Fibrinogens Kosai and Ogasa: Bbeta15Gly-->Cys (GGT-->TGT) substitution associated with impairment of fibrinopeptide B release and lateral aggregation.

We found two heterozygous dysfibrinogenemias, designated fibrinogen Kosai and fibrinogen Ogasa. Kosai was associated with arteriosclerosis obliterans but Ogasa showed no bleeding or thrombotic tendencies. The plasma fibrinogen concentrations from the two propositi (Ogasa and Kosai) were much lower when determined by the thrombin-time method (0.94 and 1.06 g L(-1), respectively) than when determined by the immunological method (2.87 and 2.72 g L(-1), respectively). We performed DNA sequencing and functional analyses to clarify the relationship between the structural and functional abnormalities. Genetic analysis of PCR-amplified DNA from the propositi identified the heterozygous substitution Bbeta15Gly-->Cys (GGT-->TGT). Western blotting analysis of purified fibrinogen revealed the existence of albumin-fibrinogen complexes. Functional analyses indicated that compared with the normal control, the propositi's fibrinogen released only half the normal amount of fibrinopeptide B and showed markedly impaired polymerization. In addition, the observation of thinner fibers in fibrin clots (by scanning electron microscopy) indicated markedly defective lateral aggregation in the variant fibrinogens. The impaired functions may be due to the substitution of Cys for Bbetao15Gly plus the existence of some additional disulfide-bonded forms.

A phylogenetic-tree analysis elucidating nosocomial transmission of hepatitis C virus in a haemodialysis unit.

Nosocomial transmission of hepatitis C virus (HCV) subtype 1b involving 11 haemodialysis patients occurred in a haemodialysis unit in Japan in March 2000. Sequencing of the HCV-E1 region (411-bp) and phylogenetic-tree analysis showed near identity between HCV isolates derived from these patients and a haemodialysis patient who was known to be HCV-positive. The mode of transmission could not be conclusively established, but retrospective analysis suggested that the sharing of contaminated multidose vials of heparin-saline solutions, which were prepared in the Haemodialysis Center using accidentally contaminated instruments such as needles, may have been responsible for the outbreak. To prevent transmission of HCV in a haemodialysis unit, it may be important to observe strictly standard precautions and to prepare all medications in the Pharmacy. After these measures were taken, no new seroconversions and no new nosocomial transmissions of HCV have been observed in our haemodialysis unit.

Hypofibrinogenemia associated with a heterozygous C-->T nucleotide substitution at position -1138 BP of the 5'-flanking region of the fibrinogen A alpha-chain gene.

We found a novel genetic abnormality, heterozygous C-->T nucleotide substitution at position -1138 bp in the 5'-flanking region of the fibrinogen A alpha gene, in patients with hypofibrinogenemia. Luciferase reporter assay using the pGL3-basic vector and CHO cells indicates that the transcriptional activity of a vector incorporated with -1138T was reduced to one-third that of a vector incorporated with -1138C. These results suggest that the region adjacent to the -1138C bp of the 5'-flanking region of the fibrinogen A alpha gene is one of the most crucial sites for the transcription of the fibrinogen A alpha gene.

Tryptophan glycoconjugate as a novel marker of renal function.

