The relationship between the activity and the activation state of RuBP carboxylase and carbon exchange rate as affected by sink and developmental changes

The purpose of this study was to explore if sink manipulations which affect leaf carbon exchange rate (CER) are mediated by ribulose 1,5-bisphosphate (RuBP) carboxylase activity. Tomato leaf (Lycopersicon esculentum Mill. cv. Vendor) RuBP carboxylase was assayed using a rapid extraction method. Over a diurnal period, leaf CER fluctuated independent of carboxylase activity. Differences in leaf CER induced by fruit pruning in one leaf-one cluster plants were not accompanied by changes in carboxylase activity.During leaf expansion, carboxylase activity and percent enzyme in the active form paralleled the increase and then decrease in leaf carbon exchange rate. Differences in leaf CER induced by root warming at ambient air temperature, were accompanied by parallel changes in carboxylase activity.These results suggest that modifications in leaf CER are not mediated exclusively through changes in carboxylase activity, but rather that modifications in carboxylase activity coincide with overall changes in leaf physiology and morphology in response to sink demand.     

Neuronal activity recorded extracellularly from in situ canine mediastinal ganglia

Spontaneous activity of 226 neurons was recorded from in situ mediastinal ganglia in 10 dogs. Forty-two percent of these were active during specific phases of the cardiac cycle, primarily during systole. Cardiovascular-related activity occurred when systolic pressure was between approximately 70 and 185 mmHg (1 mmHg = 133.3 Pa) whether the pressure was altered by positive inotropic pharmacological agents or cross clamping of the aorta. Twenty percent of the identified neurons displayed respiratory-related activity which occurred during positive pressure inflation or deflation. Thirty-eight percent of the identified neurons displayed bursts of activity or sporadic activity. The activity of 17% of the identified neurons was altered by gentle mechanical distortion of localized regions of the neck, left elbow, ventral thoracic wall, ventral abdominal wall, superior vena cava, right ventricle, or aorta. In the majority of instances cardiovascular- or respiratory-related activity persisted following acute decentralization, indicating that neurons in mediastinal ganglia can function in the absence of influences of central nervous system neurons. Five percent of the identified neurons were activated by single 1-4 ms, 10-20 V stimuli delivered at 0.5 Hz to the nerves connected with either the cranial or the caudal poles of the mediastinal ganglion or the ansae. These neurons were activated after a fixed latency when 0.5 Hz was used and in most instances when 10 Hz was used. These data indicate that 5% or less of the neurons identified projected axons out of the mediastinal ganglia investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.