Separation of regulatory and catalytic subunits of the cyclic 3',5'-adenosine monophosphate-dependent protein kinase(s) of rabbit skeletal muscle

The cyclic 3′, 5′-adenosine monophosphate-dependent (cAMP-dependent) protein kinase(s) from rabbit skeletal muscle has been separated into catalytic and regulatory subunits by affinity chromatography utilizing a casein-Sepharose column in the presence of cAMP. The isolated catalytic subunit manifests full activity in the absence of cAMP but its requirement for this nucleotide is regained when the enzyme is reconstituted by addition of the regulatory subunit. Evidence is presented for the existence of more than a single type of regulatory or cAMP-binding subunit in muscle.     

Antigen-presenting T cells. I. Class I alloantigen (bm1)-bearing T lymphoblasts efficiently stimulate a primary clonal response of Lyt-2+ cytotoxic lymphocyte precursors of B6 origin

The cytotoxic response of splenic Lyt-2+ T cells to class I H-2 alloantigen-bearing stimulator cells was analyzed under limiting dilution conditions. One of 50 to one of 200 nylon wool-nonadherent (FACS-purified), small Lyt-2+ spleen cells of B6 origin gave rise in vitro to a cytotoxic T lymphocyte clone that specifically lysed targets bearing bm1 alloantigen. This population of alloantigen-specific cytotoxic lymphocyte precursors (CLP) was activated by different types of bm1 stimulator cells with different efficiency: 2 X 10(5) nonfractionated spleen cells, 5000 normal peritoneal cells, 400 to 10(4) L3T4+ helper T blasts, or 2000 to 10(4) Lyt-2+ T blasts induced clonal growth of this CLP pool. Irradiated or mitomycin-treated small (L3T4+ or Lyt-2+) bm1-derived T cells were inefficient stimulator cells for this response. Supplementation of culture medium with (recombinant) interleukin 2 was necessary and sufficient to support clonal development of alloantigen-triggered CLP in the presence of allogeneic T blasts. Under these limiting dilution conditions, we observed comparable cloning efficiencies for (wild-type) Kb-allospecific splenic Lyt-2+ CLP from bm1 mice generated in response to either irradiated B6 spleen cells or inactivated B6-derived T cell lines (EL4 and RBL-5 lymphoma cells). The data indicate that normal T lymphoblasts as well as tumor T cell lines stimulate clonal development in vitro of class I H-2-allospecific cytotoxic T lymphocytes in the presence of interleukin 2.     

Mevinolin, a competitive inhibitor of hydroxymethylglutaryl coenzyme A reductase, suppresses enterocyte esterification of exogenous but not endogenous cholesterol.

Mevinolin (lovastatin), a competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase, directly inhibited acyl-CoA cholesteryl acyltransferase in rabbit intestinal microsomes at a dose of 20 micrograms/ml or more. Lineweaver-Burk analysis showed a competitive type of inhibition with respect to oleoyl-CoA. In cultured intestinal Caco-2 cells, mevinolin reduced [14C]oleate incorporation into cholesteryl-esters by 86% of controls at doses as low as 0.1 micrograms/ml. However, in cells whose activity of acyl-CoA cholesteryl acyltransferase was stimulated 7-fold by 10 mM mevalonolactone, a significant inhibitory effect on cholesteryl-ester formation could not be detected, even at 40 micrograms/ml of mevinolin. In contrast, cells supplied with liposomal cholesterol or cholesterol derived from low-density lipoproteins showed a marked reduction of cholesteryl-ester formation in the presence of 10 or 0.1 micrograms/ml of mevinolin, respectively. It is concluded that the observed suppressive effects of mevinolin on cholesterol esterification in cultured Caco-2 cells are indirect and possibly caused by changes in the acyl-CoA cholesteryl acyltransferase substrate pool or intracellular cholesterol transport.     

Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A.

A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae. The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis. This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C. The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer. On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration. The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid. Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines. Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50%. The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A. An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not. These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.     

The geometry of the human trochlea tali

The geometrical shape of the trochlea tali is responsible for two completely different courses of motion in the ankle joint setting out from the neutral position: dorsiflexion and plantar flexion. Dorsiflexion: The tibia leads the talus, whereas the fibula is pushed laterally by the screw-shaped lateral articular facet of the talus. The malleoli tightly embrace the trochlea tali, whilst an obvious cleft appears dorsally and medially between the superior articular surface of the talus and the tibial roof. Plantar flexion: The fibula leads the talus which withdraws from the medial malleolus by stretching the anterior talofibular ligament. At the same time the superior articular face of the talus closely contacts the tibial roof.     

Einsatz eines Hybridrechnersystems zur Prozeßführung von Antibiotikafermentationen in einer Scale-up-Linie

A hybrid computer completes a scale up line to a computer coupled fermentation (CCF) system for data processing, monitoring and operator servicing, documentation and process control. Presented experiences using this arrangement were adapted and transferred to microcomputer systems.     

Multiple cyclic nucleotide‐gated channels coordinate calcium oscillations and polar growth of root hairs

Calcium gradients underlie polarization in eukaryotic cells. In plants, a tip‐focused Ca²⁺‐gradient is fundamental for rapid and unidirectional cell expansion during epidermal root hair development. Here we report that three members of the cyclic nucleotide‐gated channel family are required to maintain cytosolic Ca²⁺ oscillations and the normal growth of root hairs. CNGC6, CNGC9 and CNGC14 were expressed in root hairs, with CNGC9 displaying the highest root hair specificity. In individual channel mutants, morphological defects including root hair swelling and branching, as well as bursting, were observed. The developmental phenotypes were amplified in the three cngc double mutant combinations. Finally, cngc6/9/14 triple mutants only developed bulging trichoblasts and could not form normal root hair protrusions because they burst after the transition to the rapid growth phase. Prior to developmental defects, single and double mutants showed increasingly disturbed patterns of Ca²⁺ oscillations. We conclude that CNGC6, CNGC9 and CNGC14 fulfill partially but not fully redundant functions in generating and maintaining tip‐focused Ca²⁺ oscillations, which are fundamental for proper root hair growth and polarity. Furthermore, the results suggest that these calmodulin‐binding and Ca²⁺‐permeable channels organize a robust tip‐focused oscillatory calcium gradient, which is not essential for root hair initiation but is required to control the integrity of the root hair after the transition to the rapid growth phase. Our findings also show that root hairs possess a large ability to compensate calcium‐signaling defects, and add new players to the regulatory network, which coordinates cell wall properties and cell expansion during polar root hair growth.