scholarly journals Synthesis and incorporation of myelin polypeptides into CNS myelin.

1982 ◽  
Vol 95 (2) ◽  
pp. 598-608 ◽  
Author(s):  
D R Colman ◽  
G Kreibich ◽  
A B Frey ◽  
D D Sabatini
Keyword(s):  
Microsomal Fraction ◽  
Proteolipid Protein ◽  
Terminal Amino Acid ◽  
Free Translation ◽  
Myelin Fraction ◽  
Old Rats ◽  
Terminal Amino ◽  
Subsequent Passage

The distribution of newly synthesized proteolipid protein (PLP, 23 kdaltons) and myelin basic proteins (MBPs, 14-21.5 kdaltons) was determined in microsomal and myelin fractions prepared from the brainstems o1 10-30 d-old rats sacrificed at different times after an intracranial injection of 35S-methionine. Labeled MBPs were found in the myelin fraction 2 min after the injection, whereas PLP appeared first in the rough microsomal fraction and only after a lag of 30 min in the myelin fraction. Cell-free translation experiments using purified mRNAs demonstrated that PLP and MBPs are synthesized in bound and free polysomes, respectively. A mechanism involving the cotranslational insertion into the ER membrane and subsequent passage of the polypeptides through the Golgi apparatus is consistent with the lag observed in the appearance of the in vivo-labeled PLP in the myelin membrane. Newly synthesized PLP and MBPs are not proteolytically processed, because the primary translation products synthesized in vitro had the same electrophoretic mobility and N-terminal amino acid sequence as the mature PLP and MBP polypeptides. It was found that crude myelin fractions are highly enriched in mRNAs coding for the MBPs but not in mRNA coding for PLP. This suggests that whereas the bound polysomes synthesizing PLP are largely confined to the cell body, free polysomes synthesizing MBPs are concentrated in oligodendrocyte processes involved in myelination, which explains the immediate incorporation of MBPs into the developing myelin sheath.

scholarly journals A synthetic glucagon-like peptide-1 analog with improved plasma stability

1998 ◽  
Vol 159 (1) ◽  
pp. 93-102 ◽  
Author(s):  
U Ritzel ◽  
U Leonhardt ◽  
M Ottleben ◽  
A Ruhmann ◽  
K Eckart ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasma Insulin ◽  
Rat Plasma ◽  
Plasma Stability ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 (GLP-1) is the most potent endogenous insulin-stimulating hormone. In the present study the plasma stability and biological activity of a GLP-1 analog, [Ser]GLP-1(7-36)amide, in which the second N-terminal amino acid alanine was replaced by serine, was evaluated in vitro and in vivo. Incubation of GLP-1 with human or rat plasma resulted in degradation of native GLP-1(7-36)amide to GLP-1(9-36)amide, while [Ser]GLP-1(7-36)amide was not significantly degraded by plasma enzymes. Using glucose-responsive HIT-T15 cells, [Ser]GLP-1(7-36)amide showed strong insulinotropic activity, which was inhibited by the specific GLP-1 receptor antagonist exendin-4(9-39)amide. Simultaneous i.v. injection of [Ser]GLP-1(7-36)amide and glucose in rats induced a twofold higher increase in plasma insulin levels than unmodified GLP-1(7-36)amide with glucose and a fivefold higher increase than glucose alone. [Ser]GLP-1(7-36)amide induced a 1.5-fold higher increase in plasma insulin than GLP-1(7-36)amide when given 1 h before i.v. application of glucose. The insulinotropic effect of [Ser]GLP-1(7-36)amide was suppressed by i.v. application of exendin-4(9-39)amide. The present data demonstrate that replacement of the second N-terminal amino acid alanine by serine improves the plasma stability of GLP-1(7-36)amide. The insulinotropic action in vitro and in vivo was not impaired significantly by this modification.

scholarly journals Energy-dependent protein folding: modeling how a protein folding machine may work

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 3
Author(s):  
Harutyun Sahakian ◽  
Karen Nazarian ◽  
Arcady Mushegian ◽  
Irina Sorokina
Keyword(s):  
Molecular Dynamics ◽  
Protein Folding ◽  
Amino Acid ◽  
Dynamics Simulation ◽  
Terminal Amino Acid ◽  
Peptide Backbone ◽  
Terminal Amino ◽  
Energy Dependent

