Prenatal diagnosis of rhombencephalosynapsis: neuroimaging features and severity of vermian anomaly.

OBJECTIVES: To describe the prenatal neuroimaging spectrum of rhombencephalosynapsis (RES) and criteria for its classification according to the severity of vermian anomaly. METHODS: In this multicenter retrospective study of fetuses with RES between 2002 and 2020, the medical records and brain ultrasound and magnetic resonance images were evaluated comprehensively to determine the severity of the vermian anomaly and the presence of associated brain findings. RES was classified, according to the pattern of vermian agenesis and the extent of the fusion of the hemispheres, as complete RES (complete absence of the vermis) or partial RES (further classified according to the part of the vermis that was missing and, consequently, the region of hemispheric fusion, as anterior, posterior, severe or mixed RES). Findings were compared between cases with complete and those with partial RES. RESULTS: Included in the study were 62 fetuses with a gestational age ranging between 12 and 37 weeks. Most had complete absence of the vermis (complete RES, 77.4% of cases), a 'round-shaped' cerebellum on axial views (72.6%) and a transverse cerebellar diameter (TCD) < 3rd centile (87.1%). Among the 22.6% of cases with partial RES, 6.5% were classified as severe partial, 6.5% as partial anterior, 8.1% as partial mixed and 1.6% as partial posterior. Half of these cases presented with normal or nearly normal cerebellar morphology and 28.5% had a TCD within the normal limits. Infratentorially, the fourth ventricle was abnormal in 88.7% of cases overall, and anomalies of the midbrain and pons were frequent (93.5% and 77.4%, respectively). Ventriculomegaly was observed in 80.6% of all cases, being more severe in cases with complete RES than in those with partial RES, with high rates of parenchymal and septal disruption. CONCLUSIONS: This study provides prenatal neuroimaging criteria for the diagnosis and classification of RES, and identification of related features, using ultrasound and magnetic resonance imaging. According to our findings, a diagnosis of RES should be considered in fetuses with a small TCD (severe cerebellar hypoplasia) and/or a round-shaped cerebellum on axial views, during the second or third trimester, especially when associated with ventriculomegaly. Partial RES is more common than previously thought, but presents an extreme diagnostic challenge, especially in cases with normal or nearly-normal cerebellar morphobiometric features. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Anticholinergic load negatively correlates with recovery of cognitive activities of daily living for geriatric patients after stroke in the convalescent stage.

WHAT IS KNOWN AND OBJECTIVE: Anticholinergic drugs are associated with risks of falls, confusion and cognitive dysfunction. However, the effect of anticholinergic drug use on rehabilitation outcomes after a stroke is poorly documented. We therefore aimed to establish whether the anticholinergic load was associated with functional recovery among geriatric patients convalescing after stroke. METHOD: Consecutive geriatric stroke patients admitted and discharged from a convalescence rehabilitation ward between 2010 and 2016 were included in this retrospective cohort study. Anticholinergic load was assessed by the Anticholinergic Risk Scale (ARS), and functional recovery was assessed by the Functional Independence Measure (FIM). The primary outcome was cognitive FIM (FIM-C) gain, but we also assessed the interaction of other putative factors identified from univariate analysis. Multivariate analyses were performed, adjusting for confounding factors. RESULTS AND DISCUSSION: We included 418 participants (171 males, 247 females) with a median age of 78 years (interquartile range, 72-84 years). Multiple regression analysis revealed that ARS change, length of stay, and epilepsy were independently and negatively correlated with cognitive FIM gain. Multiple logistic regression analysis indicated that the "Comprehension" and "Memory" items of the cognitive FIM gain were independently and negatively associated with anticholinergic load. WHAT IS NEW AND CONCLUSION: A causal relationship cannot be established, but increased ARS scores during hospitalization may predict limited cognitive functional improvement in geriatric patients after stroke. Alternatively, cognitive impairment may lead to increased use of anticholinergic drugs.

Examination of micro-superficial lesions of up to 5 mm in size in the pharyngolaryngeal region.