PURPOSE: Neither serum creatinine concentration nor creatinine clearance assess renal function accurately. Serum creatinine concentration is affected by muscle mass, and the creatinine clearance overestimates the glomerular filtration rate because of tubular secretion of creatinine. The present study was designed to determine whether serum concentrations of 2-(alpha-mannopyranosyl)-L-tryptophan (MPT), a tryptophan glycoconjugate, can be used as a marker of renal function. METHODS: Clearances of MPT and of inulin were compared in normal rats and in rats with cisplatin-induced acute renal failure. We also compared the clearances of MPT and of creatinine with inulin clearance in 25 patients with chronic renal disease. Serum concentrations of MPT and creatinine as a function of MPT clearance were determined in 108 patients with chronic renal disease. RESULTS: There was strong linear correlation between clearances of MPT and inulin in rats (r = 0.97) and humans (r = 0.87), indicating that renal handling of MPT is similar to that of inulin. In humans, linear regression analyses indicated that MPT was a better indicator of inulin clearance than was creatinine clearance. At the same level of renal function, serum creatinine concentrations tended to be lower in patients with less muscle mass (as indicated by a urinary creatinine excretion <1,000 mg in 24 hours) than in those who excreted >1,000 mg in 24 hours, whereas serum MPT concentrations were not affected by creatinine excretion. CONCLUSION: MPT clearance can replace inulin clearance in the clinical setting. The serum MPT concentration is an accurate measure of renal function even in patients with diminished muscle mass, and thus is a better indicator of renal function than is the serum creatinine concentration.

[The application of end user computing (EUC) for detection of lipoprotein lipase gene abnormality].

Lipoprotein lipase (LPL) is an enzyme digesting lipoprotein triglyceride (TG) in peripheral blood vessels. Most patients with LPL deficiency show very high plasma TG and low HDL-C. To establish an effective computer-based screening system to identify individuals with genetic LPL disorders, we selected 50 subjects whose plasma TG was over 350mg/dl and HDL-C was lower than 35mg/dl from patients at Hamamatsu University Hospital. We applied End User Computing (EUC) of our laboratory system to select high risk subjects with LPL gene abnormalities. Polymerase chain reaction (PCR) products from LPL gene exons 2-9 were screened by single-strand conformation polymorphism (SSCP), direct DNA sequence analysis and restriction fragment length polymorphism (RFLP). We found a novel missense mutation (1223C-->G, S323C) in LPL gene exon 7 from three subjects. By PCR-mediated site-directed mutagenesis and restriction digestion, the three subjects were found to be heterozygous. In addition, we identified two other common mutations in Japanese employing the RFLP method. One was the 1595C-->G (S447X) in exon 9 from six subjects, two homozygous and four heterozygous individuals. The other was a mutation of intron 3 (C-->T transition) from four heterozygous subjects. Using EUC screening method, we detected genetic LPL abnormalities more easily. The frequency of the LPL gene mutation in the 50 high-risk subjects was 26%, and was estimated to be one out of 2,000 patients at our clinic. Using the EUC system to screen for LPL mutations was established to be an effective computer-based screening system to identify individuals with genetic abnormalities.

[Evaluation of screening methods for cholinesterase variants from daily laboratory data].

The genetic variants of the cholinesterase (ChE) are frequently misdiagnosed as a liver dysfunction. We compared three extraction methods for screening of ChE abnormalities. Employing these three methods, total 31 cases were found to be genetic abnormalities from 2985 patients of Hamamatsu University Hospital. We picked up 11 of candidates with low enzyme activities less than 100U/l as group 1 using the first method and effectively detected 9 cases (82%) with genetic abnormalities. The second extraction method was based on the ratio between Albumin (Alb) and ChE and subsequently, 48 of high risk patients were picked up as group 2 and 28 cases (58%) showed genetic abnormalities. Furthermore, all cases of group 1 were contained in the second group. The third method was based on the discrimination function from Alb and total cholesterol (TC) as group 3 and 32 cases were picked up. Fourteen cases (44%) out of them showed the genetic abnormalities using this method, and surprisingly, 13 cases (93%) of them were estimated to be K-variant. Although the three methods showed the different characteristics to extract genetic abnormalities of ChE, the second extraction method could pick up the largest population with genetic abnormalities. Further phenotypic extraction methods should be compared to understand the relationship between phenotype and genotype.

Estimation of the gene frequency of aceruloplasminemia in Japan.

