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.

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.

Association of myosin light chain kinase with lymphocyte membrane-cytoskeleton complex.

A specific antibody against myosin light chain kinase (MLCK) was used to identify the presence of a Ca2+-calmodulin-activated MLCK in mouse 1-lymphoma cells. With a double immunofluorescence technique, MLCK was determined to be accumulated directly under Con A-capped structures in a manner similar to that of previously described accumulation of actomyosin. The lymphocyte MLCK was phosphorylated in the uncapped cell and, by immunoprecipitation with a specific MLCK antibody, was shown to possess a Mr of 130,000. The MLCK was also found to constitute a major fraction of the phosphoproteins present in the plasma membrane associated-cytoskeleton. Myosin light chain kinase catalyzed the phosphorylation of both endogenous lymphocyte myosin light chains and those from smooth and skeletal muscle. The enzyme activity was dependent on the presence of Ca2+-calmodulin and was inhibited by the calmodulin-binding drug, trifluoperazine. These data suggest that the membrane-cytoskeleton-associated MLCK activity may be important in regulation of the actinmyosin contraction which is believed to be required for the collection of surface receptors into capped structures.

Estrogen stimulates the transient association of calmodulin and myosin light chain kinase with the chicken liver nuclear matrix.

Previous work has demonstrated that estrogen administration to immature chickens results in a rapid but transient increase in nuclear estrogen receptor content, a large portion of which is associated with the nuclear matrix. The present studies were undertaken to determine whether estrogen produced a more generalized change in the protein composition of the nuclear matrix. High-resolution two-dimensional gel analysis of the matrix revealed a very complex protein pattern, but several major qualitative differences were observed after estrogen treatment. To simplify the number of proteins evaluated, we examined the effects of estrogen on a subset of matrix proteins, namely, calmodulin and its binding proteins. Calmodulin was measured by radioimmunoassay and the binding proteins were detected by interaction of 125I-calmodulin with matrix proteins distributed on one-dimensional polyacrylamide gels. Calmodulin and two specific Ca2+-dependent calmodulin-binding proteins were found to be associated with matrix preparations. The two binding proteins exhibited apparent Mr of 200,000 and 130,000. The Mr 130,000 protein was identified as myosin light chain kinase on the basis of enzymatic activity and immunoreactivity with a specific antibody to this enzyme. Estrogen treatment of immature chickens did not alter the hepatic content of calmodulin. However, the steroid did result in an enrichment of the proportion of calmodulin and its two binding proteins associated with the nuclear matrix within 4 h after injection. The time course of these changes paralleled those previously documented for estrogen receptor. Taken together, these data are compatible with a role for calmodulin and myosin light chain kinase in the response of chicken liver cells to steroid hormones.

Two alternative mRNAs coding for the angiogenic factor, placenta growth factor (PlGF), are transcribed from a single gene of chromosome 14.

We have previously reported on the identification of a cDNA (placenta growth factor, PlGF) coding for a novel angiogenic factor expressed in placental tissue that is similar to vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). Biochemical and functional characterization of PlGF derived from transfected COS-1 cells revealed that it is a glycosylated dimeric secreted protein able to stimulate endothelial cell growth in vitro. Here, we report the isolation and characterization of the PlGF gene located on chromosome 14. At least two different mRNAs are produced from this single-copy gene in different cell lines and tissues. Sequence comparison of the polypeptides encoded by the two different isolated cDNAs indicates that they are identical except for the insertion of a highly basic 21 amino acid stretch at the carboxyl end of the protein. RNA expression analysis of several tissues, tumors and cell lines indicates differential distribution of the two PlGF mRNAs. Finally, preliminary results indicate that the PIGF gene has been conserved in evolution, since the human PlGF cDNA hybridizes to sequences present in the genomic DNA of Drosophila, Xenopus, chicken and mouse.

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.

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.

Hormonal regulation of a chicken oviduct messenger ribonucleic acid that shares a common domain with gizzard myosin light chain kinase.

Changes in myosin light chain kinase (MLCK) and calmodulin (CaM) mRNAs have been evaluated during estrogen-mediated differentiation of the chicken oviduct. Also examined were acute changes that occur in oviduct RNA from animals stimulated with estrogen, withdrawn from hormone and then injected for 1, 2, and 4 days with synthetic estrogen [diethylstilbestrol (DES)], progesterone (P), or testosterone (T). Small changes were noted in both CaM and MLCK RNAs during primary stimulation when oviduct cells are actively dividing. On the other hand no significant changes were observed during secondary stimulation regardless of the steroid hormone injected. These data support the contention that CaM and MLCK are constitutively expressed but vary as a function of cell cycle. The MLCK mRNA is 5.5 kilobases (kb) but the MLCK cDNA also hybridizes to an oviduct RNA 2.7 kb long. This RNA species is acutely regulated by estrogen, P, and T but in a manner different from that of ovalbumin mRNA. The magnitude of stimulation of the 2.7 kb mRNA by diethylstilbestrol and T is greater than that of ovalbumin whereas changes in response to P are similar. The 12- to 16-fold increase of the 2.7 kb mRNA in response to T is the largest effect reported for this hormone acting on oviduct. The 2.7 kb mRNA encodes an unknown protein yet contains a 520 nucleotide segment that is highly homologous with the COOH-terminal coding portion of the MLCK mRNA. Since this homology does not include either catalytic or CaM-binding domains of MLCK, it is unlikely that the 2.7 kb mRNA encodes a CaM-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Dexamethasone effects on myoblast proliferation and differentiation.

