Loss of K-Cl co-transporter KCC3 causes deafness, neurodegeneration and reduced seizure threshold

K-Cl co-transporters are encoded by four homologous genes and may have roles in transepithelial transport and in the regulation of cell volume and cytoplasmic chloride. KCC3, an isoform mutated in the human Anderman syndrome, is expressed in brain, epithelia and other tissues. To investigate the physiological functions of KCC3, we disrupted its gene in mice. This severely impaired cell volume regulation as assessed in renal tubules and neurons, and moderately raised intraneuronal Cl(-) concentration. Kcc3(-/-) mice showed severe motor abnormalities correlating with a progressive neurodegeneration in the peripheral and CNS. Although no spontaneous seizures were observed, Kcc3(-/-) mice displayed reduced seizure threshold and spike-wave complexes on electrocorticograms. These resembled EEG abnormalities in patients with Anderman syndrome. Kcc3(-/-) mice also displayed arterial hypertension and a slowly progressive deafness. KCC3 was expressed in many, but not all cells of the inner ear K(+) recycling pathway. These cells slowly degenerated, as did sensory hair cells. The present mouse model has revealed important cellular and systemic functions of KCC3 and is highly relevant for Anderman syndrome.     

21st century zoo design

A review of Conservation Centres for the New Millennium. Proceedings of the 5^th International Symposium on Zoo Design, edited by Amy B. Plowman and Peter M.C. Stevens. Paignton, Devon, U.K.: Whitely Wildlife Conservation Trust, 1999, 181 pp., paperback.     

5-Aminolevulinic Acid Induced Fluorescence Is a Powerful Intraoperative Marker for Precise Histopathological Grading of Gliomas with Non-Significant Contrast-Enhancement

Background

Intraoperative identification of anaplastic foci in diffusely infiltrating gliomas (DIG) with non-significant contrast-enhancement on MRI is indispensible to avoid histopathological undergrading and subsequent treatment failure. Recently, we found that 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) fluorescence can visualize areas with increased proliferative and metabolic activity in such gliomas intraoperatively. As treatment of DIG is predominantely based on histopathological World Health Organisation (WHO) parameters, we analyzed whether PpIX fluorescence can detect anaplastic foci according to these criteria.

Methods

We prospectively included DIG patients with non-significant contrast-enhancement that received 5-ALA prior to resection. Intraoperatively, multiple samples from PpIX positive and negative intratumoral areas were collected using a modified neurosurgical microscope. In all samples, histopathological WHO criteria and proliferation rate were assessed and correlated to the PpIX fluorescence status.

Results

A total of 215 tumor specimens were collected in 59 patients. Of 26 WHO grade III gliomas, 23 cases (85%) showed focal PpIX fluorescence, whereas 29 (91%) of 33 WHO grade II gliomas were PpIX negative. In intratumoral areas with focal PpIX fluorescence, mitotic rate, cell density, nuclear pleomorphism, and proliferation rate were significantly higher than in non-fluorescing areas. The positive predictive value of focal PpIX fluorescence for WHO grade III histology was 85%.

Conclusions

Our study indicates that 5-ALA induced PpIX fluorescence is a powerful marker for intraoperative identification of anaplastic foci according to the histopathological WHO criteria in DIG with non-significant contrast-enhancement. Therefore, application of 5-ALA optimizes tissue sampling for precise histopathological diagnosis independent of brain-shift.     

Homozygous and Compound-Heterozygous Mutations in TGDS Cause Catel-Manzke Syndrome

Catel-Manzke syndrome is characterized by Pierre Robin sequence and a unique form of bilateral hyperphalangy causing a clinodactyly of the index finger. We describe the identification of homozygous and compound heterozygous mutations in TGDS in seven unrelated individuals with typical Catel-Manzke syndrome by exome sequencing. Six different TGDS mutations were detected: c.892A>G (p.Asn298Asp), c.270_271del (p.Lys91Asnfs^∗22), c.298G>T (p.Ala100Ser), c.294T>G (p.Phe98Leu), c.269A>G (p.Glu90Gly), and c.700T>C (p.Tyr234His), all predicted to be disease causing. By using haplotype reconstruction we showed that the mutation c.298G>T is probably a founder mutation. Due to the spectrum of the amino acid changes, we suggest that loss of function in TGDS is the underlying mechanism of Catel-Manzke syndrome. TGDS (dTDP-D-glucose 4,6-dehydrogenase) is a conserved protein belonging to the SDR family and probably plays a role in nucleotide sugar metabolism.     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Calcium Signaling and Arrhythmias in the Heart Evoked by β-Adrenergic Stimulation

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca²⁺-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca²⁺ release in cardiac myocytes evoked by β-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca²⁺ signals sensitive to inhibitors of both acidic Ca²⁺ stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca²⁺ transients caused by high concentrations of the β-adrenergic agonist isoproterenol. Ca²⁺ transients were recorded both as increases of the free cytosolic Ca²⁺ concentration and as decreases of the sarcoplasmic luminal Ca²⁺ concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca²⁺ transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy.     

