A Streptococcus uberis transposon mutant screen reveals a negative role for LiaR homologue in biofilm formation.

AIMS: The environmental pathogen Streptococcus uberis causes intramammary infections in dairy cows. Because biofilm growth might contribute to Strep. uberis mastitis, we conducted a biological screen to identify genes potentially involved in the regulation of biofilm growth. METHODS AND RESULTS: By screening a transposon mutant library of Strep. uberis, we determined that the disruption of 13 genes (including hasA, coaC, clpP, miaA, nox and uidA) led to increased biofilm formation. One of the genes (SUB1382) encoded a homologue of the LiaR response regulator (RR) of the Bacillus subtilis two-component signalling system (TCS). Electrophoretic mobility shift assays revealed that DNA binding by LiaR was greatly enhanced by phosphorylation. Two-dimensional differential in-gel electrophoresis analyses of the liaR mutant and the parental Strep. uberis strain revealed five differentially produced proteins with at least a 1·5-fold change in relative abundance (P < 0·05). CONCLUSIONS: The DNA-binding protein LiaR is a potential regulator of biofilm formation by Strep. uberis. SIGNIFICANCE AND IMPACT OF THE STUDY: Several molecular primary and downstream targets involved in biofilm formation by Strep. uberis were identified. This provides a solid foundation for further studies on the regulation of biofilm formation in this important pathogen.

The composition of the perinatal intestinal microbiota in horse.

The establishment of the intestinal microbiota is critical for the digestive and immune systems. We studied the early development of the rectal microbiota in horse, a hindgut fermenter, from birth until 7 days of age, by qPCR and 16S rRNA gene amplicon sequencing. To evaluate initial sources of the foal microbiota, we characterised dam fecal, vaginal and oral microbiotas. We utilised an amplicon sequence variant (ASV) pipeline to maximise resolution and reproducibility. Stringent ASV filtering based on prevalence and abundance in samples and controls purged contaminants while preserving intestinal taxa. Sampled within 20 minutes after birth, rectal meconium contained small amounts of diverse bacterial DNA, with a profile closer to mare feces than mouth. 24 hours after birth, rectum was colonised by Firmicutes and Proteobacteria, some foals dominated by single genera. At day 7, the rectal genera were still different from adult feces. The mare vaginal microbiota contributed to 24 h and 7 day microbiotas. It contained few lactobacilli, with Corynebacterium, Porphyromonas, Campylobacter and Helcococcus as the most abundant genera. In the oral mucosa, Gemella was extremely abundant. Our observations indicate that bacteria or bacterial components are present in the intestine immediately after birth, but the newborn microbiota changes rapidly.

The human laminin beta 2 chain (S-laminin): structure, expression in fetal tissues and chromosomal assignment of the LAMB2 gene.

The sequence of the human laminin beta 2 chain (previously s-laminin) was derived from cloned cDNAs. The complete translation product has 1798 amino acid residues, including a 32-residue signal peptide. The human chain lacks the tripeptide sequence LRE in domain I which is present in the rat polypeptide chain and has been shown to promote motor neuronal cell adhesion. The human gene (LAMB2) was localized to chromosome 3p21 using somatic cell hybrids and fluorescent in situ hybridization analysis. Northern and in situ hybridization analyses from numerous fetal tissues revealed that the beta 2 chain is generally widely expressed. beta 2, but not beta 1, was shown by in situ hybridization to be expressed in fetal brain and renal glomeruli. In fetal skin, beta 2 was expressed both in epidermal and dermal cells, while beta 1 was expressed only in the dermis. Expression of beta 2 in fetal liver was seen in hepatocytes, while no signals were observed for beta 1. In lung, both beta 1 and beta 2 were expressed in alveoli and bronchial smooth muscle cells, whereas only the beta 2 chain was expressed in bronchial epithelial cells. In striated muscle, however, the beta 1 chain, but not beta 2, was expressed. These results indicate different biological roles for the laminin beta 1 and beta 2 chains.

