Effects of selected vasoactive substances on adenylate cyclase activity in brain,isolated brain microvessels,and primary cultures of brain microvessel endothelial cells

The specific activity of adenylate cyclase was assayed in homogenates of gray matter, freshly isolated and primary cultured microvessel endothelial cells from bovine cerebral cortex. Specific activities for the tissues were 14.6±2.1, 15.6±2.7, and 8.4±1.5 pmol cAMP/mg protein/min±SD for gray matter, cultured microvessels, and freshly isolated microvessels, respectively. Adenylate cyclase associated with gray matter and cultured microvessels was sensitive to histamine and selected catecholamines. Perhaps due to metabolic deficiencies, adenylate cyclase of freshly isolated microvessels exhibited little or no response to either the catecholamines or histamine. Angiotensin II stimulated adenylate cyclase of both freshly isolated and cultured microvessels but had no effect on gray matter. Bradykinin did not stimulate cAMP generation in any of the tissues. Overall results support the role of cAMP in regulating brain microvessel functions and suggest that primary cultures of brain microvessels may be useful in examining cAMP-mediated biochemical pathways at the blood-brain barrier.     

Stem cells and brain cancer

An increasing body of research is showing that cancers might contain their own stem cells. In fact, cancer cells, like stem cells, can proliferate indefinitely through a deregulated cellular self-renewal capacity. This raises the possibility that some features of tumor cells may be due to cancer stem cells. Stem cell-like cancer cells were isolated from several solid tumors. Now, evidence has shown that brain cancers, such as glioblastomas, medulloblastomas and astrocytomas, also contain cells that may be multipotent neural stem cell-like cells. In this review, we discuss the results of these studies, along with the molecular pathways that could be involved in cancer stem cell physiopathology.     

Expression of phosphoprotein p19 in brain, testis, and neuroendocrine tumor cells. Developmental regulation in rat brain

We have recently purified from bovine brain a 19-kDa protein, p19, that was previously shown to undergo hormonally regulated phosphorylation in several neuroendocrine tumor cells. We now report the tissue distribution of p19, studied by immunoblotting. Using a rabbit antiserum, which binds both to the unphosphorylated form and to the two predominant phosphoforms of p19, we show that the protein is present in brain and testis but not in a variety of other mammalian tissues. High levels of p19 are also present in several cultured tumor cells expressing neuroendocrine properties. In addition, p19 was detected in HL60 promyelocytic leukemia and in Friend erythroleukemia cells, but not in several other cell lines. In rat brain, we show that the level of p19 is maximal on the first postnatal day and declines within the first 2 weeks of life to a low plateau that persists into adulthood. The concentration of translatable p19 mRNA also decreases postnatally in rat brain, suggesting that the developmental regulation of the expression of p19 occurs, at least in part, at a pretranslational level. The broad species cross-reactivity of the p19 antibody suggests that the gene encoding p19 has been highly conserved during mammalian evolution. Based on the pattern of expression of this protein, we propose that p19 plays a role in the development of neurons and neuroendocrine cell types.     

Adenosine transport systems on dissociated brain cells from mouse,guinea-pig,and rat

The kinetics and sodium dependence of adenosine transport were determined using an inhibitorstop method on dissociated cell body preparations obtained from mouse guinea-pig and rat brain. Transport affinity (K_T) values for the high affinity adenosine transport systems (K_T(H)) were significantly different between these three species; mean ±SEM values were 0.34 ±0.1 in mouse, 0.9 ±0.2 in rat, and 1.5±0.5 M in guinea-pig. The K_T values for the low affinity transport system (K_T(L)) were not different between the three species. Brain cells from rat displayed a significantly greater maximal capacity to accumulate [³H]adenosine (V_max) than did mouse or guinea-pig for the high affinity system, or than did mouse for the low affinity system. When sodium chloride was replaced in the transport medium with choline chloride, the K_T(H) values for guinea-pig and rat were both increased by approximately 100%; only in rat did the change reach statistical significance. The sodium-dependence of adenosine transport in mouse brain was clearly absent. The differences between K_T(H) values in mouse and those in guinea-pig or rat were accentuated in the absence of sodium. The differences in kinetic values, ionic requirements, and pharmacological characteristics between adenosine transporters in CNS tissues of mouse guinea-pig and rat may help account for some of the variability noted among species in terms of their physiological responses to adenosine.     

