Wind-sensitive interneurones in the spider CNS (Cupiennius salei ): directional information processing of sensory inputs from trichobothria on the walking legs

Spiders can use air particle movements to localize moving prey. We studied the responses of 32 wind-sensitive interneurones in the hunting spider Cupiennius salei to prey stimuli. Stimulation with a tethered flying fly or with artificial air pulses activated plurisegmental interneurones that responded to changes in air movement velocity and were thus well suited to represent the highly fluctuating air stream typical of prey stimuli. In most interneurones (n = 18) the responses to the stimulation of different legs were not significantly different from each other. Different interneurones had different response characteristics and their latencies largely overlapped suggesting that there is parallel processing of the signals by populations of interneurones with different response characteristics. In two interneurones the number of spikes and the spiking pattern elicited by stimulation of each of the eight legs markedly differed depending on the leg stimulated. These neurones may play an important role in directional information processing. Stimulation of the adjacent legs from front to back or from back to front revealed two interneurones sensitive to the direction of successive stimulation of the legs. These neurones may be able to detect the motion of an air movement source in a preferred direction and thus act as nearfield motion detectors to localize a moving prey item. Accepted: 28 September 1996     

Corynebacterium diphtheriae: identification and characterization of a channel-forming protein in the cell wall

The cell wall fraction of the gram-positive, nontoxic Corynebacterium diphtheriae strain C8_r(−) Tox⁻ (= ATCC 11913) contained a channel-forming protein, as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent-treated cell walls and in extracts of whole cells obtained using organic solvents. The protein had an apparent molecular mass of about 66 kDa as determined on Tricine-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and consisted of subunits having a molecular mass of about 5 kDa. Single-channel experiments with the purified protein suggested that the protein formed channels with a single-channel conductance of 2.25 nS in 1 M KCl. Further single-channel analysis suggested that the cell wall channel is wide and water filled because it has only slight selectivity for cations over anions and its conductance followed the mobility sequence of cations and anions in the aqueous phase. Antibodies raised against PorA, the subunit of the cell wall channel of Corynebacterium glutamicum, detected both monomers and oligomers of the isolated protein, suggesting that there are highly conserved epitopes in the cell wall channels of C. diphtheriae and PorA. Localization of the protein on the cell surface was confirmed by an enzyme-linked immunosorbent assay. The prospective homology of PorA with the cell wall channel of C. diphtheriae was used to identify the cell wall channel gene, cdporA, in the known genome of C. diphtheriae. The gene and its flanking regions were cloned and sequenced. CdporA is a protein that is 43 amino acids long and does not have a leader sequence. cdporA was expressed in a C. glutamicum strain that lacked the major outer membrane channels PorA and PorH. Organic solvent extracts of the transformed cells formed in lipid bilayer membranes the same channels as the purified CdporA protein of C. diphtheriae formed, suggesting that the expressed protein is able to complement the PorA and PorH deficiency of the C. glutamicum strain. The study is the first report of a cell wall channel in a pathogenic Corynebacterium strain.     

Epithelial trafficking of Sonic hedgehog by megalin.

We present here evidence of in vivo epithelial endocytosis and trafficking of non-lipid-modified Sonic hedgehog (ShhN) when infused into rat efferent ducts via microinjection. Initially, exogenous ShhN is detected in endocytic vesicles and early endosomes located near the apical plasma membrane of non-ciliated cells. Within 30-60 min following infusion, ShhN can be detected in lysosomes and at basolateral regions of non-ciliated cells. Basolaterally, ShhN was observed along the extracellular surfaces of interdigitated plasma membranes of adjacent cells and in the extracellular compartment underlying the efferent duct epithelium. Uptake and subcellular trafficking of infused ShhN by non-ciliated cells could be blocked by either anti-megalin IgG or the megalin antagonist, RAP. Ciliated cells, which do not express megalin, displayed little if any apical internalization of ShhN even though they were found to express Patched-1. However, ShhN was found in coated pits of lateral plasma membranes of ciliated cells as well as in underlying endocytic vesicles. We conclude that megalin-mediated endocytosis of ShhN can occur in megalin-expressing epithelia in vivo, and that the internalized ShhN can be targeted to the lysosome or transcytosed in the plane of the epithelium or across the epithelium. These findings highlight the multiple mechanisms by which megalin may influence Shh morphogen gradients in vivo.     

