Hiromoto Iwadate

Potential role of kallikrein in diurnal rhythms and perivascular distribution in rat pineal glands

Kallikrein hydrolyzes various biologically active peptides, other than kininogens, such as vasoactive intestinal polypeptide (VIP), in vitro. Since kallikrein and VIP have been immunohistochemically shown to be present in the perivascular areas of the pineal gland, this study was designed to determine their topographic proximity in these glands, using immunohistochemical and immunoelectron microscopic double staining methods. Furthermore, since this gland is well-known to have a circadian rhythm, the kallikrein content was measured every 4 h, using a synthetic substrate, Pro-Phe-Arg-MCA, and an enzyme-linked immunosorbent assay (ELISA) to determine whether kallikrein has a circadian rhythm. The immunoreactivities of kallikrein and VIP were highly localized in the perivascular extracellular spaces and were virtually identical in distribution. The kallikrein content changed every 4 h and was high under light and low under dark conditions. The change was more evident when the synthetic substrate was used, and this rhythm was subtle on ELISA. VIP is also said to have a circadian rhythm in the pineal glands, being low under light and high under dark conditions, i.e., opposite to that of kallikrein. Since kallikrein degrades VIP in vitro, it is reasonable to speculate that pineal gland kallikrein is involved in the processing of VIP and possibly other biologically active peptides in the perivascular areas with a discernible circadian rhythm.

Biochemical and immunohistochemical demonstration of tissue kallikrein in the neuronal nuclei of the developing rat brains

Kallikrein content and cellular localization in the prenatal, newborn and adult rat brains were determined by the enzyme-linked immunosorbent assay and immunohistochemistry. The content was the highest in the prenatal rats and highly predominant in the neuronal nuclei during the prenatal to newborn periods, whereas the immunoreactive kallikrein was mainly located around neuronal cell bodies and their processes in the adult rats. The preferential nuclear localization in the prenatal rat brains was further confirmed by the immunoblotting technique after the SDS-polyacrylamide gel electrophoresis of the lysate of the nuclei fractionated from the prenatal rat brains. The meaning(s) of this kallikrein localization in the neuronal nuclei at the prenatal and newborn stages is unknown. However, we would like to conclude that this enzyme plays an important role in the morphogenesis of brain by acting on the substance(s) in the neuronal nuclei at the developing stage.

Development of Novel C-Terminal Sequencing Methods

Development of a reliable C-terminal sequencing method has been expected from various aspects including sequencing protein, analyzing posttranslational process, confirming recombinant proteins and cloning. Carboxypeptidase digestion has been commonly used with limitations. Since a classical isothiocyanate degradation was proposed, several modifications were reported in the past two conferences (Hawkes and Boyd, 1991; Inglis et al., 1991; Miller and Shively, 1989). The present paper summarizes two related novel carboxy-terminal sequencing methods using perfluorinated carboxylic acids in monohydrate form and carboxylic acid anhydrides. These reagents produce the mixtures of C-terminal successive degraded molecules which are able to be analyzed by fast-atom-bombardment (FAB)- or electrospray ionization (ESI)-mass spectrometry (MS).