Cell proliferation and differentiation kinetics during spermatogenesis inHydra carnea

Summary Spermatogenesis inHydra carnea was investigated. The cell proliferation and differentiation kinetics of intermediates in the spermatogenesis pathway were determined, using quantitative determinations of cell abundance, pulse and continuous labelling with³H-thymidine and nuclear DNA measurements. Testes develop in the ectoderm of male hydra as a result of interstitial cell proliferation. Gonial stem cells and proliferating spermatogonia have cell cycles of 28 h and 22 h, respectively. Stem cells undergo four, five or six cell divisions prior to meiosis which includes a premeiotic S+G2 phase of 20 h followed by a long meiotic prophase (22 h).Spermatid differentiation requires 12–29 h. When they first appear, testes contain only proliferating spermatogonia; meiotic and postmeiotic cells appear after 2 and 3 days, respectively and release of mature sperm begins after 4 days. Mature testes produce about 27,000 sperm per day over a period of 4–6 days: about 220 gonial stem cells per testis are required to support this level of sperm differentiation. Further results indicate that somatic (e.g. nematocyte) differentiation does not occur in testes although it continues normally in ectodermal tissue outside testes. Our results support the hypothesis that spermatogenesis is controlled locally in regions of the ectoderm where testes develop.     

Color genes in the orchid Oncidium Gower Ramsey: identification, expression, and potential genetic instability in an interspecific cross

Orchids are one of the most unique and evolved of flowering plants, with many being valuable floricultural crops. Spatial localization of pigments within the flower of the commercially important bi-color Oncidium Gower Ramsey demonstrated a mixture of carotenoids and anthocyanins concentrated in the adaxial epidermis. Chromatography identified the predominant yellow pigment to be an equal mixture of all-trans and 9-cis isomers of violaxanthin, with esterification specific to the 9-cis isomer. Red ornamentation was comprised of the anthocyanins cyanidin and its methylated derivate, peonidin. Five key pigment biosynthesis genes encoding dihydroflavonol 4-reductase (DFR), phytoene synthase (PSY), phytoene desaturase, carotenoid isomerase, and the downstream 9-cis epoxycarotenoid dioxygenase were isolated and their expression profiles determined. Northern analyses showed both phytoene desaturase and carotenoid isomerase expression to be up-regulated in floral tissue relative to leaves whereas PSY was not. Three closely related DFR genes were isolated, including one with an insertion in the 3′ coding region. DFR expression occurred throughout flower development in Oncidium, unlike in Dendrobium and Bromheadia orchids. A number of the isolated anthocyanin and carotenoid genes showed variations due to insertion events. These findings raise questions about the genetic stability in interspecific crosses in orchids, such as the tri-specific Oncidium Gower Ramsey.     

Production of a thermostable alcohol dehydrogenase from Rhodococcus ruber in three different yeast species using the Xplor?2 transformation/expression platform

The Xplor(?)2 transformation/expression platform was employed for comparative assessment of three different yeast species as hosts for synthesis of a thermostable nicotinamide adenine dinucleotide (NAD(+))-dependent medium-chain alcohol dehydrogenase from Rhodococcus ruber strain 219. Using yeast ribosomal DNA (rDNA) integrative expression cassettes (YRCs) and yeast integrative expression cassettes (YICs) equipped with a selection-marker module and one, two or four expression modules for transformation of auxotrophic Arxula adeninivorans, Hansenula polymorpha, and Saccharomyces cerevisiae strains, quantitative comparison of the yield of recombinant alcohol dehydrogenase RR-ADH6Hp in all three species was carried out. In all cases, the RR-ADH6H gene was expressed under the control of the strong constitutive A.?adeninivorans-derived TEF1 promoter, which functions in all yeast species analyzed. Recombinant RR-ADH6Hp accumulated intracellularly in all strains tested. The best yields of active enzyme were obtained from A.?adeninivorans, with S.?cerevisiae producing intermediate amounts. Although H.?polymorpha was the least efficient producer overall, the product obtained was most similar to the enzyme synthesized by R.?ruber 219 with respect to its thermostability.