OBJECTIVE: For low-grade intraepithelial neoplasia cases, pharyngolaryngeal lesions equal to or less than 5 mm in size do not generally progress to invasive carcinoma. However, micro-superficial lesions equal to or less than 5 mm that showed rapid growth have been recently encountered. This study aimed to identify the characteristics of preferential progression of lesions equal to or less than 5 mm in size. METHOD: Gross findings, endoscopic findings and pathological results of 55 lesions measuring equal to or less than 5 mm in diameter were retrospectively reviewed to identify factors that distinguish squamous cell carcinoma or high-grade intraepithelial neoplasia from low-grade intraepithelial neoplasia or non-atypia lesions. RESULTS: The overall sensitivity, specificity, accuracy, and positive and negative predictive value of background colouration and intrapapillary capillary loop pattern in differentiation of squamous cell carcinoma or high-grade intraepithelial neoplasia from low-grade intraepithelial neoplasia or non-atypia lesions were all 100 per cent. CONCLUSION: Diagnosis based on background colouration and the intrapapillary capillary loop pattern on narrow-band imaging facilitates the pathological examination of lesions measuring equal to or less than 5 mm.

Differential expression of angiotensin-converting enzyme-2 in human paranasal sinus mucosa in patients with chronic rhinosinusitis.

OBJECTIVE: Severe acute respiratory syndrome coronavirus-2 uses angiotensin-converting enzyme-2 as a primary receptor for invasion. This study investigated angiotensin-converting enzyme-2 expression in the sinonasal mucosa of patients with chronic rhinosinusitis, as this could be linked to a susceptibility to severe acute respiratory syndrome coronavirus-2 infection. METHODS: Ethmoid sinus specimens were obtained from 27 patients with eosinophilic chronic rhinosinusitis, 18 with non-eosinophilic chronic rhinosinusitis and 18 controls. The angiotensin-converting enzyme-2 and other inflammatory cytokine and chemokine messenger RNA levels were assessed by quantitative reverse transcription polymerase chain reaction. Angiotensin-converting enzyme-2 positive cells were examined immunohistologically. RESULTS: The eosinophilic chronic rhinosinusitis patients showed a significant decrease in angiotensin-converting enzyme-2 messenger RNA expression. In the chronic rhinosinusitis patients, angiotensin-converting enzyme-2 messenger RNA levels were positively correlated with tumour necrosis factor-α and interleukin-1ß (r = 0.4971 and r = 0.3082, respectively), and negatively correlated with eotaxin-3 (r = -0.2938). Angiotensin-converting enzyme-2 immunoreactivity was mainly localised in the ciliated epithelial cells. CONCLUSION: Eosinophilic chronic rhinosinusitis patients with type 2 inflammation showed decreased angiotensin-converting enzyme-2 expression in their sinus mucosa. Angiotensin-converting enzyme-2 regulation was positively related to pro-inflammatory cytokines, especially tumour necrosis factor-α production, in chronic rhinosinusitis patients.

Oxygen radicals in influenza-induced pathogenesis and treatment with pyran polymer-conjugated SOD.

The pathogenicity of influenza virus infection in the mice involves, at least in part, overreaction of the immune responses of the host rather than a direct effect of virus multiplication. Xanthine oxidase, which is responsible for the generation of oxygen free radicals, was elevated in serum and lung tissue of mice infected with influenza virus. To test the theory that oxygen-free radicals are involved in pathogenesis, free radicals were removed by injecting superoxide dismutase (SOD), a specific superoxide radical scavenger, which was conjugated with a pyran copolymer. The conjugate protected mice against a potentially lethal influenza virus infection if administered 5 to 8 days after infection. These findings indicate that oxygen radicals are important in the pathogenesis of influenza virus infection, and that a polymer-conjugated SOD has therapeutic potential for this virus infection and other diseases associated with free radicals.

Raman spectroscopic study on the solvation of p-aminobenzonitrile in supercritical water and methanol.

