Correlation between levels of adipokines and inflammatory mediators with spirometric parameters in individuals with obesity and symptoms of asthma: Cross-sectional study.

INTRODUCTION: The adipose tissue secretes adipokines and influences the release of inflammatory mediators contributing to a state of low-grade systemic inflammation that may change lung function. OBJECTIVE: To correlate levels of adipokines and inflammatory mediators with lung function in individuals with obesity and bronchial asthma symptoms. MATERIALS AND METHODS: A cross-sectional study, including women with obesity (grade II and III) with symptoms and clinical diagnosis of asthma. Anthropometric measurements (weight, height, BMI), pulmonary function test (spirometry), asthma control test questionnaire, collection of systemic inflammatory markers (blood collection) and pulmonary markers (sputum collection) were collected and were analyzed: IL-6, IL-8, TNF-α, adiponectin, resistin, leptin and C-reactive protein (CRP). The patients were stratified into two groups according to asthma control. RESULTS: 80 women were analyzed and 40% had an ACT score greater than or equal to 18 and were classified as "controlled asthma". More than half of the patients of ACT<18 score obtaining measures of FEV1, PEF and FEF25-75% below and 80% of predicted. There was a significant and negative correlation between IL-6 in the sputum with FVC and FEF25-75% in the group ACT<18 and with FVC and FEV1 in the group ACT≥18. CONCLUSIONS: Therefore, we concluded that the increase of interleukin-6 in the sputum is related to worse pulmonary function even in patients with controlled asthma, especially in the translate airway permeability measures.

Modeling the interference between shear and longitudinal waves under high intensity focused ultrasound propagation in bone.

Magnetic resonance-guided high intensity focused ultrasound (MR-HIFU) is a noninvasive thermal technique that enables rapid heating of a specific area in the human body. Its clinical relevance has been proven for the treatments of soft tissue tumors, like uterine fibroids, and for the treatments of solid tumors in bone. In MR-HIFU treatment, MR-thermometry is used to monitor the temperature evolution in soft tissue. However, this technique is currently unavailable for bone tissue. Computer models can play a key role in the accurate prediction and monitoring of temperature. Here, we present a computer ray tracing model that calculates the heat production density in the focal region. This model accounts for both the propagation of shear waves and the interference between longitudinal and shear waves. The model was first compared with a finite element approach which solves the Helmholtz equation in soft tissue and the frequency-domain wave equation in bone. To obtain the temperature evolution in the focal region, the heat equation was solved using the heat production density generated by the raytracer as a heat source. Then, we investigated the role of the interaction between shear and longitudinal waves in terms of dissipated power and temperature output. The results of our model were in agreement with the results obtained by solving the Helmholtz equation and the frequency-domain wave equation, both in soft tissue and bone. Our results suggest that it is imperative to include both shear waves and their interference with longitudinal waves in the model when simulating high intensity focused ultrasound propagation in solids. In fact, when modeling HIFU treatments, omitting the interference between shear and longitudinal waves leads to an over-estimation of the temperature increase in the tissues.

Transcriptional control of glucoamylase synthesis in vegetatively growing and sporulating Saccharomyces species.

