Human non-secretory ribonucleases. II. Structural characterization of theN-glycans of the kidney, liver and spleen enzymes by NMR spectroscopy and electrospray mass spectrometry

The N-glylycans have been removed by peptide-N-glycosidase F(PNGase F) from purified human non-secretory RNases derivedfrom kidney, liver and spleen. The spleen RNase was purifiedby two procedures, one of which did not include the usual acidtreatment step (0.25 M H₂SO₄, 45 min, 4C), to determine ifacid treatment alters the carbohydrate moieties. TheN-glycansof the RNases were fractionated by Bio-Gel P-4 chromatographyand analysed by 600 MHz ¹H-NMR spectroscopy and electrospraymass spectrometry. All four non-secretory RNase preparationscontained the following structures: The relative amounts of the trisaccharide, pentasaccharide andhexasaccharide appeared to vary slightly in the different tissueRNases. The overall results indicate: (i) that acid treatmentduring purification does not alter the N-glycans of non-secretoryRNases; (ii) that the N-glycans from kidney, liver and spleennon-secretory RNases are very similar, if not identical, toone another, but different from the N-glycan structures reportedfor secretory RNase. N-glycans non-secretory RNases     

Biotechniques for air pollution control

This paper gives an overview of present biological techniques for the treatment of off-gases and the techniques that are being developed at the moment. The characteristics, advantages, disadvantages, costs and application area are discussed and compared. Biological off-gas treatment is based on the absorption of volatile contaminants in an aqueous phase or biofilm followed by oxidation by the action of microorganisms. Biofilters, bioscrubbers and biotrickling filters are used for elimination of odour and bioconvertable volatile organic and inorganic compounds and are enjoying increasing popularity. This popularity is a result of the low investment and operational costs involved compared to physico-chemical techniques and the elimination efficiencies that can be obtained. The operational envelop is still extending to higher concentrations and gas flow rates (exceeding 200,000 m³ h^–1) and a broader spectrum of degradable compounds. Research and development on the use of membranes and the addition of activated carbon or a second liquid phase to the biological systems may lead to a more efficient elimination of hydrophobic compounds and buffering of fluctuating loads. Shorter adaptation periods can be obtained by inoculation with specialized microorganisms. Improved design and operation are made possible by the growing insights in the kinetics and microbiology and supported by the development of models describing biological off-gas treatment. In conclusion, biotechniques are efficient and cost effective in treating off-gases with concentrations of biodegradable contaminants up to 1–5 g/m³. They could play a justified and important role in air pollution control in the coming years.Abbreviations VOC volatile organic compound - NO _x gaseous oxides of nitrogen     

Order of bovine DRB3, DYA,and PRL determined by sperm typing

The order and recombination fractions () between the bovine major histocompatibility complex DRB3, DYA, and prolactin (PRL) genes were determined by typing of 254 sperm from a triply heterozygous bull. A recently developed method, primer extension preamplification (PEP), was used to amplify the bovine sperm genome prior to amplification of specific loci by the polymerase chain reaction (PCR). At least 28 copies of the DRB3, PRL, or DYA gene were obtained from 50 cycles of PEP. For sperm typing, alleles of each locus were discriminated by restriction endonuclease cleavage of PCR products and polyacrylamide gel electrophoresis of the restriction fragments. The most likely gene order is PRL-DRB3-DYA, with =0.025 (±0.012) and =0.150 (±0.024), respectively. The odds are 128:1 in favor of this order in comparison with the second most likely order DRB3-PRL-DYA. Our results demonstrate the power of sperm typing in concert with PEP for multilocus gene mapping.     

Cloning and molecular analysis of the galE gene of Neisseria meningitidls and its role in lipopolysaccharide biosynthesis

The galE gene from Haemophilus influenzae was used as a hybridization probe for the galE gene of Neisseria meningitidis Group B, identifying two different homologous loci. Each of the loci was cloned and nucleotide sequence analysis revealed that both loci contained sequences similar to galE. One contained a functional galE gene and mapped to the capsule biosynthetic locus. The second contained only a partial galE-coding sequence, which did not express a functional gene product. A galE mutant meningococcal strain was constructed by transformation with an inactivated galE gene. Analysis of the LPS from the galE mutant strain revealed an apparent reduction in molecular weight and a loss of reactivity with monoclonal antibodies specific for structures known to contain galactose. These results are consistent with an essential role for galE in the incorporation of galactose into meningococcal lipopolysaccharide.     

