adrenomedullin (AM) specific receptor. We examined the expression of CRLR and RAMP1, 2, and 3 in several tissues from mice in a sepsis model induced by lipopolysaccharide (LPS). High expression of CRLR and RAMP2 mRNA was observed in lungs of normal mice, but it was markedly decreased in endotoxemic mice. It is suggested that the abundant binding sites of AM in lungs are formed by CRLR and RAMP2 in healthy subjects and that their reduction should contribute to the increase of plasma AM concentrations during sepsis. In contrast, LPS treatment markedly increased RAMP3 gene expression in lungs, spleen, and thymus. It is revealed that the distributions of receptor or binding sites of AM are changed in sepsis, and it is suggested that AM plays distinct roles in the clinical course of this syndrome.

Establishing the independent culture of a strictly symbiotic bacterium Symbiobacterium thermophilum from its supporting Bacillus strain.

Symbiobacterium thermophilum is a strictly symbiotic thermophile, the growth of which is dependent on the coexistence of an associating thermophilic Bacillus sp., strain S. S. thermophilum grows only in mixed culture with the Bacillus strain in liquid media, and does not form visible colonies on solid media. To measure the growth of this symbiotic bacterium and to analyze its growth requirements, we developed a quantitative PCR method by using its specific sequences in a putative membrane translocator gene tnaT as primers. According to this method, independent growth of S. thermophilum was first confirmed in a dialyzing culture physically separated from Bacillus strain S with a cellulose membrane. Independent growth of S. thermophilum was also managed by adding conditioned medium prepared from the culture filtrate of the Bacillus strain, but the growth in the conditioned medium stopped at a very limited extent with appearance of filamentous cells, suggesting the uncoupling of cellular growth and cell division. Formation of micro-colonies of S. thermophilum was observed on the conditioned agar medium under both aerobic and anaerobic conditions, but the colony-forming efficiencies remained below 1%. Several other bacterial species, such as Bacillus stearothermophilus, Bacillus subtilis, Thermus thermophilus, and even Escherichia coli, were also found to support the growth of S. thermophilum. These results indicate that S. thermophilum essentially requires some ubiquitous metabolite(s) of low molecular weight produced by various bacterial species as growth factor(s) but coexistence of the living partner cells is still required, probably to maintain an effective level of the putative factor(s) in the medium.

Newly identified Chinese hamster ovary cell mutants defective in peroxisome biogenesis represent two novel complementation groups in mammals.

We isolated peroxisome biogenesis mutants from Chinese hamster ovary (CHO) cells, using the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/ UV) method and wild-type CHO-K1 cells that had been stably transfected with cDNA encoding Pex2p (formerly peroxisome assembly factor-1, PAF-1). Three mutant cell clones, ZP110, ZP111, and ZP114, showed cytosolic localization of catalase, thereby indicating a defect in peroxisome biogenesis, whereas ZP112 and ZP113 contained fewer but larger catalase-positive particles. Mutant ZP115 displayed an aberrant, tubular structure immunoreactive to anti-catalase antibody. Mutants lacking morphologically recognizable peroxisomes also showed the typical peroxisome assembly-defective phenotype such as severe loss of catalase latency and resistance to 12-(1'-pyrene)dodecanoic acid (P12)/UV treatment. ZP110 and ZP111, and ZP114 were found to belong to two novel complementation groups, respectively, by complementation group analysis with cDNA transfection and cell fusion. Cell fusion with fibroblasts from patients with peroxisome biogenesis disorders such as Zellweger syndrome revealed that ZP110 and ZP114 could not be classified to any of human complementation groups. Thus, ZP110/ZP111 and ZP114 are the first, two peroxisome-deficient cell mutants of newly identified complementation groups distinct from the ten mammalian groups previously characterized.

Comparison of tissue distribution of two novel serine/threonine kinase genes containing the LIM motif (LIMK-1 and LIMK-2) in the developing rat.

We previously isolated two novel serine/threonine kinase genes containing the LIM motif (LIMK-1 and LIMK-2) from a rat cDNA library. To examine the functions of these genes, we performed in situ hybridization in the developing rat nervous system. LIMK-1 and LIMK-2 mRNAs mostly co-localized during development and are expressed preferentially in the central nervous system during mid-to-late gestation but the signals decreased during the post-natal period. However, differential gene expression was observed in some nuclei in the CNS; LIMK-1 mRNA was intensely expressed in the facial motor nucleus, the hypoglossal nucleus, deep nuclei of the cerebellum and the layers 3, 5 and 6 of the adult cerebral cortex while only LIMK-2 mRNA was preferentially expressed in the some parts of the epithelium. In the nasal cavity, LIMK-1 and LIMK-2 mRNAs were expressed complementarily. Our results suggest that LIMK-1 and LIMK-2 may have different functions in these regions during development.

Self-association of LIM-kinase 1 mediated by the interaction between an N-terminal LIM domain and a C-terminal kinase domain.

