Chain ordering of Stratum corneum lipids investigated by EPR slow-tumbling simulation.

We investigated the chain ordering of the lipid bilayer of Stratum corneum (SC) using an electron paramagnetic resonance (EPR) spin probe method in conjunction with slow-tumbling simulation. The ordering of SC lipids was evaluated by analysis of the signals of 5-doxylstearic acid (5-DSA) spin probe incorporated into the lamellar lipids. The result obtained with the conventional method of calculating the order parameter using hyperfine values was 0.80. The value of the order parameter obtained by spectral simulation was 0.73. It was found that the conventional method of calculating the chain ordering using hyperfine values could not differentiate subtle EPR spectral changes. However, EPR slow-tumbling simulation can differentiate such subtle spectral changes. Thus, the present EPR investigation suggests that simulation provides more detail about the structure of the lipid bilayer than the conventional method.

Sequence motif-specific assignment of two [2Fe-2S] clusters in rat xanthine oxidoreductase studied by site-directed mutagenesis.

The sequence motif-specific assignment of the two distinct [2Fe-2S] clusters in rat xanthine oxidoreductase (XOR) was unequivocally established by site-directed mutagenesis of recombinant enzymes expressed in a baculovirus-insect cell system and electron paramagnetic resonance (EPR) spectroscopy. The conserved cysteine residues, including Cys-115, in the unusual C-terminal -Cys-Xaa(2)-Cys-//-Cys-Xaa(1)-Cys- motif serve as ligands to the Fe/S I center, which is probably located in close proximity to the Mo-pterin center. Other conserved cysteine residues, including Cys-43 and Cys-51, in the N-terminal plant ferredoxin-like motif serve as ligands to the Fe/S II center, which is distantly located from the Mo-pterin center. The present sequence motif-specific assignment of the Fe/S I and II centers is discussed in the light of the structural features of XOR.

Effect of surfactants on human stratum corneum: electron paramagnetic resonance study.

Electron paramagnetic resonance (EPR) spectra of nitroxide spin probes are useful for studying biological membranes and chemical-membrane interactions. Recently, we established a stripping method to remove stratum corneum (SC) for this purpose. To assess this stripping method with EPR and correlate with standard methods, we quantified the irritant effects of three types of surfactants by measurements of visual score and transepidermal water loss (TEWL), SC hydration and chromametry and studied EPR spectra measurements of surfactant-treated cadaver SC (C-SC) and stripped off SC (S-SC) on patch tested sites. 5-Doxyl stearic acid was the spin label. The order parameter S obtained from the spectra of S-SC correlated with those of C-SC and TEWL values. The results suggest that this method is capable of evaluating the fluidity of SC and correlates with the above bioengineering parameters.

Characterization of two types of mitochondrial creatine kinase isolated from normal human cardiac muscle and brain tissue.

Two types of mitochondrial creatine kinase (Mi-CK), sarcomeric (sMi-) and ubiquitous (uMi-)CKs, were isolated from normal human cardiac muscle and brain tissue, respectively, and their heterogeneity was characterized by means of isoelectric focusing (IEF). Octameric sMi-CK and uMi-CK were electrophoresed cathodic to cytoplasmic muscle-type creatine kinase isoenzyme (CK-MM) and dimeric Mi-CKs were found at the position of CK-MM on a cellulose acetate membrane. The electrophoretic mobilities of sMi-CK were similar to those of uMi-CK. Octameric sMi-CK was focused at pI 7.1-8.0 and dimeric forms at pI 6.55, 6.75, 6.85, and 6.95. New bands appearing at pI 6.65 and 6.75 after treatment of sMi-CK with carboxypeptidase B were found to be delysined forms. sMi-CK reacted with anti-sMi-CK antibodies, and the immune complexes were focused at pI 5.8. The Km value of sMi-CK for creatine phosphate (PCr) was 1.19 +/-0.20 mmol/L (mean +/- standard error), the activation energy (Ea) was 108.3+/-1.2 kJ/ mol, and the residual enzyme activity after heating at 45 degrees C for 20 min was 79.6+/-1.9%. On the other hand, octameric uMi-CK was focused at pI 7.1-7.9 and the dimeric forms were focused at pI 6.6, 6.7, 6.8, 6.9, and 7.0. Delysined forms were focused around pI 6.3, 6.4, 6.8, and 6.9. uMi-CK reacted with anti-sMi-CK antibodies, and the immune complexes were focused at pI 5.8. The Km value of uMi-CK for PCr was 1.07+/-0.03 mmol/L, Ea of uMi-CK was 110.0+/-0.9 kJ/mol, and the residual enzyme activity after heating at 45 degrees C for 20 min was 90.3+/-0.4%. The sMi-CK and uMi-CK were hybridized and the hybrid Mi-CK appeared at pI 6.78, 6.98, and 7.1-7.95. The pIs of the hybrid Mi-CK were between those of sMi-CK and uMi-CK. As described above, sMi-CK and uMi-CK were slightly different from each other with respect to the pI and some enzyme characteristics.

