The effect of calf nutrition on the performance of dairy herd replacements.

Sixty-five Holstein-Friesian calves were randomly allocated to one of eight nutritional treatments at 4 days of age. In this factorial design study, the treatments comprised of four levels of milk replacer (MR) mixed in 6 l of water (500, 750, 1000 and 1250 g/day) × two crude protein (CP) concentrations (230 and 270 g CP/kg dry matter (DM)). MR was fed via automatic teat feeders and concentrates were offered via automated dispensers during the pre-wean period. MR and calf starter concentrate intake were recorded until weaning with live weight and body measurements recorded throughout the rearing period until heifers entered the dairy herd at a targeted 24 months of age. There was no effect of MR protein concentration on concentrate or MR intake, and no effect on body size or live weight at any stage of development. During the pre-weaning period, for every 100 g increase in MR allowance, concentrate consumption was reduced by 39 g/day. While, for every 100 g increase in the amount of MR offered, live weight at days 28 and 270 increased by 0.76 and 2.61 kg, respectively (P < 0.05). Increasing MR feed levels increased (P < 0.05) heart girth and body condition score at recordings during the first year of life, but these effects disappeared thereafter. Increasing MR feeding level tended to reduce both age at first observed oestrus and age at first service but no significant effect on age at first calving was observed. Neither MR feeding level nor MR CP content affected post-calving live weight or subsequent milk production. Balance measurements conducted using 44 male calves during the pre-weaning period showed that increasing milk allowance increased energy and nitrogen (N) intake, diet DM digestibility, true N digestibility and the biological value of the dietary protein. Increasing the MR protein content had no significant effect on the apparent digestibility of N or DM.

Effects of feeding level and protein content of milk replacer on the performance of dairy herd replacements.

It has been suggested that United Kingdom recommendations for feeding the neonatal calf (500 g milk replacer (MR)/day; 200-230 g CP/kg milk powder) are inadequate to sustain optimal growth rates in early life. The current study was undertaken with 153 high genetic merit, male and female Holstein-Friesian calves (PIN2000 = £48) born between September and March, with heifers reared and bred to calve at 24 months of age. Calves were allocated to one of four pre-weaning dietary treatments arranged in a 2 MR feeding level (5 v. 10 l/day) × 2 MR protein content (210 v. 270 g CP/kg dry matter (DM)) factorial design. MR was reconstituted at a rate of 120 g/l of water, throughout, and was offered via computerised automated milk feeders. Calves were introduced to pre-weaning diets at 5 days of age and weaned at day 56. During the first 56 days of life, calves offered 10 l MR/day had significantly higher liveweight gains (P < 0.001) than calves fed 5 l MR/day. No significant differences in liveweight gain were found between calves fed 210 g CP/kg DM MR and those fed 270 g CP/kg DM MR from birth to day 56. Differences in live weight and body size due to feeding level disappeared by day 90. Neither MR feeding level nor MR CP content affected age at first service or age at successful service, and with no milk production effects, the results indicate no post-weaning benefits of increased nutrition during the milk-feeding period in dairy heifers.

c-src tyrosine kinase is associated with the asialoglycoprotein receptor in human hepatoma cells.

The asialoglycoprotein (ASGP) receptor is expressed on hepatocytes and liver-derived cell lines and is responsible for the endocytosis of galactose-terminal glycoproteins via the coated pit pathway. Prior data showed that tyrosine kinase activity plays an important role in this endocytic process, though the critical kinase(s) responsible for this effect are unknown. We have detected a 60-kDa protein which coprecipitates with ASGP receptor in detergent-solubilized lysates of HepG2 cells. This protein autophosphorylates and binds radioactive ATP. It comigrates with authentic pp60 c-src and is recognized by a specific anti-src monoclonal antibody. The kinase associated with the ASGP receptor retains the ability to phosphorylate exogenous substrates on tyrosine. In conclusion, the tyrosine kinase c-src associates with the ASGP receptor, a protein of the coated pit pathway of endocytosis.

The development and use of electronic ruminal boluses as a vehicle for bovine identification.

