Production of human pro-relaxin H2 in the yeast Pichia pastoris.

BACKGROUND: Initially known as the reproductive hormone, relaxin was shown to possess other therapeutically useful properties that include extracellular matrix remodeling, anti-inflammatory, anti-ischemic and angiogenic effects. All these findings make relaxin a potential drug for diverse medical applications. Its precursor, pro-relaxin, is an 18 kDa protein, that shows activity in in vitro assays. Since extraction of relaxin from animal tissues raises several issues, prokaryotes and eukaryotes were both used as expression systems for recombinant relaxin production. Most productive results were obtained when using Escherichia coli as a host for human relaxin expression. However, in such host, relaxin precipitated in the form of inclusion bodies and, therefore, required several expensive recovery steps as cell lysis, refolding and reduction. RESULTS: To overcome the issues related to prokaryotic expression here we report the production and purification of secreted human pro-relaxin H2 by using the methylotrophic yeast Pichia pastoris as expression host. The methanol inducible promoter AOX1 was used to drive expression of the native and histidine tagged forms of pro-relaxin H2 in dual phase fed-batch experiments on the 22 L scale. Both protein forms presented the correct structure, as determined by mass spectrometry and western blotting analyses, and demonstrated to be biologically active in immune enzymatic assays. The presence of the tag allowed to simplify pro-relaxin purification obtaining higher purity. CONCLUSIONS: This work presents a strategy for microbial production of recombinant human pro-relaxin H2 in Pichia pastoris that allowed the obtainment of biologically active pro-hormone, with a final concentration in the fermentation broth ranging between 10 and 14 mg/L of product, as determined by densitometric analyses.

Active targeting of cancer cells by CD44 binding peptide-functionalized oil core-based nanocapsules.

Selectivity in tumor targeting is one of the major issues in cancer treatment. Therefore, surface functionalization of drug delivery systems with active moieties, able to selectively target tumors, has become a worldwide-recognized strategy. The CD44 receptor is largely used as a biomarker, being overexpressed in several tumors, and consequently as a target thanks to the identification of the CD44 binding peptide. Here we implemented the CD44 binding peptide logic onto an oil core-polymer multilayer shell, taking into account and optimizing all relevant features of drug delivery systems, such as small size (down to 100 nm), narrow size distribution, drug loading capability, antifouling and biodegradability. Besides promoting active targeting, the oil core-based system enables the delivery of natural and synthetic therapeutic compounds. Biological tests, using curcumin as a bioactive compound and fluorescent tag, demonstrated that CD44 binding peptide-functionalized nanocapsules selectively accumulate and internalize in cancer cells, compared to the control, thanks to ligand-receptor binding.

A 13-week oral dose subchronic toxicity study of gardenia yellow containing geniposide in rats.

Gardenia yellow powders A, B and C, containing geniposide at 0.284%, 0.938% and 2.783%, respectively, were administered orally to male and female SD rats as 3% feed admixtures for 13-weeks to evaluate any potential toxicity. Mean geniposide intake values were 5.72, 18.9 and 56.3mg/kg/day in groups receiving these feed admixtures, respectively. All animals survived the duration of the study. The following findings were evident in the gardenia yellow C group: chromatouria, slightly increased plasma total bilirubin, blackish brown discoloration of the kidneys and liver, brown pigments in the proximal tubular epithelium of the kidneys. Slightly increased plasma total bilirubin was considered to be due to interference of metabolite of geniposide with the system of measurement and not to be a toxic effect since there were no related changes in histopathology of the liver or in any blood chemistry parameters. Other findings were limited to pigmentations or discolorations attributable to metabolites of geniposide. No treatment-related effects were evident on body weight, food consumption, ophthalmology, hematology or organ weights in any group. Therefore, it was concluded that 3-month ingestion of the gardenia yellow powder containing geniposide at 2.783% (approximately 60 mg/kg/day as geniposide intake) does not cause any severe toxic effects.

Designing Covalently Linked Heterodimeric Four-Helix Bundles.

