To test the hypothesis that propionate can reduce hepatic capacity to detoxify ammonia, effects of the inclusion of propionate in intraruminal infusions of urea on the concentrations of ammonia, other metabolites and insulin in peripheral blood were investigated in two experiments with non-lactating dairy cows. Both experiments were of a 4 × 4 Latin square design with four animals, four treatments and four experimental periods of 7 d; feed was given in two equal meals each day, all intraruminal infusions were given for 1 h at the time of the morning feed, and propionic acid was partly neutralized with NaOH. In Expt 1, the treatments were a basal diet of pelleted lucerne and chopped hay alone or with the following infusions (g/d): urea 80, propionic acid 350, urea 80 plus propionic acid 350. The inclusion of propionate in the urea infusion markedly increased (P < 0·001) the concentration of ammonia in plasma compared with infusion of urea alone. Moreover, the inclusion of urea with the propionate infusion abolished (P < 0·01) the increase in blood insulin level seen with the infusion of propionate alone. In Expt 2, less severe treatments were imposed, the aim being to reproduce metabolic loads of propionate and ammonia that might be expected from a diet of high-protein grass silage rich in lactic acid. The treatments were a basal diet of grass silage alone or with the following infusions (g/d): NaCl 145, NaCl 145 plus urea 50, propionic acid 200, urea 50 plus propionic acid 200. Effects were less pronounced than in Expt 1 but, in the period immediately after infusion, similar effects were seen. It is concluded that propionate–ammonia interactions may have potentially important effects on milk production especially for diets with high proportions of grass silage containing high levels of protein and lactic acid.

Experiments were designed to test the hypothesis that milk ejection rate decreases during milking, thereby causing insufficient refill of the cistern and decreasing milk flow rate towards the end of milking. In a first series of experiments machine milking of the left front quarters of 11 cows was interrupted for 2 min after removal of 25, 50 or 75% of expected total milk yield, while milking was continued in the other three quarters. Milk flow was recorded during machine-on times. Intramammary pressure (IMP) was recorded during premilking teat stimulation and during interruption of milking. IMP during interruption of milking decreased with decreasing amounts of milk remaining in the udder. The IMP did not change during these interruptions when they occurred after 25 and 50% of expected total milk yield was removed. Thus, the ejection rate could keep up with the milk flow or removal rate. However, IMP increased during interruption of milking following removal of 75% of total yield, although significantly so only in cows with a high milk flow rate. Obviously, more milk was removed than was transported to the cisternal cavity. It is likely that a reduced ejection rate caused the decreased milk flow rate. In a second series of experiments the pulsation ratio of the milking machine was changed from the usual 70:30 to 50:50 with the aim of reducing the milk flow rate and thus adapting to the ejection rate at the end of milking. The changed pulsation ratio caused a reduced peak flow rate and a prolonged high milk flow period, whereas the main flow rate did not change significantly.

Milk yield and milking time were measured on one occasion for several daughters (n = 6–44) from 16 bulls at morning milkings. Blood from the bulls was collected, and platelets and mononuclear leucocytes were isolated. The α2-adrenoceptors on platelet membranes were identified by binding of [3H]rauwolscine, whereas for the determination of β2-adrenoceptors on intact mononuclear leucocytes, [3H]CGP–12177 was used. It was found that mean milk flow rate was highly correlated (P < 0·001) with the α2-adrenoceptor densities on blood platelets. No correlation was found with the β2-adrenoceptors on mononuclear leucocytes. It is concluded that estimation of the α2-adrenoceptors on blood platelets from bulls could eventually be used to investigate milking characteristics of cows, and might be useful in the future as a marker in genetic studies.

