Arginine 485 of human serum albumin interacts with the benzophenone moiety of ketoprofen in the binding pocket of subdomain III A and III B.

Arylpropionic acid nonsteroidal anti-inflammatory drusg (NSAIDs) primarily bind to subdomain III A (site II) of human serum albumin (HSA). Ketoprofen (KP), an arylpropionic acid that contains a photoreactive benzophenone moiety, was used to photolabel the binding region of site II. LC/Q-TOF mass spectrometry determination revealed that R485 was the amino acid residue that formed covalent adduct with the benzophenone moiety of KP. Point mutation of arginine 485 to alanine showed a slight decrease in the overall binding percentage of KP when compared to that of native HSA. The induced circular dichroism spectral data of KP with both R485A and native albumin confirmed the photolabeling findings. Interestingly, an increase in the extent of [14C]KP covalent adduct formation with the 11.6 kDa peptide derived from subdomain IIB-IIIA was observed for R485A. In contrast, mutation of arginine 410 caused a significant reduction of binding percentage, confirming the importance of this residue in high affinity binding of arylpropionic acid derivatives. This may indicate that while KP's carboxylate interacts electrostatically with arginine 410, the benzophenone moiety may have swung away from helix 6 in the absence of arginine 485. In this study, photolabeling of native and mutants albumins, R485A and R410C with [14C]KP confirmed that R485 involved in the non-electrostatic interaction with the benzophenone moiety of KP, but not vital to hold KP in the binding pocket of subdomain IIIA.

Distribution and species composition of juvenile and adult scombropids (Teleostei, Scombropidae) in Japanese coastal waters.

Two scombropid fishes, Scombrops boops and Scombrops gilberti, are closely related and commercially important species in Japan. These species are often confused in commercial markets because of their morphological similarity. In this study, scombropid specimens collected from various Japanese coastal waters were subjected to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and phylogenetic analysis of the 16S rRNA gene in mitochondrial DNA. These analyses showed that all the scombropid specimens collected from localities in the Sea of Japan were identified as S. boops, whereas those from the Pacific Ocean included two species, S. boops and S. gilberti. Almost all juvenile (<200 mm standard body length, S(L)) S. gilberti originated from the Pacific coastal waters of the northern Japan, whereas adults (>400 mm S(L)) were found only in deep water off the Izu Peninsula to the Izu Islands. This suggests that S. gilberti might migrate extensively during its life cycle. In addition, differences in the number of specimens and the distribution between the two species suggest that S. gilberti is less abundant than S. boops in Japanese waters.

Tissue alkalosis in cold-ischemia time.

The control of pre-analytical-factors in human biospecimens collected for health research is currently required. Only two previous reports using post-mortem brain samples have tried to address the impact of cold-ischemia on tissue pH. Here we report pH variations according to time (third-order polynomial model) in mice for liver, kidney and lung samples. Tissue alkalosis in cold-ischemia time may be an underlying mechanism of gene expression changes. Therefore, tissue-pH regulation after organ removal may minimize biological stress in human tissue samples.

Evidence for the presence of angiogenin in human follicular fluid and the up-regulation of its production by human chorionic gonadotropin and hypoxia.

Angiogenesis is an essential event during the development of the ovarian follicle and ensuing formation of the corpus luteum. We investigated the presence of angiogenin, a potent inducer of angiogenesis, and the regulatory mechanisms of its production in the human ovary. Follicular fluid (FF) and granulosa cells (GCs) were collected from women undergoing in vitro fertilization and embryo transfer. The presence of angiogenin in FF and GCs was demonstrated by Western blot analysis. The production of angiogenin by cultured GCs was stimulated with the addition of human CG or cAMP or under the hypoxic milieu. Concentrations of angiogenin in FF from an individual follicle were positively correlated with those of progesterone, but not estradiol and testosterone. Given the presence of angiogenin in FF and up-regulation of its production by human CG and hypoxia, it seems logical to assume that angiogenin may play a role as a local angiogenic factor in the human ovary.

Quantitative analysis of the spreading of the mouse trophoblast in vitro: a model for early invasion.

