Measurement of changes in membrane surface morphology associated with exocytosis using scanning ion conductance microscopy

The extent that vesicles maintain a distinct identity and morphology after fusing with the plasma membrane is controversial. We used scanning ion conductance microscopy to image changes in the surface membrane of adrenal chromaffin cells after stimulation of exocytosis with a high K(+) solution. Within several minutes after stimulation, punctate depressions, 100-600 nm wide, were noted from 16% of the cells. The depressions were not randomly distributed, but appeared in clusters of two or more within a approximately 1 microm(2) area and disappeared after several minutes. Increases in membrane surface area, consistent with the fusion and collapse of one or more vesicles into the surface membrane, were observed in 64% of the cells after high K(+) stimulation. Surface area increases did not occur if the high K(+) solution did not contain Ca(2+). We conclude that scanning ion conductance microscopy can be used to follow the time course of surface membrane changes resulting from exocytosis and endocytosis.     

Slow and selective death of spinal motor neurons in vivo by intrathecal infusion of kainic acid: implications for AMPA receptor-mediated excitotoxicity in ALS

Excitotoxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors has been proposed to play a major role in the selective death of motor neurons in sporadic amyotrophic lateral sclerosis (ALS), and motor neurons are more vulnerable to AMPA receptor-mediated excitotoxicity than are other neuronal subclasses. On the basis of the above evidence, we aimed to develop a rat model of ALS by the long-term activation of AMPA receptors through continuous infusion of kainic acid (KA), an AMPA receptor agonist, into the spinal subarachnoid space. These rats displayed a progressive motor-selective behavioral deficit with delayed loss of spinal motor neurons, mimicking the clinicopathological characteristics of ALS. These changes were significantly ameliorated by co-infusion with 6-nitro-7-sulfamobenso(f)quinoxaline-2,3-dione (NBQX), but not with d(-)-2-amino-5-phosphonovaleric acid (APV), and were exacerbated by co-infusion with cyclothiazide, indicative of an AMPA receptor-mediated mechanism. Among the four AMPA receptor subunits, expression of GluR3 mRNA was selectively up-regulated in motor neurons but not in dorsal horn neurons of the KA-infused rats. The up-regulation of GluR3 mRNA in this model may cause a molecular change that induces the selective vulnerability of motor neurons to KA by increasing the proportion of GluR2-lacking (i.e. calcium-permeable) AMPA receptors. This rat model may be useful in investigating ALS etiology.     

Inhibition of Escherichia coli respiratory enzymes by the lactoperoxidase-hydrogen peroxide-thiocyanate antimicrobial system

AIMS: The lactoperoxidase-hydrogen peroxide-thiocyanate antimicrobial system (LPAS) is known to inhibit bacterial respiration. In the present study, several respiratory enzymes of Escherichia coli were compared in terms of their susceptibility to the LPAS. METHODS AND RESULTS: Exposure of E. coli to the LPAS, upon which 99.6% of the bacteria were killed, resulted in the following percentage of inactivation of substrate-specific membrane oxidases: succinate (94.2%) > NADH (84.6%) > glycerol-3-phosphate (67.8%) > DL-lactate (64.1%). With the same treatment, substrate-specific membrane dehydrogenases were inactivated as follows: succinate (99.1%) > DL-lactate (53.8%) > glycerol-3-phosphate (45.0%) > NADH (36.8%). Terminal oxidase, however, measured using a ubiquinone analogue (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone) after reduction, was only 21.4% inactivated by the LPAS. CONCLUSION: These data suggest that dehydrogenases are the primary targets of the LPAS in the respiratory chain of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has determined for the first time the primary targets of LPAS in the bacterial respiratory chain.     

Isolation of Cytopathic Small Round Virus (Aichi Virus) from Pakistani Children and Japanese Travelers from Southeast Asia

Aichi virus was isolated in Vero cells from 5 (2.3%) of 222 Pakistani children with gastroenteritis but none was found in 91 healthy children. Aichi virus was also isolated from 5 (0.7%) of 722 Japanese travelers returned from tours to Southeast Asian countries and complained of gastrointestinal symptoms at the quarantine station of Nagoya International Airport in Japan. Of 5 Japanese travelers, 3 were returning from Indonesia, and 2 from Thailand or Malaysia. These results indicate that Aichi virus or a similar agent is endemic in Southeast Asian countries and is a cause of gastrointestinal symptoms in children in these areas or in Japanese travelers who visit there.     

