Characterization of fecal bacterial populations in canines: effects of age, breed and dietary fiber.

The effects of age, breed, and diet on fecal chemistry, enzyme activity, and bacterial populations of dogs were studied. Eighteen dogs from two age groups (young: 2.5 +/- 0.5 years, old: 10.9 +/-0.7 years) and three different breeds (German shepherds, miniature schnauzers, and English setters) were rotated through a Latin Square design such that every dog was fed each of the diets. The test diets included a low-fiber (control) diet and a 10% fiber diet which contained 5% soybean hulls and 5% beet pulp. Inclusion of 10% fiber in the diet decreased the fecal concentration of ammonia, sulfide, and indole. Fiber inclusion significantly increased acetic, propionic, and butyric acid concentrations, while fecal pH decreased by 0.4 units. Fresh fecal samples were plated on selected aerobic and anaerobic culture media and DNA extracted for denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S ribosomal DNA fragments. Plate counts showed significant effects of breed (p < or = 0.05) and age (p < or = 0.01) on selected aerobic and anaerobic bacterial counts, while no significant effect of diet was found. Analysis of PCR-DGGE banding patterns showed there was a tendency for individual dogs to cluster together according to age (young or old dogs) and also for size (large or small dogs). However, the outstanding conclusion obtained from the DGGE analysis of fecal bacterial profiles was that individual dogs had their own characteristic banding pattern which was unique and stable. The relative stability and individuality of the patterns indicates that each individual harbored a characteristic fecal bacterial community which was independent of diet.

Effect of diet on markers of intestinal health in dogs.

The effects of two popular commercial pet foods on faecal markers of microbial metabolism were investigated. Adult dogs were fed a dry, extruded diet and a moist, canned diet in a randomly assigned crossover design. Fresh faecal samples were collected for chemical and enzyme activity assays. Relative to the canned diet, the dry food resulted in decreased faecal pH and faecal indole, sulphide and ammonia concentrations and increased total short-chain fatty acid, acetic and propionic acid concentrations. Faecal beta-glucosidase, beta-glucuronidase, beta-galactosidase and nitroreductase activities were decreased in dogs fed the dry diet. These changes in microbial metabolic activity are consistent with beneficial effects of the dry diet on colonic health.

[Physician-patient relations in family medicine]. / La relation médecin-patient en médecine familiale.

PROBLEM ADDRESSED: In addition to clinical instruction, residents need "people" skills that will enable them to deal with all sorts of patients in difficult clinical situations. We planned a series of 12 seminars to teach these skills to first-year residents. OBJECTIVES OF PROGRAM: To ask relevant questions typical of the patient-centred approach; with empathy and respect, to encourage patients to express their emotions; to become more aware of one's own emotions and reactions in one's work as a physician; to negotiate with patients, taking into account both the patient's agenda and one's own. MAIN COMPONENTS OF PROGRAM: Clinical problems drawn from a list of situations likely to involve difficult contact with patients were used to achieve program objectives. Various teaching methods (discussion, brief presentation, practical demonstration, role play) were used during the four stages of skills development: information, demonstration, practice, and feedback. Various tools were used to test the program. CONCLUSION: Proper planning requires ongoing exploration of objectives, content, teaching methods, and evaluation. This discussion of the teaching principles applied in planning our seminars might inspire others to develop similar programs.

Cloning and sequencing of the gene encoding alpha-acetolactate decarboxylase from Leuconostoc oenos.

The alsD gene encoding alpha-acetolactate decarboxylase was isolated from a genomic library of Leuconostoc oenos, using a screening procedure developed on microtiter plates. The nucleotide sequence of alsD encodes a putative protein of 239 amino acids showing significant similarity with other bacterial alpha-acetolactate decarboxylases. Upstream from alsD lies an open reading frame (alsS) which is highly similar to bacterial genes coding for catabolic alpha-acetolactate synthases. Northern (RNA) blotting analyses indicated the presence of a 2.4-kb dicistronic transcript of alsS and alsD. This suggests that the alsS and alsD genes are organized in a single operon.

In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries.

A method for isolating and cloning mRNA populations from individual cells in living, intact plant tissues is described. The contents of individual cells were aspirated into micropipette tips filled with RNA extraction buffer. The mRNA from these cells was purified by binding to oligo(dT)-linked magnetic beads and amplified on the beads using reverse transcription and PCR. The cell-specific nature of the isolated mRNA was verified by creating cDNA libraries from individual tomato leaf epidermal and guard cell mRNA preparations. In testing the reproducibility of the method, we discovered an inherent limitation of PCR amplification from small amounts of any complex template. This phenomenon, which we have termed the "Monte Carlo" effect, is created by small and random differences in amplification efficiency between individual templates in an amplifying cDNA population. The Monte Carlo effect is dependent upon template concentration: the lower the abundance of any template, the less likely its true abundance will be reflected in the amplified library. Quantitative assessment of the Monte Carlo effect revealed that only rare mRNAs (< or = 0.04% of polyadenylylated mRNA) exhibited significant variation in amplification at the single-cell level. The cDNA cloning approach we describe should be useful for a broad range of cell-specific biological applications.

