Alternative splice variants of alpha 7 beta 1 integrin selectively recognize different laminin isoforms.

The integrin alpha(7)beta(1) occurs in several cytoplasmic (alpha(7A), alpha(7B)) and extracellular splice variants (alpha(7X1), alpha(7X2)), which are differentially expressed during development of skeletal and heart muscle. The extracellular variants result from the alternative splicing of exons X1 and X2, corresponding to a segment within the putative ligand binding domain. To study the specificity and affinity of the X1/X2 variants to different laminin isoforms, soluble alpha(7)beta(1) complexes were prepared by recombinant coexpression of the extracellular domains of the alpha- and beta-subunits. The binding of these complexes to purified ligands was measured by solid phase binding assays. Surprisingly, the alternative splice variants revealed different and specific affinities to different laminin isoforms. While the alpha(7X2) variant bound much more strongly to laminin-1 than the alpha(7X1) variant, the latter showed a high affinity binding to laminins-8 and -10/11. Laminin-2, the major laminin isoform in skeletal muscle, was recognized by both variants, whereas none of the two variants were able to interact with laminin-5. A specific blocking antibody inhibited the binding of both variants to all laminins tested, indicating the involvement of common epitopes in alpha(7X1)beta(1) and alpha(7X2)beta(1). Because laminin-8 and -10/11 as well as alpha(7X1) are expressed in developing skeletal and cardiac muscle, these findings suggest that alpha(7X1)beta(1) may represent a physiological receptor with novel specificities for laminin-8 and -10.     

Inhibition of K+-sensitive p-nitrophenylphosphatase activity on rhesus-positive red cells after incubation with IgG-anti-rhesus-D

The incubation of ghosts derived from human Rhesus-positive red cells with IgG-anti-Rhesus-D inhibited the K⁺-sensitive p-nitrophenylphosphatase activity. This enzyme has a partial function in the (Na⁺ + K⁺)-ATPase system related to the phosphorylation step, which is important for active potassium transport through the red cell membrane. The specificity of the impairment by the antigen-antibody reaction in the Rhesus-D system was proved by the following controls. Ghosts obtained from Rhesus-negative red cells incubated by IgG-anti-Rhesus-D and those of Rhesus-positive red cells treated with non-immune serum did not show any reduction of the K⁺-p-nitrophenylphosphatase activity. The ghost preparation with lanthanum carried out after hypotonic hemolysis of the washed red cells in 2 mM LaCl₃ at pH 6 was the most suitable procedure to explore this topic in comparison to other techniques for preparing ghosts of red cells.     

Zur Kenntnis der cytologischen Vorgänge an den Geschlechtszellen von Arianta arbustorum während ihrer Reifungs- und Reifephase,Unter besonderer Berücksichtigung des jahreszeitlichen Rhythmus

Ohne Zusammenfassung     

The glucosidic pathways and glucose production by frog muscle.

Resting muscle is generally perceived as a glucose-utilizing organ; however, we show that resting well-oxygenated frog muscle recovering from strenuous exercise can release significant amounts of glucose. The metabolic pathway responsible for this process does not involve glucose-6-phosphatase because this enzyme is undetectable in frog muscle. The participation of amylo-1,6-glucosidase in the production of glucose is also ruled out since neither marked net phosphorolytic breakdown of glycogen nor considerable cycling between glycogen and glucose 6-phosphate occur. The glucosidic pathways of glycogen breakdown are the likely source of glucose as they are the only metabolic avenues with sufficient capacity to account for the rate at which glucose is released from post-exercised muscle. This rate of glucose production is high enough to be of physiological importance. Our results clearly indicate that to measure lactate glycogenesis in muscle, the simultaneous hydrolysis of muscle glycogen by the glucosidic pathways must be taken into account to prevent marked underestimation of the rate of glycogen synthesis. The glucosidic pathways seem the predominant avenues of glycogen breakdown in post-exercised resting frog muscle and are active enough to account for the rate of glycogen breakdown in resting muscle, suggesting that these rather than the phosphorolytic pathways are the chief routes of glycogen breakdown in resting muscle.     

Determination of rifabutin by high-performance liquid chromatography using on-line concentration and column switching

A simple HPLC method has been developed that allows the sensitive determination of rifabutin (RBT) in human serum using on-line concentration and column switching. After pretreatment of the serum with acetonitrile and centrifugation, the samples were applied to a concentration column (CC) (Zorbax CN). Washing with phosphate buffer-methanol removed most of the contaminating substances. Via a six-port valve the CC was switched to the analytical mode. RBT was separated on a Chromspher RP 8 column (acetonitrile-phosphate buffer pH 7.4/sodium chloride) and determined photometrically at 278 nm. The lower limit of quantification for 200 μl serum precipitated with 200 μl acetonitrile and after injection of 2×150 μl was 33 μg/l and linearity was observed up to 27 mg/l. Different modes of sample application (single, repeated, and different injection volume portions), as well as washing time, cycle time and different CC materials were investigated.     

