Studies of the M15 β-Galactosidase Complementation Process

M15 -Galactosidase was activated by heat-denatured wild-type -galactosidase, urea, and heat-denatured wild-type -galactosidase, a peptide made up of residues 6–44 of -galactosidase and CB2, the peptide that is normally used for complementation (residues 3–92 of -galactosidase). In each case roughly equal activation levels were attained. Heat-denatured wild-type -galactosidase was present as a finely divided visible white precipitate both before and after complementation. The heat-denatured protein by itself did not migrate on native PAGE and both the protein and the activity that occurred as a result of the complementation also remained at the point of application. The N-terminal ends of the heat-denatured wild-type -galactosidase must have been available for complementation and must have been mobile enough to allow tetramer to form despite being aggregated. -Galactosidase denatured by both urea and heat resulted in a streak of interacting protein on the native PAGE. Upon activation, a streak (indicating that interaction was still occurring) was still present, but it moves more slowly. Complementation using a peptide called XP (made up of residues 6–44 plus an additional nine C-terminal amino acids) resulted in three discrete forms of active enzyme at ratios of peptide to M15 -galactosidase monomer of less than 1:1. The fastest migrating of the three bands predominated at ratios near 1:1. A single active tetrameric form of M15 -galactosidase was formed with CB2. In both of these last two cases an active slow-moving diffuse band also formed (possibly a dimer of the tetramer). A quantitation of the amount of peptide bound to M15 -galactosidase by titration with XP and with CB2 and by using gel filtration after an excess of fluorescent-labeled XP was added showed that peptide bound in a 1:1 ratio (peptide/monomer) when full activity was achieved. These fluorescent studies also showed that peptide initially bound to dimer and that the tetramer was then formed.     

Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC₅₀ to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of ¹²⁵I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

α-Synemin localizes to the M-band of the sarcomere through interaction with the M10 region of titin

α-Synemin contains a unique 312 amino acid insert near the end of its C-terminal tail. Therefore we set out to determine if the insert is a site of protein–protein interaction that regulates the sub-cellular localization of this large isoform of synemin. Yeast-two hybrid analysis indicated that this region is a binding site for the M10 region of titin. This was confirmed with GST pull-down assays. Co-immunoprecipitation of endogenous proteins indicated close association of the two proteins in vivo and immunostaining of cardiomyocytes demonstrated co-localization of the proteins at the M-band of the sarcomere.     

Different contributions of the indirect effects of γ-rays on the cytotoxicity in M10 and XRCC4 transfected M10 cells

The protective effects of dimethyl sulfoxide (DMSO) against cell killing by ¹³⁷Cs γ-rays were investigated in XRCC4-deficient cell line M10, XRCC4-complemented M10 and the parental mouse leukemia cell line L5178Y. Cell survival was determined by the colony-forming ability. M10 cells were more sensitive to γ-ray-induced cell death than L5178Y and complemented M10 cells. Cell survival was increased in both M10 and L5178Y in the presence of DMSO. However, estimation of the DMSO-protectable fraction revealed a smaller protectable fraction for M10 cells than for L5178Y cells, indicating that indirect effects contributed in a smaller extent to the cytotoxicity in M10 than that in L5178Y. This effect is due to XRCC4 deficiency, since transfection of XRCC4 cDNA into M10 cells restored the radioprotective effects of DMSO to the level seen in L5178Y. In M10 cells, the killing effects of high LET radiation (Auger electrons from ¹²⁵I-antipyrine, carbon ions with an LET of 166 keV μm⁻¹) were similar to those of low LET radiation (¹³⁷Cs γ-rays, characteristic X-rays from ¹²⁵I-bovine serum albumin). We discuss that lethal lesions produced by indirect actions in L5178Y and XRCC4-complemented M10 cells may differ, at least in part, from DNA double-strand breaks repairable by non-homologous end joining.     

