Purification and properties of N5, N10-methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum (strain Marburg)

The reduction of N5,N10-methylenetrahydromethanopterin (CH2 = H4MPT) to N5-methyltetrahydromethanopterin (CH3-H4MPT) is an intermediate step in methanogenesis from CO2 and H2. The reaction is catalyzed by CH2 = H4MPT reductase. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) was found to be specific for reduced coenzyme F420 as electron donor; neither NADH or NADPH nor reduced viologen dyes could substitute for the reduced 5-deazaflavin. The reductase was purified over 100-fold to apparent homogeneity. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed only one protein band at the 36-kDa position. The apparent molecular mass of the native enzyme was determined by gel filtration to be in the order of 150 kDa. The purified enzyme was colourless. It did not contain flavin or iron. The ultraviolet visible spectrum was almost identical to that of albumin, suggesting the absence of a chromophoric prosthetic group. Reciprocal plots of the enzyme activity versus the substrate concentration at different constant concentrations of the second substrate yielded straight lines intersecting at one point on the abscissa to the left of the vertical axis. This intersecting pattern is characteristic of a ternary complex catalytic mechanism. The Km for CH2 = H4MPT and for the reduced coenzyme F420 were determined to be 0.3 mM and 3 microM, respectively. Vmax was 6000 mumol.min-1.mg protein-1 (kcat = 3600 s-1). The CH2 = H4MPT reductase was stable in the presence of air; at 4 C less than 10% activity was lost within 24 h.     

Role of UV-inducible proteins in repair of various wild-type Escherichia coli cells

3 wild-type strains of E. coli, namely K12 AB2497, B/r WP2 and 15 555-7v proficient in excision and post-replication repair, differ markedly in their UV resistance. To elucidate this difference, the influence was investigated of induction by application of inducing fluence (IF) before lethal fluence (LF) on repair processes after LF. In cells distinguished by low UV resistance (E. coli 15 555-7; E. coli B/r WP2), dimer excision was less complete in cultures irradiated with IF + LF than in cultures irradiated with LF only. The highly resistant E. coli K12 AB2497 performed complete excision both after IF + LF or after LF alone. All 3 types of cell survived better after IF + LF than after LF only. Because, in most strains so far investigated, the application of IF reduced dimer excision and increased survival, dimer excision per se does not appear important for survival.We conclude that the rate and completeness of dimer excision can serve as a measure of efficiency of the excision system whose action is necessary for repair of another lesion. Cells of all investigated strains could not resume DNA replication and died progressively when irradiated with LF and post-incubated with chloramphenicol (LF CAP⁺). Thus, it appears that inducible proteins are necessary for repair in all wild-type E. coli cells give with potentially lethal doses of UV irradiation.     

Structures and fatty acid compositions of neutral glycosphingolipids of human plasma

Major neutral glycosphingolipids were isolated from human plasma and their structures and fatty acid compositions studied. The four neutral glycosphingolipids of plasma were characterized as Glc beta(1 leads to 1)ceramide, Gal beta(1 leads to 1)- ceramide, Gal beta(1 leads to 4) Glc beta (1 leads to 1)ceramide, Gal alpha(1 leads to 4) Gal beta(1 leads to 4) Glc beta(1 leads to 1)ceramide and GalNAc beta(1 leads to 3) Gal (1 leads to 4) Gal (1 leads to 4) Glc beta(1 leads to 1)-ceramide. The glycosphingolipids contained mostly short chain fatty acids of which most prominent was C16. Erythrocyte glucosylceramide and lactosylceramide exhibited similar fatty acid compositions as their plasma counterparts. Triglycosylceramide and globoside of erythrocytes contained almost exclusively long-chain fatty acids. In lactosylceramide obtained from "p" erythrocytes, an accumulation of long-chain fatty acids was found; this accumulation was not observed, however, in lactosylceramide isolated from "p" plasma. It was concluded that plasma and erythrocyte glycosphingolipids are synthesized at separate sites where short- and long-chain fatty acids, respectively, are available. Plasma and erythrocyte glucosylceramide, and probably a fraction of lactosylceramide, exchange between plasma and erythrocyte pools. The latter conclusion is discussed in the light of the relative roles of carbohydrate and lipid moieties of the glycosphingolipids in maintaining their association with erythrocyte membranes.     