Localized vs. systemic inflammation in guinea pigs: a role for prostaglandins at distinct points of the fever induction pathways?

In guinea pigs, dose-dependent febrile responses were induced by injection of a high (100 microg/kg) or a low (10 microg/kg) dose of bacterial lipopolysaccharide (LPS) into artificial subcutaneously implanted Teflon chambers. Both LPS doses further induced a pronounced formation of prostaglandin E(2) (PGE(2)) at the site of localized subcutaneous inflammation. Administration of diclofenac, a nonselective cyclooxygenase (COX) inhibitor, at different doses (5, 50, 500, or 5,000 microg/kg) attenuated or abrogated LPS-induced fever and inhibited LPS-induced local PGE(2) formation (5 or 500 microg/kg diclofenac). Even the lowest dose of diclofenac (5 microg/kg) attenuated fever in response to 10 microg/kg LPS, but only when administered directly into the subcutaneous chamber, and not into the site contralateral to the chamber. This observation indicated that a localized formation of PGE(2) at the site of inflammation mediated a portion of the febrile response, which was induced by injection of 10 microg/kg LPS into the subcutaneous chamber. Further support for this hypothesis derived from the observation that we failed to detect elevated amounts of COX-2 mRNA in the brain of guinea pigs injected subcutaneously with 10 microg/kg LPS, whereas subcutaneous injections of 100 microg/kg LPS, as well as systemic injections of LPS (intra-arterial or intraperitoneal routes), readily caused expression of the COX-2 gene in the guinea pig brain, as demonstrated by in situ hybridization. Therefore, fever in response to subcutaneous injection of 10 microg/kg LPS may, in part, have been evoked by a neural, rather than a humoral, pathway from the local site of inflammation to the brain.     

Peroxisomes and peroxisomal functions in muscle. Studies with muscle cells from controls and a patient with the cerebro-hepato-renal (Zellweger) syndrome

In the present study we investigated peroxisomal functions in cultured human muscle cells from control subjects and from a patient with the Zellweger syndrome, a genetic disease characterized by the absence of morphologically distinguishable peroxisomes in liver and kidney. In homogenates of cultured muscle cells from control subjects, catalase is contained within subcellular particles, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity is present and palmitoyl-CoA can be oxidized by a peroxisomal beta-oxidative pathway; these findings are indicative of the presence of peroxisomes in the cells. In homogenates of cultured muscle cells from the patient with the Zellweger syndrome, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity was deficient, peroxisomal beta-oxidation of palmitoyl-CoA was impaired and catalase was not particle-bound. These findings indicate that functional peroxisomes are absent in muscle from patients with the Zellweger syndrome. We conclude that cultured human muscle cells can be used as a model system to study peroxisomal functions in muscle and the consequences for this tissue of a generalized dysfunction of peroxisomes.     

Indications de la recherche des anticorps anti-spermatozoïdes

The clinical significance of antisperm antibodies (ASA) is highly controversial. A significant percentage of infertile men and women present immunity to spermatozoa, suggesting that ASA may interfere with the fertilizing capacity. ASA can act negatively on sperm parameters, sperm-cervical mucus interaction, gamete fusion and possibly also on the first step of embryonic development. ASA are present in approximately 2.8% to 26% of the male population and 0.2% to 1.6% of women. The pathogenesis of immunity to spermatozoa had not been fully elucidated: breakdown of normal protective mechanisms, i.e. blood-testis barrier, or epithelial barrier in women, and other mechanisms of immunological sperm tolerance, such as regulation of suppressor T lymphocytes. The indication for antisperm antibody testing is based on clinical and laboratory findings of infertile patients. In men, indictions for ASA testing include a history of genital disease, surgery for genital abnormalities, vasectomy, obstruction or injuries of the male genital tract, infection of accessory glands, long-standing infertility, alteration of semen parameters (agglutination, motility), mucus penetration, and reduced fertilizing capacity in IVF. In many cases, no etiological cause of autoimmunity is found and a genetic predisposition has been suggested. A majority of women do not develop antisperm antibodies, despite repeated contact with spermatozoa during their sexual life. Upper genital tract infection is the main cause of isoimmunization in females, although sexual practices, endometriosis, surgery for cervical neoplasia, recurrent spontaneous abortion and long-term infertility may also be involved. Sperm-cervical mucus impairment is the most obvious effect of immunization in women associated with IVF failure. Autoantibodies are frequently associated with antisperm antibodies. One of the consequences of the success of ICSI has been a decreased research effort to further the understanding of the origin and relevance of antisperm antibodies and specific antibody-antigen interactions. A better understanding of the natural history of immunological infertility would be useful for patient conseiling and to develop the most effective, efficient and safest management strategies. Such data could also be useful for the development of new tests and immunological methods of male contraception.     

