Glutamate 85 and glutamate 228 contribute to the pH-response of the soluble form of chloride intracellular channel 1

The chloride intracellular channel protein, CLIC1, is synthesised as a soluble monomer that can reversibly bind membranes. Soluble CLIC1 is proposed to respond to the low pH found at a membrane surface by partially unfolding and restructuring into a membrane-competent conformation. This transition is proposed to be controlled by strategically located “pH-sensor” residues that become protonated at acidic pH. In this study, we investigate the role of two conserved glutamate residues, Glu85 in the N-domain and Glu228 in the C-domain, as pH-sensors. E85L and E228L CLIC1 variants were created to reduce pH sensitivity by permanently breaking the bonds these residues form. The structure and stability of each variant was compared to the wild type at both pH 7.0 and pH 5.5. Neither substitution significantly altered the structure but both decreased the conformational stability. Furthermore, E85L CLIC1 formed a urea-induced unfolding intermediate state at both pH 7 and pH 5.5 compared to wild-type and E228L CLIC1 which only formed the intermediate at pH 5.5. We conclude that Glu85 and Glu228 are two of the five pH-sensor residues of CLIC1 and contribute to the pH-response in different ways. Glu228 lowers the stability of the native state at pH 5.5, while Glu85 contributes both to the stability of the native state and to the formation of the intermediate state. By putting these interactions into the context of the three previously described CLIC1 pH-sensor residues, we propose a mechanism for the conversion of CLIC1 from the soluble state to the pre-membrane form.     

How reliable are the Powell–Wetherall plot method and the maximum-length approach? Implications for length-based studies of growth and mortality

Length-based methods are the cornerstone of many population studies and stock assessments. This study tested two widely used methods: the Powell–Wetherall (P–W) plot and the L_max approach (i.e., estimating L_∞ directly from L_max). In most simulations, P–W estimates of the ratio total mortality/growth (Z/K ratio) were biased beyond acceptable limits (bias?>?30%). Bias in Z/K showed a complex behavior, without possible corrections. Estimates of asymptotic length (L_∞) were less biased than Z/K, but were very sensitive to intra-cohort variability in growth and to changes in the occurrence of large individuals in the sample. Exclusion of the largest size classes during the regression procedure or weighing by abundance does not solve these issues. Perfect linearization of the data and extremely narrow confidence intervals for Z/K will lead users to erroneous overconfidence in outputs. Clearly, the P–W method is not suitable for the assessment of Z/K ratios of natural populations. Estimation of L_∞ may be tentatively possible under very specific conditions, with necessary external verifications. Also, this study demonstrates that there is no way to estimate L_∞ directly from L_max, since there is no particular relationship to expect a priori between L_∞ and L_max. Errors in estimating L_∞ directly affect the estimate of the growth constant K and all other subsequent calculations in population studies, stock assessments and ecosystem models. New approaches are urgently needed for length-based studies of body growth (e.g., unconstrained curve fit with subsequent bootstrapping), that consider the inherent uncertainty regarding the underlying data and processes.     

Microanatomy and Development of the Dwarf Male of Symbion pandora (Phylum Cycliophora): New Insights from Ultrastructural Investigation Based on Serial Section Electron Microscopy

Cycliophorans have a complex life cycle that involves several sexual and asexual stages. One of the sexual stages is the 40 μm-long dwarf male, which is among the smallest free-living metazoans. Although the dwarf male has a highly complex body plan, this minute organism is composed of a very low number of somatic cells (~50). The developmental processes that give rise to this unique phenotype are largely unknown. Here we use high resolution serial block face—scanning electron microscopy to analyze the anatomy and morphogenesis of three cycliophoran dwarf males at different developmental stages ranging from internal bud to mature male. The anatomical and morphological features of the mature dwarf male stage reported here largely correspond to those reported in earlier studies. Interestingly, the organs that typically characterize the anatomy of the mature dwarf male, e.g., muscles, brain, testis and glands, are already formed in the young male. However, there are striking differences between the mature male and young male stages at the level of cellular architecture. Thus, while the young male stage, like the internal bud stage, possesses approximately 200 nucleated cells, the mature male stage comprises only around 50 nucleated cells; muscle and epidermal cells of the mature male lack nuclei. Moreover, the total body volume of the mature male is only 63% of the body of the young male implying that the maturation of the young male into a mature male involves a marked reduction of internal body volume, mainly by massive nuclei loss. Our comparative analysis of these dwarf male specimens reveals unprecedented insight into the striking morphological and developmental differences that characterize these highly miniaturized male stages both at the level of body organization and at the level of cellular ultrastructure.     

