Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules

A novel Mr 28,000 integral membrane protein ("28kDa") was identified in human erythrocytes and found entirely associated with the Triton X-100 insoluble membrane skeletons. Antibodies to 28kDa reacted strongly on immunoblots with 28kDa and a diffuse region of Mr 35,000-60,000 ("HMW-28kDa"). Selective proteolytic digestions of membranes demonstrated that HMW-28kDa has an extracellular domain, and both 28kDa and HMW-28kDa have intracellular domains. 28kDa and HMW-28kDa were purified to homogeneity. Quantitative immunoblots indicate that each erythrocyte contains 120,000-160,000 copies of 28kDa. Two-dimensional iodopeptide maps of 28kDa and HMW-28kDa were nearly identical; peptide-N-glycosidase digestion of purified HMW-28kDa demonstrated that it is the N-glycosylated form of 28kDa. When concentrated, 28kDa formed a series of larger oligomers which were stable in sodium dodecyl sulfate. Of several nonerythroid tissues studied with anti-28kDa immunoblots, only kidney displayed immunoreactive 28kDa. Purified rat kidney 28kDa was nearly identical to rat erythrocyte 28kDa when compared by two-dimensional iodopeptide mapping. Immunohistochemical staining of human kidney with anti-28kDa demonstrated prominent staining over the apical brush borders of proximal convoluted tubules. A novel integral membrane protein has been purified from erythrocyte and kidney membranes. This new protein may play a role in linkage of the membrane skeleton to the lipid bilayer.     

Rabbit endometrium in organ culture: Morphological evidence for progestational differentiation in vitro

Summary This communication describes conditions for long-term organotypic culture of rabbit endometrium allowing progesterone-induced transformation, as typcial for early pregnancy, to continue in vitro. This system appears to compare favorably with in vitro models so far proposed for the study of hormonal control of uterine function or for the investigation of cell-biological aspects of embryo implantation. The specific aim in the presented system is to provide approximate normal epithelium-stroma interrelationships. Fragments of endometrium consisting of epithelium and stroma were obtained during early pseudopregnancy and cultured on a gyratory shaker. Morphology was investigated by light microscopy, transmission and scanning electron microscopy. During the first two days the epithelium grows over the exposed stroma regenerating a complete epithelial lining. No central necrosis is found in the stroma for up to 6 days, and the tissue keeps its organotypic architecture although certain morphological differences can be observed between regenerated versus original epithelium. In the regenerating portion a stage-specific cell differentiation and the reformation of a basal lamina are missing. Progesterone substitution preserves cell morphology and allows to maintain, in vitro, the stage-specific pattern of cell organelles. Most characteristic is the induction of extensive fusion of epithelial cells. These large symplasms are comparable in size and structure to those formed in pregnancy in the implantation chamber in vivo. Only the superficial parts of the original (not the regenerated) epithelium are capable of progesterone-induced large-scale fusion. This organotypical culture system appears to be of potential value for in vitro studies on hormone action and on endometrial receptivity for embryo implantation.Abbreviations d.p.c. dies post coitum - d p. hCG days after injection of hCG (dies post injectionem hCG) - FBS fetal bovine serum - hCG human chorionic gonadotropin Preliminary results were presented by Denker et al. (1984) and by Hohn et al. (1984)     

Characterization and expression of a spliced leader RNA in the parasitic nematode Ascaris lumbricoides var. suum.

The parasitic nematode Ascaris spp. contains a 22-nucleotide spliced-leader (SL) sequence identical to the trans-SL previously described in Caenorhabditis elegans and other nematodes. The SL comprises the first 22 nucleotides of a approximately 110-base RNA and is transcribed by RNA polymerase II. The SL RNA contains a trimethylguanosine cap and a consensus Sm binding site. Furthermore, the Ascaris SL RNA has the potential to adopt a secondary structure which is nearly identical to potential secondary structures of similar SL RNAs in C. elegans and Brugia malayi.     