Perspectives on brain tumor formation involving macrophages, glia, and neural stem cells

The incidence of brain tumors is rising in children and the elderly, but little is known about the mechanisms underlying brain tumor initiation and progression. In the 1940s, Zimmerman and coworkers exploited the tumor-promoting potential of polycyclic hydrocarbons to produce brain tumor models in adult mice that simulated the neuropathology of human brain tumors. Based on these early findings and on recent neurobiological studies of stem cells, I propose that crystalline carcinogenic pellets surgically implanted in the central nervous system establish over time a microenvironment that fosters proliferation and genetic damage in neural stem cells and their progenitors. Moreover, activated glia (microglia and astrocytes) and recruited macrophages mediate these processes. Gradually local tissue fields, which normally restrict stem cell proliferation, become disorganized, leading to further stem cell proliferation, genetic damage, and eventual neoplasia. Depending on age, location, and the state of glial/macrophage activation, the resulting brain tumor may resemble transformed neural progenitors aborted in more or less differentiated states. This hypothesis integrates the general mechanisms by which neural stem cells, glia, and macrophages orchestrate the initiation and progression of brain cancer. Also discussed are implications of these concepts for the diagnosis and therapy of human brain tumors.     

Tales of biomaterials, molecules, and cells for repairing and treating brain dysfunction

Current therapies have limited or no capacity to restore lost function, slow ongoing neurodegeneration, or promote regeneration following damage to the brain. Biomaterials are playing an increasingly important role in the development of novel, potentially efficacious approaches to brain treatment and repair. Programmable biomaterials enable and augment the targeted delivery of drugs into the brain and allow cell/tissue transplants to be effectively delivered and integrate into the brain, to serve as delivery vehicles for therapeutic proteins, and rebuild damaged circuits. Similarly, biomaterials are being increasingly used to recapitulate specific aspects of brain niches to promote regeneration and/or repair damaged neuronal pathways with stem cell therapies. Many of these approaches are gaining momentum because nanotechnology allows greater control over material-cell interactions that induce specific developmental processes and cellular responses including differentiation, migration, and outgrowth. This review discusses the state of the art and new directions in the convergence of biomaterial science, drug delivery, and stem cell biology in the treatment of degenerative and malignant brain diseases.     

A human IAP-family gene, apollon, expressed in human brain cancer cells.

IAP is a family of protein that has baculovirus IAP repeat (BIR) domains and inhibits apoptosis. We found a human IAP family gene, which we named Apollon, encoding a huge protein (530 kDa) that contains a single BIR domain and a ubiquitin-conjugating enzyme domain, that is a human homolog of BRUCE. Apollon protein was expressed in four of six brain cancers (gliomas), and one of five ovarian cancers in 38 human cancer cell lines that we examined. Among the brain cancer cell lines, SNB-78 expressed a high level of Apollon, and this cell line shows resistance against various anticancer drugs. Treating SNB-78 cells with antisense oligonucleotide against Apollon reduced the expression of Apollon protein, and significantly sensitized the cells to apoptosis induced by cisplatin and camptothecin. These results suggest that Apollon protects SNB-78 cells from undergoing apoptosis and, at least in part, plays a role in tumorigenesis and drug resistance of this cell line.     

Epithelial cells and their neighbors. II. New perspectives on efferent signaling between brain, neuroendocrine cells, and gut epithelial cells

Surface sensory enteroendocrine cells are established mucosal taste cells that monitor luminal contents and provide an important link in transfer of information from gut epithelium to the central nervous system. Recent studies now show that these cells can also mediate efferent signaling from the brain to the gut. Centrally elicited stimulation of vagal and sympathetic pathways induces release of melatonin, which acts at MT2 receptors to increase mucosal electrolyte secretion. Psychological factors as well mucosal endocrine cell hyperplasia are implicated in functional intestinal disorders. Central nervous influence on the release of transmitters from gut endocrine cells offers an exciting area of future gastrointestinal research with a clinical relevance.     

Association of putrescine,spermidine, spermine,and GABA with structural elements of brain cells

The present experiments are the first survey of the association of endogenous and exogenous putrescine, spermidine, and spermine with subcellular structures of rat brain cortex. The differences of distribution in subfractions obtained from salt-free and salt-containing density gradients were studied, with the following results: (1) In contrast with liver preparation, putrescine and the polyamines spermidine and spermine are not distributed in parallel with RNA. (2) In salt-containing media, putrescine and the polyamines were preferentially associated with synaptosomes and with synaptosomal membranes. Significant association with myelin constituents was observed only in salt-free media. (3) Exogenous putrescine and the polyamines were less firmly attached to synaptosomes and to synaptosomal membrane fractions than the endogenous amines. There is good evidence for similar subcellular localizations of putrescine and GABA. Putrescine seems to be entrapped in the nerve endings. (4) Uptake studies with crude mitochondria under conditions of high-affinity uptake showed no temperature-sensitive component of polyamine accumulation in synaptosomes, in contrast with GABA, monoacetylputrescine, and ornithine. (5) Polyamines bound to myelin constituents or mitochondria could be displaced by a 200-fold concentration of nonradioactive amines; this was not the case with polyamines bound to synaptosomes. Mg²⁺ did not effectively compete with spermine for binding sites at synaptic regions. (6) Electrical stimulation and stimulation by mono- and bivalent cations did not change the concentrations of the polyamines and GABA in guinea pig cortex. (7) There is no evidence for a neurotransmitter role of putrescine, spermidine, or spermine, although these compounds might function as modulators of neurotransmission.     

