Augmentation and inhibition of beta-adrenergic agonists-stimulated tissue cyclic AMP accumulation by alpha-adrenergic agonists in rat parotid gland

It was found that phenylephrine and methoxamine had two effects (one was inhibitory and the other was augmentative) on isoproterenol-stimulated cyclic AMP in rat parotid slices. The augmentation was abolished by alpha-adrenergic antagonists or by omission of calcium in the medium. Cyclic AMP accumulation by norepinephrine (NE) was significantly decreased by omission of calcium in the medium. Calmodulin antagonists, trifluoperazine and W-7, decreased NE-induced cyclic AMP accumulation, but another calmodulin antagonist, carmidazolium, did not. Phorbol ester such as 4 beta-phorbol 12-myristate, 13-acetate and phorbol 12, 13-dibutyrate, did not augment the effect of isoproterenol. These results suggest that although the influx of calcium is required in the alpha-adrenergic agonists-induced augmentation, calmodulin and protein kinase C may not be intermediates in this process. Calcium ions (10(-7) and 10(-6) M) slightly increased the activity of adenylate cyclase, but calcium (10(-6)-10(-4) M) dose-dependently inhibited the effect of isoproterenol. Therefore, calcium ions do not participate in the augmentation by directly modulating the activity of adenylate cyclase. The inhibitory effect was not affected by alpha-adrenergic antagonists. The activation of adenylate cyclase by isoproterenol was inhibited by phenylephrine with higher inhibition being obtained in lower concentrations of isoproterenol. Phenylephrine in the presence of isobutylmethylxanthine increased the amount of cyclic AMP and this effect was inhibited by propranolol, but not by phentolamine. [3H]-CGP 12177 binding of the parotid membrane was inhibited by alpha-adrenergic antagonists. These results suggest that the inhibitory effect of phenylephrine and methoxamine may be mediated by beta-adrenergic receptor.     

Membrane attack complex of complement and 20 kDa homologous restriction factor (CD59) in myocardial infarction

In order to investigate the mechanism of deposition of the complement membrane attack complex (MAC) in cardiomyocytes in areas of human myocardial infarction, the 20 kDA homologous restriction factor of complement (HRF20; CD59) and complement components (C1q, C3d and MAC) were analysed immunohistochemically using specific antibodies. Myocardial tissues obtained at autopsy from nine patients who died of acute myocardial infarction were fixed in acetone and embedded in paraffin. The ages of the infarcts ranged from about 3.5 h to 12 days. In cases of myocardial infarction of 20 h or less, MAC deposition was shown in the infarcted cardiomyocytes without loss of HRF20. Where the duration was 4 days or more, the cardiomyocytes with MAC deposition in the infarcted areas also showed complete loss of HRF20. Outside the infarcts, HRF20 in the cardiomyocytes was well preserved without MAC deposition. The present study suggests that the initial MAC deposition in dead cardiomyocytes can occur as a result of degradation of plasma-membrane by a mechanism independent of complement-mediated injury to the membrane. Loss of HRF20 from dead cardiomyocytes may not be the initial cause of MAC deposition, but may accelerate the deposition process of MAC in later stages of infarction.     

