The genesis and differentiation of neurons in a frog parasympathetic ganglion

The cellular mechanisms that underlie formation of an autonomic ganglion have been investigated by studying the formation of the cardiac ganglion of the frog. Analysis of the genesis of neurons with [³H]thymidine autoradiography revealed that neuronal precursors do not divide via a “stem cell lineage” but rather divide exponentially, such that both daughter cells either re-enter the mitotic cycle or differentiate. Neurogenesis in this autonomic ganglion is prolonged, beginning during the second day after fertilization and continuing for at least 2 weeks. The use of acetylcholinesterase (AChE) as a neuronal marker showed that differentiated neurons start condensing in their target 1.5 days after the first neurons are born. Neurons accumulate, concomitant with neurogenesis, at a constant rate of approximately six neurons per day. Transplantation and organ culture demonstrated that immature neurons are present well before definitive expression of the mature phenotype and that their initial expression does not depend upon maintained contact by preganglionic axons.     

Towards an integrated system for bio-energy: hydrogen production by <Emphasis Type="Italic">Escherichia coli</Emphasis> and use of palladium-coated waste cells for electricity generation in a fuel cell

Escherichia coli strains MC4100 (parent) and a mutant strain derived from this (IC007) were evaluated for their ability to produce H₂ and organic acids (OAs) via fermentation. Following growth, each strain was coated with Pd(0) via bioreduction of Pd(II). Dried, sintered Pd-biomaterials (‘Bio-Pd’) were tested as anodes in a proton exchange membrane (PEM) fuel cell for their ability to generate electricity from H₂. Both strains produced hydrogen and OAs but ‘palladised’ cells of strain IC007 (Bio-Pd_IC007) produced ~threefold more power as compared to Bio-Pd_MC4100 (56 and 18 mW respectively). The power output used, for comparison, commercial Pd(0) powder and Bio-Pd made from Desulfovibrio desulfuricans, was ~100 mW. The implications of these findings for an integrated energy generating process are discussed.     

Fiftyfold amplification of the Lowry protein assay

The blue product of the Lowry et al. (1951, J. Biol. Chem. 193, 265-275) reaction interacts with malachite green (MG), inducing a change in the visible light spectrum. At A690 nm the absorbance of malachite green solutions increases 10-fold in the presence of Lowry blue (LB). Under the optimum conditions, 0.01 A700 nm unit of Lowry blue produces a change in A690 nm unit of malachite green of 0.5 and the delta A690 nm is a linear function of Lowry blue concentration. Conditions under which this 50-fold amplification can be exploited to detect less than 100 ng of protein (or 4 micrograms X ml-1) are described. A number of chemicals including sodium dodecyl sulfate can interfere with the assay but a strategy has been devised to overcome these problems. Amplification of the Lowry assay appears to involve a cooperative interaction between malachite green and the Lowry blue product such that about 23 molecules of malachite green undergo a spectral shift per molecule of a model reactant such as tyrosine. Malachite green can be used to amplify the molybdenum blue signal obtained in other assays. Less than 10 pmol of tyrosine can be detected using this procedure. Lowry blue also interacts with auramine O, giving a large increase in A500 nm and a 40-fold amplification of the LB signal. As with malachite green, there is a cooperative interaction between auramine O and LB. About 72 molecules of auramine O undergo a spectral shift per molecule of tyrosine. The product of this reaction is also fluorescent and could be exploited in a protein assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Signal peptide etiquette during assembly of a complex respiratory enzyme

Salmonella enterica serovar Typhimurium is a Gram‐negative pathogen capable of respiration with a number of terminal electron acceptors. Tetrathionate reductase is important for the infection process and is encoded by the ttrBCA operon where TtrA and TtrB are metallocofactor‐containing proteins targeted to the periplasmic side of the membrane by two different Tat targeting peptides. In this work, the inter‐relationship between these two signal peptides has been explored. Molecular genetics and biochemical approaches reveal that the processing of the TtrB Tat signal peptide is dependent on the successful assembly of its partner protein, TtrA. Inactivation of either the TtrA or the TtrB Tat targeting peptides individually was observed to have limited overall effects on assembly of the enzyme or on cellular tetrathionate reductase activity. However, inactivation of both signal peptides simultaneously was found to completely abolish physiological tetrathionate reductase activity. These data suggest both signals are normally active during assembly of the enzyme, and imply a code of conduct exists between the signal peptides where one can compensate for inactivity in the other. Since it appears likely that tetrathionate reductase presents itself for export as a multi‐signal complex, these observations also have implications for the mechanism of the bacterial Tat translocase.     

An Unbiased Approach to Identifying Tau Kinases That Phosphorylate Tau at Sites Associated with Alzheimer Disease

Neurofibrillary tangles, one of the hallmarks of Alzheimer disease (AD), are composed of paired helical filaments of abnormally hyperphosphorylated tau. The accumulation of these proteinaceous aggregates in AD correlates with synaptic loss and severity of dementia. Identifying the kinases involved in the pathological phosphorylation of tau may identify novel targets for AD. We used an unbiased approach to study the effect of 352 human kinases on their ability to phosphorylate tau at epitopes associated with AD. The kinases were overexpressed together with the longest form of human tau in human neuroblastoma cells. Levels of total and phosphorylated tau (epitopes Ser(P)-202, Thr(P)-231, Ser(P)-235, and Ser(P)-396/404) were measured in cell lysates using AlphaScreen assays. GSK3α, GSK3β, and MAPK13 were found to be the most active tau kinases, phosphorylating tau at all four epitopes. We further dissected the effects of GSK3α and GSK3β using pharmacological and genetic tools in hTau primary cortical neurons. Pathway analysis of the kinases identified in the screen suggested mechanisms for regulation of total tau levels and tau phosphorylation; for example, kinases that affect total tau levels do so by inhibition or activation of translation. A network fishing approach with the kinase hits identified other key molecules putatively involved in tau phosphorylation pathways, including the G-protein signaling through the Ras family of GTPases (MAPK family) pathway. The findings identify novel tau kinases and novel pathways that may be relevant for AD and other tauopathies.