The outgrowth of the mouse blastocyst in culture represents an in vitro model of trophoblastic invasion. In the present study we analysed trophoblast spreading by time lapse video microscopy. Trophoblast spreading consists of (1) the migration and (2) the giant cell transformation of trophoblast cells, (3) the proliferation of ectoplacental cone (EPC) cells and (4) the subsequent transformation of EPC cells into the secondary giant cells. During migration, ruffling of the trophoblast cell membrane is followed by the formation of lamellipodia. The mean surface areas of the spreading trophoblast, measured in more than 100 cultured blastocysts, increased linearly from 48 to 96 h of culture, while the linear migratory speed at the periphery of the outgrowth declined as the time of culture advanced. The EPC cells increased in size approximately eightfold during the giant cell transformation. The apparent nuclear:cytoplasmic ratios, i.e., ratios between the size of nucleus and that of the cytoplasm, measured as the surface areas on the photomicrographs, of EPC cells increased between 40-46 h of culture, but a sharp decline in the ratio occurred between 50 and 51 h of culture, reflecting either the sudden and tremendous increase in the cellular volume and/or spreading of the cytoplasm. The rates of trophoblast spreading varied considerably among the blastocysts of different genetic constitution examined (ICR, C57BL/6, C3H/He and (B6 x C3)F1. It was fastest in blastocysts obtained from matings of males and females of (B6 x C3)F1, and slowest in the C57BL/6 embryos. The differences in the rate of outgrowth observed may not simply be ascribed to difference in the developmental speed of the early embryos, because the rate of outgrowth reached a plateau at about 96-120 h and no "catch-up' was observed by leaving the blastocysts in culture longer. Our results strongly suggest the possible presence of genetic regulatory mechanisms underlying trophoblast outgrowth; further analysis of the phenomenon may provide clues to understand the molecular mechanisms of trophoblastic invasion during the early phase of implantation, hopefully leading to improved success rates of in vitro fertilization-embryo transfer.

Functional mapping against Escherichia coli for the broad-spectrum antimicrobial peptide, thanatin, based on an in vivo monitoring assay system.

Previously, we established for the first time an in vivo monitoring assay system conjugated with random mutagenesis in order to study the structure-function relationship of the antimicrobial peptide, apidaecin [Taguchi et al. (1996) Appl. Environ. Microbiol. 62, 4652-4655]. In the present study, this methodology was used to carry out the functional mapping of a second target, thanatin, a 21-residue peptide that exhibits the broadest antimicrobial spectrum so far observed among insect defense peptides [Fehlbaum et al. (1996) Proc. Natl. Acad. Sci. USA 93, 1221-1225]. First, a synthetic gene encoding thanatin was expressed in a fused form with Streptomyces protease inhibitor protein, SSI, under the control of tac promoter in Escherichia coli JM109. Expression of the thanatin-fused protein was found to depend on the concentration of the transcriptional inducer, isopropyl-beta-D-thio-galactopyranoside (IPTG), and to parallel the degree of growth inhibition of the transformant cells. When a PCR random mutation was introduced into the structural gene for thanatin, diminished growth inhibition of the IPTG-induced transformed cells was mostly observed in variants as measured by colony size (plate assay) or optical density (liquid assay) in comparison with the wild-type peptide, possibly depending on the decreased antimicrobial activity of each variant. Next, wild-type thanatin and three variants screened by the in vivo assay, two singly mutated proteins (C11Y and M21R) and one doubly mutated protein (K17R/R20G), were stably overproduced with a fusion partner protein resulting in the efficient formation of inclusion bodies in E. coli BL21(DE3). The products were isolated in large amounts (yield 30%) from the fused protein by successive chemical and enzymatic digestions at the protein fusion linker site. Anti-E. coli JM109 activities, judged by minimum inhibitory concentration, of the purified peptides were in good agreement with those estimated semi-quantitatively by the in vivo assay. Based on the NMR solution structure and molecular dynamics, the structure-function relationship of thanatin is discussed by comparing the functional mapping data obtained here with the previous biochemical data. The functional mapping newly suggests the importance of a hydrogen bonding network formed within the C-terminal loop joining the beta-strands arranged antiparallel to one another that are supposed to be crutial for exhibiting anti-E. coli activity.

Specificity of omega-conotoxin MVIIC-binding and -blocking calcium channel antibodies in Lambert-Eaton myasthenic syndrome.

