Quick-freeze,deep-etch studies of endothelial components,with special reference to cytoskeletons and vesicle structures

A three-dimensional study of the ultrastructure of endothelial cells is helpful in understanding important endothelial functions such as vascular transport and cell permeability. For this purpose, in addition to serial sectioning electron microscopy and high-voltage electron microscopy, the quick-freeze, deep-etching technique also enables us to analyze structures at the molecular level by its high resolution and is useful for three-dimensional morphological studies. Some modifications on the conventional deep-etching method were made in this study to reduce the undesirable aggregation of proteins and salts during etching. Using this technique, we examined the rat aortic endothelium, particularly the membrane structures and cytoskeletons. The luminal surface of the endothelium was covered with a fine filamentous coat, which was anchored to the plasma membrane. In the cytoplasm, actin filaments were prominent and were oriented randomly or in a parallel fashion near the plasma membrane. Of the vesicles seen in the endothelium, some had basket coats of clathrin, and others had striped coats on the cytoplasmic membrane surface. These surface structures of the vesicles suggest the transport mechanism of the vesicles in association with the fine filaments attached to the vesicles.     

Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction

Streptococcus mutans is an etiological agent in human dental caries. A method for the detection of S. mutans directly from human dental plaque by polymerase chain reaction has been developed. Oligonucleotide primers specific for a portion of the dextranase gene (dexA) of S. mutans Ingbritt (serotype c) were designed to amplify a 1272-bp DNA fragment by polymerase chain reaction. The present method specifically detected S. mutans (serotypes c, e and f), but none of the other mutans streptococci: S. cricetus (serotype a), S. rattus (serotype b), S. sobrinus (serotypes d and g), and S. downei (serotype h), other gram-positive bacteria (16 strains of 12 species of cocci and 18 strains of 12 species of bacilli) nor gram-negative bacteria (1 strain of 1 species of cocci and 20 strains of 18 species of bacilli). The method was capable of detecting 1 pg of the chromosomal DNA purified from S. mutans Ingbritt and as few as 12 colony-forming units of S. mutans cells. The S. mutans cells in human dental plaque were also directly detected. Seventy clinical isolates of S. mutans isolated from the dental plaque of 8 patients were all positive by the polymerase chain reaction. These results suggest that the dexA polymerase chain reaction is suitable for the specific detection and identification of S. mutans.     

High-speed and low-power operation of a resonant tunneling logicgate MOBILE

High-speed operations up to 35 Gb/s were demonstrated for a resonant tunneling (RT) logic gate monostable-bistable transition logic element (MOBILE). The test circuit consisted of a MOBILE and a DCFL-type output buffer, and it was fabricated using InP-based resonant tunneling diode/HEMT integration technology. This operation bit rate is close to the cutoff frequency of the 0.7-μm gate HEMTs used in the circuit, and was obtained after improvement of the output buffer design. This result indicates the high-speed potential of the MOBILE, though the speed is still limited by the buffer. The power dissipation of the MOBILE was also discussed based on a simple equivalent circuit model of RTDs. This revealed that the power dissipation is as small as 2 mW/gate over a wide range of operation bit rates     

Study on the concentration of an enzyme immobilized by Langmuir-Blodgett films

Urease is an enzyme which decomposes urea into NH₃ and CO₂. We can produce a urea sensor by immobilizing urease on the pH sensor, and the Langmuir-Blodgett (LB) method has been expected to be useful as one of the immobilizing methods. We have measured for the first time the amount of urease adsorbed onto the LB film and shown that the relationship between the amount of adsorbed urease and the concentration in the solution can be expressed by an equation similar to the Langmuir adsorption isotherm.     

