Regulation of 5-hydroxytryptamine-induced calcium mobilization by cAMP-elevating agents in cultured canine tracheal smooth muscle cells

The effects of increases in cellular adenosine 3′5′-cyclic monophosphate (cAMP) on 5-hydroxytryptamine-(5-HT-) induced generation of inositol phosphates (IPs) and increases in intracellular Ca²⁺ ([Ca²⁺]_i) were investigated using canine cultured tracheal smooth muscle cells (TSMCs). Cholera toxin and forskolin induced concentration- and time-dependent cAMP formation with half-maximal effects (−logEC₅₀) produced at concentrations of 7.0 ± 0.5 and 4.9 ± 0.4  respectively. Pretreatment of TSMCs with either forskolin or dibutyryl cAMP inhibited 5-HT-stimulated responses. Even after treatment for 24h, these agents still inhibited the 5-HT-induced Ca²⁺ mobilization. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration response curves of 5-HT. The water-soluble forskolin analogue L-858051 [7-deacetyl-7β-(γ-N-methylpiperazino)-butyryl forskolin] significantly inhibited the 5-HT-stimulated accumulation of IPs. In contrast, the addition of 1,9-dideoxy forskolin, an inactive forskolin analogue, had little effect on this response. Moreover, SQ-22536 [9-(tetrahydro-2-furanyl)-9-H-purin-6-amine], an inhibitor of adenylate cyclase, and both H-89 [N-(2-aminoethyl)-5-isoquinolinesulphonamide] and HA-1004[N-(2-guanidinoethyl)-5-isoquinolinesulphonamide], inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit the 5-HT-stimulated accumulation of IPs. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. The AlF₄ ⁻-induced accumulation of IPs was inhibited by forskolin, suggesting that G protein(s) are directly activated by AlF₄ ⁻- and uncoupled from phospholipase C by forskolin treatment. These results suggest that activation of cAMP/PKA might inhibit the 5-HT-stimulated phosphoinositide breakdown and consequently reduce the [Ca²⁺]_i increase or inhibit both responses independently. Received: 14 March 1996/Accepted: 10 April 1996     

ASK1 associates with troponin T and induces troponin T phosphorylation and contractile dysfunction in cardiomyocytes

There is increasing support for the idea that excessive production of proinflammatory mediators such as tumor necrosis factor (TNF) and reactive oxygen species (ROS) contribute to the pathogenesis of cardiac dysfunction. However, the mechanisms by which cytokine/ROS production mediates cardiac dysfunction have not been established. Given that apoptosis signal-regulating kinase 1 (ASK1) is highly expressed in cardiac muscle and that ASK1 is an important mediator in the signaling pathways induced by tumor necrosis factor, interleukin-1, and ROS, we used the yeast two-hybrid system with ASK1 as bait to identify ASK1 substrates from a human heart cDNA library. The cDNA encoding the cardiac troponin T (cTnT) was isolated. ASK1 specifically interacted with cTnT, but not cTnI, in vitro and in vivo via the C-terminal ASK1 domain. ASK1 specifically phosphorylated cTnT in vitro and in vivo. Mutations in cTnT (T194/S198) at an ASK1-phosphorylation consensus sequence significantly reduced phosphorylation by ASK1. ROS-induced ASK1 activation, cTnT phosphorylation, and contractile dysfunction in cardiomyocytes showed similar kinetics. Moreover, overexpression of constitutively active ASK1 induces cTnT phosphorylation and inhibits shortening and calcium transient in adult cardiomyocytes. We conclude that ASK1 plays an important role in regulation of cardiac contractile function by phosphorylating cTnT and may participate in cytokine/ROS-induced pathogenesis of cardiomyopathy and heart failure.     

Preparation and characterization of Pertussis toxin subunits

Pertussis toxin (PT), a typical A-B oligomer exotoxin of Bordetella pertussis, has been demonstrated to be an essential protective antigen for acellular pertussis vaccine against whooping cough. In order to investigate the associated functionality ascribed to its components, we have purified A and B oligomers for the activity study. The A oligomer (S1 subunit) of PT was expressed in E. coli B834 (DE3) harboring expression vector (pET-20b) with the insert of S1 coding region and purified by metal-chelating column. The B oligomer was isolated by a single-step purification procedure. Individually, recombinant S1 and B oligomer exhibited quite distinct biological activities in vivo. S1 subunit induced leukocytosis-promoting (LP) activity, but did not affect mouse body weight-gain. On the contrary, B oligomer reduced mouse body weight-gain but did not reveal LP activity. In vitro, the combination of S1 subunit and B oligomer could enhance the toxic activities as exhibited by native PT and showed an additive toxicity in CHO cell clustering test and hemagglutination assay.     

