Mutual recognition between polymerized liposomes. IV. Polysaccharide-lectin system.

As a model of cell-cell recognition processes, the association processes of a polysaccharide (mannan)-carrying liposome with a lectin (Concanavalin A, Con A)-carrying polymerized liposome were followed by turbidimetry. The association process was strongly inhibited by the addition of a low molecular weight sugar, methyl-alpha-D-mannopyranoside, which shows that the association between the liposomes is due to the specific interaction between Con A and mannan. The association rate constant obtained was much smaller than the theoretical value for a diffusion-controlled binary association process. This implies that the association rate of liposomes is limited by the recognition between complementary ligands bound on the liposome surfaces. Another reason for the smaller association rate constant in the liposome-liposome system is the repulsive hydration effect. The effect of the surface density of the lectin immobilized on the liposome on the recognition was also examined.     

Mitochondrial translation of subunits of the rotenone-sensitive NADH:ubiquinone reductase in Neurospora crassa.

The rotenone sensitive NADH:ubiquinone was isolated from mitochondria of Neurospora crassa as a monodisperse preparation with the apparent mol. wt. in Triton solution of 0.9 X 10(6). The enzyme is composed of at least 22 subunits with apparent mol. wts. in SDS between 70 and 11 kd. Six of the subunits with the mol. wts. 70, 48, 37, 25, 22 and 18 kd were radioactively labelled in the enzyme isolated from cells which had incorporated [35S]methionine in the presence of cycloheximide. These subunits are synthesized in the mitochondria. Eleven subunits were radioactively labelled in the enzyme from cells which had incorporated [35S]methionine in the presence of chloramphenicol. These subunits are synthesized in the cytoplasm. The site of translation of the other subunits could not be established by the pulse-labelling technique. The assignment of the mitochondrially synthesized subunits to unidentified reading frames on the mitochondrial DNA is discussed.     

A second gene for the African green monkey poliovirus receptor that has no putative N-glycosylation site in the functional N-terminal immunoglobulin-like domain.

Using cDNA of the human poliovirus receptor (PVR) as a probe, two types of cDNA clones of the monkey homologs were isolated from a cDNA library prepared from an African green monkey kidney cell line. Either type of cDNA clone rendered mouse L cells permissive for poliovirus infection. Homologies of the amino acid sequences deduced from these cDNA sequences with that of human PVR were 90.2 and 86.4%, respectively. These two monkey PVRs were found to be encoded in two different loci of the genome. Evolutionary analysis suggested that duplication of the PVR gene in the monkey genome had occurred after the species differentiation between humans and monkeys. The NH2-terminal immunoglobulin-like domain, domain 1, of the second monkey PVR, which lacks a putative N-glycosylation site, mediated poliovirus infection. In addition, a human PVR mutant without N-glycosylation sites in domain 1 also promoted viral infection. These results suggest that domain 1 of the monkey receptor also harbors the binding site for poliovirus and that sugar moieties possibly attached to this domain of human PVR are dispensable for the virus-receptor interaction.     

Kinetic study of a membrane enzyme reactor

A flat-membrane dialyzer was used as enzyme reactor by introducing enzyme solution into one of the membrane-separated chambers. The apparent Michaelis constant K_m(app) of urease was always larger (ten times at [urease] = 1 mg/ml) than that of free enzyme because the permeation of substrate through the membrane was rate determining. K_m(app) for urease decreased from 125 to 20mM with increasing flow rate of the substrate solution because of the turbulent flow near the membrane. In the case of glucose oxidase or creatine kinase, the reaction rate was limited by the permeation of less permeable substrates such as oxygen or ATP. Therefore, K_m(app) of more permeable substrates such as glucose or creatine became smaller than that of free enzyme. The reaction amount calculated from the permeation data agreed well with experimental results. By designing spacers for the reactor to give turbulence to the solution, the effectiveness of the reactor was improved fivefold.     

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'-->5' exonuclease activity

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3′→5′ DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.     

Overexpression, purification, and characterization of Bacillus subtilis N-acetylmuramoyl-L-alanine amidase CwlC

N-acetylmuramoyl-L-alanine amidase CwlC of Bacillus subtilis was overproduced in Escherichia coli and purified 21-fold. The amidase hydrolyzed type A cell walls such as B. subtilis. The amidase bound slightly to the Microbacterium lacticum cell wall (type B), but did not entirely hydrolyze it. The presence of calcium or magnesium ion increased the resistance of the amidase to heat denaturation.     

