Regulation of pigment organelle translocation. II. Participation of a cAMP-dependent protein kinase

In intact goldfish xanthophores, the phosphorylation of a pigment organelle (carotenoid droplet) protein, p57, appears to play an important role in adrenocorticotropin (ACTH)- or cAMP-induced pigment organelle dispersion while the dephosphorylation of this protein upon withdrawal of ACTH or cAMP is implicated in pigment aggregation. In this paper, we report the cAMP-dependent phosphorylation of this protein in cell-free extracts of xanthophores as determined by the incorporation of 32P from [gamma-32P]ATP. As is the case in intact cells, p57 is the predominant protein phosphorylated in the presence of cAMP. The cAMP-dependent protein kinase which phosphorylates p57 is not bound to the isolated organelles but is found in the soluble portion of the cell extracts. Hence, the phosphorylation of p57 requires the carotenoid droplets bearing the substrate, soluble extract containing the kinase, cAMP (half-maximal activation at 0.5 microM), and Mg2+ (optimal at 5 mM or higher). The presence of protein phosphatase(s) in these extracts was shown indirectly by the stimulation of phosphorylation by fluoride. The phosphorylation of p57 does not appear to require a cell-specific kinase as soluble extracts of goldfish dermal nonpigment cells also phosphorylate p57 associated with isolated carotenoid droplets. Furthermore, using a constant amount of carotenoid droplets, a linear relationship was demonstrated between the rate of p57 phosphorylation and the amount of extract present in the assays. These results suggest that p57 is phosphorylated directly by a cAMP-dependent protein kinase and that the activity of this enzyme is important in regulating the intracellular movement of the pigment organelles of the xanthophore.     

Decreased Muscarinic Acetylcholine Receptor Number in the Central Nervous System of the Tottering (tg/tg) Mouse

The tottering mouse (tg/tg) is a single-locus mutant, phenotypically characterized by the development of epilepsy associated with distinct electroencephalographic abnormalities. Because of reported alterations in muscarinic receptor (mAChR) number in various seizure states, mAChR density was examined in discrete brain regions of tottering (tg/tg) and coisogenic wild-type (+/+) mice. Saturation binding experiments revealed a widespread decrease in membrane mAChR density in the CNS of adult tottering (tg/tg) mice as compared with age-matched control wild-type (+/+) mice. The decrease was most pronounced in the hippocampus, where tg/tg mice exhibited a 40-60% reduction in mAChR density with no change in the affinity of the receptor for antagonists or agonists. At postnatal day 10, before the reported onset of electroencephalographic abnormalities, 114 and 65% increases in mAChR density were observed in the tg/tg hippocampus and cortex, respectively. Following the development of seizure activity at postnatal day 22, mAChR density in the tg/tg hippocampus was reduced by 29%. No change in brain mAChR density was seen in adult heterozygotes (+/tg), which do not develop electroencephalographic or seizure abnormalities. These results indicate that the development of reduced mAChR number in the CNS of the tg/tg mouse is secondary to abnormal neuronal activity, providing further support for the hypothesis that membrane depolarization can cause a decrease in neuronal mAChR density.     

Characterization of incompatibility group HI1 plasmids from Salmonella typhi by restriction endonuclease digestion and hybridization of DNA probes for Tn3, Tn9, and Tn10

Chloramphenicol resistance in Salmonella typhi is medicated by plasmids of the incompatibility group H, subgroup 1 (IncHI1). Eight IncHI1 plasmids from S. typhi strains originating in Mexico, Vietnam, Thailand, and India were examined by restriction enzyme digestion. The restriction enzymes, Apal, Xbal, and PstI were found to be most useful for comparison of plasmid DNAs. Four plasmids from S. typhi isolated in Mexico, Vietnam, and Thailand between 1972 and 1974 had identical restriction patterns with all three enzymes. The other IncHI1 plasmids showed only minor differences. However, some significant differences were noted between these IncHI1 plasmids and the prototype IncHI1 plasmid R27, which was isolated from S. typhimurium in 1961 and for which a restriction map has been constructed. Southern transfer hybridization with a nick-translated HI1 plasmid as a probe confirmed that there is a great deal of sequence homology among the IncHI1 plasmids. DNA probes were used to locate DNA sequences for ampicillin resistance (Tn3), chloramphenicol resistance (Tn9), tetracycline resistance (Tn10), and the one-way incompatibility between IncHI1 plasmids and the F factor, a characteristic property of IncHI1 plasmids. The results demonstrate that IncHI1 plasmids isolated from S. typhi from widely different geographic sources are very similar. Comparisons between the S. typhi plasmids and R27 indicated that conserved regions of DNA were those involved in conjugative transfer.     

