Kenjiro Murata

Decreased Fluidity of Polymorphonuclear Leukocyte Membrane in Streptozocin-Induced Diabetic Rats

Using flow cytometry with the excimer-forming lipid technique with pyrenedecanoic acid, we measured membrane fluidity of polymorphonuclear leukocytes (PMNs) from 20 streptozocin (STZ)-induced diabetic rats. Diabetes mellitus was induced in male Sprague-Dawley rats (body wt 243 ± 11 g) with an injection of 25 mg/kg i.v. STZ. Membrane fluidity of PMNs was significantly lower at 2 wk after the STZ injection when serum glucose reached the plateau (31.1 ± 5.8 mM), and after 3 wk, membrane fluidity remained unchanged. In 7 STZ-resistant rats for which serum glucose was <10 mM at 2 wk after the STZ injection, gradual normalization in membrane fluidity was observed. PMN membrane fluidity at each week correlated inversely with respective serum glucose levels 1 wk previously (r = −0.76) but not with serum lipid levels. Cross-incubation studies ascribed this observation to factors in the diabetic rat serum. Glycosylated protein, which was separated from diabetic rat serum, decreased membrane fluidity of control rat PMNs. Human diabetic subjects have an increased risk for infection, which may be due partly to altered membrane fluidity of their PMNs.

Difference in changes of membrane fluidity of polymorphonuclear leukocytes stimulated with phorbol myristate acetate and formyl-methionyl-leucyl-phenylalanine: role of excited oxygen species.

Polymorphonuclear leukocytes (PMN) were stimulated with phorbol myristate acetate (PMA) and N‐formyl‐methionyl‐leucyl phenylalanine (FMLP) to clarify the role of excited oxygen species in inducing changes of membrane fluidity. Membrane fluidity was assessed by the excimer‐forming lipid technique using pyrenedecanoic acid and flow cytometry. Membrane fluidity of PMN decreased following stimulation with PMA, and the extent of decrease was both time‐ and dose‐dependent. FMLP at 10‐5 M induced a decrease, while FMLP at 10‐7 M induced a rapid increase. On stimulation with 10‐7 M FMLP as well as in a resting condition, the change of membrane fluidity of PMN from patients with chronic granulomatous disease (CGD) was similar to that of normal PMN. However, on stimulation with PMA or 10‐5 M FMLP, CGD PMN did not show a significant decrease. In addition, normal PMN incubated with catalase inhibited the decrease. These findings suggest that the generation of excited oxygen species, particularly of H2O2, is important in inducing a decrease of PMN membrane fluidity.

Abnormal Membrane Fluidity as a Cause of Impaired Functional Dynamics of Chemoattractant Receptors on Neonatal Polymorphonuclear Leukocytes: Lack of Modulation of the Receptors by a Membrane Fluidizer

ABSTRACT: Membrane properties associated with chemoattractant-mediated cellular responsiveness of neonatal polymorphonuclear leukocytes (PMN) were analyzed using n-formylmethionyl-leucyl-phenylalanine. Inasmuch as aliphatic alcohols as a membrane fluidizer can enhance the chemoattractant binding and affect subsequent cellular responsiveness in adult PMN, neonatal PMN were studied for such properties by their treatment with iso-propyl alcohol, an aliphatic alcohol. The alcohol (<2.5%) treatment enhanced the N-formylmethionyl-leucyl-phenylalanine binding to adult PMN, but there were no changes in the N-formylmethionyl-leucyl-phenylalanine binding to neonatal PMN. Although the N-formylmethionyl-leucyl-phenylalanine-induced subsequent responsiveness including migration, lysosomal enzyme release and superoxide anion production were modulated by the alcohol treatment in adult PMN, there was no such modulation in neonatal PMN. Because membrane fluidity is largely involved in the regulation of the receptor functions, the membrane fluidity of neonatal PMN was next measured by an excimer-forming lipid technique in flow cytometry. The membrane fluidity value (0.45 ± 0.037) of neonatal PMN was lower than that (0.74 ± 0.072) of adult PMN (p < 0.01). Although the aliphatic alcohol enhanced the membrane fluidity of adult PMN, it did not affect the membrane fluidity of neonatal PMN. We conclude that there is abnormal membrane fluidity as a cause of impaired functional dynamics of the chemoattractant receptors, which appears to underlie the defective modulation of cell functions by the membrane fluidizer in neonatal PMN.

