Immunohistochemical localization of atrial natriuretic polypeptide (ANP) in human atrial and ventricular myocardiocytes

Summary To date, there have been few immunohistochemical investigations of atrial natriuretic polypeptide (ANP) in human cardiac tissue, especially the ventricles. In this study, myocardial tissue was obtained from two sources: the bilateral atria and ventricles at autopsy; and biopsy tissues from the right auricle and left ventricle of a patient with myocardial infarction undergoing surgery. These tissues were examined by the avidin-biotin immunoperoxidase technique using three kinds of primary ANP-antibodies. ANP-immunoreactivity was observed in the perinuclear region of myocytes of all tissues examined. The intensity of the reaction was stronger in atrial tissue, weaker in ventricular tissue. In the later tissue, the positive-staining myocytes were not part of the pulse-conducting system. Although the tissues we studied were not obtained from normal hearts, our data demonstrates that ANP-reactivity can be detected in ventricular myocytes outside the pulse-conducting system.     

Increase in nucleoside diphosphatase in rat brain striatum lesioned with kainic acid

The activity of ammoniagenesis from guanine nucleotides was found to increase significantly in rat brain after infusion of kainic acid into the striatum. Among the enzymes involved in degrading guanine nucleotides, nucleoside diphosphatase was markedly increased in the lesioned striatum. The enzyme activity began to increase 2 days after the infusion, and reached the maximum on the 13th day, the level being 4 times as high as that of the intact contralateral region. The increased activity was due to Type L enzyme, judging from its substrate specificity. Puromycin and cycloheximide inhibited this increase, indicating that the increased activity resulted from an increase in the net synthesis of the enzyme. These findings suggest that Type L NDPase might play some important roles in gliosis after neuronal lesion.     

Colchicine-binding activity of carrot tubulin at different culture age

Tubulin contents in the extract from cultured carrot cells at different growth phases were investigated by measuring colchicine-binding activity. The addition of vinblastine and dithiothreitol to the reaction mixture appreciably improved the stability of both free and colchicine-bound tubulins. Colchicine-binding activity in the cell extract obtained from stationary phase was more labile than that from log phase though the extract showed higher affinity to colchicine. After purification, however, tubulin from the cells at different growth phases showed the same affinity and its colchicine-binding activity was much more stable than in crude extract. The colchicine-binding activity in the crude extract was corrected for the decay during measurement and apparent difference in the affinity so that the activity in the cells containing different kind and amount of interefering substances could be compared. The corrected amount of colchicine that binds to the 100,000×g extract was 46 pmol/10⁵ cells at log phase. It decreased with the progression of culture age from linear to stationary phase. Combining the data with the morphological observation, it was suggested that the log phase cells contained larger free tubulin pool than the linear or stationary phase cells.     

Studies on the relationship between paracoccidioidomycosis in ddY mice and their estrous cycle

The relationship between paracoccidioidomycosis in ddY mouse and its estrous cycle was studied. Adult ddY mice of both sexes were used as experimental animals. Estrous cycle of female mice was examined before inoculation of Paracoccidioides brasiliensis yeast cells and mice were divided into 5 groups such as proestrus, estrus, metestrus-I, metestrus-II and diestrus. Each mouse was inoculated intravenously with 10⁶ P. brasiliensis yeast cell units and sacrificed on day 28 after inoculation. Their internal organs were cultured, and in addition, their histopathologies were studied. As a result, there was no difference in the organ cultures among the male and the female mice of 5 groups. However, histopathologically, the female groups at estrus, metestrus-I and metestrus-II were affected more severely than the male group, and the susceptibility of the female mice to the fungus was closely related to their estrous cycles.Abbreviations BHI-D brain heart infusion agar supplemented with 1.0% of anhydrous dextrose - PAS periodic acid-Schiff techniques - PBS phosphate buffered saline solution - SD standard deviation     

Diagnosis of a case of pulmonary carcinosarcoma by detection of rhabdomyosarcoma cells in sputum

Cytologic examination of sputum samples from an elderly patient revealed the presence of two cell populations: squamous cell carcinoma cells and rhabdomyosarcoma cells. The abnormal squamous cells showed both keratinizing and nonkeratinizing forms while some of the rhabdomyosarcoma cells showed cross striations. Sputum cytology was thus able to suggest a diagnosis of pulmonary carcinosarcoma. Histologically, the tumor was composed mainly of sarcomatous tissue showing various kinds of cells: fusiform or fibrous cells, round anaplastic cells, spindled cells with typical cross striations and myoblastic cells. A partially myxomatous degeneration was present. In addition, squamous cell carcinoma proliferated along the bronchi and formed small invasive cell nests in the sarcomatous tissue. No transition between the two components was noted. Both cellular constituents had metastasized to an interlobar lymph node.     

