Depending on Its Nano-Spacing,ALCAM Promotes Cell Attachment and Axon Growth

ALCAM is a member of the cell adhesion molecule (CAM) family which plays an important role during nervous system formation. We here show that the two neuron populations of developing dorsal root ganglia (DRG) display ALCAM transiently on centrally and peripherally projecting axons during the two phases of axon outgrowth. To analyze the impact of ALCAM on cell adhesion and axon growth, DRG single cells were cultured on ALCAM-coated coverslips or on nanopatterns where ALCAM is presented in physiological amino-carboxyl terminal orientation at highly defined distances (29, 54, 70, 86, and 137 nm) and where the interspaces are passivated to prevent unspecific protein deposition. Some axonal features (branching, lateral deviation) showed density dependence whereas others (number of axons per neuron, various axon growth parameters) turned out to be an all-or-nothing reaction. Time-lapse analyses revealed that ALCAM density has an impact on axon velocity and advance efficiency. The behavior of the sensory axon tip, the growth cone, partially depended on ALCAM density in a dose-response fashion (shape, dynamics, detachment) while other features did not (size, complexity). Whereas axon growth was equally promoted whether ALCAM was presented at high (29 nm) or low densities (86 nm), the attachment of non-neuronal cells depended on high ALCAM densities. The attachment of non-neuronal cells to the rather unspecific standard proteins presented by conventional implants designed to enhance axonal regeneration is a severe problem. Our findings point to ALCAM, presented as 86 nm pattern, for a promising candidate for the improvement of such implants since this pattern drives axon growth to its full extent while at the same time non-neuronal cell attachment is clearly reduced.     

Axon Guidance of Sympathetic Neurons to Cardiomyocytes by Glial Cell Line-Derived Neurotrophic Factor (GDNF)

Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF) promotes cardiac sympathetic innervation in vitro and in vivo. In vitro, ventricular myocytes (VMs) and sympathetic neurons (SNs) isolated from neonatal rat ventricles and superior cervical ganglia were cultured at a close distance. Then, morphological and functional coupling between SNs and VMs was assessed in response to GDNF (10 ng/ml) or nerve growth factor (50 ng/ml). As a result, fractions of neurofilament-M-positive axons and synapsin-I-positive area over the surface of VMs were markedly increased with GDNF by 9-fold and 25-fold, respectively, compared to control without neurotrophic factors. Pre- and post-synaptic stimulation of β₁-adrenergic receptors (BAR) with nicotine and noradrenaline, respectively, resulted in an increase of the spontaneous beating rate of VMs co-cultured with SNs in the presence of GDNF. GDNF overexpressing VMs by adenovirus vector (AdGDNF-VMs) attracted more axons from SNs compared with mock-transfected VMs. In vivo, axon outgrowth toward the denervated myocardium in adult rat hearts after cryoinjury was also enhanced significantly by adenovirus-mediated GDNF overexpression. GDNF acts as a potent chemoattractant for sympathetic innervation of ventricular myocytes, and is a promising molecular target for regulation of cardiac function in diseased hearts.     

Cultivation of Methanogens under Low-Hydrogen Conditions by Using the Coculture Method