Morphology and distribution of Müller cells in the retina of the toad Bufo marinus

We have previously shown that an antibody against neuron-specific enolase (NSE) selectively labels Müller cells (MCs) in the anuran retina (Wilhelm et al. 1992). In the present study the light- and electron-microscopic morphology of MCs and their distribution were described in the retina of the toad, Bufo marinus, using the above antibody. The somata of MCs were located in the proximal part of the inner nuclear layer and were interconnected with each other by their processes. The MCs were uniformly distributed across the retina with an average density of 1500 cells/mm². Processes of MCs encircled the somata of photoreceptor cells isolating them from each other by glial sheath, except for those of the double cones. Some of the photoreceptor pedicles remained free of glial sheath. Electron-microscopic observations confirmed that MC processes provide an extensive scaffolding across the neural retina. At the outer border of the ganglion cell layer these processes formed a non-continuous sheath. The MC processes traversed through the ganglion cell layer and spread beneath it between the neuronal somata and the underlying optic axons. These processes formed a continuous inner limiting membrane separating the optic fibre layer from the vitreous tissue. Neither astrocytic nor oligodendrocytic elements were found in the optic fibre layer. The significance of the uniform MC distribution and the functional implications of the observed pattern of MC scaffolding are discussed.     

A commentary on the G₂/M transition of the plant cell cycle

Background

The complex events of mitosis rely on precise timing and on immaculate preparation for their success, but the G₂/M transition in the plant cell cycle is currently steeped in controversy and alternative models.

Scope

In this brief review, the regulation of the G₂/M transition in plants is commented on. The extent to which the G₂/M transition is phosphoregulated by WEE1 kinase and CDC25 phosphatase, as exemplified in yeasts and animals, is discussed together with an alternative model that excludes these proteins from this transition. Arabidopsis T-DNA insertional lines for WEE1 and CDC25 that develop normally prompted the latter model. An argument is then presented that environmental stress is the norm for higher plants in temperate conditions. If so, the repressive role that WEE1 has under checkpoint conditions might be part of the normal cell cycle for many proliferative plant cells. Arabidopsis CDC25 can function as either a phosphatase or an arsenate reductase and recent evidence suggests that cdc25 knockouts are hypersensitive to hydroxyurea, a drug that induces the DNA-replication checkpoint. That other data show a null response of these knockouts to hydroxyurea leads to an airing of the controversy surrounding the enigmatic plant CDC25 at the G₂/M transition.     

Characterization of Group A Streptococcal M23 Protein and Comparison of the M3 and M23 Protein’s Ligand-Binding Domains

The present study concerns the properties for binding of human plasma and extracellular matrix proteins and the relationship between M3 and M23 molecules. Here, it is demonstrated that M23 protein shows a multiple binding to fibrinogen (FG), fibronectin (FN), human serum albumin (HSA), immunoglobulin G (IgG), kininogen, and collagen type I (CI) in Western blot analysis. Some sets of truncated-recombinant M3 or M23 protein fragments were assayed for their capacity to bind FN, FG, IgG, HSA, and CI. The HSA binding activity resided in the C-repeat region of M3 protein, whereas fibrinogen-binding activity resided in the A-repeat region. The FG, FN, and IgG binding sites were mapped to the N-terminal portion of M23 protein, whereas HSA binding was localized in the B-repeat domain, which has homology with C-repeat domain in M3 molecule. Therefore, it is concluded that the FN, FG, and IgG binding regions in the M3 and M23 proteins are quite dissimilar at the amino acid sequence level, whereas HSA binding is localized to the conserved C-repeat domain in the M3 and M23 proteins.     

M?ssbauer spectroscopy applied to the oxidized and semi-reduced states of the iron-molybdenum cofactor of nitrogenase

M?ssbauer parameters at 125K for both the oxidized and semi-reduced states of FeMoco isolated from the MoFe protein of Azotobacter vinelandii nitrogenase of delta/Fe = 0.32 and 0.37 mm/s and delta Eq = 0.84 and 0.71 mm/s, respectively, are reported. FeMoco(ox) fits the Debye model perfectly from 4.2-125K and has a S = 0 ground state. FeMoco(ox) apparently contains 10-20% FeMoco(s-r) and vice versa, possibly as a result of the spontaneous oxidation phenomenon. Quantitation of the spectra indicates a Fe:Mo ratio of 5 +/- 1:1 and the similar quadrupole splittings and isomer shifts suggest a similar environment for all iron atoms.     