Stem cells and neonatal brain injury

Recent advances in regenerative medicine and in our understanding of neurogenesis may lead to new ways of recovering neuronal function lost or damaged during the perinatal period; such injuries are not amenable to conventional therapies. We review recent experimental studies based on immature rodental models of neonatal brain injury, especially hypoxic-ischemic encephalopathy. The developing brain is revealed to have considerable potential with respect to proliferation and migration to the injured site. However, the generation of fully differentiated neurons is extremely limited after brain injuries. Aggressive efforts to adjust the environment of the damaged brain in which tissue regeneration is occurring or more cautious stem cell transplantation will be required for the successful treatment of developmental brain injury. This work was supported by a Research Grant for Cardiovascular Diseases (18C-1) from the Ministry of Health, Labour and Welfare.     

Viridiflorol induces anti-neoplastic effects on breast,lung, and brain cancer cells through apoptosis

All active natural molecules are not fully exploited as therapeutic agents, causing delays in the advancement of anticancer drug discovery. Viridiflorol is a natural volatile element that may work as anti-cancer compound. We tested the anticancer properties of viridiflorol at different concentrations ranging from 0.03 to 300 μM in vitro on three cancer cells including breast (MCF-7), lung (A549) and brain (Daoy). The cancer cells responses were documented after treatment using MTT and Annexin V assays. Viridiflorol showed cytotoxic effects against all tested cell lines, reducing cell viability in a concentration-dependent manner with variable IC₅₀ values. Daoy and A549 cell lines were more sensitive to viridiflorol when compared with temozolomide and doxorubicin, respectively. Viridiflorol demonstrated the highest anticancer activity against the Daoy cells with an estimated IC₅₀ of 0.1 µM followed by MCF-7 at 10 µM, and A549 at 30 µM. In addition, upon exposure to concentrations ranging from 30 µM to 300 µM of viridiflorol, early and late apoptotic cell death was induced in a concentration dependent manner in Daoy (55.8%-72.1%), MCF-7 (36.2%-72.7%) and A459 (35%-98.9%) cell lines, respectively. In conclusion, viridiflorol demonstrates cytotoxic and apoptotic ability in three different cancer cell lines (brain, breast and lung).     

Triethyllead treatment of cultured brain cells

Cultured cells from chick embryo brains were studied for their sensitivity to triethyllead. Triethyllead chloride (3.16 M) was added to the nutrient medium and incubated for 48 hr with the cells. Morphological changes in light microscope and radioactive labeling of galactolipids were assayed. Triethyllead treatment reduced the number of neuronal cells with processes. Morphological changes were not observed in glial cells. The [³⁵S]sulfate labeling of sulfatides was reduced to 50%. The [³H]serine labeling of cerebrosides with alpha-hydroxy fatty acids was not influenced, while the [³H]serine labeling of cerebrosides with nonhydroxy fatty acids was inhibited 40% in one- and two- but not in three-week-old cultures. The results indicate that the nerve cell response to triethyllead in cultures is selective, since the neurons are more sensitive than the glia cells and the labeling of sulfatides is more sensitive than that of cerebrosides.     

Mast cells in the human brain

Mast cells, as adjudged by the metachromatic staining of their cytoplasmic granules, were found in 79% of the 97 humans brains studied. They were most numerous and most consistently present in the infundibulum, pineal organ, area postrema and choroid plexuses. They were also numerous in the leptomeninges surrounmding the pineal organ and infundibulum. Occasional mast cells were also seen within the supraoptic crest, the subfornical organ, the ventricles and the leptomeninges at sites other than over the infundibulum and pineal organ. They were not detectable elsewhere in the brain or spinal cord. In the infundibulum, pineal organ, area postrema and telencephalic choroid plexuses mast cells were most numerous in young individuals (i.e., 0-19 years of age); thereafter, their numbers progressively decreased with aging. Elsewhere mast cell numbers remained about the same with aging. Except in the area postrema where mast cells were more numerous and more consistently present in males, sex-related differences in mast cell number or distribution were not detected. No differences in either the abundance, the distribution or the percentage of individuals possessing mast cells at any of these sites were apparent between 'normative' brains, lesioned brains ('stroke', lobotomy, etc.) or those from individuals with either congenital or acquired encephalopathies.