Choline acetyltransferase activity of spinal cord cell cultures increased by co-culture with muscle and by muscle-conditioned medium

Activity of the enzyme choline acetyltransferase (CAT), which mediates the synthesis of the neurotransmitter, acetylcholine, was increased up to 20- fold in spinal cord (SC) cells grown in culture with muscle cells for 2 wk. This increase was directly related to the duration of co-culture as well as to the cell density of both the SC and muscle involved and was not affected by the presence of the acetylcholine receptor blocking agent, α-bungarotoxin. Glutamic acid decarboxylase (GAD) activity was often markedly decreased in SC-muscle cultures while the activities of acetylcholinesterase and several other enzymes were little changed. Increased CAT activity was also observed when SC cultures were maintained in medium which had been conditioned by muscle cells or by undifferentiated cells from embryonic muscle. Muscle-conditioned medium (CM) did not affect the activities of SC cell GAD or acetylcholinesterase. Dilution or concentration of the CM directly affected its ability to increase SC CAT activity , as did the duration and timing of exposure of the SC cells to the CM. The medium could be conditioned by muscle cells in the presence or absence of serum, and remained effective after dialysis or heating to 58 degrees C. Membrane filtration data were consistent with the conclusion that the active material(s) in CM had a molecular weight in excess of 50,000 daltons. We conclude that large molecular weight material that is released by muscle cells is capable of producing a specific increase in CAT activity of SC cells.     

Mucin secretion by the human colon cell line LS174T is regulated by bile salts

We recently reported that bile salts play a role in the regulation of mucin secretion by cultured dog gallbladder epithelial cells. In this study we have examined whether bile salts also influence mucin secretion by the human epithelial colon cell line LS174T. Solutions of bile salts were applied to monolayers of LS174T cells. Mucin secretion was quantified by measuring the secretion of [3H]GlcNAc labeled glycoproteins. Both unconjugated bile salts as well as taurine conjugated bile salts stimulated mucin secretion by the colon cells in a dose-dependent fashion. Hydrophobic bile salts were more potent stimulators than hydrophilic bile salts. Free (unconjugated) bile salts were more stimulatory compared with their taurine conjugated counterparts. Stimulation of mucin secretion by LS174T cells was found to occur at much lower bile salt concentrations than in the experiments with the dog gallbladder epithelial cells. The protein kinase C activators PMA and PDB had no stimulatory effect on mucin secretion. We conclude that mucin secretion by the human colon epithelial cell line LS174T is regulated by bile salts. We suggest that regulation of mucin secretion by bile salts might be a common mechanism, by which different epithelia protect themselves against the detergent action of bile salts, to which they are exposed throughout the gastrointestinal tract.      

Tritaenia Maegdefrau et Rudolf, Mesozoic 'Sciadopitys-like' leaves in mass accumulations

The Late Jurassic to Early Cretaceous genus Tritaenia Maegdefrau et Rudolf 1969 is problematic because of: (1) missing authentic material of its type species, T. linkii (Roemer 1839) Maegdefrau et Rudolf; and (2) Watson and Harrison's (1998) synonymization of T. linkii with Pseudotorellia heterophylla Watson. This paper: (1) rectifies the status of T. linkii on the basis of newly recovered specimens carrying the original author's authentication; and (2) gives the basis for rejecting Watson and Harrison's claim that T. linkii and T. crassa (Seward) Bose et Manum 1991 represent linear leaves of the heterophyllous taxon Pseudotorellia heterophylla. The three species of Tritaenia known to date (T. crassa, T. linkii, T. scotica) are reviewed, and the genus is compared with other Mesozoic so-called 'Sciadopitys-like' hypostomatic leaves with a median stomatal zone, many of which occur in mass accumulations such as T. linkii. Deciduousness is indicated for T. linkii and T. crassa by their occurrence in mass accumulations and the possession of well-developed abscission scars. Known mass accumulations of fossil foliage are reviewed and their implications for palaeoenvironmental interpretations discussed.     

Nutrient-induced signal transduction through the protein kinase A pathway and its role in the control of metabolism, stress resistance, and growth in yeast

Yeast cells growing in the presence of glucose or a related rapidly-fermented sugar differ strongly in a variety of physiological properties compared to cells growing in the absence of glucose. Part of these differences appear to be caused by the protein kinase A (PKA) and related signal transduction pathways. Addition of glucose to cells previously deprived of glucose triggers cAMP accumulation, which is apparently mediated by the Gpr1-Gpa2 G-protein coupled receptor system. However, the resulting effect on PKA-controlled properties is only transient when there is no complete growth medium present. When an essential nutrient is lacking, the cells arrest in the stationary phase G0. At the same time they acquire all characteristics of cells with low PKA activity, even if there is ample glucose present. When the essential nutrient is added again, a similar PKA-dependent protein phosphorylation cascade is triggered as observed after addition of glucose to glucose-deprived cells, but which is not cAMP-mediated. Because the pathway involved requires a fermentable carbon source and a complete growth medium, at least for its sustained activation, it has been called “fermentable growth medium (FGM)-induced pathway.”     

