In vivo phosphorylation sites in fetal and adult rat tau.

Protein kinase C (PKC) modulates cardiomyocyte function by phosphorylation of intracellular targets including myofilament proteins. Data generated from studies on in vitro heart preparations indicate that PKC phosphorylation of troponin I (TnI), primarily via PKC-ε, may slow the rates of cardiac contraction and relaxation (+dP/d t and −dP/d t). To explore this issue in vivo, we employed transgenic mice [mutant TnI (mTnI) mice] in which the major PKC phosphorylation sites on cardiac TnI were mutated by alanine substitutions for Ser43 and Ser45 and studied in situ hemodynamics at baseline and increased inotropy. Hearts from mTnI mice exhibited increased contractility, as shown by a 30% greater +dP/dt and 18% greater −dP/d t than FVB hearts, and had a negligible response to isoproterenol compared with FVB mice, in which +dP/d t increased by 33% and −dP/d t increased by 26%. Treatment with phenylephrine and propranolol gave a similar result; FVB mouse hearts demonstrated a 20% increase in developed pressure, whereas mTnI mice showed no response. Back phosphorylation of TnI from mTnI hearts demonstrated that the mutation of the PKC sites was associated with an enhanced PKA-dependent phosphorylation independent of a change in basal cAMP levels. Our results demonstrate the important role that PKC-dependent phosphorylation of TnI has on the modulation of cardiac function under basal as well as augmented states and indicate interdependence of the phosphorylation sites of TnI in hearts beating in situ.

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Stable incorporation of a bacterial gene into adult rat skeletal muscle in vivo

AJP Cell Physiology ◽

10.1152/ajpcell.1990.258.3.c578 ◽

1990 ◽

Vol 258 (3) ◽

pp. C578-C581 ◽

Cited By ~ 33

Author(s):

D. B. Thomason ◽

F. W. Booth

Keyword(s):

Skeletal Muscle ◽

Leukemia Virus ◽

Rat Skeletal Muscle ◽

Bacterial Gene ◽

Muscle Gene Expression ◽

Adult Rat ◽

Foreign Genes ◽

Muscle Gene ◽

Beta Galactosidase

We have developed a novel technique to incorporate and stably express foreign genes in adult rat skeletal muscle in vivo. Endogeneous satellite cells in skeletal muscle regenerating from bupivacaine damage were infected with an injected retrovirus containing the Escherichia coli beta-galactosidase gene under the promoter control of the Moloney murine leukemia virus long-terminal repeat. Constitutive and stable expression of beta-galactosidase activity was observed in muscle fibers after 6 days and 1 mo of muscle regeneration. Two patterns of expression were observed, diffuse expression within fibers and focal expression associated with the sarcolemma. This technique will allow future experiments with muscle-specific genes and promoters to study the physiological regulation of skeletal muscle gene expression in the intact adult mammal. Furthermore, the technique of stimulating stem cell proliferation to allow retroviral-mediated gene transfer may be generally applicable to other tissues.

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Effects of hypophysectomy and subsequent FSH and testosterone treatment on inhibin production by adult rat testes

Journal of Endocrinology ◽

10.1677/joe.0.1050001 ◽

1985 ◽

Vol 105 (1) ◽

pp. 1-6 ◽

Cited By ~ 26

Author(s):

C. L. Au ◽

D. M. Robertson ◽

D. M. de Kretser

Keyword(s):

Seminiferous Tubule ◽

Hormonal Control ◽

Human Chorionic Gonadotrophin ◽

Experimental Conditions ◽

Adult Rats ◽

Adult Rat ◽

Fluid Production ◽

Tubule Fluid

ABSTRACT The hormonal control of inhibin production by adult rat testes was investigated using an in-vitro inhibin bioassay validated for the measurement of inhibin activity in charcoal-treated rat testicular extracts. The effect of hypophysectomy examined at 16 h, 3, 7 and 42 days after surgery showed a decrease in testicular inhibin content and seminiferous tubule fluid production by 7 days and a decrease in inhibin production by 42 days. Serum FSH and LH were suppressed 3 days after surgery. In 30-day chronically hypophysectomized adult rats treated for 3 days with twice daily s.c. injections of (a) human FSH (hFSH, 22 i.u./rat per day), (b) testosterone (5 mg/rat per day), (c) hFSH + testosterone (same doses as a and b), or (d) human chorionic gonadotrophin (hCG, 12 i.u./rat per day), hFSH or hFSH and testosterone stimulated an increase in testicular inhibin content but not in inhibin production or tubule fluid production. Testosterone and hCG had no effect on these parameters. It is concluded that in vivo, FSH alone stimulates an increase in testicular inhibin content. The failure to observe an increase in inhibin production in vivo is attributed to the suppression of seminiferous tubule fluid production under the same experimental conditions. J. Endocr. (1985) 105, 1–6

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Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii

Eukaryotic Cell ◽

10.1128/ec.00067-09 ◽

2009 ◽

Vol 8 (7) ◽

pp. 922-932 ◽

Cited By ~ 37

Author(s):

Jens Boesger ◽

Volker Wagner ◽

Wolfram Weisheit ◽

Maria Mittag

Keyword(s):

Chlamydomonas Reinhardtii ◽

Protein Phosphorylation ◽

Proteome Analysis ◽

Physiological Status ◽

Phosphorylation Sites ◽

Cell Organelles ◽

Flagellar Proteins ◽

Reversible Protein Phosphorylation ◽

Cilia And Flagella

ABSTRACT Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.

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