LIM-kinase 1 (LIMK1) and 2 (LIMK2) are members of a novel class of protein kinases containing two LIM motifs at the N-terminus. The LIM motif is thought to be involved in protein-protein interactions. We report here evidence that LIMK1 self-associates and also associates with LIMK2. In vivo and in vitro binding analyses using variously deleted mutants of LIMKI revealed that the self-association of LIMK1 was caused by interaction between the N-terminal LIM domain and the C-terminal kinase domain. The association of LIMK1 with itself and with LIMK2 is important for understanding how activities and functions of LIMK family kinases are regulated.

Zinc-ion-dependent acid phosphatase exhibits magnesium-ion-dependent myo-inositol-1-phosphatase activity.

We have purified bovine brain Zn(2+)-dependent acid phosphatase (Zn(2+)-APase), which requires Zn2+ ions to hydrolyze the substrate p-nitrophenyl phosphate (pNPP) in an acidic environment. The substrate specificity and metal requirement of Zn(2+)-APase at a physiological pH was also studied. The enzyme exhibited hydrolytic activity on myo-inositol-1- and -2-monophosphates, 2'-adenosine monophosphate, 2'-guanosine monophosphate, and the alpha- and beta-glycerophosphates, glucose-1-phosphate, and fructose-6-phosphate in 50 mM Tris-HCl buffer (pH 7.4) in the presence of Mg2+ ions, but not on pNPP and phosphotyrosine. Zn2+, Mn2+ and Co2+ ions were less effective for activation. Among the above substrates, myo-inositol-1-phosphate was the most susceptible to hydrolysis by the enzyme in the presence of 3 mM Mg2+ ions. The enzyme exhibited an optimum pH at around 8 for myo-inositol-1-phosphate in the presence of 3 mM Mg2+ ions. The Mg(2+)-dependent myo-inositol-1-phosphatase activity of the enzyme was significantly inhibited by Li+ ions. The Zn(2+)-dependent p-nitrophenyl phosphatase activity and Mg(2+)-dependent myo-inositol-1-phosphatase activity of the purified enzyme fraction exhibited similar behavior on Sephadex G-100 and Mono Q colomns. These findings suggest that Zn(2+)-APase also exhibits Mg(2+)-dependent myo-inositol-1-phosphatase activity under physiological conditions.

Identification and characterization of a novel family of serine/threonine kinases containing two N-terminal LIM motifs.

We previously isolated human cDNA coding for LIMK1 (LIM motif-containing protein kinase-1), a putative protein kinase containing two LIM motifs at the N terminus and an unusual protein kinase domain at the C terminus. In the present study, we isolated human cDNA encoding LIMK2, a second member of a LIMK family, with a domain structure similar to LIMK1 and 50% overall amino acid identity with LIMK1. The protein kinase domains of LIMK1 and LIMK2 are unique in that they contain an unusual sequence motif Asp-Leu-Asn-Ser-His-Asn in subdomain VIB and a highly basic insert between subdomains VII and VIII. Expression patterns of LIMK1 and LIMK2 mRNAs in human tissues differ significantly. Chromosomal localization of human LIMK1 and LIMK2 genes was assigned to 7q11.23 and 22q12, respectively, by fluorescence in situ hybridization. The Myc epitope-tagged LIMK1 and LIMK2 proteins transiently expressed in COS cells exhibited serine/threonine-specific kinase activity toward myelin basic protein and histone in in vitro kinase assay. Immunofluorescence and subcellular fractionation analysis revealed that Myc-tagged LIMK1 and LIMK2 were localized mainly in the cytoplasm. The "native" LIMK1 protein endogenously expressed in A431 epidermoid carcinoma cells also exhibited serine/threonine kinase activity. The specific activity of native LIMK1 from A431 cells was apparently much higher than that of "recombinant" LIMK1 ectopically expressed in COS cells, hence, it is likely that there is a mechanism, by which native LIMK1 is activated. A 140-kDa tyrosine-phosphorylated protein (pp140) was co-immunoprecipitated with native LIMK1 form A431 cell lysates; therefore, pp140 may be a LIMK1-associated protein involved in the regulation of LIMK1 function.

Identification and characterization of a novel protein kinase, TESK1, specifically expressed in testicular germ cells.

We have isolated cDNA clones encoding the rat and human forms of a novel protein kinase, termed TESK1 (testis-specific protein kinase 1). Sequence analysis indicates that rat TESK1 contains 628 amino acid residues, composed of an N-terminal protein kinase consensus sequence followed by a C-terminal proline-rich region. Human TESK1 contains 626 amino acids, sharing 92% amino acid identity with its rat counterpart. The protein kinase domain of TESK1 is structurally similar to those of LIMK (LIM motif-containing protein kinase)-1 and LIMK2, with 49-50% sequence identity. Phylogenetic analysis of the protein kinase domains revealed that TESK1 is most closely related to a LIMK subfamily. Chromosomal localization of human TESK1 gene was assigned to 9p13. Anti-TESK1 antibody raised against the C-terminal peptide of TESK1 recognized two polypeptides of 68 and 80 kDa in cell lysates of COS cells transfected with human TESK1 cDNA expression plasmid. TESK1 protein expressed in COS cells exhibited serine/threonine kinase activity, when myelin basic protein was used as a substrate. Northern blot analysis revealed that TESK1 mRNA was specifically expressed in rat and mouse testicular germ cells. The TESK1 mRNA in the testis was detectable only after the 18th day of postnatal development of mice and was mainly expressed in the round spermatids. These observations suggest that TESK1 has a specific function in spermatogenesis.