Central nervous control of hindleg coordination in stridulating grasshoppers

 Stridulation in many gomphocerine grasshoppers is characterized by specific phase shifts between the two hindlegs as well as different movement patterns produced by the left and the right leg. The underlying neuronal excitation patterns are generated by networks on either side of the metathoracic ganglion. The role of the intraganglionic commissures in right-left coordination and the production of differing movement patterns was investigated by transecting the metathoracic ganglion mediosagittally in Omocestus viridulus, Chorthippus biguttulus and Chorthippus mollis. In all three species, after this operation both hindlegs produced the same pattern and no longer different movements. The effects of transection on coordination differed: rapid movement rhythms, like those typical of Ch. biguttulus and the vibratory parts of the song of Ch. mollis, on the two sides drifted with respect to one another. In contrast, the slow rhythms characteristic of O. viridulus and the song subunits of Ch. mollis were completely synchronized. It is inferred that in intact animals the pathways for coordination of the rapid stridulatory rhythms are exclusively intraganglionic, whereas the phase relations of the slow rhythms are additionally influenced by way of anterior right-left connections, perhaps within the suboesophageal ganglion. Accepted: 15 October 1996     

Aminergic neuron systems of lobsters: morphology and electrophysiology of octopamine-containing neurosecretory cells

In the American lobster (Homarus americanus) the biogenic amines serotonin and octopamine appear to play important and opposite roles in the regulation of aggressive behavior, in the establishment and/or maintenance of dominant and subordinate behavioral states and in the modulation of the associated postural stances and escape responses. The octopamine-containing neurosecretory neurons in the thoracic regions of the lobster ventral nerve cord fall into two morphological subgroups, the root octopamine cells, a classical neurohemal group with release regions along second thoracic roots, and the claw octopamine cells, a group that selectively innervates the claws. Cells of both subgroups have additional sets of endings within neuropil regions of ganglia of the ventral nerve cord. Octopamine neurosecretory neurons generally are silent, but when spontaneously active or when activated, they show large overshooting action potentials with prominent after-hyperpolarizations. Autoinhibition after high-frequency firing, which is also seen in other crustacean neurosecretory cells, is readily apparent in these cells. The cells show no spontaneous synaptic activity, but appear to be excited by a unitary source. Stimulation of lateral or medial giant axons, which excite serotonergic cells yielded no response in octopaminergic neurosecretory cells and no evidence for direct interactions between pairs of octopamine neurons, or between the octopaminergic and the serotonergic sets of neurosecretory neurons was found.     

A familial mutation in the testis-determining gene SRY shared by both sexes

A familial mutation in SRY, the gene coding for the testis-determining factor TDF, was identified in an XY female with gonadal dysgenesis, her father, her two brothers and her uncle. The mutation consists of a T to C transition in the region of the SRY gene coding for a protein motif known as the high mobility group (HMG) box, a protein domain known to confer DNA-binding specificity on the SRY protein. This point mutation results in the substitution, at amino acid position 109, of a serine residue for phenylalanine, a conserved aromatic residue in almost all HMG box motifs known. This F109S mutation was not found in 176 male controls. When recombinant wildtype SRY and SRY^F109S mutant protein were tested in vitro for binding to the target site AAC AAAG, no differences in DNA-binding activity were observed. These results imply that the F109S mutation either is a rare neutral sequence variant, or produces an SRY protein with slightly altered in vivo activity, the resulting sex phenotype depending on the genetic back-ground or environmental factors.This paper is dedicated by G. S. to Professor Ulrich Wolf on the occasion of his 60th birthday     

