Hight performance gel permeation chromatography of proteins

Proteins in the molecular weight range of 10 000–170 000 were separated by high performance gel permeation chromatography. Silica particles with 30 nm or 50 nm pores were derivatized with glycidoxy-propyltrimethoxysilane and used as support. The proteins were eluted with 50% formic acid. A protein fraction which induces endodermal and mesodermal tissues in amphibian gastrula ectoderm was purified by this method.     

Mechanisms of cell differentiation: Affinity of a morphogenetic factor to DNA

Summary A morphogenetic factor which induces inTriturus gastrula ectoderm tissues which are derived from mesoderm and endoderm has been extracted from chicken and amphibian embryos. The factor which is protein in nature has been obtained from chicken embryos in a highly purified state.The biological activity of the chicken factor is partially inhibited when the factor is combined with chicken DNA or sonicated chicken DNA.When the 3H-labelled factor is combined with sonicated DNA and then centrifuged on a sucrose gradient the factor migrates in part with the DNA. This indicates that the factor is bound to DNA.The inferences from these results are discussed with regard to the possible mechanism of action of the factor and the molecular mechanism of differentiation.     

CAM variations in the leaf-succulent Delosperma tradescantioides (Mesembryanthemaceae), native to southern Africa

Drought responses of diurnal gas exchange, malic acid accumulation and water status were examined in Delosperma tradescantioides , a succulent that grows in drought-prone microenvironments in summer rainfall and all-year rainfall regions of southern Africa. When well-watered, this species exhibited Crassulacean acid metabolism (CAM)-cycling, but its carbon fixation pattern changed during the development of drought, shifting to either low-level CAM or to CAM-idling. The rate and pattern of this change depended on environmental conditions, duration of water stress and leaf age. At the onset of drought, diurnal malate fluctuation increased, but was strongly depressed (by ca 70%) as drought continued, and when leaf water content and water potential were low (ca 35 and 50% of the initial levels, respectively). When rewatered, rates of growth and photosynthesis, gas exchange and water status recovered fully to pre-stressed values within two days. Whole-shoot carbon uptake rates suggested that leaf growth had continued unabated during a short-term (∼ one week) drought. This emphasises that CAM-idling allows the maintenance of active metabolism with negligible gas exchange when soil water is limiting. It is possible that old or senescent leaves may provide water for the expansion of developing leaves during initial periods of drought. Regardless of the water regime and environmental conditions, leaf nocturnal malate accumulation and water content were positively correlated and increased with leaf age. Thus the gradual loss of water from older mature leaves may induce CAM-idling, which reduces water loss. An important ecological consequence of this combination of CAM modes is the potential to switch rapidly between fast growth via C₃ gas exchanges when well-watered to water-conserving CAM-idling during drought.     

Renin immunohistochemistry in the mesonephros and metanephros of the pig embryo

Summary The occurrence and distribution of renin was investigated in meso- and metanephric kidneys of pig embryos in various gestational stages. The immunohistochemical peroxidase-antiperoxidase-method (PAP) was used on paraffin sections after application of an antiserum against mouse renin which cross reacts with pig renin. Renin immunoreactivity was already found in the mesonephros of 21 day pig embryos (crown-rump(CR)-length 12 mm) with the strongest reaction in the media of the juxtaglomerular afferent arteriole. Efferent vessels, mesonephric arteries, and the aortic wall also contained scattered renin-positive cells. In the definitive kidney, renin was not detected prior to the 25 mm CR-length-stage. In 45 mm embryos, immunocytochemical staining was observed not only in the media of kidney arteries and arterioles, but also in proximal tubules after pinocytic absorption of filtered renin. TEM-studies revealed that the media of both the mesonephric and the developing metanephric arteries and arterioles contains epithelioid cells whose ultrastructure is very similar to that of renin-producing cells in the adult organ. The observed distribution of renin-producing cells along the entire renal arterial tree points to the possibility that the major function of the renin-angiotensin system in the fetal animal is to participate in the stabilization of renal perfusion pressure.     

On the yolk sac of the cat

Summary The phase of primitive erythropoiesis in the feline yolk sac lasts from the 14th to the 20th day after mating. The globular nucleated primitive erythroblasts are formed extravascularly to some extent, but they can be clearly distinguished from the endoderm. They do not undergo a denucleation and are still present in the circulating blood on the 45th day. Aging primitive erythroblasts are characterized by a loss of polysomes, by the appearance of long intracytoplasmic electron-lucent channels, and by a nuclear pyknosis which can turn into a karyolysis. Definitive erythropoiesis begins around the 17th day but, even by the 19th day, it is not particularly prominent. It ends around the 45th day. It is almost exclusively intravascular. The distinction of immature primitive erythroblasts from erythroblasts of the definitive series is difficult, because it is based upon only slight differences in the heterochromatinization, in the nuclear-cytoplasmic ratio, and in the organelle content of the cells. In the definitive series, the nuclear divisions follows the law of the rhythmical halving of the nuclear volume. The cells exhibit more clearly identifiable maturation stages here, and the checkerboard nucleus is more distinct. The vascular endothelium is largely attenuated and moderately fenestrated; it lacks a distinct basement membrane. Organelle-rich adventitial cells are found in close apposition.     