De novo design has proven a powerful methodology for understanding protein folding and function, and for mimicking or even bettering the properties of natural proteins. Extensive progress has been made in the design of helical bundles, simple structural motifs that can be nowadays designed with a high degree of precision. Among helical bundles, the four-helix bundle is widespread in nature, and is involved in numerous and fundamental processes. Representative examples are the carboxylate bridged diiron proteins, which perform a variety of different functions, ranging from reversible dioxygen binding to catalysis of dioxygen-dependent reactions, including epoxidation, desaturation, monohydroxylation, and radical formation. The "Due Ferri" (two-irons; DF) family of proteins is the result of a de novo design approach, aimed to reproduce in minimal four-helix bundle models the properties of the more complex natural diiron proteins, and to address how the amino acid sequence modulates their functions. The results so far obtained point out that asymmetric metal environments are essential to reprogram functions, and to achieve the specificity and selectivity of the natural enzymes. Here, we describe a design method that allows constructing asymmetric four-helix bundles through the covalent heterodimerization of two different α-helical harpins. In particular, starting from the homodimeric DF3 structure, we developed a protocol for covalently linking the two α2 monomers by using the Cu(I) catalyzed azide-alkyne cycloaddition. The protocol was then generalized, in order to include the construction of several linkers, in different protein positions. Our method is fast, low cost, and in principle can be applied to any couple of peptides/proteins we desire to link.

Expression of a Soybean (Glycine max [L.] Merr.) Seed Storage Protein Gene in Transgenic Arabidopsis thaliana and Its Response to Nutritional Stress and to Abscisic Acid Mutations.

Among the three subunits of [beta]-conglycinin, the 7S seed storage protein of soybean (Glycine max [L.] Merr.), expression of the [beta] subunit gene is unique. Accumulation of the [beta] subunit is enhanced in sulfate-deficient soybean plants, and its mRNA levels increase when abscisic acid (ABA) is added to the in vitro cotyledon culture medium. Transgenic Arabidopsis thaliana lines carrying a gene encoding the [beta] subunit was constructed and grown under sulfate deficiency. Accumulation of both [beta] subunit mRNA and protein were enhanced in developing A. thaliana seeds. Accumulation of one of the A. thaliana seed storage protein mRNAs was also enhanced by sulfate deficiency, although the response was weaker than that observed for the soybean [beta] subunit mRNA. When the aba1-1 or abi3-1 mutations were crossed into the transgenic A. thaliana line, accumulation of the [beta] subunit was significantly reduced, whereas accumulation of the A. thaliana seed storage protein was not greatly affected. These results indicate that soybean and A. thaliana share a common mechanism for response to sulfate deficiency and to ABA, although the sensitivity is different between the species. The transgenic A. thaliana carrying the [beta] subunit gene of [beta]-conglycinin will be a good system to analyze these responses.

Isolation of an Arabidopsis thaliana Mutant, mto1, That Overaccumulates Soluble Methionine (Temporal and Spatial Patterns of Soluble Methionine Accumulation).

We isolated Arabidopsis thaliana mutants that are resistant to ethionine, a toxic analog of methionine (Met). One of the mutants was analyzed further, and it accumulated 10- to 40-fold more soluble Met than the wild type in the aerial parts during the vegetative growth period. When the mutant plants started to flower, however, the soluble Met content in the rosette region decreased to the wild-type level, whereas that in the inflorescence apex region and in immature fruits was 5- to 8-fold higher than the wild type. These results indicate that the concentration of soluble Met is temporally and spatially regulated and suggest that soluble Met is translocated to sink organs after the onset of reproductive growth. The causal mutation, designated mto1, was a single, nuclear, semidominant mutation and mapped to chromosome 3. Accumulation profiles of soluble amino acids suggested that the mutation affects a later step(s) in the Met biosynthesis pathway. Ethylene production of the mutants was only 40% higher than the wild-type plants, indicating that ethylene production is tightly regulated at a step after Met synthesis. This mutant will be useful in studying the translocation of amino acids, as well as regulation of Met biosynthesis and other metabolic pathways related to Met.

Idiopathic cardiomyopathy: the pathologic roles of arteriolopathy.