Raman spectra of the C[triple bond]N stretching vibration of p-aminobenzonitrile (ABN) have been investigated in water, methanol, and cyclohexane under sub- and supercritical conditions, and in acetonitrile under subcritical condition. In all solvent fluids covering the supercritical region, the vibrational frequency of the C[triple bond]N stretching mode decreased with increasing solvent density from the gaseous region to the medium density region rho(r) approximately = 2, where rho(r) is the reduced density by the critical density of the solvent. However, from the medium density region to the higher density region, the vibrational frequency turned to increase with the solvent density. The temperature-induced low frequency shift of the C[triple bond]N stretching Raman band was also ascertained by the measurement of the temperature dependence of Raman spectrum of ABN vapor above 543 K. The electronic absorption spectra in the UV region of ABN were also measured under the same experimental conditions. The absorption peak energies decreased with an increase of the solvent density, except in water above rho(r) = 2.8. The vibrational frequency shift in cyclohexane was explained by a sum of contributions of the repulsive interaction, the mean field attractive interaction, and the pure temperature effect probably due to the hot-band contribution. The residual frequency shift after the subtraction of the repulsive and temperature effects in water and methanol showed the low frequency shift with increasing solvent density from rho(r) congruent with 0 to 2.8. However, above rho(r) congruent with 2.8 in water, the residual shift showed a high frequency shift with increasing solvent density. The electronic state calculations based on the PCM model using the density functional theory (DFT) indicated that the solvent polarity change caused the low frequency shift of the C[triple bond]N stretching mode, which was also correlated with the shift of the electronic absorption spectrum. The results of the DFT calculations on the cluster of ABN with water molecules and the molecular dynamics simulations indicated that the high frequency shift of the C[triple bond]N stretching mode in water above rho(r) congruent with 2.8 could be due to the hydrogen bonding between water and ABN.

Expression of transient receptor potential vanilloid (TRPV) families 1, 2, 3 and 4 in the mouse olfactory epithelium.

We investigated the expression of transient receptor potential vanilloid (TRPV)-1-4 in the mouse olfactory epithelium (OE) in comparison to its expression in respiratory epithelium. CBA/J mice were used. The localization of TRPV-1, -2, -3 and -4 in the nasal mucosa was investigated using immunohistochemistry and a double staining study for TRPV-1 and -2 and SP was also carried out. TRPV-1-4, were expressed variably in the OE with a diffuse pattern in lamina propria, and were expressed in respiratory epithelium with strong positive expression in glandular cells of lamina propria. The double-staining study revealed coexpression of TRPV-1 and -2 and substance P (SP) in the trigeminal nerve fibers of the OE. Coexpression of TRPV-1 and SP was marked around the blood vessels and seromucinous gland of respiratory epithelium while TRPV-2 showed no co-localization. TRPV-1-4 were found to be localized in the mouse OE and respiratory epithelium. Our results suggest that TRPVs may play multiple roles in the OE, contributing to olfactory adaptation, olfactory/trigeminal interactions in nasal chemoreception and OE homeostasis; they may also be involved in olfactory transduction as well as olfactory dysfunction secondary to sinonasal inflammatory disease. TRPVs in respiratory mucosa may play a significant role in nasal nociception, ciliary movement and the regulation of mucous secretion.

Bayesian reconstruction of a vancomycin-resistant Enterococcus transmission route using epidemiologic data and genomic variants from whole genome sequencing.

BACKGROUND: Outbreaks of vancomycin-resistant enterococcus (VRE) are a serious problem in hospitals. Inferring the transmission route is an important factor to institute appropriate infection control measures; however, the methodology has not been fully established. AIM: To reconstruct and evaluate the transmission model using sequence variants extracted from whole genome sequencing (WGS) data and epidemiological information from patients involved in a VRE outbreak. METHODS: During a VRE outbreak in our hospital, 23 samples were collected from patients and environmental surfaces and analysed using WGS. By combining genome alignment information with patient epidemiological data, the VRE transmission route was reconstructed using a Bayesian approach. With the transmission model, evaluation and further analyses were performed to identify risk factors that contributed to the outbreak. FINDINGS: All VREs were identified as Enterococcus faecium belonging to sequence type 17, which consisted of two VRE genotypes: vanA (N = 8, including one environmental sample) and vanB (N = 15). The reconstruction model using the Bayesian approach showed the transmission direction with posterior probability and revealed transmission through an environmental surface. In addition, some cases acting as VRE spreaders were identified, which can interfere with appropriate infection control. Vancomycin administration was identified as a significant risk factor for spreaders. CONCLUSION: A Bayesian approach for transmission route reconstruction using epidemiologic data and genomic variants from WGS can be applied in actual VRE outbreaks. This may contribute to the design and implementation of effective infection control measures.

Modulation of dendritic cell development by immunoglobulin G in control subjects and multiple sclerosis patients.

