GABAB receptor transduction mechanisms, and cross-talk between protein kinases A and C, in GABAergic terminals synapsing onto neurons of the rat nucleus basalis of Meynert

The transduction mechanisms underlying presynaptic GABA_B receptor-mediated inhibition of transmitter release have been characterized for a variety of synapses in the central nervous system (CNS). These studies have suggested a range of transduction mechanisms, including a role for second messengers such as protein kinases A (PKA) and C (PKC). In the present study, we have examined the intracellular signalling pathways underlying baclofen-induced inhibition of GABA release from terminals synapsing onto rat basalis of Meynert neurons using patch-clamp recordings. Baclofen, a selective GABA_B receptor agonist, reversibly decreased both evoked and spontaneous, miniature, GABAergic inhibitory postsynaptic currents (eIPSCs and mIPSCs, respectively). Such baclofen actions were completely abolished by CGP55845A, a selective GABA_B receptor antagonist, and by staurosporine, a non-selective PKA and PKC inhibitor. The mIPSC frequency was still decreased by baclofen even in the presence of 4 AP, a K⁺ channel blocker, and Cd²⁺, a voltage-dependent calcium channel blocker. Pharmacological activation or inhibition of PKC activity affected basal GABA release and mildly affected the response to baclofen. Inhibition of the cAMP/PKA cascade also affected basal GABA release and, in a subset of neurons, occluded the effects of baclofen, suggesting that the GABA_B receptor-mediated inhibitory action on GABA release was mediated via decreases in PKA activity. In addition, PKA inhibition occluded the effects of PKC modulation on both basal GABA release and on the response to baclofen. Our results characterize the transduction pathway of baclofen at these nucleus basalis of Maynert (nBM) synapses and show, for the first time, some cross-talk between the cAMP/PKA and PKC pathways in mammalian presynaptic nerve terminals.     

Angiopoietins and angiopoietin-like proteins in angiogenesis.

Vascular network formation requires several endothelial cell growth factors. These factors have a potent angiogenic effect, and their precise coordination is essential for vascular development. Among them, angiopoietins function through the Tie2 receptor, whose signaling is critical to regulate vascular stabilization and remodeling. It has been reported that the angiopoietin/Tie2 signal is involved in survival and migration of endothelial cells and regulates vascular remodeling and maintenance of vascular integrity. More recent studies demonstrate that angiopoietin/Tie2 signaling is also required for lymphangiogenesis. The authors and several other groups have identified six angiopoietin-like proteins (Angptls) containing a coiled-coil domain and a fibrinogen-like domain, both of which are characteristic of angiopoietins. Interestingly, Angptls also function in angiogenesis through regulating survival and migration of endothelial cells, although Angptls do not bind the angiopoietin receptor Tie2. Currently, Angptls are orphan ligands, but they have been reported to have pleiotropic effects not only on vascular cells but also on metabolism and tumor biology. Here, the authors review current findings relating to the roles of angiopoietins and Angptls in vascular biology and discuss molecular mechanisms relevant to these factors and angiogenesis.     

Total hematological analysis system as a screening test for hematological malignancy

Measurement of complete blood cell count and white blood cell differentiation is an essential laboratory test and the most important screening test for hematological malignancy. Recently, several automated blood cell analyzers have been developed to improve accuracy and precision. When flag messages generated in the presence of morphological abnormalities of the samples are displayed, manual revision is necessary. In our laboratory, the manual revision rate has been 35-40%. Therefore blood cell analyzers are useful in screening for abnormalities as well as greatly reducing expensive and time-consuming manual differential procedures. In addition, automated blood cell analyzers can provide several types of useful information including the leukocyte distribution scattergram. However, most such information is not utilized in the clinical field. In the future, a total hematological analysis system will be constructed so that all information provided by automated blood cell analyzer and by manual methods are available.     

Expression and secretion of an Arthrobacter dextranase in the oral bacterium Streptococcus gordonii.

We have constructed a plasmid to express and secrete dextranase in the oral bacterium Streptococcus gordonii. The dextranase gene from Arthrobacter sp. strain CB-8 was linked to a promoter and a DNA sequence encoding the signal peptide of Streptococcus downei glucosyltransferase I (gtfI) followed by the Escherichia coli rrnBt1t2 terminator and inserted in the shuttle vector pVA838. S. gordonii transformed with this plasmid (pMNK-4) expressed and secreted mature Arthrobacter dextranase. The transformant was found to repress the firm adherence of water-insoluble glucan in a coculture experiment with cariogenic bacteria, Streptococcus sobrinus, in the presence of sucrose. Such genetically engineered oral bacteria could provide a therapy to prevent dental caries.     