Traceability of meat to the farm of origin is becoming increasingly important to consumers and producers. Traceability systems would be greatly facilitated by electronic animal identification which, for example, would eliminate errors associated with the manual transcription of data. Additionally, the level of production supports provided to the bovine sectors within the European Union demands a high level of security in bovine identification within this region. An electronic rumen bolus provides a safe, tamper-proof method of electronic animal identification. The success of the electronic rumen bolus is facilitated by having an applicator for young calves which delivers the bolus directly to the rumen/reticulum, a bolus with a specific density of 3 g/cm3 or greater and portable or static readers which are capable of reading the passive transponder in the boluses. In Europe, the different methods of electronic identification are compared in a trial organised by the Joint Research Centre (JRC) in Ispra, Italy. The JRC has developed a list of approved boluses used to electronically identify cattle with a unique number on the transponder which cannot be changed. The choice between a rumen bolus and ear tag as a method of electronic animal identification will depend on the degree of security required. If tampering with ear tags is thought to be possible, the rumen bolus would offer a secure alternative method of electronic animal identification.

Effects of dietary supplementation with vitamin E and organic selenium on the oxidative stability of beef.

The effects of supplementation of beef cattle diets with organic Se (0.3 mg/kg) and vitamin E (300 I.U. alpha-tocopheryl acetate/kg feed), for 55 d preceding slaughter, on the antioxidant status and oxidative stability of muscle was examined. Dietary vitamin E supplementation led to an increase (P < 0.05) in plasma, and longissimus muscle alpha-tocopherol levels. In minced longissimus muscle stored in 80% 02:20% CO2, lipid and oxymyoglobin oxidation were lower (P < 0.05) in muscle from vitamin E-supplemented animals compared with unsupplemented animals. Dietary Se supplementation did not significantly affect muscle Se levels, glutathione peroxidase activity, or susceptibility to lipid and oxymyoglobin oxidation in the presence or absence of vitamin E. Covariance analysis indicated that, in addition to muscle alpha-tocopherol, differences in muscle glutathione peroxidase activity, and pH could account for variation in the susceptibility of muscle to lipid and oxymyoglobin oxidation.

T-lymphopoietic capacity of cord blood-derived CD34+ progenitor cells.

Umbilical cord blood contains an abundance of CD34+ hematopoietic progenitor cells and has been used in transplantation as an alternative to adult bone marrow or mobilized peripheral blood. Although efficient myelomonocytic, erythroid, and B lymphoid differentiation has been shown in highly purified cord blood CD34+ mononuclear cells lacking expression of lineage-specific antigens, generation of functional T cells has not been previously documented. Exploiting two recently developed, complementary thymic stromal monolayer systems, we show here that immature hematopoietic progenitor cells (CD34+lineage-) from human and rhesus monkey cord blood mononuclear cells undergo T lymphopoiesis in a manner that recapitulates T cell ontogeny in vivo. After 2 weeks of proliferation, cultures contained myeloid [corrected] cells and discrete populations of CD4+CD8+ (double-positive) immature T lymphocytes, followed after an additional 2 weeks by the appearance of single-positive CD4+CD8- and CD4-CD8+ T cells that coexpressed CD3. The T lymphoid phenotype was confirmed at the transcriptional level by the presence of the lymphoid-restricted genes RAG-2 and T cell receptor. T cells generated from cord blood progenitors in these systems exhibited immunofunction as assessed by alloreactive responses in mixed lymphocyte reactions. These findings were comparable between human and rhesus progenitor cells and closely resemble previous data using adult bone marrow CD34+ cells in these models. Together, these observations demonstrate that cord blood contains abundant lymphoid progenitors that undergo T lymphopoiesis in vitro, suggesting the full multipotentiality of this stem cell source and its validity in investigating T lymphoid differentiation.

Sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease) presenting with skeletal lesions.

A 2-year-old child presenting with bone pain and bone lesions was found to have sinus histiocytosis with massive lymphadenopathy (SHML). SHML presenting with skeletal symptoms is unusual. Management has been conservative and the child has been symptom free for 30 months, although the bone lesions have not completely regressed.

Clenbuterol plasma pharmacokinetics in cattle.

