Motoi Kashiwagi

Inhibition of apoptosis by the actin-regulatory protein gelsolin

Gelsolin is an actin‐regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. In addition to its actin‐regulatory function, gelsolin has also been proposed to affect cell growth. Our present experiments have tested the possible involvement of gelsolin in the regulation of apoptosis, which is significantly affected by growth. When overexpressed in Jurkat cells, gelsolin strongly inhibited apoptosis induced by anti‐Fas antibody, C2‐ceramide or dexamethasone, without changing the F–actin morphology or the levels of Fas or Bcl‐2 family proteins. Upon the induction of apoptosis, an increase in CPP32(‐like) protease activity was observed in the control vector transfectants, while it was strongly suppressed in the gelsolin transfectants. Pro‐CPP32 protein, an inactive form of CPP32 protease, remained uncleaved by anti‐Fas treatment in the gelsolintransfectants, indicating that gelsolin blocks upstream of this protease. The tetrapeptide inhibitor of CPP32(‐like) proteases strongly inhibited Fas‐mediated apoptosis, but only partially suppressed both C2‐ceramide‐ and dexamethasone‐induced apoptosis. These data suggest that the critical target responsible for the execution of apoptosis may exist upstream of CPP32(‐like) proteases in Jurkat cells and that gelsolin acts on this target to inhibit the apoptotic cell death program.

A correlation study between serum adenosine deaminase activities and peripheral lymphocyte subsets in Parkinson's disease

Adenosine deaminase (ADA) and its isozyme activities in serum were measured together with peripheral lymphocyte subsets in 42 patients with idiopathic Parkinsons disease. The total and ADA 2 activities were significantly higher than normal controls (p < 0.01). As regards the peripheral lymphocyte subsets, the proportion of OKT 10+ cells (activated T lymphocytes) and the proportions of interleukin-2 receptor+ and HLA-DR+ cells (mainly activated T lymphocytes) were significantly higher than normal controls (p < 0.05, 0.01, 0.01, respectively). On the other hand, OKT 10+ cells demonstrated a significant correlation not only with total ADA but also with ADA 2 activity. These results suggest that high serum ADA activity may be involved in the pathogenesis of Parkinsons disease through peripheral T lymphocyte activation.

Phrenic nerve conduction in infancy and early childhood.

Diaphragmatic action potentials (DAPs) were mapped on the thorax bilaterally in 16 neurologically normal infants and 8 boys aged 1 to 4 years during artificial ventilation after thoracic surgery. Transcutaneous stimulation was used to activate the phrenic nerve at the supraclavicular fossa at the end of an artificial inspiration. The DAPs were of positive polarity and were recorded on the ipsilateral anterolateral chest wall over the sixth to the eighth intercostal spaces, with a maximal peak at the seventh intercostal space. The DAP latencies gradually decreased from 6 to 8 ms at birth to about 5 ms at the age of 1 year, despite an increase of conduction distance. Statistical analyses revealed that DAP amplitude did not correlate with age. The latencies and amplitudes of the DAPs displayed little interside variation. The results are valuable not only as a reference for the diagnosis of patients with phrenic nerve palsy, but also as an indicator of the normal development of the phrenic nerve.

High serum adenosine deaminase activity and its correlation with lymphocyte subsets in myasthenia gravis

Serum adenosine deaminase (ADA) activity and peripheral lymphocyte subsets of patients with myasthenia gravis (MG) were simultaneously measured. The ADA activity in MG (n = 30) was significantly higher as compared with normal control (n = 150) and multiple sclerosis (n = 12) (P less than 0.05). The ADA activity of generalized MG was higher than that of ocular MG, while a significant elevation of ADA activity was observed in grade IIB as compared with grade I of Ossermans classification (P less than 0.05). A trend of high ADA activity was demonstrated in those whose disease had advanced to a severe degree associated with unstable clinical features (P less than 0.05). In addition, there was a significant elevation of ADA activity in patients who disclosed positive anti-Ach-receptor-antibody as compared with negative one (P less than 0.05). There was no specific trend among the proportions of the subsets of peripheral lymphocytes which could reflect the severity of MG, however, the proportion of OK Ia1+ tended to be higher with advancing the grade of MG. Interestingly enough, a close correlation was found between the ADA activity and the proportion of OK Ia1+ cells (P less than 0.05). From the above results, it was concluded that high ADA may be responsible for the pathophysiology of MG through the alteration of peripheral lymphocyte function.

Antigenicity of the carbohydrate moiety of ganglioside GM3 having 3-O-acetyl ceramide

To elucidate the effect of a modification of ceramide on antigenicity of the carbohydrate of ganglioside, the reactivity of O-acetyl GM3 having 3-O-acetyl ceramide, which has been characterized as a gliomarelated ganglioside, with monoclonal antibody M2590 was examined in comparison to that of non-acetylated GM3, by means of quantitative enzyme-linked immunosorbent assay, TLC-immunostaining and liposome immune lysis assay. In all these assay systems, O-acetyl GM3 showed less activity than GM3 as follows: GM3 was detected till 0.1 nmol in TLC-immunostaining, whereas O-acetyl GM3 could not be detected even at 0.25 nmol; the GM3 reaction was approximately twofold that of O-acetyl GM3 at each diluted point in the enzyme-linked immunosorbent assay; and 20% of the liposomes containing GM3 were lysed at 6 mol%, while liposomes containing O-acetyl GM3 did not lyse at that concentration. The lesser antigenicity of the sugar moiety of O-acetyl GM3 could be ascribed to the presence of an acetyl group in the ceramide at the 3-position of sphingosine.

Occurrence of nonenzymatic N-acetylation of sphinganine with acetyl coenzyme A producing C2-H2-ceramide and its inconvertibility to apoptotic C2-ceramide.

Sphinganine, a biosynthetic precursor of ceramide, was non‐enzymatically acetylated with acetyl coenzyme A at the C‐2‐amino residue to produce C2‐H2‐ceramide (N‐acetyl sphinganine) in an organic solvent and in an aqueous solution with a high yield, where as sphingenine was only acetylated slightly. The structure of the N‐acetyl sphinganine was identified with mass spectrum, and with chromatography using an authentic N‐acetylated substance. Furthermore, the C2‐H2‐ceramide was examined for enzymatic desaturation to determine whether C2‐ceramide, a cell‐permeable ceramide responsible for apoptosis of cells, was produced, revealing an inferior substrate for H2‐ceramide desaturase of horse brain microsomes.

New blocking method for the hydroxyl group on carbohydrate. Determination of the O-acylated position of the modified glycolipid

To determine O-esterified positions, a rapid and complete acetalization to prepare an intermediate was established using ethyl vinyl ether as a new reagent. The new method was applied to O-esterified glycolipids followed by GC-MS analysis of the monosaccharide derivatives after methylation and methanolysis, revealing the derivatives with correctly substituted positions. This method was superior in terms of its shorter reaction time and complete acetalization, particularly of the N-glycolyl hydroxyl residue, to previously reported methods using methyl vinyl ether.