Intravenous immunoglobulin (IVIg) preparations are reportedly effective in inhibiting the relapse of multiple sclerosis (MS), but few reports have investigated the effect of IVIg on dendritic cells (DCs), which are thought to be involved in such relapses. In the system that uses monokines to differentiate DCs from peripheral blood monocytes (Mo-DCs), we investigated the effect of immunoglobulin G (IgG) on these antigen-presenting cells. Using monocytes derived from healthy volunteers, IgG partially inhibited the expression of CD1a, a marker of immature DCs (imDCs), and CD40 and CD80, which are markers associated with T cell activation. In contrast, IgG enhanced the expression of CD83, a marker of mature DCs (mDCs). Furthermore, IgG markedly inhibited the expression of CD49d [very late activation antigen (VLA)-4 alpha4-integrin], the adhesion molecule required for mDCs to cross the blood-brain barrier. We obtained similar results on all the aforementioned cell surface molecules investigated in both healthy controls and MS patients. In addition, IgG treatment of cells from both healthy controls and MS patients inhibited the production of interleukin (IL)-12, a cytokine associated with mDC differentiation, but did not inhibit the production of IL-10. These results suggested the possibility that IgG treatment, apart from its known ability to regulate inflammation, may help to prevent relapses of MS by controlling DC maturation, consequently inhibiting invasion of immune cells into the central nervous system and affecting the cytokine profile.

Cytochrome c-551 of the thermophilic bacterium PS3, DNA sequence and analysis of the mature cytochrome.

The structural gene for cytochrome c-551 was isolated from genomic DNA of the thermophilic bacterium PS3. The amino acid sequence of cytochrome c-551 as deduced from the DNA sequence consists of 111 amino acid residues and contains one heme c-binding site (-CASCH-) located approximately in the middle of the polypeptide. The N-terminus of isolated cytochrome c-551 was blocked, but treatment with Rhizopus lipase and molecular weight measurement of the mature and lipase-treated forms by ion spray mass spectroscopy suggest that the mature c-551 may have 93 or 94 amino acid residues with a diacylated glycerol-cysteine at the N-terminal region. The first 17 or 18 amino acid residues in the N-terminal region of the nascent polypeptide, rich in hydrophobic and basic amino acid residues, may be a signal peptide to translocate the major portion of cytochrome c-551 to the extracellular surface and to be processed. Similarity of amino acid sequence of this protein is discussed in relation to other c-type cytochromes of bacilli as well as bacterial small cytochromes c such as Pseudomonas aeruginosa cytochrome c-551 and cytochrome c6 of cyanobacteria.

Molecular cloning and expression of chick Gal beta 1,3GalNAc alpha 2,3-sialyltransferase.

A cDNA clone encoding chick Gal beta 1,3GalNAc alpha 2,3-sialyltransferase (ST3Gal I) was isolated from a chick embryo brain cDNA library. The cDNA sequence included an open reading frame coding for 342 amino acids, and the deduced amino acid sequence showed 64% identity with that of the mouse enzyme. Northern blot analysis of chick embryos revealed that the ST3Gal I gene was expressed in early embryonic stages. The identity of the enzyme was confirmed by construction of a recombinant sialyltransferase in which the N-terminal part including the cytoplasmic tail and signal anchor domain was replaced with an immunoglobulin signal peptide sequence. This enzyme expressed in COS-7 cells exhibited transferase activity similar to that of mouse ST3Gal I.

Solubilization and reconstitution of voltage-dependent calcium channel from bovine cardiac muscle. Ca2+ influx assay using the fluorescent dye Quin2.

Highly purified sarcolemmal membranes, prepared from fresh bovine heart left ventricle, were solubilized by n-octyl beta-D-glucopyranoside and reconstituted into proteoliposomes with soybean phospholipids by the detergent-dialysis method. Ca2+ flux into the proteoliposomes was determined using the fluorescent probe Quin2. A membrane potential (negative in the proteoliposome interior) that was created by K+ diffusion mediated by valinomycin accelerated the Ca2+ influx. The voltage-dependent Ca2+ influx was dependent on pretreatment of the sarcolemmal membranes with Bay K 8644 and was inhibited by various calcium antagonists including nicardipine (K0.5 = 4.5.10(-7) M), verapamil (K0.5 = 9.2.10(-9) M), diltiazem (K0.5 = 26.10(-8) M) and omega-conotoxin (K0.5 = 9.5.10(-9) M).

