Specific Human Astrocyte Subtype Revealed by Affinity Purified GFAP+1 Antibody; Unpurified Serum Cross-Reacts with Neurofilament-L in Alzheimer

The human GFAP splice variants GFAPΔ164 and GFAPΔexon6 both result in a GFAP protein isoform with a unique out-of-frame carboxy-terminus that can be detected by the GFAP⁺¹ antibody. We previously reported that GFAP⁺¹ was expressed in astrocytes and in degenerating neurons in Alzheimer''s disease brains. In this study we aimed at further investigating the neuronal GFAP⁺¹ expression and we started by affinity purifying the GFAP⁺¹ antibody. The purified antibody resulted in a loss of neuronal GFAP⁺¹ signal, although other antibodies directed against the amino- and carboxy-terminus of GFAPα still revealed GFAP-immunopositive neurons, as described before. With an in-depth analysis of a western blot, followed by mass spectrometry we discovered that the previously detected neuronal GFAP⁺¹ expression was due to cross-reactivity of the antibody with neurofilament-L (NF-L). This was confirmed by double-label fluorescent immunohistochemistry and western blotting with the unpurified GFAP⁺¹ antibody and an antibody against NF-L. Our data imply that NF-L can accumulate in some tangle-like structures in Alzheimer brains. More importantly, the purified GFAP⁺¹ antibody clearly revealed a specific subtype of astrocytes in the adult human brain. These large astrocytes are present throughout the brain, e.g., along the subventricular zone, in the hippocampus, in the striatum and in the spinal cord of controls, Alzheimer, and Parkinson patients. The presence of a specific GFAP-isoform suggests a specialized function of these astrocytes.     

The predation costs of symmetrical cryptic coloration

In psychological studies of visual perception, symmetry is accepted as a potent cue in visual search for cryptic objects, yet its importance for non-human animals has been assumed rather than tested. Furthermore, while the salience of bilateral symmetry has been established in laboratory-based search tasks using human subjects, its role in more natural settings, closer to those for which such perceptual mechanisms evolved, has not, to our knowledge, been investigated previously. That said, the salience of symmetry in visual search has a plausible adaptive rationale, because biologically important objects, such as prey, predators or conspecifics, usually have a plane of symmetry that is not present in their surroundings. We tested the conspicuousness to avian predators of cryptic artificial, moth-like targets, with or without bilateral symmetry in background-matching coloration, against oak trees in the field. In two independent experiments, symmetrical targets were predated at a higher rate than otherwise identical asymmetrical targets. There was a small, but significant, fitness cost to symmetry in camouflage patterns. Given that birds are the most commonly invoked predators shaping the evolution of defensive coloration in insects, this raises the question of why bilateral asymmetry is not more common in cryptic insects.     

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo . The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo . The device can be sterilized for cell culture using common methods without loss of function. The device''s applicability to studying the in vitro wound healing response will be demonstrated.Open in a separate windowClick here to view.^{(69M, flv)}     

Sox2 and Fgf interact with Atoh1 to promote sensory competence throughout the zebrafish inner ear

Atoh1 is required for differentiation of sensory hair cells in the vertebrate inner ear. Moreover, misexpression of Atoh1 is sufficient to establish ectopic sensory epithelia, making Atoh1 a good candidate for gene therapy to restore hearing. However, competence to form sensory epithelia appears to be limited to discrete regions of the inner ear. To better understand the developmental factors influencing sensory-competence, we examined the effects of misexpressing atoh1a in zebrafish embryos under various developmental conditions. Activation of a heat shock-inducible transgene, hs:atoh1a, resulted in ectopic expression of early markers of sensory development within 2 h, and mature hair cells marked by brn3c:GFP began to accumulate 9 h after heat shock. The ability of atoh1a to induce ectopic sensory epithelia was maximal when activated during placodal or early otic vesicle stages but declined rapidly thereafter. At no stage was atoh1a sufficient to induce sensory development in dorsal or lateral non-sensory regions of the otic vesicle. However, co-misexpression of atoh1a with fgf3, fgf8 or sox2, genes normally acting in the same gene network with atoh1a, stimulated sensory development in all regions of the otic vesicle. Thus, expression of fgf3, fgf8 or sox2 strongly enhances competence to respond to Atoh1.     

