A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1.

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7-Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4(+) and CD8(+) lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.

Transumbilical laparoscopic treatment of Congenital Infantile Fibrosarcoma of the Ileum.

Congenital-Infantile Fibrosarcoma (CIF) is a malignant mesenchymal tumor representing 10-20% of soft-tissue tumors. Complete surgical resection is generally the treatment of choice. The most recurrent cytogenetic abnormality was identified as the traslocation t(12;15)(p13:q25), which bears the fusion of Tel gene EVT6 with TrkC gene. This study describes a case of infantile fibrosarcoma of the ileum in a female newborn examined for intestinal occlusion and its laparoscopic treatment.

Alteration of digestive tract microbiome in neonatal Holstein bull calves by bacitracin methylene disalicylate treatment and scours.

The effects of bacitracin methylene disalicylate (BMD) and scours on the fecal microbiome, animal performance, and health were studied in Holstein bull calves. Holstein bull calves (n = 150) were obtained from a single source at 12 to 24 h of age. Bull calves were randomly assigned to 1 of 2 treatments including CON (no BMD; n = 75 calves) and BMD (n = 75 calves). Starting 3 d after arrival, BMD was added into milk replacer (0.5 g/feeding; twice daily) and fed to the calves for 10 consecutive d. No differences (P > 0.10) were observed in ADG for d 0 to 28 and d 0 to 56, DMI for d 0 to 28, d 29 to 56, and d 0 to 56, or G:F for d 0 to 28, d 29 to 56, and d 0 to 56; ADG for d 29 to 56 tended to increase (P < 0.10) for BMD-treated calves compared with controls. Fecal samples were collected from 15 scouring calves and 10 cohorts (nonscouring calves received on the same day and administered the same treatment as the scouring calves). Animal morbidity and fecal score did not vary between the 2 treatments. Mortality was not influenced by the treatments in the BMD administration period or throughout the experiment. Fecal samples were subjected to pyrotagged 454 FLX pyrosequencing of 16S rRNA gene amplicon to examine compositional dynamics of fecal microbes. Escherichia, Enterococcus, and Shigella had greater (P < 0.05) populations in the BMD group whereas Dorea, Roseburia, Fecalibacterium, Papillibacter, Collinsella, Eubacterium, Peptostreptococcus, and Prevotella were decreased (P < 0.05) by BMD treatment. Genus populations were also compared between scouring and nonscouring calves. Streptococcus was the only genus that had notable increase (P < 0.05) in fecal samples from scouring calves whereas populations of Bacteroides, Roseburia, and Eubacterium were markedly (P < 0.05) greater in nonscouring calves. These results show that BMD has the ability to alter the composition of the fecal microbiome but failed to improve performance in Holstein bull calves. Discrepancy of microorganism profiles between scouring and nonscouring calves might be associated with the occurrence of scours and bacterial genera identified might be potential target of treating diarrhea.

Heat shock protein gene expression profile may differentiate between rheumatoid arthritis, osteoarthritis, and healthy controls.

OBJECTIVE: Heat shock proteins (Hsps) have been repeatedly implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this work was to study Hsp mRNA and protein levels to determine whether they can be used to differentiate between RA, osteoarthritis (OA), and healthy controls. METHODS: Hsp27, Hsp60, Hsp70, Hsp90α, and HspBP1 mRNA expression was analysed using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 24 RA, 11 OA, and 21 healthy controls. Hsp70 and HspBP1 protein levels were measured in serum using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Hsp gene expression profiles differ significantly between inflammatory (RA) and non-inflammatory (OA) joint diseases, showing significantly increased Hsp27 and Hsp90α mRNA levels in RA synovial tissues. Up-regulated Hsp60 and Hsp90α together with down-regulated Hsp70 and elevated HspBP1/Hsp70 mRNA ratios can be used to differentiate between RA patients and healthy individuals through analysis of peripheral blood samples. Despite increased HspBP1 levels in RA sera, Hsp70 levels and the HspBP1/Hsp70 protein ratio remained identical in the RA patients and healthy individuals, which may contribute to the inhibition of Hsp70 anti-apoptotic activity. CONCLUSION: Hsp gene expression analysis can be implemented as a new diagnostic approach to facilitate differentiation between RA, OA, and healthy controls.

[Small ovarian cysts in postmenopause: assessment of their malignant potential with vaginal ultrasonography and tumor marker Ca125 titration]. / Piccole cisti ovariche in postmenopausa: valutazione del loro potenziale maligno mediante l'uso di ultrasonografia vaginale e titolazione del marcatore tumorale Ca125.