Primary structure and expression of a novel human laminin alpha 4 chain.

The complete primary structure of a novel human laminin alpha 4 chain was derived from cDNA clones. The translation product contains a 24-residue signal peptide preceding the mature alpha 4 chain of 1792 residues. The domain structure is similar to that of the recently described alpha 3 chain [Ryan, Tizard, Van Devanter and Carter (1994) J. Biol. Chem. 269, 22779-22787]. Northern analysis of RNA from human fetal and adult tissues revealed developmental regulation of expression. In adult, strong expression was observed in heart as well as lung, ovary, small and large intestines, placenta and liver, whereas weak or no expression was detected in skeletal muscle, kidney, pancreas, testis, prostate or brain. In contrast, fetal lung and kidney revealed high expression. In situ hybridization analysis of human fetal and newborn tissues showed expression of the laminin in alpha 4 chain in certain mesenchymal cells in tissues such as smooth muscle and dermis.

Natural history of aortic valve stenosis of varying severity in the elderly.

In a population sample of 501 persons aged 75 to 86 years, Doppler echocardiography uncovered moderate or severe aortic valve stenosis in 8.8% of women and 3.6% of men. Severe aortic valve stenosis predicted a four-fold-age- and sex-adjusted risk of death within 4 years of diagnosis, and mortality tended to be increased also with moderate lesions; mild aortic valve stenosis had a favorable outcome.

The effects of colour adaptation and stimulus size on white perception as a function of eccentricity in man.

We studied how much desaturating complementary colour had to be added to blue, green or red after homochromatic adaptation of short duration in order to perceive white or grey at eccentricities of 0-15 deg in the nasal visual field. The CIE 1931 (x,y) chromaticity coordinates corresponding to achromatic perception were subtracted from the chromaticity coordinates of blue, green and red in order to obtain the threshold differences (dx, dy) in chromaticity coordinates. When the stimulus size was constant at all visual field locations, the absolute values of dx and dy decreased with increasing eccentricity, which means that the desaturation threshold for white or grey perception was higher at the fovea than in the periphery. However, when the stimulus size was M-scaled magnifying its size with increasing eccentricity in inverse proportion to the lowest local sampling density of the human retina (cones and ganglion cells at eccentricities 0-10 and above 10 deg, respectively), dx and dy with blue became independent of visual field location except for the very centre of the fovea. However, M-scaling was unsuccessful with green and red. Although M-scaling with green reduced the change in dx and dy as a function of eccentricity, it had no effect on dx and dy with red colour.

Detection of chromatic deviations from white across the human visual field.

We studied how much blue, green, or red light had to be added to or subtracted from white to obtain veridical hue perception (blue, green, red, or their complementary colours) at various locations in the temporal visual field. The CIE 1931 (x, y) chromaticity coordinates corresponding to a veridical hue perception were subtracted from the chromaticity coordinates of the white (0.35, 0.35) in order to obtain the threshold differences (dx, dy) in chromaticity coordinates. When stimulus size was constant at all visual field locations, dx and dy changed with eccentricity. However, when the stimulus was M-scaled by magnifying its size with increasing eccentricity in inverse proportion to the lowest local sampling density across the human retina (cones and ganglion cells at eccentricities 0-10 and above 10 deg, respectively), dx and dy remained constant at all eccentricities.

Detection of blue under chromatic adaptation: the effects of stimulus size and eccentricity.

We measured thresholds for the perception of blue under chromatic adaptation to white, green, yellow or red at the eccentricities of 0-70 deg in the temporal visual field of four subjects. We used a series of stimulus sizes at each eccentricity, without a prior assumption of any peripheral size-scaling factor. The CIE 1976 UCS (u',v') chromaticity coordinates corresponding to blue perception were subtracted from the chromaticity coordinates of the adaptation field in order to obtain the threshold differences (du',dv') in chromaticity coordinates. Spatial scaling factors for the perception of blue were obtained by non-linear regression (E2 + 5 deg) refers to the eccentricity at which stimulus diameter had to be doubled in order to maintain performance found at the eccentricity of 2.5 deg. E2 for the perception of blue tint varied from 1.2 to 36 deg depending on the state of chromatic adaptation and subject. For the perception of blue tint in yellow three subjects and for the perception of blue tint in red one subject had no spatial scaling factor that would make performance independent of eccentricity. Thus, spatial scaling does not always work.