CO2 acquisition in Chlamydomonas acidophila is influenced mainly by CO2, not phosphorus,availability

The extremophilic green microalga Chlamydomonas acidophila grows in very acidic waters (pH 2.3–3.4), where CO₂ is the sole inorganic carbon source. Previous work has revealed that the species can accumulate inorganic carbon (Ci) and exhibits high affinity CO₂ utilization under low-CO₂ (air-equilibrium) conditions, similar to organisms with an active CO₂ concentrating mechanism (CCM), whereas both processes are down-regulated under high CO₂ (4.5 % CO₂) conditions. Responses of this species to phosphorus (Pi)-limited conditions suggested a contrasting regulation of the CCM characteristics. Therefore, we measured external carbonic anhydrase (CA_ext) activities and protein expression (CAH1), the internal pH, Ci accumulation, and CO₂-utilization in cells adapted to high or low CO₂ under Pi-replete and Pi-limited conditions. Results reveal that C. acidophila expressed CA_ext activity and expressed a protein cross-reacting with CAH1 (the CA_ext from Chlamydomonas reinhardtii). Although the function of this CA remains unclear, CA_ext activity and high affinity CO₂ utilization were the highest under low CO₂ conditions. C. acidophila accumulated Ci and expressed the CAH1 protein under all conditions tested, and C. reinhardtii also contained substantial amounts of CAH1 protein under Pi-limitation. In conclusion, Ci utilization is optimized in C. acidophila under ecologically relevant conditions, which may enable optimal survival in its extreme Ci- and Pi-limited habitat. The exact physiological and biochemical acclimation remains to be further studied.     

Tuber morphology and starch accumulation are independent phenomena: Evidence from ipt-transgenic potato lines

Tuber formation and carbohydrate metabolism in potatoes were studied using transgenic potato plants carrying the Agrobacterium tumefaciens ipt gene, involved in cytokinin biosynthesis. Three independent transformants, viz. clones 1, 11 and 13, whose cytokinin and auxin content had previously been shown to be different from each other and from the wild-type, were analysed in vitro. Clones 11 and 13 showed a higher ability to form stolons and tubers, as evident from: (1) stolon development in whole plants grown under non-inductive conditions, (2) total number and weight of tubers formed by cuttings of this clone in darkness, (3) tubers appeared earlier than tubers of wild-type plants and at a lower sucrose concentration in the medium. Clone 1 did not form stolons or tubers under any conditions tested, but rather formed short shoots. A series of metabolic changes, known to be characteristic for tubers, were analysed in leaves, stems and developing buds. It was found that the short type of shoots, formed by clone 1, had metabolic characteristics very similar to tubers formed in wild-type or clones 11 and 13, including glucose, fructose, sucrose, and starch levels, and activities of invertase, sucrose synthase and fructokinase. It is concluded that the regulation of the stolon swelling and of carbohydrate metabolism, normally occurring simultaneously, can be uncoupled, and are thus, at least partly independent phenomena. The present data obtained with a high-cytokinin line indicate that cytokinins (probably in concert with auxins) might be mainly involved in the regulation of tuber morphology.     

Newt Myotubes Reenter the Cell Cycle by Phosphorylation of the Retinoblastoma Protein

Withdrawal from the cell cycle is an essential aspect of vertebrate muscle differentiation and requires the retinoblastoma (Rb) protein that inhibits expression of genes needed for cell cycle entry. It was shown recently that cultured myotubes derived from the Rb^−/−mouse reenter the cell cycle after serum stimulation (Schneider, J.W., W. Gu, L. Zhu, V. Mahdavi, and B. Nadal-Ginard. 1994. Science (Wash. DC). 264:1467– 1471). In contrast with other vertebrates, adult urodele amphibians such as the newt can regenerate their limbs, a process involving cell cycle reentry and local reversal of differentiation. Here we show that myotubes formed in culture from newt limb cells are refractory to several growth factors, but they undergo S phase after serum stimulation and accumulate 4N nuclei. This response to serum is inhibited by contact with mononucleate cells. Despite the phenotypic parallel with Rb^−/− mouse myotubes, Rb is expressed in the newt myotubes, and its phosphorylation via cyclin-dependent kinase 4/6 is required for cell cycle reentry. Thus, the postmitotic arrest of urodele myotubes, although intact in certain respects, can be undermined by a pathway that is inactive in other vertebrates. This may be important for the regenerative ability of these animals.     

