Hiroshi Abe

Measurement of Hemoperfusion and Oxygen Sufficiency in Gastric Mucosa In Vivo: Evidence of Mucosal Hypoxia as the Cause of Hemorrhagic Shock-Induced Gastric Mucosal Lesion in Rats

Reflectance spectrophotometry was applied to the rat gastric mucosa in vivo to determine the hemodynamics as well as the intracellular oxygen metabolism. In the spectra obtained, the hemoglobin absorbances gave information as to mucosal blood volume and average oxy- and deoxyhemoglobin equilibrium, while the oxidation-reduction levels of the respiratory chain cytochromes determined the tissue oxygen saturation. In resting, air-breathing rats, the reflectance spectra showed a predominant oxyhemoglobin absorbance with almost fully oxidized cytochrome absorbances, suggesting a balanced oxygen supply and utilization. The mucosal blood volume was greater in the corpus region than in the antral region. The hemorrhagic shock caused a significant reduction of hemoglobin absorbances and induced an almost full reduction of the respiratory chain cytochromes in the corpus mucosa, indicating a severe reduction of blood supply resulting in severe hypoxia in the corpus mucosal cells. In contrast, the reflectance spectra of antral mucosa showed only a slight fall in the hemoglobin absorbance with no appreciable change of redox levels of the respiratory chain cytochromes. The mucosal hemorrhage and lesion appeared only in the corpus region, where the reduced cytochrome absorbances had been manifest. Thus, the present paper presents direct evidence supporting the concept that disturbed energy metabolism in the gastric mucosa is the cause of acute mucosal lesion during hemorrhagic shock. Further, this study represents a noninvasive methodology for measuring the intramucosal oxygen insufficiency that would precede the formation of the gastric mucosal lesion in the face of hemorrhagic shock.

Intracellular pH modulation of ADF/cofilin proteins.

The ADF/cofilin (AC) proteins are necessary for the high rates of actin filament turnover seen in vivo. Their regulation is complex enough to underlie the precision in filament dynamics needed by stimulated cells. Disassembly of actin by AC proteins is inhibited in vitro by phosphorylation of ser3 and pH<7.1. This study of Swiss 3T3 cells demonstrates that pH also affects AC behavior in vivo: (1) Wounded cells show pH-dependent AC translocation to alkaline-induced ruffling membrane; (2) The Triton extractable (soluble) ADF from Swiss 3T3 cells decreases from 42+/-4% to 23+/-4% when the intracellular pH (pH(i)) is reduced from 7.4 to 6.6; (3) Covariance and colocalization analyses of immunostained endogenous proteins show that ADF partitions more with monomeric actin and less with polymeric actin when pH(i) increases. However, the distribution of cofilin, a less pH-sensitive AC in vitro, does not change with pH; (4) Only the unphosphorylatable AC mutant (A3), when overexpressed as a GFP chimera, uniquely produces aberrant cellular phenotypes and only if the pH is shifted from 7.1 to 6.6 or 7.4. A mechanism is proposed that explains why AC(A3)-GFP and AC(wt)-GFP chimeras generate different phenotypes in response to pH changes. Phospho-AC levels increase with cell density, and in motile cells, phospho-AC increases with alkalization, suggesting a homeostatic mechanism that compensates for increased AC activity and filament turnover. These results show that the behavior of AC proteins with pH-sensitivity in vitro is affected by pH in vivo.

SITE-DIRECTED MUTAGENESIS OF THE PHOSPHORYLATION SITE OF COFILIN : ITS ROLE IN COFILIN-ACTIN INTERACTION AND CYTOPLASMIC LOCALIZATION

It has been demonstrated that the activity of ADF and cofilin, which constitute a functionally related protein family, is markedly altered by phosphorylation, and that the phosphorylation site is Ser 3 in their amino acid sequences [Agnew et al., 1995: J. Biol. Chem. 270:17582-17587; Moriyama et al., 1996: Genes Cells 1:73-86]. In order to clarify the function of the phosphorylated and unphosphorylated forms of cofilin in living cells especially in the process of cytokinesis, we generated analogs of the unphosphorylated form (A3-cofilin) and phosphorylated form (D3-cofilin) by converting the phosphorylation site (Ser 3) of cofilin to Ala and Asp, respectively. The mutated proteins were produced in an Escherichia coli expression system, and conjugated with fluorescent dyes. In in vitro functional assay, labeled A3-cofilin retained the authentic ability to bind to and sever F-actin, while labeled D3-cofilin failed to interact with actin. They were then injected into living cells to examine their cellular distribution. They exhibited distinct localization patterns in the cytoplasm; A3-cofilin was highly concentrated at the membrane ruffles and cleavage furrow, where endogenous cofilin is also known to be enriched. In contrast, D3-cofilin showed only diffuse distribution both in the cytoplasm and nucleus. These results suggest that the subcellular distribution of cofilin as well as its interacting with actin in vivo is regulated by its phosphorylation and dephosphorylation.