High mitochondrial haplotype diversity of Coleps sp. (Ciliophora: Prostomatida)

To date the awareness of the temporal population structure in eukaryotic microbes is very limited. This is exemplified in the scarce knowledge about the intraspecific genetic variation in ciliates. To elucidate the genetic variation of Coleps (Ciliophora: Prostomatida), we employed the analysis of the mitochondrial apocytochrome b gene of the Coleps community in a young lake in Germany. The analysis of 111 isolates, sampled from April 2005 to September 2006, revealed a high genetic variation for the two dominant Coleps species (11 mitochondrial haplotypes in Coleps spetai , nine in Coleps hirtus hirtus ). The study represents one of the largest datasets of intraspecific diversity in a microbial eukaryote and demonstrates for the first time the suitability of a mitochondrial gene for the detection of genetic variation within populations of eukaryotic microbes. However, the results of our study warrant caution in the application of such an approach, as we amplified some non-orthologous cob -like sequences, whose uncritical acceptance would have led to the erroneous discovery of cryptic species.     

Toward a role of dendritic cells in the germinal center reaction: triggering of B cell proliferation and isotype switching

We have reported previously that in vitro generated dendritic cells (DC) can directly regulate B cell responses. Recently, germinal center DC (GCDC) were identified within B cell follicles. Due to their particular localization, we have tested in the present study whether GCDC could contribute to key events characteristic of the GC reaction. Our present results demonstrate that 1) ex vivo GCDC induce a dramatic GC B cell expansion upon CD40 and IL-2 activation and drive plasma cell differentiation, 2) this property is shared by GCDC and blood DC, but not by Langerhans cells, 3) IL-12 production by GCDC is critical in GC B cell expansion and differentiation, and 4) importantly, GCDC also induce IL-10-independent isotype switching toward IgG1. These observations support the novel concept that GCDC directly contribute to the germinal center reaction.     

P-O bond destabilization accelerates phosphoenzyme hydrolysis of sarcoplasmic reticulum Ca2+ -ATPase

The phosphate group of the ADP-insensitive phosphoenzyme (E2-P) of sarcoplasmic reticulum Ca2+ -ATPase (SERCA1a) was studied with infrared spectroscopy to understand the high hydrolysis rate of E2-P. By monitoring an autocatalyzed isotope exchange reaction, three stretching vibrations of the transiently bound phosphate group were selectively observed against a background of 50,000 protein vibrations. They were found at 1194, 1137, and 1115 cm(-1). This information was evaluated using the bond valence model and empirical correlations. Compared with the model compound acetyl phosphate, structure and charge distribution of the E2-P aspartyl phosphate resemble somewhat the transition state in a dissociative phosphate transfer reaction; the aspartyl phosphate of E2-P has 0.02 A shorter terminal P-O bonds and a 0.09 A longer bridging P-O bond that is approximately 20% weaker, the angle between the terminal P-O bonds is wider, and -0.2 formal charges are shifted from the phosphate group to the aspartyl moiety. The weaker bridging P-O bond of E2-P accounts for a 10(11)-10(15)-fold hydrolysis rate enhancement, implying that P-O bond destabilization facilitates phosphoenzyme hydrolysis. P-O bond destabilization is caused by a shift of noncovalent interactions from the phosphate oxygens to the aspartyl oxygens. We suggest that the relative positioning of Mg2+ and Lys684 between phosphate and aspartyl oxygens controls the hydrolysis rate of the ATPase phosphoenzymes and related phosphoproteins.