The pharmacokinetics of clenbuterol (Cb) were investigated to determine the extent to which analysis of plasma concentration can be used to discriminate between therapeutic and illicit growth promoting treatment of cattle. Analysis of plasma concentration enabled assessment of the extent of differences in pharmacokinetics between such dosing regimens. Cattle were treated with Cb using either a therapeutic (20 calves, 0.8 microgram Cb kg-1, twice daily in feed for 10 days), or growth promoting (30 calves, 10 micrograms Cb kg-1, twice daily by drench for 20 days) dosing regimens. Blood samples were collected by jugular venepuncture, and plasma Cb concentrations determined by direct enzyme immunoassay. To determine plasma pharmacokinetics, use of a two compartment model was applied to the data and revealed that steady state kinetics were reached after 3 and 5 days following initiation of therapeutic and growth promoting dosing regimens, respectively. Tolerance limit analysis of concentrations during the therapeutic regimen indicated that a plasma Cb concentration greater than 1.63 ng ml-1 would be indicative (p < 0.01) of a growth promoting dose.

Sequence variation in class 1 outer membrane protein in Neisseria meningitidis isolated from patients with meningococcal infection and close household contacts.

The meningococcal porA gene encodes the class 1 outer membrane protein which contains the VR1 and VR2 regions responsible for sero-subtype specificity. However, sequence variations may occur within these regions which are not recognised by the currently available subtype antibodies. Since this "silent" microheterogeneity represents a potential hidden source of information, in the current study we have used porA gene sequence analysis to study strains isolated from cases of meningococcal infection and close household contacts. With each of the three subtypes studied, the index cases could be differentiated from each other by sequence variations within at least one of the VR1, VR2 and SV1 regions. In addition, although isolates from close household contacts showed a high degree of homology significant differences could be detected within some family groups. These data demonstrate that it is possible to use sequence information to differentiate between potential sources of infection which appear identical using conventional serological methods.

Genomic heterogenicity amongst phenotypically similar Legionella micdadei strains.

Nine unrelated Legionella micdadei strains isolated from clinical and environmental samples have been characterized biochemically, serologically using polyclonal and monoclonal antibodies and by macrorestriction analyses using pulsed-field gel electrophoresis. All strains were positive in the Bromocresol purple spot test and grew as blue colonies on dye-containing media. They were positive for catalase, weakly positive for oxidase, and negative for sodium-hippurate hydrolysis, beta-lactamase and gelatinase. None of the strains showed autofluorescence under long-wave ultraviolet light. A panel of six monoclonal antibodies raised against the ATCC strain TATLOCK revealed no significant differences in the surface antigen composition of the L. micdadei strains. None of these monoclonal antibodies reacted with L. maceachernii and L. longbeachae serogroup 2, the only species that cross-react with polyclonal antisera. Each of the nine L. micdadei strains showed individual restriction patterns of the genomic DNA when using both SfiI and NotI restriction enzymes in the pulsed-field gel electrophoresis. Macrorestriction analysis is a valuable tool for studies on the molecular epidemiology of L. micdadei.

Detection of mip gene by PCR for diagnosis of Legionnaires' disease.

The mip gene of Legionella pneumophila was demonstrated by PCR and probing in paired acute-phase and convalescent-phase sera from five patients with Legionnaires' disease but not in the acute-phase sera of 100 patients with pneumonia that showed no serological evidence of Legionella infection. PCR may help in cases presenting diagnostic difficulty.

The asialoglycoprotein receptor is associated with a tyrosine kinase in HepG2 cells.

The asialoglycoprotein (ASGP) receptor undergoes constitutive endocytosis in HepG2 cells, which is regulated by tyrosine kinase activity (Fallon, R. J., Danaher, M., Saylors, R. L., and Saxena, A. (1994) J. Biol. Chem. 269, 11011-11017). In this study we show that the receptor copurifies with a tyrosine kinase activity, as defined by tyrosine phosphorylation of an exogenous substrate (reduced carboxyamidomethylated and maleylated lysozyme). Analysis of cells transfected with one subunit of the ASGP receptor showed that signals in the cytoplasmic domain of the H1 subunit are sufficient for receptor kinase association. In addition, receptor kinase association is not dependent on the single cytoplasmic tyrosine at position 5. Analysis of the components of anti-ASGP receptor immunoprecipitates revealed the presence of a 127-kDa protein (p127), which becomes phosphorylated on tyrosine upon addition of gamma-[32P]ATP and which is capable of binding ATP.p127 was also demonstrated in anti-transferrin receptor immunoprecipitates but not in immunoprecipitates of a resident membrane protein, human HLA. In conclusion, these data demonstrate that the ASGP receptor, a protein that participates in constitutive, rapid endocytosis, is associated with a cellular tyrosine kinase.

