A Role for the acrA Gene in the Protection of Escherichia coli Cells against Excess Sodium

The acrA gene determines the sensitivity of Escherichia coliK-12 cells to acriflavine, which is one of the acridine dyesand which effectively eliminates certain plasmids from the bacterialcells. The acriflavine-sensitivity mutation leads to instabilityof plasmids, such as sex (F)- and drug-resistance (R)-factorsand to loss of a membrane protein with molecular weight about60 kDa (Nakamura 1974, 1976, Nakamura et al. 1975, 1981). Wehave found that cells with a mutant acrA gene were also moresensitive to an excess of sodium ions in the medium than werethe wild-type acrA⁺ cells (Nakamura 1977). The product of theacrA gene hindered the accumulation of sodium by the cells.Although mutations in theproA or proB genes, which determinethe synthesis of the enzymes y-glutamyl phosphate reductaseand y-glutamyl kinase, respectively, in the proline-biosyntheticpathway also led to sensitivity to and accumulation of excesssodium, the presence of the acrA⁺ allele decreased both theseparameters. (Received November 1, 1989; Accepted April 27, 1990)     

An anti-proliferative gene BTG1 regulates angiogenesis in vitro

B-cell translocation gene 1 (BTG1) is a member of the anti-proliferative gene family that regulates cell growth and differentiation. To clarify the role of BTG1 in angiogenesis, we examined the regulation of BTG1 expression in cultured endothelial cells and characterized its function in in vitro models of angiogenesis. BTG1 mRNA was abundantly expressed in quiescent endothelial cells. Addition of serum and angiogenic growth factors decreased BTG1 mRNA levels in endothelial cells. In contrast, BTG1 mRNA was up-regulated in tube-forming endothelial cells on Matrigel. This up-regulation was partially blocked by neutralizing antibody against transforming growth factor-beta (TGF-beta), and TGF-beta increased BTG1 mRNA levels. Inhibition of endogenous BTG1 by overexpression of antisense BTG1 resulted in inhibited network formation, and overexpression of sense BTG1 augmented tube formation in these cell lines. BTG1-overexpressing endothelial cells displayed increased cell migration. These findings suggest that BTG1 may play an important role in the process of angiogenesis.     

Detection of regional allozyme divergence in the rocky intertidal goby Chaenogobius annularis

Genetic differentiation in the intertidal goby Chaenogobius annularis was studied using allozyme markers. Samples were collected from six localities along the coasts of Japanese islands and the Korean Peninsula. Six out of 13 loci showed allelic variation in at least one population. Although the predominant alleles of 12 loci were the same among all populations, Mdh-1 showed clear differences among the populations located along the coasts of the Pacific Ocean and the Sea of Japan. These two possible geographic groups, the Pacific Ocean and the Sea of Japan groups, were characterized by diagnostic alleles of Mdh-1, namely Mdh-1 ¹⁰⁰ and Mdh-1 ⁷⁰.     

n-Butanol induces depolymerization of microtubules in vivo and in vitro

The effects of butanol on microtubules (MTs) were examined by immunofluorescence microscopy. Fragmentation of cortical MTs was induced by n-butanol, but not by s- and t-butanols, in cultured tobacco BY-2 cells. Taxol prevented n-butanol-induced MT fragmentation. Fragmented cortical MTs were still attached to the inner face of the plasma membrane when n-butanol-treated protoplasts were ruptured on the slide glass. Moreover, MTs were depolymerized in the presence of n-butanol in vitro. Therefore, n-butanol is not only an activator of phospholipase D but also an effective MT-depolymerizing agent.     