Enrichment and characterization of marine anammox bacteria associated with global nitrogen gas production

Microbiological investigation of anaerobic ammonium oxidizing (anammox) bacteria has until now been restricted to wastewater species. The present study describes the enrichment and characterization of two marine Scalindua species, the anammox genus that dominates almost all natural habitats investigated so far. The species were enriched from a marine sediment in the Gullmar Fjord (Sweden) using a medium based on Red Sea salt. Anammox cells comprised about 90% of the enrichment culture after 10 months. The enriched Scalindua bacteria displayed all typical features known for anammox bacteria, including turnover of hydrazine, the presence of ladderane lipids, and a compartmentalized cellular ultrastructure. The Scalindua species also showed a nitrate-dependent use of formate, acetate and propionate, and performed a formate-dependent reduction of nitrate, Fe(III) and Mn(IV). This versatile metabolism may be the basis for the global distribution and substantial contribution of the marine Scalindua anammox bacteria to the nitrogen loss from oxygen-limited marine ecosystems.     

Studies on decomposition ofCeratophyllum demersum litter under laboratory and field conditions: losses of dry mass and nutrients,qualitative changes in organic compounds and consequences for ambient water and sediments

A study was made of decomposition ofCeratophyllum demersum litter over a 17-day period under controlled conditions of temperature and oxygen (5, 10 and 18 °C; aerobic and anaerobic) and over a 169-day period in the field (Lake Vechten, The Netherlands). Litter, water and sediment were sampled on the 0, 2, 4, 7 and 17th day under controlled conditions and on the 0, 17, 49, 127 and 169th day in the field. The litter was analyzed quantitatively for dry mass, ash, carbon, nitrogen, phosphorus and qualitatively of organic composition by pyrolysis mass spectrometry. The water was analyzed for the elemental concentrations of organic carbon (total and dissolved), nitrogen (total, ammonia and particulate) and phosphorus (total and orthophosphate) and for the concentrations of photosynthetic pigments and bacteria. The sediment was analyzed for the elemental concentrations of nitrogen, carbon and phosphorus, and for bacterial numbers.The pattern of litter mass loss fitted an exponential model fairly well. Mass decreased faster under controlled aerobic than under anaerobic conditions and the decrease was stimulated by increasing temperature, relatively more in the range of 5 to 10 °C (by 20%) than in the range of 10 of 18 °C (by 2%). The residual mass ranged from 73 to 43% of initial under controlled aerobic conditions and from 84 to 65% under anaerobic conditions after 17 days. It decreased far less in the field, to 38% of initial mass in the field after 169 days.The litter initially lost a carbohydrate fraction by leaching in all treatments. The protein content decreased initially as well but increased subsequently at increasing temperature stimulated under anaerobic conditions. The changes in organic composition were correlated with those in nitrogen but not with those in carbon and phosphorus contents. The organic composition of litter incubated in the field differed from that of litter incubated in the laboratory. The field residues contained less proteinaceous material than the laboratory residues.The changes in carbon, nitrogen and phosphorus concentrations in the litter showed different patterns. The carbon concentration generally increased, the nitrogen concentration initially dropped and increased subsequently, and the phosphorus concentration initially dropped and remained relatively constant subsequently. Chemical immobilization of the decomposition process may have occurred in the laboratory, but was unlikely in the field.Carbon, nitrogen and phosphorus left the litter initially largely in particulate form and were recovered in the water. The ratio dissolved: total nutrient concentration was lower under controlled aerobic than under anaerobic conditions. Increasing temperature stimulated bacterial use of dissolved organic carbon and nitrogen. A rapid nutrient flow occurred from macrophyte litter, via water to sediment.The phytoplankton biomass in the water was greatly stimulated by substances freed from the decomposing litter. Diatoms increased generally relatively more than green algae, predominating alternatively with green algae under aerobic conditions and continuously under anaerobic conditions. Bacterial numbers in the water initially increased, partly due to transgression of bacteria from the sediment-water interface to the water and partly due to an actual increase in community biomass. The bacteria returned largely to the sediment-water interface, stimulated by increasing temperature, as most of the substrate readily usable by them had left the litter in the litter-bag and was associated with the upper sediment layers.It is feasible that the annual die-off of theC. demersum population of Lake Vechten barely affects nutrient cycling in the lake, because the contribution to the nutrient pools of the lake when fully mixed is only small. However, small particles originating from decomposingC. demersum litter may influence the lake considerably by decreasing water transparency and serving as a food source for filter-feeders and detritivorous macrofauna.