Antigen-specific,CD4+CD25+ regulatory T cell clones induced in Peyer's patches

Since intestine is exposed to numerous exogenous antigens such as food and commensal bacteria, the organ bears efficient mechanisms for establishment of tolerance and induction of regulatory T cells (T(reg)). Intestinal and inducible T(reg) include T(r)1-like and T(h)3 cells whose major effector molecules are IL-10 and transforming growth factor (TGF)-beta. These antigen-specific T(reg) are expected to become clinical targets to modify the inflammatory immune response associated with allergy, autoimmune diseases and transplantation. In the present study, we characterized the antigen-specific T(reg) induced in the intestine by orally administering high-dose beta-lactoglobulin (BLG) to BALB/c mice. Seven days after feeding, only Peyer's patch (PP) cells among different organs exerted significant suppressive effect on antibody production upon in vitro BLG stimulation. This suppressive effect was also prominent in six BLG-specific CD4(+) T cell clones (OPP1-6) established from PP from mice orally administered with high doses of BLG and was partially reversed by antibodies to TGF-beta. Intravenous transfer of OPP2 efficiently suppressed BLG-specific IgG1 production in serum following immunization, indicating the role of such T(reg) in the systemic tolerance after oral administration of antigen (oral tolerance). OPP clones secrete TGF-beta, IFN-gamma and low levels of IL-10, a cytokine pattern similar to that secreted by anergic T cells. OPP clones bear a CD4(+)CD25(+) phenotype and show significantly lower proliferative response compared to T(h)0 clones. This lower response is recovered by the addition of IL-2. Thus, antigen-specific CD4(+)CD25(+) T(reg), which have characteristics of anergic cells and actively suppress antibody production are induced in PP upon oral administration of protein antigen.     

Neuropsin is essential for early processes of memory acquisition and Schaffer collateral long-term potentiation in adult mouse hippocampus in vivo

Long-term potentiation (LTP) is thought to be particularly important in the acquisition of hippocampus-associated memory, in part because it develops quickly and persists for indefinite periods. Extracellular proteolysis has been hypothesized to contribute to LTP by modifying adhesive relations of synapses and thus the morphology of excitatory synapses. Here we report that neuropsin (NP), an extracellular serine protease, is critically involved in the formation of both the potentiation effect and hippocampus-dependent forms of memory. NP-knockout mice were significantly impaired in the Morris water maze and Y-mazes and failed to exhibit early phase LTP induced by a single tetanus. Potentiation was also impaired or completely blocked by in vivo application of a specific inhibitor or a neutralizing monoclonal antibody for NP. Intriguingly, recombinant (r-) NP alone, without tetanic stimulation, elicited either long-lasting potentiation or depression, depending on the applied dose. The r-NP-elicited potentiation was occluded by prior induction of LTP, while theta-burst-elicited LTP was occluded by application of r-NP alone, suggesting that the two forms of plasticity have a common signalling pathway. r-NP-elicited potentiation and depression increased phosphorylation at different sites on the GluR1 subunit of the AMPA receptor that had previously been associated with LTP or long-term depression. Thus, we conclude that NP is necessary for establishment of LTP and has a significant role in memory acquisition.     

Boy with a chromosome del (3)(q12q23) and Blepharophimosis syndrome

We report on a 6-year-old boy with de novo 46, XY, del(3)(q12q23) and bilateral blepharo-phimosis, ptosis, epicanthus inversus, in addition to multiple other anomalies. Since 4 previously reported cases of interstitial deletion of 3q involving 3q23 band are clinically similar, we propose this blepharophimosis sequence due to 3q23 deletion as a further “contiguous gene syndrome”.     

Loss of Hrs in the Central Nervous System Causes Accumulation of Ubiquitinated Proteins and Neurodegeneration

The endosomal sorting complex required for transport (ESCRT) proteins form multimolecular complexes that control multivesicular body formation, endosomal sorting, and transport ubiquitinated membrane proteins (including cell-surface receptors) to the endosomes for degradation. There is accumulating evidence that endosomal dysfunction is linked to neural cell degeneration in vitro, but little is known about the relationship between neural disorders and ESCRT proteins in vivo. Here we specifically deleted the hrs gene, ESCRT-0, in the neurons of mice by crossing loxP-flanked hrs mice with transgenic mice expressing the synapsin-I Cre protein (SynI-cre). Histological analyses revealed that both apoptosis and a loss of hippocampal CA3 pyramidal neurons occurred in the hrs^flox/flox;SynI-cre mice. Notably, the hrs^flox/flox;SynI-cre mice accumulated ubiquitinated proteins, such as glutamate receptors and an autophagy-regulating protein, p62. These molecules are particularly prominent in the hippocampal CA3 neurons and cerebral cortex with advancing age. Accordingly, we found that both locomotor activity and learning ability were severely reduced in the hrs^flox/flox;SynI-cre mice. These data suggest that Hrs plays an important role in neural cell survival in vivo and provide an animal model for neurodegenerative diseases that are known to be commonly affected by the generation of proteinaceous aggregates.     