We previously reported the isolation of novel methanogens by using a new cultivation method, referred to as the coculture method. Here, we extended our coculture method to various anaerobic environmental samples. As a result, we successfully cultivated some uncharacterized methanogens in coculture enrichments and eventually isolated a new methanogen, within the order Methanomicrobiales.So far, almost all cases of the cultivation and isolation of H₂-utilizing methanogenic Archaea (methanogens) have been performed under high-H₂ concentrations (e.g., around 100 kPa), even though the concentrations in their natural habitat are far lower (10 to 100 Pa) than in laboratory cultures. This difference between in vitro and in situ physicochemical conditions very likely means that fast-growing methanogens that may prefer high concentrations of H₂ will have to be specifically selected; thus, laboratory cultures under such high-H₂ conditions result in the growth of a very limited range of species. To avoid this situation, we have proposed a new cultivation method, which we named the coculture method, for cultivating H₂-utilizing methanogens (14).Under anaerobic conditions, methanogens often partner with heterotrophic H₂-producing bacteria, which catalyze the oxidation of a variety of substrates (fatty acids, alcohols, and aromatic compounds). The methanogens use the H₂ produced by these heterotrophic bacteria, and in return, the bacteria benefit from the removal of the H₂ that would otherwise inhibit their growth. This lifestyle is commonly referred to as interspecies H₂ transfer, and the heterotrophic H₂-producing bacteria relying on H₂-utilizing methanogens are called syntrophs (11). In our previous studies, cultivation was performed with propionate as an indirect precursor substrate that is converted to H₂ by syntrophs, with the expectation that methanogens would grow as a result of interspecies H₂ transfer. Based on this strategy, two novel methanogens representing new genera, Methanocella paludicola strain SANAE and Methanolinea tarda strain NOBI-1, were successfully isolated (7, 13, 14).In this study, we extended the method to various types of environmental samples to cultivate and isolate uncharacterized methanogens. Moreover, we also extended the H₂-supplying substrates to include ethanol and butyrate in addition to propionate, because these substances are also known to be decomposed by a syntrophic association of substrate-oxidizing H₂-producing bacteria and H₂-utilizing methanogens (15).Nine anaerobic environmental samples (marine coastal sediment from Kashiwazaki, Niigata, Japan [KO]; freshwater lake sediment from Lake Suwa, Nagano, Japan [SL]; freshwater pond sediment from Shouzuma Pond, Nagano, Japan [SP]; river sediment of the Azusa River, Nagano, Japan [AR]; sediment from a lotus field located in Nagaoka, Niigata, Japan [LF]; rice field soil from Matsumoto, Nagano, Japan [NR]; rice field soil from Nagaoka, Niigata, Japan [SRP]; rice field soil from Tainan, Taiwan [TNR]; and methanogenic granular sludge obtained from a lab-scale upflow anaerobic sludge blanket reactor treating wastewater from the manufacture of palm oil in our laboratory [MP]) were anaerobically incubated with ethanol (10 mM), butyrate (20 mM), or propionate (20 mM) as the sole carbon and energy source. Additionally, we prepared propionate enrichment cultures with the addition of a pure culture of anaerobic syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans strain MPOB (DSMZ 10017) cells (inoculum size, 5% [vol/vol]) to obtain stable cultures (8, 18), except for enrichments from the marine sediment and granular sludge samples because the NaCl resistance of S. fumaroxidans was unknown (6) and the granular sludge was expected to contain a large amount of indigenous syntrophic bacteria (5, 16). Moreover, as control experiments, the same environmental samples were used in enrichments by the canonical cultivation method in the presence of high concentrations of H₂ (ca. 150 kPa in headspace) or formate (40 mM). All cultivations were performed anaerobically at 37°C without shaking. In total, 52 primary enrichment cultures were prepared for this study (see Table S1 in the supplemental material).When primary enrichment was made using high concentrations of H₂ and formate, the growth of methanogen-like microbes was confirmed within 3 to 5 days of incubation (as examples, photomicrographs of the enrichment cultures from TNR are shown in Fig. S1A to D in the supplemental material). After three consecutive transfers, 16S rRNA gene-based clone analysis was performed using an archaeal universal primer pair, Ar109f/1490R (14). Twenty-nine phylotypes were detected and were closely related to previously isolated methanogens (Fig. ​(Fig.11 and 2A and B; see Table S1 in the supplemental material). Among them, 26 phylotypes were classified into the genus Methanobacterium, two phylotypes in the genus Methanospirillum, and one in the genus Methanogenium. Moreover, 20 phylotypes showed high similarities (>97%) with the 16S rRNA genes of previously isolated methanogens, whereas the remaining nine phylotypes showed 95 to 96% similarities with the 16S rRNA genes of previously characterized methanogens (see Table S1 in the supplemental material). The methanogens possessing these sequences may be taxonomically novel at least at the species level (17), but they were all affiliated with the well-studied genera Methanobacterium and Methanospirillum.Open in a separate windowFIG. 