Naomi Yamakawa

Differential gene expression of β‐1,4‐galactosyltransferases I, II and V during mouse brain development

Since most brain glycoproteins from β‐1,4‐galactosyltransferase (β‐1,4‐GalT) I knockout mice were galactosylated without apparent reduction the gene expression of novel β‐1,4‐GalTs II and V which are involved in N‐linked oligosaccharide biosynthesis in addition to β‐1,4‐GalT I was studied during mouse brain development. Isolation and characterization of β‐1,4‐GalT II and V cDNAs from mouse brains indicates that they are also functioning in the brain. Northern blot analysis revealed that the β‐1,4‐GalT I gene is expressed mainly in mid‐embryonic stages, while the expression level of β‐1,4‐GalT II transcript remains constant and of β‐1,4‐GalT V transcript increases during mouse brain development after birth. In situ hybridization revealed that β‐1,4‐GalT II and V signals are present in most neural cells, with a marked difference between them in the hippocampus of adult mouse brain tissue. The differential gene expression of β‐1,4‐GalTs I, II and V during mouse brain development could affect the differential galactosylation of brain glycoproteins, as revealed by lectin blot analysis.

Analysis of the role of egg integrins in sperm-egg binding and fusion.

Sperm‐egg fusion is believed to be mediated via specific molecular interactions. Integrin α6β1 is a strong candidate for a sperm receptor on the egg plasma membrane. However, the ability of the egg integrin α6β1 to interact with molecules on intact sperm has not yet been proven. In this report, possible involvement of integrin α6β1 in sperm‐egg interactions was examined by biochemical and immunocytochemical analyses. To identify egg molecules that specifically interact with sperm, we first incubated sperm with biotin‐labeled egg surface proteins. Under this condition, solubilized proteins from eggs inhibited sperm‐egg fusion. Western blot analysis under reducing conditions indicated that a major‐labeled band of 135 kDa bound to sperm. An immunodepletion experiment using the anti‐integrin α6 antibody GoH3 indicated that the 135 kDa egg surface molecule that bound to sperm was the integrin α6 subunit. To investigate the potential involvement of integrin α6β1 in sperm‐egg fusion, we next examined the localization of integrin α6 and β1 subunits before and after fertilization by confocal laser microscopy. At an early stage of sperm‐egg fusion, the integrin α6 and β1 subunits were accumulated at the sperm binding site. The frequency of cluster formation was closely related to that of sperm‐egg fusion, indicating that integrin receptors are accumulated by sperm destined for fusion. Taken together, these results strongly suggest that the integrin α6β1 is involved in sperm‐egg binding leading to fusion via direct association of the integrin α6 with sperm. Mol. Reprod. Dev. 56:412–423, 2000.

Age-associated changes in the subcellular localization of phosphorylated p38 MAPK in human granulosa cells.

p38 MAPK (p38) plays pivotal roles in aging and reproductive physiology. Nevertheless, involvement of p38 in female reproductive aging is uncertain. To improve knowledge of the role of p38 in age-associated reproductive failure, the expression and subcellular localization of phosphorylated p38 was investigated in human granulosa cells. p38 was 7-fold more activated in cells from older subjects than in those from younger subjects. Similar results were obtained in human granulosa-like KGN cells treated with hydrogen peroxide (H(2)O(2)). Interestingly, phosphorylated p38 was detected in the nucleus less frequently in older cells than in younger cells (Younger: 58.6%; Older: 29.8%, P< 0.01). Similarly cytoplasmic localization of phosphorylated p38 in KGN cells was observed after treatment with H(2)O(2). The activation and cytoplasmic localization of p38 in H(2)O(2)-treated KGN cells were blocked by N-acetylcysteine and SB203580. Although the p38 activators, FSH and tumor necrosis factor-α, induced a similar localization of phosphorylated p38 in KGN cells, the expression and localization patterns of p38 were distinct from those in older granulosa cells and H(2)O(2)-treated KGN cells. These results indicate that the characteristic localization of p38 in older granulosa cells is induced by oxidative stress.