A novel mass spectrometric procedure for the rapid determination of the types of carbohydrate chains present in glycoproteins: application to alpha-galactosidase I from Vicia faba seeds

A fast atom bombardment mass spectrometric protocol has been developed to determine the type of oligosaccharide chain present in glycoproteins. The procedure is based on acetolysis of the intact glycoconjugate, extraction of the peracetylated carbohydrate fragments and analysis by fast atom bombardment mass spectrometry. The molecular ions present in the FAB spectra uniquely define the composition of the oligosaccharides with respect to hexose, aminohexose and sialic acid content. High mannose oligosaccharides yield a series of peracetylated hexose oligomers whereas complex-type oligosaccharides afford a series of N-acetyl-lactosamine containing species. Fucosylation is usually not detected but sialylated oligosaccharides are readily identified and the type of sialic acid is also defined. The method has been tested on three glycoproteins of known structure - fetuin, ribonuclease B and erythrocyte Band 3 - and on a glycoprotein of unknown structure - alpha-galactosidase I, an enzyme lectin from Vicia faba. The latter is shown to contain high mannose carbohydrate chains.     

Immunoreactive luteinizing hormone, estradiol, progesterone, testosterone, and androstenedione levels during the breeding season and anestrus in Siberian tigers

Seasonal analysis of 1239 captive births of Siberian tigers (Panthera tigris altaica) indicated a peak in April to June (P less than 0.001). Studies on seven animals in Minnesota indicated that behavioral heat cycles and ovarian follicular phase cycles began in late January and ceased in early June. Behavioral observation of 12 heat cycles in four tigers yielded an estrous length of 5.3 +/- 0.2 days and an interestrous interval of 25.0 +/- 1.3 days. Hormone assays on weekly blood samples (N = 180) from three female tigers indicated 16 cycles in two breeding seasons. Peak estradiol-17 beta levels were 46.7 +/- 6.0 pg/ml (N = 17) and interestrous concentrations were 8.7 +/- 0.66 pg/ml (N = 28) during the breeding season. Anestrous estradiol levels were 4.2 +/- 0.5 pg/ml (N = 70). The interestrous interval between estradiol peaks was 24.9 +/- 1.3 days (N = 9) with two outliers of 42 days. Serum progesterone concentrations from February to June were 1.2 +/- 0.15 ng/ml (N = 32), providing no evidence for ovulation or corpus luteum formation. Luteinizing hormone (LH) levels were 0.56 +/- 0.04 ng/ml (N = 180). Serum testosterone (r=0.71, P less than 0.001) and androstenedione levels (r=0.75, P less than 0.001) were correlated with estradiol during the breeding season. The duration of anestrus was 8 mo in two of these tigers. The interval was shortened in one tiger by exposure to a 16L:8D photoperiod. The Siberian tiger appears to be a polyestrous seasonal breeder and an induced ovulator whose breeding season may be synchronized by photoperiod.     

Coordinate induction of alcohol dehydrogenase 1, aldolase, and other anaerobic RNAs in maize

Anaerobiosis results in the selective synthesis of a particular set of polypeptides in the maize root including the two alcohol dehydrogenases (Sachs, M. M., Freeling, M., and Okimoto, R. (1980) Cell 20, 761-768), pyruvate decarboxylase (Wignarajah, K., and Greenway, H. (1976) New Phytol. 77, 575-584; Laszlo, A., and St. Lawrence, P. (1983) Mol. Gen. Genet. 192, 110-117), glucose phosphate isomerase (Kelley, P. M., and Freeling, M. (1984) J. Biol. Chem. 259, 673-677) and aldolase (Kelley, P. M., and Freeling, M. (1984) J. Biol. Chem. 259, 14180-14183). This report describes the identification and characterization of cDNA clones to five different mRNA species induced upon anaerobic shock. Immunoprecipitation of hybrid-selected translation polypeptides has determined the identity of the cDNA clone for fructose-1,6-diphosphate aldolase mRNA. Quantitative hybridization analysis of anaerobic mRNAs using the cDNA clones has shown that there is not a simultaneous accumulation of anaerobic mRNAs. Upon reintroduction of air, the anaerobic mRNAs disappear rapidly and at approximately the same rate. A translocation line that generates progeny that contain 1, 2, and 3 doses of the long arm of chromosome one (1L) allowed us to test for clustering of the anaerobic genes; two of the anaerobic genes tested do not reside with Adh 1 and Phi 1 on the long arm of chromosome 1.     