Effects of glucocorticoids on myoblast proliferation and differentiation were determined in cell culture. Dexamethasone (1 X 10(-7) M) decreased myoblast doubling time by 7 h and increased cell densities as much as 800% after 8 days of exposure of the cells to dexamethasone. Stimulation of myoblast proliferation by glucocorticoids was dose dependent, with half-maximal activity exhibited at concentrations of 3 X 10(-9) M dexamethasone or 2.3 X 10(-8) M corticosterone. In differentiating cultures, the rate of accumulation and final levels of DNA and creatine phosphokinase were highly stimulated by dexamethasone. However, normalization of creatine phosphokinase levels (a marker for myoblast differentiation) per microgram DNA demonstrated a small (30%) decrease in differentiation in cultures exposed to glucocorticoids. These results indicate that glucocorticoids stimulate early myogenesis by increasing the number of myoblasts but do not stimulate the differentiation of these cells.

Quantitation of Hsp70 in tissues using a competitive enzyme-linked immunosorbent assay.

A competitive enzyme-linked immunosorbent assay has been developed to quantitate the Hsp70 levels in bovine tissues. Antibodies that show specificity to the low molecular weight form of Hsp70 (72 kDa) were developed in chickens, isolated from egg yolks, and characterized using Western blotting. Using this assay, we were able to verify quantitatively the previous observation that the low molecular weight form of Hsp70 exists at elevated levels in bovine skeletal muscle. Also, we report that the skeletal muscle contains the majority of the Hsp70 in the sarcoplasm, but a small amount of Hsp70 is localized in the sarcoplasmic reticulum. The amount of Hsp70 in bovine skeletal muscle is not at a maximal level because it can be increased by heat stress. The reliability, specificity, and use of enzyme-linked antibodies instead of radioactive materials make this assay preferable over previous quantitation techniques.

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.

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.

Synthesis of heat stress proteins in lymphocytes from livestock.

Cultured bovine, equine, ovine and chicken lymphocytes responded to heat stress by the increased synthesis of a specific set of proteins known as heat stress proteins (HSP). Proteins with molecular weights of 70 and 90 kDa were synthesized in all species. Additional proteins were found in bovine, ovine and chicken lymphocytes. A time course of induction showed an increased synthesis of some of these proteins with only 30 min of heat stress and of several proteins with 60 min of heat stress. A specific monoclonal antibody was used to identify HSP70 as one of the stress proteins in bovine lymphocytes. Cells from these animals are capable of responding to heat stress by the production of heat stress proteins. These may be of physiological importance.

Responses of bovine lymphocytes to heat shock as modified by breed and antioxidant status.

We tested whether resistance of lymphocytes to heat stress is modified by breed, intracellular glutathione content, and extracellular antioxidants. In the first experiment, lymphocytes from Angus (Bos taurus, non-heat-tolerant), Brahman (B. indicus, heat-tolerant), and Senepol (B. taurus, heat-tolerant) heifers (12 heifers per breed) were cultured at 45 degrees C for 3 h to evaluate thermal killing, at 42 degrees C for 12 h in a 60-h phytohemagglutinin-induced proliferation test, and at 42 degrees C for 1 h to measure induction of heat shock protein 70 (HSP70). Killing at 45 degrees C was affected by breed x temperature (P < .01); the decrease in viability caused by a temperature of 45 degrees C was greater for Angus than for Brahman or Senepol. For phytohemagglutinin-stimulated lymphocytes, heating to 42 degrees C reduced [3H]thymidine incorporation equally for all breeds. Viability at the end of culture was affected (P < .001) by a breed x temperature interaction because the decrease in viability caused by culture at 42 degrees C was greatest for lymphocytes from Angus heifers. Heat shock for 1 h at 42 degrees C caused a two- to threefold increase in intracellular concentrations of HSP70, but there was no interaction of temperature with breed. In another experiment (with lymphocytes harvested from three Holstein cows), buthionine sulfoximine, a glutathione synthesis inhibitor, inhibited (P < .01) proliferation of phytohemagglutinin-stimulated lymphocytes at 38.5 and 42 degrees C. Addition of the antioxidants glutathione or thioredoxin to culture did not reduce the effects of heating to 42 degrees C on proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

[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.

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.