BACKGROUND: The aim of this study was to determine the risk of malignancy in cystic ovarian tumors < 10 cm in diameter in asymptomatic postmenopausal women. METHODS: All cystic ovarian tumors, detected by abdominal and transvaginal sonography screening, in asymptomatic postmenopausal women were evaluated with respect to size and morphology. Follow-up data were available both on patients undergoing surgery and on those who elected to be followed without operative intervention. Titration of the tumoral marker Ca125 was carried out, too. RESULTS: Unilocular cystic tumors were detected in 32 of 352 postmenopausal patients (9%), of 45-65 years of age arrived at the "Centre for diagnosis and therapy of menopausal diseases" of the III Divisione di Ginecologia e Ostetricia della Seconda Università degli Studi di Napoli from the 1st January to the 31st December 1999. All tumors were < 10 cm in diameter and 98% were < 5 cm in diameter; just one tumor was hardly > 5 cm in diameter (5.8), 14 of these cystic ovarian tumors (49%) resolved spontaneously within 60 days while 18 (51%) persisted. Seven patients with persistent cystic ovarian tumors underwent operative tumor removal. Five of these patients had serous cystadenoma and 2 other women had cystoadenofibroma. Not even one case of ovarian carcinoma was found in this group. The remaining 11 patients with unilocular cystic ovarian tumors underwent sonography control every 3 months for one year and no one of these patients developed ovarian carcinoma. In all these patients the dosage of the tumoral marker Ca125 remained under the suspicious threshold of malignant ovaric tumor (Ca125 = 35 U/ml). CONCLUSIONS: Unilocular cystic ovarian tumors < 5 cm in diameter in asymptomatic postmenopausal women were associated with minimal risk for ovarian cancer. In contrast, complex ovarian cysts wall abnormalities or solid areas are associated with a significant risk for malignancy. These date are important in determining therapeutic optimal strategies in these patients.

[Role of Ca 125 in pelvic masses. Our experience]. / Ruolo del Ca 125 nelle masse pelviche. Nostra esperienza.

BACKGROUND: The goal of our study is to plan a screening program for early detection of ovarian cancer through clinical examination, pelvic ultrasonography and serum Ca 125 dosage. METHODS: Between January 1993 and June 1999, 436 patients have been submitted to ovarian cancer screening at the Obstetrics and Gynecology Institute of the Second University of Naples. All women were in postmenopausal period, older than 50 years and didn't show any gynecologic disease. RESULTS: Clinical examination selected 41 patients (9.4%) with a pelvic mass; pelvic ultrasonography revealed ovarian or uterine mass (only subserous myoma) in 87 cases (19.9%). These patients were submitted to Ca 125 serum dosage; in three cases Ca 125 was higher than 65 U/ml and in 26 cases its value was between 35 and 65 U/ml. The remaining 58 patients showed Ca 125 values lower than 35 U/ml. Four patients with ovarian cancer have been detected with our screening program. CONCLUSIONS: The authors conclude that pelvic ultrasonography and serum Ca 125 dosage are useful for the assessment of an early screening program of ovarian cancer.

[Role of echo-guided aspiration of ovarian cysts. Our experience]. / Ruolo dell'aspirazione eco-guidata delle cisti ovariche. Nostra esperienza.

BACKGROUND: Ultrasound-guided puncture is a simple and easy to perform procedure. This study was undertaken to verify the role of fine-needle aspiration (FNA) followed by cytological examination as a possible alternative to surgery in case of cystic pelvic masses. Ovarian cysts are conventionally managed by laparoscopy or laparotomy. METHODS: From January 1993 to December 1997, 224 patients with a proven cystic pelvic mass underwent surgical intervention and have been retrospectively analysed for FNA under sonographic guidance. The sediment aspirated was examined by a cytological method and when possible it was also correlated to a histological test. RESULTS: Eight patients (34.8%) had been submitted to one needle cyst aspiration before surgical intervention and 15 (65.2%) to more than one aspiration. Patients with an history of only one aspiration were submitted to surgical intervention with urgency statistically more than the group with an history of more than one aspiration. Anatomo-pathologic examination showed a significative relevance of serous and endometriotic cysts. CONCLUSIONS: We conclude that FNA might be proposed in young women with a unilocular ovarian cyst to avoid a surgical procedure. In all instances the ultrasonographic appearance of the cyst and the characteristics of aspirated fluid are the most important findings.

