Towards quantitative in situ hybridization

In situ hybridization analysis of tissue mRNA concentrations remains to be accepted as a quantitative technique, even though exposure of tissue sections to photographic emulsion is equivalent to Northern blot analysis. Because of the biological importance of in situ quantification of RNA sequences within a morphological context, we evaluated the quantitative aspects of this technique. In calibrated microscopic samples, autoradiographic signal (density of silver grains) was proportionate to the radioactivity present, to the exposure time, and to time of development of the photographic emulsion. Similar results were obtained with tissue sections, showing that all steps of the in situ hybridization protocol, before and including the detection of the signal, can be reproducibly performed. Furthermore, the integrated density of silver grains produced in liver and intestinal sections by the in situ hybridization procedure using 35S-labeled riboprobes is directly proportionate to the signal obtained by quantitative Northern blot analysis. The significance of this finding is that in situ quantification of RNA can be realized with high sensitivity and with the additional advantage of the possibility of localizing mRNA within the cells of interest. Application of this procedure on fetal and adult intestinal tissue showed that the carbamoylphosphate synthetase (CPS)-expressing epithelial cells of both tissues accumulated CPS mRNA to the same level but that whole-organ CPS mRNA levels decreased four-to fivefold in the same period, owing to a comparable decrease in the number of CPS-expressing cells in total intestinal tissu

Anomalous Power Law Distribution of Total Lifetimes of Branching Processes Relevant to Earthquakes

We consider a branching model of triggered seismicity, the ETAS (epidemic-type aftershock sequence) model which assumes that each earthquake can trigger other earthquakes (``aftershocks''). An aftershock sequence results in this model from the cascade of aftershocks of each past earthquake. Due to the large fluctuations of the number of aftershocks triggered directly by any earthquake (``productivity'' or ``fertility''), there is a large variability of the total number of aftershocks from one sequence to another, for the same mainshock magnitude. We study the regime where the distribution of fertilities $\mu$ is characterized by a power law $\sim 1/\mu^{1+\gamma}$ and the bare Omori law for the memory of previous triggering mothers decays slowly as $\sim 1/t^{1+\theta}$, with $0 < \theta <1$ relevant for earthquakes. Using the tool of generating probability functions and a quasistatic approximation which is shown to be exact asymptotically for large durations, we show that the density distribution of total aftershock lifetimes scales as $\sim 1/t^{1+\theta/\gamma}$ when the average branching ratio is critical ($n=1$). The coefficient $1<\gamma = b/\alpha<2$ quantifies the interplay between the exponent $b \approx 1$ of the Gutenberg-Richter magnitude distribution $\sim 10^{-bm}$ and the increase $\sim 10^{\alpha m}$ of the number of aftershocks with the mainshock magnitude $m$ (productivity) with $\alpha \approx 0.8$. More generally, our results apply to any stochastic branching process with a power-law distribution of offsprings per mother and a long memory.Comment: 16 pages + 4 figure

Marked differences in tissue-specific expression of chitinases in mouse and man

FWN – Publicaties zonder aanstelling Universiteit Leide

Ebstein's anomaly may be caused by mutations in the sarcomere protein gene MYH7

Ebstein's anomaly is a rare congenital heart malformation characterised by adherence of the septal and posterior leaflets of the tricuspid valve to the underlying myocardium. Associated abnormalities of left ventricular morphology and function including left ventricular noncompaction (LVNC) have been observed. An association between Ebstein's anomaly with LVNC and mutations in the sarcomeric protein gene MYH7, encoding β -myosin heavy chain, has been shown by recent studies. This might represent a specific subtype of Ebstein's anomaly with a Mendelian inheritance pattern. In this review we discuss the association of MYH7 mutations with Ebstein's anomaly and LVNC and its implications for the clinical care for patients and their family members

Expression of the cholinergic signal-transduction pathway components during embryonic rat heart development