Characterization of cortical neuron outgrowth in two- and three-dimensional culture systems

To improve the ability of regeneration by grafting living cells or by adding growth factor to a lesion site, it is important to find good biomaterials for neuron survival and regeneration. This study focused on two- and three-dimensional cultures in a matrix using biomaterials such as agarose, collagen, fibrin, and their mixtures, because these are considered to be suitable biomaterials for neuron outgrowth. Cortical neurons were dissected from E17 rat embryos and cultured in agarose gel, collagen gel, fibrin glue, and mixtures of collagen and fibrin. Results showed that neurons cultured in collagen gel and fibrin glue had longer periods of survival (more than 3 weeks) and better neurite extension than those observed in agarose gels. As to the survival rate according to the MTT and lactate dehydrogenase assays, fibrin glue was the most suitable biomaterial for neuron survival among the biomaterials examined. With two-dimensional fibrin plating, neuron cells exhibited cell aggregation and stress fibers, but the same results were not observed with collagen gel. There were no differences in neurite extension and survival in the mixtures of collagen and fibrin. The results suggest that collagen and fibrin can provide a suitable substrate for a three-dimensional culture matrix for neuronal survival and differentiation.     

Serum hepatitis B virus DNA in healthy HBsAg-negative Chinese adults evaluated by polymerase chain reaction

Serum hepatitis B virus (HBV) DNA was assayed using polymerase chain reaction, in 107 HBsAg-negative normal Chinese subjects. The results showed that eight subjects (7.5%) had HBV DNA. In the subgroup with antibody to hepatitis B surface antigen (anti-HBs) and to hepatitis B core antigen (anti-HBc), 7.3% (5/68) were positive for HBV DNA; HBV DNA was not detected in six individuals with anti-HBs only and in nine with anti-HBc only. In four persons with anti-HBc and anti-HBe, one had HBV DNA. In 20 subjects negative for all hepatitis B serological markers, two (10%) were found to have HBV DNA. This study indicates that serological markers are not adequate to rule out HBV infection, and it further implies that present blood donor screening methods may need improving.     

Semen parameters and testicular pathology in men with testicular cancer and contralateral carcinoma in situ or bilateral testicular malignancies

We evaluated 14 patients with bilateral testicular tumour, one-sided tumour and contralateral carcinoma in situ (CIS) of the testis or testis tumour in single testis with respect to their fertility. We analysed semen parameters, serum hormones [follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone], testicular sonography, testicular volumes and testicular histology prior to further anti-cancer treatment. Ten out of 14 patients showed normal or reduced sperm concentrations, while 4/14 patients were azoospermic. Serum FSH levels showed a significant negative correlation with sperm concentrations in patients with testicular malignancies (r = -0.64, P = 0.025). Testicular volumes revealed a significant positive correlation with semen parameters in patients with testes that were affected by CIS (r = 0.733, P = 0.038). We conclude that even bilateral testicular cancer and/or CIS do not preclude fertility and, therefore, patients should be offered andrological investigation and therapy, including possibly surveillance strategy or the chance for cryopreservation of the semen prior to further treatment in order to preserve their chances for paternity.      

Hyperplasia and tumors of the parathyroid glands

Zusammenfassung Die verschiedenen Formen des Hyperparathyreoidismus werden überwiegend von Hyperplasien und Adenomen der Nebenschilddrüsen verursacht. Während früher die Diagnose des Pathologen (meist schon im intraoperativen Gefrierschnitt) für das chirurgische Vorgehen maßgebend war, wird das Operationsausmaß heute überwiegend durch die wesentlich verbesserte präoperative Diagnostik bestimmt (bildgebende Verfahren) und durch den nach der Entfernung der vergrößerten Nebenschilddrüse(n) intraoperativ gemessenen Abfall des Parathormons kontrolliert. Nichtsdestotrotz sollte dem Pathologen die morphologische Differenzialdiagnose der Hyperplasie, des Adenoms und Karzinoms der Nebenschilddrüsen geläufig sein.

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