Colostral growth factors. Possible role in bovine udder epithelial cell regeneration.

Growth of the secretory epithelium during prepartum time, and for a short period after calving, is under hormonal control by estrogen, progesterone and prolactin. The mechanism(s) by which these hormones act is not known but colostrum and milk have been shown to contain different growth promoting substances. In an attempt to unravel these relationships the effect of bovine colostrum on cellular proliferation in vitro have been characterized. Colostral thermostable factors not present in milk nor associated with fat, potently induce the proliferation associated enzyme, ornithine decarboxylase, in fibroblast cell lines. However, mammary epithelial cells appear to proliferate in response to different colostral heat sensitive factor(s) that await further characterization.

Activation-induced cytidine deaminase (AID) is strongly expressed in the fetal bovine ileal Peyer's patch and spleen and is associated with expansion of the primary antibody repertoire in the absence of exogenous antigens.

Due to a limited range of immunoglobulin (Ig) genes, cattle and several other domestic animals rely on postrecombinatorial amplification of the primary repertoire. We report that activation-induced cytidine deaminase (AID) is strongly expressed in the fetal bovine ileal Peyer's patch and spleen but not in fetal bone marrow. The numbers of IGHV (immunoglobulin heavy chain variable) mutations correlate with AID expression. The mutational profile in the fetuses is similar to postnatal and immunized calves, with targeting of complementarity-determining region (CDR) over framework region (FR), preference of replacement over silent mutations in CDRs but not in FRs, and targeting of the AID hotspot motif RGYW/WRCY. Statistical analysis indicates negative selection on FRs and positive selection on CDRs. Our results suggest that AID-mediated somatic hypermutation and selection take place in bovine fetuses, implying a role for AID in the diversification of the primary antibody repertoire in the absence of exogenous antigens.

Effects of colour adaptation and stimulus size on the detection of chromatic deviations from achromatic as a function of eccentricity in man.

By using constant size and M-scaled stimuli (the stimulus size was magnified towards the visual field periphery in inverse proportion to the lowest local sampling density of the human retina) we measured the thresholds for perceiving the complementary colours of blue, green and red (i.e. yellow, purple or blue-green) under chromatic adaptation at the eccentricities of 0-15 degrees in the nasal visual field. The CIE 1931 (x, y) chromaticity coordinates corresponding to complementary hue perception were subtracted from the chromaticity coordinates of achromatic threshold. The difference was found to be constant irrespective of stimulus size and eccentricity. This means that the perception of chromatic deviation from achromatic under chromatic adaptation is independent of stimulus size and eccentricity.

Perception of green and red under chromatic adaptation: the effects of stimulus size and eccentricity.

We measured thresholds for the perception of green and red light added to white, blue, green, yellow, or red adaptation fields presented at eccentricities of 0 to 70 degrees in the temporal visual field of two subjects. A series of stimulus sizes was used at each eccentricity to determine size-scaling factor E2--the eccentricity at which stimulus diameter has to be doubled in order to maintain foveal performance. For green light added to white or yellow, E2 varied from 3.1 to 41.5 degrees. When red light was added to white, yellow, or blue, E2 varied from 0.1 to 11.8 degrees. For red color in green and green color in blue or red there was no spatial scaling factor that would make performance independent of eccentricity. Our results suggest that E2 value for green and red perception depends on the state of chromatic adaptation and also shows marked interindividual differences.

Molecular cloning and tissue-specific expression of a novel murine laminin gamma3 chain.