LIMK-1 and LIMK-2, two members of a LIM motif-containing protein kinase family.

We previously isolated human cDNA encoding LIM-kinase (LIMK), a putative protein kinase which contains two repeats of the LIM motif at the N-terminus and a protein kinase consensus sequence at the C-terminus. Using as a probe a cDNA fragment of human LIMK, we isolated from a rat brain cDNA library cDNA clones encoding two distinct protein kinases (termed LIMK-1 and LIMK-2) related to human LIMK. LIMK-1 shares with human LIMK 95% of the total 647 amino acids and is probably a rat equivalent of human LIMK. LIMK-2 has an overall sequence and a domain structure similar to that of human LIMK and rat LIMK-1, but overall identity is 50-51% at the amino acid level. Like human LIMK, the protein kinase domains of rat LIMK-1 and -2 contain a characteristic sequence DLNSHN in subdomain VIB and a highly basic insert between subdomain VII and VIII. LIMK-1 and -2 are therefore closely related but distinct members of a novel LIM-containing protein kinase subfamily. Several forms of LIMK-2 transcripts encoding proteins that are N-terminally modified and/or C-terminally truncated are generated by alternative splicing or alternative initiation. Northern blot analysis revealed the expression of LIMK-1 mRNA predominantly in the brain and the expression of LIMK-2 mRNA in various tissues in the rat. Antibody raised against LIMK-1 specifically immunoprecipitated and identified in Rat2 fibroblast cells a 72 kDa protein, which has no detectable autophosphorylating activity but is capable of phosphorylating serine and threonine residues of myelin basic protein, by in vitro kinase reaction. As the LIMK family kinases have unique structural features, they are likely to have specific functions in previously uncharacterized signaling pathways.

Identification of a human cDNA encoding a novel protein kinase with two repeats of the LIM/double zinc finger motif.

By low-stringency screening of a human hepatoma HepG2 cell cDNA library, using the genomic fragment of chick c-sea receptor tyrosine kinase as a probe, we isolated overlapping cDNAs encoding a novel protein kinase, which we termed LIM-kinase (LIMK).* The predicted open reading frame encodes a 647-amino-acid polypeptide containing a putative protein kinase structure in the C-terminal half. In addition, LIMK has two repeats of cysteine-rich LIM/double zinc finger motif at the most N-terminus. To our knowledge, this is the first protein kinase seen to contain the LIM motif(s) in the molecule. Although the protein kinase domain of LIMK has highly conserved sequence elements of protein kinases, phylogenetic analysis revealed that LIMK cannot be classified into any subfamily of known protein kinases. Northern blot analysis revealed that the single species of LIMK mRNA of 3.3 kb was expressed in various human epithelial and hematopoietic cell lines. In rat tissues, LIMK mRNA was expressed in the brain, at the highest level. LIM is suggested to be involved in protein-protein interactions by binding to another LIM motif. As the LIM domain is frequently present in the homeodomain-containing transcriptional regulators and oncogenic nuclear proteins, LIMK may be involved in developmental or oncogenic processes through interactions with these LIM-containing proteins.

Purification and characterization of hepatocyte growth factor (HGF)-converting enzyme: activation of pro-HGF.

Hepatocyte growth factor (HGF) is a heterodimer protein, derived from an inactive single-chain precursor (pro-HGF) by the proteolytic cleavage at Arg-Val site. Using a fluorogenic substrate Ac-Lys-Thr-Lys-Gln-Leu-Arg-MCA corresponding to the sequence around the cleavage site of pro-HGF, HGF-converting enzyme was purified from fetal bovine serum. The enzyme is a heterodimer molecule with an apparent molecular weight of about 90-kDa and is composed of a 65-kDa heavy-chain and a 32-kDa light-chain. The enzyme belongs to the serine-protease family and has an optimal pH around 8. The enzyme preferentially cleaved MCA-substrates containing the processing site of pro-HGF. The purified enzyme converted pro-HGF to a two-chain mature form of HGF. The enzyme-treated pro-HGF had mitogenic activity on primary cultured hepatocytes. Thus, the enzyme is likely to be involved in pro-HGF activation in vivo. The enzyme activity in rat serum was 9-fold higher than that in the plasma. This, together with the heterodimeric structure of the enzyme, suggests that the HGF-converting enzyme is activated by another protease in response to a trigger such as blood coagulation or tissue injury.

Cloning of CRP2, a novel member of the cysteine-rich protein family with two repeats of an unusual LIM/double zinc-finger motif.

The cDNA coding for a novel member of the cysteine-rich protein family was isolated from a rat brain cDNA library. It encodes a protein, denoted cysteine-rich protein 2 (CRP2), of 208 amino acid residues containing two tandem repeats of an unusual LIM/double zinc-finger-like motif. The ubiquitous tissue distribution and high level of expression of CRP2 mRNA suggest an important role for CRP2 in cell functions.