Frequency entrainment of squid axon membrane

Summary Sinusoidally varying stimulating currents were applied to space-clamped squid giant axon membranes in a double sucrose gap apparatus. Stimulus parameters varied were peak-to-peak current amplitude, frequency, and DC offset bias. In response to these stimuli, the membranes produced action potentials in varying patterns, according to variation of input stimulus parameters. For some stimulus parameters the output patterns were stable and obviously periodic with the periods being simple multiples of the input period; for other stimulus parameters no obvious periodicity was manifest in the output. The experimental results were compared with simulations using a computer model which was modified in several ways from the Hodgkin-Huxley model to make it more representative of our preparation. The model takes into account K⁺ accumulation in the periaxonal space, features of Na⁺ inactivation which are anomalous to the Hodgkin-Huxley model, sucrose gap hyperpolarization current, and membrane current noise. Many aspects of the experiments are successfully simulated but some are not, possibly because some very slow process present in the preparation is not included in the model.     

The direct influence of electromagnetic fields on nerve- and muscle cells of man within the frequency range of 1 Hz to 30 MHz

Summary By using several biophysical approximations and considering man as free space model limiting order-of-magnitude values for external electric and magnetic field strengths which may be hazardous for human beings were calculated. Danger may occur by excitation processes below 30 kHz for field strengths exceeding these limiting values; for frequencies larger than 30 kHz, thermal effects are predominant before excitation occurs. The external electric field strength necessary for causing action potentials in the central nervous system exceeds by far the corona forming level. But excitation is possible by strong alternating magnetic fields.Furthermore, by comparing the electrically and magnetically induced currents with the naturally flowing currents in man caused by the brain's and heart's electrical activity, a lower boundary-line was estimated. Regarding electric or magnetic field strengths undercutting this boundary-line, direct effects on the central nervous system may be excluded. Other mechanisms should be responsible for demonstrated biological effects.     

Evidence for multiple sites of ferricyanide reduction in chloroplasts

Various sites of ferricyanide reduction were studied in spinach chloroplasts. It was found that in the presence of dibromothymoquinone a fraction of ferricyanide reduction was dibromothymoquinone sensitive, implying that ferricyanide can be reduced by photosystem I as well as photosystem II. To separate ferricyanide reduction sites in photosystem II, orthophenanthroline and dichlorophenyl dimethylurea inhibitions were compared at various pH's. It was noted that at low pH ferricyanide reduction was not completely inhibited by orthophenanthroline. At high pH's, however, inhibition of ferricyanide reduction by orthophenanthroline was complete. It was found that varying concentration of orthophenanthroline at a constant pH showed different degrees of inhibition. In the study of ferricyanide reduction by photosystem II various treatments affecting plastocyanin were performed. It was found that Tween-20 or KCN treatments which inactivated plastocyanin did not completely inactivate ferricyanide reduction. These data support the conclusion that ferricyanide accepts electrons both before and after plastoquinone in photosystem II.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyurea - MV methyl viologen - DBMIB 2,5-dibromothymoquinone - DMBQ 2,6-dimethyl benzoquinone - OP 1,10-orthophenanthroline - TMPD tetramethyl-p-phenylenediamine - PS 1 photosystem I - PS II photosystem II - SN sucrose-sodium chloride chloroplasts Supported by NSF Grant BMS 74-19689.     

A 4-methoxybenzoate O-demethylase from Pseudomonas putida. A new type of monooxygenase system.

A strain of Pseudomonas putida grown on 4-methoxybenzoate as sole carbon source contains an enzyme system for the O-demethylation of this substrate. The enzyme system is purifiable and can be separated into two components: an NADH-dependent reductase and an iron-containing and acid-labile-sulfur-containing monooxygenase. The reductase, of molecular weight 42000 and containing two chromophores, an FMN and an iron-sulfur complex (EPR at g = 1.95), reduces both one-electron and two-electron acceptors (i.e., ferricyanide, 2,6-dichloroindophenol, cytochrome c, and cytochrome b5) at an optimum pH of 8.0. Increasing ionic strength affects these activities differently. The absolute spectrum of the oxidized displays distinct absorption peaks at 409 and 463 nm and a small shoulder between 538 and 554 nm. Treatment with dithionite or NADH reduces the absorbance throughout the visible range, yielding a spectrum with small maxima at 402 and 538 nm. Spectroscopic characteristics of the reductase indicate a tight coupling between its two chromophores. The iron-containing and acid-labile-sulfur-containing monooxygenase, which has a molecular weight of about 120000, contains an iron-sulfur chromophore with an EPR signal at g = 1.90. This protein is a dimer whose subunits each have a molecular weight of about 50000 and are perhaps identical. The optical absorption properties are somewhat unusual. In contrast to other iron-sulfur proteins, there is no significant peak near 415 nm in the absorption spectrum of the oxidized protein, but rather one at 455 nm. The presence of the substrate 4-methoxybenzoate increases both the NADH-dependent reductase. Hydroxylation can be achieved by the monooxygenase also in absence of the reductase with artifical reductants. This enzyme opens a new group of oxygenases within the classification scheme, i.e., iron-containing and labile-sulfur-containing monooxygenases. From the reported data, a scheme for the interaction of the isolated pigments and their relationship to various acceptors is proposed.     

