Utilization of AFLP markers for PCR-based identification of Aspergillus carbonarius and indication of its presence in green coffee samples.

AIMS: The objective of this work was to test whether ochratoxin A (OTA) production of Aspergillus niger and A. carbonarius is linked to a certain genotype and to identify marker sequences with diagnostic value aiding identification of A. carbonarius, a fungus of major concern regarding OTA production in food and food raw materials. METHODS AND RESULTS: Aspergillus niger and A. carbonarius were isolated mainly from Brazilian coffee sources. The ability of isolates to produce OTA was tested by thin layer chromatography (TLC). Strains were genetically characterized by AFLP fingerprinting and compared with each other and with reference strains. Cluster analysis of fingerprints showed clear separation of A. niger from A. carbonarius strains. To obtain marker sequences, AFLP fragments were isolated from silver stained polyacrylamide gels, cloned and sequenced. Sequences obtained were used to develop species- specific PCR primers for the identification of A. carbonarius in pure culture and in artificially and naturally infected samples of green coffee. CONCLUSIONS: No clear correlation between genetic similarity of the strains studied and their potential to produce OTA was found. The PCR assays designed are a useful and specific tool for identification and highly sensitive detection of A. carbonarius. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assays allow specific and sensitive detection and identification of A. carbonarius, a fungus considered to be one of the major causative agents for OTA in coffee and grape-derived products. Assays may provide powerful tools to improve quality control and consumer safety in the food processing industry.

Characterization of the pressure-induced intermediate and unfolded state of red-shifted green fluorescent protein--a static and kinetic FTIR, UV/VIS and fluorescence spectroscopy study.

The green fluorescence proteins (GFP) are widely used as reporters in molecular and cell biology. For their use it in high-pressure microbiology and biotechnology studies, their structural properties, thermodynamic parameters and stability diagrams have to be known. We investigated the pressure stability of the red-shifted green fluorescent protein (rsGFP) using Fourier-transform infrared spectroscopy, fluorescence and UV/Vis spectroscopy. We found that rsGFP does not unfold up to approximately 9kbar at room temperature. Its unique three-dimensional structure is held responsible for the high-pressure stability. At higher temperatures, its secondary structure collapses below 9kbar (e.g. the denaturation pressure at 58 degrees C is 7.8kbar). The analysis of the IR data shows that the pressure-denatured state contains more disordered structures at the expense of a decrease of intramolecular beta-sheets. As indicated by the large volume change of DeltaV degrees (u) approximately -250(+/-50)mlmol(-1) at 58 degrees C, this highly cooperative transition can be interpreted as a collapse of the beta-can structure of rsGFP. For comparison, the temperature-induced unfolding of rsGFP has also been studied. At high temperature (T(m)=78 degrees C), the unfolding resulted in the formation of an aggregated state. Contrary to the pressure-induced unfolding, the temperature-induced unfolding and aggregation of GFP is irreversible. From the FT-IR data, a tentative p,T-stability diagram for the secondary structure collapse of GFP has been obtained. Furthermore, changes in fluorescence and absorptivity were found which are not correlated to the secondary structural changes. The fluorescence and UV/Vis data indicate smaller conformational changes in the chromophore region at much lower pressures ( approximately 4kbar) which are probably accompanied by the penetration of water into the beta-can structure. In order to investigate also the kinetics of this initial step, pressure-jump relaxation experiments were carried out. The partial activation volumes observed indicate that the conformational changes in the chromophore region when passing the transition state are indeed rather small, thus leading to a comparably small volume change of -20 ml mol(-1) only. The use of the chromophore absorption and fluorescence band of rsGFP in using GFP as reporter for gene expression and other microbiological studies under high pressure conditions is thus limited to pressures of about 4kbar, which still exceeds the pressure range relevant for studies in vivo in micro-organisms, including piezophilic bacteria from deep-sea environments.

Characterization of lactobacilli towards their use as probiotic adjuncts in poultry.