Structural alterations and pathologic features of arteriolopathy of the heart were studied in 12 patients with idiopathic cardiomyopathy, five with hypertrophic cardiomyopathy (HOCM), and seven with congestive cardiomyopathy (COCM). The patients, six men and six women, ranged in age from 25 to 66 years. Diagnoses of idiopathic cardiomyopathy were confirmed clinically and at autopsy. Heart weights ranged from 350 to 700 g. Dilatative hypertrophy of both ventricles was observed in four patients, dilatation of both ventricles in six patients, and concentric hypertrophy of the left ventricle in two patients. The ventricular septa ranged in thickness from 7 to 27 mm, and the septal:free wall ratios ranged form 1.0 to 1.8. The lumens of extramural coronary arteries showed less than 30 per cent narrowing. Sections of the heart and all body organs were examined. The myocardia showed microscopic areas of hypertrophy, degeneration and disorganization of muscle cells, interstitial fibrosis, and various features of arteriolopathy. Histometric studies showed that hypertrophy of muscle cells was greater in HOCM than in COCM, that the content of connective tissue in the myocardium was higher in COCM, and that interstitial fibrosis was greater in the epicardial zone than in the endocardial zone in COCM but was greater in the endocardial zone in HOCM. Of the arteriolar changes, those of smooth muscle cells and internal elastic lamina were observed most frequently; no arteriolar narrowing or stenosis was observed in either HOCM or COCM. The correlation between the frequency of arteriolopathy (loss of smooth muscle cells in the media) and myocardial interstitial fibrosis was significant in COCM (P less than 0.05) but not in HOCM. No significant correlations were found between arteriolopathy and age, the duration of symptoms, the cause of death, the heart weight, the thickness of the septum or posterior free wall, the septal:free wall ratio, the presence of disorganized cells in the septum, mural thrombi, the mean muscle cell diameter, or the extent of very hypertrophic cells (P greater than 0.05) in either HOCM or COCM.

Tigloylcholine: a new choline ester toxin from the hypobranchial gland of two species of muricid gastropods (Thais clavigera and Thais bronni).

Crude extracts from hypobranchial glands of two species of muricid gastropods, Thais clavigera and Thais bronni, were highly lethal to mice. Regardless of species, a new choline ester was isolated as the major toxic principle and elucidated to be tigloylcholine, a structural isomer of senecioylcholine widely found in gastropod hypobranchial glands, by 1H- and 13C-NMR as well as FAB-MS. The i.v. LD50 (mouse) of tigloylcholine was estimated to be 0.92 mg/kg.

Convulsant action of systemically administered glutamate and bicuculline methiodide in immature rats.

Developmental changes of transport of drugs into the brain play an important role in ontogenetic neuropharmacology. Two convulsant drugs with different mechanisms of action (glutamate and bicuculline methiodide) were chosen to demonstrate these changes in developing rats. High dose of glutamate (4 g/kg i.p.) induced both minimal (predominantly clonic) and generalized tonic-clonic seizures in rat pups 7, 12, and 18 days old. In contrast, seizures were only exceptionally observed in 25 and 90 days old animals. Bicuculline methiodide was administered in a dose of 2 or 20 mg/kg i.p. The first sign of bicuculline methiodide action in all age groups was represented by automatisms, a symptomatology never seen after bicuculline hydrochloride administration. Minimal seizures were induced in 12-day-old and in a few 18-day-old and adult rats. Generalized seizures were common after the higher dose of bicuculline methiodide in 7- and 12-day-old rat pups, seldom in 18-day-old ones and never seen in 25-day-old and adult animals. Both glutamate and bicuculline methiodide enter the brain in immature rats but the mechanisms are probably different - glutamate is transported actively through the blood-brain barrier whereas no similar system is known for bicuculline methiodide.

Distribution and characterization of enzymes causing starch degradation in rice (Oryza sativa cv. koshihikari).

The thermal dependency and stability of enzymes producing reducing sugar (RS) were examined in bran, the exterior 13% part (outer endosperm), and the remaining inner endosperm of rice grains. RS-producing enzymes in the inner endosperm showed a higher optimum temperature than those in other parts of the rice grain. Diethylaminoethyl-Sephacel chromatography of crude extracts revealed two peaks of RS-producing activity with different optimum temperatures (60 and 37 degrees C) in all three parts. alpha-Glucosidase (EC 3.2.1.20) and alpha-amylase (EC 3.2.1.1) isoform G were thought to be major components of the RS-producing activities with high and low optimum temperatures, respectively. The peak with a high optimum temperature was a more abundant component in the inner endosperm, compared with other parts of the rice grain. Thus, different parts of rice were found to have distinct enzyme sets having different thermal dependency and to be involved in starch degradation to various sugars.

