Ca2+-dependent contractility of isolated and demembranated macronuclei in the hypotrichous ciliate Euplotes aediculatus.

The hypotrichous ciliated protozoan Euplotes aediculatus possesses a characteristic C-shaped somatic nucleus (macronucleus) within the cytoplasm, which shows dynamic shape change during the cell cycle. It is shown that isolated macronuclei possess Ca(2+)-dependent contractility. Macronuclei were isolated, stuck fast on the glass surface, and subjected to different concentrations of Ca(2+) in a Ca(2+)-EGTA buffer. The nuclei became expanded at [Ca(2+)]<10(-7)M, and they contracted on subsequent addition of higher concentrations of Ca(2+). Cycles of expansion and contraction of the nucleus could be repeated many times by alternate addition of EGTA and Ca(2+), indicating that the size of isolated nuclei can be regulated by [Ca(2+)] alone. The nuclear contraction was observed in all phases of the cell cycle, but contractility was less evident around replication bands in the S phase. In addition to the hypotrichous ciliate Euplotes, similar Ca(2+)-dependent nuclear contractility was found to exist in other cell types, including protozoans of different taxa (a heliozoon Actinophrys sol and a peniculine ciliate Paramecium bursaria), and also mammalian culture cells (HeLa cells). Our findings suggest a possibility that Ca(2+)-dependent nuclear contractility may be shared among diverse eukaryotic organisms.

The ultrastructure of contractile tubules in the heliozoon Actinophrys sol and their possible involvement in rapid axopodial contraction.

We employed an improved fixation procedure for electron microscopy using ruthenium red, and found a bundle of contractile tubules inside the axopodia of the heliozoon Actinophrys sol. Upon food uptake, the tubules shorten and transform into a mass of small granules when rapid axopodial contraction occurs, suggesting that these structures are involved in the process of axopodial contraction. The relationship between transformation of the contractile tubules and accompanying disassembly of the axonemal microtubules was studied by examining the ultrastructure of the contractile tubules after disassembly of the microtubules was artificially induced by cold or colchicine treatment. Granulation of the contractile tubules was induced by cold but not by colchicine treatment. During recovery from cold treatment, granular forms of the contractile tubules became re-elongated and their initial tubular appearance was restored. These results suggest that the contractile tubules in heliozoon axopodia play a role in repetitive cytoplasmic contraction.

Involvement of a 40-kDa glycoprotein in food recognition, prey capture, and induction of phagocytosis in the protozoon Actinophrys sol.

A 40-kDa glycoprotein (gp40) was identified as a Con A-binding adhesive substance of the heliozoon Actinophrys sol for immobilizing and ingesting prey flagellates. Isolation and partial characterization of gp40 showed that: 1) gp40 is a major Con A-binding protein of Actinophrys with a molecular weight of 40 kDa, and is stored in secretory granules called extrusomes; 2) gp40 was purified by Con A-affinity chromatography, and the N-terminal amino acid sequence was determined as H2N-KVLK-FEDDFDTFDLQ; 3) prey flagellates became adhered to gp40-immobilized agarose beads; 4) phagocytosis of Actinophrys was induced against gp40-immobilized agarose beads; and 5) solubilized gp40 induced exocytosis of extrusomes and cell fusion of heliozoons. These results indicate that gp40 is a multi-functional secretory protein of Actinophrys, which is required in correct targeting of the heliozoon to food organisms as well as in self-recognition.

Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli.

The suppressor mutation, named sfhC21, that allows Escherichia coli ftsH null mutant cells to survive was found to be an allele of fabZ encoding R-3-hydroxyacyl-ACP dehydrase, involved in a key step of fatty acid biosynthesis, and appears to upregulate the dehydrase. The ftsH1(Ts) mutation increased the amount of lipopolysaccharide at 42 degrees C. This was accompanied by a dramatic increase in the amount of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase [the IpxC (envA) gene product] involved in the committed step of lipid A biosynthesis. Pulse-chase experiments and in vitro assays with purified components showed that FtsH, the AAA-type membrane-bound metalloprotease, degrades the deacetylase. Genetic evidence also indicated that the FtsH protease activity for the deacetylase might be affected when acyl-ACP pools were altered. The biosynthesis of phospholipids and the lipid A moiety of lipopolysaccharide, both of which derive their fatty acyl chains from the same R-3-hydroxyacyl-ACP pool, is regulated by FtsH.

Experimental free-space optical network for massively parallel computers.