Three unlinked, homologous genes, STA1, STA2, and STA3, encode the extracellular glycosylated glucoamylase isozymes I, II, and III, respectively, in Saccharomyces species. S. cerevisiae, which is sta0 (absence of functional STA genes in haploids), does carry a glucoamylase gene, delta sta, expressed only during sporulation (W. J. Colonna and P. T. Magee, J. Bacteriol. 134:844-853, 1978; I. Yamashita and S. Fukui, Mol. Cell. Biol. 5:3069-3073, 1985). In this study we examined some of the physiological and genetic factors that affect glucoamylase expression. It was found that STA2 strains grown in synthetic medium produce glucoamylase only in the presence of either Maltrin M365 (a mixture of maltooligosaccharides) or starch. Maximal levels of glucoamylase activity were found in cells grown in rich medium supplemented with glycerol plus ethanol, starch, or Maltrin. When various sugars served as carbon sources they all supported glucoamylase synthesis, although at reduced levels. In any given growth medium glucoamylase isozyme II synthesis was modulated by functionality of the mitochondria. Synthesis of glucoamylase is continuous throughout the growth phases, with maximal secretion taking place in the early stationary phase. In the various regimens, the differences in enzyme accumulation are accounted for by differences in the levels of glucoamylase mRNA. Both glucoamylase mRNA and enzyme activity were drastically and coordinately inhibited in MATa/MAT alpha diploids and by the presence of the regulatory gene STA10. Both effects were partially overcome when the STA2 gene was present on a multicopy plasmid. The STA2 mRNA and glucoamylase were coinduced in sporulating STA2/STA2 diploids. A smaller, coinduced RNA species was also detected by Northern blotting with a STA2 probe. The same mRNA species was detected in sporulating sta0 diploids and is likely to encode the sporulation-specific glucoamylase.

Degrees of malignancy in human primary central nervous system tumors: ornithine decarboxylase levels as better indicators than adenosylmethionine decarboxylase levels.

The levels of activity to ornithine decarboxylase (ODC) and adenosylmethionine decarboxylase (AMD) were measured in various types of primary human tumors of the central nervous system (CNS) and whenever possible were related to the malignancy of the tumor graded according to histopathologic criteria. In astrocytomas ODC levels increased linearly and progressively from infratentorial pilocytic astrocytoma (grade I) to glioblastoma multiforme (grade IV) and corrected well with the degree of histologic malignancy of the tumor. AMD activity levels, however, correlated with tumor malignancy only up to grade III astrocytoma. Medulloblastomas exhibited an unusual dichotomy with regard to the levels of polyamine biosynthetic decarboxylases (PBD): Medulloblastomas had the highest ODC activities of all the CNS tumors tested but had low AMD activities. In tumors of neuroepithelial tissue ODC level increases and, when present, AMD level increases were not due to proliferation of new blood vessels, because CNS hemangioblastomas had very low levels of both PBD activities. No significant differences in either of the PBD levels were observed among the several variants of meningiomas tested, the meningotheliomatous, the transitional, and the fibrous meningiomas. However, atypical forms of meningioma, i.e., those with mitotic figures, whatever the histologic variants, had higher levels of ODC, but not of AMD, than the typical forms, i.e., those without mitotic figures.

Changes in th circadian rhythm of ornithine decarboxylase in rat liver during chemical hepatocarcinogenesis.

The chronobiology of ornithine decarboxylase (ODC) activity in livers was investigated in noninbred Sprague-Dawley rats fed for 5 months with a basal diet or diets with 3-methyl-4'-(dimethylamino)azobenzene (3-Me-DAB) that were oncogenic or caused bile duct hyperplasia (1-naphthylisothiocyanate) (NIT). After a transient disappearance of the ODC circadian rhythm during month 1 on the oncogenic diet, this rhythm in the livers of the rats was reestablished at 60 and 90 days and then disappeared for the next 2 months. When present, the ODC rhythm in 3-Me-DAB-treated rats had the same daily temporal pattern as that of the controls. In the livers of rats treated with NIT, the ODC circadian rhythm was never detectable, even after only 1 month of feeding. Generally, the 3-Me-DAB feeding induced higher levels of ODC activity than did the NIT feeding. The alternation of the appearance and the disappearance of ODC circadian rhythm might reflect changes in the cell population during neoplastic transformation. Te chronobiologic differences in ODC rhythm between the group fed 3-Me-DAB and the group fed NIT could be related to the different types of proliferating cells involved in the hepatic responses to the two drugs.

Delivery of saporin to human B-cell lymphoma using bispecific antibody: targeting via CD22 but not CD19, CD37, or immunoglobulin results in efficient killing.

