Changes of epidermal cell morphology and keratin expression induced by inhibitors of protein kinase C.

Several lines of evidence show protein kinase C as being involved in various regulatory processes in keratinocyte biology, e.g. proliferation and differentiation. In the present study, we investigated the effects of three different inhibitors of protein kinase C, staurosporine, CP 46'665-1, and tiflucarbine, on cell morphology and keratin expression in a non-tumorigenic human keratinocyte cell line (HaCaT cells). Staurosporine, being the most potent inhibitor of protein kinase C activity in vitro, and CP 46'665-1 induced morphological transformation to a fibroblast-like cell shape. In contrast, no changes in cell morphology were observed after exposure to tiflucarbine. The investigation of keratin expression in HaCaT cells grown in the presence of the different compounds revealed the following changes: After 72 h of cultivation, keratins 8 and 18 were still expressed in treated cells, whereas expression of keratin 13 was decreased as compared to control cells. Immunoblotting to detect vimentin demonstrated its absence in treated and control cells. Since tiflucarbine is known as a dual protein kinase C/calmodulin inhibitor whereas staurosporine and CP 46'665-1 do not antagonize calmodulin function, it might be possible that not only protein kinase C but also calmodulin is involved in the process leading to the morphological changes.     

Proliferation and production of hemopoietic cells in two stages of disease: Preleukemia and overt leukemia

Summary The kinetics of erythropoietic and granulocytopoietic cell proliferation have been investigated in the same patient at two distinct stages: firstly in preleukemia presenting as pancytopenia with ineffective erythropoiesis, and secondly 2 years later in acute myelogenous leukemia. The method of investigation is based on determining the DNA synthesis rate of individual cells by means of quantitative¹⁴C-autoradiography after short-term incubation with¹⁴C-thymidine and fluorodeoxyuridine.Erythropoiesis was equally ineffective in the two stages, the rate of proliferation, however, slowed down towards the leukemic state. The production rate of myeloblasts was markedly reduced in preleukemia accompanied by a very low labelling index. In leukemia on the other hand the production rate was increased to such a degree that the leukemic myeoblast compartment is to be considered as prevailingly self-reproductive. The proliferation rate of myeloblasts was reduced already in preleukemia, and there was a further decrease in leukemia. From the point of view of cell kinetics the manifestation of leukemia in this patient is explained best by a change in the mode of proliferation: the myeloblasts change from steady state growth to behaving like an exponentially expanding population.Study performed under the association contract for hematology between EURATOM and GSF no. 089-72-I-BIAD Supported by the Deutsche Forschungsgemeinschaft: SFB 51/E-3     

Antigenic and molecular analyses of different Chlamydia pneumoniae strains.

Chlamydia pneumoniae is an important human respiratory pathogen. Classification of C. pneumoniae isolates into distinguishable serovars or genotypes has not yet been reported. To determine whether antigenic or molecular variants among C. pneumoniae isolates exist, six strains were studied via immunoblot analysis and DNA sequence determination of the entire major outer membrane protein (MOMP) gene omp1. The strains included four prototype strains and two clinical isolates from our laboratory. Immunoblot analysis of sera from patients infected with C. pneumoniae revealed antigenic differences between the C. pneumoniae strains. Strong reactivity of one serum sample with a 65-kDa protein in two C. pneumoniae strains which was not observed with the other strains was the most prominent finding. All sera reacted with the 40-kDa MOMP. Comparison of the omp1 DNA sequences revealed that the omp1 genes of all strains were identical and were 100% identical to the sequence of the omp1 gene of C. pneumoniae AR-39. The results of this study demonstrate that unlike C. trachomatis, the omp1 gene is conserved in C. pneumoniae. Furthermore, it was shown that C. pneumoniae strains are antigenically different. This finding indicates that more than one serovar of C. pneumoniae exist.     

IR-und UV-spektroskopische Eigenschaften einer homologen Reihe von molekulareinheitlichen, 12gliedrige Ringe enthaltenden Leiteroligomeren

The reactions of multiple acrylates of oligo[(hydroxyphenylene)methylene] s ( 2a–2f ), strongly diluted in boiling benzene, with 2,2′-azoisobutyronitrile in a mole ratio of 1:10, were investigated. The elemental analysis and the determination of relative molar masses of the resulting products ( 3, 4, 5, 6a, 6b, 7a and 7b ) demonstrate that their molecules contain two nitrile groups. These data, together with the IR and UV spectra, show that the products are molecularly uniform ladder oligomers with two 1-cyano-1-methylethyl groups on both ends.     