Bone morphogenetic protein 4 (BMP-4), a member of the TGF-beta family, in early embryos of Xenopus laevis: analysis of mesoderm inducing activity

We have screened a Xenopus ovary cDNA library using a synthetic oligonucleotide derived from that part of the inhibin beta A sequence, which is highly conserved within the TGF-beta family. Out of several clones yielding autoradiographic signals four turned out to represent Xenopus counterparts to the human bone morphogenetic protein 4 (BMP-4). Each two of the four sequences are nearly identical and probably account for different alleles whereas the two pairs showing 5% divergence may have arisen by genome duplication in this tetraploid species. The amino acid sequence of the Xenopus protein is 80% homologous to the human sequence showing no single exchange within the last 100 amino acids at the C-terminus. This region, which constitutes the main part of the mature, biologically active protein, also exhibits substantial homologies to other representatives of the TGF-beta family, especially to the Drosophila DPPC protein. Transfection of COS-1 cells with the Xenopus BMP-4 sequence under control of the CMV-promoter leads to the secretion of a protein which exhibits mesoderm inducing activity when tested with animal cap explants from Xenopus blastula stage embryos.     

The pig yolk sac I

Summary Yolk sacs from pig embryos ranging between 18 mm and 55 mm in length were investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and histochemistry. The organ was no longer present in embryos of 70 mm length. The endoderm proliferates in embryos of about 20 mm length with gland-like endodermal cell columns and finally becomes stratified, representing over 90% of the yolk sac mass. The endodermal cells show a high activity of oxidoreductases and lysosomal enzymes; their luminal surface bears few absorptive specializations. The mesothelium is inert, as judged from its surface ultrastructure, organelle composition and enzyme content. TEM reveals the endodermal cells to be polarized even in stratified areas. They resemble liver parenchymal cells with respect to their basal villi, which are exposed to capillaries with discontinuous or fenestrated endothelium. Giant mitochondria with crystalline inclusions in the mature endodermal cytoplasm are outnumbered by large stacks of the rough ER, which can amount to 60% of the cytoplasm. This conspicuous RER is suspected to be the production site of serum proteins which are discharged into the vascular bed. Close to the time of the organ's regression, an unusual storage of material in terminal buds of the ER was found. Intercellular canaliculi and the endocytic apparatus of the endoderm are thought to serve regression.     

Formation of mesodermal pattern by secondary inducing interactions

Summary A highly purified vegetalizing factor induces endoderm preferentially in amphibian gastrula ectoderm. After combination of this factor with less pure fractions, a high percentage of trunks and tails with notochord and somites are induced. The induction of these mesodermal tissues depends on secondary factors which may act on plasma membrane receptors of the target cells. The secondary factors are probably proteins as they are inactivated by trypsin or cellulose-bound proteinase K. They are not inactivated by thioglycolic acid.The implication of these findings for tissue determination and differentiation in normal development in relation to the anlageplan for endoderm and mesodermal tissues is discussed.     

Sleep Apnea-Hypopnea Quantification by Cardiovascular Data Analysis

Sleep disorders are a major risk factor for cardiovascular diseases. Sleep apnea is the most common sleep disturbance and its detection relies on a polysomnography, i.e., a combination of several medical examinations performed during a monitored sleep night. In order to detect occurrences of sleep apnea without the need of combined recordings, we focus our efforts on extracting a quantifier related to the events of sleep apnea from a cardiovascular time series, namely systolic blood pressure (SBP). Physiologic time series are generally highly nonstationary and entrap the application of conventional tools that require a stationary condition. In our study, data nonstationarities are uncovered by a segmentation procedure which splits the signal into stationary patches, providing local quantities such as mean and variance of the SBP signal in each stationary patch, as well as its duration . We analysed the data of 26 apneic diagnosed individuals, divided into hypertensive and normotensive groups, and compared the results with those of a control group. From the segmentation procedure, we identified that the average duration , as well as the average variance , are correlated to the apnea-hypoapnea index (AHI), previously obtained by polysomnographic exams. Moreover, our results unveil an oscillatory pattern in apneic subjects, whose amplitude is also correlated with AHI. All these quantities allow to separate apneic individuals, with an accuracy of at least . Therefore, they provide alternative criteria to detect sleep apnea based on a single time series, the systolic blood pressure.