Effect of hyperglycemia on the changes of intracellular [Ca2+]i in heart myoblast.

A rise in cytosolic free Ca2+ is the immediate trigger for contraction in heart muscle. In the present study, we investigated changes of intracellular Ca2+ increased by potassium chloride (KCl) and phenylephrine (PE) under hyperglycemia in rat heart myoblast H9c2 cells (BCRC 60096), respectively. We employed the fluorescent Ca2+-indicator, fura-2, and digital imaging microscopy to measure [Ca2+]i in H9c2 cells. Cells were cultured in hyperglycemic (30 mM glucose) Dulbecco's Modified Eagle's Medium. The variation of [Ca2+]i induced by KCI and PE in hyperglycemia was examined, respectively. Moreover, tiron, one of the antioxidants, was pretreated in hyperglycemia-treated H9c2 cells to measure the role of free radicals in the changes of intracellular [Ca2+]i. An influx in intracellular Ca2+ induced by KCl or PE was observed in a dose-dependent manner and reached the highest concentration of 434 +/- 42.3 nM and 443 +/- 42.8 nM (n = 24 cells), respectively. Moreover, this increase of intracellular [Ca2+]i induced by KCl or PE was markedly reduced in cells exposed to hyperglycemia (434 +/- 42.3 vs. 1.26 +/- 0.21 nM and 443 +/- 42.8 vs. 2.54 +/- 0.25 nM, n = 24 cells, P < 0.001, respectively). Similar changes were not observed in cells received mannitol showing same osmolarity. However, the reduction of intracellular [Ca2+]i induced by hyperglycemia was abolished significantly in the presence of tiron. Our results suggest that an increase of intracellular Ca2+ by KCl or PE in heart cell was markedly reduced by hyperglycemic treatment; mediation of free radicals in this action can be considered because it was reversed in the presence of tiron.

The antihypertensive activity of angiotensin-converting enzyme inhibitory peptide containing in bovine lactoferrin.

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the bovine lactoferrin hydrolysate using peptic hydrolysis by 2-step of reverse-phase high-performance liquid chromatography. This peptide was identified as Leu-Arg-Pro-Val-Ala-Ala and it produced a concentration-dependent inhibition of ACE activity in vitro with an IC50 value of about 4.14 microM. Also, this inhibition was identified as noncompetitive from the Lineweaver-Burk plot. Moreover, the antihypertensive activity of Leu-Arg-Pro-Val-Ala-Ala was investigated by the intravenous injection into spontaneously hypertensive rats (SHRs). A dose-dependent reduction of systolic blood pressure by this peptide was observed at 60 min after injection and it maximally decreased the blood pressure at a rate of 1 nmol/ml/kg. The blood pressure lowering activity of this peptide was calculated as 210% of captopril (10 pmol/ml/kg) that was used as positive control. Otherwise, identification of this peptide in the blood of SHRs was carried out chromatographically. Reduction of blood pressure coincides with the peak peptide concentration in the serum. Thus, we conclude that this peptide inhibits ACE activity in vitro and lowers systolic blood pressure in spontaneously hypertensive rat.

Susceptibilities against bovine lactoferrin with microorganisms isolated from mastitic milk.

Antibacterial effects of bovine lactoferrin were studied in vitro against microorganisms isolated from mastitic milk in Tokachi area, Hokkaido, Japan. Microorganisms isolated were Escherichia coli (11 isolates), Klebsiella pneumoniae (5 isolates), enterococci (8 isolates), Staphylococcus aureus (10 isolates), coagulase negative staphylococci (CNS, 13 isolates), streptococci (11 isolates), Prototheca zopfii (7 isolates) and yeast-like fungi (9 isolates). Lactoferrin has been known as a multifunctional protein and its antimicrobial effect is one of the most essential function of it. In order to compare their susceptibilities against lactoferrin, the minimal inhibitory concentration values were estimated by a microplate assay method using 96-well microplate, which involved measuring the optical density of the cultures. Prototheca zopfii was highly sensitive to bovine lactoferrin and complete inhibition of this microorganism was observed even at the low concentration of 7 mug/ml. On the other hand, E. coli and enterococci showed resistance against lactoferrin action and staphylococci showed strain-dependent resistance.

Expression of bovine lactoferrin and lactoferrin N-lobe by recombinant baculovirus and its antimicrobial activity against Prototheca zopfii.

Lactoferrin (LF) is a multifunctional, iron-binding glycoprotein found in secretory fluids of mammals. In this study, DNA encoding bovine lactoferrin (bLF) or the N-terminal half of bLF (bLF N-lobe) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLF or bLF N-lobe was isolated. An 80-kDa bLF-related protein expressed by the recombinant baculovirus was detected by monoclonal antibodies against bLF N-lobe and the C-terminal half of bLF (bLF C-lobe). A 43-kDa bLF N-lobe-related protein expressed by the recombinant baculovirus was detected by anti-bLF N-lobe monoclonal antibody, but not by anti-bLF C-lobe monoclonal antibody. These proteins were also secreted into the supernatant of insect cell cultures. Recombinant bLF (rbLF) and bLF N-lobe (rbLF N-lobe) were affected by tunicamycin treatment, indicating that rbLF and rbLF N-lobe contain an N-linked glycosylation site. Antimicrobial activity of these recombinant proteins against Prototheca zopfii (a yeast-like fungus that causes bovine mastitis) was evaluated by measuring the optical density of the culture microplate. Prototheca zopfii was sensitive to rbLF and rbLF N-lobe, as well as native bLF. There was no difference in antimicrobial activity between rbLF N-lobe and bLF C-lobe.