Calcium activated proteolysis of fibrous proteins in central nervous system

A Ca²⁺ activated protease(s) capable of hydrolyzing several polypeptides at neutral pH including cytoskeletal proteins, actin, myosin, tubulin and neurofilament triplet was identified in calf brain cortex. The enzyme activity precipitates at 75 mM KCl, pH 6.5 – 7.0 and is inhibited by the sulfhydryl inhibitors, N-ethylmaleimide and para-chloromercuribenzoate and the protease inhibitors, antipain, pepstatin and leupeptin, leupeptin being the most effective.     

Teratogenic effects of diazepam in the hamster.

Pregnant hamsters were treated with different doses of oral and intravenous diazepam during the period of organogenesis. Teratogenic effects of diazepam were observed following oral treatment on days 8 and 10 and following intravenous treatment on day 11 of gestation. Types of malformations included cleft palate, exencephaly, limb anomalies, and hemorrhage. A dose-effect relationship was not observed. Comparison with reported literature seems to indicate that diazepam may be a mild teratogen in some species.     

Viral Pollution of Surface Waters Due to Chlorinated Primary Effluents

The role of chlorinated primary effluents in viral pollution of the Ottawa River (Ontario) was assessed by examining 282 field samples of wastewaters from two different sewage treatment plants over a 2-year period. The talc-Celite technique was used for sample concentration, and BS-C-1 cells were employed for virus detection. Viruses were detected in 80% (75/94) of raw sewage, 72% (68/94) of primary effluent, and 56% (53/94) of chlorinated effluent samples. Both raw sewage and primary effluent samples contained about 100 viral infective units (VIU) per 100 ml. Chlorination produced a 10- to 50-fold reduction in VIU and gave nearly 2.7 VIU/100 ml of chlorinated primary effluent. With a combined daily chlorinated primary effluent output of approximately 3.7 × 10⁸ liters, these two plants were discharging 1.0 × 10¹⁰ VIU per day. Because the river has a mean annual flow of 8.0 × 10¹⁰ liters per day, these two sources alone produced a virus loading of 1.0 VIU/8 liters of the river water. This river also receives at least 9.0 × 10⁷ liters of raw sewage per day and undetermined but substantial amounts of storm waters and agricultural wastes. It is used for recreation and acts as a source of potable water for some 6.0 × 10⁵ people. In view of the potential of water for disease transmission, discharge of such wastes into the water environment needs to be minimized.     

Is alcoholic acidic silver nitrate reagent really specific for the histochemical localization of ascorbic acid?

Summary The histochemical localization of ascorbic acid in plant tissues with the alcoholic acidic silver nitrate reagent is shown here to be not specific for ascorbic acid, since some of the polyphenolic substances, including flavonoids, which are known to be widely distributed in plant tissues, are also able to reduce the acidic alcoholic silver nitrate reagent at low temperature (0–4°C) and at pH 2 to 2.5 in dark. This method may perhaps be used for animal tissues where flavonoid pigments do not occur in such large quantities as they do in plants. I therefore, come to the inevitable conclusion that the use of alcoholic acidic silver nitrate reagent in localizing ascorbic acid in plant tissues may be highly misleading.     

Nitrogenase. VII. Effect of component ratio, ATP and H2 on the distribution of electrons to alternative substrates.

Some kinetic properties of purified component I (Mo-Fe protein) and component II (Fe protein) of nitrogenase (EC 1.7.99.2) from Azotobacter vinelandii have been examined. The apparent Km values for reducible substrates (0.1 atm for N2, 0.01 atm for acetylene) and dithionite (0.5 mM) are similar for osmotically shocked cell lysates and purified components. However, the ATP dependence of acetylene and N2 reduction varies sigmoidally with ATP concentration and as a function of the relative and absolute concentration of components I and II in the assay. Acetylene is reduced in preference to N2 in competitive assays when component I is in relative excess. Acetylene reduction is not as dependent upon ATP concentration as is N2 reduction, so that acetylene is also a preferred substrate at lower ATP levels. Hydrogen specifically inhibits N2 reduction, diverting electrons to acetylene when both substrates are present in the assay. We propose a model of the enzyme activity, in which the substrates for reduction are bound to component I with electrons being activated by component II. ATP may be involved in activating electrons and in maintaining the appropriate conformation or reduction state of components to allow effective reduction of substrates. The relative rate of reduction of alternative substrates is dependent on the concentration of the particular state(s) capable of reacting with each substrate. The concentration of a particular state of component I is a function of components I, II and ATPL     

Cytochrome oxidase and ascorbic acid in the normal and regenerating tail of the scincid lizard, Mabuya carinata. A histophysiological study.

The concentration of ascorbic acid (AA) and the histochemical distribution of this vitamin together with cytochrome oxidase have been investigated in the normal and regenerating tail of the Scincid lizard, Mabuya carinata. An interesting aspect of this investigation is the observation of a total lack of cytochrome oxidase in both the normal and regenerating tail of the lizard, except for the differentiating phase. On the other hand, AA has been found to be present in the normal and regenerating tail with above normal levels during wound healing (twofold) and differentiation (fivefold). In the light of the poor cytochrome oxidase activity, the higher content of AA noted during regeneration has been construed to play a possible role in the respiratory mechanics of the regenerating lizard tail. Further, the importance of AA in cellular metabolism and the wound healing and differentiative processes have also been discussed.     

Division of labor within the DNA damage tolerance system reveals non-epistatic and clinically actionable targets for precision cancer medicine

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine.     

Comparing Accuracy of Wrist Intra-articular Needle Placement Via Ulnocarpal Approach by Training Level: A Cadaveric Study

BackgroundIntra-articular injections are a standard therapy and diagnostic tool for a variety of wrist conditions. Accurate needle placement is crucial for proper therapeutic benefit and prevention of complications. While some studies claim accurate needle placement requires imaging, others conclude that anatomical guidance is sufficient. This study aimed to evaluate the accuracy of intra-articular wrist needle placement with the ulnocarpal approach across differing levels of training using clinical anatomy alone.MethodsFourteen fresh-frozen, above-elbow cadaveric specimens were used. Intra-articular needle placement into the wrist via an ulnocarpal approach was attempted by nine study participants: two interns, two junior-level residents, two senior-level residents, two hand fellows, and one attending hand surgeon. Each injection was performed based on clinical examination and landmarks alone. The number of attempts and total time taken for each injection was recorded.ResultsOverall success rate was 71%, (89 of 126 attempts) and did not vary significantly across levels of training. Average time for needle placement among all participants was 10.9 ± 6.5 seconds. Timing of successful intra-articular needle placement (10.4 ± 5.2 seconds) significantly differed between levels. However, timing did not trend in any direction with more or less training. No significant difference was noted in total attempts or attempts with successful outcomes when comparing level of training.ConclusionThe ulnocarpal approach is a viable option for injection or aspiration of the wrist without image guidance. We were unable to show any relevant trends with timing or number of attempts in comparison to level of training. Level of Evidence: V     

Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10–12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.Subject terms: Cancer metabolism, CNS cancer