Adsorption of Vibrio parahaemolyticus onto Chitin and Copepods

Vibrio parahaemolyticus was observed to adsorb onto chitin particles and copepods. The efficiency of adsorption was found to be dependent on pH and on the concentration of NaC1 and other ions found in seawater. Highest efficiency was observed in water samples collected from Chesapeake Bay and lowest in water from the open sea. V. parahaemolyticus was found to adborb onto chitin with the highest efficiency of the several bacterial strains tested. Escherichia coli and Pseudomonas fluorescens did not adsorb onto chitin. The adsorption effect is considered to be one of the major factors determining the distribution of this species and affecting the annual cycle of V. parahaemolyticus in the estuarine system.     

PURIFICATION AND PROPERTIES OF BRAIN N-ACETYL-β-HEXOSAMINIDASE A

—N-Acetyl-β-hexosaminidase A was purified to homogeneity from human and monkey brains by the conventional procedures followed by concanavalin A–Sepharose affinity chromatography. The optimal activity was observed at pH 4·5 for both enzyme preparations with both the aglycones N-acetylglucosamine and N-acetylgalactosamine derivatives. The K_m values for hexosaminidase A from monkey brain were 0·26 mm and 0·04 mm respectively for N-acetylglucosamine and N-acetylgalactosamine. K_m values obtained for glucosamine and galactosamine derivatives for the human brain hexosaminidase A were of the same order. The glycoprotein nature of the enzymes was established by the affinity towards concanavalin A as well as by the presence of sialic acid, galactose, glucose, mannose and hexosamines in the enzyme molecule from monkey brain.     

JmjC-KDMs KDM3A and KDM6B modulate radioresistance under hypoxic conditions in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC), the most frequent esophageal cancer (EC) subtype, entails dismal prognosis. Hypoxia, a common feature of advanced ESCC, is involved in resistance to radiotherapy (RT). RT response in hypoxia might be modulated through epigenetic mechanisms, constituting novel targets to improve patient outcome. Post-translational methylation in histone can be partially modulated by histone lysine demethylases (KDMs), which specifically removes methyl groups in certain lysine residues. KDMs deregulation was associated with tumor aggressiveness and therapy failure. Thus, we sought to unveil the role of Jumonji C domain histone lysine demethylases (JmjC-KDMs) in ESCC radioresistance acquisition. The effectiveness of RT upon ESCC cells under hypoxic conditions was assessed by colony formation assay. KDM3A/KDM6B expression, and respective H3K9me2 and H3K27me3 target marks, were evaluated by RT-qPCR, Western blot, and immunofluorescence. Effect of JmjC-KDM inhibitor IOX1, as well as KDM3A knockdown, in in vitro functional cell behavior and RT response was assessed in ESCC under hypoxic conditions. In vivo effect of combined IOX1 and ionizing radiation treatment was evaluated in ESCC cells using CAM assay. KDM3A, KDM6B, HIF-1α, and CAIX immunoexpression was assessed in primary ESCC and normal esophagus. Herein, we found that hypoxia promoted ESCC radioresistance through increased KDM3A/KDM6B expression, enhancing cell survival and migration and decreasing DNA damage and apoptosis, in vitro. Exposure to IOX1 reverted these features, increasing ESCC radiosensitivity and decreasing ESCC microtumors size, in vivo. KDM3A was upregulated in ESCC tissues compared to the normal esophagus, associating and colocalizing with hypoxic markers (HIF-1α and CAIX). Therefore, KDM3A upregulation in ESCC cell lines and primary tumors associated with hypoxia, playing a critical role in EC aggressiveness and radioresistance. KDM3A targeting, concomitant with conventional RT, constitutes a promising strategy to improve ESCC patients’ survival.Subject terms: Predictive markers, Cancer     

Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection

Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation.