The effect of heat stress on performance, fertility, and adipokines involved in regulating systemic immune response during lipolysis of early lactating dairy cows.

The aim of this study was to assess the potential effect of heat stress on dairy cow productivity, fertility, and biochemical blood indices during the early lactation stage in a temperate climate. Additionally, the study aimed to determine the role of leptin and adiponectin in regulating the immune response accompanying lipolysis after calving in dairy cows. The study included 100 clinically healthy Polish Holstein-Friesian dairy cows selected based on parity and 305 d of milk yield from 5 commercial farms with similar herd management and housing systems. Prospective cohort data were recorded from calving day until 150 d in milk, and microclimate loggers installed inside the barns were used to record temperature and relative humidity data to calculate daily temperature-humidity index (THI) on the calving day, through +7, +14, and +21 d during early lactation. Additionally, monthly productive performance parameters such as milk yield, chemical composition, fatty acids composition, and fertility indices were analyzed. Results showed that the THI from calving day through +7, +14, and +21 d during early lactation was negatively associated with fertility parameters such as delayed first estrus postpartum and an elongated calving interval, respectively, by 29, 27, 25, and 16 d. Furthermore, an increase in THI value during early lactation was associated with an elongated artificially inseminated service period, days open, and intercalving period. Increasing THI from calving day (0 d) through +7, +14, and up to +21 d during early lactation was also linked to decreased milk yield by 3.20, 4.10, 5.60, and 5.60 kg, respectively. The study also found that heat stress during early lactation was associated with a lower body condition score in dairy cows and higher concentrations of leptin, nonesterified fatty acids, and ß-hydroxybutyrate, accompanied by a drastic reduction in adipose tissue-secreted adiponectin levels after calving. Additionally, heat stress-induced lipolysis in adipose tissue caused an inflammatory response that increased biochemical blood indices associated with immune responses such as cytokines, acute phase proteins, and heat shock protein. These findings suggest that exposing dairy cows to heat stress during early lactation can negatively affect their productive performance, fertility, and biochemical blood indices in subsequent lactations. Thus, farm management changes should be implemented during early lactation to mitigate the negative consequences of heat stress occurrence.

Yeast fructose-2,6-bisphosphate 6-phosphatase is encoded by PHO8, the gene for nonspecific repressible alkaline phosphatase.

Yeast fructose-2,6-bisphosphate 6-phosphatase has been purified 7000-fold by heat treatment, poly(ethylene glycol) precipitation, ion-exchange chromatography with Q-Sepharose Fast Flow and Mono Q followed by affinity chromatography with concanavalin-A-Sepharose and gel filtration with Superose 12. The purified dimeric enzyme contains 1.5 mol zinc and 1.3 mol copper/mol subunit. It reacts with fructose 2,6-bisphosphate [Fru(2,6)P2] as well as with p-nitrophenyl phosphate (NpP) showing a pH optimum at pH 6-6.5 with Fru(2,6)P2 [Plankert, U., Purwin, C. & Holzer, H. (1988) FEBS Lett. 239, 69-72] and above pH 9.0 with NpP. The following observations suggest that activity with both substrates depends on the same protein. (a) During 7000-fold purification, the ratio of activity with NpP to that with Fru(2,6)P2 remained constant. (b) The time course of inactivation of enzyme activity in dilute solution at 30 degrees C is similar for both substrates. (c) At increasing temperatures, inactivation of enzyme activity measured with both substrates proceeds at nearly identical rates. (d) Activity with both substrates is found preferentially in the vacuoles. (e) Mutants defective in the nonspecific alkaline phosphatase coded by the PHO8 gene are also defective in Fru(2,6)P2 6-phosphatase activity. (f) A proteinase A mutant, defective in processing and activation of nonspecific alkaline phosphatase coded by the PHO8 gene, also fails to activate Fru(2,6)P2 6-phosphatase.

Characterization of yeast fructose-2,6-bisphosphate 6-phosphatase.

To obtain information on the biological significance of yeast fructose-2,6-bisphosphate 6-phosphatase, kinetic data of the purified enzyme [(1987) Eur. J. Biochem. 164, 27-30] have been measured. Maximal activity was found between pH 6 and 7, the apparent Michaelis constant with fructose 2,6-bisphosphate was 7.2 microM at pH 6.0 and 79 microM at pH 7.0. Concentrations required for 50% inhibition of the enzyme at pH 6.0 were 8 microM Fru2P, 45 microM G1c6P, 80 microM Fru6P and 200 microM inorganic phosphate. The known intracellular steady-state level of about 10 microM fructose 2,6-bisphosphate in the presence of glucose is likely to be the result of a balance between the rapid synthesis of fructose 2,6-bisphosphate catalyzed by 6-phosphofructose 2-kinase and a fructose 2,6-bisphosphate degrading activity. The biological function of fructose-2,6-bisphosphate 6-phosphatase with an apparent Michaelis constant between 7 and 79 microM fructose 2,6-bisphosphate at pH 6-7 is therefore suggested to participate in the maintenance of a steady-state level of fructose 2,6-bisphosphate in a concentration range that fits well with the Michaelis constant of the enzyme.

Mechanism of stimulation of endogenous fermentation in yeast by carbonyl cyanide m-chlorophenylhydrazone.

Addition of the uncoupler and protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to starved yeast cells starts endogenous alcoholic fermentation lasting about 20 min. Hexose 6-phosphates, fructose 2,6-bisphosphate, and pyruvate accumulate in less than 2 min after addition of CCCP from almost zero concentration to concentrations which correspond to 1/5-1/10 of the steady-state concentrations during fermentation of glucose. CCCP immediately causes a decrease of the intracellular cytosolic pH from 6.9 to 6.4. This change activates adenylate cyclase (Purwin, C., Nicolay, K., Scheffers, W.A., and Holzer, H. (1986) J. Biol. Chem. 261, 8744-8749) and leads to the previously observed transient increase of cyclic AMP. It is shown here that the following enzymes known from in vitro experiments to be activated by cyclic AMP-dependent phosphorylation are activated in the CCCP-treated starved yeast cells in vivo: glycogen phosphorylase, trehalase (pH 7), 6-phosphofructo-2-kinase. The activation of 6-phosphofructo-2-kinase leads to an accumulation of fructose 2,6-bisphosphate, which is known from in vitro experiments to activate 6-phosphofructo-1-kinase and to inhibit fructose-1,6-bisphosphatase. All effects observed in the intact yeast cells fit with the idea that the CCCP-initiated activation of adenylate cyclase leads to a sequence of events which by protein phosphorylation and allosteric effects initiates endogenous alcoholic fermentation.

Fructofuranose 2-phosphate is the product of dephosphorylation of fructose 2,6-bisphosphate.

Using comparative ion-exchange chromatography on Dowex 1X4, the product of dephosphorylation of fructose 2,6-bisphosphate with purified yeast fructose-2,6-bisphosphate 6-phosphohydrolase, was shown to be identical to the furanose form of fructose 2-phosphate prepared by chemical synthesis according to Pontis and Fischer [Biochem. J. 89, 452-459 (1963)]. As expected for the furanose form of fructose 2-phosphate, the enzymatically formed product consumes 1 mol periodate/mol fructose 2-phosphate, whereas the chemically synthesized pyranose form consumes 2 mol periodate/mol. In addition, it is shown that the enzymatic product behaves identically to the furanose, not the pyranose, form of fructose 2-phosphate in hydrolysis of the ester bond at pH 4 and 37 degrees C, as described previously for the chemically synthesized compounds [Pontis and Fischer (1963) vide supra].

Fructose 2-phosphate, an intermediate of the dephosphorylation of fructose 2,6-bisphosphate with a purified yeast enzyme.

A fructose-2,6-bisphosphate dephosphorylating enzyme was 3000-fold purified to electrophoretic homogeneity from Saccharomyces cerevisiae. Half-maximal activity was obtained at pH 6.0 with 6 microM fructose 2,6-bisphosphate and 0.15 mM Mg2+. On incubation for 90 min with fructose 2,6-bisphosphate, about 80% of the substrate appears with an almost linear time dependence as fructose. In the first 30 min a substance accumulates to about 40% of the consumed fructose 2,6-bisphosphate which forms free fructose on mild acid treatment. Formation of fructose 6-phosphate was negligible. The mild-acid-labile intermediate was identified as fructose 2-phosphate by comparative ion-exchange chromatography with authentic fructose 2-phosphate synthesized from fructose 1-phosphate [Pontis, H.G. & Fischer, C.L. (1963) Biochem. J. 89, 452-459]. The data suggest the reaction sequence fructose 2,6-bisphosphate----fructose 2-phosphate----fructose. The designation fructose-2,6-bisphosphate 6-phosphohydrolase is proposed for the enzyme described here.

Mechanism of control of adenylate cyclase activity in yeast by fermentable sugars and carbonyl cyanide m-chlorophenylhydrazone.

The phosphorylation of fructose-1,6-bisphosphatase is preceded by a transient increase in the intracellular level of cyclic AMP which activates a cyclic AMP-dependent protein kinase (Pohlig, G., and Holzer, H. (1985) J. Biol. Chem. 260, 13818-13823). Possible mechanisms by which sugars or ionophores might activate adenylate cyclase and thereby lead to an increase in cyclic AMP concentrations were studied. Studies with permeabilized yeast cells demonstrated that neither sugar intermediates nor carbonyl cyanide m-chlorophenylhydrazone are able to increase adenylate cyclase activity. In the light of striking differences of the effects of fermentable sugars and of carbonyl cyanide m-chlorophenylhydrazone on parameters characterizing the membrane potential, it seems not reasonable that the activity of adenylate is under control of the membrane potential. Rapid quenching of 9-aminoacridine fluorescence after addition of fermentable sugars to starved yeast cells indicated an intracellular acidification. The 31P NMR technique showed a fast drop of the intracellular pH from 6.9 to 6.55 or 6.4 immediately after addition of glucose or carbonyl cyanide m-chlorophenylhydrazone. The time course of the decrease of the cytosolic pH coincides with the transient increase of cyclic AMP concentration and the 50% inactivation of fructose-1,6-bisphosphatase under the conditions of the NMR experiments. Kinetic studies of adenylate cyclase activity showed an approximately 2-fold increase of activity when the pH was decreased from 7.0 to 6.5, which is the result of a decrease in the apparent Km for ATP with no change in Vmax. These studies suggest that activation of adenylate cyclase by decrease in the cytosolic pH starts a chain of events leading to accumulation of cyclic AMP and phosphorylation of fructose-1,6-bisphosphatase.

How does glucose initiate proteolysis of yeast fructose-1,6-bisphosphatase?

Glucose-induced proteolysis of fructose-1,6-bisphosphatase (FBPase) in yeast (Funayama, S. et al. (1980) Eur. J. Biochem. 109, 61-66) is preceded by rapid phosphorylation of the enzyme (Müller, D., and Holzer, H. (1981) Biochem. Biophys. Res. Commun. 103. 926-933; Mazón, M. J. et al. (1982) J. Biol. Chem. 257, 1128-1130). The postulated sequence of events is as follows. Glucose causes a decrease of the cytosolic pH from 7.0 to about 6.5 (Purwin, C. et al. (1986) J. Biol. Chem. 261, in press). The change in pH activates adenylate cyclase which is at pH 6.5 2-3 times more active than at pH 7.0 The concentration of cAMP increases 2-5 times (Purwin, C. et al. (1982) Biochem. Biophys. Res. Commun. 107, 1482-1489) and thereby activates a protein kinase which phosphorylates FBPase (Pohlig, G., and Holzer, H. (1985) J. Biol. Chem. 260, 13818 to 13823). The phosphorylated enzyme is either proteolyzed in the cytosol or taken up into the vacuoles, the compartment where unspecific proteolysis takes place.

Induction of enzymes of phytoalexin synthesis in cultured soybean cells by an elicitor from Phytophthora megasperma f. sp. glycinea.

The glucan elicitor from cell walls of the fungal pathogen, Phytophthora megasperma f. sp. glycinea, induced rapid but transient increases in enzyme activities of general phenylpropanoid metabolism (phenylalanine ammonia-lyase and 4-coumarate: CoA ligase) and of the flavonoid pathway (chalcone synthase) in cell suspension cultures of soybean (Glycine max). After transferring cells into fresh medium, two peaks of inducibility for the enzymes by elicitor were observed, one shortly after transfer (stage I), and one at the end of the linear growth phase (stage II). Only one of the two isoenzymes of 4-coumarate: CoA ligase ("isoenzyme 2"), for which a specific involvement in flavonoid biosynthesis has been postulated, was affected by the elicitor. For two of the induced enzymes, phenylalanine ammonia-lyase and chalcone synthase, the changes in activity at stage I were shown to be preceded by large changes in their rates of synthesis, as determined by in vivo labelling with [(35)S] methionine and immunoprecipitation.

Rapid Response of Suspension-cultured Parsley Cells to the Elicitor from Phytophthora megasperma var. sojae: INDUCTION OF THE ENZYMES OF GENERAL PHENYLPROPANOID METABOLISM.

Large and rapid increases in the activities of two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, occurred in suspension-cultured parsley cells (Petroselinum hortense) treated with an elicitor preparation from Phytophthora megasperma var. sojae. Highest enzyme activities were obtained with an elicitor concentration similar to that required for maximal phenylalanine ammonialyase induction in cell suspension cultures of soybean, a natural host of the fungal pathogen.The changes in phenylalanine ammonia-lyase activity in parsley cells were caused by corresponding changes in the mRNA activity for this enzyme. Phenylalanine ammonia-lyase mRNA activity increased much faster and transiently reached a much higher level in elicitor-treated than in irradiated cell cultures. In contrast to irradiation, treatment of the cells with the elicitor did not induce the enzymes of the flavonoid glycoside pathway, as demonstrated for acetyl-CoA carboxylase and chalcone synthase. Induction of these enzymes by light was abolished by simultaneous application of the elicitor.