Complement-dependent cytotoxic factor to megakaryocyte progenitors in sera from patients with idiopathic thrombocytopenic purpura

The influence of sera from patients with idiopathic thrombocytopenic purpura (ITP) was examined on colony formation from megakaryocyte (M) progenitors. Though incubation of marrow cells in Iscove's modified Dulbecco's medium (IMDM) containing 50% sera from several ITP patients stimulated M-colony formation in 8 of 13 cases, incubation in the sera from the patients and in baby rabbit serum as a source of complement significantly suppressed the colony formation. Experiments showed that sera of immunoglobulin G from ITP patients had significant complement-dependent cytotoxicity to M-progenitors in normal marrow cells or in the marrow cells from corresponding patients, but not to CFU-e, BFU-e or CFU-gm. Cytospin preparations of individually collected M-colonies from marrow cells treated with ITP patients' sera and complement revealed a reduction of megakaryocyte colonies containing cells of multilineages. These results indicate that autoantibodies detected in ITP patients can bind not only to platelets and megakaryocytes, but may also bind to M-progenitors.     

N-2′-Acetoxybenzoyl derivatives of chitosan, N-desulphated heparin and 2-amino-2-deoxy-d-glucose

N-2′-Acetoxybenzoyl (aspirin) derivatives (degree of substitution 0·35–1·00) of chitosan, N-desulphated heparin and 2-amino-2-deoxy-d-glucose were prepared by methods that gave yields in the range 65–86%. The salicylate of chitosan was isolated with a 98% yeild. Aspirin or salicylic acid was released much more slowly from N-(2′-acetoxybenzoyl)-chitosan than from the salicylate of chitosan, and much faster at 37°C in 0·1 m NaOH solution than in 2% aqueous acetic acid solution. Salicylic acid was isolated from the dialysate (0·1 m NaOH solution) of N-(2′-acetoxybenzoyl)-chitosan.     

Studies on the heterotropic interaction of hemoglobin. I. Mass spectrometric method for determination of the pKa of the beta-146 histidine residue in human hemoglobin.

A mass spectrometric method was developed to determine pH-dependent hydrogen-deuterium exchange at the C-2 position of the imidazole ring of histidine, after converting the amino acid to the methylthiohydantoin derivative. The amount of deuterium exchange in N-acetyl-histidine estimated by the present method was confirmed to be in good agreement with that determined by NMR spectrometry. N-Acetylhistidine was deuterated at various pH's. From the amount of deuterium exchange, a pseudo-first order rate constant (kpsi) was calculated. A pKa value of 7.2 for the amino acid was obtained from the relation between kpsi and pH. This method was applied to estimate the pKa value of beta-146 histidine in human hemoglobin. Human hemoglobin deuterated at various pH's was digested with carboxypeptidase A [EC 3.4.12.2] to release the beta-146 histidine. The amount of deuterium exchange in the isolated histidine was determined to obtain kpsi. From these measurements pKa values of 7.0 for the histidine in oxyhemoglobin and of 8.2 for that in deoxyhemoglobin were found at 36.5 degrees, respectively.     

Block of acid secretion by amytal and its partial reversal by menadione with ascorbate in the gastric mucosa of the guinea-pig

The effect of amytal on energy metabolism and acid secretion in an isolated gastric mucosa of the guinea-pig were studied. Determination of adenine nucleotides, creatine phosphate, pyruvate and lactate in the gastric mucosa showed that amytal depressed the levels of ATP, creatine phosphate and energy charge with elevation of the AMP and pyruvate levels. This treatment inhibited concomitantly acid secretion and active chloride transport detected by short circuit current. The addition of menadione with ascorbate to the medium in the presence of amytal partially restored ATP and energy charge levels and also induced a partial recovery of acid secretion and active chloride transport. These results suggest that ATP is a direct energy donor for acid secretion in the gastric mucosa of the guinea-pig.     

Low frequency of (5''-3'') -C-G- connection in 70S RNA from simian sarcoma virus.

The frequency of oligonucleotides obtained from simian sarcoma virus RNA by digestion with ribonuclease T1 was compared with the frequency expected of an RNA molecule in which nucleotides are arranged in random distribution. Oligonucleotides containing C-residue attached to 3'-Gp were found significantly less in simian sarcoma virus 70S RNA than expected by random distribution.     

Identification of Biological Properties of Intralymphatic Tumor Related to the Development of Lymph Node Metastasis in Lung Adenocarcinoma

Background

Intralymphatic tumors in the extratumoral area are considered to represent the preceding phase of lymph node metastasis. The aim of this study was to clarify the biological properties of intralymphatic tumors susceptible to the development of lymph node metastasis, with special reference to the expression of cancer initiating/stem cell (CIC/CSC) related markers in cancer cells and the number of infiltrating stromal cells.