A novel laminin gamma3 chain was identified from the expressed sequence tag data base at the National Center for Biotechnology Information. A complete cDNAderived peptide sequence reveals a 1592-amino acid-long primary translation product, including a tentative 33-amino acid-long signal peptide. Comparison with the laminin gamma1 chain predicts that the two polypeptides have equal spatial dimensions. In addition, the well conserved domains VI and III(LE4) predict that gamma3 containing laminins are able to integrate to the laminin network and also via nidogen connect to other protein networks in the basement membranes. Combination of Northern analysis and in situ hybridization experiments indicate that expression of the gamma3 chain is highly tissue- and cell-specific, being significantly strong in capillaries and arterioles of kidney as well as in interstitial Leydig cells of testis.

myo-Inositol reverses Li+-induced inhibition of phosphoinositide turnover and ornithine decarboxylase induction during early lymphocyte activation.

One of the earliest detectable responses by T lymphocytes in vitro to various mitogenic ligands is the rapid induction of ornithine decarboxylase (ODC) activity (Scott et al., Eur. J. Immunol. 1985. 15: 783). This early activation is measurable within minutes after the addition of the mitogen. The T cell mitogen concanavalin A also induces rapid breakdown of phosphoinositides in T lymphocytes. The inositol phosphates formed are recycled and used in synthesis of new phosphoinositides. Li+ interrupts this cycle by inhibiting the enzyme inositol-1-phosphatase, which produces the inositol needed for phosphoinositide synthesis. Here we report that when human blood lymphocytes are kept in an inositol-deficient medium for 30 min in the presence of 1 mM Li+, the cells become unable to respond to mitogens by inositide breakdown and rapid induction of ODC activity. Addition of 1 mM myo-inositol restores the inducibility of these responses within a few minutes. These findings represent a novel aspect of the activation of resting lymphocytes and implies a new role for the inositol turnover during signal transduction.

Associations between atrial natriuretic peptides, echocardiographic findings and mortality in an elderly population sample.

OBJECTIVES: To examine associations of N-terminal and C-terminal components of the proatrial natriuretic peptide [ANP (1-98) and ANP (99-126), respectively], with echocardiographic measurements of left ventricular structure and performance and with the function of the aortic and mitral valves in old age. To compare the predictive value of the atrial peptides and echocardiographic data for short-term mortality. DESIGN: A population based survey with 1.5-year mortality follow-up. SETTING: University hospital. SUBJECTS: Three-hundred and thirty-three people aged 78-88 years. MAIN OUTCOME MEASURES: (i) Plasma ANP (1-98) and ANP (99-126); (ii) M-mode and Doppler echocardiographic measurements of left atrial diameter; left ventricular diameters, mass and fractional shortening; peak transmitral velocities; aortic valve area, aortic regurgitation jet length and mitral regurgitant jet area; (iii) total and cardiovascular 1.5-year mortality. RESULTS: ANP (1-98) correlated with left atrial diameter (r = 0.33; P < 0.001), left ventricular mass (r = 0.19; P < 0.001), fractional shortening (r = -0.16; P < 0.01) and the early-to-atrial peak transmitral velocity ratio (r = 0.23; P < 0.001). Also, ANP (1-98) predicted the degree of aortic valve obstruction and the severity of aortic and mitral regurgitation. Associations of ANP (99-126) with echocardiographic data were much weaker. Aortic valve stenosis and ANP (1-98) were independent predictors of age- and sex-adjusted total and cardiovascular mortality at 1.5 years of entry. CONCLUSIONS: Circulating ANP (1-98) correlates with left atrial size, with left ventricular mass and performance and with the severity of aortic and mitral valve dysfunction in persons representing the general elderly population. ANP (1-98) also predicts both total and cardiovascular mortality.

Congestive heart failure in old age: prevalence, mechanisms and 4-year prognosis in the Helsinki Ageing Study.

