d-Ribulokinase from Klebsiella pneumoniae for continuous production of d-(−)-ribulose-5-phosphate

Summary The production of d-ribulose-5-phosphate in an enzyme membrane reactor was examined. Phosphoryl transfer from ATP to d-ribulose was catalysed by d-ribulokinase isolated from Klebsiella pneumoniae. For production of d-ribulose-5-phosphate the phosphoryl donor ATP was used either in stoichiometric or in catalytic amounts. Using catalytic amounts of ATP requires a second enzyme, e.g. pyruvate kinase, to regenerate ATP. The kinetic parameters for d-ribulokinase and pyruvate kinase were determined to calculate the performance of an enzyme membrane reactor for continuous production of d-ribulose-5-phosphate. Both processes operated for more than 200 h. Regardless of whether ATP was used in catalytic or stoichiometric amounts, about the same production parameters were determined. In continuous production space/time yields of 117 g (with ATP regeneration) and 103 g (without ATP regeneration) of d-ribulose-5-phosphate 1^–1 per day were reached.Offprint requests to: D. Gygax     

Anaerobic degradation of benzaldehyde in methanogenic granular sludge: the influence of additional substrates

Summary The degradation of benzaldehyde in methanogenic granular sludge was investigated in batch and in upflow anaerobic sludge blanket (UASB) reactors. The effect due to the presence of co-substrates, such as H₂, sodium butyrate and sucrose, was studied using formaldehyde as a reference compound. The additional substrates enhanced the activity of benzaldehyde- and formaldehyde-degrading microorganisms (ACT_bdm and ACT_fdm, respectiveky) and increased the transient production of benzyl alcohol and methanol. As a consequence, the concentrations of benzaldehyde and formaldehyde that caused 50% inhibition of the methanogenic activity (50% IC_m) on sucrose were 3133 and 254 mg chemical oxygen demand (COD)/l respectively, three times higher than the literature data values on acetate. Experiments performed in UASB reactors on benzaldehyde showed that the replacement of volatile fatty acids with sucrose as co-substrate improved the treatment capacity of the system from 0.73 to 4.36 kg COD benzaldehyde·m^–13·day^–1. Correspondence to: O. Todini     

Ricerche Sull'infrastruttura del Coleottile di Avena

Abstract

Researches on ultrastructure of Avena coleoptile. 3. The sieve elements. — A study on the ultrastructural organization of the mature sieve elements of Avena coleoptile has been carried out. Data suggest that functional phloem tubes are alive and remain alive until they are working. Judging on morphological basis, the metabolic activity of sieve elements should be of peculiar type and low in comparison to that of the companion cells. In fact the cytoplasm is located in a narrow parietal strand, mitochondria, Golgi apparatus and endoplasmic reticulum are present, but they appear very modified; plastids and nucleus are absent. The cytoplasm is bounded externally by a normal plasmalemma, whilst the vacuole has no visible limits: a tonoplast is, therefore not identifiable.

The strands connecting the superimposed sieve elements with one another through the sieve plate result to be made by a double membrane system very similar to the endoplasmic reticulum, which we believe to realize cytoplasmic continuity between phloem tubes.

The data reported are more favorable to the existence in the sieve tubes of an active mechanism of translocation of organic solutes than a passive mass-flow.

The collaboration of companion cells in the translocation mechanism has been discussed.     

Quantitative Phosphoproteome Analysis Unveils LAT as a Modulator of CD3ζ and ZAP-70 Tyrosine Phosphorylation

Signaling through the T cell receptor (TCR) initiates adaptive immunity and its perturbation may results in autoimmunity. The plasma membrane scaffolding protein LAT acts as a central organizer of the TCR signaling machinery to activate many functional pathways. LAT-deficient mice develop an autoimmune syndrome but the mechanism of this pathology is unknown. In this work we have compared global dynamics of TCR signaling by MS-based quantitative phosphoproteomics in LAT-sufficient and LAT-defective Jurkat T cells. Surprisingly, we found that many TCR-induced phosphorylation events persist in the absence of LAT, despite ERK and PLCγ1 phosphorylation being repressed. Most importantly, the absence of LAT resulted in augmented and persistent tyrosine phosphorylation of CD3ζ and ZAP70. This indicates that LAT signaling hub is also implicated in negative feedback signals to modulate upstream phosphorylation events. Phosphorylation kinetics data resulting from this investigation is documented in a database (phosphoTCR) accessible online. The MS data have been deposited to the ProteomeXchange with identifier PXD000341.     

