Protein synthesis and secretion by the epididymis of the tammar wallaby, Macropus eugenii (Macropodidae: Marsupialia)

1992 ◽  
Vol 4 (5) ◽  
pp. 533 ◽  
Author(s):  
G Chaturapanich ◽  
RC Jones ◽  
J Clulow
Keyword(s):  
Protein Synthesis ◽  
Protein Secretion ◽  
Tammar Wallaby ◽  
Secreted Proteins ◽  
Blood Proteins ◽  
Macropus Eugenii ◽  
Cauda Epididymidis

The objectives were to assess the following in a marsupial: which proteins are synthesized by the different regions of the epididymis and secreted into the lumen of the ductus; the effect of the experimental method on the detection of protein secretion; the role of the testis in regulating the protein synthesis and secretion; and whether any of the secreted proteins may associate with spermatozoa. Samples from untreated animals were collected for examination by perfusing Krebs-bicarbonate through the ductus epididymidis in vivo (microperfusion), and after incorporation of [35S]methionine during incubation of minced duct in vitro. Electrophoresis of the samples showed that the caput and corpus epididymidis (initial segments) secreted most of the proteins that were synthesized and secreted by the epididymal mucosa, and that the cauda epididymidis secreted mainly blood proteins. Also, many more proteins were secreted in vitro than into the microperfusates in vivo, or were found by Jones (1987) in micropuncture samples of epididymal plasma. The synthesis and secretion of five proteins was androgen dependent (M(r) 75,700, 30,000, 18,700, 17,400 and 12,800). Also, the luminal fluids from the testis stimulated the secretion of two proteins (M(r) 46,300 and 36,100) and inhibited the secretion of three proteins (M(r) 43,000, 32,300 and 21,400). Examination of detergent extracts of spermatozoa indicated that they lose three proteins (M(r) 28,000, 30,000 and 47,000) and gain one (M(r) 30,400) during passage through the epididymis. The method of determining protein secretion affected the findings. Protein secretion, its control and its association with spermatozoa are broadly similar in the tammar wallaby to the processes described in eutherian mammals.

scholarly journals Citrulline directly modulates muscle protein synthesis via the PI3K/MAPK/4E-BP1 pathway in a malnourished state: evidence from in vivo, ex vivo, and in vitro studies

2017 ◽  
Vol 312 (1) ◽  
pp. E27-E36 ◽  
Author(s):  
Servane Le Plénier ◽  
Arthur Goron ◽  
Athanassia Sotiropoulos ◽  
Eliane Archambault ◽  
Chantal Guihenneuc ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ex Vivo ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Fold Increase ◽  
Underlying Mechanism ◽  
Muscle Homeostasis

Citrulline (CIT) is an endogenous amino acid produced by the intestine. Recent literature has consistently shown CIT to be an activator of muscle protein synthesis (MPS). However, the underlying mechanism is still unknown. Our working hypothesis was that CIT might regulate muscle homeostasis directly through the mTORC1/PI3K/MAPK pathways. Because CIT undergoes both interorgan and intraorgan trafficking and metabolism, we combined three approaches: in vivo, ex vivo, and in vitro. Using a model of malnourished aged rats, CIT supplementation activated the phosphorylation of S6K1 and 4E-BP1 in muscle. Interestingly, the increase in S6K1 phosphorylation was positively correlated ( P < 0.05) with plasma CIT concentration. In a model of isolated incubated skeletal muscle from malnourished rats, CIT enhanced MPS (from 30 to 80% CIT vs. Ctrl, P < 0.05), and the CIT effect was abolished in the presence of wortmannin, rapamycin, and PD-98059. In vitro, on myotubes in culture, CIT led to a 2.5-fold increase in S6K1 phosphorylation and a 1.5-fold increase in 4E-BP1 phosphorylation. Both rapamycin and PD-98059 inhibited the CIT effect on S6K1, whereas only LY-294002 inhibited the CIT effect on both S6K1 and 4E-BP1. These findings show that CIT is a signaling agent for muscle homeostasis, suggesting a new role of the intestine in muscle mass control.

scholarly journals The effects of 6 hours of hypoxia on protein synthesis in rat tissues in vivo and in vitro