Gap junction formation in rabbit uterine epithelium in response to embryo recognition

Gap junction formation was studied in the uterine epithelium of nonpregnant, pregnant, and pseudopregnant rabbits in the periimplantation phase (6, 7, 8 days post coitum/post human gonadotropin injection) using freeze-fracture and immunocytochemistry as well as intracellular Lucifer yellow injection. At implantation (7 days post coitum) the uterine epithelial cells of the implantation chamber become junctionally coupled as evidenced by all three methods used. Gap junction protein (26K) becomes detectable immunocytochemically with a monoclonal antibody at 6 days post coitum in the epithelium surrounding the blastocyst, i.e., in the forming implantation chamber. The same sequence of events, starting with the presence of the gap junction protein before cell-to-cell coupling becomes evident, was observed in the blastocyst-free segments 1 day later. In contrast, uterine epithelium of nonpregnant and pseudopregnant animals in comparable phases shows an extremely low degree of coupling. The presence of the blastocyst is a necessary condition for the induction of gap junctions as demonstrated by unilateral pregnancy produced by tubal ligation. Thus, gap junction formation is one of the first maternal responses to a locally acting signal of the blastocyst.     

Variation von PCB-Gemischen in Eiern und V?geln des Wattenmeeres

Zusammenfassung Bei Flußseeschwalbe und Austernfischer wurde der Einfluß von Standort und Jahr auf die Zusammensetzung der PCB-Gemische im Ei untersucht. Die Gesamtkonzentration, der Metabolisierungsgrad und die Ringstabilitätszahlen des Gemisches von 45 bestimmten PCB-Kongeneren wurden erfaßt. In den Mündungsbereichen von Elbe, Weser und in der Inneren Deutschen Bucht traten nicht nur die höchsten PCB-Konzentrationen auf, sondern auch die am weitesten metabolisierten Gemische. 1987–1989 zeigte sich im Metabolisierungsgrad bei beiden Vogelarten ein linearer Anstieg. Flußseeschwalbeneier enthielten zwar deutlich höhere PCB-Konzentrationen als Austernfischereier, doch war das Gemisch geringer metabolisiert. Deutliche Artunterschiede traten auch in den Ringstabilitätszahlen auf. Zur Beurteilung der PCB-Belastung sollte deshalb in einem Monitoring-Programm der Metabolisationsgrad auch mitberücksichtigt werden.

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Variation of PCB mixtures in eggs of birds of the Wadden Sea

Summary The influence of location and year on the composition of PCB mixtures found in eggs was examined in the two wadden sea bird species Common Tern (Sterna hirundo) and Oystercatcher (Haematopus ostralegus). By means of 45 PCB congeners the total concentration, the degree of metabolism and the ring stability values were analysed. The highest PCB concentrations as well as the highest degree of metabolism were found in the estuaries of the rivers Elbe and Weser and in the Deutsche Bucht. The degree of metabolism of both species increased linearly from 1987 to 1989. The total PCB concentration in the eggs of Common Terns were clearly higher than those found in the eggs of Oystercatchers but the mixture was less metabolised. Furthermore, there are significant differences of the ring stability values between the two species. To assess the PCB incrimination the degree of metabolism should be considered as well in a monitoring program.

     

Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission

Aged proteins can become hazardous to cellular function, by accumulating molecular damage. This implies that cells should preferentially rely on newly produced ones. We tested this hypothesis in cultured hippocampal neurons, focusing on synaptic transmission. We found that newly synthesized vesicle proteins were incorporated in the actively recycling pool of vesicles responsible for all neurotransmitter release during physiological activity. We observed this for the calcium sensor Synaptotagmin 1, for the neurotransmitter transporter VGAT, and for the fusion protein VAMP2 (Synaptobrevin 2). Metabolic labeling of proteins and visualization by secondary ion mass spectrometry enabled us to query the entire protein makeup of the actively recycling vesicles, which we found to be younger than that of non‐recycling vesicles. The young vesicle proteins remained in use for up to ~ 24 h, during which they participated in recycling a few hundred times. They were afterward reluctant to release and were degraded after an additional ~ 24–48 h. We suggest that the recycling pool of synaptic vesicles relies on newly synthesized proteins, while the inactive reserve pool contains older proteins.     

A Reassessment of the Population Size,Demography, and Status of Tanzania’s Endemic Kipunji Rungwecebus kipunji 13 Years on: Demonstrating Conservation Success

International Journal of Primatology - Long-term population data on endangered species are fundamental to measure conservation implementation objectively, but they are rare, especially in remote...     