The effects of culture supernatants conditioned by the growth of Staphylococcus aureus M60 on in vitro growth and functional properties of bovine mammary myoepithelial cells were examined. Myoepithelial cell proliferation was reduced by Staph, aurexis M60 culture supernatants. Exposure of myoepithelial cells to culture supernatants of isogeneic mutants of Staph, aureus M60 that produced either α or β toxins reduced proliferation, but to a lesser extent than supernatants from the wild type strain. Thus, α and β toxins may play some role in affecting myoepithelial cell proliferation. Of the cells tested, 42% contracted following addition of oxytocin (10–7 M) in the culture medium. Treatment of myoepithelial cells for 15 min with Staph, aureus M60 supernatants, prior to addition of oxytocin in the culture medium, increased the number of cells that contracted to 92%. Exposure of cells for 3 h to the same supernatant, prior to addition of oxytocin in the culture medium, abolished oxytocin responsiveness, had no effect on immunolocalization of actin and vimentin, but affected the localization of α-actinin within myoepithelial cells. Treatment of myoepithelial cells for 3 h with a combination of purified staphylococcal proteinases XVI and XVII-B abolished oxytocin responsiveness and mimicked the effect of the Staph, aureus culture supernatant. We conclude that Staph, aureus M60 culture supernatant affected proliferation and functional properties of myoepithelial cells.

Peptides that inhibit calmodulin-dependent cyclic nucleotide phosphodiesterase were isolated from a pepsin digest of α-casein. Analysis of these peptides showed that they corresponded to the αs2-casein sequences 164–179 (Leu–Lys–Lys–Ile–Ser–Gln–Arg–Tyr–Gln–Lys–Phe–Ala–Leu–Pro–Gln–Tyr), 183–206 (Val–Tyr–Gln–His–Gln–Lys–Ala–Met–Lys–Pro–Trp–Ile–Gln–Pro–Lys–Thr–Lys–Val–Ile–Pro–Tyr–Val–Arg–Tyr) and 183–207 (C-terminus, Val–Tyr–Gln–His–Gln–Lys–Ala–Met–Lys–Pro–Trp–Ile–Gln–Pro–Lys–Thr–Lys–Val–Ile–Pro–Tyr–Val–Arg–Tyr–Leu). These peptides inhibited calmodulin-induced cyclic nucleotide phosphodiesterase activity over the range 1–50 μM without affecting the basal enzyme activity. These results demonstrated that the affinities of these peptides for calmodulin are comparable to the affinities of certain endogenous neurohormones and proteins that interact with calmodulin.

Heat coagulation time-pH curves at 140°C were obtained for 43 blended skim milk samples from Holstein cows to determine the effects of genetic variants of κ-casein and β-lactoglobulin on milk heat stability. The blended milk samples were similar in terms of protein content and milk salts, but were genotypically different for κ-casein (AA, AB) and β-lactoglobulin (AA, AB, BB). Type A curves were obtained for all milks. Maximum heat stability was affected by the κ-casein genotype (AB > AA, P < 0·01) but the influence of the β-lactoglobulin genotype was only significant when the κ-casein AA genotype was present (β-lactoglobulin AA > BB, P < 0·0001). Minimum heat stability was significantly higher (P < 0·0001) for milk genotyped κ-casein AB:β-lactoglobulin BB. The effects of milk genotyped κ-casein BB on maximum and minimum heat stability were determined by analysing individual milks: κ-casein BB:β-lactoglobulin AB (n=8) and reconstituted milks: κ-casein BB:β-lactoglobulin AA, AB and BB (n = 17). Type B curves were obtained on three occasions for individual κ-casein BB:β-lactoglobulin AB milk and on five occasions in the case of reconstituted milks with κ-casein BB:β-lactoglobulin AA, AB and BB. This suggests a relationship between the type B curve and the κ-casein B genetic variant. Comparison of the mean values of heat stability at the pH of maximum heat stability of each individual and reconstituted milk genotype suggested that the best genotype for κ-casein in terms of heat stability was BB.