1985 ◽  
Vol 228 (1) ◽  
pp. 179-185 ◽  
Author(s):  
V R Preedy ◽  
D M Smith ◽  
P H Sugden
Keyword(s):  
Protein Synthesis ◽  
Gastric Emptying ◽  
Skeletal Muscles ◽  
Rat Tissues ◽  
Tissue Nitrogen ◽  
The Brain ◽  
H Protein

Rates of protein synthesis were measured in vivo in several tissues (heart, skeletal muscles, liver, tibia, skin, brain, kidney, lung) of fed rats exposed to O2/N2 (1:9) for 6 h starting at 08:00-11:00 h. Protein synthesis rates were depressed by 15-35% compared with normoxic controls in all of the tissues studied. The decreases were greatest in the brain and the skin. Although hypoxia inhibited gastric emptying, its effects on protein synthesis could probably not be attributed to its induction of a starved state, because protein-synthesis rates in brain and skin were not decreased by a 15-18 h period of starvation initiated at 23:00 h. Furthermore, we showed that protein synthesis was inhibited by hypoxia in the rat heart perfused in vitro, suggesting a direct effect. The role of hypoxia in perturbing tissue nitrogen balance in various physiological and pathological states is discussed.

249 Targeting IFNβ-regulated secretory profiles to overcome acquired anti-PD-L1 resistance

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A270-A270
Author(s):  
Yuhao Shi ◽  
Melissa Dolan ◽  
Michalis Mastri ◽  
Kevin Eng ◽  
John Ebos
Keyword(s):  
Treatment Failure ◽  
Acquired Resistance ◽  
Secreted Proteins ◽  
Tumor Growth Inhibition ◽  
Type I ◽  
Altered Expression ◽  
The Impact

BackgroundTherapeutic targeting of programmed cell death protein ligand 1 (PD-L1) has led to durable benefits for many cancer patients; however, the development of acquired resistance is common. Dysregulated type II interferon (IFN) signaling on tumor cells can contribute to resistance via altered expression of IFN stimulated genes (ISGs), which include cytokines and growth factors capable of immune-suppression and tumor promotion. However, the role of type I IFNs, including IFNα and IFNβ, in acquired resistance remain understudied. Here we examined the impact of chronic PD-L1 blockade to evaluate the role of IFN-related secretory changes in preclinical models of resistance.MethodsUsing a mouse breast EMT6 orthotopic tumor model, we selected PD-L1 drug resistant (PDR) cells from tumors initially responsive to PD-L1 blockade, but that later relapsed. Using transcriptomic and proteomic approaches, we evaluated secreted proteins associated with IFN signaling. To test for direct connections between PD-L1 and IFN signaling in secretory profile modulation, genetic and therapeutic disruption of PD-L1/IFNAR1 were conducted in vitro.ResultsWe identified a unique gene signature for secreted proteins following acquired resistance to PD-L1 blockade that associated with IFN signaling. This secretory signature was validated using publicly available datasets derived from preclinical tumors and clinical biopsies after anti-PD-L1 treatment failure. Interestingly, genetic and antibody inhibition of PD-L1 in vitro enhanced PDR secretory signatures following IFNβ stimulation suggesting PD-L1 tumor-intrinsic functions may regulate IFN responses following acquired resistance. To test whether secretory profiles impact tumor growth, inhibition of specific ISGs (IL-6) or ISG regulators (IFNAR1) were examined and found to inhibit PDR tumors in vivo, compared to parental controls.ConclusionsTogether, these findings identify a secretory profile associated with acquired resistance to PD-L1 blockade that may be modulated, at least in part, by IFNβ. Selective targeting of secreted ISGs may provide a benefit for patients after anti-PD-L1 treatment failure.

