Influence of denervation on the molecular forms of junctional and extrajunctional acetylcholinesterase in fast and slow muscles of the rat.

Acetylcholinesterase (AChE) molecular forms in denervated rat muscles, as revealed by velocity sedimentation in sucrose gradients, were examined from three aspects: possible differences between fast and slow muscles, response of junctional vs extrajunctional AChE, and early vs late effects of denervation. In the junctional region, the response of the asymmetric AChE forms to denervation is similar in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle: (a) specific activity of the A12 form decreases rapidly but some persists throughout and even increases after a few weeks; (b) an early and transient increase of the A4 AChE form lasting for a few weeks may be due to a block in the synthetic process of the A12 form. In the extrajunctional regions, major differences with regard to AChE regulation exist already between the normal EDL and SOL muscle. The extrajunctional asymmetric AChE forms are absent in the EDL because they became completely repressed during the first month after birth, but they persist in the SOL. Differences remain also after denervation and are, therefore, not directly due to different neural stimulation patterns in both muscles: (a) an early but transient increase of the G4 AChE occurs in the denervated EDL but not in the SOL; (b) no significant extrajunctional activity of the asymmetric AChE forms reappears in the EDL up till 7 wk after denervation. In the SOL, activity of the asymmetric AChE forms is decreased early after denervation but increases thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iodide, thiocyanate and cyanide ions as capturing reagents in one-step copper-thiocholine method for cytochemical localization of cholinesterase activity.

The necessity of the presence of iodide in Cu-ThCh reaction was investigated by following the precipitate formation "in vitro" and by evaluating the ultrastructural localization of the precipitate in sympathetic ganglion cells of the frog and in the end-plate regions of the rat diaphragm. It was found that thiocyanate or cyanide is the only anion that can be substituted for iodide as the capturing agent in precipitation. The optimal concentration in the preincubation and incubation media of any one of the three anions is from 2 to 5 mM. At a concentration below 1 mM precipitation "in vitro" is considerably delayed as a result of which in electron microscopy diffusion artefacts appear in tissue sections. The unconverted primary precipitate obtained in the presence of iodide had been used for ultrastructural localization of ChE activity and now this use has been extended to precipitates obtained in the presence of CN- or CNS-. Better-quality localization in the presence of either one of the latter anions suggests that they, and particularly CN-, should be substituted for I- in the one-step Cu-ThCh method for the cytochemistry of cholinesterases.     

Recovery of Acetylcholinesterase in the Diaphragm, Brain, and Plasma of the Rat After Irreversible Inhibition by Soman: A Study of Cytochemical Localization and Molecular Forms of the Enzyme in the Motor End Plate

Abstract Recovery of AChE activity in the motor end plate region and end plate free region of the rat diaphragm was studied after irreversible inhibition by soman. Recovery was slow during the first 2 days and only 4 S and 10 S molecular forms of AChE were present in the end plate region. However, cytochemical evidence indicates that synaptic AChE has already started to accumulate and that the synthesis of AChE in muscle and Schwann cell might even be enhanced. Tubular structures, observed underneath the motor end plate, may serve to transport the enzyme from its sites of synthesis in the sarcoplasmic reticulum. Asymmetric molecular forms of AChE in the end plate region appeared later during recovery and, one week after poisoning, their activity was only about 50% of normal value. The limited ability of newly synthesized AChE to attach to the subcellular structures and, therefore, to be retained in the muscle, may explain the phase of slow recovery. In accordance with this view, AChE activity in brain recovered in a similar way as in muscle, whereas soluble plasma cholinesterases recovered faster, apparently without a slow initial phase.     

Effects of training on fibre composition in rat gastrocnemius muscle

Forty-eight adult Wistar rats were divided into four groups for experimentation. The group I was used as a control and groups II, III and IV underwent progressive treadmill training. Samples of the red and mixed portions of m. gastrocnemius (lateral head) were stained with the histochemical technique of m-ATPase to determine the percentage of type I, IIA and IIB fibres, and with NADH-TR, in order to quantify variations in "low-oxidative" fibre percentages. The results showed that progressive training did not lead to statistical variations in the percentage of type I fibres. However, the proportion of type IIA fibres rose, while that of IIB fibres fell, in both cases significantly. Variations were more marked in mixed than in red Gastrocnemius muscle. A clear decrease was noted in "low-oxidative" fibres, which were virtually absent from red portion. This decrease was more marked, and occurred more rapidly, than in type IIB fibres.     

Induction of natural competence in Bacillus cereus ATCC14579

Natural competence is the ability of certain microbes to take up exogenous DNA from the environment and integrate it in their genome. Competence development has been described for a variety of bacteria, but has so far not been shown to occur in Bacillus cereus. However, orthologues of most proteins involved in natural DNA uptake in Bacillus subtilis could be identified in B. cereus. Here, we report that B. cereus ATCC14579 can become naturally competent. When expressing the B. subtilis ComK protein using an IPTG‐inducible system in B. cereus ATCC14579, cells grown in minimal medium displayed natural competence, as either genomic DNA or plasmid DNA was shown to be taken up by the cells and integrated into the genome or stably maintained respectively. This work proves that a sufficient structural system for DNA uptake exists in B. cereus. Bacillus cereus can be employed as a model system to investigate the mechanism of DNA uptake in related bacteria such as Bacillus anthracis and Bacillus thuringiensis. Moreover, natural competence provides an important tool for biotechnology, as it will allow more efficient transformation of B. cereus and related organisms, e.g. to knockout genes in a high‐throughput way.     