A panel of bispecific F(ab')2 antibodies (BsAb) have been constructed for delivering the ribosome-inactivating protein saporin to human B cell lymphoma. Each derivative was prepared with specificity for saporin and CD19, CD22, CD37, or immunoglobulin. In vitro studies measuring inhibition of [3H]leucine uptake by cultured Daudi and Raji cells demonstrated that, despite all BsAb capturing saporin on the cell surface, BsAb targeting through CD22 were far more cytotoxic than those functioning via CD19, CD37, or surface immunoglobulin. This exceptional activity of the CD22-specific BsAb appears to derive from its ability to deliver and accumulate saporin inside the target cells. Further studies showed that four CD22-specific BsAb all performed with equal potency and were able to increase saporin toxicity (50% inhibitory concentration) up to 1000-fold, from 2 x 10(-7) M to 2 x 10(-10) M. Pairs of anti-CD22 BsAb which recognized different nonblocking epitopes on the saporin molecule were able to bind saporin more avidly to the target cell and, as a consequence, increased cytotoxicity by at least an additional 10-fold, resulting in 50% inhibitory concentration for protein synthesis of 2 x 10(-11) M. These results suggest that selected combinations of BsAb which bind cooperatively to a toxin and the cell surface may provide an efficient way of delivering toxins to unwanted cells in patients.

Modelling the temperature evolution of bone under high intensity focused ultrasound.

Magnetic resonance-guided high intensity focused ultrasound (MR-HIFU) has been clinically shown to be effective for palliative pain management in patients suffering from skeletal metastasis. The underlying mechanism is supposed to be periosteal denervation caused by ablative temperatures reached through ultrasound heating of the cortex. The challenge is exact temperature control during sonication as MR-based thermometry approaches for bone tissue are currently not available. Thus, in contrast to the MR-HIFU ablation of soft tissue, a thermometry feedback to the HIFU is lacking, and the treatment of bone metastasis is entirely based on temperature information acquired in the soft tissue adjacent to the bone surface. However, heating of the adjacent tissue depends on the exact sonication protocol and requires extensive modelling to estimate the actual temperature of the cortex. Here we develop a computational model to calculate the spatial temperature evolution in bone and the adjacent tissue during sonication. First, a ray-tracing technique is used to compute the heat production in each spatial point serving as a source term for the second part, where the actual temperature is calculated as a function of space and time by solving the Pennes bio-heat equation. Importantly, our model includes shear waves that arise at the bone interface as well as all geometrical considerations of transducer and bone geometry. The model was compared with a theoretical approach based on the far field approximation and an MR-HIFU experiment using a bone phantom. Furthermore, we investigated the contribution of shear waves to the heat production and resulting temperatures in bone. The temperature evolution predicted by our model was in accordance with the far field approximation and agreed well with the experimental data obtained in phantoms. Our model allows the simulation of the HIFU treatments of bone metastasis in patients and can be extended to a planning tool prior to MR-HIFU treatments.

Identification and cloning of human chaperonin 10 homologue.

We have identified a heat-shock-inducible 10 kDa protein in the human hepatoma cell line HepG2. The total RNA extracted from the heat-shocked cells was amplified by reverse transcription PCR (polymerase chain reaction) using 21 5' and 18 3' oligonucleotides of rat cpn10 (chaperonin10) cDNA as primers. Sequencing of the above PCR fragment showed a very high homology between human, bovine and rat cpn10 cDNA. The predicted amino acid sequence revealed a 100% identity with the bovine homologue.

Nucleotide sequence of cDNA coding for dianthin 30, a ribosome inactivating protein from Dianthus caryophyllus.

Rabbit antibodies raised against dianthin 30, a ribosome inactivating protein from carnation (Dianthus caryophyllus) leaves, were used to identify a full length dianthin precursor cDNA clone from a lambda gt11 expression library. N-terminal amino acid sequencing of purified dianthin 30 and dianthin 32 confirmed that the clone encoded dianthin 30. The cDNA was 1153 basepairs in length and encoded a precursor protein of 293 amino acid residues. The first 23 N-terminal amino acids of the precursor represented the signal sequence. The protein contained a carboxy-terminal region which, by analogy with barley lectin, may contain a vacuolar targeting signal.