Analysis of the Angiotensinogen Gene T174M Polymorphism in Populations of the Volga–Ural Region

The method of polymerase chain reaction was used to analyze T174M polymorphism at the angiotensinogen (AGT) gene in a number of populations of the Volga–Ural region, belonging to Finno–Ugric (Komi-Permyaks, Maris, Mordovians, and Udmurts), Turkic (Chuvashes, Tatars, and Bashkirs), and Eastern-Slavic (Russians) ethnic groups. Population-specific patterns of the polymorphic alleles and genotypes frequency distribution were established. Comparison of the results with the literature data on the AGT gene polymorphism in different world populations provided identification of specific trends in the changes of genotype frequency of the AGT gene depending on the ethnicity of the populations.     

Detection of symmetrical decomposition of molecules—isotopomeric analysis of the M/2 clusters

The interpretation of mass spectra (ms) of molecules containing poly-isotopic elements (e.g. Ge, Se, W, Os, Sn, Te, Zn, Yb) can be difficult due to the occurrence of fragments resulting from isotopomeric composition. MS-clusters located in the range lower than or equal to M/2 are very difficult to interpret. In this area many perturbations may be observed. The coincidence of different fragmentation pathways, the existence of multiply charged ions, background levels, etc. can all contribute to this problem. The present paper reports the application of multi-isotopomeric analysis methods for low-resolution ms. We present a solution that may be useful for detection of the symmetrical decomposition of a molecule and for elucidation of cluster ion genesis. The complex character of the cluster does not perturb determination of the contents of the investigated pattern. In such cases the dominated component is applied in subsequent computations.

The influence of the reference electrode location on the M−wave characteristics in the quadriceps

IntroductionIn the compound muscle action potential (M wave) recorded using the belly-tendon configuration, the contribution of the tendon electrode is assumed to be negligible compared to the belly electrode. We tested this assumption by placing the reference electrode at a distant (contralateral) site, which allowed separate recording of the belly and tendon contributions.MethodsM waves were recorded at multiple selected sites over the right quadriceps heads and lower leg using two different locations for the reference electrode: the ipsilateral (right) and contralateral (left) patellar tendon. The general parameters of the M wave (amplitude, area, duration, latency, and frequency) were measured.Results(1) The tendon potential had a small amplitude (<30%) compared to the belly potential; (2) Changing the reference electrode from the ipsilateral to the contralateral patella produced moderate changes in the M wave recorded over the innervation zone, these changes affecting significantly the amplitude of the M−wave second phase (p = 0.006); (3) Using the contralateral reference system allowed recording of short-latency components occurring immediately after the stimulus artefact, which had the same latency and amplitude (p = 0.18 and 0.25, respectively) at all recording sites over the leg.ConclusionsThe potential recorded at the “tendon” site after femoral nerve stimulation is small (compared to the belly potential), but not negligible, and makes a significant contribution to the second phase of belly-tendon M wave. Adopting a distant (contralateral) reference allowed recording of far-field components that may aid in the understanding of the electrical formation of the M wave.     

Immunoreactivity of the hermaphroditic gonad of the snail Helix aspersa Müller towards antibodies raised to fragments of pre-pro-opio-melanocortin

Summary Male and female germinal cells of the active hermaphroditic gonad of a snail (Helix aspersa Müller) reveal a positive immunoreactivity to some antibodies raised against biologically active peptides related to pre-pro-opiomelanocortin of vertebrates. All but the oldest cells of the different spermatogenetic and oogenetic stages are methionine-enkephalin-immunopositive, whereas only the young oocytes are -MSH- and 17–39 ACTH-positive. Sometimes some male cells show an 1–24 ACTH positivity. Structures other than germinal cells also react with some antibodies: for example, the nurse cells are -MSH-immunoreactive, the nerve fibers surrounding each acinus and the hermaphrodite duct are both 17–39 ACTH and FMRF amide positive.     