AIMS: A total of 112 strains of lactic acid bacteria of duck origin were studied for their use as a probiotic feed supplement. METHODS AND RESULTS: In vitro studies included aggregation, co-aggregation, cell surface hydrophobicity and adhesion activities on poultry crop cells and human Hep2-cells. Additionally, growth with bile acids (chicken bile, ox gall and taurocholic acid) and tolerance to acidic pH were tested. Among all the isolates, two strains (Lactobacillus animalis TMW 1.972 and Lactobacillus salivarius TMW 1.992) were selected for a survival test in poultry. Monitoring and differentiation of these strains was achieved by selective detection as rifampicin and erythromycin double-resistant mutants. After a single feed administration, both micro-organisms were shown to persist in the crop and caecum of ducks for a period of 18 and 22 days, respectively. For identification of Lact. animalis and Lact. salivarius, two specific PCRs targeted against 16S rDNA were developed. CONCLUSIONS: Within the autochtoneous microflora of ducks, two strains of lactobacilli exhibited strong potential as probiotic adjuncts. The results indicate that the natural gut microflora of poultry serves as an excellent source for optimal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: A general strategy for the selection of probiotic strains is presented. The suggested sequence of tests allows identification of the most promising candidates within complex ecosystems or large strain collections with minimal expenditure.

In wounded sugar beet (Beta vulgaris L.) tap-root, hexose accumulation correlates with the induction of a vacuolar invertase isoform.

Wounding of sugar beet tap-root causes an induction of invertase activity, which contributes to post-harvest sucrose losses. In this first comprehensive monitoring of wound-induced invertase mRNAs, proteins, enzyme activities, and tissue hexose concentrations, the VI isoform responsible for wound-induced hexose accumulation in mature tap-root could be identified.

Should all patients with cardiovascular disease receive statin therapy?

With the strong correlation between the development of coronary heart disease and elevated levels of total cholesterol and low-density lipoprotein cholesterol (LDL-C), therapies that significantly lower lipid levels will be widely prescribed. Within the past 15 years, major studies have shown the statins to be very effective in lowering LDL-C levels. Are the effects of statin therapy powerful enough to justify administering one of these drugs to every patient with cardiovascular disease? Among the arguments favoring its general usage are its safety record, its high rate of patient compliance (especially in comparison to alternative therapies such as diet and exercise), and its cost effectiveness. On the other hand, elevated levels of LDL-C are not the only cause of atherosclerosis, so simply lowering the LDL-C level is not the sole answer to reducing the risk of mortality and morbidity from coronary heart disease.

In vitro germ cell models for the detection of fertility impairment.

Pluripotent embryonic carcinoma cells and pluripotent embryonic stem cells established from undifferentiated cells of an early mouse embryo were investigated for induction of proliferation inhibition, sister chromatid exchanges (SCE) and single-strand breaks by treatment with various germ cell mutagens. The comparison of malignant cells with nonmalignant cells showed an increased sensitivity of nonmalignant cells independent of their state of differentiation. Mitomycin C (MMC) inhibited the proliferation of nonmalignant cells at a concentration of 10(-6) M but did not affect growth of the teratocarcinoma cell line P19. There were no differences between the investigated cell lines at a lower MMC concentration. At the concentration of 10(-6) M MMC the sister chromatid exchanges of P19 were enhanced up to 41 SCE per metaphase. Testing of another germ cell mutagen, ethylnitrosourea (ENU), gave similar results: a decreasing generation time of nonmalignant cell lines after treatment with 1 mM ENU and no effect on the teratocarcinoma cells. This concentration also induced a high number of SCE. Single-strand breaks could be produced by exposure to methanmethylsulphonate (MMS). 56.3% of embryonic stem cell DNA was passing through the filter after MMS treatment. In contrast to the embryonic stem cells, only 35.6% of teratocarcinoma DNA was affected.

Human protein tyrosine phosphatase-sigma: alternative splicing and inhibition by bisphosphonates.

Two forms of the transmembrane human protein tyrosine phosphatase (PTP sigma), generated by alternative splicing, were identified by cDNA cloning and Northern hybridization with selective cDNA probes. The larger form of PTP sigma is expressed in various human tissues, human osteosarcoma, and rat tibia. The hPTP sigma cDNA codes for a protein of 1911 amino acid residues and is composed of a cytoplasmic region with two PTP domains and an extracellular region that can be organized into three tandem repeats of immunoglobulin-like domains and eight tandem repeats of fibronectin type III-like domains. In the brain, the major transcript of PTP sigma is an alternatively spliced mRNA, in which the coding region for the fibronectin type III-like domains number four to seven are spliced out, thus coding for a protein of 1502 amino acid residues similar to the rat PTP sigma and rat PTP-NE3. Using in situ hybridization, we assigned hPTP sigma to chromosome 6, arm 6q and band 6q15. The bacterial-expressed hPTP sigma exhibits PTPase activity that was inhibited by orthovanadate (IC50 = 0.02 microM) and by two bisphosphonates used for the treatment of bone diseases, alendronate (ALN) (IC50 = 0.5 microM) and etidronate (IC50 = 0.2 microM). In quiescent calvaria osteoblasts, micromolar concentrations of vanadate, ALN and etidronate stimulate cellular proliferation. These findings show tissue-specific alternative splicing of PTP sigma and suggest that PTPs are putative targets of bisphosphonate action.