A free-space optical interconnection scheme is described for massively parallel processors based on the interconnection-cached network architecture. The optical network operates in a circuit-switching mode. Combined with a packet-switching operation among the circuit-switched optical channels, a high-bandwidth, low-latency network for massively parallel processing results. The design and assembly of a 64-channel experimental prototype is discussed, and operational results are presented.

Focal nodular hyperplasia of the liver without central scar.

An unusual case of focal nodular hyperplasia (FNH) of the liver is presented. A 31-year-old male was admitted to our hospital for the evaluation of an hepatic mass 10 cm in diameter located in segment 4. Routine examinations including liver function tests were all normal. Hepatitis B surface antigen, hepatitis C virus antibody, alpha-fetoprotein were all negative. Imagining studies could not detect specific findings of FNH such as a central scar structure or a spoke-wheel appearance due to dilated tumor vessels coursing centrally followed by radiating peripheral coursing vessels. Extensive left lobectomy of the liver was performed. Histological examination of the specimen cut into 1-cm-thick slices revealed no central stellate scars. Preoperative diagnosis of FNH with imaging studies was difficult because of the absence of central scars.

Histopathological assessment of lymph node metastasis in patients with gastric cancer.

BACKGROUND/AIMS: Lymph node dissection has been carried out for two decades in Japan, resulting in improved survival of patients with gastric cancer. By analyzing dissected nodes of resected advanced gastric cancers, we intend to determine which parameter(s) are more concerned in node metastasis. PATIENTS AND METHODS: Curative gastrectomy with dissection of lymph nodes was performed for 78 of the 108 patients with advanced gastric cancer. Metastases were found in 171 of 1359 nodes (13%) dissected. In each node, the maximum and minimum diameters were measured, and the ratio of the former to the latter was calculated. RESULTS: The minimum diameter of node was closely related to node metastasis. When the minimum diameter in node metastatic criteria was "3 mm or more", a sensitivity of 81% and specificity of 54% were calculated. In false negative node, signet-ring cell carcinoma (SIG) (33%) and poorly differentiated adenocarcinoma (POR) (22%) were more frequently found as primary lesions. Half or more of these nodes were invaded by only a few carcinoma cells. When the primary lesion is histologically SIG or POR, endoscopic treatment is contraindicated for possible nodes metastasis. CONCLUSIONS: It is impossible to distinguish nodes with micrometastasis from nodes without metastasis. The minimum diameter of node is more important in node metastasis of gastric cancer than the maximum diameter of node or the ratio of both.

Eosinophilic gastroenteritis involving the entire digestive tract.

Eosinophilic gastroenteritis, an inflammatory disease of unknown etiology, commonly involves the stomach and small intestine with eosinophilic infiltration. Herein we describe an unusual case of eosinophilic gastroenteritis involving the entire digestive tract. A 24-yr-old woman came to our hospital presenting with diarrhea with marked peripheral eosinophilia. Stool specimens were negative for parasites, ova, bacteria, and fungi. Barium and endoscopic studies showed thickened edematous folds of the duodenum and small intestine. Biopsy specimens of the esophagus, stomach, duodenum, ileum, and colon showed eosinophilic infiltration. A diagnosis of eosinophilic gastroenteritis involving the entire digestive tract was made, and therapy with prednisone was started. Symptoms and peripheral eosinophilia rapidly resolved, and biopsies of the digestive tract demonstrated no eosinophilic infiltration. This case illustrates the expanded spectrum of the disease to the entire digestive tract and emphasizes the diagnostic usefulness of endoscopic biopsies. The initial presentation of patients with eosinophilia requires the evaluation of the entire digestive tract. It is worth noting that eosinophilic gastroenteritis can involve the entire digestive tract.

Quantitative analysis of superoxide anion generation in living cells by using chemiluminescence video microscopy.

Superoxide anions (O2-) generated by rabbit neutrophils were detected and quantified by a video microscope equipped with a photon-counting camera. One count obtained by this system was equivalent to 59 amol of O2-. Maximum O2- production was observed at 6-8 min after stimulation and was estimated as 1.9 fmol/min per cell on the average.

Intussusception associated with Salmonella typhimurium enterocolitis.

Intussusception in the adult is unusual, and its etiology is mostly neoplastic. In most cases of intussusception, a polypoid tumor located in the terminal ileum invaginates within the cecum. We described an unusual case of adult intussusception associated with Salmonella typhimurium enterocolitis. Preoperative diagnosis of intussusception in the ileocecal region was made by ultrasonography, and the ileocecal region containing the mass in the cecum, causing the intussusception, was resected. Histological features of the mass included an edematous focal wall thickening. Later, the diagnosis of S. typhimurium enterocolitis was made by the preoperative stool cultures. This case demonstrated intussusception as a complication of S. typhimurium enterocolitis. It is worth noting that S. typhimurium enterocolitis is in fact a potential cause of intussusception.