Use of M1–M5 Muscarinic Receptor Knockout Mice as Novel Tools to Delineate the Physiological Roles of the Muscarinic Cholinergic System

In this review we report recent findings on the physiological role of the five known muscarinic acetylcholine receptors (mAChRs) as shown by gene targeting technology. Using knockout mice for each mAChRs subtype, the role of mAChRs subtypes in a number of physiological functions was confirmed and new activities were discovered. The M₁ mAChRs modulate neurotransmitter signaling in cortex and hippocampus. The M₃ mAChRs are involved in exocrine gland secretion, smooth muscle contractility, pupil dilation, food intake, and weight gain. The role of the M₅ mAChRs involves modulation of central dopamine function and the tone of cerebral blood vessels. mAChRs of the M₂ subtype mediate muscarinic agonist-induced bradycardia, tremor, hypothermia, and autoinhibition of release in several brain regions. M₄ mAChRs modulate dopamine activity in motor tracts and act as inhibitory autoreceptors in striatum. Thus, as elucidated by gene targeting technology, mAChRs have widespread and manifold functions in the periphery and brain.     

Anatomy and uses of the mature leaves of three species of Sabal (Arecaceae) of the Yucatan, México

This paper describes the leaf anatomy of Sabal mauritiiformis (Karst.) Griseb. & H. Wendl., Sabal mexicana Mart. and Sabal yapa Wright ex Becc., three of the four most representative species of the Yucatán Península, in Mexico. These species are locally used: in the roofing of traditional homes, as food (fruits and apical buds), and in the production of hats, brooms and handicrafts. Leaf samples were collected in secondary growth of lower montane rainforest in the state of Quintana Roo and in two home gardens in the state of Yucatán. Herbarium samples were obtained, and samples of blade and petiole were fixed in formaline-acetic acid-alcohol. Cross incisions were made on the blade and petiole, and were dyed with safranin and toluidine blue O. The results show that S. mauritiiformis and S. yapa are morphologically alike: both are tall, slim palm trees; the leaf in S. mauritiiformis is a shorter palm-like structure compared with the other two species. The shape of the main nerve, as seen in cross section, is rectangular in the three species. The hastula in the three species is acuminate and adaxial. The foliar anatomic structure is similar in the three species, although there are some differences. The adaxial an abaxial epidermis of the blade consist of one layer and, superficially, the anticlinal walls are straight; the stomata are intercostal, of the tetracytic type, present on both surfaces in S. mexicana and S. yapa and only on the abaxial surface on S. mauritiiformis. The hypodermis is one layer thick in S. yapa and in S. mexicana and two layers thick in S. mauritiiformis. In the three species the palisade parenchyma consists of several undefined strata as the cells are similar-in shape and size--to the cells in the spongy parenchyma, so there is no marked difference between these strata and the spongy parenchyma seems almost continuous. Both fibrous and vascular bundles are distributed between the hypodermis and the palisade parenchyma; the fiber bundles can be found towards the abaxial surface while the vascular and fiber bundles are located towards the adaxial surface. The fibers, in the three species, are elongated, with the pointed tips, undivided and unseptated. One to three wide vessels of metaxilem can be seen in the vascular bundles, those in S. yapa being the widest in diameter. The vascular bundles are surrounded by thick fiber sheaths which come in pairs. The anatomic structure of the petiole is similar to that of the blade, and is characterized by the many vascular and fiber bundles dispersed in the parenchymatous tissue, and which are very resistant. The histological structure of the blade and petiole reflects strength and flexibility, qualities which make these plants adequate in the construction of roofs for rural housing and other buildings.