Endotoxin rejection by ultrafiltration through high-flux, hollow fiber filters

The efficacy of endotoxin (ET) rejection of four hollow fiber membranes with comparable sieving properties was evaluated in an ultrafiltration experiment. The solution conditioned with type I lipopolysaccharide (LPS) from Escherichia coli, 80,000 endotoxin units (EU)/L, was filtered through polyesterpolymer alloy (PEPA), polymethyl methacrylate (PMMA), polyacrylonitrile (PAN), and polysulfone (PS) membranes. The ET activity of the filtrate was not detectable in PEPA and PMMA, 6.4 +/- .04 (mean +/- SD) EU/L in PAN, and 10.3 +/- 1.1 EU/L in PS. The ET activity of the filtrate of type II LPS from Acinetobacter solution, 80,000 EU/L, was not detectable in PEPA, 3.7 +/- 0.4 EU/L in PMMA, 16.5 +/- 1.5 EU/L in PAN, and 20.7 +/- 1.4 EU/L in the PS filter. The order of the rejection capability coincided with the adsorptive capacity as shown by the decrement in ET levels of solutions filled within the filter modules in the adsorption equilibrium experiment. In conclusion complete rejection of ET molecules can be achieved by ultrafiltration through hydrophobic membranes having a high adsorptive capacity in addition to an appropriate sieving property for ET molecules.     

Cloning and sequencing of the gene encoding a novel lysine-specific cysteine proteinase (Lys-gingipain) in Porphyromonas gingivalis: structural relationship with the arginine-specific cysteine proteinase (Arg-gingipain)

Lys-gingipain (KGP), so termed due to its peptide cleavage specificity for lysine residues, is a cysteine proteinase produced by the Gram-negative anaerobic bacterium Porphyromonas gingivalis. Mixed oligonucleotide primers designed from the NH2-terminal sequence of the purified enzyme were used to clone the KGP-encoding gene (kgp) from the organism. The nucleotide sequence of kgp had a 5,169-bp open reading frame encoding 1,723 amino acids with a calculated molecular mass of 218 kDa. As the extracellular mature enzyme had an apparent molecular mass of 51 kDa in gels, the precursor of KGP was found to comprise at least four domains, the signal peptide, the NH2-terminal prodomain, the mature proteinase domain, and the COOH-terminal hemagglutinin domain, and to be proteolytically processed during its transport. Importantly, the COOH-terminal region contained three direct repeats of two different amino acid sequences, LKWD(or E)AP and YTYTVYRDGTKI, and the subdomains located between the two repeats exhibited strong similarity to those of Arg-gingipain (RGP), another major cysteine proteinase produced by the organism and having cleavage specificity for arginine residues, although the arrangement of the subdomains was not necessarily identical in the two enzymes. Since the KGP activity was greatly decreased in RGP-deficient mutants and since the most probable site of the propeptide cleavage was present in the homologous sequence highly susceptible to proteolysis by RGP, the precursor of KGP is likely to be processed by RGP to form the mature enzyme.     

Oxidation-reduction equilibrium of Cu2+/Cu+ in binary alkaline sulfate melts

Oxidation-reduction equilibria for a Cu²⁺/Cu⁺ couple in binary alkaline sulfate melts, Li₂SO₄ + R₂SO₄ (R = Na, K, Rb, Cs), were determined at temperatures of 973, 1023, and 1073 K by equilibrating these melts with gas mixtures of Ar + O₂ + SO₂. RedOx equilibria are well interpreted by the average ionic radii of alkaline metals: r(average) = X(Li₂SO₄) r(Li) + X(R₂SO₄) r(R), wherer andX denote, respectively, mole fraction and the ionic radii of alkaline metal. The oxygen anion activities would increase with an increase in R₂SO₄ mole fractions of binary sulfates Li₂SO₄ + R₂SO₄.     

Liver volume in children measured by computed tomography

Primary malignant melanoma of the urethra is very rare. In the male, the distal urethra is the most common site. The histopathology does not usually differ from that of melanoma at other body sites. This report describes a case of urethral malignant melanoma which closely resembled urethral carcinoma. It showed both papillary and solid growth, and the diagnosis only became apparent from special stains. Pathologists should be aware of this rare occurrence.