Isolation and characterization of isoforms of HspBP1, inhibitors of Hsp70.

The protein sequences derived from cDNA sequences for Hsp70 binding proteins from human (HspBP2) and rat tissues (HspBPR) are presented in this paper. The derived amino acid sequences of these proteins differ from human HspBP1 in the number of consecutive glycines near the amino-terminus. These differences, however, do not alter the inhibitory activity.

Recognition of a dominant epitope in bovine heat-shock protein 70 in inner ear disease.

OBJECTIVE: To investigate the specificity of antibodies to heat-shock protein 70 (HSP70) in patients with idiopathic, progressive, bilateral sensorineural hearing loss (IPBSNHL) and Meniere disease. STUDY DESIGN: Test immunoreactivity of patients' sera using recombinant human (rh) and bovine (rb) HSP70, as well as segments representing different regions of bovine HSP70 as antigen. METHODS: Sera were tested by Western blotting. RESULTS: Of 52 patients with IPB-SNHL, 40 sera reacted only with rbHSP70; 12 reacted with both rbHSP70 and rhHSP70. Sera from 13 patients with IPBSNHL and from 8 with Meniere disease were tested on the panel of bovine HSP70 segments. Eleven and 7 samples, respectively, reacted with amino acid segment 427-461 from the carboxy (C)-terminal region of the molecule. CONCLUSION: In IPBSNHL and Meniere disease, antibodies are directed primarily against an epitope(s) within the C-terminal region of HSP70 where diversity in sequence among different species, including possible pathogens, is greatest. These findings may provide clues to the pathogenesis or specific serodiagnosis (or both) of diseases of the inner ear.

Inhibition of Hsp70 ATPase activity and protein renaturation by a novel Hsp70-binding protein.

A cDNA that codes for an Hsp70-interacting protein (HspBP1) was isolated from a human heart cDNA library using the yeast two-hybrid system. The derived amino acid sequence is unique and therefore represents a new regulator of Hsp70. Northern blots of RNA from human tissues indicate that HspBP1 mRNA has a size of approximately 1.7 kilobase pairs and is present in all tissues analyzed but is most abundant in heart and skeletal muscle. Western blot analysis revealed a protein of approximately 40 kilodaltons detected in cell extracts. The ATPase domain of Hsp70 demonstrated binding to HspBP1. Further experiments showed binding of HspBP1 to Hsp70 and Hsc70 in a total heart extract. HspBP1 (8 microM) inhibited approximately 90% of the Hsp40-activated Hsp70 ATPase activity. HspBP1 prevented ATP binding to Hsp70, and therefore this is the likely mechanism of inhibition. Hsp40-activated ATPase activity is essential for the renaturation activity of Hsp70; therefore, the effects of HspBP1 on renaturation of luciferase in a reticulocyte lysate and a defined system were examined. HspBP1 inhibited renaturation with half-maximal inhibition at 2 microM. These data indicate that we have identified a novel Hsp70-interacting protein that inhibits Hsp70 chaperone activity.

Molecular structure of a proteolytic fragment of TLP20.

Myosin light-chain kinase is responsible for the phosphorylation of myosin in smooth muscle cells. In some tissue types, the C-terminal portion of this large enzyme is expressed as an independent protein and has been given the name telokin. Recently, an antibody directed against telokin was found to interact with a protein derived from the baculovirus Autographa californica nuclear polyhedrosis virus. This protein was biochemically characterized and given the name TLP20 for telokin-like protein of 20 000 molecular weight. The amino-acid sequence of TLP20 was determined on the basis of a cDNA clone and subsequent alignment searches failed to reveal any homology to telokin or to other known proteins. The three-dimensional structure of a proteolytic portion of TLP20 is reported here. Crystals employed in the investigation were grown from ammonium sulfate solutions at pH 6.0 and belonged to the space group P2(1)3 with unit-cell dimensions of a = b = c = 76.3 A and one molecule per asymmetric unit. The structure was determined by multiple isomorphous replacement with three heavy-atom derivatives. Least-squares refinement of the model reduced the crystallographic R factor to 18.1% for all measured X-ray data from 30.0 to 2.2 A. The overall fold of the molecule may be described as a seven-stranded antiparallel beta-barrel flanked on the bottom by two additional beta-strands and on the top by an alpha-helix. Quite surprisingly, the three-dimensional structure of this beta-barrel is not similar to telokin or to any other known protein.

Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability.

