Comparative studies in induction of micronuclei by three genetically recombinant and urinary human erythropoietins

Comparative studies on the induction of micronucleated erythrocytesby three recombinant human erythropoietins (rhEPOs) such asepoetin     

Differential expression of PD-L1 and PD-L2, ligands for an inhibitory receptor PD-1, in the cells of lymphohematopoietic tissues

PD-1 is a member of the immunoglobulin superfamily expressed on immune cells, including T and B cells, and is involved in the delivery of inhibitory signal upon engagement of its ligands, PD-L1 and PD-L2. While the expression profile of PD-1 has been well documented, the analysis of PD-L1 and PD-L2 distributions on a protein basis has not been carried out because of the lack of available monoclonal antibodies specific for the molecules. In this study, we established two monoclonal antibodies, 1-111A and 122, specific for murine PD-L1 and PD-L2, respectively, and examined their expression profiles. Based on flow cytometric analyses, the expression of PD-L1 was detected in a variety of lymphohematopoietic cell types, including a minor proportion of T and B cells in the spleen, majority of pre-B cells and myeloid cells in bone marrow and subsets of thymocytes, while the expression of PD-L2 was not observed in the lymphohematopoietic cells at all. Notably, a significant proportion of the most immature lineage-marker-negative and c-Kit-positive bone marrow cells containing stem cells did express PD-L1. Following mitogenic stimulation, essentially all lymphocytes expressed PD-L1. Furthermore, a variety of leukemic lines also expressed PD-L1, while none of them did PD-L2. Thus, present results demonstrate the distinct expression patterns of PD-L1 and PD-L2 with the cells of lymphohematopoietic tissues exclusively expressing the former.     

Short half-lives of ataxia-associated aprataxin proteins in neuronal cells

Early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH)/ataxia with oculomotor apraxia type 1 (AOA1) is caused by mutations in the gene encoding aprataxin (APTX). Although several in vitro findings proposed that impaired enzymatic activities of APTX are responsible for EAOH/AOA1, potential instability of mutant proteins has also been suggested as the pathogenesis based on in vivo finding that mutant proteins are almost undetectable in EAOH/AOA1 tissues or cells. The present study aimed to experimentally prove instability of mutant proteins in neuronal cells, the cell type preferentially affected by this disease. Results of pulse-chase experiments demonstrated that all of the disease-associated mutants had extremely shorter half-lives than the WT. We further found that mutants were targeted for rapid proteasome-mediated degradation. These results help establish pathogenic and physiological protein characteristics of APTX in neuronal cells.     

Three Kinds of Foamy Cells in the Spleen: Comparative Histochemical and Ultrastructural Studies

By light and electron microscopy, we observed foamy cells in the spleens from a patient with hemolytic anemia due to red cell adenosine deaminase (ADA) overproduction, a patient with rheumatoid arthritis (RA) treated with gold, and patients with idiopathic thrombocytopenic purpura (ITP)

The foamy cells associated with red cell ADA overproduction were essentially similar to Gaucher-like cells described in patients with thalassemia, and it was suggested that the accelerated destruction of red cells was one of the factors responsible for the development of foamy cells. Foamy cells in ITP and RA were closely associated with an increased destruction of platelets in the spleen. Morphologic transitions between phagocytosed platelets and myelinlike materials were traced in these disorders. In RA, however, foamy cells were heterogeneous from an ultrastructural standpoint, with different cytoplasmic inclusions. In addition to myelinlike materials, dense bodies, vacuoles with flocculent materials, and gold were noted in most of foamy cells. As gold compounds are known to inhibit lysosomal enzymes, we surmise that an acquired disturbance in lysosomal digestion is partially responsible for the accumulation of intermediate metabolites.

In the pathogenesis of foamy cells associated with blood cell dyscrasia, the accelerated destruction of blood cells and/or acquired disorders in catabolic pathways within the macrophages are suggested to be the underlying mechanism of an intralysosomal accumulation of incompletely degraded cellular debris.     

Malignant Cystosarcoma Phyllodes Of Prostate

A case of malignant cystosarcoma phyllodes of the prostate is reported in a 45-year-old male. This tumor was composed of benign columnar or squamous cystic folds and sarcomatous stroma including rhabdomyomatous elements. The prostatic origin of the tumor was clearly proved by the unlabeled immunoperoxidase method. ACTA PATHOL. JPN. 34: 663–668, 1984.     

Microanatomical localization of PD-1 in human tonsils

PD-1 is an immunoinhibitory receptor, which belongs structurally to the CD28 family. PD-1-deficient mice show breakdown of peripheral tolerance and manifest multiple autoimmune symptoms. We previously described expression of PD-1 on activated T and B lymphocytes and myeloid cells. However, little is known about the microanatomical distribution of PD-1 in lymphoid organs. In this study, we performed immunohistochemistry using monoclonal antibodies against human PD-1. In human tonsils, PD-1 was expressed on most of T cells and a small subset of centrocytes in the light zone of germinal centers (GCs), where clonal selection of centrocytes takes place. These results suggest that PD-1 may play an important role in GC reaction.     