Phospholipid Membrane-Mediated Hemozoin Formation: The Effects of Physical Properties and Evidence of Membrane Surrounding Hemozoin

Phospholipid membranes are thought to be one of the main inducers of hemozoin formation in Plasmodia and other blood-feeding parasites. The “membrane surrounding hemozoin” has been observed in infected cells but has not been observed in in vitro experiments. This study focused on observing the association of phospholipid membranes and synthetic β-hematin, which is chemically identical to hemozoin, and on a further exploration into the mechanism of phospholipid membrane-induced β-hematin formation. Our results showed that β-hematin formation was induced by phospholipids in the fluid phase but not in the gel phase. The ability of phospholipids to induce β-hematin formation was inversely correlated with gel-to-liquid phase transition temperatures, suggesting an essential insertion of heme into the hydrocarbon chains of the phospholipid membrane to form β-hematin. For this study, a cryogenic transmission electron microscope was used to achieve the first direct observation of the formation of a monolayer of phospholipid membrane surrounding β-hematin.     

Cerebral Blood Flow Modulation by Basal Forebrain or Whisker Stimulation Can Occur Independently of Large Cytosolic Ca2+ Signaling in Astrocytes

We report that a brief electrical stimulation of the nucleus basalis of Meynert (NBM), the primary source of cholinergic projection to the cerebral cortex, induces a biphasic cerebral cortical blood flow (CBF) response in the somatosensory cortex of C57BL/6J mice. This CBF response, measured by laser Doppler flowmetry, was attenuated by the muscarinic type acetylcholine receptor antagonist atropine, suggesting a possible involvement of astrocytes in this type of CBF modulation. However, we find that IP3R2 knockout mice, which lack cytosolic Ca2+ surges in astrocytes, show similar CBF changes. Moreover, whisker stimulation resulted in similar degrees of CBF increase in IP3R2 knockout mice and the background strain C57BL/6J. Our results show that neural activity-driven CBF modulation could occur without large cytosolic increases of Ca2+ in astrocytes.     

Ensemble patterns of hippocampal CA3-CA1 neurons during sharp wave-associated population events

Transfer of neuronal patterns from the CA3 to CA1 region was studied by simultaneous recording of neuronal ensembles in the behaving rat. A nonlinear interaction among pyramidal neurons was observed during sharp wave (SPW)-related population bursts, with stronger synchrony associated with more widespread spatial coherence. SPW bursts emerged in the CA3a-b subregions and spread to CA3c before invading the CA1 area. Synchronous discharge of >10% of the CA3 within a 100 ms window was required to exert a detectable influence on CA1 pyramidal cells. Activity of some CA3 pyramidal neurons differentially predicted the ripple-related discharge of circumscribed groups of CA1 pyramidal cells. We suggest that, in SPW behavioral state, the coherent discharge of a small group of CA3 cells is the primary cause of spiking activity in CA1 pyramidal neurons.     

Multiphoton stimulation of neurons

We pulsed the activation of neurons using a femtosecond laser. Pyramidal neurons are depolarized and fire action potentials when high intensity mode-locked infrared light irradiates somatic membranes and axon initial segments. This depolarization is reversible, does not occur with CW laser light, and appears to be due to multiphoton excitation. We describe two regimes of multiphoton optical stimulation. Low intensity, long duration laser irradiation produces a sustained depolarization, insensitive to sodium channel blockers yet sensitive to antioxidants. On the other hand, high intensity, short duration irradiation can induce fast depolarizations, which appear due to different mechanism. The combination of multiphoton stimulation and optical probing could enable systematic analysis of circuits.     

Calcium dynamics of cortical astrocytic networks in vivo

Large and long-lasting cytosolic calcium surges in astrocytes have been described in cultured cells and acute slice preparations. The mechanisms that give rise to these calcium events have been extensively studied in vitro. However, their existence and functions in the intact brain are unknown. We have topically applied Fluo-4 AM on the cerebral cortex of anesthetized rats, and imaged cytosolic calcium fluctuation in astrocyte populations of superficial cortical layers in vivo, using two-photon laser scanning microscopy. Spontaneous [Ca²⁺]_i events in individual astrocytes were similar to those observed in vitro. Coordination of [Ca²⁺]_i events among astrocytes was indicated by the broad cross-correlograms. Increased neuronal discharge was associated with increased astrocytic [Ca²⁺]_i activity in individual cells and a robust coordination of [Ca²⁺]_i signals in neighboring astrocytes. These findings indicate potential neuron–glia communication in the intact brain.