Donor substrate specificities of Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase and Gal beta 1,3GalNAc alpha 2,3-sialyltransferase: comparison of N-acetyl and N-glycolylneuraminic acids.

Using cloned sialyltransferases, Gal beta 1,3GalNAc alpha 2,3-sialyltransferase (ST3Gal I) and Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase (ST6Gal I) from both chicken and mouse, CMP-NeuAc and CMP-NeuGc were compared as donor substrates with pyridylamino-oligo-saccharides as acceptors. ST6Gal I showed 4-7-times higher activity toward CMP-NeuGc than CMP-NeuAc, while for ST3Gal I there was no significant difference between them, irrespective of the origin of the enzymes. Also, the difference in donor substrate (i.e., NeuAc and NeuGc) had little effect on the preference to acceptor substrates of these enzymes. Thus, the results showed that the cloned sialyltransferases can utilize both CMP-NeuAc and CMP-NeuGc as donor substrates, and that the preference difference between the sialyltransferases to CMP-NeuGc and CMP-NeuAc could, at least partly, explain the discrepancy in the ratio of NeuAc and NeuGc in glycolipids and glycoproteins in individual tissues.

Sequence and over-expression of subunits of adenosine triphosphate synthase in thermophilic bacterium PS3.

The primary structures of all the subunits of thermophilic ATP synthase were determined, and its alpha, beta and gamma subunits could be over-expressed in Escherichia coli, because these subunits were stable and reconstitutable. DNA of 7500 base pairs in length was found to contain a cluster of nine genes for subunits of ATP synthase. The order of their reading frames (size in base pairs) was: I(381): a(630): c(216): b(489): delta(537): alpha(1507): gamma(858): beta(1419): epsilon(396), I being a gene for a small hydrophobic, basic protein expressed in vitro. All the termini of TF0F1 subunits were confirmed by peptide sequencing. Large quantities of the overexpressed thermophilic alpha, beta and gamma subunits were prepared from the extract of E. coli, by a few purification steps.

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)

The alpha/beta interfaces of alpha(1)beta(1), alpha(3)beta(3), and F1: domain motions and elastic energy stored during gamma rotation.

ATP synthase (F(o)F(1)) consists of F(1) (ATP-driven motor) and F(o) (H(+)-driven motor). F(1) is a complex of alpha(3)beta(3)gammadeltaepsilon subunits, and gamma is the rotating cam in alpha(3)beta(3). Thermophilic F(1) (TF(1)) is exceptional in that it can be crystallized as a beta monomer and an alpha(3)beta(3) oligomer, and it is sufficiently stable to allow alphabeta refolding and reassembly of hybrid complexes containing 1, 2, and 3 modified alpha or beta. The nucleotide-dependent open-close conversion of conformation is an inherent property of an isolated beta and energy and signals are transferred through alpha/beta interfaces. The catalytic and noncatalytic interfaces of both mitochondrial F(1) (MF(1)) and TF(1) were analyzed by an atom search within the limits of 0.40 nm across the alphabeta interfaces. Seven (plus thermophilic loop in TF(1)) contact areas are located at both the catalytic and noncatalytic interfaces on the open beta form. The number of contact areas on closed beta increased to 11 and 9, respectively, in the catalytic and noncatalytic interfaces. The interfaces in the barrel domain are immobile. The torsional elastic strain applied through the mobile areas is concentrated in hinge residues and the P-loop in beta. The notion of elastic energy in F(o)F(1) has been revised. X-ray crystallography of F(1) is a static snap shot of one state and the elastic hypotheses are still inconsistent with the structure, dyamics, and kinetics of F(o)F(1). The domain motion and elastic energy in F(o)F(1) will be elucidated by time-resolved crystallography.

Nonoxidative metabolism of ethanol in the pancreas; implication in alcoholic pancreatic damage.

