Effect of the storage period of paraffin sections on the detection of mRNAs by in situ hybridization.

In this study we evaluated whether storing non-deparaffinized sections can affect the detection of specific mRNAs by radioactive in situ hybridization (ISH). Using a standard ISH protocol, we hybridized serial sections of paraffin blocks stored for different periods of time with (33)P-labeled riboprobes specific for rat Type III collagen and matrix metalloproteinase-2 (MMP-2). Signal intensities were evaluated using a phosphorimager and by blinded microscopic examination. For slides hybridized with the Type III collagen riboprobe, signal intensities measured with the phosphorimager or evaluated by microscopic examination were negatively correlated with the storage period of the sections. For slides hybridized with the MMP-2 riboprobe, differences in signal intensity could be detected, albeit inconsistently, with the phosphorimager, although microscopic examination consistently indicated stronger signals in freshly sectioned slides compared to slides stored for 2 weeks or more. We concluded that it was preferable to use recently prepared sections for trying to locate mRNAs in paraffin-embedded tissues by ISH. In addition, our results suggest that quantifying signal intensity using a phosphorimager is feasible for abundant mRNAs or when large differences in expression are anticipated.(J Histochem Cytochem 49:927-928, 2001)     

Biotransformation of nitriles by rhodococci

Rhodococci have been shown to be capable of a very wide range of biotransformations. Of these, the conversion of nitriles into amides or carboxylic acids has been studied in great detail because of the biotechnological potential of such activities. Initial investigations used relatively simple aliphatic nitriles. These studies were quickly followed by the examination of the regio- and stereoselective properties of the enzymes involved, which has revealed the potential synthetic utility of rhodococcal nitrile biotransforming enzymes. Physiological studies on rhodococci have shown the importance of growth medium design and bioreactor operation for the maximal conversion of nitriles. This in turn has resulted in some truly remarkable biotransformation activities being obtained, which have been successfully exploited for commercial organic syntheses (e.g. acrylamide production from acrylonitrile).The two main types of enzyme involved in nitrile biotransformations by rhodococci are nitrile hydratases (amide synthesis) and nitrilases (carboxylic acid synthesis with no amide intermediate released). It is becoming clear that many rhodococci contain both activities and multiple forms of each enzyme, often induced in a complex way by nitrogen containing molecules. The genes for many nitrile-hydrolysing enzymes have been identified and sequenced. The crystal structure of one nitrile hydratase is now available and has revealed many interesting aspects of the enzyme structure in relationship to its catalytic activity and substrate selectivity.     

Sheep-goat hybrid karyotypes

A “Spanish” goat female was bred to a Barbados sheep ram and gave birth to twin offspring, one of each sex. The F_l female was backcrossed to a Barbados ram and also gave birth to twins, one of each sex. The F_l female has goat-like characters and the coloration of the Barbados. Although the female Barbados is polled, the F_l female hybrid has goat-like horns. The F_l male was lost before assessments could be made. The F_l hybrid possesses a diploid chromosome number of 57 and a karyotype containing 3 unpaired metacentrics, 5 unpaired and 23 pairs of acrocentric autosomes. The male and female backcrosses have Barbados characters and coloration; however, the female has goat-like horns. The male has Barbados or sheep-like horns. The backcrosses have diploid chromosome numbers of 55 and karyotypes containing 1 unpaired and 2 pairs of metacentrics and 2 unpaired and 23 pairs of acrocentric autosomes. The sex chromosomes consist of a large acrocentric X and a small bi-armed Y in the male and 2 large acrocentric Xs in the female.     

Regulation of the thdF gene, which is involved in thiophene oxidation by Escherichia coli K-12

The thdF gene of Escherichia coli encodes a 48 kD protein which is involved in the oxidation of derivatives of the sulphur-containing heterocycle thiophene and which appears to be induced during stationary phase. In this work the upstream regulatory region of the thdF gene was isolated by polymerase chain reaction and inserted in front of the lacZ structural gene. Examination of the resulting thdF-lacZ operon fusions showed that expression of the thdF gene increased as E. coli entered the stationary phase. However, the expression of thdF was not dependent on RpoS (KatF), the stationary phase sigma factor. The thdF gene was subject to substantial catabolite repression by glucose and its expression was also greatly decreased in the absence of oxygen. The thdF-lacZ fusions were not significantly affected by elevated temperature or medium of high osmolarity, nor by mutations in thdA, fadR, arcA, arcB, or fnr. Both multicopy, plasmid-borne fusions and single-copy fusions gave similar results in all of the above cases except that the plasmid-borne fusions still showed substantial expression in the absence of oxygen. The heterocyclic compounds thiophene carboxylic acid, furan carboxylic acid and proline increased expression of the thdF gene by 2- to 3-fold, but only during the stationary phase. Tryptophan, indole, and several indole derivatives had no effect.     