Effect of a novel antibiotic, heliquinomycin, on DNA helicase and cell growth.

Heliquinomycin, a novel microbial product, was found to inhibit a human DNA helicase enzyme isolated from HeLa S3 cells at concentrations of 5 to 10 micrograms/ml. In contrast, adriamycin, etoposide and cisplatin did not inhibit this enzyme at the concentrations tested. Furthermore, the replication and repair of SV40 chromosome were not affected at heliquinomycin concentration of 50 micrograms/ml. The topoisomerase II and I enzymes were inhibited at 30 micrograms/ml and 100 micrograms/ml of heliquinomycin, respectively. Heliquinomycin inhibited the growth of HeLa S3, KB, LS180, K562 and HL60 human tumor cell lines at IC50 values of 0.96 to 2.8 micrograms/ml. In addition, the growth of adriamycin and cisplatin resistant P388 cell lines were inhibited at similar concentrations. Heliquinomycin inhibited both DNA and RNA synthesis in cell culture but did not inhibit protein synthesis. HeLa S3 cells were arrested at the G2/M phase by heliquinomycin. These studies suggest that heliquinomycin is a selective inhibitor of a cellular DNA helicase and in turn, inhibits growth of tumor cell lines.

Heliquinomycin, a new inhibitor of DNA helicase, produced by Streptomyces sp. MJ929-SF2 II. Structure determination of heliquinomycin.

The structure of heliquinomycin which was isolated from the culture broth of Streptomyces sp. MJ929-SF2 was studied by NMR spectroscopies, X-ray crystallographic analysis and degradation experiments. Heliquinomycin is the first member of glycosylated rubromycins and griseorhodins group antibiotics.

Heliquinomycin, a new inhibitor of DNA helicase, produced by Streptomyces sp. MJ929-SF2 I. Taxonomy, production, isolation, physico-chemical properties and biological activities.

Heliquinomycin was isolated as a part of a program designed to find inhibitors of DNA helicase from microbial sources. It was purified from the culture broth of Streptomyces sp. MJ929-SF2 by solvent extraction and serial chromatographies of centrifugal partition chromatography, Sephadex LH-20 and Capcell Pak C18 (HPLC). The isolated red powder was analyzed to have the molecular formula of C33H30O17. It inhibited partially purified DNA helicase from HeLa cell in a non-competitive manner with the inhibition constant (Ki) of 6.8 mM. Heliquinomycin exhibited biological activity against microorganisms including MRSA, and cultured cell lines.

Diperamycin, a new antimicrobial antibiotic produced by Streptomyces griseoaurantiacus MK393-AF2. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities.

Antibacterial antibiotics, diperamycin (1) was produced in the culture broth of Streptomyces griseoaurantiacus MK393-AF2. Various spectroscopic analyses of 1 suggested that 1 belonged to a member of cyclic hexadepsipeptide antibiotic. Antibiotic 1 had potent inhibitory activity against various Gram-positive bacteria including Enterococcus seriolicida and methicillin-resistant Staphylococcus aureus.

Prognostic significance of 24-hour ambulatory electrocardiographic monitoring in patients with dilative cardiomyopathy: a prospective study.

We studied 33 patients with dilative cardiomyopathy to evaluate the predicting factors for sudden death occurring within one year. The information on each of the patients included history, physical examinations, two-dimensional echocardiograms, 24-h ambulatory electrocardiograms, and cardiac catheterization or autopsy. Patients were followed up for one year. Univariate analysis showed maximum number of premature ventricular complexes per hour (PVCs/h) (p = .0012), maximum beats per episode of ventricular tachycardia (VTmax) (p = .0012), and left ventricular end-diastolic pressure (p = .046) to be significant prognostic risk indicators of sudden death within one year. To select the best combination of factors that predict sudden death, multivariate stepwise logistic regression analysis was performed. By this method, only PVCs/h and VTmax were selected as the best combination. Probability of sudden cardiac death within 1 year = 1/(1 + exp[6.65-1.78 (log PVCs/h)-0.71 (VTmax)]). The equation showed 85.7% sensitivity and 69.2% specificity at a probability cutoff point of p = .124, with accuracy of 72.7%. The incidence of sudden death was 80% in patients showing both frequent (greater than 100/h) PVCs and presence of VT (VTmax greater than or equal to 3), and 6% in patients with neither or both. We concluded that PVCs/h and VTmax are independent and significant prognostic factors in patients with dilative cardiomyopathy.