An immunoprecipitation assay was used to measure omega-conotoxin MVIIC (P/Q-type) binding and blocking calcium channel antibodies in 67 patients with Lambert-Eaton myasthenic syndrome (LEMS) and in a large control population. We first showed the presence of omega-conotoxin MVIIC-blocking antibody in LEMS patients. Binding antibodies were detected in 55 of 67 (82.1%) LEMS patients and in 2 of 296 (0.7%) controls. In contrast, blocking antibodies were positive in 14 of 67 (20.9%) LEMS patients and 8 of 171 (4.7%) controls. No LEMS patient had negative binding antibodies and positive blocking antibodies. The immunoprecipitation assay detected no antibodies against the whole P/Q-type calcium channel in either the paraneoplastic cerebellar degeneration or the amyotrophic lateral sclerosis sera. Neither the omega-conotoxin MVIIC-binding nor the -blocking calcium channel antibodies were correlated with clinical severity across the individuals, but longitudinal studies of some LEMS patients showed an inverse relation between binding antibody titre and disease severity. We concluded that the 125I-omega-conotoxin MVIIC assay for anti-P/Q-type voltage-gated calcium channel antibodies is highly specific for LEMS and that this sensitive binding antibody assay could be more valuable than the blocking antibody assay in the diagnosis of LEMS.

Seronegative myasthenia gravis associated with atonic urinary bladder and accommodative insufficiency.

We report a 20-year-old female with generalized myasthenia gravis (MG) who developed atonic urinary bladder and accommodative insufficiency. Although her sera did not contain antibodies to either nicotinic acetylcholine receptor (AChR) or voltage-gated calcium channels, a positive intravenous edrophonium test and a waning phenomenon on electromyographic studies indicated a diagnosis of seronegative MG. Myasthenic symptoms as well as urinary incontinence and impaired near vision disappeared with slight sequelae after corticosteroid therapy and total removal of the hyperplastic thymus. These symptoms recurred during a tapering course of corticosteroids, and improved again with an increased dose. Therefore, in this patient, the neuromuscular transmission of muscarinic type of AChR in the smooth muscles was also affected. This patient provides insight into the mechanisms by which some smooth muscles are involved in MG.

Antibodies to recombinant synaptotagmin and calcium channel subtypes in Lambert-Eaton myasthenic syndrome.

Several proteins have been postulated as possible targets of immune attack in Lambert-Eaton myasthenic syndrome (LEMS). Heterogeneity of autoantibodies in sera from 20 LEMS patients was studied by comparing their reactivity to synaptotagmin, a synaptic vesicle protein, and voltage-gated calcium channels (VGCCs). Six patients' sera (1 with small cell lung carcinoma (SCLC)) contained antibodies specifically recognizing the recombinant synaptotagmin on immunoblots. Thirteen (11 with SCLC) and 16 (11 with SCLC and 1 with poorly differentiated cell carcinoma in the lung) patients' sera immunoprecipitated omega-conotoxin GVIA-labeled N-type and omega-conotoxin MVIIC-labeled Q-type VGCCs, respectively. Three of 6 synaptotagmin-positive sera had cross-reactivity with N and/or Q subtypes of VGCC; the remaining 3 showed no cross-reactivity with VGCCs. Results indicate that LEMS sera are heterogeneous in the spectrum of containing antibodies, and suggest that this heterogeneity reflects the immune response to various synaptic proteins including not only multiple VGCCs but also synaptosecretory complex proteins.

Successful application of pentoxifylline in the treatment of HTLV-I associated myelopathy.

Fifteen patients with human T-cell lymphotropic virus type-I (HTLV-I)-associated myelopathy (HAM) were treated in an uncontrolled preliminary trial by oral administration of pentoxifylline (PTX). Motor function, neurological evaluation, immunological markers and parameters were evaluated after four weeks. In 13 of the 15 patients, motor disability, especially spasticity, improved substantially. PTX suppressed spontaneous proliferation of peripheral blood mononuclear cells in 14 of the 15 patients at four weeks. No adverse effect was observed. We concluded that PTX may be a safe and beneficial agent for the treatment of HAM.

Computational observation of an ion permeation through a channel protein.

The ion permeation process, driven by a membrane potential through an outer membrane protein, OmpF porin of Escherichia coli, was simulated by molecular dynamics. A Na+ ion, initially placed in the solvent region at the outer side of the porin channel, moved along the electric field passing through the porin channel in a 1.3 nsec simulation; the permeation rate was consistent with the experimentally estimated channel activity (10(8)-10(9)/sec). It this simulation, it was indicated that the ion permeation through the porin channel proceeds by a "push-out" mechanism, and that Asp113 is an important residue for the channel activity.

A comparative study of the interaction of warfarin with human alpha 1-acid glycoprotein and human albumin.