Conversion of Salmonella typhimurium to L-forms contributes to the maintenance of acquired immunity against murine typhoid.

Conversion of Salmonella typhimurium to L-forms, both in vitro and in vivo, resulted in the expression of proteins cross-reacting to the mycobacterial 65,000 MW heat-shock protein (hsp). Immunization of C3H/HeJ mice with a protective dose of stable L-form S. typhimurium induced gamma delta T cells in the liver, in accordance with the multiplication of L-form Salmonella in Kupffer cells. The number of gamma delta T cells decreased after the intracellular growth of L-form Salmonella plateaued. Persistance of the L-forms in Kupffer cells, however, allowed hepatic gamma delta T cells to increase within 48 hr of infection with virulent S. typhimurium. Thus, the intrahepatic colonization of L-form Salmonella seems to keep gamma delta T cells on standby, but the emergence of these T cells does not correlate with the expression of L-form hsp. In addition, Kupffer cells colonized by L-forms constitutively synthesized mRNA for interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha). These results suggest that conversion of S. typhimurium to L-forms in phagocytic cells builds up and maintains acquired resistance, conferred by live-cell vaccines of S. typhimurium, against murine typhoid.     

Cloning of TPA-inducible early (TIE) genes by differential hybridization using TPA-Nonresponsive variant of mouse 3T3-L1 cells

The tumor promoter 12-O -tetradecanoylphorbol-13-acetate (TPA) induces DNA synthesis in quiescent 3T3-L1 cells but not in its variant VT-1 cells. A gt10 cDNA library was constructed using poly(A)⁺ RNA from 3T3-L1 cells that were stimulated by TPA for 20 min. Radioactive cDNA probes were prepared from mRNAs of TPA-treated 3 T3-L1 and VT-1 cells and used for screening of the 3T3-L1 cDNA library by differential hybridization. Nine of 6000 phage plaques hybridized only to the 3T3-L1 cDNA probe. Analysis of the nucleotide sequence of five of these clones indicated a high degree of homology with human or mouse type I and type III collagen genes. Three other independent clones showed no homology with any known DNA sequences. These isolated clones of TPA-inducible early (TIE) genes may be useful to study the signal transduction pathway of phorbol esters.     

Estrogen affects vascular tone differently according to vasoactive substances in ovariectomized Sprague-Dawley rat

The favorable effects of estrogen on cardiovascular diseases can be explained by several mechanisms such as changes in serum lipid profiles and thrombogenecity. Estrogen also affects the vascular tone, but there has been no report in which the effect of estrogen was tested comprehensively for several vasoactive substances, especially after long-term administration. Two weeks after bilateral ovariectomy in 8-week old female Sprague-Dawley rats, placebo or 17 beta-estradiol (E2) pellets (0.5 mg; released over 3 weeks) were implanted subcutaneously. Two weeks after pellet implantation, organ chamber experiments were performed using aortae. Compared with control, E2-treated vessels showed impaired endothelium-dependent relaxation to acetylcholine. E2 enhanced the contraction to norepinephrine and U46619 and had no effect on endothelin-1-induced contraction. In contrast, the contraction to angiotensin (AT)-II was inhibited by E2. Northern blot analysis for AT1 receptor expression using cultured aortic smooth muscle cells showed no difference between control and E2-treated cells, suggesting that AT1 receptor downregulation is not the likely mechanism. These results suggest that E2 affects the vascular tone variably according to vasoactive substances.     

Genetics of cell-surface receptors for bioactive polypeptides: A variant of mouse BALBc/3T3 fibroblasts possessing altered insulin-binding ability

An insulin-nonresponsive variant was isolated from mutagenized mouse BALBc/3T3 fibroblasts. Selection was based on the insulin's mitogenic action upon quiescent cells and subsequent arrest at mitosis by vinblastine sulfate to remove insulin-responsive cells. Among four surviving colonies, one, designated IN-2, exhibited no binding for [¹²⁵I] insulin at 2× 10^–10 M and at 4° C. The binding ability, however, recovered substantially at 15° C and increased with higher temperature and at higher ligand concentrations. The binding profiles, Scatchard plot analysis, and the dissociation kinetics indicated that the receptors expressed on IN-2 cells possess lower affinity than the parental 3T3 cells. The IN-2 cells were negative for stimulating effects of insulin on 2-deoxyglucose uptake, thymidine incorporation, and cell growth. The IN-2 cells were also negative for cross-reactivity to antibodies which react with insulin receptors on 3T3 cells and for the susceptibility to a cytotoxic chimeric insulin which was cross-linked to diphtheria toxin fragment A. This negative response of IN-2 cells can be attributed to a deficiency in high-affinity receptors for insulin. The insulin bound to the low-affinity binding sites of IN-2 cells, however, undergoes internalization and intracellular degradation. Therefore, such processing by itself does not account for insulin's mitogenic action.