CAG repeat analyses in frozen and formalin-fixed tissues following primer extension preamplification for evaluation of mitotic instability of expanded SCA1 alleles

Neurodegenerative disorders, including spinocerebellar ataxias (SCA), Huntington disease (HD) and dentatorubral-pallidoluysian atrophy (DRPLA), are associated with unstable CAG repeats. To investigate the mitotic stability of the repetitive element in the genes for SCA1, SCA3, HD, and DRPLA we extracted DNA from up to 13 tissue samples from four deceased individuals with progressive neurological disorders and neuropathological signs. Due to the formalin fixation of some tissues the genomic DNA was highly degraded and unsuitable for amplification of fragments longer than 150 bp. After size selection and primer extension preamplification, specific analyses could be performed even for expanded alleles. In all four patients the SCA1 mutation could be demonstrated, in one case with remarkable somatic heterogeneity of the elongated allele, whereas alleles of the normal range were stable in all tissues examined. Received: 3 February 1997 / Accepted: 9 April 1997     

Watershed ‘chemical cocktails’: forming novel elemental combinations in Anthropocene fresh waters

In the Anthropocene, watershed chemical transport is increasingly dominated by novel combinations of elements, which are hydrologically linked together as ‘chemical cocktails.’ Chemical cocktails are novel because human activities greatly enhance elemental concentrations and their probability for biogeochemical interactions and shared transport along hydrologic flowpaths. A new chemical cocktail approach advances our ability to: trace contaminant mixtures in watersheds, develop chemical proxies with high-resolution sensor data, and manage multiple water quality problems. We explore the following questions: (1) Can we classify elemental transport in watersheds as chemical cocktails using a new approach? (2) What is the role of climate and land use in enhancing the formation and transport of chemical cocktails in watersheds? To address these questions, we first analyze trends in concentrations of carbon, nutrients, metals, and salts in fresh waters over 100 years. Next, we explore how climate and land use enhance the probability of formation of chemical cocktails of carbon, nutrients, metals, and salts. Ultimately, we classify transport of chemical cocktails based on solubility, mobility, reactivity, and dominant phases: (1) sieved chemical cocktails (e.g., particulate forms of nutrients, metals and organic matter); (2) filtered chemical cocktails (e.g., dissolved organic matter and associated metal complexes); (3) chromatographic chemical cocktails (e.g., ions eluted from soil exchange sites); and (4) reactive chemical cocktails (e.g., limiting nutrients and redox sensitive elements). Typically, contaminants are regulated and managed one element at a time, even though combinations of elements interact to influence many water quality problems such as toxicity to life, eutrophication, infrastructure corrosion, and water treatment. A chemical cocktail approach significantly expands evaluations of water quality signatures and impacts beyond single elements to mixtures. High-frequency sensor data (pH, specific conductance, turbidity, etc.) can serve as proxies for chemical cocktails and improve real-time analyses of water quality violations, identify regulatory needs, and track water quality recovery following storms and extreme climate events. Ultimately, a watershed chemical cocktail approach is necessary for effectively co-managing groups of contaminants and provides a more holistic approach for studying, monitoring, and managing water quality in the Anthropocene.     

Formation of the peroxisome lumen is abolished by loss of Pichia pastoris Pas7p, a zinc-binding integral membrane protein of the peroxisome.

We have cloned and sequenced PAS7, a gene required for peroxisome assembly in the yeast Pichia pastoris. The product of this gene, Pas7p, is a member of the C3HC4 superfamily of zinc-binding proteins. Point mutations that alter conserved residues of the C3HC4 motif abolish PAS7 activity and reduce zinc binding, suggesting that Pas7p binds zinc in vivo and that zinc binding is essential for PAS7 function. As with most pas mutants, pas7 cells exhibit a pronounced deficiency in import of peroxisomal matrix proteins that contain either the type 1 peroxisomal targeting signal (PTS1) or the type 2 PTS (PTS2). However, while other yeast and mammalian pas mutants accumulate ovoid, vesicular peroxisomal intermediates, loss of Pas7p leads to accumulation of membrane sheets and vesicles which lack a recognizable lumen. Thus, Pas7p appears to be essential for protein translocation into peroxisomes as well as formation of the lumen of the organelle. Consistent with these data, we find that Pas7p is an integral peroxisomal membrane protein which is entirely resistant to exogenous protease and thus appears to reside completely within the peroxisome. Our observations suggest that the function of Pas7p defines a previously unrecognized step in peroxisome assembly: formation of the peroxisome lumen. Furthermore, because the peroxisomal intermediates in the pas7 delta mutant proliferate in response to peroxisome-inducing environmental conditions, we conclude that Pas7p is not required for peroxisome proliferation.     