A total of 169 Lactobacillus strains from 12 species (Lb. acidophilus, Lb. brevis, Lb. buchneri, Lb. casei, Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. delbrueckii, Lb. delbrueckii subsp. lactis, Lb. fermentum, Lb. helveticus, Lb. paracasei subsp. paracasei, Lb. plantarum and Lb. rhamnosus), isolated from raw milk and various milk products, and 9 Lactococcus lactis strains were evaluated for peptidase activities with five chromogenic substrates and a tryptic digest of casein. Within each species, the peptidase activity of the cell-free extracts of the strains varied. Furthermore, differences were observed between the Lactobacilhis species and Lc. lactis. Lb. helveticus had by far the highest hydrolysing activities towards all substrates, indicating the presence of powerful aminopeptidases, X-prolyl-dipeptidyl aminopeptidases and proline iminopeptidases. Lb. delbrueckii subsp. bulgaricus possessed high hydrolysing activities towards substrates containing proline, alanylprolyl–p–nitroanilide and prolyl–p–nitroanilide. On the other hand, Lb. fermentum and Lb. brevis could be considered as weakly proteolytic species. A more detailed study with highly proteolytic Lactobacillus strains indicated that at least three different proteinases or endopeptidases were present. Compared with Lc. lactis, the Lactobacillus strains had a much lower hydrolytic action on glutamyl-glutamic acid, suggesting that glutamyl aminopeptidase was absent in lactobacilli.

Cell extracts of various lactobacilli and two Lactococcus strains were investigated for their immunoresponse with monoclonal and polyclonal antibodies raised against various proteolytic enzymes from Lc. lactis. Except for Lactobacillus casei SBT 2233, none of the lactobacilli proteins showed immunoresponse with the monoclonal antibodies. With polyclonal antibodies raised against aminopeptidases N and C and endopeptidase of Lc. lactis an immunoresponse was observed. However, the molecular masses of the reactive bands on the blot were considerably different from those of the corresponding lactococcal peptidases, except for the band that reacted with polyclonal antibodies against aminopeptidase C. The polyclonal antibodies raised against X-prolyl-dipeptidyl aminopeptidase and tripeptidase did not show any immunoreaction. As a control, all antibodies reacted with the lactococcal proteins on the blot, with molecular masses corresponding to those reported for the proteinases and peptidases. The results clearly showed that most of the proteolytic enzymes of lactobacilli were immunologically different from those of lactococci. The proteolytic enzymes in the cell-free extracts were separated by non-denaturing PAGE and visualized by zymogram staining. The electrophoretic pattern of the proteolytic enzymes of lactobacilli was different from that of Lc. lactis. Both experiments indicate that the enzymes of the proteolytic system of lactobacilli are different from those of lactococci.

Two polysaccharides produced concurrently by Lactococcus lactis subsp. cremoris strain LC330 have been identified. One had a high molecular mass (> 1 × 106 Da) and was neutral. The second was smaller (∼ 10000 Da), charged and had a high phosphorus content. Sugar composition also differed. In chemostat culture the neutral polysaccharide was influenced by temperature and by nitrogen limitation. This polysaccharide was branched with terminal galactose moieties and contained galactose, glucose and glucosamine. The phosphopolysaccharide was more complex with glucose, rhamnose, galactose and glucosamine in an approximate ratio of 6:5:4:1.

The water-soluble extract of Cheddar cheese was fractionated by diafiltration using 10 kDa cut-off membranes. Peptides were isolated from the diafiltrate retentate by chromatography on DEAE-cellulose with a linear NaCl gradient in 50 mM-Tris-HCl, pH 8·6, and reversed-phase HPLC or electroblotting from urea-PAGE gels. Peptides were identified by determining N-terminal amino acid sequences and mass spectrometry. Most (45) of the total 51 peptides identified in the diafiltrate retentate originated from β-casein, especially from a short region in the N-terminal half of the molecule. Only six peptides originated from αs1-casein; β-lactoglobulin was also identified in the retentate. The origin of most of these peptides could be explained on the basis of known specificities of lactococcal cell envelope proteinases.

Owing to a production error, Figure 8 of the paper by Plowman & Creamer that appeared in the previous issue (Journal of Dairy Research 62 451–467) was printed with the lower part of the Figure reversed. We regret this error, and reproduce the correct version of Figure 8 (from p. 462) opposite.