Nafamostat mesilate, a noncalcium compound, as an anticoagulant, induces calcium-dependent haemolysis when infused with packed erythrocytes.

BACKGROUND: Nafamostat mesilate (NM), a protease inhibitor, is available for acute pancreatitis and disseminated intravascular coagulopathy and is used as an anticoagulant for haemodialysis in Japan. Co-infusion of red cell concentrates (RCC) and intravenous drugs is usually contraindicated. Because of limited venous access, adherence to the guidelines may be compromised in some clinical settings. Therefore, we investigated the influence of co-infusion of RCC and various anticoagulants on haemolysis in vitro. METHODS: We investigated the effect of co-incubation of RCC and various anticoagulant drugs [NM, gabexate mesilate (GM), heparin] in packed erythrocytes. We evaluated haemolysis using lactate dehydrogenase and free haemoglobin. In addition, we also evaluated the influence of co-incubation on phosphatidylserine (PS) expression on the erythrocyte membrane. RESULTS: GM and NM induced haemolysis in a dose-dependent manner, which was inhibited by removal of citrate and pretreatment with the calcium chelator, ethylenediaminetetraacetic acid. In a dynamic experiment using an infusion pump, NM not only induced haemolysis during co-infusion with RCC but also elevated PS expression dependent on extracellular calcium. CONCLUSION: NM and GM induce haemolysis in packed erythrocytes in the presence of citrate that is dependent on extracellular calcium.

Passive near-field imaging via grating-based spectroscopy.

Passive scattering-type scanning near-field optical microscopy (s-SNOM) has recently been developed for studying long-wavelength infrared (LWIR) waves. It detects surface-localized waves without any external illumination or heating and enables the imaging of hot-electron energy dissipation and nanoscale Joule heating. However, the lack of a wavelength selection mechanism in the passive LWIR s-SNOM makes it difficult to perform a thorough analysis of the surface-localized waves. Here, we develop a novel passive scanning near-field optical spectroscopy with a diffraction grating. The spectroscopic optics are designed to exhibit a high signal efficiency and mechanical performance at the temperature of liquid helium (4.2 K). Using the developed passive LWIR near-field spectroscopy, the spectral information of thermally excited evanescent waves can be directly obtained without any influence from the external environment factors, including environmental heat. We have detected the thermally excited evanescent waves on a SiC/Au micropatterned sample at room temperature with a spatial resolution of 200 nm and a wavelength resolution of 500 nm at several wavelengths in the range of 14-15 µm. The obtained spectra are consistent with the electromagnetic local density of states calculated based on the fluctuation-dissipation theorem. The developed passive LWIR near-field spectroscopy enables the spectral analysis of ultrasmall surface-localized waves, making it a high-performance surface analysis tool.

Passive dual-probe near-field microscopy.

Accurate and simultaneous multiposition near-field measurements are essential to study the time-dependent local dynamics, including heat and carrier transfer. The existing passive long-wavelength infrared (LWIR) scattering-type scanning near-field optical microscopy (s-SNOM) systems with a single probe cannot perform precise near-field measurements of the heat or carrier transporting process at the nanoscale level. Therefore, in this study, we developed a passive LWIR s-SNOM system with two probes. To test the effectiveness of the proposed passive LWIR dual-probe s-SNOM system, each probe was precisely controlled using a shear-force feedback system, and the mechanical interference between the probes was used to monitor the distance between the probes. We achieved simultaneous near-field measurements at two different positions 500 nm apart using the proposed passive LWIR dual-probe s-SNOM system. The simultaneously detected near-field signals from two different points were extracted individually, making this technique an effective nanoscale analysis tool for local carrier dynamics.

Identification of oral cavity abnormalities in pre-term and full-term newborns: a cross-sectional and comparative study.