Background: Proteins fold robustly and reproducibly in vivo, but many cannot fold in vitro in isolation from cellular components. Despite the remarkable progress that has been achieved by the artificial intelligence approaches in predicting the protein native conformations, the pathways that lead to such conformations, either in vitro or in vivo, remain largely unknown. The slow progress in recapitulating protein folding pathways in silico may be an indication of the fundamental deficiencies in our understanding of folding as it occurs in nature. Here we consider the possibility that protein folding in living cells may not be driven solely by the decrease in Gibbs free energy and propose that protein folding in vivo should be modeled as an active energy-dependent process. The mechanism of action of such a protein folding machine might include direct manipulation of the peptide backbone. Methods: To show the feasibility of a protein folding machine, we conducted molecular dynamics simulations that were augmented by the application of mechanical force to rotate the C-terminal amino acid while simultaneously limiting the N-terminal amino acid movements. Results: Remarkably, the addition of this simple manipulation of peptide backbones to the standard molecular dynamics simulation indeed facilitated the formation of native structures in five diverse alpha-helical peptides. Steric clashes that arise in the peptides due to the forced directional rotation resulted in the behavior of the peptide backbone no longer resembling a freely jointed chain. Conclusions: These simulations show the feasibility of a protein folding machine operating under the conditions when the movements of the polypeptide backbone are restricted by applying external forces and constraints. Further investigation is needed to see whether such an effect may play a role during co-translational protein folding in vivo and how it can be utilized to facilitate folding of proteins in artificial environments.

Role of the N-terminal Amino Acid for the Biological Activities of Angiotensin and Inhibitory Analogues

1974 ◽  
Vol 52 (1) ◽  
pp. 39-49 ◽  
Author(s):  
D. Regoli ◽  
F. Rioux ◽  
W. K. Park ◽  
C. Choi
Keyword(s):  
Smooth Muscles ◽  
Rabbit Aorta ◽  
Intrinsic Activity ◽  
Vascular Smooth Muscles ◽  
Terminal Amino Acid ◽  
Duration Of Action ◽  
Metabolic Degradation ◽  
Terminal Amino

Aspartic acid was replaced in position 1 of angiotensin II (ATII) with several amino acids, to assess the possible influence of the N-terminal amino acid for (a) the intrinsic activity, (b) the affinity, and (c) the metabolic degradation of agonist analogues of ATII. Some of the substitutions in position 1 were used in combination with replacement of Phe by Gly or Leu in position 8, to obtain the corresponding antagonist.The compounds were tested in vivo (rat blood pressure) and in two in vitro preparations (rat stomach and rabbit aorta strips). The oil immersion technique, described by Kalsner and Nickerson (1968) (Can. J. Physiol. Pharmacol. 46, 719–730), was used to study the disposition of the peptides by vascular smooth muscles (rabbit aorta strips). Degradation of the peptides by purified aminopeptidases was evaluated in vitro by measuring the fragments on paper chromatography. Potency of antagonists was estimated in vivo (ID50) and in vitro (pA2 values): duration of action was established by infusing the inhibitors intravenously into anesthetized rats and testing the effect of standard doses of angiotensin before and after.The results indicate that replacement of Asp with other amino acids does not influence the intrinsic activity, but can either increase or decrease the affinity in vitro or the potency in vivo. 1-Sar-ATII, and 1-D-Ala-ATII are more potent and longer acting than 1-Asp-ATII on isolated intestinal and vascular smooth muscles, but not in vivo. On the contrary, 1-β-Asp-ATII and 1-β-D-Asp-ATII are more potent than 1-Asp-ATIIin vivo, but not on rabbit aorta strips. Rate of relaxation of rabbit aorta strips suspended in oil, after contraction with submaximal doses of several analogues of ATII, are significantly slower than relaxation after 1-Asn2-ATII and 1-Asp-ATII. A close parallelism between the diminution of the relaxation rate in oil and the degradation by aminopeptideses in vitro was observed, suggesting that metabolic degradation may be the major factor determining relaxation of rabbit aorta in oil after contraction with one of these peptides. Potencies of antagonists in vivo and in vitro are increased by replacing Asp with Sar. Substitution of Asp with β-Asp or β-D-Asp brings about a slight increase of potency in vivo but not in vitro. It appears that firm binding and prolonged occupation of receptors by sarcosyl derivatives are the primary factors contributing to increase the potency and to prolong the duration of action of antagonists, while prevention or reduction of metabolic breakdown by aminopeptidases is much less efficient.

scholarly journals Energy-dependent protein folding: modeling how a protein folding machine may work

2020 ◽  
Author(s):  
Harutyun K. Sahakyan ◽  
Karen B. Nazaryan ◽  
Arcady R. Mushegian ◽  
Irina N. Sorokina
Keyword(s):  
Molecular Dynamics ◽  
Protein Folding ◽  
Amino Acid ◽  
Dynamics Simulation ◽  
Terminal Amino Acid ◽  
Peptide Backbone ◽  
Terminal Amino ◽  
Energy Dependent