Values of electrocardiography and two-dimensional echocardiography to identify myocardial infarction due to left circumflex and right coronary artery disease.

To investigate the value of the 12-lead ECG and two-dimensional echocardiography (2DE) in the distinction of left circumflex (LCX) from right coronary artery (RCA) disease, we analyzed the location of Q waves, infarct lesions, and coronary artery narrowings in 26 patients with angiographically documented single-vessel disease. Q waves in leads II, III, and aVF were associated with the posterior wall (PW) lesions at the papillary muscle level. Extensive lesions from the PW to the posterior septum (PS) identified RCA disease, while extension to the lateral wall (LW) identified LCX disease. Eleven of 12 patients with high posterior infarction (tall R wave in V1) were found to have extensive LW lesions and 10 of these had coronary narrowings in or proximal to the obtuse marginal branch of LCX. All 6 patients with high posterior infarction and high lateral infarction (Q in I or aVL) had infarct lesions extending from the LW to the anterior wall (AW) and were associated with LCX disease with a large obtuse marginal branch. Of 10 patients with Q waves in V6, the apical LW and PW were involved in 7 and either segment in 3. Nine of these 10 patients had LCX disease. It is concluded that the location of Q waves in inferior infarction could aid in recognizing infarct extension and underlying coronary artery disease.

Cultivation of rat hepatocytes in a medium based on commercially available infusate solutions.

The possibility of using commercially available infusate solutions as a culture medium for hepatocytes was investigated in primary monolayer cultures of rat hepatocytes. The addition of Ca2+ to the infusate medium was necessary for hepatocytes to express their albumin secreting ability. The infusate medium supplemented with hormones (10(-7) M insulin and 10(-7) M dexamethasone) and Ca2+ (72.5 mg/l) allowed hepatocytes to produce albumin of an amount comparable to that produced in Williams' E medium. The activity of released lactate dehydrogenase (LDH) was kept at a low level throughout the cultivation in the infusate medium.

Systemic lupus erythematosus with pulmonary hypertension.

A 50-year-old Japanese female with a long history of Raynaud's phenomenon presented with progressive dyspnea due to pulmonary hypertension. The diagnosis of systemic lupus erythematosus was confirmed by proteinuria, lymphocytopenia, bilateral pleurisy, and a seizure of convulsion which was consistent with neurological manifestations of systemic lupus erythematosus, whereas the antinuclear antibody showed a low titer. Despite improvement in the activity of systemic lupus erythematosus, steroid treatment did not alter the progression of pulmonary hypertension, which increased in severity, eventually resulting in her death. We believe pulmonary hypertension to be an unusual but critical complication of systemic lupus erythematosus.

Intercostal-to-pulmonary arterial anastomosis, complicated by high-output heart failure: case report.

A patient who had high-output heart failure resulting from anastomosis of the intercostal and bronchial arteries to the right pulmonary artery is reported. The patient was a fifty-one year-old man who was admitted to our hospital with left heart failure. A radionuclide angiocardiogram revealed blood flow from the aorta to the right pulmonary artery. A descending aortogram revealed abnormally dilated right intercostal and bronchial arteries, which formed the anastomosis. The right pulmonary artery was visualized via that anastomosis. Tuberculous pleurisy in the patient's history was considered to be the cause of the anastomosis. The therapeutic procedure used was embolization via a catheter for the anastomosis, but most anastomoses could not be embolized. The present report is considered the first on anastomosis of the intercostal artery to the pulmonary artery to the extent that high-output heart failure occurs.

Aortitis syndrome with fatal acute aortic regurgitation due to aortic dilatation and aortic valve perforation--a case report.

This is a case report of a fifteen-year-old female with aortitis syndrome complicated by acute fatal aortic regurgitation due to the aortic valve perforation, as well as to aortic dilatation, which was confirmed by the autopsy. Aortic valve perforation was first recognized as the important cause of acute aortic regurgitation in the aortitis syndrome in this report.