PURPOSE: Compare maxillary labial frenum and lingual frenum topography, the ridges relationship and oral inclusion cysts occurrence between pre-term (PT) and full-term newborns (FT). METHODS: This cross-sectional and comparative study was conducted through the evaluation of 74 PT and 100 FT. Data were collected from medical records: mother age, gestational age, gender, height, weight, and delivery type. The variables were verified by Chi-square test and Mann-Whitney U test, at 5% significance level. RESULTS: Bohn's nodules and dental lamina cysts were more frequent in FT (P = 0.000). Epstein pearls occurrence was similar between FT and PT (P = 0.243). The lower alveolar ridge in distal position to the upper one was more prevalent in both groups. Abnormal upper labial frenum anatomy had been observed in 10.0% of FT. Upper labial frenum was attached in piriform papilla in 90.5% of PTG, whereas in FT, 61.0% were attached from crest of alveolar ridge to mucogingival line. CONCLUSIONS: PT oral cavity presented some peculiarities when compared with FT: maxillary labial frenum insertion in the Piriform papilla and palatal cysts more prevalent than alveolar cysts.

Diffusion of nodal signaling activity in the absence of the feedback inhibitor Lefty2.

The role of Lefty2 in left-right patterning was investigated by analysis of mutant mice that lack asymmetric expression of lefty2. These animals exhibited various situs defects including left isomerism. The asymmetric expression of nodal was prolonged and the expression of Pitx2 was upregulated in the mutant embryos. The absence of Lefty2 conferred on Nodal the ability to diffuse over a long distance. Thus, Nodal-responsive genes, including Pitx2, that are normally expressed on the left side were expressed bilaterally in the mutant embryos, even though nodal expression was confined to the left side. These results suggest that Nodal is a long-range signaling molecule but that its range of action is normally limited by the feedback inhibitor Lefty2.

Quasiparticle band structure of vanadium dioxide.

Vanadium dioxide is insulating below 340 K in experiments, whereas the band structure calculated in the local density approximation (LDA) is gapless. We study the self-energy effects using the ab initio GW method. We found that the self-energy depends strongly on the energy, and proper treatment of the dynamical effect is essential for getting precise quasiparticle energies. Off-diagonal matrix elements in the Kohn-Sham basis are also important for disentangling bands. Inclusion of the two effects opens up a direct gap. Our results also suggest that one-shot GW on top of LDA is not enough, and the impact of self-consistency is significant.

Packageable antiviral therapeutics against human immunodeficiency virus type 1: virion-targeted virus inactivation by incorporation of a single-chain antibody against viral integrase into progeny virions.

To determine their activities as an antiviral agent packageable within virions and suitable for continued expression in cells, we tested a single-chain antibody (scAb) against human immunodeficiency virus type 1 (HIV-1) integrase and its three fusion proteins: fused to viral protein R (scab-Vpr), a double-cassette of the WXXF motif binding to Vpr (scAb-WXXF), and viral major capsid protein (scAb-CA), respectively. Cotransfection of human 293T cells with expression plasmid for scAb-Vpr or -WXXF along with HIV-1 clone pLAI resulted in the production of a normal amount of progeny virions with infectivity decreased by more than 10(3)-fold. Immunoblot analyses showed that scAb-Vpr or -WXXF was associated with virions, whereas scAb or scAb-CA was not, suggesting that scAb-Vpr or -WXXF was incorporated into virions. The incorporation of scAb-WXXF appeared to be Vpr dependent, because the fusion protein was associated with the wild-type but not with Vpr-truncated HIV-1 virions. Since G418-selected HeLa clones carrying expression plasmid for scAb-WXXF were obtained much more frequently than those for scAb-Vpr, scAb-WXXF was inferred to be less toxic to cells than scAb-Vpr. These results suggest that scAb-WXXF may serve as a novel class of antiviral therapeutic that inactivates progeny HIV virions from within.

Role of asymmetric signals in left-right patterning in the mouse.

Left-right asymmetric signaling molecules in mammals include three transforming growth factor beta (TGFbeta)-related factors, Nodal, Lefty1 and Lefty2. They are all expressed on the left half of developing mouse embryos. Nodal acts as a left-side determinant by transducing signals through Smad and FAST and by inducing Pitx2 expression on the left side. Lefty proteins are antagonists that inhibit Nodal signaling. There are positive and negative transcriptional regulatory loops between nodal and lefty2 genes. Thus, Nodal activates its own gene and lefty2. Lefty2 protein produced then inhibits Nodal signaling and terminates expression of both genes. This feedback mechanism can restrict the range and duration of Nodal signaling in developing embryos.

A catalogue of genes in mouse embryonal carcinoma F9 cells identified with expressed sequence tags.