Isolation of histamine-producing Lactobacillus buchneri from Swiss cheese implicated in a food poisoning outbreak.

A histamine-producing strain of Lactobacillus buchneri was isolated from Swiss cheese that had been implicated in an outbreak of histamine poisoning. It produced up to 4,070 nmol of histamine per ml in MRS broth supplemented with 0.1% histidine. The identification of this isolate was based on its biochemical, bacteriological, and DNA characterizations.     

Subcellular localization of a membrane-associated transglutaminase activity in rat liver

Fractionation of rat liver by homogenization and differential centrifugation revealed that only about 83% of the transglutaminase activity in the tissue is in a soluble form, and that the remainder is associated with the particulate fraction. This latter activity remained with the membranes even after they were extensively washed to remove 99% of such soluble enzymes as lactate dehydrogenase and aldolase. Subsequent fractionation of the membranes by isopycnic density gradient centrifugation in sucrose resulted in a single band of transglutaminase activity at a density of 1.194 g/cm3. This activity was coincident with the major band of plasma membranes, which was identified by its content of 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and leucine aminopeptidase activities. After treatment with digitonin and fractionation on sucrose gradients, the transglutaminase activity and the plasma membrane marker enzyme activities were found at a new density of 1.210 g/cm3, while the enzyme markers for the other membrane fractions remained unchanged. From these data, we conclude that approximately 17% of the transglutaminase activity in rat liver is specifically associated with the plasma membranes.     

Behavior of a fluorescent analogue of calmodulin in living 3T3 cells

We have prepared and partially characterized a lissamine-rhodamine B fluorescent analogue of calmodulin, LRB-CM. The analogue had a dye/protein ratio of approximately 1.0 and contained no free dye or contaminating labeled proteins. LRB-CM was indistinguishable from native calmodulin upon SDS PAGE and in assays of phosphodiesterase and myosin light chain kinase. The emission spectrum of LRB-CM was insensitive to changes in pH, ionic strength, and temperature over the physiological range, but the apparent quantum yield was influenced somewhat by divalent cation concentration. LRB-CM injected into living Swiss 3T3 fibroblasts became associated with nitrobenzoxadiazole-phallacidin staining stress fibers in some interphase cells. LRB-CM and acetamidofluorescein-labeled actin co-injected into the same cell both became associated with fibers in some cells, but in most cases association of the two analogues with fibers was mutually exclusive. This suggests that calmodulin may differ from actin in the timing of incorporation into stress fibers or that we have distinguished distinct populations of stress fibers. We were able to detect no direct interaction of LRB-CM with actin by fluorescence photobleaching recovery (FRAP) of aqueous solutions. Interaction of LRB-CM with myosin light chain kinase also was not detected by FRAP. This suggests that the mean lifetime of the calmodulin-myosin light chain kinase complex is too short to affect the diffusion coefficient of calmodulin. We examined various fluorescent derivatives of proteins and dextrans as suitable control molecules for quantitative fluorescent analogue cytochemistry in living cells. Fluorescein isothiocyanate-dextrans were found to be preferable to all the proteins tested, since their mobilities in cytoplasm were inversely dependent on molecular size and there was no evidence of binding to intracellular components. In contrast, FRAP of LRB-CM in the cytoplasm of living 3T3 cells suggested that the analogue interacts with intracellular components with a range of affinities. The mobility of LRB-CM in the cytoplasm was sensitive to treatment of the cells with trifluoperazine, which suggests that at least some of the intracellular binding sites are specific for calmodulin in the calcium-bound form. FRAP of LRB-CM in the nuclei of living 3T3 cells indicated that the analogue was highly mobile within the nucleus but entered the nucleus from the cytoplasm much more slowly than fluorescein isothiocyanate-dextran of comparable molecular size and much more slowly than predicted from its mobility in cytoplasm.