Vergleichende Untersuchungen über das Wachstum von Ratten- und Mäuseembryonen

Zusammenfassung Das Wachstum von Ratten- und Mäuseembryonen verläuft verschieden. Wie Volumen- und Gewichtsbestimmungen gezeigt haben, ist der Mäuseembryo nach Ablauf von 65% der Tragzeit noch größer und schwerer als der Rattenembryo entsprechenden Alters, obwohl die Ratte bei der Geburt etwa viermal schwerer ist als die Maus. Während die Maus innerhalb der gesamten Embryonalzeit mehr oder weniger stetig heranwächst, liegt die Hauptwachstumsphase der Ratte im letzten Drittel der Tragzeit.Das rasche Wachstum der Ratte im letzten Drittel der Embryonalzeit beruht nicht auf einer verstärkten Wassereinlagerung, da auch die Trockensubstanz nahezu in dem gleichen Verhältnis zunimmt wie das Frischgewicht.Die Zellen des 17tägigen Mäuseembryos sind etwas kleiner als diejenigen des 19tägigen Rattenembryos, doch sind die Unterschiede gering.Dagegen liegt die Mitoserate beim 19 tägigen Rattenembryo um etwa 20% höher als bei dem vergleichbaren Mäuseembryo, entspricht also dem zu dieser Zeit einsetzenden schnelleren Wachstum.Im Gegensatz zum Wachstum setzen die Differenzierungsvorgänge bei der Ratte z. T. früher ein als bei der Maus. Dies trifft z. B. für die Entstehung der Dünndarmkrypten und der Haaranlagen zu. Die Verknöcherung des Knorpelskelettes beginnt jedoch bei den beiden Nagetieren etwa gleichzeitig.

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Comparative investigation of the growth of rat and mouse embryos

Summary The growth of rat and mouse embryos follows a different course. Volume and weight studies have shown that after 65% of the gestation period has elapsed, the mouse embryo is larger and heavier than the rat embryo. At birth, however, the rat is about 4 times as heavy as the mouse. The mouse has a more or less uniform growth over the entire gestation period, whereas the chief growth of the rat occurs in the last third of the gestation period. The rapid growth of the rat during this time does not result from an increment in water content; the dry matter undergoes an increase nearly proportional to that of the total body weight.The cells of the 17-day mouse embryo are somewhat smaller than those of the 19-day rat embryo, but the difference is small. On the other hand, the rate of mitosis in the 19-day rat is 20% higher than that of the comparable mouse embryo, in accordance with the faster growth which becomes established at this time in the rat.In contrast to growth, certain of the processes of differentiation occur earlier in the rat than in the mouse. This holds true, for example, for the formation of the crypts in the small intestine and the hair anlagen. The ossification of the cartilaginous skeleton begins at about the same time in the two rodents.

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Für die Bereitstellung der finanziellen Mittel zur Durchführung dieser Arbeit danke ich der Deutschen Forschungsgemeinschaft.     

Influence of prostaglandin F autoantibodies on the estrous cycle of the guinea pig

Adult female guinea pigs were actively immunized with prostaglandin F_2α conjugated to bovine serum albumin (BSA). Control animals, immunized against BSA continued to cycle normally, while the animals immunized against prostaglandin F_2α stopped cycling after one to three normal cycles. Laparotomy at 30 days after the last estrus revealed no recently formed corpora lutea. During the remaining 70 days of observation the antibody titer increased to 1:700, accompanied by increasing total serum estrogens (136 pg/ml at day 100) and a slow decline in circulating progesterone levels (0.6 ng/ml at day 100). The ovaries at day 100 contained degenerated corpora lutea and luteinized follicles. The suppression of the estrous cycle in the present experiments was interpreted as resulting from prolongation of luteal function as well as from inhibition of ovulation.     

Rab8 Binding to Immune Cell-Specific Adaptor LAX Facilitates Formation of trans-Golgi Network-Proximal CTLA-4 Vesicles for Surface Expression

Despite playing a central role in tolerance, little is known regarding the mechanism by which intracellular CTLA-4 is shuttled from the trans-Golgi network to the surfaces of T cells. In this context, Ras-related GTPase Rab8 plays an important role in the intracellular transport, while we have previously shown that CTLA-4 binds to the immune cell adaptor TRIM in T cells. In this study, we demonstrate that CTLA-4 forms a multimeric complex comprised of TRIM and related LAX that in turn binds to GTP bound Rab8 for post-Golgi transport to the cell surface. LAX bound via its N terminus to active GTP-Rab8, as well as the cytoplasmic tail of CTLA-4. TRIM required LAX for binding to Rab8 in a complex. Wild-type LAX or its N terminus (residues 1 to 77) increased CTLA-4 surface expression, whereas small interfering RNAs of Rab8 or LAX or disruption of LAX/Rab8 binding reduced numbers of CTLA-4-containing vesicles and its coreceptor surface expression. LAX also promoted the polarization of CTLA-4 and the reorientation of the microtubule-organizing center to the site of T-cell receptor engagement. Our results identify a novel CTLA-4/TRIM/LAX/Rab8 effector complex in the transport of CTLA-4 to the surfaces of T cells.