Tumoral calcinosis: a case report with an electron microscopic study.

A 68-year-old woman developed large subcutaneous masses on her abdomen and thighs after a bruise sustained in a traffic accident. She had severe pain when sitting up straight. Histological examination revealed calcified tissues in the entire dermis of the injured areas. On electron microscopy, crystalline materials were observed in the dermis, which seemed to be formed by the deposition of hydroxyapatite on unusual proteoglycan. In a vessel wall, a thick, layered basement membrane was observed. This suggests that vascular injury and subsequent hypoxia play a role in the process of calcinosis. We performed a partial resection with good results in alleviating the patient's pain.

Inter-laboratory difference among eleven clinical laboratories in the Okayama City area.

The aim of the present study was to find the cause of inter-laboratory differences in laboratory test data and to examine whether control assessment helps to reduce inter-laboratory differences. Blood and serum samples of one healthy subject and one subject with liver cirrhosis were analyzed by 11 laboratories in the Okayama City area. No differences were found in the assay units of 26 tests surveyed. However, considerable differences were observed in test data, reference interval, and clinical level (CL), though most laboratories pointed out that the test data for the normal subject was within the reference intervals and those for the patient with liver cirrhosis showed abnormalities in tests for liver function. The difference in reference intervals was serious in the tests of direct bilirubin (D-Bil), thymol turbidity test (TTT), alkaline phosphatase (ALP), gamma-glutamyltranspeptidase (GGTP) and choline sterase. Marked differences in CLs were found in the tests of D-Bil, TTT, ALP, GGTP, creatine phosphokinase, amylase, heavy density lipoprotein cholesterol and white blood cell count. However, three hepatologists independently suggested that such inter-laboratory differences would not seriously affect a clinical decision on the disease status of the cirrhotic patient. Most tests that showed a trend error in a recent quality control survey appeared to have the same trend in the present study. These results indicate that inter-laboratory differences occur at various levels and control assessment are helpful in establishing, and therefore reducing, the level of inter-laboratory differences.

Expression of Xenopus laevis translation initiation factor 4E (eIF-4E) by baculovirus-insect cell system.

A gene encoding Xenopus laevis eIF-4E was cloned into a transfer vector, and its gene expression was attempted in cells of E. coli, yeast and insect. Effective expression of the active eIF-4E was achieved in the soluble fraction of the insect cell Sf9, which was infected with the recombinant baculovirus. Overexpression of the eIF-4E protein caused remarkable change in the shape of the cells.

A gamma Gly-268 to Glu substitution is responsible for impaired fibrin assembly in a homozygous dysfibrinogen Kurashiki I.

A new type of gamma Gly-268 (GGA) to Glu (GAA) substitution has been identified in a homozygous dysfibrinogen by analyses of the affected polypeptide and its encoding gene derived from a 58 year-old man manifesting no major bleeding or thrombosis. The functional abnormality was characterized by impaired fibrin assembly most likely due to failure to construct properly aligned double-stranded fibrin protofibrils. This presumption was deduced from the following findings: (1) Factor XIIIa-catalyzed cross-linking of the fibrin gamma-chains progressed in a normal fashion, indicating that the contact between the central E domain of one fibrin monomer and the D domain of another took place normally; (2) Nevertheless, factor XIIIa-catalyzed cross-linking of the fibrinogen gamma-chains was obviously delayed, suggesting that longitudinal association of D domains of different fibrin monomers, ie, D:D association was perturbed; (3) Plasminogen activation catalyzed by tissue-type plasminogen activator was not as efficiently facilitated by polymerizing fibrin monomer derived from the patient as by the normal counterpart. Therefore, gamma Gly-268 would not be involved in the 'a' site residing in the D domain, which functions as a complementary binding site with the thrombin-activated 'A' site in the central E domain, but would be rather involved in the D:D self association sites recently proposed for human fibrinogen. Thus, the gamma Glu-268 substitution newly identified in this homozygous dysfibrinogen seems to impair proper alignment of adjacent D domains of neighboring fibrin molecules in the double-stranded fibrin protofibril, resulting in delayed fibrin gel formation.