The interaction of warfarin with human alpha 1-acid glycoprotein (alpha 1-AGP) and human albumin (HA) has been investigated using fluorescence and circular dichroism techniques. The fluorescence of warfarin is greatly enhanced following binding to alpha 1-AGP or HA, the binding constant for a single site being estimated by the Scatchard method. The binding constants for the two serum proteins are similar, but the thermodynamic parameters differ. The binding constants increase as the pH is raised to 9.0. Various basic drugs, such as chlorpromazine, propranolol and imipramine, markedly inhibited the binding of warfarin to alpha 1-AGP. But, some acidic drugs, including phenylbutazone, effectively displaced warfarin bound to HA. The difference in CD spectra observed for alpha 1-AGP and HA indicated that the drug-binding sites of the two proteins might have different asymmetries. It thus appears that the mode of interaction of warfarin with the two proteins differs.

Arg133Cys mutation of Notch3 in two unrelated Japanese families with CADASIL.

OBJECTIVE: More than 80 unrelated, but all Caucasian, patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), originating from various communities around the world, have been molecularly identified. To clarify the occurrence of CADASIL in Orientals, we investigated Japanese families presenting as CADASIL. METHODS: We performed the PCR-SSCP and sequence analyses using genomic DNA, isolated from venous blood of participants under informed consent. PATIENTS: We identified two unrelated Japanese families with CADASIL, including 5 affected members through 2 generations. RESULTS: Each of the affected individuals developed recurrent strokes without risk factors resulting in progressive dementia, pseudobulbar palsy, and gait disturbances which started after the fifth decade of life. Although affected individuals had no vascular risk factors, they showed various degrees of narrowing of retinal arteries. Their MRI/CTs showed characteristics of the disease; bilateral small infarcts in the thalamus, basal ganglia, brain stem, and deep white matter in addition to the findings of leukoaraiosis. On SPECT imaging, there was severe hypoperfusion in the cortex as well as in the white matter. Ultrastructural studies revealed an abnormal deposition of granular osmiophilic materials (GOM) within the basal lamina of pericytes in muscular capillaries. On PCR-SSCP and sequence analyses, a heterozygous Arg133Cys mutation was present, in the affected individuals, in the exon 4 of Notch3 gene which is the hot spot region for CADASIL mutations in Caucasian families. None of the non-affected members nor the 50 Japanese normal controls revealed this mutation. CONCLUSION: Thus, our results confirm that CADASIL is a geographically widespread disorder caused by a Notch3 mutation.

[Differential effect of sodium bicarbonate on disposition of sulfadimethoxine and sulfisoxazole in rabbits].

The orally co-administered sodium bicarbonate significantly enhanced the blood concentration of sulfadimethoxine at the early stage after oral administration to rabbits, by increasing its intestinal absorption. On the other hand, the sodium bicarbonate significantly reduced the blood concentration of sulfisoxazole at the elimination phase after oral administration to rabbits, by increasing its urinary excretion. The fact that sodium bicarbonate exhibits different effects in the disposition of these two sulfonamides is an interesting example to gain a better understanding for the complexity of drug interaction.

[Anti-voltage-gated calcium channel antibodies in the Lambert-Eaton myasthenic syndrome].

We have tested 29 patients with the Lambert-Eaton myasthenic syndrome (LEMS) for serum antibodies to voltage-gated calcium channels (VGCC) using an immunoprecipitation assay in which [125]-omega-conotoxin GVIA is used to label calcium channels extracted from IMR-32, a human neuroblastoma cell line. Fifty-five percent of these patients had significant levels of antibody [31.6 (26.5, 48.2) (med., Q1, Q3) pmol/L, n = 29], compared with healthy controls [21.5 +/- 3.4 (mean +/- SD) pmol/L, n = 30] and other neurological disorders [25.2 +/- 4.2 (mean +/- SD) pmol/L, n = 10]. These antibodies were found in 43% of the patients with small cell lung carcinoma (SCLC) without signs and symptoms of LEMS [32.2 +/- 7.2 (mean +/- SD) pmol/L n = 30] and 7% of the myasthenic patients [21.4 +/- 6.8 (mean +/- SD) pmol/L n = 14]. Anti-VGCC antibody titers did not correlate with presence of SCLC, disease duration, or an electromyographic index of disease severity. Our results suggest that the antibodies detected in this assay are specific to some patients with LEMS, but not all. This assay is a useful aid in diagnosing LEMS but has much room for improvement.