Mycobacterial Cpn10 promotes recognition of the mammalian homologue by a mycobacterium-specific antiserum.

Self-tolerance, a key feature of the immune system, is still a matter of intense debate. We give here evidence for a peculiar behavior of an antiserum against Mycobacterium tuberculosis chaperonin 10 (m-Cpn10), which could have implications for the mechanism of self-recognition by antibodies against non-self. We show that this antiserum can interact in terms of both inhibition of biological activity and physical association (immunoprecipitation), with the mammalian homologue of m-Cpn10, but only if the bacterial protein is present. Several lines of evidence led us to exclude that the two proteins physically associate to form heterocomplexes: (1) the behavior of the antiserum was not shared by a monoclonal antibody against m-Cpn10; (2) a matrix selective for human Cpn10 (h-Cpn10) did not co-purify m-Cpn10; (3) the distribution pattern in non-denaturing isoelectric focusing of labeled m-Cpn10 was not altered by the presence of the unlabeled h-Cpn10. We conclude therefore that the antiserum against M. tuberculosis Cpn10 also recognizes mammalian Cpn10, with an affinity/avidity regulated by the mycobacterial protein, or by the promotion of hetero-oligomerization. This emergence of self-recognition in the presence of M. tuberculosis Cpn10 could imply a breaking of self-tolerance in situations of infection or vaccination.

Levels of activity of the polyamine biosynthetic decarboxylases as indicators of degree of malignancy of human cutaneous epitheliomas.

The activities of ornithine decarboxylase and of S-adenosyl-L-methionine decarboxylase in human normal epidermis, in basal cell epitheliomas, and in squamous cell carcinomas of human skin have been compared. All 3 types of tissues have characteristic levels of each of these enzymes. The normal epidermis had the lowest levels of both ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase activities. The levels of the polyamine biosynthetic decarboxylases in basal cell epitheliomas were significantly higher than those of normal epidermis, but at the same time significantly lower than those present in squamous cell carcinomas. These results support the conclusion that in epithelial malignant tumors of human skin the extent of the increase in the activities of polyamine biosynthetic decarboxylases is well correlated with the neoplasm's growth rate, which is faster in the squamous cell carcinomas than in the basal cell epitheliomas.

Expression in Escherichia coli, purification and functional activity of recombinant human chaperonin 10.

We have recently reported the cloning of a cDNA coding for a stress inducible human chaperonin 10. The protein was shown to possess 100% identity with the bovine homologue and a single amino acid replacement (glycine to serine at position 52) compared to rat chaperonin 10. Here we report the heterologous expression of human chaperonin 10 in Escherichia coli, its purification and its functional characterization. The recombinant protein was purified to homogeneity as judged by different analytical techniques, and mass spectrometry analysis showed a MW of 10,801 Da in agreement with the predicted sequence. This molecular weight accounts for a protein which is not modified post-translationally. In fact, natural rat chaperonin 10 has been shown to be acetylated at the N-terminus, a feature suggested to be important for targeting and functional activity. Here we show that recombinant human chaperonin 10 is fully active in assisting the chaperonin 60 GroEL in the refolding of denatured yeast enolase, thereby showing that, at least in the present system, post-translational acetylation is not necessary for its activity.

Increased p21ras-specific Guanine-nucleotide exchange causes tumor-formation in nude-mice.

RAS activation state is set by GTPase Activating Proteins (GAP) and Guanine Nucleotide Releasing Proteins (GNRP). The latter were discovered in yeast as the products of the CDC25 and SDC25 genes; two protein families with homologous catalytic domains but different structural organization exist also in mammals. We show that the C-terminal, catalytic domain of a mouse homologue of CDC25 transactivates the ras-responsive fos promoter in vivo. The increased p21ras-specific guanine nucleotide releasing activity of fibroblasts expressing CDC25Mm catalytic domain correlates with tumor induction in nude mice, suggesting that deregulation of these proteins may be important in tumor development.