Alcohol dehydrogenase activity and fatty acid ethyl ester synthase activity were measured in various organs of male Wistar strain rats. The mean (+/- SE) values of alcohol dehydrogenase activity in liver, testis, pancreas and brain were 223 +/- 34, 35 +/- 13, 27 +/- 17 and 24 +/- 15 nmol/hr/mg protein, respectively, but not detectable in heart and skeletal muscle. Fatty acid ethyl ester synthase activity in pancreas, liver, testis and heart were 1348 +/- 263, 23 +/- 14, 17 +/- 3 and 2 +/- 1 nmol/hr/mg protein, respectively, but not detectable in brain and skeletal muscle. Alcohol dehydrogenase activity, fatty acid ethyl ester synthase activity, fatty acid ethyl ester content and amylase activity were measured in pancreas of rat after 7 weeks of ethanol feeding. Compared with control rats, ethanol-fed rats had normal fatty acid ethyl ester synthase activity and alcohol dehydrogenase activity. However, fatty acid ethyl ester content increased five-fold and amylase activity decreased up to 20% of the control group. Fatty acid ethyl ester content was inversely correlated with amylase activity. These results suggest that fatty acid ethyl ester may be responsible for the development of pancreatic damage by ethanol.

Net adenosine triphosphate synthesis driven by an external electric field in rat liver mitochondria.

Rat liver mitochondria irradiated with external electric pulses (760 V/cm, 30 ms, rectangular) catalyzed net ATP synthesis of 0.055 nmol/mg protein/pulse. The amount of ATP synthesized increased on increasing the number, voltage and duration of the electric pulses. There was no apparent threshold voltage or duration for ATP synthesis in the ranges tested (0-760 V/cm and 0.05-30 ms). The energy applied to the mitochondrial membrane within a much shorter time than the turnover time of H+-ATPase must be stored in the membrane until it is utilized for ATP synthesis. Synthesis of ATP was inhibited by the specific H+-ATPase inhibitor aurovertin and by high concentrations of uncouplers. The energy transfer inhibitors oligomycin and dicyclohexylcarbodiimide had no effect on synthesis of ATP in mitochondria by electric pulses, but inhibited oxidative phosphorylation under the same conditions. In contrast to the pulse-driven ATP synthesis in subchloroplast particles, that in intact mitochondria required hexokinase-glucose, higher ADP concentration, lower osmolarity and protection against the pH effect of the electrodes.

Ouabain-resistant cells cultured in a synthetic medium.

Several strains of kidney and liver cells cultured in a synthetic medium were found to be resistant to ouabain. These cell strains were characterized because this resistance may serve as a good marker in genetic studies on somatic cells in chemically defined conditions in the absence of Na+ related growth factors and hormones. The phenotype was stable in the absence of selection for at least two years, and the original strains before adaptation to the synthetic medium were found to have ouabain sensitivity equal to the corresponding cells in the synthetic medium. The resting membrane potential, Na+,K+-ATPase activity, and growth rate of the resistant cells were similar to those of ouabain-sensitive cells. The resistance of the cells was not affected by serum or antibodies against some cytoskeletal proteins and the sensitivity of the Na+,K+-ATPase was not restored by partial purification of the membranes. Western blotting of the Na+,K+-ATPase of the ouabain-resistant cells showed that the molecular weights of its two subunits and its immunoreactivity were similar to those of the enzyme from the ouabain-sensitive strain. Thus the ouabain resistance is caused not by ouabain-like hormone produced by the cells or change in the cytoskeletal system, but by a mutation resulting in expression of an ouabain-resistant ATPase gene.

A novel human metalloprotease synthesized in the liver and secreted into the blood: possibly, the von Willebrand factor-cleaving protease?

We identified a novel metalloprotease, which could be responsible for cleaving the Tyr842-Met843 peptide bond of von Willebrand factor (vWF). This metalloprotease was purified from Cohn Fraction-I precipitate of human pooled plasma by the combination of gel filtration, DEAE chromatography, and preparative polyacrylamide gel electrophoresis in the presence of SDS. The NH2-terminal amino acid sequence of the isolated protein was: AAGGILHLELLVAVGPDVFQAHQEDTRRY. Based on this sequence, we searched human genomic and EST databases, and identified compatible nucleotide sequences. These results suggested that this protein is a novel metalloprotease, a member of the family of a disintegrin and metalloprotease with thrombospondin type-1 motifs (ADAMTS), and its genomic DNA was mapped to human chromosome 9q34. Multiple human tissue northern blotting analysis indicated that the mRNA encoding this protease spanned approximately 5 kilobases and was uniquely expressed in the liver. Furthermore, we determined the cDNA sequence encoding this protease, and found that this protease was comprised of a signal peptide, a proregion followed by the putative furin cleavage site, a reprolysin-type zinc-metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 (TSP1) motif, a cysteine-rich region, a spacer domain, and COOH-terminal TSP1 motif repeats.