PURIFICATION OF FACTOR XIa INHIBITOR FROM HUMAN PLATELETS

An inhibitor of activated factor XI (FXIa) in human platelets was recently identified as an amyloid beta-protein precursor (APP). We purified an FXIa inhibitor (XIaI) from the supernatant of activated human platelets, and assessed its inhibitor activity toward FXIa amidolytic activity. Approximately 90 micrograms of XIaI that cross-reacted with anti-APP antibody was obtained from two hundred units of platelet suspension by employing a six-step column chromatography procedure. The molecular weight of the purified XIaI was 94,000. The Ki value of XIaI to factor XIa was 526 +/- 120 pM, and the inhibition was enhanced by the addition of ZnCl2. The amino-terminal sequence of XIaI was L-E-V-P-T-D-G-N-A-, which is identical to that for the leucine (N18) to alanine (N26) sequence of APP751 and the amino-terminal sequence of protease nexin-2.

Elevation of Factor Xla—α1-Antitrypsin Complex Levels in NIDDM Patients With Diabetic Nephropathy

Excess activated FXIa in plasma indicates hypercoagulability in the early contact phase. We have already developed methods for detecting the hypercoagulable state in clinical samples by our ELISA for complexed FXIa and α1AT, which has been confirmed to be the predominant inhibitor of FXIa. Indiabetes, whether the activation of FXI is associated with the development of vascular complications remains unknown, although various hemostatic abnormalities have been described. We tested the complexed FXIa-α1AT level in 45 NIDDM patients, who were divided into three groups according to the development of diabetic nephropathy, as assessed by UAE. Normoalbuminuria was defined as UAE <15 μg/min, microalbuminuria as UAE in the range of 15–200 μg/min, and albuminuria as UAE >200 μg/min. In the patients as a whole, FXIa-α1AT and TAT levels were significantly increased compared with these levels in age-matched control subjects (17.3 ± 5.7 vs. 12.4 ± 2.4 ng/ml and 2.67 ± 1.23 vs. 1.93 ± 0.45 ng/ml, respectively). No significant difference was observed between FXIa-α1AT levels in the control subjects and in the normoalbuminuric group (13.0 ± 2.1 ng/ml; n = 19). However, in the microalbuminuric (17.9 ± 3.9 ng/ml; n = 16) and albuminuric (24.1 ± 5.4 ng/ml; n = 10) groups, FXIa-α1AT levels were significantly increased compared with those in the control and normoalbuminuric group. The TAT level was not correlated with FXIa-α1AT, and no significant differences in its levels were found among these diabetic groups. We agree with the widely held belief that diabetic nephropathy is strongly related to widespread microangiopathy, and suggest that the FXIa-α1AT level reflects vascular complications that occur concomitantly with such nephropathy. Therefore, we conclude that plasma FXIa-α1AT levels may be of potential pathophysiological or clinical importance for detecting early diabetic microangiopathy.

Measurement of membrane fluidity of polymorphonuclear leukocytes by flow cytometry

A method was established for measuring membrane fluidity of polymorphonuclear leukocytes (PMN) by the excimer-forming lipid technique with pyrenedecanoic acid in flow cytometry. When cells were labeled, the use of 2-25 microM of pyrenedecanoic acid provided similar results. Neither the removal of the unincorporated pyrenedecanoic acid nor adjustment of PMN counts exhibited any effect. By the gate analysis method, membrane fluidity of PMN could be measured with 100 microliters of heparinized whole blood in a short time and results with PMN in whole blood was similar to those with purified PMN. Therefore, purification and count adjustment of PMN could be omitted. By this method, membrane fluidity of PMN, which were treated with membrane fluidizer, was measured successfully. This method could be applied to the study of PMN function in various diseases.