Technology platform development for targeted plasma metabolites in human heart failure

Background

Heart failure is a multifactorial disease associated with staggeringly high morbidity and motility. Recently, alterations of multiple metabolites have been implicated in heart failure; however, the lack of an effective technology platform to assess these metabolites has limited our understanding on how they contribute to this disease phenotype. We have successfully developed a new workflow combining specific sample preparation with tandem mass spectrometry that enables us to extract most of the targeted metabolites. 19 metabolites were chosen ascribing to their biological relevance to heart failure, including extracellular matrix remodeling, inflammation, insulin resistance, renal dysfunction, and cardioprotection against ischemic injury.

Results

In this report, we systematically engineered, optimized and refined a protocol applicable to human plasma samples; this study contributes to the methodology development with respect to deproteinization, incubation, reconstitution, and detection with mass spectrometry. The deproteinization step was optimized with 20% methanol/ethanol at a plasma:solvent ratio of 1:3. Subsequently, an incubation step was implemented which remarkably enhanced the metabolite signals and the number of metabolite peaks detected by mass spectrometry in both positive and negative modes. With respect to the step of reconstitution, 0.1% formic acid was designated as the reconstitution solvent vs. 6.5 mM ammonium bicarbonate, based on the comparable number of metabolite peaks detected in both solvents, and yet the signal detected in the former was higher. By adapting this finalized protocol, we were able to retrieve 13 out of 19 targeted metabolites from human plasma.

Conclusions

We have successfully devised a simple albeit effective workflow for the targeted plasma metabolites relevant to human heart failure. This will be employed in tandem with high throughput liquid chromatography mass spectrometry platform to validate and characterize these potential metabolic biomarkers for diagnostic and therapeutic development of heart failure patients.     

长春市儿童医院1998～2001年婴幼儿杯状病毒腹泻流行病学研究

人类杯状病毒(human calicivirus,HuCV)是引起儿童和成人非菌性胃肠炎的主要病原之一.为了掌握HuCV在我国的流行情况,1998年7月至2001年6月,从长春市儿童医院2343例5岁以下腹泻患儿中共收集粪便标本1264份,其中1056份来自2135例住院患儿.对轮状病毒检测为阴性的588份标本,经多价酶免疫试验(EIA)和两组引物反转录-聚合酶链反应(RT-PCR)检测HuCV,202份为阳性,其中住院患儿标本178份,HuCV检出率为16.9%.HuCV腹泻以2岁以下儿童为主(占96%),流行高峰季节为11月至次年3月.选择17株HuCV进行分子鉴定,15株属GⅡ-4群,1株属GⅡ-3群,另1株属GⅠ-2群,表明GⅡ-4群HuCV是我国流行的优势株.根据HuCV住院患儿的监测资料初步估计,HuCV腹泻住院率约为0.5‰～2.4‰.讨论了长春地区HuCV的流行趋势和疾病负担.以上结果为我国HuCV腹泻的预防和控制提供了科学依据.     

Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger

Background

The integration of biotechnology into chemical manufacturing has been recognized as a key technology to build a sustainable society. However, the practical applications of biocatalytic chemical conversions are often restricted due to their complexities involving the unpredictability of product yield and the troublesome controls in fermentation processes. One of the possible strategies to overcome these limitations is to eliminate the use of living microorganisms and to use only enzymes involved in the metabolic pathway. Use of recombinant mesophiles producing thermophilic enzymes at high temperature results in denaturation of indigenous proteins and elimination of undesired side reactions; consequently, highly selective and stable biocatalytic modules can be readily prepared. By rationally combining those modules together, artificial synthetic pathways specialized for chemical manufacturing could be designed and constructed.

Results

A chimeric Embden-Meyerhof (EM) pathway with balanced consumption and regeneration of ATP and ADP was constructed by using nine recombinant E. coli strains overproducing either one of the seven glycolytic enzymes of Thermus thermophilus, the cofactor-independent phosphoglycerate mutase of Pyrococcus horikoshii, or the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase of Thermococcus kodakarensis. By coupling this pathway with the Thermus malate/lactate dehydrogenase, a stoichiometric amount of lactate was produced from glucose with an overall ATP turnover number of 31.

Conclusions

In this study, a novel and simple technology for flexible design of a bespoke metabolic pathway was developed. The concept has been testified via a non-ATP-forming chimeric EM pathway. We designated this technology as “synthetic metabolic engineering”. Our technology is, in principle, applicable to all thermophilic enzymes as long as they can be functionally expressed in the host, and thus would be potentially applicable to the biocatalytic manufacture of any chemicals or materials on demand.