1.Phylogenetic tree showing the placement of 16S rRNA gene sequences/clones obtained in this study. The colored phylotypes were obtained in this study. The difference in color among phylotypes indicates the different substrates used for the enrichment cultures (blue, hydrogen; green, formate; orange, ethanol; pink, butyrate; red, propionate and propionate plus S. fumaroxidans strain MPOB). The name of each phylotype is composed of the sample name, an abbreviation of the substrate for cultivation (H2, hydrogen; For, formate; Eth, ethanol; Buty, butyrate; Pro, propionate; ProM, propionate plus S. fumaroxidans), and the phylotype (for example, SRP-Pro-A is phylotype A recovered from the propionate enrichment culture cultivated from the environmental sample SRP). The number in parentheses indicates the number of identical clones obtained per number of clones analyzed for each phylotype. The accession numbers are also shown after each phylotype name. The phylotypes indicated by the same accession numbers have the same sequences (e.g., SP-For-A and SRP-Buty-C, AR-ProM-A and NR-ProM-A). All of the clonal sequences were greater than 1,000 nucleotides in length, with the exception of Methanospirillum sp. TM20-1 (GenBank acc. no. AB062404; 789 bp). Therefore, the initial tree was constructed with sequences greater than 1,000 nucleotides using the neighbor-joining method. Subsequently, the Methanospirillum sp. TM20-1 sequence was inserted into the tree by using the parsimony insertion tool of the ARB program. The scale bar indicates the estimated number of base changes per nucleotide sequence position. The symbols at the branch nodes indicate bootstrap values.Open in a separate windowFIG. 2.Phylogenetic affiliation of the identified phylotypes based on their cultivation substrates. The panels indicate the results of enrichment cultures with the following substrates: H₂ (A), formate (B), ethanol (C), butyrate (D), propionate (E), and propionate with the addition of the pure culture of S. fumaroxidans (F). The identified phylotypes were classified into their respective genera according to their 16S rRNA gene similarity with previously characterized methanogens. Phylotypes possessing sequence similarity greater than 92% were treated as the same genus. The number of phylotypes for each group is indicated in parentheses.In the coculture enrichments, substrate degradation concomitant with methane formation was confirmed after 1 week and more than 1 to 3 months of incubation in ethanol enrichment cultures and butyrate and propionate enrichment cultures, respectively. In particular, the growth of microbes in the propionate enrichments without the addition of S. fumaroxidans cells, except for the enrichments constructed from the RF and SRP samples, was very slow and unstable; the growth and methane production stopped unexpectedly and often made successive passages to fresh medium difficult. Additionally, two propionate enrichments in the absence of S. fumaroxidans inoculated from the KO and NR samples did not show methane production after a year of incubation. On the other hand, all of the propionate enrichments in the presence of S. fumaroxidans cells showed stable growth. During the incubation of the coculture enrichments, the H₂ partial pressures in the cultures were kept at <100 Pa in the ethanol enrichments and at <30 Pa in the butyrate and propionate cultures. Methane, H₂, short-chain fatty acids, and ethanol were measured as described previously (14). Microscopic observation after three to four transfers showed that those enrichments were comprised mainly of F₄₂₀-autofluorescent methanogen-like cells and oval- or rod-shaped bacterial cells, possibly syntrophs (see Fig. S1E to L in the supplemental material). These observations suggested that ethanol, butyrate, and propionate degradation were carried out by syntrophic association between syntrophic substrate-oxidizing H₂-producing bacteria and H₂-utilizing methanogens. To identify the methanogens present in those enrichments, archaeal 16S rRNA gene-based clone analyses were performed. A total of 52 phylotypes were obtained (Fig. ​(Fig.11 and 2C to F; see Table S1 in the supplemental material). Of these, 23 phylotypes were classified into the genera Methanobacterium (19 phylotypes) and Methanospirillum (4 phylotypes), which were very similar to those obtained from the H₂ and formate enrichments. On the other hand, the remaining 29 phylotypes were comprised of the orders Methanomicrobiales (20 phylotypes), Methanocellales (5 phylotypes), and Methanosarcinales (4 phylotypes, all belonging to the genus Methanosaeta). Within the order Methanomicrobiales, some phylotypes were affiliated with the genera Methanoculleus (nine phylotypes), Methanofollis (two phylotypes), Methanocalculus (one phylotype), and Methanoplanus (one phylotype). Additionally, sequences very closely related to the recently isolated methanogens Methanolinea tarda (six phylotypes) (7, 14) and “Candidatus Methanoregula boonei” (one phylotype) (1) were also obtained. Both M. tarda and “Ca. Methanoregula boonei” represent a family-level clade, which had long been recognized as an uncultured archaeal lineage called the group E1/E2 (1) (Fig. ​(Fig.1).1). Regarding the five phylotypes within the order Methanocellales, all were obtained from SRP and SP enrichments. Though the order Methanocellales had been recognized as the clone cluster rice cluster I, one strain has been isolated very recently (13, 14) and the rice cluster I methanogens are now being unveiled.Of the 52 phylotypes obtained from the coculture enrichments, 38 phylotypes (73% of the total phylotypes) were >97% similar to the 16S rRNA genes of the previously characterized (cultivated) methanogens. In contrast, 14 phylotypes (27%) had <96% sequence similarity with those of known methanogens. The organisms represented by these phylotypes were considered to be taxonomically novel at the species or even the genus level. Most of these phylotypes were affiliated with the orders Methanomicrobiales and Methanocellales with 92 to 96% sequence similarity (see Table S1 in the supplemental material). According to the 16S rRNA gene-based clone analysis, taxonomically novel methanogens were found in abundance in one ethanol, two butyrate, and eight propionate enrichments (from the KO, SP, SL, TNR, LF, and SRP samples). Especially, the ethanol and six propionate