Aceruloplasminemia is a newly recognized autosomal recessive disorder of iron metabolism that causes neurodegeneration of the retina and basal ganglia as well as diabetes mellitus. We screened the serum ceruloplasmin concentrations of 4,990 healthy adult individuals. Subsequent sequence determination of the mutant alleles showed three mutations (5-bp insertion in exon 7, one heterozygote, one-bp deletion in exon 14, two heterozygotes, nonsense mutation in exon 15, one homozygote and two heterozygotes). The gene frequency was 70/100,000. In Japan, the incidence of aceruloplasminemia was estimated to be approximately 1 per 2,000,000 in the case of nonconsanguineous marriages.

[Recurrent pulmonary infarction associated with familial protein S deficiency type III].

A 38-year-old woman was admitted to our hospital because of recurrent chest pain and fever. Chest X-ray films and computed tomograms showed subpleural consolidation containing small cavity-like opacities. Open lung biopsy revealed non-infectious abscess and vessels with organizing thrombus. The patient was given a diagnosis of pulmonary infarction due to the existence of deep venous thrombosis. Coagulation studies demonstrated that she had decreased plasma protein S activity, whereas her free and total protein S antigen levels were normal. Because her mother and maternal uncle and aunt also demonstrated decreased protein S activity with normal plasma protein S antigen levels, the patient was considered to be affected by familial protein S deficiency type III.

[Difference of blood cell counts with reference blood cell counters in different makers: Part II].

This is the second report of evaluation on the difference of blood cell counting among different automated blood cell counters in Japan. We tested reference blood cell counters of 6 different companies: Coulter, Sysmex, Bayer-Sankyo, Nihon Kohden, Horiba and Dainabot. Forty ml of whole blood were taken from 3 healthy persons and EDTA-2K anticoagulated blood samples (Sample 1, 2 and 3) were sent to each company to determine blood cell counts with a reference automated counter. As a result, the following items showed more than 10% difference among makers: RBC between Dainabot and Horiba in Sample 3, hematocrit values between Coulter and Dainabot in Sample 2, WBC between Nihon Kohden and each of three makers (Sysmex, Horiba and Dainabot) in all 3 samples and that between Bayer-Sankyo and each of two makers (Sysmex and Horiba) in Sample 3 and platelet count between Dainabot and each of 3 makers (Bayer-Sankyo, Nihon Kohden and Horiba) in all 3 samples. The following items showed difference between 5 and 10%: MCV between Coulter and each of two makers (Bayer-Sankyo and Horiba), WBC between each of two makers (Coulter and Nihon Kohden) and each of other 4 makers, and platelet count between each of two makers (Nihon Kohden and Horiba) and each of 3 makers (Coulter, Sysmex and Bayer-Sankyo). Recently Japanese Committee for Clinical Laboratory Standards proposed minimum clinical allowance of blood cell count as follows: hemoglobin 3%, RBC 4%, MCV 4%, WBC 7% and platelet count 10%. It is suggested that all of the items showing the difference more than above allowance among makers should be improved for clinical use to have good external quality control in blood cell counting by automated instruments.

Automated homogeneous liposome-based assay system for total complement activity.

We developed an automated homogeneous immunoassay, based on immune lysis of dinitrophenyl (DNP)-labeled liposomes, for measuring total complement activity. Liposome lysis caused by complement activity was detected spectrophotometrically from entrapped glucose-6-phosphate dehydrogenase activity. Complement activity in human sera was quantified by comparison with a calibration curve. For ease of application to fully automated routine clinical analyzers, we adopted a two-reagent system, one reagent containing a homogeneous population of small DNP-labeled liposomes and one containing antibody/substrate. This system required calibration only once a week. Within-run and between-run CVs were 0.4-1.3% (n = 10) and 1.8-4.7% (n = 10), respectively. Serum results were linear upon dilution (with saline) over a twofold range. Bilirubin, hemoglobin, Intrafat, and serum proteins such as rheumatoid factor, M protein, IgG, and IgA did not affect the assay results. The results (y) correlated well with those from a hemolytic complement activity test (x): y = 1.05x - 1.14, r = 0.92, on 66 samples in the range < 10- > 50 kU/L. This method should therefore be of great use for the determination of complement activity.