NER, a new member of the gene family encoding the human steroid hormone nuclear receptor.

NER, a new member of the steroid hormone nuclear receptor (NR)-encoding gene family, was isolated from a human osteosarcoma SAOS/B10 cell line cDNA library. NER codes for a polypeptide of 461 amino acids which contains the conserved sequences of the DNA-binding and ligand-binding domains of typical steroid hormone NR. It has highest homology with the retinoic acid receptors: 55% at the DNA-binding domain and 38-40% at the ligand-binding domain. A single transcript of 2.3 kb was detected in all cells and tissues tested. Although no ligand was identified for NER-I, its wide distribution may indicate that this novel steroid hormone NR may play a basic role in cell function.

Effect of magnesium sulphate infusion on ex vivo platelet aggregation in swine.

The effect of magnesium sulphate infusion on ex vivo platelet aggregation in 11 female Yorkshire swine was observed using platelet-rich plasma and different agonists (ADP 5 mM; ADP 10 mM and collagen 1 mg/ml). Infusion of 1 g MgSO4 over 1 h produced a significant decrease in platelet aggregability. A dose-dependent effect of different ADP concentrations on platelet aggregation was noticed. Platelet inhibition was most consistent when using ADP 5 mM. We estimate this concentration of agonist as optimal in swine. The swine model is a good choice for investigation of in vivo platelet activation, especially with regard to cardiovascular research. We conclude that there is an inhibitory effect of supplemental magnesium on ex vivo platelet aggregation in swine with initial normomagnesaemia.

Myoelectric prostheses. A long-term follow-up and a study of the use of alternate prostheses.

Forty-four patients who had had a total of forty-seven amputations of an upper extremity and who had had a myoelectric prosthesis for more than two years were evaluated retrospectively for the amount of use of the prosthesis, the use of any other prosthesis, and the demographic factors that might be related to use of the prosthesis. The average duration of follow-up was five years (range, twenty-five months to seventeen years). Forty of the forty-four patients also had a conventional prosthesis. Twenty-two patients (50 per cent) rejected the myoelectric prosthesis completely; thirteen (32 per cent) of the forty patients who also had a conventional prosthesis rejected the conventional prosthesis completely. The patients who used the myoelectric device the least were employed in occupations that required high-demand use of the prosthesis (lifting of more than 4.5 kilograms [ten pounds] or repetitive manual labor) or were receiving or seeking Workers' Compensation, or both.

In vitro approach to fertility research: genotoxicity tests on primordial germ cells and embryonic stem cells.

In vitro screening tests for reproductive toxicology are required by the 7th amendment of the directive 67/548 EEC and the OECD-programme on existing chemicals. Unfortunately, appropriate methods for testing developmental toxicity or impairment of fertility are not at hand. Therefore, we have tried to design test method based on mutagenic effects in germ cells that may be used as a test of fertility impairment as well. Embryonic stem cells (ESC) derived from mouse blastocysts can be kept in culture routinely. Establishment of ESC and improvement of their culture conditions are described and special properties of ESC are discussed in relation to germ cells. Because some properties of germ cells and pluripotent embryonic stem cells (ESC) are found to be comparable, permanent lines of ESC hold promise to be used as an in vitro test of impairment of fertility.

A review: "a comparison of treated and untreated glaucoma suspects" (4).

OBJECTIVES: This study was conducted to evaluate the effect of long-term intraocular pressure reduction with timolol on the incidence of glaucomatous visual field defects and optic nerve cupping in individuals with ocular hypertension and to compare this incidence with that of a similar group on no treatment.

Topically administered norfloxacin compared with topically administered gentamicin for the treatment of external ocular bacterial infections. The Worldwide Norfloxacin Ophthalmic Study Group.