Das elektronenmikroskopische Bild Menschlicher Endometrialer Drüsenzellen Während des Menstruellen Zyklus

Zusammenfassung Die elektronenmikroskopisch sichtbaren Veränderungen menschlicher endometrialer Drüsenzellen im Verlauf des menstruellen Zyklus werden beschrieben.In der Proliferationsphase zeichnen sich die Drüsenzellen durch reichliche Ergastoplasmamembranen und Paladegranula aus, besonders in den basalen Zytoplasmaanteilen. Daneben sieht man, fast ausschließlich supranukleär, zahlreiche Sekretgranula von etwa 0,7 Durchmesser, deren Zahl am Ende der Proliferationsphase ein Maximum erreicht. Außerdem findet man noch am basalen Kernpol ein Sekret, das aus einem elektronenoptisch schwach konturierten Material besteht und aus Glykogen sowie Glyk- ound Mucoproteiden aufgebaut ist. Gleichzeitig werden die hier liegenden Paladegranula und Ergastoplasmamembranen aufgelöst. Die hier liegenden Mitochondrien vergrößern sich auf ein Mehrfaches, die Zahl ihrer Cristae nimmt zu. Sobald die Sekretproduktion abgeschlossen ist, verkleinern sie sich wieder.Zur Zeit der mittleren Sekretionsphase ist dieses Sekret in das apikale Zytoplasma gewandert. Dabei verschwinden die in den vorangehenden Subphasen reichlich vorhandenen Mikrovilli weitgehend. Gegen Ende des menstruellen Zyklus erscheinen die Zellen durch Abstoßung der apikalen Zytoplasmateile im ganzen niedriger. Kurz vor der Desquamation lösen sie sich dann voneinander, wobei sich der Interzellularraum auf ein Mehrfaches verbreitert. Gleichzeitig treten im Zytoplasma Degenerationszeichen wie vakuoläre Umwandlungen von Mitochondrien, Ergastoplasmaräume und Golgizone auf. Außerdem verlieren die Zellorganellen ihre scharfen Konturen, und die bis dahin runden oder ovalen Zellkerne zeigen eine unregelmäßige, teilweise sogar gelappte Begrenzung.Die seitlichen Zellgrenzen verlaufen in den dem Drüsenlumen nahen Abschnitten gerade oder leicht gewunden und besitzen zahlreiche Desmosomen. Weiter basal hingegen weisen sie starke Verzahnungen mit den Naehbarzellen auf, wobei die Desmosomen nur noch sehr selten zu finden sind. Nach Abstoßung der Zellspitzen in der späten Sekretionsphase reicht die Verzahnungszone bis an das Drüsenlumen heran.Die Basalmembran der Drüsen ist zu Beginn des Zyklus relativ schmal (etwa 300 Å). Sie wächst dann in den späteren Subphasen weiter an und erreicht am Ende des Zyklus eine Dicke von etwa 800 Å.Neben den Drüsenzellen begegnet man hin und wieder in allen Subphasen cilientragenden Zellen (Flimmerzellen), die relativ arm an Zytoplasmaorganellen sind. Die Cilien besitzen den typischen Aufbau mit 9 auf einem Kreisbogen liegenden und einem zentralen Filament, die aus je 2 Subfilamenten bestehen.Außerdem sieht man mitunter zwischen den Drüsenzellen einen weiteren Zelltyp, der reich an Paladegranula und Ergastoplasmastrukturen ist. Art und Funktion dieser Zellen, bei denen es sich nicht um Wanderzellen wie Plasmazellen, Lympho- oder Leukozyten handelt, ist noch unklar.Herrn Prof. Dr. med. H. Siebke und Herrn Oberarzt Doz. Dr. Puck, Universitäts-Frauenklinik Bonn, danke ich für Überlassung des Untersuchungsgutes, Herrn Prof. Dr. med. Piekarski, Hygiene-Institut der Universität Bonn, für die Benutzung des Siemens-Elmiskops.     

Structure and expression of the polyubiquitin gene in sea urchin embryos.

A cloned Lytechinus pictus cDNA has been identified, which includes seven direct repeats of a 228 bp sequence encoding ubiquitin and about 450 bp of 3' noncoding sequence. The deduced amino acid sequence is identical to that of ubiquitins of other animals (though repeats 3 and 5 each have single amino acid substitutions at different positions). Southern blot analysis revealed that the sea urchin genome contains a single copy of the polyubiquitin gene, and the number of 228 bp repeat units appears to vary from seven to ten among different alleles; no other ubiquitin coding sequences were detected. The size distribution of polyubiquitin mRNA is polymorphic among different individuals, probably corresponding to the differences in copy number of the repetitive coding sequence. The abundance of cytoplasmic polyubiquitin RNA is constant throughout embryogenesis and is similar in ectoderm, endoderm, and mesoderm cells. The constant prevalence of polyubiquitin mRNA apparently results from a balance between ontogenetic changes in its rate of synthesis and its stability in the presence of actinomycin D. Accumulation of polyubiquitin RNA was not heat shock-inducible during embryogenesis.