Primary meningococcal conjunctivitis and the need for prophylaxis in close contacts.

We present three cases of primary meningococcal conjunctivitis associated with systemic sepsis. The management of such patients should include combined topical and parenteral therapy with appropriate chemoprophylaxis for close contacts of cases.

Expression of an endogenous asialoglycoprotein receptor in a human intestinal epithelial cell line, Caco-2.

We have previously shown that rat asialoglycoprotein receptor expressed in the intestine and liver differ in mRNA size, cell surface distribution, and ratio of compositional protein subunits. In this study, we examined a well characterized intestinal epithelial cell line, Caco-2, as a potential model for studying endogenous receptor in a polarized cell line. Both subunits H1 and H2 of human asialoglycoprotein receptor were detected in Caco-2 cells by Western blots using subunit-specific antisera raised against the hepatic receptor. Antigenic receptor level in fully differentiated Caco-2 cells was approx. 1/3 to 1/2 the level of hepatic HepG2 cells H1 was the dominant subunit in both cell lines. The apparent size of H1 and H2 in Caco-2 cells was not the same as that in HepG2 cells, due to differences in N-linked glycosylation. Consistent with this finding, Northern blot analysis showed that receptor mRNA in the two cell types was of identical size. In pulse-chase experiments H1 was first detected as a 'high-mannose' precursor (40 kDa) in Caco-2 cells that was converted to mature H1 (43 kDa) with a half-life of approx. 60 min. Antigenic levels of H1 and H2 in undifferentiated Caco-2 cells were low, but increased rapidly during cell differentiation, reaching a peak level at 7 days after confluence. Immunocytochemical staining and domain-selective cell surface biotinylation assays showed that the ASGP-R was predominantly localized in the basolateral domain. The receptor in Caco-2 cells was capable of mediating specific uptake and degradation of [125I]asialoorosomucoid. The ligand uptake capacity of the basolateral surface of was approx. 10-fold higher than the apical. These characteristics (H1 subunit and basolateral predominance) of the receptor in Caco-2 cells, resembles the hepatic receptor. We conclude that Caco-2 cells endogenously express in ectopic hepatic-type functional asialoglycoprotein receptor.

Bile acid efflux mediated by the rat liver canalicular bile acid transport/ecto-ATPase protein requires serine 503 phosphorylation and is regulated by tyrosine 488 phosphorylation.

Transfection of cDNA for a hepatocyte canalicular phosphoprotein, the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105, confers bile acid efflux and ecto-ATPase activities on heterologous cells (Sippel, C. J., Suchy, F. J., Ananthanarayanan, M., and Perlmutter D. H. (1993) J. Biol. Chem. 268, 2083-2091). Our previous studies have also indicated that there is a positive correlation between the degree of phosphorylation of this transporter and its bile acid efflux activity. In this study, we introduced site-specific mutations of amino acid residues within a protein kinase C-dependent (T502A, S503A) and a tyrosine kinase-dependent (Y488F) phosphorylation consensus sequence in the cytoplasmic tail of this transporter in order to map the sites that are phosphorylated in vivo and to examine the functional significance of each. COS cells were transfected with mutant and wild type constructs using the pCDM8 expression vector. Metabolic labeling and cell surface labeling showed that the mutant proteins were synthesized and delivered to the cell surface as efficiently as the wild type. Phosphoamino acid analysis using lysates of transfected cells showed that the T502A, S503A mutant contained [32P]phosphotyrosine, the Y488F mutant contained [32P]phosphoserine, and the wild type contained both 32P-labeled amino acids, proving that Ser503 and Tyr488 are the only amino acids phosphorylated in this system under control conditions. Bile acid transport activity was completely abrogated in cells transfected with the T502A, S503A mutant cDNA and was retained but altered in kinetic characteristics in cells transfected with the Y488F mutant cDNA, even though both of these constructs conferred ecto-ATPase activity to the same extent as the wild type cDNA. Taken together, these data show that the bile acid efflux activity of this transporter requires site-specific phosphorylation of Ser503 and is regulated by site-specific phosphorylation of Tyr488.