Muscle spindle supply to the masticatory muscles in the Japane ermine (Carnivora).

Histological examination of the jaw muscles of the Japanese ermine showed that 4 jaw-closing muscles have 13 muscle spindles on one side of the face. The temporal muscle has 99 muscle spindles, 68 being in the anterior vertical and 31 in the posterior horizontal belly. The masseter muscle has 33 muscle spindles, 23 being in the profound and 10 in the superficial belly. The medial pterygoid muscle has 7 muscle spindles and the zygomaticomandibular muscle contains 4 muscle spindles. The lateral pterygoid and the jaw-opening muscles have no spindles.     

Conversion of highly malignant colon cancer from an aggressive to a controlled disease by oral administration of a metalloproteinase inhibitor

In this study, we describe the activity of CT1746, an orally-active synthetic MMP inhibitor that has a greater specificity for gelatinase A, gelatinase B and stromelysin than for interstitial collagenase and matrilysin, in a nude mouse model that better mimics the clinical development of human colon cancer. The model is constructed by surgical orthotopic implantation (SOI) of histologically-intact tissue of the metastatic human colon tumor cell line Co-3. Animals were gavaged with CT1746 twice a day at 100 mg/kg for 5 days after the SOI of Co-3 for 43 days. In this model CT1746 significantly prolonged the median survival time of the tumor-bearing animals from 51 to 78 days. Significant efficacy of CT1746 was observed on primary tumor growth (32% reduction in mean tumor area at day 36), total spread and metastasis (6/20 treated animals had no detectable spread and metastasis at autopsy compared to 100% incidence of secondaries in control groups). Efficacy of CT1746 could also be seen on reducing tumor spread and metastasis to individual organ sites such as the abdominal wall, cecum and lymph nodes compared to vehicle and untreated controls. We conclude that chronic administration of a peptidomimetic MMP inhibitor via the oral route is feasible and results in inhibition of solid tumor growth, spread and metastasis with increase in survival in this model of human cancer, thus converting aggressive cancer to a more controlled indolent disease.     

Detection of local abnormalities in ventricular activation sequence by body surface isochrone mapping in patients with previous myocardial infarction

Body surface isochrone mapping was performed in 36 normal subjects and in 85 patients with previous myocardial infarction. Eighty-seven unipolar electrocardiograms distributed over the anterior chest and the back were recorded simultaneously. For each lead, activation time was measured as the time from the onset of QRS to the peak of the R wave. The lead points where R waves were not observed were designated the "no R wave area" (NR area). Isochrone maps of normal subjects had a consistent pattern, with isochrone lines extending from the right upper anterior chest to the left anterior chest and then to the back. NR area was small and was located only on the right upper chest or the upper back. On the isochrone maps of patients with myocardial infarction, abnormal findings were observed; NR area was found in 26 of 28 patients with anterior infarction on the upper to middle anterior chest, in 13 of 22 patients with inferior infarction on the lower chest, and in 24 of 25 patients with anterior and inferior infarction on the upper to lower anterior chest. Activation time was delayed near the NR area (peri-NR area delay) in 37 patients. In patients with apical infarction, an islandlike zone of delayed activation was typically found on the left precordium. These abnormal patterns are considered to indicate local abnormalities in the activation of infarcted myocardium; the NR area indicates dead unexcitable scar, and the peri-NR area delay and islandlike zone of delayed activation indicate partially infarcted myocardium of slow activation. Patients with NR area had greater degree of left ventricular asynergy and lower ejection fraction than those without.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fraction of pluripotent hemopoietic stem cells in DNA synthesis varies with generation age

The fraction of pluripotent hemopoietic stem cell (CFU-S) in DNA synthesis (S phase) was evaluated for day 7 through 13-spleen colonies. The CFU-S in S phase in normal bone marrow decreased progressively, from 43% on day 7, to 23% on day 11, and 4% on day 13. The cycling fraction of 5-fluorouracil (5FU)-treated CFU-S was also evaluated. The S phase fraction for CFU-S surviving 5FU was 100.0, 58.8, 54.7, and 25.6% at day 7, 9, 11, and 13, respectively, and the proportion of the fraction also was not constant. The S phase fraction for CFU-S surviving 5FU was much higher than that for normal bone marrow cells; this might be attributed to the extensive regenerating ability of CFU-S surviving 5-FU. The results obtained suggest that a stem cell population in S-phase may exhibit a hierarchy based on the "generation-age hypothesis" (Rosendaal, Hodgson, and Bradley 1979) of the stem cell compartment.