Dermatofibrosarcoma protuberans of the sole.

A 42-year-old Chinese man presented with a dome-shaped, red, tender, bony-hard tumor on his right sole. Histological examination revealed tumor nests which consisted mainly of fibroblast-like cells with large, spindle-shaped nuclei and histiocyte-like cells extending from the upper dermis to the subcutaneous tissue. The tumor cells showed both a storiform and a cartwheel pattern. The location of a dermatofibrosarcoma protuberans on the plantar aspect of the foot is exceptional.

Basal cell carcinoma of the vulva with lymph node and skin metastasis--report of a case and review of 20 Japanese cases.

A 79-year-old Japanese woman who had basal cell carcinoma presenting as a large ulcer on her vulva with lymph node and skin metastasis is described. Histological examination revealed that tumor nests with peripheral palisading invaded deeply into the subcutaneous tissue and were accompanied by marked mucinous changes and fibrous reaction. Vascular invasion was also observed. There were inguinal lymph node metastases and two papular skin metastases on her right thigh. The primary tumor and the metastases were excised. The defect was repaired by bilateral gracilis musculo cutaneous flaps and a skin graft. We surveyed the literature and found 20 cases of metastasizing basal cell carcinoma in Japan.

A case of giant cell tumor of the tendon sheath developing on the palm.

A 22-year-old Japanese woman with a giant cell tumor of the tendon sheath on the palm is described. The location of the tumor is rare. Light microscopically, the tumor consisted of round or polygonal cells, giant cells, and foam cells. Electron microscopically, histiocyte-like cells and fibroblast-like cells were mainly observed, suggesting that the tumor may have originated from the synovial membrane.

Atypical melanosis of the foot. A report of three cases in Japanese populations.

BACKGROUND: Acral lentiginous melanoma is the most common type of malignant melanoma in the Japanese population. In most instances, classic acral lentiginous melanoma or acral lentiginous melanoma in situ can be readily distinguished from other benign pigmented lesions by virtue of its atypical clinical appearance. OBSERVATION: We present three cases of seemingly malignant pigmented lesions on the foot, all arising in Japanese females. Clinically, the lesions are characterized by irregular borders and variegated pigmentation closely mimicking those of acral lentiginous melanoma in situ. However, the histologic findings revealed only focal slight melanocytic hyperplasia with minimal cytologic atypia along the basal layer. Despite the malignant clinical features, thorough histologic examination failed to disclose any area with significant melanocytic atypia or evidence of malignancy. CONCLUSIONS: To the best of our knowledge, similar lesions with the clinical appearance of melanoma in situ and completely lacking histologic evidence of malignancy have not been reported. We, therefore, prefer to designate these lesions as atypical melanosis of the foot to highlight the clinically apparent atypical findings and to distinguish them from malignant melanoma in situ of the foot.

Electrophoretic and kinetic characterization of three variants of soluble cytoplasmic L-alanine:2-oxoglutarate aminotransferase in human liver tissue.

Three common variants of soluble cytoplasmic L-alanine:2-oxoglutarate aminotransferase (ALT, EC 2.6.1.2), sALT 1, 2-1 and 2, were isolated from normal human liver, and characterized by electrophoretic and kinetic analyses. The isoelectric point of sALT 1 was pH 6.45. sALT 2-1 was focused into three bands with pl 6.1, 6.2 and 6.45; sALT was focused into one band with pl 6.1. The electrophoretic mobilities of sALTs altered to the fast beta-globulin fraction after aging or papain treatment. Ammonia was produced during the latter, and the altered migration was considered to be caused by deamidation of sALT. The relative molecular mass of each of the enzymes was 110,000. Minor differences in the apparent Km values among the multiple forms for both L-alanine and 2-oxoglutarate were observed after incubation with 100 mumol/L of pyridoxal phosphate (PALP). PALP stimulation of the enzyme activities was also different. sALT 1 was more stable than sALT 2-1 and 2 after heat and urea treatments. In human sera from 1065 adult Japanese, sALT 2-1, a heterozygote form of sALT 1 and 2, was dominant.

Mitochondrial creatine kinase as a tumor-associated marker.