Characterization of translucent segments observed in an smbA mutant of Escherichia coli.

The smbA gene of Escherichia coli is essential for cell proliferation. The smbA2 mutant shows cold-sensitive colony formation at 22 degrees C. A novel morphological phenotype, formation of a translucent segment at midcell or at a cell pole, was observed by phase-contrast microscopy at a high frequency in the smbA2 mutant cells incubated in L medium lacking NaCl at 22 degrees C, but not observed in L medium containing 1% NaCl or 20% sucrose at the same temperature. No translucent segment was observed in the wild-type cells in any of the media used. Electron microscopic observation revealed that the translucent segments resulted from the enlargement of a periplasmic space by separation of the inner membrane from the peptidoglycan layer and the outer membrane.

Topology and subcellular localization of FtsH protein in Escherichia coli.

FtsH protein in Escherichia coli is an essential protein of 70.7 kDa (644 amino acid residues) with a putative ATP-binding sequence. Western blots (immunoblots) of proteins from fractionated cell extracts and immunoelectron microscopy of the FtsH-overproducing strain showed exclusive localization of the FtsH protein in the cytoplasmic membrane. Most of the FtsH-specific labeling with gold particles was observed in the cytoplasmic membrane and the adjacent cytoplasm; much less was observed in the outer membrane and in the bulk cytoplasm. Genetic analysis by TnphoA insertions into ftsH revealed that the 25- to 95-amino-acid region, which is flanked by two hydrophobic stretchs, protrudes into the periplasmic space. From these results, we concluded that FtsH protein is an integral cytoplasmic membrane protein spanning the membrane twice and that it has a large cytoplasmic carboxy-terminal part with a putative ATP-binding domain. The average number of FtsH molecules per cell was estimated to be approximately 400.

[Endosonographic diagnosis of metastatic tumors in the recto-uterine or recto-vesical space by transrectal scan].

A 6-year experience of transrectal ultrasonography (TRUS) in 75 patients with advanced gastrointestinal cancers was reviewed regarding the diagnostic yield of metastatic extrarectal tumors. A diagnostic capability of TRUS (a sensitivity of 82.9%, a specificity of 97.5% and an accuracy of 90.7%) was statistically superior to that of digital rectal examination (a sensitivity of 62.9%, a specificity of 75% and an accuracy of 69.3%). However, TRUS could hardly correctly diagnose a miliary type of extrarectal tumors in contrast to nodular and thickened types. TRUS also had an advantage over conventional ultrasonography in detecting a small amount of ascites with a sensitivity of 75%, a specificity of 93% and an accuracy of 86.6%. Patients with positive signs of extrarectal tumors or ascites on TRUS survived significantly shorter than those with negative signs. TRUS renders a convenient means in the objective diagnosis of peritoneal carcinomatosis, and predicts patient's prognosis.

Isolation and properties of the axopodial cytoskeleton of a heliozoan, Echinosphaerium akamae.

The axopodia of a large heliozoan, Echinosphaerium akamae, were efficiently liberated from the cell body by treatment with 65% D(2)O solution containing 5 mM MgCl(2),2.1 mM EGTA, 1 mM KCl and 5 mM HEPES (pH 6.9). After D20 treatment, the cell bodies were removed by centrifugation at a low speed for 30 sec and the resulting supernatant was recentrifuged at 10,000 × g for 10 min. The axopodia were obtained as the pellet fraction without any contamination from the cell body. The isolated axopodia maintained their regular arrangement of cytoskeletal microtubules and were shortened by treatment with Ca(2+). When the isolated axopodia was subjected to SDS-polyacrylamide gel electrophoresis, two major protein bands were detected. The molecular weights of the proteins, tentatively identified as the heliozoan tubulins, were estimated to be about 46 and 50 kD, and an antibody against rat brain tubulin reacted with only the 46 kD protein species.

Ultrastructure and calcium-dependent contraction of the myonemal network in a heterotrich ciliate, Blepharisma japonicum.

This laboratory has previously demonstrated that shortening of the cell body of a heterotrich ciliate, Blepharisma japonicum, could be induced as a step-down photophobic response. Here, we examined the structure and contractility of the myonemes in detergent-extracted cell models and in isolated cortical fragments. Ultrastructural observation showed that the myoneme was connected to the basal ends of the posterior kinetosomes and constructed a systematic network as a whole. Shortening of the cell model was induced by > 10(-4) M Ca(2+), while the rounded cell model did not re-elongate even when it was washed in a calcium-free solution either with or without addition of ATP. Fluffy fibrils, which were tentatively identified as aggregated bundles of the myonemes, were isolated with the kinetosomal complex and showed calcium-dependent and ATP-independent contraction. The minimum concentration of Ca(2+) required for inducing contraction was at the level of 10(-6) M. These results suggest that the cell body shortening in Blepharisma is caused by the Ca(2+)-dependent contraction of the myonemal network.