OBJECTIVE: To examine the prevalence, underlying diseases, abnormalities of left ventricular function and prognosis in congestive heart failure (CHF) of old age. DESIGN: A population-based clinical and echocardiographic study with a 4-year mortality follow-up. SETTING: University hospital. SUBJECTS: Five hundred and one individuals born in 1904. 1909 and 1914 (367 women). MAIN OUTCOME MEASURES: Presence of CHF by clinical and chest radiograph criteria: left ventricular size and systolic function by echocardiography; grade of aortic and mitral valve lesion by Doppler echocardiography; 4-year total and cardiovascular mortality. RESULTS: Forty-one of 501 participants (8.2%) had CHF. Ischaemic heart disease (54%), hypertension (54%) and moderate-to-severe mitral or aortic valve disease (51%) were the main underlying conditions; 90% of patients had one or more of these diseases. Most individuals with CHF (28 of 39 patients, 72%) had normal left ventricular contractions at echocardiography. 'Diastolic CHF', defined as CHF with normal systolic left ventricular function and no regurgitant valve disease, was found in 51% (20 of 39 patients). The relative 4-year risk for death associated with CHF, adjusted for age and sex, was 2.1 (95% confidence interval 1.3-3.4) for all-cause mortality and 4.2 (CI 1.9-5.6) for cardiovascular mortality. CONCLUSIONS: The prevalence of CHF in a population aged 75-86 years is approximately 8%. Ischaemic or valvular heart disease and hypertension are the main underlying conditions. At echocardiography, about 50% of the elderly with CHF have normal left ventricular systolic contractions in the absence of valve disease and an additional 20% have normal systolic function with mitral regurgitation. The presence of CHF doubles the age- and sex-adjusted risk of death from all causes, and quadruples the risk of cardiovascular death during 4-year follow-up.

Phosphotyrosine phosphatases are involved in reversion of T lymphoblastic proliferation.

Resting T lymphocytes can be activated by mitogens or antigens to become T blasts, which revert spontaneously both in vivo and in vitro in extended cultures to secondary, memory T lymphocytes. We have studied the role of phosphotyrosine phosphatases (PTPase) in the reversion of lymphoblasts in extended, phyto-hemagglutinin-stimulated cultures of human T lymphocytes. Membrane-associated PTPase activity is high in resting T cells, but decreased during mitogen-induced blast transformation. When the blasts were reverting to lymphocytes, the PTPase activity increased more than twofold concomitantly with an elevated surface expression of CD45. When T blasts from phytohemagglutinin-activated cultures were kept in the presence of sodium orthovanadate, an inhibitor of PTPase, they maintained their lymphoblastic proliferation and did not revert to resting lymphocytes. This was accompanied by retention of a 48-kDa phosphotyrosine-containing protein. Our data indicate an important role for PTPase in the transition of lymphocytes from an activated to a resting stage.

Primary structure, developmental expression, and immunolocalization of the murine laminin alpha4 chain.

The complete primary structure of the mouse laminin alpha4 chain was derived from cDNA clones. The translation product contains a 24-residue signal peptide preceding the mature alpha4 chain of 1,792 residues. Northern analysis on whole mouse embryos revealed that the expression was weak at day 7, but it later increased and peaked at day 15. In adult tissues the strongest expression was observed in lung and cardiac and skeletal muscles. Weak expression was also seen in other adult tissues such as brain, spleen, liver, kidney, and testis. By in situ hybridization of fetal and newborn tissues, expression of the laminin alpha4 chain was mainly localized to mesenchymal cells. Strong expression was seen in the villi and submucosa of the developing intestine, the mesenchymal stroma surrounding the branching lung epithelia, and the external root sheath of vibrissae follicles, as well as in cardiac and skeletal muscle fibers. In the developing kidney, intense but transient expression was associated with the differentiation of epithelial kidney tubules from the nephrogenic mesenchyme. Immunohistologic staining with affinity-purified IgG localized the laminin alpha4 chain primarily to lung septa, heart, and skeletal muscle, capillaries, and perineurium.