Artifact Interpretation of Spectral Response Measurements on Two‐Terminal Multijunction Solar Cells

Multijunction solar cells promise higher power‐conversion efficiency than the single‐junction. With respect to two‐terminal devices, an accurate measurement of the spectral response requires a delicate adjustment of the light‐ and voltage‐biasing; otherwise it can result in artifacts in the data and thus misinterpretation of the cell properties. In this paper, the formation of measurement artifacts is analyzed by modeling the measurement process, that is, how the current–voltage characteristics of the component subcells evolve with the photoresponse to the incident spectrum. This enables the examination on the operation conditions of the subcells, offering additional information for the study of artifacts. In particular, the influence of shunt resistance, bias‐light intensity, and bias voltage on the measurement is examined. Having observed the dynamics and vulnerability of the measurement, the proper ways to configure and interpret a measurement are discussed in depth. As a practical example, simulations of the measurements on a quadruple‐junction thin‐film silicon solar cell demonstrate that the modeling can be used to interpret eventual irregularities in the measured spectral response. The application of such tool is especially meaningful taking account of the diverse and rapid development of novel hybrid multijunction solar cells, in which the role of reliable characterizations is essential.     

THE APPEARANCE OF ACETYLCHOLINESTERASE IN THE DORSAL ROOT NEUROBLAST OF THE RABBIT EMBRYO : A Study by Electron Microscope Cytochemistry and Microgasometric Analysis with the Magnetic Diver

In the nine day old embryo, acetylcholinesterase (AChE) is found in the reticulum, i.e. the nuclear envelope, endoplasmic reticulum, and Golgi complex, of a few cells in the neural crest. When the neurite first enters the neural tube, reticulum-bound enzyme is present also in the varicosity of the growth cone of the bipolar neuroblast. At later stages, AChE in the neuroblast has a dual distribution; in addition to the reticulum, activity also appears at the axolemmal surface. The axolemmal activity is found initially on the distal portions of axons in the posterior fasciculus and then progressively appears along the nerve roots in a distal to proximal direction. Very little reticulum-bound enzyme is present within the axon proper. After the 13th day the levels of AChE activity in the posterior fasciculus greatly exceed those in the dorsal root or in the ganglion. Enzymatic activity in the dorsal root equals or exceeds that in the posterior fasciculus by day 16, and both areas are considerably more active than the ganglion.     

Plasma cell survival is mediated by synergistic effects of cytokines and adhesion-dependent signals

Recent results suggest that plasma cell longevity is not an intrinsic capacity, but depends on yet unknown factors produced in their environment. In this study, we show that the cytokines IL-5, IL-6, TNF-alpha, and stromal cell-derived factor-1alpha as well as signaling via CD44 support the survival of isolated bone marrow plasma cells. The cytokines IL-7 and stem cell factor, crucially important for early B cell development, do not mediate plasma cell survival, indicating that plasma cells and early B cells have different survival requirements. As shown in IL-6-deficient mice, IL-6 is required for a normal induction, but not for the maintenance of plasma cell responses in vivo, indicating that the effects of individual survival factors are redundant. Optimal survival of isolated plasma cells requires stimulation by a combination of factors acting synergistically. These results strongly support the concept that plasma cell survival depends on niches in which a combination of specific signals, including IL-5, IL-6, stromal cell-derived factor-1alpha, TNF-alpha, and ligands for CD44, provides an environment required to mediate plasma cell longevity.     

Persistent nuclear accumulation of protein kinase CK2 during the G1-phase of the cell cycle does not depend on the ERK1/2 pathway but requires active protein synthesis

Protein kinase CK2 and phosphorylated ERK1/2 accumulated in nucleus after serum stimulation of quiescent HepG2 cells. Nonetheless, phospho-ERK1/2 accumulated mainly in the nuclease-extracted fraction (NE) whereas the increases in nuclear CK2 (either CK2alpha or CK2beta) occurred initially in the nuclease-resistant fraction (NR). Transient decreases in CK2 were observed in cytoplasm and NE in the first 3h but thereafter they either reverted (cytoplasm) or increased above the control (NE). CK2 levels in both NE and NR were high in cells arrested at G1/S. Maximal nuclear accumulation of CK2 was blocked by cycloheximide but little affected by PD98059, SB203580 or apigenin, all of which affected nuclear phopho-ERK1/2. Thus, nuclear accumulation of CK2 during G1 phase is independent of ERK1/2 pathway. Although this process may initially relay on intracellular redistribution of the preexisting enzyme, active protein synthesis is required to attain maximal nuclear CK2 levels.     

Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1

Anti-human immunodeficiency virus type 1 (HIV-1) antibodies whose binding to gp120 is enhanced by CD4 binding (CD4i antibodies) are generally considered nonneutralizing for primary HIV-1 isolates. However, a novel CD4i-specific Fab fragment, X5, has recently been found to neutralize a wide range of primary isolates. To investigate the precise nature of the extraordinary neutralizing ability of Fab X5, we evaluated the abilities of different forms (immunoglobulin G [IgG], Fab, and single-chain Fv) of X5 and other CD4i monoclonal antibodies to neutralize a range of primary HIV-1 isolates. Our results show that, for a number of isolates, the size of the neutralizing agent is inversely correlated with its ability to neutralize. Thus, the poor ability of CD4i-specific antibodies to neutralize primary isolates is due, at least in part, to steric factors that limit antibody access to the gp120 epitopes. Studies of temperature-regulated neutralization or fusion-arrested intermediates suggest that the steric effects are important in limiting the binding of IgG to the viral envelope glycoproteins after HIV-1 has engaged CD4 on the target cell membrane. The results identify hurdles in using CD4i epitopes as targets for antibody-mediated neutralization in vaccine design but also indicate that the CD4i regions could be efficiently targeted by small molecule entry inhibitors.