We used expressed sequence tags (ESTs) to identify genes expressed in mouse embryonal carcinoma F9 cells and prepared 2132 ESTs from undifferentiated F9 cDNA libraries: 1026 were prepared after randomly selecting clones from one of the libraries and the remaining 1106 ESTs were prepared after classifying 2896 clones of the libraries into four classes, according to the levels and patterns of expression. Among the former 1026 ESTs, 797 (78%) matched known genes, 61 (6%) matched database sequences of uncharacterized cDNAs, and 168 (16%) represented novel genes. The ESTs matching known genes were catalogued according to putative structural and cellular functions. As many as 53% were related to transcription and translation, and 19% were related to energy metabolism, including transcripts of mitochondrial DNA. These percentages were significantly higher in F9 cells than in the human heart and brain, and a human liver cell line, HepG2. We found that approximately 7% of the ESTs corresponding to low-abundance mRNAs are either related to retinoic acid-regulated genes or mammalian development- and/or differentiation-related genes. Cataloguing of the genes expressed in the F9 cells paves the way for isolating genes involved in early mammalian development.

Optic-spinal form of multiple sclerosis and anti-thyroid autoantibodies.

The optic-spinal form of multiple sclerosis (OSMS), characterized by recurrent involvement of optic nerve and spinal cord with rare brain magnetic resonance imaging lesions, is relatively common among Asians. While individual cases of OSMS with anti-thyroid autoantibodies (ATABs) have been reported, the frequency of ATAbs in OSMS and classical multiple sclerosis has not been studied. We studied serum ATAbs and anti-nuclear antibodies (ANA) in 46 Japanese patients with multiple sclerosis: 14 with OSMS, and 32 with non-OSMS. Six patients were positive for ATAbs: five women with OSMS and one man with non-OSMS. The frequency of ATAbs in OSMS (5/14) was significantly higher than that in non-OSMS (1/32; P = 0.007), but the frequency of ANA did not differ between OSMS (3/14) and non-OSMS (6/32; P = 0.99). There may be a pathogenetic link between anti-thyroid autoimmunity and a subgroup of OSMS in Japanese.

A novel two-base mutation in the Cu/Zn superoxide dismutase gene associated with familial amyotrophic lateral sclerosis in Japan.

We have identified a novel two-base mutation in exon 1 of the Cu/Zn superoxide dismutase (SOD1) gene (TGC to TTT), which resulted in Cys6 to Phe substitution in a Japanese family with amyotrophic lateral sclerosis (ALS). This is the first case of familial ALS-associated two-base change of the SOD1 gene. Similar to several mutations in exon 1 of the SOD1 gene such as Ala4 to Val, Ala4 to Thr and Val14 to Met, affected members of the present family showed a rapid progression of motor dysfunction. Although Ala4, Cys6 and Val7 reside in the middle of the first beta-strand of the SOD1, a family with a mutation of Val7 to Glu associates with slow progression of the disease. These findings suggest that clinical courses are variable with each mutation, even in the same exon.

Familial spinal arachnoiditis with secondary syringomyelia: clinical studies and MRI findings.

We report the clinical and MRI findings of two patients with familial spinal arachnoiditis. Although their initial symptoms were various, they both showed spastic paraparesis and sensory disturbance below the thoracic level. Cytokines and WBC in the CSF were studied, but they were not elevated at all. The spinal magnetic resonance images of each showed extensive arachnoiditis and a cystic structure. The other impressive features included: (i) an enhancement within the thickened arachnoid and an adhesion between the spinal cord and the dura mater, (ii) deformation of the thoracic cord where the arachnoid adhered, and (iii) secondary syrinx formation. Laminectomy may have an adverse outcome for such patients.

Screening for adenine and hypoxanthine phosphoribosyltransferase deficiencies in human erythrocytes by high-performance liquid chromatography.