BACKGROUND: Previous studies showed that acetylcholinesterase (AChE) activity is present in the downstream (arterial) part of the embryonic chick and rat heart, but its functional significance was unclear. To establish whether other components of a cholinergic signal-transduction pathway are present in the embryonic heart, we localised the mRNAs encoding choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and the muscarinic receptor isoforms (mAChRs; m1-m5). METHODS: Messenger RNA detection and localisation by in situ hybridisation and reverse transcriptase-polymerase chain reaction were employed. RESULTS: Expression of ChAT and AChE mRNAs was observed from 15 embryonic days onward in the neural tissue covering the dorsocranial wall of the atria. Muscarinic receptors (m1, m2, m4) were observed at the same localisation as AChE and ChAT mRNAs, both during embryogenesis and after birth. In addition, m1 and m4 mAChRs showed a low level of expression in the atrial myocardium during the fetal period. No expression of the m3 or the m5 mAChRs was observed in or near the embryonic hearts. ChAT, AChE, and mAChRs (m1, m2, m4) mRNAs always colocalised in the cardiac ganglia. However, none of these mRNAs was found at a detectable level in the outflow tract and/or the ventricular trabeculations. CONCLUSIONS: The AChE activity in the arterial part of the embryonic heart is probably synthesised elsewhere and subserves a function different from the hydrolysis of locally produced acetylcholin

Developmental appearance of ammonia-metabolizing enzymes in prenatal murine liver

To resolve an apparent discrepancy in the developmental appearance of glutamine synthetase (GS) protein in rat [Gaasbeek Janzen et al. (1987) J. Histochem, Cytochem., 35:49-54] and mouse [Bennett et al. (1987) J. Cell Biol., 105:1073-1085] liver, we have investigated its expression during liver development in the mouse and compared it with that of carbamoylphosphate synthetase I (CPS). The expression of glutamate dehydrogenase was used as a marker to identify all hepatocytes in these strongly hematopoietic livers. GS protein accumulation starts in mouse hepatocytes at embryonic day (ED) 15. The first hepatocytes in which the enzyme accumulates were found around the major hepatic veins. CPS protein was found to accumulate in mouse hepatocytes from ED 13 onward: first, at the center of the median and lateral lobes, but temporarily not at the periphery of these lobes and not at the caudate lobe. The initial phase of accumulation of GS and CPS protein was characterized by a heterogeneity in enzyme content between hepatocytes. By ED 17, both enzymes were detectable in all hepatocytes at the center of the median and lateral lobes. This event marked the onset of the development of the complementary distribution of the enzymes typical of zonal heterogeneity in the adult mammalian liver. However, during the perinatal period, the pericentral hepatocytes temporarily accumulated CPS protein. We also observed heterochrony between species in the appearance of CPS protein in the small intestin

Development of the pacemaker tissues of the heart

Pacemaker and conduction system myocytes play crucial roles in initiating and regulating the contraction of the cardiac chambers. Genetic defects, acquired diseases, and aging cause dysfunction of the pacemaker and conduction tissues, emphasizing the clinical necessity to understand the molecular and cellular mechanisms of their development and homeostasis. Although all cardiac myocytes of the developing heart initially possess pacemaker properties, the majority differentiates into working myocardium. Only small populations of embryonic myocytes will form the sinus node and the atrioventricular node and bundle. Recent efforts have revealed that the development of these nodal regions is achieved by highly localized suppression of working muscle differentiation, and have identified transcriptional repressors that mediate this process. This review will summarize and reflect new experimental findings on the cellular origin and the molecular control of differentiation and morphogenesis of the pacemaker tissues of the heart. It will also shed light on the etiology of inborn and acquired errors of nodal tissues

Neerslag-afvoerrelatie solide basis voor keuzen in hoogwaterbeheer

Voor deelstroomgebieden van de rivieren de Dommel en de Aa zijn neerslagafvoerrelaties bepaald. Dit is gedaan met een neerslagafvoermodel dat het toenemen en afnemen van afvoer uit de deelstroomgebieden in de tijd beschrijft. Dit artikel behandelt de modelkeuze, -kalibratie en -validatie en de constructie van een langjarige afvoerreeks voor één van de deelstroomgebieden. De berekende afvoeren worden in een hydrodynamisch model in SOBEK-1D2D gebruikt om stroming in open waterlopen en de daarbij optredende waterstanden en inundaties te analyseren. De bouw van het model is geheel geautomatiseerd vanuit GIS-bestande