Clinical Report on the First Prototype of a Photoacoustic Tomography System with Dual Illumination for Breast Cancer Imaging

Photoacoustic tomography is a recently developed imaging modality that can provide high spatial-resolution images of hemoglobin distribution in tissues such as the breast. Because breast cancer is an angiogenesis-dependent type of malignancy, we evaluated the clinical acceptability of breast tissue images produced using our first prototype photoacoustic mammography (PAM) system in patients with known cancer. Post-excisionally, histological sections of the tumors were stained immunohistochemically (IHC) for CD31 (an endothelial marker) and carbonic anhydrase IX (CAIX) (a marker of hypoxia). Whole-slide scanning and image analyses were used to evaluate the tumor microvessel distribution pattern and to calculate the total vascular perimeter (TVP)/area for each lesion. In this clinical study, 42 lesions were primarily scanned using PAM preoperatively, three of which were reported to be benign and were excluded from statistical analysis. Images were produced for 29 out of 39 cancers (visibility rate = 74.4%) at the median depth of 26.5 (3.25–51.2) mm. Age, menopausal status, body mass index, history of neoadjuvant treatment, clinical stage and histological tumor angiogenesis markers did not seem to affect the visibility. The oxygen saturation level in all of the measured lesions was lower than in the subcutaneous counterpart vessels (Wilcoxon test, p value<0.001), as well as in the counterpart contralateral normal breast region of interest (ROI) (Wilcoxon test, p value = 0.001). Although the oxygen saturation level was not statistically significant between CAIX-positive vs. -negative cases, lesional TVP/area showed a positive correlation with the oxygen saturation level only in the group that had received therapy before PAM. In conclusion, the vascular and oxygenation data obtained by PAM have great potential for identifying functional features of breast tumors.     

Structure and dynamics of phytoplankton in an Amazon lake, Brazil

Natural lake systems represent important reservoirs for residential water supply, fish production, recreational activities and enjoyment of their natural beauty. Nevertheless, human impacts may affect their health status resulting in degradation and loss of biodiversity. The aim of the present study was to obtain data on the health status of a natural lake located in an indigenous reservation in the Brazilian Amazon, using the phytoplankton community changes along the rainy (June) and dry (November) seasons of 2006. We collected water (temperature, pH, Secchi depth and conductivity) and phytoplankton samples from the subsurface, middle of the water column, and approximately 30 cm above the bottom, over 24-hour sampling periods, from a central station in the lake. Samples taken from biotic and abiotic variables were correlated using canonical correspondence analysis (CCA). Results showed that the lake exhibited high temperatures in both seasons, and showed thermal stratification only during the rainy season. Dissolved oxygen exhibited a clinograde pattern in the rainy season and high oxygen in the hypolimnion in the dry season. In the rainy season, the water near the bottom was acidic, turbid and had a greater concentration of phosphorus. Dissolved oxygen, conductivity, pH, nitrite, total phosphorus and total dissolved phosphorus exhibited diel variations in the rainy season, whereas water temperature, dissolved oxygen, total nitrogen and total dissolved phosphorus exhibited significant differences between hours of the day in the dry season. The phytoplankton was represented by 39 taxa, and Chlorophyta showed the greatest species richness, totaling 25 taxa. Among Chlorophyta, desmids were the most diverse, accounting 52%. Bacillariophyta (nine species) was the second most diverse group. Cyanophyta was represented by three species, including Merismopedia tenuissima, the most abundant taxon. Despite the occurrence of taxa that indicate organic pollution, their biomass and frequency indicate that the system is not currently threatened. Lake Caracaran? is an oligotrophic system, with low algal density and isolated blooming episodes due to its shallow depth.     

Involvement of prostaglandin E receptor EP3 subtype in duodenal bicarbonate secretion in rats

We investigated the involvement of prostaglandin E (PGE) receptor subtype EP3 in the regulatory mechanism of duodenal HCO₃⁻ secretion in rats. A proximal duodenal loop or a chambered stomach was perfused with saline, and HCO₃⁻ secretion was measured using a pH-stat method and by adding 2 mM HCl. Mucosal acidification was achieved through 10 min of exposure to 10 mM HCl in the duodenum or 100 mM HCl in the stomach. Various EP agonists or the EP4 antagonist were given i.v., while the EP1 or EP3 antagonist was given s.c. or i.d., respectively. Sulprostone (EP1/EP3 agonists) stimulated duodenal HCO₃⁻ secretion in a dose-dependent manner, and this response was inhibited by AE5-599 (EP3 antagonist) but not AE3-208 (EP4 antagonist). AE1-329 (EP4 agonist) also increased duodenal HCO₃⁻ secretion, and this action was inhibited by AE3-208 but not AE5-599. The response to PGE₂ or acidification in the duodenum was partially attenuated by AE5-599 or AE3-208 alone but completely abolished by the combined administration. Duodenal damage caused by mucosal perfusion with 150 mM HCl for 4 h was worsened by pretreatment with AE5-599 and AE3-208 as well as indomethacin and further aggravated by co-administration of these antagonists. Neither the EP3 nor EP4 antagonist had any effect on the gastric response induced by PGE₂ or acidification. These results clearly demonstrate the involvement of EP3 receptors, in addition to EP4 receptors, in the regulation of duodenal HCO₃⁻ secretion as well as the maintenance of the mucosal integrity of the duodenum against acid injury.