A screening method using high-performance liquid chromatography (HPLC) for the simultaneous detection of deficiencies of adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT) activities in human erythrocytes is described. Both enzyme reactions of APRT and HPRT in lysates treated with a charcoal-dextran were simultaneously carried out in the same reaction tube and the enzyme activities were determined by measuring the increases in absorbance at 260 nm of adenosine and inosine converted from adenosine-5'-monophosphate and inosine-5'-monophosphate with alkaline phosphatase. Adenosine and inosine were separated from adenine and hypoxanthine by a reversed-phase column. The method could detect 1% of normal APRT activity and 0.3% of normal HPRT activity. The within-run coefficients of variation for APRT and HPRT activities were 3.2 and 3.4%, respectively.

Phosphoribosylpyrophosphate synthetase in human erythrocytes: assay and kinetic studies using high-performance liquid chromatography.

A method using high-performance liquid chromatography (HPLC) for determination of phosphoribosylpyrophosphate (PRPP) synthetase activity in human erythrocytes has been developed and PRPP synthetase activity on purine and pyrimidine metabolic disorders has been studied. Kinetic properties of erythrocyte PRPP synthetase of patients with gout and of a patient with pyrimidine 5'-nucleotidase deficiency were compared with those of healthy subjects. The mean of PRPP synthetase activity of gouty patients was a little higher (P less than 0.01) than that of healthy subjects. The response of the enzyme for ATP of gouty patients was different from that of healthy subjects. The shapes of activation curve of the enzyme for inorganic phosphate were hyperbolic in gouty patients and in a patient with pyrimidine 5'-nucleotidase deficiency.

The structure of the core polyol of the ether lipids from Sulfolobus acidocaldarius.

The major ether-type lipid structures of Sulfolobus acidocaldarius (ATCC33909) were composed of caldarchaeol and calditoglycerocaldarchaeol. However, the characterization by nuclear magnetic resonance spectroscopy and mass spectrometry showed that the structure of calditol in calditoglycerocaldarchaeol is not nonitol, 2-(1',2',3'-trihydroxypropyl)1,2,3,4,5,6-hexahydroxyhexane, but 2-hydroxymethyl-1-(2,3-dihydroxypropoxy)2,3,4,5-cyclopentanetet raol with an ether linkage in the molecule. Such an intermolecular ether linkage was resistant to BCl3 treatment, but nonresistant to 57% HI degradation treatment conducted at 100 degrees C for 60 h, producing 2-hydroxymethyl-1,2,3,4,5-cyclopentanepentaol from calditol as reaction product. Further, it was confirmed that the structure of calditol is essentially a derivative of glycerol, and hydrocarbon chains were conjugated to the glycerol-like site in the structure. The calditol with an ether linkage in the molecule suggested an important role regarding the properties of heat-resistance and acid-resistance observed in Sulfolobales.

Two siblings with classical xanthinuria type 1: significance of allopurinol loading test.

Two brothers with classical xanthinuria who lacked xanthine dehydrogenase activity were encountered. Their hypouricemia was caused by underproduction of uric acid. In their duodenal mucosa, no xanthine dehydrogenase (oxidase) activity was detected. The patients had no symptoms except for duodenal ulcer in one case. The conversion of allopurinol to oxipurinol during an allopurinol loading test for determining the type of classical xanthinuria revealed that the patients had classical type 1 xanthinuria, because aldehyde oxidase activity was present. Furthermore, the allopurinol loading test was conducted to determine the optimal examination times and specimens required for this test.

Partial deficiency of hypoxanthine-guanine phosphoribosyltransferase manifesting as acute renal damage.

A 32-year-old man who had had frequent gouty arthritis over the past 17 years, was admitted for acute renal failure. Acute renal failure was improved rapidly after medication was resumed and the patient was sufficiently hydrated. The hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity in the patient had been reduced to about 30% of the normal control. Therefore we considered that this patient suffered from a partial deficiency of HPRT. A point mutation of HPRT gene 68G (guanine) to T (thymine) was detected. This is a mutation that has not been previously reported. Familial analysis indicated that his mother and sister were heterozygotes.

[A case of multiple intracranial tuberculoma diagnosed by open brain biopsy].