The binding of heparin to spike glycoprotein inhibits SARS-CoV-2 infection by three mechanisms

Heparin, a naturally occurring glycosaminoglycan, has been found to have antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. To elucidate the mechanistic basis for the antiviral activity of heparin, we investigated the binding of heparin to the SARS-CoV-2 spike glycoprotein by means of sliding window docking, molecular dynamics simulations, and biochemical assays. Our simulations show that heparin binds at long, positively charged patches on the spike glycoprotein, thereby masking basic residues of both the receptor-binding domain (RBD) and the multifunctional S1/S2 site. Biochemical experiments corroborated the simulation results, showing that heparin inhibits the furin-mediated cleavage of spike by binding to the S1/S2 site. Our simulations showed that heparin can act on the hinge region responsible for motion of the RBD between the inactive closed and active open conformations of the spike glycoprotein. In simulations of the closed spike homotrimer, heparin binds the RBD and the N-terminal domain of two adjacent spike subunits and hinders opening. In simulations of open spike conformations, heparin induces stabilization of the hinge region and a change in RBD motion. Our results indicate that heparin can inhibit SARS-CoV-2 infection by three mechanisms: by allosterically hindering binding to the host cell receptor, by directly competing with binding to host heparan sulfate proteoglycan coreceptors, and by preventing spike cleavage by furin. Furthermore, these simulations provide insights into how host heparan sulfate proteoglycans can facilitate viral infection. Our results will aid the rational optimization of heparin derivatives for SARS-CoV-2 antiviral therapy.     

Rat carotid artery dilation by PTCA balloon catheter induces neointima formation in presence of IEL rupture

The best animal angioplasty model is the porcine model, which is expensive and not available in all laboratories. The aim of this study was to describe a new rat model of angioplasty. An injury was induced with the use of a standard percutaneous transluminal coronary angioplasty (PTCA) 1.5-mm balloon catheter. The neointimal tissue, arterial dimensions, and the injury index were assessed following angioplasty. Ki-67 expression was detected to evaluate cell turnover after balloon angioplasty. In contrast with the standard Clowes model, a significant neointimal formation was detected only in the presence of ruptured internal elastic lamina (IEL). A positive correlation between the percentage of ruptured IEL and the amount of neointimal tissue was also demonstrated. The percentage of IEL fracture correlates with the proliferation index by anti-Ki-67 immunolabeling 7 and 14 days after the angioplasty. Significant arterial negative remodeling was observed following PTCA balloon dilation. In conclusion, our inexpensive animal model of restenosis after angioplasty may have great relevance toward a better understanding of the mechanisms and toward assessment of new therapeutical strategies for this phenomenon.     

Ascorbic acid: much more than just an antioxidant

Vitamin C (ascorbic acid (AA)) is very popular for its antioxidant properties. Consequently, many other important aspects of this multifaceted molecule are often underestimated or even ignored. In the present paper, we have tried to bring to the foreground some of these aspects, including the peculiarities of the AA biosynthetic pathway in different organisms, the remarkable function of AA as a co-substrate of many important dioxygenases, the role of AA-regenerating enzymes and the known pathways of AA catabolism, as well as the intriguing function of AA in gene expression.     

Plasma and bile antibodies of the teleost Trematomus bernacchii specific for the nematode Pseudoterranova decipiens

We investigated the occurrence of antibodies against protein antigens of the nematode parasite Pseudoterranova decipiens in the plasma and bile of the Antarctic teleost Trematomus bernacchii. Three different P. decipiens protein solutions were prepared: excreted/secreted proteins from live larvae (ESP); surface-associated proteins obtained by mild extraction of larval bodies (SAP); and cuticular soluble proteins recovered by extraction in strong reducing conditions (CSP). Using different immunoassays, these 3 preparations were tested for their ability to bind fish antibody. As determined by ELISA, the specific antibody binding activity was higher in SAP than in CSP. As determined by dot-blot immunoassay, the specific antigen binding activity versus SAP was higher in bile than in plasma antibodies. A different number of antigenic components of SAP and ESP were identified by immunoblotting performed with plasma or bile antibodies. These results led to the conclusion that T. bernacchii parasitism by nematodes involves plasma and bile anti-parasite antibodies. Furthermore bile antibodies were found to be more reactive and more heterogeneous than plasma.     

Effects of Blood Transfusion on Exercise Capacity in Thalassemia Major Patients

Anemia has an important role in exercise performance. However, the direct link between rapid changes of hemoglobin and exercise performance is still unknown.To find out more on this topic, we studied 18 beta-thalassemia major patients free of relevant cardiac dysfunction (age 33.5±7.2 years,males = 10). Patients performed a maximal cardiopulmolmonary exercise test (cycloergometer, personalized ramp protocol, breath-by-breath measurements of expired gases) before and the day after blood transfusion (500 cc of red cell concentrates). After blood transfusion, hemoglobin increased from 10.5±0.8 g/dL to 12.1±1.2 (p<0.001), peak VO₂ from 1408 to 1546mL/min (p<0.05), and VO₂ at anaerobic threshold from 965 to 1024mL/min (p<0.05). No major changes were observed as regards heart and respiratory rates either at peak exercise or at anaerobic threshold. Similarly, no relevant changes were observed in ventilation efficiency, as evaluated by the ventilation vs. carbon dioxide production relationship, or in O₂ delivery to the periphery as analyzed by the VO₂ vs. workload relationship. The relationship between hemoglobin and VO₂ changes showed, for each g/dL of hemoglobin increase, a VO₂ increase = 82.5 mL/min and 35 mL/min, at peak exercise and at anaerobic threshold, respectively. In beta-thalassemia major patients, an acute albeit partial anemia correction by blood transfusion determinates a relevant increase of exercise performance, observed both at peak exercise and at anaerobic threshold.