Simultaneous inhibition of two tomato fruit cell wall hydrolases, pectinmethylesterase and polygalacturonase, with antisense gene constructs.

The cloning and sequencing of two cDNAs representing pectinmethylesterase (PME) RNAs from tomato fruit is reported. The clones were used to construct chimeric antisense PME genes designed for high-level constitutive expression in plants. A full-length antisense PME gene construct, in conjunction with a chimeric antisense polygalacturonase gene, was introduced into tomato plants via Agrobacterium-mediated transformation. Simultaneous and significant reduction in the mRNA and protein levels of these normally highly abundant cell wall hydrolases of the pectin degradation pathway were observed in ripe fruit of transformants. Thus, antisense gene constructs in plants can be used to block multiple steps in metabolic pathways simultaneously.

Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase.

Pseudomonas sp. strain ACP is capable of growth on 1-aminocyclopropane-1-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase and the subsequent conversion of ACC to alpha-ketobutyrate and ammonia (M. Honma, Agric. Biol. Chem. 49:567-571, 1985). The complete amino acid sequence of purified ACC deaminase was determined, and the sequence information was used to clone the ACC deaminase gene from a 6-kb EcoRI fragment of Pseudomonas sp. strain ACP DNA. DNA sequence analysis of an EcoRI-PstI subclone demonstrated an open reading frame (ORF) encoding a polypeptide with a deduced amino acid sequence identical to the protein sequence determined chemically and a predicted molecular mass of 36,674 Da. The ORF also contained an additional 72 bp of upstream sequence not predicted by the amino acid sequence. Escherichia coli minicells containing the 6-kb clone expressed a major polypeptide of the size expected for ACC deaminase which was reactive with ACC deaminase antiserum. Furthermore, a lacZ fusion with the ACC deaminase ORF resulted in the expression of active enzyme in E. coli. ACC is a key intermediate in the biosynthesis of ethylene in plants, and the use of the ACC deaminase gene to manipulate this pathway is discussed.

Regulation of metallocarboxypeptidase inhibitor gene expression in tomato.

Tomato fruits contain a metallocarboxypeptidase inhibitor (MCPI) the sequence of which has already been determined. Here we report the isolation of a tomato cDNA clone that encodes the mature MCPI protein as well as an N-terminal signal peptide for entry into the secretory system and an eight amino acid carboxyterminal extension. MCPI RNA is present at very high levels in anthesis stage ovaries and decreases quite rapidly during fruit development. MCPI protein accumulation reflects the pattern of MCPI RNA accumulation in fruit, consistent with a transcriptional control of MCPI gene activity. In leaves, the levels of MCPI RNA and protein are very low. Wounding of the leaves causes a dramatic (100-fold) increase in steady-state level of MCPI RNA without a concomitant increase in MCPI protein level suggesting a control at the post-transcriptional or translational level of gene expression. Genomic DNA blot hybridization data indicate that MCPI in tomato may be encoded by a single gene.

Research resources in academic radiology.

Research resources in academic radiology were investigated by analyzing the responses to a survey from 72 North American institutions. The questionnaire addressed five general areas: department size, departmental resources committed to research, availability of research training, research quality control, and research productivity. The highest correlates of grant productivity included measures of departmental resources committed to research, for example, space devoted to research, size of research budget, and full-time employee support for engineers, physicists, and chemists. In a regression model, measures of the number of engineers employed by a department, the number of attending staff, and the number of training lectures given by engineers were found to be most highly associated with dollar value of grant support. The average level of research resources available at responding institutions was generally low, despite a seemingly strong desire to do quality research. This is evidenced by a strong sentiment among respondents in favor of research training and quality control of research.

Two cases of Mycobacterium haemophilum infection in Canada.

In 1987, Mycobacterium haemophilum was isolated from cutaneous lesions, a lymph node, and the right eye of a male patient with acquired immunodeficiency syndrome and also from a cervical lymph node in a 3-year-old girl. These two cases are the first M. haemophilum infections to be reported in Canada.

[Not Available]. / La publication: un indice d'intérêt pour la recherche.

Research is just one part of an academic physician's activities, which also include teaching, patient care, and administration. Research productivity, however, is still expected for academic advancement and to enhance family medicine as an academic discipline. The five units of Laval University's Department of Family Medicine were surveyed to determine the effect of family physicians trained in research on the number and type of publications produced by all of their teachers between 1982 and 1987. A total of 55 articles were published. Forty-eight per cent of the teachers had participated in the generation of at least one publication during the study period. The presence of family physicians trained in research proved to be the most significant factor influencing the number and type of articles published in each unit.

Expression of a C3 plant Rubisco SSU gene in regenerated C4 Flaveria plants.

We report the successful transformation, via Agrobacterium tumefaciens infection, and regeneration of two species of the genus Flaveria: F. brownii and F. palmeri. We document the expression of a C3 plant gene, an abundantly expressed ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit gene isolated from petunia, in these C4 plants. The organ-specific expression of this petunia gene in Flaveria brownii is qualitatively identical to its endogenous pattern of expression.

Cell-specific photosynthetic gene expression in maize determined using cell separation techniques and hybridization in situ.