enrichments (from the SP, TNR, LF, and SRP samples) contained novel methanogens belonging to the group E1/E2 and/or the order Methanocellales (formerly known as rice cluster I), both of which contain only a few cultivated representatives so far. Therefore, we attempted to isolate these methanogens from the enrichments. After several attempts were performed over a year, a novel methanogen, designated strain TNR, was successfully isolated from the propionate enrichment culture (TNR) by serial dilution in liquid medium with H₂ (ca. 150 kPa) as the substrate.Strain TNR was a nonmotile, rod-shaped methanogen, which utilized H₂/CO₂ and formate for growth and methane production (see Fig. S2 in the supplemental material). The doubling time was 1.2 days at 37°C and pH 7. The most closely related methanogen cultivated so far was Methanolinea tarda that we have recently isolated (7), but the similarity of the 16S rRNA genes between the two was only 95% (Fig. ​(Fig.1).1). On the other hand, the isolation of methanogens from the other enrichments was not successful, i.e., when the coculture enrichments were inoculated into the serial dilution cultures with high concentrations of H₂ or formate, nontargeted methanogens, almost all of which had >97% sequence similarities to the 16S rRNA genes of known Methanobacterium and Methanoculleus species, outgrew in the cultures. The conventional method for final purification (i.e., using high concentrations of H₂ or formate as a direct substrate) has, therefore, a clear limitation, and new methods to overcome this will be needed.By using the coculture method, we successfully enriched methanogens that were absent in previous cultivation attempts and were only detected as environmental clones. In addition, we were able to isolate a methanogen belonging to the group E1/E2 of the order Methanomicrobiales. Our study clearly demonstrated that the coculture method is an effective way to cultivate hitherto uncharacterized methanogens. Interestingly, the taxonomic compositions of the phylotypes were clearly different depending on the substrates used in the coculture method (Fig. ​(Fig.11 and ​and2).2). When conventional cultivation was employed using high concentrations of H₂ and formate, only very limited phylotypes were obtained, namely, Methanobacterium- and Methanospirillum-related phylotypes in the H₂ cultures and Methanobacterium- and Methanogenium-related phylotypes in the formate cultures. When using the coculture method with ethanol or butyrate, Methanobacterium-related phylotypes were also dominant, accounting for 64.3% of the total phylotypes, whereas more diverse methanogen phylotypes than those in the H₂ and formate cultures were retrieved. Contrary to these results, propionate (with and without S. fumaroxidans) enrichments allowed quite a different pattern of methanogen phylotypes to become established. The most abundant phylotypes obtained from the propionate enrichments belonged to the orders Methanocellales and Methanomicrobiales, accounting for 72.8 and 84.7% of the clones examined. The addition of S. fumaroxidans cells into the propionate enrichments seemed to have no significant effect on the methanogenic community compositions that emerged, but it helped the stability of the whole community and the capability of the propionate degradation. The theoretical ranges of H₂ partial pressure that allow the anaerobic oxidation of ethanol, butyrate, and propionate to occur are 0.5 to 27,000 Pa; 0.5 to 60 Pa; and 0.5 to 28 Pa, respectively. These values were calculated based on the review on energy conservation by Thauer et al. (19), in which the concentrations of products and reactants were 0.35 atm , 0.65 atm , and 20 mM substances at 37°C and pH 7. For the calculation, a temperature correction was made using the van''t Hoff equation. Theoretically, the H₂ partial pressures in the various cultures differ depending on the substrates used, becoming lower in the order of substrates: ethanol > butyrate > propionate. Actually, the H₂ partial pressures measured during substrate degradation in the coculture enrichments remained within these theoretical ranges (data not shown). Given the above theoretical values, the apparent H₂ partial pressure that could be generated from a particular substrate would be the crucial factor affecting the change in the compositions of H₂-utilizing methanogens in the community. In fact, the relative abundance of members of the genera Methanobacterium and Methanospirillum increased as the given H₂ partial pressure became higher (propionate → butyrate → ethanol → H₂), and conversely, the relative abundance of members of the orders Methanomicrobiales (except for the genus Methanospirillum) and Methanocellales increased as the H₂ partial pressure became lower (Fig. ​(Fig.11 and ​and2).2). We assume that Methanocellales spp. and Methanomicrobiales spp. (except for Methanospirillum spp.) have higher affinities for H₂ than Methanobacterium spp. and Methanospirillum spp. Several previous studies also support this prediction. Lu et al. reported that Methanocellales methanogens incorporated ¹³C when rice roots were incubated in a low-H₂ atmosphere in the presence of ¹³CO₂, while Methanobacteriales and Methanosarcinales methanogens preferentially incorporated ¹³C in a high-H₂ atmosphere (10). Also, Methanocellales phylotypes were detected from methanogenic environments, usually with a low concentration of H₂, such as rice fields, fens, and peat bogs (e.g., see references 3, 4, and 9). In addition to the Methanocellales methanogens, members of the order Methanomicrobiales were frequently found in abundance in low-H₂-concentration methanogenic environments, such as peat bogs, fens, lake sediments, and rice fields (e.g., see references 2, 12, and 20). Detailed substrate affinity information will provide insight into the relevance between the population structures of methanogens and the H₂ concentrations of their habitats.     