Production and characterization of monoclonal antibodies against the ribosome inactivating proteins dianthin32 and momochin.

Female BALB/c mice were immunized with either dianthin32 or momochin, type 1 ribosome-inactivating proteins (RIPs) derived from Dianthus charyophyllus and Momordica cochinchinensis, respectively. Five anti-dianthin32 and 6 anti-momochin secreting hybridomas were obtained by somatic fusion of lymphocytes with myeloma cell line NS0. The monoclonal antibodies (MAbs) produced were highly specific, as demonstrated by cross-reactivity assays performed with taxonomically related and unrelated type 1 RIPs, and recognized different epitopes of the antigen. The affinity constant of anti-RIPs MAbs ranged between 10(8) M-1 and 10(10) M-1.

Production and characterization of murine monoclonal antibodies to polypeptide hormones and their fragments.

The diagnostic importance of monoclonal antibodies assays for peptide hormones in biological fluids and for histological peptide localization is rapidly increasing. In our laboratory a general protocol has been developed for immunization and fusion that has been proven very useful, with minor modifications, for generating monoclonal antibodies against insulin, calcitonin, adrenocorticotropin and parathyroid hormone. Our procedure offers the following advantages: 1) it requires a relatively low amount of antigen; 2) it takes only 16-20 days from the first immunization to the time of fusion; 3) it mostly generates IgG producing hybrids; 4) it involves few manipulations of the animals and no i.v. injections. The widely used methods utilized for peptide carrier conjugation, few guidelines for the choice of peptide fragments suitable as immunogens and some applications of antipeptide monoclonal antibodies will be briefly discussed.

Molecular cloning and characterization of the STA2 glucoamylase gene of Saccharomyces diastaticus.

The Saccharomyces diastaticus structural gene STA2, encoding an exracellular glucoamylase (1,4-alpha-D-glucan glycohydrolase, EC 3.2.1.3.), has been cloned by complementation of a stao strain. A genomic library was initially constructed from a STA2 yeast strain in the yeast Escherichia coli shuttle cosmid vector pYCl. The Sta+ complementing function was further delimited to an 8.3 kb BglII fragment whose restriction map was found to be similar to related genomic regions of STA1 and STA3. Fusions of several DNA fragments derived from the 8.3 kb BglII fragment with a truncated E. coli beta-galactosidase gene resulted in two overlapping fragments that could direct the production of large fusion proteins in E. coli. These fusion proteins were immunoprecipitable by anti-glucoamylase II antibodies, confirming that the Sta+ complementing fusion was due to the expression of a gene that coded for a yeast glucoamylase. Measurements of the STA1, STA2 and STA3 RNA transcripts by RNA-DNA hybridization using an internal fragment of the cloned STA2 gene as the probe indicated that a common transcript of 2.5 kb is produced by each of the STA genes. Integrative disruption of the STA2 gene through homologous recombination was achieved by transforming a STA2 yeast strain to Sta- using an in vitro constructed donor DNA fragment that has the URA3 gene inserted within the coding region of the cloned glucoamylase gene. This was confirmed by tetrad analysis of crosses between strains carrying a disrupted STA2 and a functional STA2. Southern blot analysis using BamHI digested genomic DNA from 15 tetrads demonstrated consistent co-segregation and Mendelian inheritance of the Sta- phenotype with STA2::URA3. These data further confirm that the cloned DNA that showed Sta+ complementing activity carries a functional STA2 gene that encodes the yeast extracellular glucoamylase II.

Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus.