In this double-masked study, we randomly assigned 488 patients with clinical signs of acute bacterial conjunctivitis or blepharitis, or both, to treatment with either norfloxacin ophthalmic solution 0.3% (245) or gentamicin ophthalmic solution 0.3% (243) for one week. Of the patients with positive cultures, 71% (85 of 120) of the norfloxacin-treated patients and 65% (86 of 133) of the gentamicin-treated patients were clinically cured. An additional 25% (30 of 120) of norfloxacin-treated patients and 32% (43 of 133) of gentamicin-treated patients were clinically improved. On the basis of posttreatment cultures, 89% of all cultured bacteria were eradicated (146 of 179 organisms) or suppressed (14 of 179 organisms) after treatment with norfloxacin. The condition of five norfloxacin-treated patients did not clinically improve, compared with the condition of eight gentamicin-treated patients. Both antibiotics had similar efficacy against gram-positive and against gram-negative organisms. One norfloxacin-treated patient and two gentamicin-treated patients withdrew from the study because of local intolerance. Norfloxacin appears to be an effective and relatively safe agent for the treatment of bacterial infections of the eyelids or conjunctiva, or both. In this study, norfloxacin was clinically and microbiologically similar in activity to gentamicin.

[Selenium poisoning in fattening swine]. / Selenintoxikation bei Mastschweinen.

A case of selenium toxicosis was observed in fattening pigs. Intoxication was caused by high levels of selenium in a commercial mineral premix. Instead of the recommended dose of 16 ppm Se, the mineral feed contained selenium at concentrations of 657 and 1059 ppm. The ration in use was found to contain more than 14 ppm selenium. Clinical symptoms were observed 5 to 6 weeks after the pigs began consuming the contaminated feed mixture. Feed intake was markedly reduced and animals showed severe lameness due to separation and necrosis of the hoof wall at the coronary band. Some pigs were reluctant to stand. In some cases alopecia was detected. At histopathological examination one animal with paralysis of the hind limbs revealed a focal bilaterally symmetric poliomyelomalacia in the lumbar segment of the spinal cord. Diagnosis was confirmed by high selenium contents of liver, kidneys and blood. After removing the incriminated feed no further pigs developed signs of intoxication. New horn growth was present and lame animals recovered slowly.

Evaluation of edetate and thiamine for treatment of experimentally induced environmental lead poisoning in cattle.

Twenty mature Holstein cows were randomized into 5 treatment groups. Cows of groups 2 to 5 were given 2 mg of elemental Pb/kg of body weight for 28 days. Clinical signs of plumbism were scored, and blood for Pb, progesterone, and hematologic analyses was collected weekly. Cows also were examined weekly for anomalous ovarian cycles. Starting on study day 28, cows in group 3 were treated once daily with 2 mg of thiamine HCl/kg (IM) for 13 days, cows in group 4 were treated twice daily with 62 mg of Na2,Ca-EDTA/kg (IV) for 4 days, and cows in group 5 were given thiamine (dosage regimen the same as for group 3) plus Na2,Ca-EDTA (dosage regimen the same as for group 4). On study days 96 through 139, cows were slaughtered in a commercial abattoir and samples of blood, skeletal muscles, bones, liver, and kidneys were collected and assayed for Pb concentration. Thiamine was not effective in reducing blood Pb concentration, and treatment with Na2,Ca-EDTA and thiamine plus Na2,Ca-EDTA was effective in reducing the concentration of Pb in blood. However, treatment with thiamine was more effective than treatment with Na2,Ca-EDTA or thiamine plus Na2,Ca-EDTA in inducing remission of clinical signs of plumbism. The concentration of Pb in blood was significantly (P less than 0.05) correlated to the concentration of Pb in liver, kidneys, skeletal muscles, and bones. Significant (P less than 0.05) relationship existed between number of days from Pb exposure to slaughter and concentration of Pb in blood, liver, and skeletal muscles. Exposure to Pb did not significantly alter CBC values.(ABSTRACT TRUNCATED AT 250 WORDS)

Methotrexate-induced leukoencephalopathy is treatable with high-dose folinic acid: a case report and analysis of the literature.