Giant Glial Cell: New Insight Through Mechanism-Based Modeling

The paper describes a detailed mechanism-based model of a tripartite synapse consisting of P- and R-neurons together with a giant glial cell in the ganglia of the medical leech (Hirudo medicinalis), which is a useful object for experimental studies in situ. We describe the two main pathways of the glial cell activation: (1) via IP₃ production and Ca^2?+? release from the endoplasmic reticulum and (2) via increase of the extracellular potassium concentration, glia depolarization, and opening of voltage-dependent Ca^2?+? channels. We suggest that the second pathway is the more significant for establishing the positive feedback in glutamate release that is critical for the self-sustained activity of the postsynaptic neuron. This mechanism differs from the mechanisms of the astrocyte-neuron signaling previously reported.     

Novel Parachlamydia acanthamoebae Quantification Method Based on Coculture with Amoebae

Parachlamydia acanthamoebae, belonging to the order Chlamydiales, is an obligately intracellular bacterium that infects free-living amoebae and is a potential human pathogen. However, no method exists to accurately quantify viable bacterial numbers. We present a novel quantification method for P. acanthamoebae based on coculture with amoebae. P. acanthamoebae was cultured either with Acanthamoeba spp. or with mammalian epithelial HEp-2 or Vero cells. The infection rate of P. acanthamoebae (amoeba-infectious dose [AID]) was determined by DAPI (4′,6-diamidino-2-phenylindole) staining and was confirmed by fluorescent in situ hybridization. AIDs were plotted as logistic sigmoid dilution curves, and P. acanthamoebae numbers, defined as amoeba-infectious units (AIU), were calculated. During culture, amoeba numbers and viabilities did not change, and amoebae did not change from trophozoites to cysts. Eight amoeba strains showed similar levels of P. acanthamoebae growth, and bacterial numbers reached ca. 1,000-fold (10⁹ AIU preculture) after 4 days. In contrast, no increase was observed for P. acanthamoebae in either mammalian cell line. However, aberrant structures in epithelial cells, implying possible persistent infection, were seen by transmission electron microscopy. Thus, our method could monitor numbers of P. acanthamoebae bacteria in host cells and may be useful for understanding chlamydiae present in the natural environment as human pathogens.     

Acanthamoeba-Campylobacter Coculture as a Novel Method for Enrichment of Campylobacter Species

In this study, we present a novel method to isolate and enrich low concentrations of Campylobacter pathogens. This method, Acanthamoeba-Campylobacter coculture (ACC), is based on the intracellular survival and multiplication of Campylobacter species in the free-living protozoan Acanthamoeba polyphaga. Four of the Campylobacter species relevant to humans and livestock, Campylobacter jejuni, C. coli, C. lari, and C. hyointestinalis, were effectively enriched by the coculture method, with growth rates comparable to those observed in other Campylobacter enrichment media. Studying six strains of C. jejuni isolated from different sources, we found that all of the strains could be enriched from an inoculum of fewer than 10 bacteria. The sensitivity of the ACC method was not negatively affected by the use of Campylobacter-selective antibiotics in the culture medium, but these were effective in suppressing the growth of seven different bacterial species added at a concentration of 10⁴ CFU/ml of each species as deliberate contamination. The ACC method has advantages over other enrichment methods as it is not dependent on a microaerobic milieu and does not require the use of blood or other oxygen-quenching agents. Our study found the ACC method to be a promising tool for the enrichment of Campylobacter species, particularly from water samples with low bacterial concentrations.     

Cell Growth Suppression of Astrocytoma C6 Cells by Glial Fibrillary Acidic Protein cDNA Transfection

Abstract: The cellular functions of the intermediate filament family including glial fibrillary acidic protein (GFAP) are not well known yet beyond their roles as structural elements of cells. Expression of GFAP, which is specific in astrocytes and regulated developmentally, suggests its involvement in cell growth and differentiation of astrocytes. We transfected murine GFAP cDNA into a rat astrocytoma C6 cell line to assess the specific effect of GFAP on cells. Two stable GFAP-transfected cell lines, GFC6-5 and GFC6-6, exhibited a series of morphological and growth characteristics that distinguish them from their counterparts, i.e., NeoC6 cells transfected only with the neomycin-resistant gene, and native C6 cells. Both GFC6-5 and GFC6-6 cells showed elongated cell shapes with extended processes rich in GFAP, markedly suppressed cell growth, and decreased bromodeoxyuridine uptake. Western blot analysis revealed a remarkable increase of GFAP expression in GFC6-5 and GFC6-6 compared with that in NeoC6 and C6, in contrast to similar vimentin expression in all cell lines. The results indicate that the expression of GFAP has dramatic effects on cell morphology and cell growth suppression in C6 cells, suggesting that GFAP may function as a tumor suppressor in astrocytoma.     