An epidemiological study of methicillin-resistant Staphylococcus aureus (MRSA) isolated from medical staff, inpatients, and hospital environment in one ward at our hospital.

Methicillin‐resistant Staphylococcus aureus (MRSA) is one of the most important causative microorganisms for nosocomial infections. Recently, the incidence of isolation of MRSA has been increasing every year in Japan and is, notably, much more frequently found in inpatients than in outpatients. Therefore, we have done epidemiological studies of MRSA isolated from medical staff, inpatients, and the hospital environment in one ward of our hospital. Thereafter, we examined the antibiotic susceptibility (ABPC, DMPPC, CET, CMZ, IPM, GM, MINO, OFLX, EM, CLDM, VCM), phage typing, and coagulase typing of these MRSA MRSA were isolated more frequently from anterior nares of inpatients than from doctors and nurses. MRSA were isolated more frequently from the environment near carriers of MRSA

Murine monoclonal antibodies to human factor XI

Murine monoclonal antibodies to human factor XI (F.XI) are described. The monoclonal antibodies (2-1, 4-1, 7-1 and 10-1) consisted of IgG1. 4-1 inhibited the activation of F.XI completely in the presence of high molecular weight kininogen and kaolin and the others did so partially, whereas these antibodies had no effect on the activation of F.XI with activated factor XII (beta-XIIa). Four antibodies had no effect directly on the amidolytic activity of activated F.XI (F.XIa). 10-1 inhibited the activation of factor IX in coagulant assay for F.XIa by Mannhalter. And 4-1 and 7-1 did so partially, whereas 2-1 did not. In immunoblotting analysis, all antibodies bound to F.XI, its reduced form and F.XIa. All were directed against the heavy chain of F.XI. All antibodies recognized F.XIa-alpha 1 antitrypsin complex.

Increased accumulation of nonenzymatically glycated fibrinogen in the renal cortex in rats

We have determined that the nonenzymatic glycation of fibrinogen altered its biological functions in vitro. Thrombin clottability of rat fibrinogen incubated with glucose decreased with increasing incubation time, but was not affected by the glucose concentration. Fibrin prepared from glycated fibrinogen showed a significant resistance in susceptibility to plasmin degradation. We also examined the in vivo distribution of glycated fibrinogen in renal cortex. Iodine labeled rat glycated or unglycated fibrinogen was injected into streptozocin-induced diabetic and control rats. No appreciable difference in the plasma disappearance rate in control rats was observed (half-lives in hours for glycated, 25.6 +/- 0.37; unglycated, 26.1 +/- 0.74). The radioactivity of fibrinogen retained to the renal cortex was calculated 24-hours after injection. In both control and diabetic rats, the retention rate of glycated fibrinogen in renal cortex was significantly higher than that of the unglycated. These results suggest that glycated fibrinogen may occur in a more resistant form to plasmin digestion with fibrin deposition as confirmed in in vitro studies. Therefore, we suggest that glycated fibrinogen may partly contribute to the development of diabetic microangiopathic lesions such as glomerulosclerosis.

An increase in polymorphonuclear leucocyte chemotaxis accompanied by a change in the membrane fluidity with age during childhood

Leucocyte membrane fluidity affects cell deformability as well as the accessibility of receptors and the degree of their exposure on the membrane. These effects modulate the subsequent cellular responsiveness. We have assayed membrane fluidity of polymorphonuclear leucocytes of children, using an excimer‐forming lipid technique in flow cytometry. and evaluated its relation to their chemotaxis to a formyl peptide. We report that membrane fluidity and chemotaxis of polymorpho nuclear leucocytes are increased with age. These findings may have important implications for the physiological processes in polymorphonuclear leucocyte motility during childhood.