Colocalization of ADF and cofilin in intranuclear actin rods of cultured muscle cells

SummaryImmunofluorescence microscopy revealed that two actin-binding proteins of low molecular weight with different functional activity. ADF and cofilin, are transported into nuclei of cultured myogenic cells to form rod structures there together with actin, when the cells were incubated in medium containing dimethylsulfoxide. In most cases, ADF and cofilin colocalized in the same nuclear actin rods, but ADF appeared to predominate in mononucleated cells, while cofilin was present in multinucleated myotubes. In some mononucleated cells, the nuclear actin rods were composed of ADF and actin but devoid of cofilin. An ADF homologue in mammals, destrin, was also translocated into nuclear actin rods under similar conditions. As a nuclear transport signal sequence exists in cofilin and ADF but not in actin, ADF and/or cofilin may be responsible for the nuclear import of actin in myogenic cells under certain conditions.

Generation of functional beta-actinin (CapZ) in an E. coli expression system.

Abstractβ-actinin (CapZ) is a heterodimeric actin-binding protein which caps the barbed end of actin filaments and nucleates actin-polymerization in a Ca2+-independent manner. In myofibrils it is localized in the Z-lines. As judged by these properties of β-actinin, it is conceivable that β-actinin is involved in the regulation of actin assembly, especially in the formation of I-Z-I complex during myofibrillogenesis. In this study, we devised a system to produce functional β-actinin in E. Coli.The cDNAs of βI′ and βII subunits of β-actinin were obtained by RT-PCR methods using the published sequence as references, and subcloned in a pET vector. When the proteins were produced with the cDNA of either βI′ or βII in E. coli, the proteins were insoluble and non-functional. However, when the cDNAs encoding the two subunits were cloned into a single vector and␣both proteins were expressed simultaneously, the proteins became soluble and purified as a functional heterodimer. The␣activity of the purified proteins was not distinguishable from that of β-actinin purified from skeletal muscle.

Normalization of the Paradoxic Secretion of Glucagon in Diabetics Who Were Controlled by the Artificial Beta Cell

Since it is important to elucidate the precise significance of pancreatic A-cell hypersecretion in the pathogenesis of diabetes mellitus, the change in the immunoreactive glucagon (IRG) response to 100 g oral glucose challenges was studied in diabetics whose blood glucose responses and plasma immunoreactive insulin concentrations (IRI) simulated those in normal subjects with the aid of the artificial beta cell system that we developed originally. In six nonobese adult-onset and four insulin-dependent diabetics whose blood glucose responses and plasma insulin concentrations after 100 g oral glucose load were made equivalent to those seen in normal subjects by the artificial beta cell, the glucagon release was similar to the response in normal subjects. In one insulin-dependent diabetic with high anti-insulin-binding capacity, the blood glucose response after an oral glucose challenge was not normalized by the artificial beta cell and the glucagon secretion was paradoxically increased. This fact suggested that the paradoxic rise in glucagon, seen in response to an oral glucose load in some diabetics, is secondary to insulin deficiency.

Detection of a sequence involved in actin-binding and phosphoinositide-binding in the N-terminal side of cofilin

Cofilin is an actin-binding protein of low molecular weight which is widely distributed in eukaryotes and is deeply involved in the dynamics of actin assembly in the cytoplasm. The actin-binding ability of cofilin is inhibited by inositol phosphates (PIP2), and the PIP2- and actin-binding site(s) has been localized in residues W104 - M115 of the cofilin primary sequence (Yonezawa et al. 1991). In the present study, in order to further clarify the functional domains in cofilin molecule, we constructed expression vectors containing cDNAs of different size with deletion at the 3′-region of the open reading frame. The truncated cofilin molecules produced in E. coli were purified and examined for their actin-binding and PIP2-binding ability. We found that the truncated cofilin molecule without C-terminal residues #100-#166 including the previously-described actin-binding site could be cross-linked with actin by EDC, a zero-length cross-linker. In addition, these truncated peptides as well as synthetic peptides corresponding to the N-terminal sequence of cofilin suppressed the inhibitory action of PIP2 on actin-cofilin interaction. These results strongly suggest that additional actin- and PIP2-binding sites exist in the N-terminal region of cofilin.