Kinetics of Oxygen Release from ORC

Oxygen release compounds (ORC) are one possibility to enhance aerobic degradation in contaminated aquifers. However, some applications have been reported where oxygen concentrations did not meet expectations, this was attributed to ground water composition, e.g., high pH. Column experiments have been performed and the measurements were interpreted using a numerical model to investigate oxygen release kinetics from ORC in more detail. Because the zero-order rate law recommended by the manufacturer did not reflect the measurements, a more complex kinetic scheme was developed. The simulations show a minor influence of inorganic ground water constituents on oxygen release from ORC in the columns due to buffering by mineral precipitation, but an enhanced oxygen release if aerobic degradation takes place. If ORC is applied as socks, the impact of inorganic ground water composition increases compared to the application in column experiments. A simple quadratic equation is provided to estimate oxygen release rate from the buffer capacity of the ground water versus increasing pH—a parameter easily determinable in the laboratory. For slightly mineralized waters with high pH, this equation forecasts decreased oxygen release, but no total inhibition of oxygen release.     

Inactivation of glutamine synthetase by adenylylation in intact cells of E. coli

Summary The adenine pool of a purineless mutant of E. coli was radioactively labelled by short incubation with ¹⁴C-adenine.The glutamine synthetase was inactivated in vivo by incubation of the cell suspension with 2x10^-3 M NH₄ ⁺ for 2 min. The inactivated glutamine synthetase was extracted from the cells and purified 20-fold.Incubation of the purified glutamine synthetase with phosphodiesterase regenerated the biosynthetic activity of the enzyme paralleled by the liberation of ¹⁴C-adenine and ¹⁴C-adenosine. ¹⁴C-adenine and ¹⁴C-adenosine were also obtained when inactivated glutamine synthetase, prepared in vitro by use of ¹⁴C-ATP and purified adenylylating enzyme, was incubated with phosphodiesterase under the same conditions.The similar liberation of adenine derivatives by phosphodiesterase from glutamine synthetase inactivated in a cell-free system as well as in intact cells, demonstrates that in both cases the inactivation consists in an adenylylation of the enzyme.     

K+(Rb+) and Ca2+ fluxes in young winter wheat plants exposed to sub-zero temperatures

Ice crystal formation temperature was determined in the region of the crown in one group of 7-day-old intact unhardened high-salt plants of winter wheat (Triticum aestivum L. cv. Weibulls Starke II) with TA (Thermal Analysis) and DTA (Differential Thermal Analysis) methods. After exposure of another group of plants, grown for the first 7 days in the same way as the first group, to various sub-zero temperatures (-1 to 5°C), influx in roots of Rb⁺(⁸⁶Rb⁺) and Ca²⁺(⁴⁵Ca²⁺) and contents of K⁺ and Ca²⁺ were determined at intervals during 7 days of recovery. Ice crystal formation in the crown tissue was probably extracellular and took place at about -4°C. There was a large loss of K⁺ from the roots after treatment at sub-zero temperatures. This loss increased as the temperature of the sub-zero treatment decreased. During recovery, roots of plants exposed to -1, -2 and -3°C gradually reabsorbed K⁺. Reabsorption of K⁺ in roots of plants exposed to -4°C was greatly impaired. Rb⁺ influx decreased and Ca²⁺ influx increased after sub-zero temperature treatments of the plants. Active Rb⁺ influx mechanisms and active extrusion of Ca²⁺ were impaired or irreversibly damaged by the exposure. While Rb⁺ influx mechanisms were apparently repaired during recovery in plants exposed to temperatures down to -3°C, Ca²⁺ extrusion mechanisms were not. The temperature for ice crystal formation in the region of the crown tissue coincides with the temperature at which the plants lost the ability to reabsorb K⁺ and to repair Rb⁺ influx mechanisms during the recovery period. Plants were lethally damaged at temperatures below ?4°C.