Type B nucleoside-diphosphatase of rat brain. Purification and properties of an enzyme with high thiamin pyrophosphatase activity

Type B nucleoside-diphosphatase was purified from membranes of rat brain by solubilization with a non-ionic detergent and successive column chromatographies on DEAE-cellulose DE-52, concanavalin-A-Sepharose, Bio-Gel HT, blue-Sepharose CL-6B, chelating Sepharose 6B, Ultrogel AcA44 and TSK gel G3000 SW. The purified enzyme gave a single protein band on SDS/polyacrylamide gel electrophoresis and its molecular mass was estimated to be 75 kDa. It hydrolyzed thiamin diphosphate as well as GDP, IDP and UDP. Thiamin diphosphate (TPP) was hydrolyzed twice as efficiently as nucleoside diphosphates in the presence of Mn2+ at pH 7.4. The Km values for TPP, GDP, IDP and UDP were 0.66, 0.40, 0.54 and 1.06 mM respectively. ATP, ADP and pyridoxal 5'-phosphate inhibited thiamin-pyrophosphatase activity competitively and their Ki values were 2.3 mM, 1.0 mM and 0.59 mM respectively. The optimum pH of thiamin-pyrophosphatase activity was 7.4 in the presence of Mn2+ and that of GDP-hydrolytic activity was 6.5 in the presence of Mg2+.     

Generation of cytotoxic T lymphocytes from thymocyte precursors to trinitrophenyl-modified self antigens. II. Establishment of a T cell hybrid clone constitutively producing killer-helper factor(s) (KHF) and functional analysis of released KHF

T cell hybridoma lines were constructed by fusion of Mycobacterium tuberculosis-primed and boosted BALB/c T cells with the AKR-derived T lymphoma cell line BW5147. Certain of the hybridomas prepared in this manner secreted constitutively into their culture supernatants biologically active molecules that displayed precursors of cytotoxic T cell activating properties characteristic of killer-helper factor (KHF). Cell surface analysis revealed that the hybridomas were indeed somatic cell hybrids between the two respective partner cells used for fusion. KHF properties of these hybridoma supernatants were verified by their capacity to stimulate peanut agglutinin-binding (PNA+) C3H/He thymocytes to respond in vitro to 2,4,6-trinitrophenyl(TNP)-modified syngeneic stimulator cells in conjunction with suboptimal doses (10 U/ml) of interleukin 2 (IL 2) for the generation of H-2-restricted, TNP-reactive cytotoxic T cells. The biologically active molecules secreted by a T cell hybrid clone (2Y4) were, like conventional KHF, distinct from IL 1, IL 2, or immune interferon (IFN-gamma). The partially purified KHF derived from 2Y4 cells shows activity at apparent m.w. range of 34,000 to 60,000 on gel permeation, and is relatively homogeneous with respect to isoelectric point, which was approximately 4.5 to 4.7. The partially purified 2Y4-KHF is able to augment proliferation of as well as the expression of IL 2 receptors on PNA+ thymocytes in conjunction with IL 2. Finally, addition of 2Y4-KHF on day 0, followed by the addition of IL 2 on day 2 for 7 days of culture was effective in generating potent CTL responses, whereas addition of IL 2 on day 0, followed by the addition of 2Y4-KHF on day 2 to the culture was ineffective.     

Suppressive effect of human natural killer cells on Epstein-Barr virus-induced immunoglobulin synthesis

The suppressive effect of human natural killer (NK) cells on Epstein-Barr virus (EBV)-induced immunoglobulin (Ig) synthesis by autologous B cells was investigated. By Percoll discontinuous density gradient centrifugation, low-density fractions enriched for NK cells were isolated from human peripheral blood lymphocytes. These NK-enriched fractions were added to purified autologous B cells in the presence of EBV, were cultivated for 8 days, and were examined for their suppressive effect on Ig synthesis by an enzyme-linked immunosorbent assay. The fractions markedly suppressed both IgM and IgG synthesis induced by EBV. It was possible to reduce the suppressive effect of NK-enriched cells by complement-dependent lysis of NK cells and Leu-11, but not by OKT3 monoclonal antibody, indicating that NK cells may be responsible for the suppression of Ig synthesis. Upon close examination of interferon (IFN) activity, it was revealed that the co-cultures of NK-enriched cells and EBV-infected B cells generated production of IFN-alpha, which might be produced by NK cells in response to EBV-stimulated B cells. Addition of anti-IFN-alpha but not anti-IFN-gamma serum almost completely abrogated the suppressive effect of NK-enriched cells on Ig synthesis, indicating that IFN-alpha produced are required for the NK cell-mediated suppression of Ig synthesis. However, addition of IFN-alpha into purified B cells showed no direct suppressive effect on EBV-induced Ig synthesis by B cells in the absence of NK cells. Nevertheless, NK cells when previously incubated with IFN-alpha and added to B cells showed a suppressor activity on Ig synthesis to a level higher than that of untreated NK controls. These results strongly suggest the possibility that NK cells display an interaction with EBV-infected B cells and produce IFN-alpha, which in turn activates NK cells. These activated NK cells suppress the Ig synthesis by B cells, which undergo transformation induced by EBV.     

Isolation and Identification of α-(γ-Aminobutyryl)-Hypusine

A new dipeptide, alpha-(gamma-aminobutyryl)-hypusine, was identified in bovine brain. This compound was isolated from trichloroacetic acid-soluble fraction of bovine brain with five steps of ion-exchange chromatography. Its structure was postulated by routine chemical analyses and determined by synthesis. The amount of the compound isolated from 1.2 kg of bovine brain was 870 nmol.