Microcontact Peeling as a New Method for Cell Micropatterning

Micropatterning is becoming a powerful tool for studying morphogenetic and differentiation processes of cells. Here we describe a new micropatterning technique, which we refer to as microcontact peeling. Polydimethylsiloxane (PDMS) substrates were treated with oxygen plasma, and the resulting hydrophilic layer of the surface was locally peeled off through direct contact with a peeling stamp made of aluminum, copper, or silicon. A hydrophobic layer of PDMS could be selectively exposed only at the places of the physical contact as revealed by water contact angle measurements and angle-resolved X-ray photoelectron spectroscopy, which thus enabled successful micropatterning of cells with micro-featured peeling stamps. This new micropatterning technique needs no procedure for directly adsorbing proteins to bare PDMS in contrast to conventional techniques using a microcontact printing stamp. Given the several unique characteristics, the present technique based on the peel-off of inorganic materials may become a useful option for performing cell micropatterning.     

Growth Response to Hormones by a New Rat Ovary Cell Line

SEVERAL endocrine cell lines established in recent years show a functional response to hormones in vitro¹ but, except for one mammary cell line², none of them exhibits the normal hormone requirement for growth in vivo. We have now isolated a rat ovarian cell line whose growth in vitro is markedly stimulated by bovine luteinizing hormone (LH-NIH-B7), a pituitary gonadotrophin and by dexamethasone, a synthetic glucocorticoid. This cell line provides the first permanent in vitro system for studying the growth stimulation of gonadal cells by hormones.     

Glial Cell Markers in the Reeler Mutant Mouse: A Biochemical and Immunohistological Study

The glial cell contents of S100 protein, 2',3'-cyclic AMP, 3'-phosphohydrolase (CNP), isoenzyme II of carbonic anhydrase (CAII) and butyrylcholinesterase (BuChE) were biochemically determined in the cerebellum and cerebrum of the reeler mutant mouse. Astrocytes and oligodendrocytes, shown by this study, contain abnormal amounts of these components. The CAII concentration was significantly increased in the particulate fraction of the reeler cerebellum and cerebrum (by 50% and 89%, respectively). The BuChE specific activity was greatly increased in the reeler, by 120% for cerebellum and by 40% in cerebrum. In contrast, the S100 protein concentration was reduced in the reeler cerebellum by 40% and by 25% in cerebrum, while the CNP specific activity increased by 30% in the reeler cerebellum. In addition, the glial cell distribution was studied by immunohistological techniques with antibodies directed against S100 protein, glial fibrillary acidic protein (GFA) and CAII. Apparently the density of glial cells is not significantly affected. However, the Golgi epithelial cells were usually abnormally placed and their Bergmann fibres were less well developed.     

Short- and Long-Term Effects of LRRK2 on Axon and Dendrite Growth

Mutations in leucine-rich repeat kinase 2 (LRRK2) underlie an autosomal-dominant form of Parkinson''s disease (PD) that is clinically indistinguishable from idiopathic PD. The function of LRRK2 is not well understood, but it has become widely accepted that LRRK2 levels or its kinase activity, which is increased by the most commonly observed mutation (G2019S), regulate neurite growth. However, growth has not been measured; it is not known whether mean differences in length correspond to altered rates of growth or retraction, whether axons or dendrites are impacted differentially or whether effects observed are transient or sustained. To address these questions, we compared several developmental milestones in neurons cultured from mice expressing bacterial artificial chromosome transgenes encoding mouse wildtype-LRRK2 or mutant LRRK2-G2019S, Lrrk2 knockout mice and non-transgenic mice. Over the course of three weeks of development on laminin, the data show a sustained, negative effect of LRRK2-G2019S on dendritic growth and arborization, but counter to expectation, dendrites from Lrrk2 knockout mice do not elaborate more rapidly. In contrast, young neurons cultured on a slower growth substrate, poly-L-lysine, show significantly reduced axonal and dendritic motility in Lrrk2 transgenic neurons and significantly increased motility in Lrrk2 knockout neurons with no significant changes in length. Our findings support that LRRK2 can regulate patterns of axonal and dendritic growth, but they also show that effects vary depending on growth substrate and stage of development. Such predictable changes in motility can be exploited in LRRK2 bioassays and guide exploration of LRRK2 function in vivo.     