Contractile events resulting from phosphorylation of the 20-kDa myosin light chain (MLC20) have been implicated in the regulation of epithelial tight junction permeability. To address this question, Madin-Darby canine kidney cells were transfected with a murine leukemia retroviral vector containing DNA encoding either the catalytic domain of myosin light chain kinase (tMK) or the beta-galactosidase gene (beta-gal). Autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of myosin immunoprecipitated from 32Pi-labeled transfected cells demonstrated that MLC20 phosphorylation was increased 3.1 +/- 0.9-fold in cells expressing tMK compared with cells expressing beta-gal. Phosphopeptide mapping confirmed that myosin light chain kinase was responsible for the increased MLC20 phosphorylation. Transepithelial electrical resistance, a measurement of barrier function, of tMK cell monolayers was consistently < 10% (123 +/- 20 omega.cm2) of that of monolayers comprised of wild-type cells (1,456 +/- 178 omega.cm2) or cells expressing beta-gal (1,452 +/- 174 omega.cm2). Dual 22Na+ and [3H]mannitol flux studies indicated that the decrease in resistance in tMK cells was attributable to increased paracellular flow. These data support the idea that MLC20 phosphorylation by myosin light chain kinase is involved in regulating epithelial tight junction permeability.

HSP70 accumulation in tissues of heat-stressed rats is blunted with advancing age.

To determine whether aging results in reduced accumulation of the 70-kDa heat shock protein (HSP70) in response to a thermal challenge, experiments were conducted in conscious and freely moving mature (12-mo-old) and senescent (24-mo-old) male Fischer 344 rats. Rats were assigned to a euthermic control group or a nonexertionally heated group that was exposed to an ambient temperature of 42 degrees C until colonic temperature reached 41 degrees C. Samples were subsequently obtained from the liver and myocardium, and absolute levels of both the constitutive and inducible forms of HSP70 were quantitated. Heat-stressed rats had significantly elevated HSP70 levels in the liver compared with the euthermic groups. Post hoc comparisons revealed that heat stress elicited marked elevations in liver HSP70 in mature rats compared with age-matched control animals. In contrast, HSP70 values were unchanged in the senescent heated group vs. the control group. In the myocardium, heat stress produced marked increases in HSP70 levels in both the mature and senescent groups compared with age-matched control animals, with accumulation significantly blunted in the senescent vs. mature rats. Thus the increases in liver and myocardial HSP70 accumulation in response to nonexertional heat stress are attenuated with senescence. Because these proteins are postulated to protect cells from injury and enhance cellular recovery from heat stress, the data suggest that an aging organism has a reduced ability to properly maintain cellular function and integrity after a thermal challenge.

Fibroblast contractility without an increase in basal myosin light chain phosphorylation in wild type cells and cells expressing the catalytic domain of myosin light chain kinase.

We investigated the role of myosin light chain (MLC20) phosphorylation (MLC-P) in non-muscle contractility by comparing MLC-P and the contractile properties of wild type 3T3 fibroblasts and 3T3 fibroblasts expressing the catalytic domain of myosin light chain kinase (tMK). MLC-P is 0.96 MOL of PO4/mol of MOL20 in cell expressing tMK compared to 0.20 mol of PO4/mol of MLC20 in control cells. Expressing tMK also results in a 2-fold increase in cortical stiffness compared to control cells. Contractile properties were quantified by growing wild type and transfected fibroblasts in collagen and attaching the ensuing fibers to an apparatus for performing mechanical measurements. Serum stimulation resulted in a dose-dependent increase in force with maximal force generated in the presence of 30% (v/v) serum. Surprisingly, MLC-P did not increase in wild type cells following stimulation with 30% serum, and tMK expression did not affect the contractile properties of fibers made from these cells. Moreover, the dose responses to serum, maximal force, force-velocity relationships, and dynamic stiffness were similar in the wild type cells and fibroblasts expressing tMK. These data demonstrate that non-muscle cells can generate force without an increase in MLC-P, and that an increase in MLC-P does not affect the contractile properties of fibroblast fibers.

Chemical modifications of a recombinant bovine stress-inducible 70 kDa heat-shock protein (Hsp70) mimics Hsp70 isoforms from tissues.