Using ecological networks to answer questions in global biogeography and ecology

Ecological networks have classically been studied at site and landscape scales, yet recent efforts have been made to collate these data into global repositories. This offers an opportunity to integrate and upscale knowledge about ecological interactions from local to global scales to gain enhanced insights from the mechanistic information provided by these data. By drawing on existing research investigating patterns in ecological interactions at continental to global scales, we show how data on ecological networks, collected at appropriate scales, can be used to generate an improved understanding of many aspects of ecology and biogeography—for example, species distribution modelling, restoration ecology and conservation. We argue that by understanding the patterns in the structure and function of ecological networks across scales, it is possible to enhance our understanding of the natural world.     

Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.     

Biotin carboxylases in mitochondria and the cytosol from skeletal and cardiac muscle as detected by avidin binding

Biotin carboxylases in mammalian cells are regulatory enzymes in lipogenesis and gluconeogenesis. In this study, endogenous biotin in skeletal and cardiac muscle was detected using avidin conjugated with alkaline phosphatase and applied in high concentrations to muscle sections. The avidin binding was subsequently visualized by histochemical demonstration of the alkaline phosphatase activity. All cardiac muscle cells showed high affinity for avidin with only the nuclei and the intercalated discs remaining unstained. In skeletal muscle a diffuse reaction could be detected in the sarcoplasm of the muscle fibres. A granular reaction was noted in the same fibres that showed activity for succinic dehydrogenase. The specificity of the coloured reaction product in the muscle sections was investigated and is suggested to be caused by avidin binding to biotin moieties in mitochondria and the cytosol. Mitochondrial and cytosolic preparations of skeletal muscle were electrophoresed in sodium dodecyl sulphate gels. After blotting and incubation with conjugated avidin, two bands with molecular weights of 75 kDa and 130 kDa respectively were evident in the mitochondrial preparation. It is suggested that the 75-kDa band represents comigration of the biotin-containing subunits of propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The 130-kDa band may represent the biotin-containing pyruvate carboxylase. In the cytosolic preparation a 270-kDa band was stained in blots that had been incubated with conjugated avidin; this band is suggested to represent acetyl-CoA carboxylase. A 190-kDa cytosolic band might be a cleavage product of acetyl-CoA carboxylase. We propose that using alkaline phosphatase-conjugated avidin it is possible to detect the mitochondrial and cytosolic biotin-dependent carboxylases in striated muscle.     

Inhibition of bacteriophage Mu transposition by Mu repressor and Fis

In this paper we show that the Escherichia coli protein Fis has a regulatory function in Mu transposition in the presence of Mu repressor. Fis can lower the transposition frequency of a mini-Mu 3–80-fold, but only if the Mu repressor is expressed simultaneously. In this novel type of regulation of transposition by the concerted action of Fis and repressor, the IAS, the internal activating sequence, is also involved as deletion of this site leads to the loss of the Fis effect. As the IAS contains strong repressor binding sites these are probably the target for the repressor in the observed negative regulation by Fis and repressor. However, the role of Fis and repressor is not only to inactivate the IAS, since a 4bp insertion in the IAS, which changes the spacing of the repressor-binding site, abolishes the enhancing function of the IAS but leaves the repressor-Fis effect intact. A likely target for Fis in this regulation is a strong Fis-binding site, which is located adjacent to the L2 transposase-binding site. However, when this Fis-binding sequence was substituted by a random sequence and Fis no longer showed specific binding to this site, the Fis effect was still observed. Although it is still possible that Fis can function by binding to this non-specific site in a particular complex, it seems more likely that Fis is directly or indirectly involved in determining the level of the repressor.     

A Mycobacterium leprae-specific gene encoding an immunologically recognized 45 kDa protein

By screening a Mycobacterium leprae lambda gt11 expression library with a serum from an Ethiopian lepromatous leprosy (LL) patient a clone was isolated (LL4) belonging to hybridization group III of a panel of previously isolated M. leprae clones. Members of this hybridization group encode a serologically recognized 45 kDa protein. The complete DNA sequences of the partially overlapping clones LL4 and L1 (hybridization group III) are presented and these revealed the presence of an open reading frame (ORF) predicting a protein with a molecular size of 42 448 Da. Southern hybridizations on total genomic DNA of M. Ieprae, Mycobacterium tuberculosis and eight atypical mycobacteria showed that the LL4 DNA fragment is specific for M. Ieprae DNA even under low-stringency conditions. The M. Ieprae specificity of LL4 DNA was further confirmed by the polymerase chain reaction using four different sets of primers. Western blotting analyses showed that the M. Ieprae 45 kDa protein is frequently recognized by antibodies from leprosy patients and that this recognition is specific since no antibodies could be detected in sera of tuberculosis patients. T-cell proliferation assays also demonstrated T-cell recognition by leprosy patients and healthy contacts of the M. Ieprae 45 kDa protein. The specificity of the LL4 DNA region and the 45 kDa antigen that is encoded by hybridization group III could provide unique tools for the development of M. Ieprae-specific immunological and DNA reagents.