Study of Glial Fibrillary Acidic Protein in a Human Glioma Cell Line Grown in Culture and as a Solid Tumor

Abstract: A continuous human glioma cell line grown in culture and as a solid tumor was analyzed for glial fibrillary acidic (GFA) protein. This material provided a rich source for GFA protein that could also be manipulated and controlled. Immunoperoxidase staining at the light and electron microscopic levels revealed that the cell culture and tumor specimens were strongly positive for GFA protein. When aqueous soluble fractions of the cell culture and tumor were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose and stained immunochemically, they contained exclusively low molecular weight (41–43 K-dalton) GFA peptides. SDS (0.15%)-soluble fractions contained either low molecular weight only (culture) or a mixture of peptides ranging from 41 to 49K daltons. SDS (1%) extracts of either cell culture or tumor contained only 49K dalton GFA protein. Two-dimensional gel separation revealed that the GFA protein extracted from either the culture or tumor with 1% SDS resolved to two or three spots at pH 5.8. Low molecular weight GFA peptides (<49K daltons) in aqueous and 0.15% SDS-soluble extracts became increasingly more acidic with decreasing molecular weight. The extremely rapid degradation seen suggests that this cell line may be a valuable system for further study of intermediate filament protein turnover.     

An Accurate Prostate Cancer Prognosticator Using a Seven-Gene Signature Plus Gleason Score and Taking Cell Type Heterogeneity into Account

One of the major challenges in the development of prostate cancer prognostic biomarkers is the cellular heterogeneity in tissue samples. We developed an objective Cluster-Correlation (CC) analysis to identify gene expression changes in various cell types that are associated with progression. In the Cluster step, samples were clustered (unsupervised) based on the expression values of each gene through a mixture model combined with a multiple linear regression model in which cell-type percent data were used for decomposition. In the Correlation step, a Chi-square test was used to select potential prognostic genes. With CC analysis, we identified 324 significantly expressed genes (68 tumor and 256 stroma cell expressed genes) which were strongly associated with the observed biochemical relapse status. Significance Analysis of Microarray (SAM) was then utilized to develop a seven-gene classifier. The Classifier has been validated using two independent Data Sets. The overall prediction accuracy and sensitivity is 71% and 76%, respectively. The inclusion of the Gleason sum to the seven-gene classifier raised the prediction accuracy and sensitivity to 83% and 76% respectively based on independent testing. These results indicated that our prognostic model that includes cell type adjustments and using Gleason score and the seven-gene signature has some utility for predicting outcomes for prostate cancer for individual patients at the time of prognosis. The strategy could have applications for improving marker performance in other cancers and other diseases.     

Psychiatric Illness in General Practice: A Detailed Study Using a New Method of Case Identification

A self-administered questionary (the General Health Questionnaire) aimed at detecting current psychiatric disturbance was given to 553 consecutive attenders to a general practitioner''s surgery. A sample of 200 of these patients was given an independent assessment of their mental state by a psychiatrist using a standardized psychiatric interview. Over 90% of the patients were correctly classified as “well” or “ill” by the questionary, and the correlation between questionary score and the clinical assessment of severity of disturbance was found to be +0·80.The “conspicuous psychiatric morbidity” of a suburban general practice assessed by a general practitioner who was himself a psychiatrist and validated against independent psychiatric assessment was found to be 20%. “Hidden psychiatric morbidity” was found to account for one-third of all disturbed patients. These patients were similar to patients with “conspicuous illnesses” in terms both of degree of disturbance and the course of their illnesses at six-month follow-up, but were distinguished by their attitude to their illness and by usually presenting a physical symptom to the general practitioner.When 87 patients who had been assessed as psychiatric cases at the index consultation were called back for follow-up six months later, two-thirds of them were functioning in the normal range. Frequency of attendance at the surgery in the six months following index consultation was found to have only a modest relationship to severity of psychiatric disturbance.It is argued that minor affective illnesses and physical complaints often accompany each other and usually have a good prognosis.     

Influence of the Rate of Cell Growth and Cell Density on Interferon Action in Chick Embryo Cells

Chick embryo cells became more sensitive to the action of interferon the longer they remained in culture. This phenomenon was found even before confluency had been reached. The relative insensitivity of newly seeded cells was not due to a loss of receptors. Cells synthesizing deoxyribonucleic acid (DNA) at a high rate were less sensitive to interferon action than cells synthesizing DNA at a low rate, but the inhibition of DNA synthesis had no effect on interferon action. An increase in the number of cells used for seeding resulted in an earlier appearance of increased sensitivity to interferon action. These results are discussed in relation to the induction process in animal cells.     

细菌诱导对昆虫细胞系NIH-SaPe-4生长与抗菌蛋白活性的影响

离体昆虫细胞系在昆虫免疫、抗菌肽及蛋白研究和药物开发方面具有较好的应用前景。该文对双翅目麻蝇科麻蝇成虫卵巢胚细胞系NIH-SaPe-4在藤黄微球菌诱导和非诱导条件下,细胞密度和活力的变化、诱导对细胞生长的影响、抗菌活性及其活性随时间的变化关系等进行了研究,并对所得抗菌蛋白进行了初步分离纯化和稳定性评估。结果表明,诱导使得细胞密度增长减缓,活力变弱。诱导和非诱导组细胞均可产生对3种革兰氏阳性菌具有抑菌活性的抗菌蛋白,其中对藤黄微球菌的抑菌活性最明显;诱导组细胞抗菌蛋白活性出现时间、稳定期抑菌活性均大于非诱导组,诱导菌消失一段时间后抗菌活性恢复到同等水平。抗菌蛋白具有酸碱稳定性和热稳定性。2组抗菌蛋白粗提液经凝胶、反相分离纯化后均得到一种60 kDa左右的抗菌蛋白,诱导组电泳后条带亮度大于非诱导组。该研究为昆虫细胞抗菌蛋白性质、分离纯化等研究奠定了科学基础。     