239. Expression of PAF-R and p53 in the endometrium during entry into and reactivation from diapause in the tammar wallaby

2008 ◽  
Vol 20 (9) ◽  
pp. 39
Author(s):  
J. C. Fenelon ◽  
G. Shaw ◽  
M. B. Renfree
Keyword(s):  
Tammar Wallaby ◽  
Early Embryo ◽  
Human Reproduction ◽  
Embryonic Diapause ◽  
P53 Expression ◽  
Cellular Location ◽  
Macropus Eugenii ◽  
A Cell

Embryonic diapause is widespread amongst mammals, but is especially common in the kangaroos and wallabies. In the tammar, Macropus eugenii, the sequence of endocrine events leading to embryonic diapause and reactivation are well defined and the blastocyst can remain in diapause for up to 11 months without cell division or apoptosis occurring (Renfree and Shaw 2000). The ovarian hormones exert their effects on the blastocyst by alterations in the endometrial secretions, but the molecular cross-talk between the endometrium and blastocyst is unknown. One possible regulator of diapause is the phospholipid PAF, an embryotrophin that acts as a trophic/survival factor for the early embryo (O'Neill 2005) partly by inactivating the expression of p53, a cell cycle inhibitor, via the PI3-K pathway. PAF is released from the tammar endometrium around the time of reactivation from diapause (Kojima et al. 1993). This study examined the expression of PAF-R and p53 in the tammar endometrium at entry into, and reactivation from, diapause. PAF-R and p53 were highly conserved with orthologueues in human and mouse. PAF-R and p53 expression was assessed by RT–PCR and both genes were expressed in the endometrium at all stages examined. Quantitative PCR (QPCR) studies performed for PAF-R in the endometrium show that levels of PAF-R vary depending on the stage examined and appear to be increasing at entry into diapause and decreasing at exit from diapause. Immunohistochemical (IHC) studies are in progress to determine the cellular location of PAF-R in the endometrium and confirm the QPCR results. QPCR and IHC studies are in progress to determine if there is any change in levels of expression or cellular location of p53 between the stages examined and how this relates to PAF-R availability. These results suggest that the control of diapause in the tammar involves interactions between multiple factors. (1) Renfree MB, Shaw G (2000) Diapause. Annu Rev Physiol 62, 353–375 (2) O'Neill C (2005) The role of Paf in embryo physiology. Human Reproduction Update 11, 215–228 (3) Kojima T et. al. (1993) Production and secretion of progesterone in vitro and presence of PAF in early pregnancy of the marsupial, Macropus eugenii. Reproduction Fertility Development 5, 15–25.

scholarly journals The Reverse Side of a Coin: “Factor-Free” Ribosomal Protein Synthesis In Vitro is a Consequence of the In Vivo Proofreading Mechanism

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 588
Author(s):  
Finkelstein
Keyword(s):  
Quality Control ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ribosomal Protein ◽  
Elongation Factor ◽  
Ribosomal Protein Synthesis

This paper elucidates a close connection between two well-known facts that until now have seemed independent: (i) the quality control (“proofreading”) of the emerging amino acid sequence, occurring during the normal, elongation-factor-dependent ribosomal biosynthesis, which is performed by removing those Aa-tRNAs (aminoacyl tRNAs) whose anticodons are not complementary to the exhibited mRNA codons, and (ii) the in vitro discovered existence of the factor-free ribosomal synthesis of polypeptides. It is shown that a biological role of proofreading is played by a process that is exactly opposite to the step of factor-free binding of Aa-tRNA to the ribosome-exposed mRNA: a factor-free removal of that Aa-tRNA whose anticodon is not complementary to the ribosome-exhibited mRNA codon.

Studies on the Regulatory Role of trans-Cinnamic Acid on the Activity of the Phenylalanine Ammonia-Lyase (PAL) in Suspension Cultures of Daucus carota L.

1983 ◽  
Vol 38 (5-6) ◽  
pp. 408-412 ◽  
Author(s):  
W. Noé ◽  
H. U. Seitz
Keyword(s):  
Protein Synthesis ◽  
Cinnamic Acid ◽  
Phenylalanine Ammonia Lyase ◽  
Competitive Inhibitor ◽  
Regulatory Role ◽  
Coumaric Acid ◽  
Daucus Carota L