Material and Methods

Primary lung adenocarcinomas with lymphatic permeation in the extratumoral area were retrospectively examined (n = 107). We examined the expression levels of CIC/CSC related markers including ALDH1, OCT4, NANOG, SOX2 and Caveolin-1 in the intralymphatic cancer cells to evaluate their relationship to lymph node metastasis. Moreover, the number of infiltrating stromal cells expressing CD34, α-smooth muscle actin, and CD204 were also evaluated.

Results

Among the intralymphatic tissues, low ALDH1 expression in cancer cells, high SOX2 expression in cancer cells, and a high number of CD204(+) macrophages were independent predictive factors for lymph node metastasis (P = 0.004, P = 0.008, and P = 0.028, respectively). Among these factors, only low ALDH1 expression in cancer cells was significantly correlated with the farther spreading of lymph node metastasis (mediastinal lymph node, pathological N2) (P = 0.046) and the metastatic lymph node ratio (metastatic/resected) (P = 0.028). On the other hand, in the primary tumors, ALDH1 expression in the cancer cells was not associated with lymph node metastasis. Intralymphatic cancer cells expressing low ALDH1 levels exhibited lower E-cadherin expression levels than cancer cells with high levels of ALDH1 expression (P = 0.015).

Conclusions

Intralymphatic cancer cells expressing low levels of ALDH1 and infiltrating macrophages expressing CD204 have a critical impact on lymph node metastasis. Our study also highlighted the significance of evaluating the biological properties of intralymphatic tumors for tumor metastasis.     

Crystal Structure of Silkworm Bombyx mori JHBP in Complex With 2-Methyl-2,4-Pentanediol: Plasticity of JH-Binding Pocket and Ligand-Induced Conformational Change of the Second Cavity in JHBP

Juvenile hormones (JHs) control a diversity of crucial life events in insects. In Lepidoptera which major agricultural pests belong to, JH signaling is critically controlled by a species-specific high-affinity, low molecular weight JH-binding protein (JHBP) in hemolymph, which transports JH from the site of its synthesis to target tissues. Hence, JHBP is expected to be an excellent target for the development of novel specific insect growth regulators (IGRs) and insecticides. A better understanding of the structural biology of JHBP should pave the way for the structure-based drug design of such compounds. Here, we report the crystal structure of the silkworm Bombyx mori JHBP in complex with two molecules of 2-methyl-2,4-pentanediol (MPD), one molecule (MPD1) bound in the JH-binding pocket while the other (MPD2) in a second cavity. Detailed comparison with the apo-JHBP and JHBP-JH II complex structures previously reported by us led to a number of intriguing findings. First, the JH-binding pocket changes its size in a ligand-dependent manner due to flexibility of the gate α1 helix. Second, MPD1 mimics interactions of the epoxide moiety of JH previously observed in the JHBP-JH complex, and MPD can compete with JH in binding to the JH-binding pocket. We also confirmed that methoprene, which has an MPD-like structure, inhibits the complex formation between JHBP and JH while the unepoxydated JH III (methyl farnesoate) does not. These findings may open the door to the development of novel IGRs targeted against JHBP. Third, binding of MPD to the second cavity of JHBP induces significant conformational changes accompanied with a cavity expansion. This finding, together with MPD2-JHBP interaction mechanism identified in the JHBP-MPD complex, should provide important guidance in the search for the natural ligand of the second cavity.     

Amino acid sequence variations of signaling lymphocyte activation molecule and mortality caused by morbillivirus infection in cetaceans

Morbillivirus infection is a severe threat to marine mammals. Mass die‐offs caused by this infection have repeatedly occurred in bottlenose dolphins (Turiops truncatus) and striped dolphins (Stenella coeruleoalba), both of which belong to the family Delphinidae, but not in other cetaceans. However, it is unknown whether sensitivity to the virus varies among cetacean species. The signaling lymphocyte activation molecule (SLAM) is a receptor on host cells that allows morbillivirus invasion and propagation. Its immunoguloblin variable domain‐like (V) region provides an interface for the virus hemagglutinin (H) protein. In this study, variations in the amino acid residues of the V region of 26 cetacean species, covering almost all cetacean genera, were examined. Three‐dimensional (3D) models of them were generated in a homology model using the crystal structure of the marmoset SLAM and measles virus H protein complex as a template. The 3D models showed 32 amino acid residues on the interface that possibly bind the morbillivirus. Among the cetacean species studied, variations were found at six of the residues. Bottlenose and striped dolphins have substitutions at five positions (E68G, I74V, R90H, V126I, and Q130H) compared with those of baleen whales. Three residues (at positions 68, 90 and 130) were found to alternate electric charges, possibly causing changes in affinity for the virus. This study shows a new approach based on receptor structure for assessing potential vulnerability to viral infection. This method may be useful for assessing the risk of morbillivirus infection in wildlife.