Sex pheromone-releasing behaviour in females of the dermestid beetle,Trogoderma glabrum

Adult Trogoderma glabrum females assume stationary postures characterized by abdominal elevation and partial exposure of the ovipositor segments. The postural activity was studied in relation to sex pheromone production and was designated calling, or sex pheromone-releasing behaviour because it was accompanied by an increase in the rate of release of sex pheromone(s). Calling was largely restricted to an 8 hr interval centered toward the middle of a 16 hr photophase. Mating frequency and female pheromone content increased during the same interval. Because females extracted during the calling period yielded at least 10 times more pheromone than extracts prepared at other times, and because pheromone activity released during one calling period was estimated to be at least 10-fold higher than that which could be extracted from whole females several hours prior to the start of calling, heightened production of active pheromone(s) was postulated to occur during calling. Functional significance of the calling posture is discussed in relation to sex pheromone gland location.     

Metabolic actions of vasopressin, glucagon and adrenalin in the intact rat.

Metabolic effects of vasopressin, glucagan and adrenalin were compared, in intact rats, especially in regard to time courses of effects. Hyperglycaemia was transient in response to vasopressin, prolonged following adrenalin, and, suprisingly, was not discernible after glucagon, except in response to a very large dose. Vasopressin decreased and adrenalin increased, the plasma free fatty acid concentration; both hormones decreased the triacylglycerol level. Muscle glycogen concentrations, measured in heart, diaphragm and skeletal muscle, exhibited small changes, with complex time courses, following hormone administration. Vasopressin brought about a rapid but transient activation of heaptic glycogen phosphorylase which resembled that due to adrenalin. The activation by glucagon of phosphorylase was greater and more prolonged, despite the absence of hyperglycaemia. In response to vasopressin, there was in increase in plasma insulin. Incorporation of 14C from [14C]glucose into glycogen or fatty acids was not influenced by vasopressin. Taken together, these results may be explained by rapid metabolic action of vasopressin on hepatic glycogenolysis, whereas adrenalin has multiple prolonged actions.     

Models of learning based on the plastic properties of the "placing reaction" in cats]

A possibility of functional reorganization of initial sensorimotor connections of the forepaw has been shown on seven cats. The main initial relationships between the afferent tactile input and motor output for the ulnar joint of the cat forepaw are as follows: tactile stimulation of the dorsal surface of the paw produces a flexion in the ulnar joint ("placing reaction"), and that of the ventral surface, an extension of the paw in the ulnar joint ("magnetic reflex"); simultaneous tactile stimulation of the ventral surface of the paw blocks the "placing reaction" evoked by a touch of the dorsal side. Extinction was produced of the above unconditioned connections and elaboration of a new "cross" connection consisting in that tactile stimulation of the ventral side of the paw resulted in flexion in the ulnar joint.     

Study of the functional activity of the macrophages of animals immunized with staphylococcal alpha-anatoxin]

A study was made by histochemical methods of the activity of the enzymatic systems of macrophages from normal rabbits and those immunized with staphylococcus alpha-toxoid per se and infected with the strains of staphylococcus--producers of alpha-toxin or leukocydin. Immunization of rabbits was accompanied by a reduction in macrophages of the activity of the group of lysosomal enzymes and by an increase in the activity of the redox enzymes. In infection of "immune" macrophages with the living culture of the alpha-toxigenic strains the mentioned changes were more pronounced; no such changes were found after the infection with the leukocydin-active strain. The data obtained suggested that the lysosomal enzymes played a definite role in the process of phagocytosis.     

Single-cell transcriptomic atlas of primate cardiopulmonary aging

Aging is a major risk factor for many diseases,especially in highly prevalent cardiopulmonary comorbidities and infectious diseases including Coronavirus Diseas...     

Enhanced Orai1-mediated store-operated Ca2+ channel/calpain signaling contributes to high glucose-induced podocyte injury

Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca²⁺ entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca²⁺-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)–induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca²⁺ channels, which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-Cas9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca²⁺ pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/calpain-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/SOCE/Calpain axis contributes to HG-induced podocyte injury.