In vivo and in vitro experiments were performed in order to study the regulatory role of trans- cinnamic acid and its hydroxylated derivatives (p-coumaric acid, caffeic acid) on the deamina­tion of phenylalanine catalyzed by PAL (EC 4.3.1.5). Trans-cinnamic acid inhibits growth and reduces the content of soluble proteins of anthocyanin-containing carrot cells grown in suspen­sion. There is strong evidence from the polysomal patterns and from the effect of trans-cinnamic acid on protein synthesis in vitro that protein synthesis is inhibited. The kinetic data of PAL clearly demonstrate that trans-cinnamic acid inhibits the enzyme by a noncompetitive mecha­nism. On the contrary, ʟ-α-aminooxy-β-phenylpropionic acid (ʟ-AOPP), a competitive inhibitor of PAL, does not affect protein metabolism.

scholarly journals Changes in a-Lactalbumin, Total Lactose, UDP-Galactose Hydrolase and other Factors in Tammar Wallaby (Macropus eugenii) Milk during Lactation

1987 ◽  
Vol 40 (1) ◽  
pp. 37 ◽  
Author(s):  
MichaeI Messer ◽  
Christine Elliott
Keyword(s):  
Post Partum ◽  
Rabbit Antiserum ◽  
Tammar Wallaby ◽  
Macropus Eugenii ◽  
Milk Samples ◽  
In Vitro Hydrolysis ◽  
Nucleotide Pyrophosphatase ◽  
Hydrolysis Of

a-Lactalbumin was isolated from milk of M. eugenii and its concentration in milk samples taken at various times during lactation (0-40 we.eks post partum) was determined by single radial immunodiffusion using rabbit antiserum to the purified protein. The a-lactalbumin concentration remained almost constant throughout lactation even though the concentration of total lactose (free �lactose plus lactose contained in oligosaccharides) feli to zero after 34 weeks post partum. This fall in lactose was accompanied by a rise in the free galactose and glucose concentrations and marked increases in UDPgalactose hydrolase, nucleotide pyrophosphatase, alkaline phosphatase and acid fj-galactosidase activities. It is suggested that the in vitro hydrolysis of UDP-galactose was due to nucleotide pyrophosphatase and that this enzyme may also playa role in vivo late in lactation by making UDP-galactose unavailable for the synthesis of lactose. Alternatively, lactose and lactose-containing oligosaccharides might be degraded by the acid fj-galactosidase during or after secretion.

scholarly journals Protein synthesis regulation by leucine

2010 ◽  
Vol 46 (1) ◽  
pp. 29-36 ◽  
Author(s):  
Daiana Vianna ◽  
Gabriela Fullin Resende Teodoro ◽  
Francisco Leonardo Torres-Leal ◽  
Julio Tirapegui
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Mrna Translation ◽  
Protein Translation ◽  
Cellular Effects ◽  
Cellular Processes ◽  
High Protein Diets

In vivo and in vitro studies have demonstrated that high protein diets affect both protein synthesis and regulation of several cellular processes. The role of amino acids as substrate for protein synthesis has been established in the literature. However, the mechanism by which these amino acids modulate transcription and regulate the mRNA translation via mTOR-dependent signaling pathway has yet to be fully determined. It has been verified that mTOR is a protein responsible for activating a cascade of biochemical intracellular events which result in the activation of the protein translation process. Of the aminoacids, leucine is the most effective in stimulating protein synthesis and reducing proteolysis. Therefore, it promotes a positive nitrogen balance, possibly by favoring the activation of this protein. This amino acid also directly and indirectly stimulates the synthesis and secretion of insulin, enhancing its anabolic cellular effects. Therefore, this review aimed to identify the role of leucine in protein synthesis modulation and to discuss the metabolic aspects related to this aminoacid.

Interactions of sperm and the reproductive ducts of the male tammar wallaby, Macropus eugenii (Macropodidae: Marsupialia)

1994 ◽  
Vol 6 (4) ◽  
pp. 437 ◽  
Author(s):  
RC Jones ◽  
J Clulow
Keyword(s):  
Animal Model ◽  
Inorganic Ions ◽  
Tammar Wallaby ◽  
Duct System ◽  
Sperm Production ◽  
Structural Differentiation ◽  
Macropus Eugenii ◽  
Cauda Epididymidis ◽  
Good Animal Model

This review compares sperm production in the tammar wallaby and eutherian mammals, particularly the rat. The capacity of sperm to fertilize an ovum when they leave the testis and the changes they undergo in the epididymidis are considered. The structural differentiation and regulation of the extratesticular duct system is assessed and related to the reabsorption and secretion of water, inorganic ions and proteins, and the interaction of sperm and proteins synthesized and secreted by the epididymidis. Adaptations of the cauda epididymidis for storing spermatozoa are also considered. It is suggested that the tammar may be a good animal model to study the suppression of sperm motility and metabolism in the cauda epididymidis as it is possible to collect from them luminal samples of sperm which are initially immotile and then spontaneously activate during incubation in vitro.

