Phosphorylation of myosin light chain kinase: a cellular mechanism for Ca2+ desensitization

Phosphorylation of the regulatory light chain of myosin by the Ca²⁺/calmodulin-dependent myosin light chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin light chain kinase is also phosphorylated, and recent results from experiments designed to satisfy the criteria of Krebs and Beavo for establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca²⁺/calmodulin-dependent protein kinase II phosphorylates myosin light chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca²⁺/calmodulin required for activation and hence increases the Ca²⁺ concentrations required for myosin light chain kinase activity in cells. However, the concentration of cytosolic Ca²⁺ required to effect myosin light chain kinase phosphorylation is greater than that required for myosin light chain phosphorylation. Phosphorylation of myosin light chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca²⁺ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin light chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin light chain phosphorylation.     

Genetics of methane and methanol oxidation in Gram-negative methylotrophic bacteria

Within the past few years, considerable progress has been made in the understanding of the molecular genetics of methane and methanol oxidation. In order to summarize this progress and to illustrate the important genetic methods employed, this review will focus on several well-studied organisms. These organisms include the gramnegative faculative methylotrophsMethylobacterium extorquens, Methylobacterium organophilum andParacoccus denitrificans. In addition, the obligate methanotrophsMethylococcus capsulatus andMethylosinus trichosporium are discussed. We have chosen not to discuss the genetics of methanol oxidation in the yeasts or in gram-positive bacteria. Likewise, the genetics of related topics (for example, methylamine oxidation and carbon assimilation pathways) are not reviewed here. Broad host range conjugatable plasmids have enabled researchers to complement mutations and clone genes from gram-negative methylotrophic bacteria. More recently, promoter probe derivative plasmids have been used to elucidate aspects of gene regulation. Also, alternative gene-cloning techniques are proving useful in circumventing problems in the genetic studies of the obligate methanotrophs, the group of bacteria that is the most refractory to traditional methods.     

Small differences in Drosophila tropomyosin expression have significant effects on muscle function.

The effects of promoter deletions on Drosophila tropomyosin I (TmI) gene expression have been determined by measuring TmI RNA levels in transformed flies. Decreases in RNA levels have been correlated with rescue of flightless and jumpless mutant phenotypes in Ifm(3)3 mutant transformed flies and changes in muscle ultrastructure. The results of this analysis have allowed us to identify a region responsible for 20% of maximal TmI expression, estimate threshold levels of TmI RNA required for indirect flight and jump muscle function, and obtain evidence suggesting that sarcomere length may be an important determinant of flight muscle function.     

Efficient isolation of thermophilic and thermotolerant mucoralean fungi

Rhizomucor miehei (Cooney & Emerson) Schipper, R. pusillus (Lindt) Schipper, Rhizopus rhizopodiformis (Cohn) Zopf, and Absidia corymbifera (Cohn) Sacc. & Trotter were isolated from numerous materials immersed in YpSs agar or 2% malt extract agar containing 50 g/ml Benlate, 100 U/ml penicillin G, and 40 g/ml streptomycin sulfate; agar plates were incubated at 45 °C. Alternative media and procedures useful for special circumstances are described. These four pathogenic thermophilic or thermotolerant species were readily isolated from spices, herbal teas, sunflower seeds, and other materials.     

Functional mapping of receptor specificity domains of glial cell line-derived neurotrophic factor (GDNF) family ligands and production of GFRalpha1 RET-specific agonists

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) (GDNF, neurturin, artemin, and persephin) are critical regulators of neurodevelopment and support the survival of midbrain dopaminergic and spinal motor neurons in vitro and in animal disease models making them attractive therapeutic candidates for treatment of neurodegenerative diseases. The GFLs signal through a multicomponent receptor complex comprised of a high affinity binding component (GDNF-family receptor alpha-component (GFRalpha1-GFRalpha4)) and the receptor tyrosine kinase RET. To begin characterization of GFL receptor specificity at the molecular level, we performed comprehensive homologue-scanning mutagenesis of GDNF, the prototypical member of the GFLs. Replacing short segments of GDNF with the homologous segments from persephin (PSPN) (which cannot bind or activate GFRalpha1.RET or GFRalpha2.RET) identified sites along the second finger of GDNF critical for activating the GFRalpha1.RET and GFRalpha2.RET receptor complexes. Furthermore, introduction of these regions from GDNF, neurturin, or artemin into PSPN demonstrated that they are sufficient for activating GFRalpha1. RET, but additional determinants are required for interaction with the other GFRalphas. This difference in the molecular basis of GFL-GFRalpha specificity allowed the production of GFRalpha1. RET-specific agonists and provides a foundation for understanding of GFL-GFRalpha.RET signaling at the molecular level.     

12-O-tetradecanoylphorbol-13-acetate induces transient cell cycle arrest in G1 and G2 in metastatic melanoma cells: inhibition of phosphorylation of p34cdc2.

The growth of Demel human metastatic melanoma cells was inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA) and other nonphorbol tumor promoters including palytoxin and okadaic acid. Using flow cytometry, we have demonstrated that the cells arrested growth in G1 and G2 phases of the cell cycle. Detailed analysis of the kinetics of the growth arrest in unsynchronized cells showed that (a) the growth arrest was transient and peaked 16-20 h following addition of TPA; (b) effects of TPA on cell growth began within 1-2 h after the addition; and (c) cells completed S phase and arrested in G2. In addition, TPA induced a pronounced morphological change, which peaked by 1 h and gradually subsided over 24 h. In populations of cells synchronized in G1 using lovastatin, (a) addition of TPA blocked the onset of DNA synthesis up to the end of G1; (b) the lag between addition of the drug and onset of DNA synthesis was less than 30 min; and (c) addition of TPA at the end of G1 prevented the increased phosphorylation of p34cdc2, as determined by immunoprecipitation. The experiments reported here show that TPA transiently blocked the proliferation of Demel melanoma cells at the G1-S border and in G2, thus preventing cells from progressing through the cell cycle. These experiments suggest that pathways involving protein kinase C interact with and rapidly alter the molecular pathways involving p34cdc2 which regulate the onset of DNA synthesis and the G2-M transition.     

A physiologically discrete population of chromatophres in Octopus vulgaris (Mollusca)

Specimens of Octopus vulgaris (Cuvier) immersed in cold sea-water (4°C) exhibit a flashing display of brown chromatophores that we call "brown spots". The spots, which flashed approximately once every three seconds, are distributed over the dorsal skin of the head, mantle and arms and correspond mainly with the distribution of the white spots and white streaks previously described, and appear to act as a screen for the patches composing the white spots. Flashing brown spots could be evoked in animals with the supraoesophageal brain removed but not in animals with local denervation of the skin.
The precise site of action of the cold is unknown but it is proposed that the nerves supplying the brown spot units are normally tonically inhibited and that the cooling process removes this inhibition and allows the spots to flash. They are particularly useful for the in vivo study of the control of chromatophores because they can be reliably activated and isolated from responses of other chromatophores.