Autoradiographic analyses of35S-sulfate uptake in regenerating limbs of larvalAmbystoma

Summary Autoradiographic and histochemical techniques were used to determine whether chondrocytes continue to synthesize chondroitin sulfate or closely related compounds during morphological dedifferentiation of these cells in regenerating limbs of larvalAmbystoma. Forelimbs were amputated either through the mid-diaphysis or the distal epiphysis of the humerus and each animal was subsequently injected with Na₂ ³⁵SO₄ at an appropriate stage of regeneration. Incorporation of the isotope and metachromatic staining responses were used as indices of cell specialization.In autoradiographs of unamputated limbs, epiphyseal chondrocytes exhibited moderate sulfate incorporation, whereas isotope uptake was slight in diaphyseal regions. Accordingly, in early stages of regeneration, limbs amputated through the diaphysis showed a low level of sulfate incorporation by cartilage-derived cells; since these cells dispersed during blastema formation, they were not identifiable in later stages. When limbs were amputated through the epiphysis, the matrix here underwent slow dissolution and epiphyseal-derived chondrocytes and their progeny consequently remained identifiable as they contributed to the blastema. These cells continued to exhibit isotope uptake, even during early and middle stages of regeneration —results which support the idea of tissue-specific regeneration of cartilage.Further inspection of the stained autoradiographs revealed that in addition to chondrocytes and blastema cells derived from chondrocytes, fibroblast-like cells located lateral to the limb skeleton and seemingly derived from muscle or muscle-associated cells also exhibited a moderate label during certain stages in the restoration of the limb. In several respects isotope incorporation and related metachromatic responses by these two types of cells during blastemal and early redifferentiating stages of regeneration were seen to parallel results reported in the literature of histochemical and autoradiographic studies of differentiating chick fimb buds. These observations, which may be added to previous analogies concerning developing and regenerating limbs, suggest a similar mechanism of cytodifferentiation in the two systems. The possibility is also considered that the observed isotope uptake by cells of non-cartilaginous origin may indicate the synthesis of sulfated glycosaminoglycans which alfect cell interactions during the regenerative processes.A portion of a dissertation submitted to the University of New Hampshire in partial fulfillment of the requirements for the degree of Doctor of Philosophy.NASA Predoctoral Trainee during the course of this work.The authors wish to thank Nr. Carl Paulitz for his assistance in photography of the autoradiographs.     

Changes in the activity and subcellular distribution of cyclic-AMP-dependent protein kinases with retinoic-acid-induced differentiation of embryonal carcinoma cells

We investigated changes in the activity and subcellular distribution of cyclic-AMP-dependent protein kinases (cAMP-PKs) in response to treatment with retinoic acid in three different embryonal carcinoma cell lines derived from the same teratoma 6050. After retinoic-acid treatment, F9 and PCC4 cells gave rise to parietal-like endoderm, while PC13 cells differentiated into visceral endoderm. Retinoid treatment of F9 and PCC4 cells caused an increase in cAMP-PK activity as measured by histone phosphorylation, as well as increases in the amount of the RI and RII regulatory subunits of the cAMP-PKs, as quantitated by photoaffinity labeling with 8-azido-cyclic-32P-AMP, in both the soluble and plasma-membrane fractions. The increases in membrane cAMP-PK activity and RI and RII levels reached their maximum within 18 h of retinoid treatment, and then dropped to intermediate levels after 3 days of treatment. The cytosolic activity and the levels of the regulatory subunits exhibited a progressive increase during the 3 days of exposure to retinoic acid. The relative RI/RII ratios in the cytosol and membrane fractions of the treated cells were comparable to those found in established PYS-2 parietal-endoderm cells. PC13 stem cells had high levels of cAMP-PK activity and cAMP binding to the regulatory subunits in both the cytosol and plasma membranes, while also exhibiting very low levels of type-II cAMP-PK. Retinoid treatment induced a progressive increase in cAMP-PK activity in the cytosol, and a decrease in activity at the membrane level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trisomy 14 in spontaneous abortus

Summary Chromosome analysis of the amnion from a spontaneous abortus revealed a 47,XX,D+karyotype. Giemsa banding pattern technique identified the extra chromosome as a No. 14.

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Zusammenfassung Die Chromosomenanalyse der Amnionzellen eines Spontanaborts zeigte einen Karyotyp 47,XX,D+. Mit der Giemsamusterfärbung wurde das Extrachromosom als Nr. 14 identifiziert.

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This study was supported by the Ford Foundation Population Program, Grant No. 640-0411B and by the World Health Organisation.     

Analysis of bacterial polyhydroxybutyrate production by multimodal nanoimaging

In this paper, we will employ two microscopy techniques, transmission electron microscopy and infrared nanospectromicroscopy, to study the production of polyhydroxybutyrate in Rhodobacter capsulatus and to evaluate the influence of glucose and acetone on the production yield. The results overlap which leads us to a consistent conclusion, highlighting that each technique brings specific and complementary information. By using electron microscopy and infrared nanospectromicroscopy we have proved that both glucose and acetone had a positive effect on the biopolymer production, although the first study done by Fourier transform infrared spectroscopy only identified the effect of acetone. In conclusion, we have now established a method to be able to perform fast diagnostic for PHB production.