To provide histological diagnoses of brain diseases, CT-guided stereotactic brain biopsy (CT-SBB) has been widely used because of its less invasive technique compared with open brain biopsy (OBB). However, CT-SBB is not always diagnostic. We report a case of multiple intracranial tuberculoma whose diagnosis was not made by CT-SBB but by OBB. The patient is a 46-year-old man with insulin-dependent diabetes mellitus who had been receiving immunosuppressive agents (azathioprine, cyclosporin, and prednisolone) after renal transplantation for diabetic renal failure for 9 years. He gradually developed febrile, headache and unsteady gait. Brain MRI demonstrated multiple intracranial lesions involving left fronto-temporal and right parietal lobes, left cerebellar hemisphere, and the fourth ventricle. Although the MRI findings were consistent with those of previously reported cases of intracranial tuberculoma, other conditions, such as malignant lymphoma and toxoplasmosis, were not ruled out. Therefore, CT-SBB targeting the left temporal lobe lesion was done for definitive diagnosis, but it revealed only mild perivascular infiltration of mononuclear cells and hemorrhage. He was transferred to our clinic for further evaluation. On examination, mild truncal and limb ataxia on the left were noted in addition to the neurological findings corresponding to diabetic retinopathy and neuropathy. Despite vigorous laboratory examinations, including repeated bacterial cultures and PCR of cerebrospinal fluid, no evidence of tuberculous infection was obtained. A tentative diagnosis of multiple intracranial tuberculoma was made, and anti-tuberculous drugs (isoniazid 400 mg, ethambutol 750 mg, and pyrazinamide 1.5 g) were administered. Since his symptoms deteriorated because of ventricular dilatation resulting from the enlarged lesion in the fourth ventricle after a temporary clinical improvement, VP-shunting and OBB from the left temporal lobe lesion were done. The excised lesion was firmly encapsulated and the histological examination revealed typical pathology of tuberculoma. Ziehl-Neelsen staining and PCR for Mycobacterium tuberculosis of the biopsied specimen were also positive. Further administration of increased doses of anti-tuberculous drugs (isoniazid 600 mg, ethambutol 500 mg, pyrazinamide 2.0 g and intramuscular injection of streptomycin 0.3 g twice a week) eventually ameliorated the symptoms and shrank the lesions. In case of intracranial tuberculoma, the needle of CT-SBB may not penetrate the firm capsule of tuberculoma and only the surrounding brain tissue may be obtained as in the present case. Therefore, it is recommended to consider OBB from the beginning for definitive diagnosis of intracranial tuberculoma. Paradoxical worsening of the clinical and laboratory findings of tuberculosis in spite of appropriate anti-tuberculous therapy as seen in the present case has been described in both pulmonary and extra-pulmonary tuberculosis. The phenomenon, called transient worsening, could happen and we have to keep it in mind during the treatment of intracerebral tuberculoma.

[Steroid-responsive diffuse cerebral white matter lesions in a case of intractable fungal meningoencephalitis].

We report a case of fungal meningoencephalitis with steroid-responsive diffuse cerebral white matter lesions. A 49-year-old male developed auditory hallucination, confusion and fever, on April, 1994. He was diagnosed as having cryptococcal meningoencephalitis based on the detection of cryptococcal antigens in the cerebrospinal fluid (CSF). Intravenous administration of fluconazole resulted in improvement of his neurologic symptoms and CSF findings. For the next seven months, he was treated with oral fluconazole and the neurological status was stable. However, soon after the dose of fluconazole was tapered, he became confused and febrile, which made him admitted to our hospital. Neurological examination on admission showed disturbance of consciousness, disorientation and meningeal irritation. The CSF examination revealed mild pleocytosis (mostly lymphocytes), elevated protein and normal glucose levels, although fungus was not detected. The T2-weighted image of brain MRI demonstrated diffuse hyperintense lesions in the bilateral cerebral white matters. GD-DTPA enhanced MRI showed spotty enhanced lesions in the periventricular white matters. The neurologic symptoms were once relieved after intravenous administration of fluconazole was started, but two months later, he became comatose and needed ventilatory support, despite amphotericine B therapy. Then, a needle brain biopsy targeting the white matter lesion was done. Histopathology of the specimen showed chronic inflammation with granuloma formation and T lymphycyte infiltrate around the small vessels, though fungus was not detected in the tissue. Combined therapy with corticosteroid and antifungal agents remarkably improved the neurological symptoms as well as the MRI findings. In the present case, fungal infection possibly induced an altered immune reactions which resulted in the steroid responsive diffuse cerebral white matter lesions.