An episode of leukoencephalopathy is reported in a 13-year-old girl who, after standard radiotherapy for a posterior fossa medulloblastoma, received 8 treatments with a protocol containing a 4-hour infusion of 500 mg/m2 methotrexate and 12 mg intrathecal methotrexate. The leukoencephalopathy, documented clinically and by CT and EEG, cleared after 2350 mg of leucovorin (citrovorum factor, folinic acid) was given in addition to the 135 mg given as part of the therapy. A review of the literature suggests that leukoencephalopathy may be prevented by high doses of leucovorin and can be treated by high doses, if lower doses were used initially. When high dose leucovorin was not used, residual neurological damage is not unusual.

Beneficial effects of ascorbic acid on preimplantation mouse embryos after exposure to cyclophosphamide in vivo.

To study mechanisms of embryotoxicity in early pregnancy, we have evaluated the genotoxic and embryolethal effects of ascorbic acid (AA) alone or in combination with cyclophosphamide (CPA). Female mice were exposed on day 3 of pregnancy. Embryotoxicity was investigated at term and genotoxicity shortly after treatment using the chromosomal aberration test and the sister chromatid exchange (SCE) assay as sensitive end points. Additionally, cytotoxic effects were determined by a proliferation test. AA was not found to be embryotoxic, cytotoxic, or genotoxic when given alone. In combination with 10 mg/kg CPA, however, which induced 50% aberrant metaphases, 100% increase SCE frequency, and a strong inhibition of cell proliferation, AA in a dose range of 25-1,600 mg/kg did not change SCE and proliferation, but reduced the rate of aberrant metaphases significantly. This anticlastogenic effect was clearly correlated to a beneficial effect on embryolethality at term when 200 mg/kg ascorbic acid was given in combination with 40 mg/kg CPA. The results suggest that during early pregnancy AA is not genotoxic even at so-called megadoses doses, but it seems to protect early embryos against damage induced by genotoxic agents like CPA.

Proteinase-sensitive regions in the heavy chain of low molecular weight kininogen map to the inter-domain junctions.

Low molecular weight kininogen from human plasma was subjected to limited proteolysis with trypsin, chymotrypsin, elastase, and bromelain, and the resulting fragments of 20,000 or 40,000 Da were isolated. Amino-terminal sequence analysis of the fragments disclosed for the various proteinases eight independent cleavage sites distinct from the typical kallikrein cleavage sites flanking the kinin region. All the identified cleavage sites cluster in two stretches of 11-12 residues of the kininogen heavy chain. These short segments represent the primary attack sites for proteinases ("proteinase-sensitive regions") in the heavy chain portion of human low molecular weight kininogen. The amino acid sequences of the two proteinase-sensitive regions are mutually homologous; they are further characterized by the presence of a single copy each of the consensus tetrapeptide Cys-X-Gly-Cys known to form a narrow disulfide loop (Kellermann, J., Thelen, C., Lottspeich, F., Henschen, A., Vogel, R., and Müller-Esterl, W. (1987) Biochem. J. 247, 15-21). The proteinase-sensitive regions are located at the junctions of the three cystatin-like domains constituting the kininogen heavy chain. Proteolytic cleavage at the sensitive regions dissects the kininogen heavy chain and releases single domains of 20,000 Da and combined domains of 40,000 Da which can function as cysteine proteinase inhibitors. The presence of kininogen heavy chain domains in plasma samples under pathologic conditions suggests that cleavage of the proteinase-sensitive regions might also occur in vivo.

Induction of sister-chromatid exchanges and exchange aberrations by UV light and quinacrine mustard in relation to chiasma formation in a standard line and two oligochiasmatic mutants of Vicia faba L.

The frequencies of sister-chromatid exchanges (SCE) and exchange aberrations (EA) in root tip cells were compared to chiasma formation in pollen mother cells of a standard line and two oligochiasmatic lines of Vicia faba L. SCE and EA were induced by UV light and quinacrine mustard. Between the lines SCE frequencies were not different. The background level of SCE was doubled after UV irradiation and 4 times higher after exposure to quinacrine mustard in all Vicia lines analysed. However, the induced frequencies of EA were found to be different under the same treatment conditions for the standard line and the oligochiasmatic mutants. Between the frequencies of induced EA and the frequencies of chiasmata a correlation could be shown. The relationship between the formation of SCE and EA due to the reduced ability of meiotic recombination in the mutants of Vicia faba is discussed.