Enzym-Topochemie von Frühentwicklung und Implantation des Kaninchens

Zusammenfassung An Frühstadien der Entwicklung des Kaninchens (Keim, Tube und Uterus von der Ovulation bis zur Implantation) wird die histochemische Verteilung von Proteinasen und LAP untersucht.Zum Proteinasennachweis dient ein neuer Substratfilmtest. Sein Wert wird mit dem anderer Methoden verglichen. Der Test demonstriert eine hohe proteolytische Aktivität des Trophoblasten, die örtlich und zeitlich korreliert ist mit der Auflösung der Blastozystenhüllen und der Implantation; ein Kausalzusammenhang wird vermutet.Im Uterusepithel tritt beim Übertritt des Keims aus der Tube eine hohe LAP-Aktivität auf, die noch vor der Implantation wieder abfällt. Eine Bedeutung für die Bereitstellung der spezifischen Sekretproteine wird diskutiert.

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Enzyme topochemistry of the early development and implantation in the rabbitIII. Proteases

Summary Proteinases and LAP are demonstrated histochemically in early stages of development in the rabbit: in the egg, the Fallopian tube, and the uterus.For proteinases a new substrate film technique is used. Its value is compared with that of various other methods. It demonstrates a high proteolytic activity of the trophoblast, which is timed to the dissolution of blastocyst coverings and to implantation.A high LAP activity is observed in the epithelium of the uterus rising at the time when the eggs enter the uterus and dropping at the time of implantation. A physiologic role in the preparation of specific uterine secretion proteins is discussed.

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Abkürzungen EDTA Äthylendiamintetraazetat - FA Formol-Alkohol - FAE Formol-Alkohol-Eisessig - HgBPB Hg-Bromphenolblau (-färbung) - LAP Leucinaminopeptidase - MPS Mukopolysaccharid, -e - MS Mukosubstanz, -en - NA Neuraminsäure, Sialinsäuren - NAase Neuraminidase - nMS neutrale Mukosubstanz, -en - PAS Perjodsäure-Schiff-Reaktion - p.c. post coitum - h p.c., d p.c. Stunden p.c., Tage p.c. - sMS saure Mukosubstanz, -en - v/v Mischungsverhältnis zweier Volumina - w/v Gewicht pro Endvolumen     

Enzym-Topochemie von Frühentwicklung und Implantation des Kaninchens

Zusammenfassung An Frühstadien der Entwicklung des Kaninchens von der Ovulation bis zur Implantation (einschließlich der umgebenden mütterlichen Gewebe) wird die Topochemie von Enzymen des Glykogenstoffwechsels untersucht.Für den Phosphorylase-Nachweis kommt eine neue methodische Variante zur Anwendung. Furchungsstadien sind gekennzeichnet durch eine hohe Aktivität in Keim und Tubenepithel. Beim Übergang in das Blastozytenstadium scheint der Glykogenabbau von besonderer Bedeutung zu sein. Gegen den Zeitpunkt der Implantation beginnt, gemessen an Phosphorylaseaktivität und Glykogenkonzentration, erneut ein reger Glykogenstoffwechsel, jetzt im Uterusepithel. Glykogensynthetase war in den hier interessierenden Strukturen nicht nachweisbar.Eine eventuelle Bedeutung von Amylase wird diskutiert (Tubenepithel, Blastozystenhöhle).

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Enzyme topochemistry of the early development and implantation in the rabbitI. Metabolism of glycogen

Summary The early stages of development from the ovulation to implantation (including the Fallopian tube and uterus) are studied in the rabbit. The distribution of enzymes of the metabolism of glycogen is demonstrated.For phosphorylase, a new method is used. A high activity is found in the epithelium of the Fallopian tube and in the egg during cleavage. In blastocyst formation, the degradation of glycogen seems to be important, whereas phosphorylase activity in the epithelium of the uterus at the time of implantation is correlated with a storage of glycogen. Glycogen synthetase could not be demonstrated in these tissues.The possibility of a physiologic role of amylase is discussed.

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Abkürzungen EDTA Äthylendiamintetraazeta - PAS Perjodsäure-Schiff-Reaktion - p.c post coitum - h p.c., d p.c. Stunden p.c., Tage p.c. - PVP Polyvinylpyrrolidon - UDP Uridindiphosphat