Structure and function of the cytoskeleton in heliozoa: 2. Measurement of the force of rapid axopodial contraction in Echinosphaerium.

The large heliozoan Echinosphaerium extends a number of needle-like axopodia by which it captures food organisms. Every axopodium contains a bundle of several hundreds of axonemal microtubules as a cytoskeletal element. When the tip of a poly-L-lysine-coated glass micro-needle came into contact with the distal part of an axopodium, a rapid axopodial contraction (2.6 mm/s) occurred with a concomitant bending of the needle toward the cell body. In this report, we measured the force of the axopodial contraction by utilizing the relation between force and bending displacement of the micro-needle, and examined a possibility that the axopodial contraction is ascribed to the axopodial tension (surface tension and/or cytoplasmic elasticity) that is developed as a result of microtubule degradation. The force of the axopodial contraction was estimated in the order of 10(-9) N. Treatment with 10 mM colchicine induced disassembly of the axopodial microtubules and a resulting slow retraction of the axopodia (0.1 µm/s) occurred. The force of the slow retraction was also measured by the same procedure to estimate the axopodial tension, and was in the order of 10(-11) N. It was thus demonstrated that the motive force for axopodial contraction cannot be explained as an axopodial tension generated as a result of disassembly of the microtubules.

Successful treatment of adult Henoch-Schönlein purpura with factor XIII concentrate.

We report the cases of three adult patients with severe abdominal complications of Henoch-Schönlein purpura who had low activity of factor XIII during the acute phase of the disease. In all three cases, abdominal symptoms and purpura immediately responded to heat-treated, placenta-derived factor XIII concentrate. No adverse effects were experienced. Factor XIII concentrate replacement should be considered as the initial treatment for severe abdominal symptoms in adult Henoch-Schönlein purpura associated with a decreased level of factor XIII activity.

Dielectric behavior of the frog lens in the 100 Hz to 500 MHz range. Simulation with an allocated ellipsoidal-shells model.

In an attempt to correlate the passive electrical properties of the lens tissue with its structure, we measured ac admittances for isolated frog lenses, lens nuclei, and homogenate of cortical fiber cells, over the frequency range 10(2)-5.10(8) Hz. The whole lenses molded into discoid shape show a characteristic "two-step" dielectric dispersion with a huge permittivity increment of the order of 10(5) at 1 kHz. Of the two subdispersions disclosed, dispersion 1 has a permittivity increment (delta epsilon) of 2.10(5) with a characteristic frequency (fc) of 2 kHz, and dispersion 2 has a delta epsilon of 400 with an fc of 2 MHz. In terms of loss tangent, these dispersions are more clearly located as two separate peaks. Data are analyzed using an allocated ellipsoidal-shells model which has been developed by taking into account fiber orientation inside the lens tissue. Dispersion 1 is assigned to the equatorial cortex, where fiber cells run parallel to the applied electric field, and dispersion 2 to the nucleus with a complex fiber arrangement and also to the polar cortex, in which the fiber alignment is predominantly perpendicular. In addition, the model analysis reveals that, in the frog lens, the nucleus occupies approximately 30% in volume and that relative permittivity and conductivity for the cell interior are, respectively, 45 and 3 mS/cm for the cortical cells, and 28 and 0.3 mS/cm for the nuclear cells.

Studies on the mechanism of cell elongation in Blepharisma japonicum: 4. Three dimensional construction of the kinetosomal complex and its functional role on cell elongation.

The kinetosomal complex of a heterotrich ciliate Blepharisma japonicum was investigated at ultrastructural level, with special reference to its cell elongation in response to light stimulation. In serial sections, vacuole-associated microtubules were found to be originated from both anterior fiber sheet and left surface of the anterior kinetosome. These microtubules form a bundle and extend toward the anterior end of the organism. The postciliary microtubular sheet is attached to the proximal half of the posterior kinetosome. Two types of transverse microtubules are present: the anterior 8-9 microtubules arising at the base of the anterior kinetosome and the posterior 2-3 microtubules arising between the paired kinetosomes. Based on these results, a three dimensional model of the kinetosomal complex was proposed and the mechanism of cell elongation in Blepharisma was discussed.