Cytogenetic analysis of diploidy in cloned bovine embryos using an improved air-dry karyotyping method

Of the few published studies on the cytogenetic analyses of bovine nuclear transferred (NT) embryos, results differ between air-dry and fluorescent in situ hybridization (FISH) procedures. A modified air-dry procedure is reported in this study that provides more metaphase plates for analysis. Day 5 and Day 7 bovine NT embryos were cultured in colcemid-containing CR1aa for 10-12 or 16-18 h, then treated in hypotonic sodium citrate for 3-5 min. The standard procedure of 5h in colcemid and 15-20 min in hypotonic solution was the control. A much higher (P<0.01) percent of mitotic nuclei was observed in the experimental groups. The 33 and 41% mitotic nuclei were obtained from 10 to 12 h and 16 to 18 h-colcemid-treated Day 5 embryos, respectively, which was higher (P<0.001) than the control (15%). The mitotic nuclei in Day 7 NT embryos were 24% in 10-12 h- and 28% in 16-18 h-colcemid-treated groups, which also was higher (P<0.05) than the control (10%). The majority of analyzable embryos were diploid. Analyses of mixoploid embryos showed on average that 70% of the cells were diploid. Day 5 mixoploid embryos contained numerically higher polyploid cells than Day 7 embryos, although statistically there were no differences. We concluded that the modified air-dry method provided a larger source of mitotic nuclei for chromosome analyses of cloned bovine embryos.     

Synthesis and receptor binding affinity of new selective GluR5 ligands

Two hybrid analogues of the kainic acid receptor agonists, 2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA) and (2S,4R)-4-methylglutamic acid ((2S,4R)-4-Me-Glu), were designed, synthesized, and characterized in radioligand binding assays using cloned ionotropic and metabotropic glutamic acid receptors. The (S)-enantiomers of E-4-(2,2-dimethylpropylidene)glutamic acid ((S)-1) and E-4-(3,3-dimethylbutylidene)glutamic acid ((S)-2) were shown to be selective and high affinity GluR5 ligands, with Ki values of 0.024 and 0.39 microM, respectively, compared to Ki values at GluR2 of 3.0 and 2.0 microM. respectively. Their affinities in the [3H]AMPA binding assay on native cortical receptors were shown to correlate with their GluR2 affinity rather than their GluR5 affinity. No affinity for GluR6 was detected (IC50 > 100 microM).     

A novel mechanism for localizing membrane proteins to yeast trans-Golgi network requires function of synaptojanin-like protein

Localization of resident membrane proteins to the yeast trans-Golgi network (TGN) involves both their retrieval from a prevacuolar/endosomal compartment (PVC) and a "slow delivery" mechanism that inhibits their TGN-to-PVC transport. A screen for genes required for the slow delivery mechanism uncovered INP53, a gene encoding a phosphoinositide phosphatase. A retrieval-defective model TGN protein, A(F-->A)-ALP, was transported to the vacuole in inp53 mutants approximately threefold faster than in wild type. Inp53p appears to function in a process distinct from PVC retrieval because combining inp53 with mutations that block retrieval resulted in a much stronger phenotype than either mutation alone. In vps27 strains defective for both anterograde and retrograde transport out of the PVC, a loss of Inp53p function markedly accelerated the rate of transport of TGN residents A-ALP and Kex2p into the PVC. Inp53p function is cargo specific because a loss of Inp53p function had no effect on the rate of Vps10p transport to the PVC in vps27 cells. The rate of early secretory pathway transport appeared to be unaffected in inp53 mutants. Cell fractionation experiments suggested that Inp53p associates with Golgi or endosomal membranes. Taken together, these results suggest that a phosphoinositide signaling event regulates TGN-to-PVC transport of select cargo proteins.     

Expression of a modified Neocallimastix patriciarum xylanase in Butyrivibrio fibrisolvens digests more fibre but cannot effectively compete with highly fibrolytic bacteria in the rumen

AIMS: This study investigated the competitive abilities of two Neocallimastix patriciarum-derived xylanases constructs in Butyrivibrio fibrisolvens H17c (xynA and pUMSX) and their ability to compete in vivo. METHODS AND RESULTS: The digestibility of neutral detergent fibre (NDF) increased during co-culture of xynA or pUMSX and weakly cellulolytic, but not with highly cellulolytic, ruminococci. Competition studies among xynA, pUMSX and cellulolytic consortia demonstrated that xynA was the fittest. XynA did not persist at high levels in the rumen and was undetectable after 22 days. CONCLUSION: The construction of recombinant xylanolytic B. fibrisolvens does improve the digestibility of fibre above that of the native, but digestibility is still less than that of the most potent fibre digesters such as ruminococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Fibre digestion may be improved by genetic manipulation of ruminal bacteria but ecological parameters, such as persistence in vivo and the niche of the organism, must be taken into account.