Analysis of the Relationship between Cellular Thymidine Kinase Activity and Virulence of Thymidine Kinase-Negative Herpes Simplex Virus Types 1 and 2

The virulence of thymidine kinase-negative herpes simplex virus type 1 (HSV-1; VRTK^? strain) and type 2 (HSV-2; UWTK^? strain) was studied in comparison with that of their parental strains (VR-3 and UW-268, respectively) in an encephalitis model of adult (4-week-old) and newborn (3-day-old) mice. Viral thymidine kinase (TK) activity was essential for the maximum expression of virulence of HSV-1, because the 50% lethal dose (LD₅₀) of VRTK^? was 60 times higher than that of VR-3 in the brains of newborn mice expressing high levels of cellular TK activity. However, the UWTK^? strain showed the same virulence as the parental strain in newborn mice, despite the lack virulence in adults, suggesting that replication of the UWTK^? strain was completely supported by cellular TK activity. This difference in the role of viral and cellular TKs for virus growth between HSV-1 and HSV-2 was confirmed with the one-step growth of virus strains in L-M and L-M(TK^?) cells.     

Axon growth and guidance genes identify T-dependent germinal centre B cells

Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4alpha. This is consistent with formation of long neurite (axon and dendrite)-like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD-GC B cells including Basp1, Shh, Sult4alpha, Sc4mol are also preferentially expressed in post-GC B cell neoplasms.     

Ran-binding protein 3 phosphorylation links the Ras and PI3-kinase pathways to nucleocytoplasmic transport

The major participants of the Ras/ERK and PI3-kinase (PI3K) pathways are well characterized. The cellular response to activation of these pathways, however, can vary dramatically. How differences in signal strength, timing, spatial location, and cellular context promote specific cell-fate decisions remains unclear. Nuclear transport processes can have a major impact on the determination of cell fate; however, little is known regarding how nuclear transport is regulated by or regulates these pathways. Here we show that RSK and Akt, which are activated downstream of Ras/ERK and PI3K, respectively, modulate the Ran gradient and nuclear transport by interacting with, phosphorylating, and regulating Ran-binding protein 3 (RanBP3) function. Our findings highlight an important link between two major cell-fate determinants: nuclear transport and the Ras/ERK/RSK and PI3K/Akt signaling pathways.     

Beneficial effect of silkworm hemolymph on a CHO cell system: Inhibition of apoptosis and increase of EPO production

To produce erythropoietin (EPO), Chinese hamster ovary (CHO) cells were first cultured in a medium containing FBS (growth medium) and then in a serum-free medium containing sodium butyrate (production medium). Sodium butyrate increases recombinant protein production, but also induces apoptosis, which reduces cell viability and productivity. In a previous study, we found that silkworm hemolymph (SH), an insect serum, inhibits the apoptosis of insect and mammalian cells. To overcome sodium butyrate-induced apoptosis, we added SH to growth medium. This pretreatment with SH inhibited the sodium butyrate-induced apoptosis of CHO cells and consequently increased their longevity and their ability to produce EPO. As a result, the volumetric productivity of EPO was increased five-fold. SH was found to inhibit cytochrome c release from mitochondria into the cytosol, and prevented the activation of caspase-3 and other subsequent caspase reactions.     

The usefulness of semi‐solid medium in the isolation of highly virulent Leptospira strains from wild rats in an urban area of Fukuoka,Japan

Leptospirosis is a worldwide zoonosis. The importance of urban leptospirosis is recognized in Japan: urban rats carry pathogenic leptospires and people acquire these pathogens through contact with surface water or soil contaminated by the urine of the infected animals. To determine the current Leptospira carriage rate in urban rats, 29 wild rats were trapped in the central area of Fukuoka and strains isolated from their kidneys and urine analyzed. When semi‐solid Korthof's medium containing 0.1% agar was used for isolation, 72.2% and 30.8% of the kidney and urine cultures, respectively, were found to be Leptospira‐positive. The isolates belonged to Leptospira interrogans, and were classified into two groups (serogroups Pomona and Icterohaemorrhagiae) based on the results of gyrB sequence analysis and microscopic agglutination testing (MAT). Strains belonging to serogroup Icterohemorrhagiae grew well in liquid medium. On the other hand, serogroup Pomona isolates multiplied very little in liquid medium, but did grow in a semi‐solid medium. Although strains belonging to serogroup Pomona have not been recognized as native to Japan, this strain may be widely distributed in urban rats. Representative strains from each group were found to be highly pathogenic to hamsters. Our findings should serve as a warning that it is still possible to become infected with leptospires from wild rats living in inner cities of Japan. Furthermore, the use of semi‐solid medium for culture will improve the isolation rate of leptospires from the kidneys of wild rats.