Osteoplastic frontal sinusotomy and extradural microsurgical repair of frontobasal cerebrospinal fluid fistulas

Summary The choice of the surgical approach and operative technique for the management of cerebrospinal fluid (CSF) fistulas of the anterior cranial fossa are still a controversially discussed topic. Although extracranial approaches through the paranasal sinuses are becoming increasingly more popular among otolaryngologists and maxillo-facial surgeons, most neurosurgeons traditionally prefer the intracranial repair of CSF fistulas by a craniotomy.We present an approach through the frontal sinus for the repair of dural defects behind the posterior wall of the frontal sinus and at the floor of the anterior cranial fossa. The operative procedure comprises the following main steps: 1) exposure of the anterior wall of the frontal sinus by a bicoronal incision; 2) excision of the anterior wall without frontal burr holes; 3) bilateral removal of the posterior wall of the fronal sinus; 4) extradural inspection of the dura behind the frontal sinus and above the cribriform plate, ethmoidal roof, and orbital roof bilaterally; 5) closure of dural tears by direct suture and a periosteal graft; 6) reinsertion of the anterior wall of the frontal sinus and fixation with titanium micro plates.Twenty-five patients operated upon using this technique are described. The aetiology of the frontobasal lesion was traumatic in 23, and an ethmoid carcinoma in two. In all patients, the dural fistulas were successfully repaired during the initial procedure. One patient died from sudden circulatory arrest after an uneventful postoperative course of nine days. Otherwise, there were no postoperative complications.This technique affords atraumatic extradural inspection and repair of dural fistulas bilaterally behind the frontal sinus, and above the cribriform plate and the ethmoidal and orbital roofs with none or minimal brain retraction. It therefore allows early repair of CSF fistulas also in patients with severe brain injury. Although we consider the extradural closure of fistulas the method of choice, this approach also allows for a combined extradural-intradural procedure, thus enabling the surgeon to treat associated intradural pathologies, such as traumatic lesions or tumours of the frontal cranial base.     

Reticuloendotheliomatosen

Ohne Zusammenfassung     

Homozygous and compound heterozygous mutations in the ATP6V1B1 gene in patients with renal tubular acidosis and sensorineural hearing loss

Mohebbi N, Vargas‐Poussou R, Hegemann SCA, Schuknecht B, Kistler AD, Wüthrich RP, Wagner CA. Homozygous and compound heterozygous mutations in the ATP6V1B1 gene in patients with renal tubular acidosis and sensorineural hearing loss. Distal renal tubular acidosis (dRTA) is characterized by the inability to excrete acid in the renal collecting ducts resulting in inappropriately alkaline urine and hyperchloremic (normal anion gap) metabolic acidosis in the context of a normal (or near‐normal) glomerular filtration rate. Inborn dRTA can be due to autosomal dominant or recessive gene defects. Clinical symptoms vary from mild acidosis, incidental detection of kidney stones or renal tract calcification to severe findings such as failure to thrive, severe metabolic acidosis, and nephrocalcinosis. The majority of patients with recessive dRTA present with sensorineural hearing loss (SNHL). Few cases with abnormal widening of the vestibular aqueduct have been described with dRTA. Mutations in three different genes have been identified, namely SLC4A1, ATP6V1B1, and ATP6V0A4. Patients with mutations in the ATP6V1B1 proton pump subunit develop dRTA and in most of the cases sensorineural hearing loss early in childhood. We present two patients from two different and non‐consanguineous families with dRTA and SNHL. Direct sequencing of the ATP6V1B1 gene revealed that one patient harbors two homozygous mutations and the other one is a compound heterozygous. To our knowledge, this is the first case in the literature describing homozygosity in the same dRTA gene on both alleles.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near −65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor–based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure–function relationships of the light-gated pore.Discovery and engineering of the microbial opsin genes not only has stimulated basic science investigation into the structure–function relationships of proteins involved in light-triggered ion flow but also has opened up opportunities for biological investigation (reviewed in ref. 1) via the technique of optogenetics, which involves targeting these genes and corresponding optical stimuli to control activity within specified types of cells within intact and functioning biological systems. For example, optogenetics has been used to identify causally the brain cells and projections involved in behaviors relevant to memory formation, affective states, and motor function, among many other discoveries (2–4). For the channelrhodopsins, an important member of this protein family widely used in optogenetics (5, 6), the light-activated cation-conducting channel pore has been the subject of structural investigation, both because of curiosity regarding the physical properties of its ion conduction and because the creation of inhibitory channels had been sought for optogenetic applications. Converging lines of work recently achieved the latter goal; resolving the high-resolution structure of channelrhodopsin (7) allowed a principled structure-guided approach to engineering for chloride selectivity by testing an electrostatic model for pore function (8, 9). Subsequently, by screening the genome of the Guillardia theta microbe, two naturally occurring light-gated chloride-conducting channelrhodopsins (10) were identified.Because optogenetic control of behavior has not yet been demonstrated with chloride channelrhodopsins, and to test further integrative ideas regarding pore function from structural considerations as shown here, we sought to design and test the next generation of enhanced chloride channels (iC++ and SwiChR++). Along the way, we provide the initial test of the hypothesis that light-activated channels will be more efficient tools than pumps for optogenetic neuronal inhibition at the cellular level, demonstrate the initial utility of light-gated chloride channels in controlling behavior in freely moving animals, and reveal key principles regarding the functional selectivity of light-gated ion channel pores.     

Drüsen (ekkrine und endokrine)

Ohne Zusammenfassung