Bundle sheath strands and mesophyll cell extracts have been isolated from maize (Zea mays L.) leaves using a mechanical disruption-filtration technique. Northern blot analysis showed that phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) mRNA accumulates only in mesophyll cells. The mechanisms regulating the cell-specific expression of this gene must, therefore, be at either the level of RNA transcription or that of mRNA turnover. The first successful application of hybridization to mRNA molecules in photosynthetic plant tissue sections is described. Results obtained from this in situ study corroborate our finding that PEPCase mRNA accumulates only in mesophyll cells as well as the previously reported (Link, G, DM Coen, L Bogorad 1978 Cell 15: 725-731) finding that the accumulation of ribulose 1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) large subunit mRNA is restricted to bundle sheath cells. Demonstrating the differential accumulation of PEPCase mRNA and RuBPCase mRNA by utilizing the in situ hybridization technique paves the way for its use as a powerful tool in relating cellular differentiation to gene expression during plant development.

Sanitary study of surface water and of the beach of a water sports and leisure complex.

This report presents the parasitological, bacteriological, mycological and physicochemical data obtained from both surface water and beach sand of a lake used for water sports. These show that the lake is contaminated in both winter and spring by water which overflows from the River Maine, and is self-purified by a mechanism of 'lagunage'. In summer signs of pollution are at their lowest level although use of the complex is at its peak. Conversely, the amoebic flora, which is independent of the usual criteria of pollution, predominates in summer, and serves as a marker for the need for increased surveillance. The sand of the beaches does not appear to show any infectious hazard. Environmental pressure will doubtless change these data over a period of time, and it is planned to monitor this.

Differentiation and development of bundle sheath and mesophyll thylakoids in maize. Thylakoid polypeptide composition, phosphorylation, and organization of photosystem II.

Photosynthetic electron flow, polypeptide pattern, presence of chlorophyll-protein complexes, and phosphorylation of thylakoid polypeptides have been investigated in differentiated mesophyll (M) and bundle sheath (B) thylakoids of the C4 plant Zea mays. The polypeptide pattern of M thylakoids and their photosynthetic electron flow are comparable to those of other green plants. B thylakoids exhibit only photosystem I (PSI) activity, contain only traces of the PSII light harvesting (LHCII) polypeptide, do not bind [3H] diuron, and lack polypeptides of the water-oxidation complex of PSII and the herbicide binding 32-kDa polypeptide, as detected by specific antibodies. However, B thylakoids possess a partially active PSII reaction center, as demonstrated by light-dependent reduction of silicomolybdate with 1,5-diphenylcarbazide (DPC) as an electron donor, and the presence of the PSII reaction center polypeptides of 44-47 kDa. Only one chlorophyll a-protein complex, corresponding to the PSI reaction center-core antenna, was detectable in B thylakoids, as opposed to chlorophyll a and chlorophyll a,b-protein complexes present in M thylakoids. The light-dependent, membrane-bound kinase activity present in M thylakoids could not be detected in B thylakoids which, nevertheless, contain a protein kinase able to phosphorylate casein. A total of 19 differences between the electrophoretic pattern of B and M thylakoid polypeptides were observed. The mRNA coding for the LHCII polypeptide is primarily, if not exclusively, localized in M cells. The development of PSII complex precedes that of PSI during the differentiation of B and M chloroplasts in expanding leaves of light-grown plants and during the greening of dark-grown etiolated seedlings. The differentiation of the maize leaf into cells programmed to form B or M chloroplasts does not require light. In light-grown plants, the differentiation of B and M thylakoids occurred progressively from the base of the leaf and was completed at 4-5 cm from the leaf base.

Photosynthetic gene expression and cellular differentiation in developing maize leaves.

We have exploited the positional gradient of cellular differentiation in Zea mays leaves to study the accumulation of mRNAs encoding subunits of the two CO(2)-fixing enzymes and the major chlorophyll-binding protein. These three proteins are differentially compartmentalized in the two photosynthetically active cell types of the leaf. Previous studies have shown that accumulation of the two carboxylases commences 2 to 4 cm from the base of the leaf (Mayfield SP, WC Taylor Planta 161: 481-486) at a position where bundle sheath and mesophyll cells show morphological evidence of maturation. The light-harvesting chlorophyll a/b protein accumulates progressively from the leaf base, as does its mRNA, in spite of its localization in mesophyll cells after cellular differentiation occurs. While small quantities of phosphoenolpyruvate carboxylase mRNA are detectable in the basal region of the leaf, significant mRNA accumulation is coincident with that of the polypeptide at 4 to 6 cm from the leaf base, the region where bundle sheath and mesophyll cells exhibit fully differentiated morphologies. mRNAs encoding the small and large subunits of ribulose 1,5-bisphosphate carboxylase accumulate to significant levels before bundle sheath cells are fully differentiated and before their polypeptides are detectable. Cytological examination indicates that this is the position at which the maturation of intermediate vascular bundles is first evident. Cytosolically localized small subunit mRNA and chloroplast-localized large subunit mRNA are complexed with polyribosomes at all positions of the leaf.