Regulation of prolyl oligopeptidase activity in regenerating rat liver

We have previously shown that the naturally occurring polyamines, spermidine and spermine, reverse effectively the in vitro inhibition of prolyl oligopeptidase (POPase) by its endogenous inhibitor by forming a kinetically significant complex (Soeda et al., J. Neurochem. (1986) 46, 1304-1307). In this study, we examined changes in the activities of POPase and its endogenous inhibitor and in the concentrations of polyamines during the regeneration of rat liver. POPase activity in the liver cytosol peaked 2 days after partial hepatectomy and then decreased near to control activity by 9 days, without its altered synthetic levels. Total polyamine concentrations also peaked at 2 days and remained elevated by 9 days, while cytosolic POPase inhibitor activity was minimal (56% of control) at 2 days. Treatment of the animals with a synthetic POPase inhibitor, Z-Gly-Pro-CHN2 (4 mg/kg), resulted in an obvious suppression of the liver regeneration. These results imply that the activity of POPase involved in nonlysosomal proteolytic pathway is exquisitely regulated by changes not only in its endogenous inhibitor levels but also in intracellular cationic potentials such as polyamines, and that POPase plays a crucial role for the growth and differentiation of liver cell.

Inhibition of proline endopeptidase activity by acyl-coenzyme A esters.

Coenzyme A (CoA), its related compounds and acylcarnitine non-competitively inhibited the activity of proline endopeptidase (PEPase) purified from rat liver cytosol. The degree of inhibition was in the order of acyl-CoA greater than CoA greater than dephospho-CoA greater than or equal to acylcarnitine. However, carnitine did not inhibit the enzyme activity. Among the compounds examined, n-decanoyl-CoA showed the highest inhibitory activity (Ki = 9 microM). These results suggest that both the acyl group and CoA contribute to the inhibition of PEPase by acyl-CoA. The abilities of n-decanoyl-CoA and its related compounds to quench the intrinsic fluorescence at 332 nm from PEPase excited at 280 nm, was used as a probe for the binding affinity of the enzyme for these compounds. The quenching of fluorescence by CoA was nearly equal to that by n-decanoyl-CoA. n-Decanoylcarnitine and carnitine were unable to quench the fluorescence. These results indicate that n-decanoyl-CoA at least binds to PEPase through its CoA portion.

Effects of polyamines on proline endopeptidase activity in rat brain.

Abstract: The in vitro effects of polyamines on the activity of proline endopeptidase (PEPase) in rat brain cytosol, which contains an endogenous PEPase inhibitor, have been studied. Of the three amines tested (spermine, spermidine, and putrescine), spermine and spermidine markedly enhanced the enzyme activity in brain cytosol. At 6.25 mM spermine or 25 mM spermidine, a 13‐ or 14‐fold enhancement of the enzyme activity was observed. When Mg2+ was used, an approximately fourfold enhancement of the enzyme activity was observed at 50 mM. The enhancement produced by spermine or spermidine was unaffected by Mg2+ up to 50 mM. The activity of purified PEPase was only slightly affected by each polyamine, but it was inhibited 50% by 50 mM Mg2+. On the other hand, 50% inhibition of the enzyme produced by the purified PEPase inhibitor (Mr 7,000; Ki 0.67 mM) was completely restored by addition of 0.7 mM spermine, 3.5 mM spermidine, or 28 mM putrescine. This restoration of inhibition by polyamines was reversed by increasing the inhibitor concentration. These data suggest that polyamines effectively reverse the inhibition of PEPase by its endogenous inhibitor by the reversible formation of a kinetically significant complex. The possible functions of polyamines in the regulation of PEPase in vivo are discussed.

Molecular Alterations During Female Reproductive Aging: Can Aged Oocytes Remind Youth?

Misa Imai1, Junwen Qin2, Naomi Yamakawa3, Kenji Miyado4, Akihiro Umezawa4 and Yuji Takahashi4 1Department of Biochemistry, Tufts University School of Medicine 2Institute of Reproductive Immunology, College of Life Science and Technology, Jinan University 3Research Team for Geriatric Disease, Tokyo Metropolitan Institute of Gerontology 4Department of Reproductive Biology, National Center for Child Health and Development 1USA 2China 3,4Japan