Expression of multiple troponin T variants in neonatal chicken breast muscle

The types of troponin-T (TNT) expressed in neonatal chicken breast muscle were examined by two-dimensional gel electrophoresis (2-D PAGE), immunoblotting, and peptide mapping. When troponin from neonatal chicken breast muscle or whole lysate of the muscle was displayed on 2-D PAGE, multiple spots were observed in the TNT region on the gel. They differed slightly from those in adult breast- and leg-type TNT, but were positively stained with the antibody specific for TN-T. These results indicate that multiple spots observed in the TNT region are all TNT isoforms. The TNT isoforms in the neonatal breast muscle were classified into two groups, based on size. Each group contained about five variants. The first group with a larger size was in the molecular weight range of adult breast TNT, while the smaller-sized second group was in the molecular weight range of adult leg TNT. Overall peptide map patterns of variants in the first group and also that of adult breast TNT resembled each other, whereas those of variants in the second group were similar to that of adult leg TNT. The TNT of adult breast-type appeared at about 2- to 3-weeks posthatch, and thereafter became a major TNT isoform.

Quantitative analysis of low molecular weight G-actin-binding proteins, cofilin, ADF and profilin, expressed in developing and degenerating chicken skeletal muscles

SummaryA large amount of G-actin is pooled in the cytoplasm of young embryonic skeletal muscle and, although its concentration is reduced as muscle develops, the total amount of actin in muscle cells increases remarkably. Three G-actin-binding proteins, cofilin, ADF and profilin, are known to be involved in creating the G-actin pool in the embryonic muscle. To better understand how they are responsible for the regulation of assembly and disassembly of actin in developing and degenerating muscles, we measured the amounts of the three G-actin-binding proteins by means of quantitative immunoblotting and compared them with that of G-actin. The sum of the amounts of the three actin-binding proteins was insufficient at early developmental stages but sufficient at later stages to account for the pool of G-actin in young muscle cells. It decreased in parallel with the decrease in the G-actin pool as muscle developed. Expression of thymosin β4, which is known to be extremely important for G-actin-sequestering in a variety of non-muscle cells, was detected at a considerable level in young embryonic but not in adult skeletal muscles according to Northern and Western blotting. In degenerating denervated and dystrophic muscles, cofilin and profilin, but not ADF, were significantly increased in amount. From these results, we conclude that the G-actin pool in young embryonic skeletal muscle is mainly due to cofilin, ADF, profilin and thymosin β4, but thymosin β4 as well as ADF becomes less important as muscle develops. Cofilin and profilin may also be involved in the redistribution of actin during myofibrillogenesis and in the process of actin disassembly in degenerating muscles.

Involvement of Slingshot in the Rho-mediated Dephosphorylation of ADF/Cofilin during Xenopus Cleavage

Abstract ADF/cofilin is a key regulator for actin dynamics during cytokinesis. Its activity is suppressed by phosphorylation and reactivated by dephosphorylation. Little is known, however, about regulatory mechanisms of ADF/cofilin function during formation of contractile ring actin filaments. Using Xenopus cycling extracts, we found that ADF/cofilin was dephosphorylated at prophase and telophase. In addition, constitutively active Rho GTPase induced dephosphorylation of ADF/cofilin in the egg extracts. This dephosphorylation was inhibited by Na3VO4 but not by other conventional phosphatase-inhibitors. We cloned a Xenopus homologue of Slingshot phosphatase (XSSH), originally identified in Drosophila and human as an ADF/cofilin phosphatase, and raised antibody specific for the catalytic domain of XSSH. This inhibitory antibody significantly suppressed the Rho-induced dephosphorylation of ADF/cofilin in extracts, suggesting that the dephosphorylation at telophase is dependent on XSSH. XSSH bound to actin filaments with a dissociation constant of 0.4 μM, and the ADF/cofilin phosphatase activity was increased in the presence of F-actin. When latrunculin A, a G-actin-sequestering drug, was added to extracts, both Rho-induced actin polymerization and dephosphorylation of ADF/cofilin were markedly inhibited. Jasplakinolide, an actin-stabilizing drug, alone induced actin polymerization in the extracts and lead to dephosphorylation of ADF/cofilin. These results suggest that Rho-induced dephosphorylation of ADF/cofilin is dependent on the XSSH activation that is caused by increase in the amount of F-actin induced by Rho signaling. XSSH colocalized with both actin filaments and ADF/cofilin in the actin patches formed on the surface of the early cleavage furrow. Injection of inhibitory antibody blocked cleavage of blastomeres. Thus, XSSH may reorganize actin filaments through dephosphorylation and reactivation of ADF/cofilin at early stage of contractile ring formation.