MicroRNA-145 Is Downregulated in Glial Tumors and Regulates Glioma Cell Migration by Targeting Connective Tissue Growth Factor

Glioblastomas (GBM), the most common and aggressive type of malignant glioma, are characterized by increased invasion into the surrounding brain tissues. Despite intensive therapeutic strategies, the median survival of GBM patients has remained dismal over the last decades. In this study we examined the expression of miR-145 in glial tumors and its function in glioma cells. Using TCGA analysis and real-time PCR we found that the expression of miR-145/143 cluster was downregulated in astrocytic tumors compared to normal brain specimens and in glioma cells and glioma stem cells (GSCs) compared to normal astrocytes and neural stem cells. Moreover, the low expression of both miR-145 and miR-143 in GBM was correlated with poor patient prognosis. Transfection of glioma cells with miR-145 mimic or transduction with a lentivirus vector expressing pre-miR 145 significantly decreased the migration and invasion of glioma cells. We identified connective tissue growth factor (CTGF) as a novel target of miR-145 in glioma cells; transfection of the cells with this miRNA decreased the expression of CTGF as determined by Western blot analysis and the expression of its 3′-UTR fused to luciferase. Overexpression of a CTGF plasmid lacking the 3′-UTR and administration of recombinant CTGF protein abrogated the inhibitory effect of miR-145 on glioma cell migration. Similarly, we found that silencing of CTGF decreased the migration of glioma cells. CTGF silencing also decreased the expression of SPARC, phospho-FAK and FAK and overexpression of SPARC abrogated the inhibitory effect of CTGF silencing on cell migration. These results demonstrate that miR-145 is downregulated in glial tumors and its low expression in GBM predicts poor patient prognosis. In addition miR-145 regulates glioma cell migration by targeting CTGF which downregulates SPARC expression. Therefore, miR-145 is an attractive therapeutic target for anti-invasive treatment of astrocytic tumors.     

Glial Cell Line-Derived Neurotrophic Growth Factor Inhibits Apoptotic Death of Postnatal Substantia Nigra Dopamine Neurons in Primary Culture

Abstract: Glial cell line-derived neurotrophic factor (GDNF) was identified on the basis of its ability to enhance the development of embryonic mesencephalic dopamine neurons. It remains unknown whether GDNF is a physiologically relevant trophic factor for these neurons. We have shown that natural cell death among dopamine neurons of the substantia nigra occurs largely postnatally. To investigate whether GDNF may have the ability to support these neurons during their period of natural cell death, we have used a postnatal primary culture model. We find that GDNF is able to support the viability of postnatal nigral dopamine neurons by inhibiting apoptotic death. This ability of GDNF shows both regional specificity for the nigra and cellular specificity for the dopamine phenotype. Among eight other neurotrophic factors previously reported to support embryonic dopamine neurons, GDNF was unique in this ability. Thus, GDNF meets this criterion for a physiologically relevant trophic factor for dopamine neurons of the substantia nigra.     

Radial Glial Cells: New Views on Old Questions

Neurochemical Research - Radial glial cells (RGC) are at the center of brain development in vertebrates, acting as progenitors for neurons and macroglia (oligodendrocytes and astrocytes) and as...     

Neuronal and Glial Properties Coexist in a Novel Mouse CNS Immortalized Cell Line

A mes-c-myc A1 (A1) cell line was generated by retroviral infection of cultured embryonic mesencephalic cells and selected by neomycin resistance. A1 cells cease to divide and undergo morphological differentiation after serum withdrawal or addition of c-AMP. Proliferating or morphologically differentiated A1 cells are all positive for vimentin and nestin, a marker of neural precursor, and show neuronal markers such as microtubule-associated protein 1, neuron-specific enolase and peripherin, and the glial marker glial fibrillary acidic protein. Neuronal and glial markers coexist in single cells. Furthermore, A1 cells show presence of glutamic acid decarboxylase 67 mRNA and its embryonic form EP10 and accumulate the neurotransmitter GABA. Electrophysiological studies demonstrate that morphologically differentiated A1 cells display voltage-gated sodium and potassium channels in response to depolarizing stimuli. A1 cells thus represent a novel, bipotent neural cell line useful for studying CNS differentiation and plasticity, as well as the molecular mechanisms underlying development of GABAergic neurotransmission.