Sera from 167 patients with malignant tumor were examined with regard to mitochondrial creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2, CK). The CK (CKm) was detected in 66 cases (40%), in 42 of 96 men and in 24 of 71 women. Seventeen patients had hepatic carcinomas and 14 gastric carcinoma. CKm was also present in sera from patients with malignancy of the lung, the mammary gland etc. In the CKm positive group, about 85% had metastatic and/or infiltrating malignancy, and about 90% were undergoing chemotherapy. The figures resembled those in the CKm negative group. Mortality rates in the CKm positive group were 1.7 times higher than in the CKm negative group. CKm may be a marker of some biological factors leading to and/or resulting from a poor prognosis and not simply of primary or metastatic tumors. Most of the total serum CK activities in the CKm positive group were within normal limits. CKm activities in the gastric carcinoma group were higher than those in other groups, particularly in comparison to the liver carcinoma group.

A new creatine kinase found in mitochondrial extracts from malignant liver tissue.

A new creatine kinase (CK, ATP: creatine N-phosphotransferase, EC 2.7.3.2) band was found in mitochondrial extracts prepared from malignant liver tissue. By agar gel electrophoresis the new band (CKmLT) migrated between CK-MM and a sample application point. The CK band was not found in mitochondrial extracts of normal liver, cytosolic extracts of normal and malignant liver homogenates. We thus presume that the origin of CKmLT is from mitochondria of malignant liver tumors. CKmLT reacted with anti-mitochondrial CK antibodies so that its antigenicity was similar to that of mitochondrial CK. Physico-chemical characteristics were similar to those of the polymeric form of the normal liver mitochondrial CK (CKmL1); i.e. relative molecular mass (320 000 to 350 000), heat stability, and the behavior in 2 mol/l urea. CKmLT migrated to the CKmL1 and the MM position after long-term storage at -20 degrees C. CKmLT did not demonstrate affinity to concanavalin A, and there was no significant change in the electrophoretic mobility of CKmLT following neuraminidase treatment.

The origin of a cathode-migrating creatine kinase found in serum from a cancer patient.

The origin of an atypical creatine kinase (CK, ATP:creatine N-phosphotransferase, EC 2.7,3.2) migrating cathodic to the MM position found in the serum of a cancer patient was studied. The electrophoretic mobility of the atypical CK is similar to that of the fast-moving cathodal mitochondrial CK. The relative molecular mass was estimated to be approximately 350000, and was similar to that of the fast-moving cathodal mitochondrial CK. The atypical CK reacted with anti-human mitochondrial CK antibody. It is therefore suggested that the atypical CK is of mitochondrial origin. After incubation in 2 mol/l urea, the enzyme was converted into a new form migrating to the MM position. The conversion was observed in liver mitochondrial CK but not heart mitochondrial CK. The residual CK activity after heating at 56 degrees C for 60 s was 77%, and the apparent Km value for creatine phosphate at 30 degrees C was about 0.27 mmol for the atypical CK. These characteristics were very similar to those of the liver mitochondrial CK, because the data from the enzyme determined at the same time were 75% for residual enzyme activity to heat, and 0.24 mmol for apparent Km value. Therefore liver mitochondria are suggested to be the source of the atypical CK.

Characteristics of mitochondrial creatine kinases from normal human heart and liver tissues.

Mitochondrial creatine kinases (CKs, ATP:creatine N-phosphotransferases, EC 2.7.3.2) were isolated from normal human heart and liver, and their characteristics were compared. The electrophoretic patterns of the extracted enzymes exhibited two forms both migrating cathodic to CK-MM. The fast-moving cathodal form is the major form and the slow-moving cathodal form is the minor one. Incubation of the heart mitochondrial CK at 37 degrees C in normal human serum for 7 h and of the liver mitochondrial CK at 26 degrees C for 1 h in 2 mol/l urea, converted the fast-moving form into the slow-moving one, and finally into a third form migrating in the MM position. The relative molecular masses were estimated to be approximately 350,000 for the major form, and 80,000 for the minor and the third forms. The electrophoretic mobility and molecular weight of the third form were identical to those of the CK-MM; however, the third form was distinguished from CK-MM by its different antigenicity. Thus, three forms were ultimately recognized as mitochondrial CKs by electrophoretic mobilities and molecular weights. The liver mitochondrial CK reacted with anti-human heart mitochondrial CK antibody, thus these two isoenzymes could not be discriminated by their antigenicities. The liver mitochondrial CK was more stable to heat and had higher apparent affinity for creatine phosphate than the heart mitochondrial CK.