[A case of hereditary ceruloplasmin deficiency with hemosiderosis].

We report a 49-year-old female with hereditary ceruloplasmin deficiency with hemosiderosis. There was a family history of the same symptoms; her brother showed hypoceruloplasminemia and decrease of the serum copper content. On physical examinations, dementia, dysarthria, downbeat nystagmus, sensorineural hearing disturbance, orthostatic hypotension, retinitis pigmentosa, diffuse goiter, and cerebellar ataxia were noted. Laboratory examinations disclosed leukopenia, diabetes mellitus, hypothyroidism, decrease of copper content in the serum and urine. Serum ferritin concentration was remarkably increased. Serum ceruloplasmin could not be detected. Biopsy of the liver showed that iron content in the liver was increased. On MRI study, dentate nucleus of the cerebellum, basal ganglia, and the liver showed low intensity in both T1 and T2 weighted images. A nonsense mutation in the ceruloplasmin gene was found in this patient. Systemic iron deposition and tissue damage were considered as caused by deficiency of function of ceruloplasmin as ferroxidase. To our knowledge, the characteristic combination of the clinical signs in this patient has not been reported.

[CADASIL: clinical analysis of CADASIL and CADASIL-like disorders in Japan].

To clarify the characteristics of CADASIL in Japan, we performed clinical and genetic investigations for six patients from 5 Japanese families diagnosed as CADASIL. We identified that the onset of focal neurologic deficits ranged from 38 to 63 years old (mean 49 +/- 9.4 yrs) and the occurrence rates of main neurologic symptoms and signs were 1/6 for migraine, 3/6 for recurrent stroke episodes, 6/6 for dementia, and 4/6 for pseudobulbar palsy. The marked narrowing of retinal arteries were observed in 3/6. The notch 3 mutations were all found in exon 4. Although other several families shared similar phenotype of CADASIL, there were no deposition of granular osmiophilic materials within the basal lamina of smooth muscle cells in the arterioles of biopsied muscle and no mutations in the cording regions of notch 3 gene. We investigated prospectively the incidence of CADASIL and CADASIL-like disease in Kumamoto district from 1999 to 2000. One thousand and thirty four patients with stroke were hospitalized in 6 hospitals which have stroke care unit. Among them, 7 patients fulfilled the criteria that were less than 60 years old, lacunar strokes and/or TIA, presence of a family history, and no risk factors such as hypertension, diabetes mellitus, and hyperlipidemia. One of seven patients was diagnosed as CADASIL by DNA analysis. It was suspected the incidences of CADASIL and CADASIL-like disease were not so rare in Japan.

[A family of hereditary neuropathy with liability to pressure palsies with a proband who developed right and left foot drop successively following the left radial nerve palsy].

A 41-year-old man developed multifocal mononeuropathies manifesting right and left foot drop successively, following the left radial nerve palsy as an initial symptom. Based on the neurological findings and the results of the genetic study of peripheral myelin protein (PMP) 22 gene and the histological study of the sural nerve on biopsy, the diagnosis of hereditary neuropathy with liability to pressure palsies (HNPP) was made. Two asymptomatic carriers were found among his family members based on the genetic study. The diagnosis of HNPP can be definitely established by the genetic study and this disease is relatively rare. In this report it is important to note that there are a few patients who show radial nerve palsy as an initial symptom, that we should carefully study the family members to obtain the prevalence of HNPP because asymptomatic carriers may be present, and that the carriers should be advised to avoid strenuous exercises and works which may produce excessive extension or compression of nerve trunks with the subsequent development of clinical symptoms.

[Effects of polychlorinated biphenyls on regeneration of the peripheral nerve in rats].

The effects of polychlorinated biphenyls (PCB) on regeneration of the peripheral nerves were investigated in rats. The sciatic nerves were crushed at the mid-thigh level on the last day of 32 days of oral administration of PCB. The sciatic nerves were biopsied from the crushed regions at 1, 2, 4, 8 and 12 weeks after crushing. Myelin thickness on the axon diameter was smaller in the PCB administered group than in the control group. There was no difference between the experimental group and the control group in the density of regenerating fibers and distribution of fiber diameters in unmyelinated fibers. After 12 weeks the number of large diameter myelinated fiber densities was lower in the experimental group than in the control group. These results indicate that PCB may affect the remyelination of the regenerating nerve.