Dedifferentiated chondrosarcoma of the rib with a malignant mesenchymomatous component: An autopsy case report

A rare variant of dedifferentiated chondrosarcoma wlth malignant mesenchymomatous component in a 57-year-old male is reported. The patient presented with a posterior mediastinal mass arising from the left eighth and ninth ribs showing well differentiated, low-grade chondrosarcoma. Five years later, local recurrence occurred and an excised specimen also showed the same histological features as the primary tumor. Another 6 years later, the tumor recurred and metastasized to the multiple organs, the patient dying 4 months later. Autopsy revealed that the recurrent and metastatic tumors showed malignant mesenchymomatous 'dedifferentiation' of chondrosarcoma composed of rhab domyosarcoma, angiosarcoma, chondrosarcoma, osteosarcoma, and leiomyosarcoma, in addition to fibrosarcomatous areas. Although the less differentiated component of dedifferentiated chondrosarcoma usually shows the histological features of malignant fibrous histiocytoma and fibrosarcoma, multilineage differentiation can occur in that component. The phenomenon of 'dedifferentiation' in chondrosarcoma and the relationship to and distinction from malignant mesenchymoma of soft tissue and bone are discussed.     

Viability and osteogenic potential of cryopreserved human bone marrow-derived mesenchymal cells

Human bone marrow-derived mesenchymal cells contain mesenchymal stem cells (MSCs), which are well known for their osteo/chondrogenic potential and can be used for bone reconstruction. This article reports the viability of cryopreserved human mesenchymal cells and a comparison of the osteogenic potential between noncryopreserved and cryopreserved human mesenchymal cells with MSC-like characteristics, derived from the bone marrow of 28 subjects. The viability of cryopreserved mesenchymal cells was approximately 90% regardless of the storage term (0.3 to 37 months). It is clear by fluorescence-activated cell sorter analysis that the cell surface antigens of both noncryopreserved and cryopreserved mesenchymal cells were negative for hematopoietic cell markers such as CD14, CD34, CD45, and HLA-DR but positive for mesenchymal characteristics such as CD29 and CD105. To monitor the osteogenic potential of the cells, such as alkaline phosphatase (ALP) activity and in vitro mineralization, a subculture was conducted in the presence of dexamethasone, ascorbic acid, and glycerophosphate. No difference in osteogenic potential was found between cells with or without cryopreservation treatment. In addition, cells undergoing long-term cryopreservation (about 3 years) maintained high osteogenic potential. In conclusion, cryopreserved as well as noncryopreserved human mesenchymal cells could be applied for bone regeneration in orthopedics.     

A rapid isolation technique of unmodified human T cells on a polystyrene resin column

An easy and rapid isolation technique of human T cells on a polystyrene resin particle column has been developed. The cells of the effluent fraction contained more than 90% sheep erythrocyte (SRBC) rosette-forming cells and less than 1% of cells bearing surface immunoglobulin (Ig) or peroxidase. The T cell (SRBC rosette-forming cells) recovery rate was 80%. The distribution of OKT antigen T cell subsets was essentially the same as that of T cells separated by rosette sedimentation. Cell functions such as tritiated thymidine uptake by T cells and helper activity in Ig production were also the same as that of T cells separated by SRBC rosette sedimentation. Natural killer-like activity of the T cells isolated by the present method increased more than that of T cells obtained by the conventional method. Moreover, it was free from functional modification which tends to result from stimulation such as by the SRBC antigen in the SRBC centrifugation method. The combination of a T cell population offered by the present method and B cells depleted of SRBC-binding B cells minimized background plaque formation and enabled us to quantify the plaque-forming cell number in an antigen-specific plaque-forming assay. Furthermore, these populations produced relatively pure interleukin 2 (IL 2) by stimulation of an autologous mixed lymphocyte reaction without any absorption of IL 2 produced in the same culture. It seemed to be useful to evaluate the ability of lymphocytes from normal individuals and patients to produce IL 2.