Inducible Silencing Of eIF4E Using a Tet-On System Results In Myeloma Growth In Vivo That Correlates With eIF4E Expression

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3164-3164
Author(s):  
Shirong Li ◽  
Jing Fu ◽  
Jordan M. Schecter ◽  
Caisheng Lu ◽  
Markus Y. Mapara ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Synthesis ◽  
Cell Growth ◽  
Tumor Growth ◽  
Critical Role ◽  
Myeloma Cell ◽  
Control Shrna

Abstract Introduction Overexpression and/or activation of eukaryotic initiation factor 4E (eIF4E) is critical for oncogenic protein synthesis. Mutations in genes related to mRNA translation are involved in the pathogenesis of multiple myeloma (Chapman, Lawrence et al. 2011). Recently, we found that MM cells express high levels of eIF4E protein compared to normal plasma cells and overexpression of eIF4E induces transcription factors such as c-myc critical for the growth of multiple myeloma cells (Li, Fu et al. 2011,2012). The understanding of the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. Here we show the critical role of eIF4E driven protein synthesis by using an inducible knockdown system to silence eIF4E gene expression and confirm the critical role of eIF4E in multiple myeloma growth in vivo and in vitro. Methods and Results We stably infected U266, RPMI-8226, IM-9 and MM.1S cells with a robust inducible single-lentiviral knockdown vector pLKO-Tet-On containing either control non-targeting shRNA or eIF4E targeting shRNA sequences. Doxycycline-induced eIF4E shRNA expression resulted in significant decrease of eIF4E mRNA and protein in eIF4E-shRNA but not the control shRNA infected MM cells. To determine the effects of eIF4E knockdown on MM cell growth and viability, stably transfected cell lines were grown in the presence or absence of doxycycline. Silencing of eIF4E by doxycycline induction of eIF4E shRNA in RPMI-8226 cells significantly inhibited (>72%,P<0.01) cell growth accompanied by a decrease of c-myc, cyclin D1, C/EBP beta and IRF4 all critical for myeloma cell growth. Cell cycle analysis revealed increased cells population in G0/G1 phase (62% vs 80%) in doxycycline-induced eIF4E shRNA cells with a significant reduction (P<0.001) of clonogenic tumor growth reflected by a decrease in colony numbers (27.6 ± 4.2 vs 5.3 ± 3.4) and size. To determine the role of high expression of eIF4E in MM tumor growth in vivo, we generated subcutaneous MM xenografts in severe combined immunodeficient x beige (SCID/bg) mice using the inducible U266-Tet-CT-shRNA and U266-Tet-eIF4E-shRNA cells. In contrast to vehicle or doxycycline-treated control shRNA tumors, doxycycline treated animals bearing U266-Tet-eIF4E-shRNA xenografts showed a significant inhibition (P<0.001) of tumor growth by 80% after 21 days. The transient inhibition of tumor growth correlated with the transient doxycycline-induced eIF4E knockdown further confirming the critical role of eIF4E. Immunohistochemical staining of tumors confirmed the decreased of eIF4E expression in doxycycline-treated mice bearing U266-Tet-eIF4E-shRNA tumors compared with tumors of vehicle-treated or non-doxycyclin treated mice. Conclusion Here we show that eIF4E, a key player in the translational machinery, promotes multiple myeloma cell growth. We found that high eIF4E expression is indispensable for the growth of MM cells both in vitro and in vivo. Silencing of eIF4E decreases protein expression of a subset of transcripts encoding regulators of the cell cycle and proliferation, and resulted in tumor inhibition. Our study indicated that targeting transcriptional initiating factor eIF4E may represent a novel therapeutic strategy for MM treatment. Disclosures: Schecter: Seattle Genetics: Honoraria, Research Funding. Lentzsch:Celgene: Research Funding.

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