In Saccharomyces diastaticus each one of three unlinked genes (STA1, STA2, STA3) encodes a glucoamylase (alpha-1,4 glucanglucohydrolase, EC 3.2.1.3) that allows yeast to grow on starch. The enzyme encoded by the STA2 gene (glucoamylase II) has been purified from culture medium to near homogeneity by ethanol precipitation, Trisacryl M DEAE chromatography, and HPLC gel filtration. Glucoamylase II consists of two identical subunits whose average size is 300 kDa. Under denaturing conditions, the native dimeric enzyme readily dissociates to a monomer. Enzymatic deglycosylation of denatured enzyme gives rise to intermediate, partially glycosylated forms and to a 56-kDa completely deglycosylated protein. Glucoamylase releases glucose units by cleaving alpha-1,4 bonds from the nonreducing end of different oligosaccharides, but has only a barely detectable alpha-1,6 hydrolyzing activity. The pH optimum for the purified enzyme was found to be 5.1. The enzyme has a greater affinity for maltohexaose (Km = 0.98 mM, V/Km = 2.39) than for maltotriose (Km = 2.38, V/Km = 0.68) or maltose (Km = 3.20, V/Km = 0.39). Both polyclonal and monoclonal antibodies have been raised against glucoamylase II. The polyclonal antibodies specifically inhibit yeast glucoamylase II activity in a dose-dependent manner, but are found to immunoblot other yeast glycoproteins as well. This oligosaccharide-specific reaction can be competed out by adding excess mannan without affecting glucoamylase reactivity. The cross-reactivity of the polyclonal antibodies with other amylolytic enzymes correlates well with evolutionary distance. Evidence is presented that monoclonal antibodies specific for either carbohydrate or protein epitopes have been obtained.

Restoration of normal ornithine decarboxylase antizyme activity in rat liver after acute carcinogen treatment.

This study was undertaken to see whether or not the decrease in ornithine decarboxylase antizyme activity caused in rat liver by a hepatocarcinogen could be reversed. Thioacetamide was administered only once, in a single i.p. injection and at a non-carcinogenic, non-necrogenic dose. The activities of both hepatic ornithine decarboxylase and hepatic ornithine decarboxylase antizyme were measured at intervals of hours after the injection of thioacetamide. The hepatic ornithine decarboxylase antizyme in thioacetamide-treated rats was minimal at 40 and 80 h after carcinogen administration. The reversal process requires a very long time, namely 450 h for normal levels of hepatic ornithine decarboxylase antizyme activity to be restored in treated rats. This time is much longer than that required to restore normal ornithine decarboxylase activity in liver of thioacetamide-treated rats. The results of this study, combined with those of the preceding paper, demonstrate that hepatocarcinogens cause a relative inability of rat liver cells to make the ornithine decarboxylase antizyme and that the irreversibility of this defect in cellular control of ornithine decarboxylase activity may be a constant feature in the neoplastic transformation of the rat liver.

Permanent decrease in activity of ornithine decarboxylase antizyme in rat liver during chemical hepatocarcinogenesis.

This study was undertaken to determine whether or not there is failure of cellular control of L-ornithine decarboxylase activity by its antizyme, the only known natural intracellular inhibitor protein for L-ornithine decarboxylase activity, in rat liver during hepatocarcinogenesis induced by 3'-methyl-4-dimethylaminoazobenzene. The formation of hepatic ornithine decarboxylase antizyme was elicited by i.p. injections of putrescine into rats fed a basal diet and rats fed the carcinogenic diet. The activities of both hepatic ornithine decarboxylase and hepatic ornithine decarboxylase antizyme were measured every month for five months, i.e., until hepatoma was fully developed. During azo-dye hepatocarcinogenesis and in fully developed hepatoma the activity of hepatic ornithine decarboxylase antizyme was always significantly lower than in normal resting liver, with minima at the second and the third months. The hepatoma does not synthesize ornithine decarboxylase antizyme more slowly than normal liver, since the difference could be neither abolished nor lessened by lengthening the time available for antizyme formation. Our results strongly suggest that the high intracellular putrescine levels in the livers of rats during 3'-methyl-4-dimethylaminoazobenzene hepatocarcinogenesis do not exert their normal control on hepatic ornithine decarboxylase activity because of a relative inability of these preneoplastic or neoplastic cells to make the ornithine decarboxylase antizyme.