A cDNA clone for the stress-inducible 70 kDa heat-shock protein (Hsp70) has been isolated from a bovine skeletal-muscle cDNA library. This mRNA encodes a protein with a calculated molecular mass of 70250 Da. The cDNA has one continuous open reading frame capable of encoding a 641-amino-acid protein. Expression of this cDNA in a bacterial expression system produced a protein with a mobility identical with that of the inducible Hsp70 protein from bovine skeletal muscle as determined by SDS/PAGE. Two-dimensional gel electrophoresis demonstrated this protein to have focusing properties identical with that of a minor isoform from bovine skeletal muscle. Upon carbamylation of this bacterially expressed protein, a train of charged proteins with charge differences of -1 were produced. These carbamylated proteins were shown to have similar focusing mobilities to the Hsp70 isoforms isolated from bovine skeletal muscle. These results demonstrate the identification of a skeletal-muscle inducible Hsp70 gene and suggest that the presence of multiple Hsp70 isoforms may be the product of post-translational modifications to the Hsp70 proteins.

HSP70 induction during exercise and heat stress in rats: role of internal temperature.

The purpose of this study was to determine if the accumulation of the 72-kDa heat shock protein (HSP70) is elevated in response to a prolonged bout of submaximal exercise in which colonic temperature (Tco) remained at control levels. Sprague-Dawley rats were randomly assigned to one of four testing groups [n = 8 per group; ambient temperatures (Ta) for each condition are included]: 1) control (cool/rest; Ta = 24 degrees C); 2) cool and exercise (cool/exercise; Ta = 14 degrees C); 3) nonexertional heating (heat/rest; Ta = 42 degrees C); 4) heat and exercise (heat/exercise; Ta = 32 degrees C). All interventions were approximately 60 min in duration. An exercise bout consisted of treadmill running at 17 m/min and 0% grade, while the heat/rest and heat/exercise experiments consisted of heat exposure that was terminated when Tco reached 41 degrees C. Baseline Tco was similar for all four groups. In the cool/rest and cool/exercise groups, final Tco was not different from the baseline values, nor was it different between these two groups. In the heat/rest and heat/exercise groups, heating rates were similar. Tissue samples were obtained from the gastrocnemius, soleus, and extensor digitorum longus (EDL) muscles of the left hindlimb and the left ventricle 30 min after a trial was completed. An enzyme-linked immunosorbent assay specific for HSP70 was used to directly quantitate absolute levels of HSP70 in tissues. There were significant main effects of both heating and exercise for HSP70 levels in the gastrocnemius, soleus, and left ventricle (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Relative abundance of bovine Hsp70 mRNA and protein.

A solution hybridization assay to measure bovine Hsp70 mRNA levels is used to demonstrate that skeletal muscle contains the highest amount of this mRNA while brain has the lowest amount and the level of Hsp70 mRNA is heat inducible in bovine skeletal muscle. Furthermore, this assay allowed for the comparison of relative Hsp70 protein and mRNA levels. Relative transcript levels compared to relative Hsp70 protein levels in tissues demonstrated 50- to 200-fold differences. These results, then, approximate the magnitude of the previously reported preferential translation of Hsp70 mRNA.

Mutants of smooth muscle myosin light chain kinase at tryptophan 800.

The importance of Trp 800 in the calmodulin-binding site of myosin light chain kinase was investigated. Truncation mutants from Leu 447 to the C-terminus were expressed in E. coli and these were modified by point mutations of Trp 800 to Gly, Cys, Leu and Tyr. Trp at this position was more effective than any of the other residues. The Leu mutant was partially active and its Km for calmodulin decreased from about 10 nM to 175 nM. The Tyr mutant had detectable activity but the other two mutants were inactive and did not bind calmodulin. Thus Trp at position 800 is critical. The activity of the Leu mutant at high calmodulin concentrations was less than the wild-type mutant, about 20%. This suggests that the binding of calmodulin does not release inhibition in an all-or-none mechanism and that other intramolecular interactions are important.

Sequence and expression of a baculovirus protein with antigenic similarity to telokin.

A protein from baculovirus-infected cells reacted with an antibody against the smooth muscle protein telokin. Because of this unusual similarity, the protein, termed telokin-like protein-20 (TLP20), was isolated and characterized. Its M(r) on denaturing polyacrylamide gels was 28K and the protein contained a high proportion of beta structure. A cDNA for TLP20 was isolated and sequenced. The 3' non-coding sequence contained a region of high identity with the 5' end of two other baculovirus genes. The 5' non-coding region contains several baculovirus regulatory elements. Surprisingly, the derived amino acid sequence showed no homologies to telokin. The cDNA was cloned into a bacterial expression vector and the subsequently expressed protein had a slightly lower M(r) than the native protein, but cross-reacted with telokin antibody. This paper reports the characterization of a new baculovirus protein that shares some antigenic similarities to the smooth muscle protein telokin.