A hydrophilic tetrahydro-beta-carboline in human urine.

A substance that exhibited a tryptophan-like fluorescence peak at 354 nm on excitation at 295 nm at neutral pH was isolated from human urine. This compound was determined by visible-light absorption spectroscopy, fluorescence spectroscopy, 1H and 13C NMR spectroscopies, and FAB-MS to be 1-(1',2',3',4',5'-pentahydroxypentyl)-1,2,3,4-tetrahydro-2-carboli ne-3- carboxylic acid. This compound, named tetrahydropentoxyline, is a new type of hydrophilic tetrahydro-beta-carboline, and its elution position was between those of 4-pyridoxic acid and kynurenic acid on C18 reversed-phase HPLC. The amount of tetrahydropentoxyline excreted in the urine of normal subjects [n = 21; age, 45 (SD 20) years] was about 5.2 (SD 1.0) mg per day.

Localization in the fibrinogen gamma-chain of a new site that is involved in the acceleration of the tissue-type plasminogen activator-catalysed activation of plasminogen.

In previous publications [e.g. Voskuilen, Vermond, Veeneman, Van Boom, Klasen, Zegers & Nieuwenhuizen (1987) J. Biol. Chem. 262, 5944-5946] we have shown that fibrin(ogen) chain fragment A alpha-(148-160) contains a site that contributes to the acceleration of Glu-plasminogen activation by tissue-type plasminogen activator (t-PA). In contrast with fibrin, this peptide, however, does not enhance the rate of mini-plasminogen activation. Therefore, possibly more stimulatory sites than A alpha-(148-160) are present in fibrin. In the present investigation we have localized a possible second type of stimulatory site in the fibrin(ogen) molecule. A whole CNBr digest of fibrinogen was applied to a Bio-Gel P-2 column run in water, pH 4. Two peaks with stimulatory activity were observed, one at the void volume and one between the void volume and the total volume. The former contained the previously described stimulating fragment FCB-2 [which comprises A alpha-(148-160)]; the latter had not been observed before and was characterized further. The stimulating material in the low-M(r) fraction of the Bio-Gel P-2 column was precipitated at pH 8.3 in a virtually pure form. It has a high tryptophan content, and an M(r) of 6500 as assessed by SDS/PAGE. On reduction, a main band of M(r) 2500 is seen, plus a weakly staining band of M(r) 4000. These properties plus the amino acid sequence data identify the fragment as FCB-5. FCB-5 consists of two chains, i.e. gamma-(311-336) and gamma-(337-379), linked by a single disulphide bond between Cys-gamma-326 and Cys-gamma-339. Both these chains and the disulphide bond appear to be essential for rate enhancement. FCB-5 enhances the activation rates of Glu-, mini- and micro-plasminogen, with all five kringles, only kringle V and without kringles respectively. FCB-5 binds t-PA, but none of the plasminogen forms binds to FCB-5. This indicates that the rate enhancements induced by FCB-5 are due to an effect on t-PA.

Cysteine proteinase inhibitor levels in semen.

New proteinase inhibitors have recently been found in seminal plasma which specifically inhibit cysteine proteinases such as ficin, papain, cathepsin H and cathepsin B. The inhibitors consist of two components, In-A and In-B, both of which are effective in native semen. In the present work, a method of assay is devised to determine activities of both components separately. Under conditions in which In-B is denatured completely while In-A is not, inhibitory activity of In-A alone is determined. The mean In-A inhibitory level is 1.52 +/- 0.28 inhibitor unit/mL and the level is independent of the number of spermatozoa. In-B level is calculated by subtracting In-A level from the total ficin inhibition level of the semen. The mean In-B level is 1.47 +/- 0.52 inhibitor unit/ml. In-B level has a tendency to increase with increasing number of spermatozoa. The correlation coefficient is 0.307 and statistical significance p less than 0.005.