AbstractProteins fold robustly and reproducibly in vivo, but many cannot fold in vitro in isolation from cellular components. The pathways to proteins’ native conformations, either in vitro or in vivo, remain largely unknown. The slow progress in recapitulating protein folding pathways in silico may be an indication of the fundamental deficiencies in our understanding of folding as it occurs in nature. Here we consider the possibility that protein folding in living cells may not be driven solely by the decrease in Gibbs free energy and propose that protein folding in vivo should be modeled as an active energy-dependent process. The mechanism of action of such protein folding machine might include direct manipulation of the peptide backbone. To show the feasibility of a protein folding machine, we conducted molecular dynamics simulations that were augmented by the application of mechanical force to rotate the C-terminal amino acid while simultaneously limiting the N-terminal amino acid movements. Remarkably, the introduction of this simple manipulation of peptide backbones to the standard molecular dynamics simulation indeed facilitated the formation of native structures in five diverse alpha-helical peptides. Such effect may play a role during co-translational protein folding in vivo: considering the rotating motion of the tRNA 3’-end in the peptidyltransferase center of the ribosome, it is possible that this motion might introduce rotation to the nascent peptide and influence the peptide’s folding pathway in a way similar to what was observed in our simulations.

scholarly journals Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems.

1989 ◽  
Vol 9 (11) ◽  
pp. 5073-5080 ◽  
Author(s):  
M Kozak
Keyword(s):  
Context Effects ◽  
Consensus Sequence ◽  
Negative Regulator ◽  
Late Event ◽  
Free Translation ◽  
Initiator Codon ◽  
Translation Systems ◽  
Short Versions

The context requirements for recognition of an initiator codon were evaluated in vitro by monitoring the relative use of two AUG codons that were strategically positioned to produce long (pre-chloramphenicol acetyl transferase [CAT]) and short versions of CAT protein. The yield of pre-CAT initiated from the 5'-proximal AUG codon increased, and synthesis of CAT from the second AUG codon decreased, as sequences flanking the first AUG codon increasingly resembled the eucaryotic consensus sequence. Thus, under prescribed conditions, the fidelity of initiation in extracts from animal as well as plant cells closely mimics what has been observed in vivo. Unexpectedly, recognition of an AUG codon in a suboptimal context was higher when the adjacent downstream sequence was capable of assuming a hairpin structure than when the downstream region was unstructured. This finding adds a new, positive dimension to regulation by mRNA secondary structure, which has been recognized previously as a negative regulator of initiation. Translation of pre-CAT from an AUG codon in a weak context was not preferentially inhibited under conditions of mRNA competition. That result is consistent with the scanning model, which predicts that recognition of the AUG codon is a late event that occurs after the competition-sensitive binding of a 40S ribosome-factor complex to the 5' end of mRNA. Initiation at non-AUG codons was evaluated in vitro and in vivo by introducing appropriate mutations in the CAT and preproinsulin genes. GUG was the most efficient of the six alternative initiator codons tested, but GUG in the optimal context for initiation functioned only 3 to 5% as efficiently as AUG. Initiation at non-AUG codons was artifactually enhanced in vitro at supraoptimal concentrations of magnesium.

scholarly journals In vitro analysis of the origin and maintenance of O-2Aadult progenitor cells.

1992 ◽  
Vol 116 (1) ◽  
pp. 167-176 ◽  
Author(s):  
D Wren ◽  
G Wolswijk ◽  
M Noble
Keyword(s):  
Adult Life ◽  
Cell Types ◽  
Adult Rats ◽  
Optic Nerves ◽  
Limited Life ◽  
Old Rats ◽  
Vitro Analysis

We have been studying the differing characteristics of oligodendrocyte-type-2 astrocyte (O-2A) progenitors isolated from optic nerves of perinatal and adult rats. These two cell types display striking differences in their in vitro phenotypes. In addition, the O-2Aperinatal progenitor population appears to have a limited life-span in vivo, while O-2Aadult progenitors appear to be maintained throughout life. O-2Aperinatal progenitors seem to have largely disappeared from the optic nerve by 1 mo after birth, and are not detectable in cultures derived from optic nerves of adult rats. In contrast, O-2Aadult progenitors can first be isolated from optic nerves of 7-d-old rats and are still present in optic nerves of 1-yr-old rats. These observations raise two questions: (a) From what source do O-2Aadult progenitors originate; and (b) how is the O-2Aadult progenitor population maintained in the nerve throughout life? We now provide in vitro evidence indicating that O-2Aadult progenitors are derived directly from a subpopulation of O-2Aperinatal progenitors. We also provide evidence indicating that O-2Aadult progenitors are capable of prolonged self renewal in vitro. In addition, our data suggests that the in vitro generation of oligodendrocytes from O-2Aadult progenitors occurs primarily through asymmetric division and differentiation, in contrast with the self-extinguishing pattern of symmetric division and differentiation displayed by O-2Aperinatal progenitors in vitro. We suggest that O-2Aadult progenitors express at least some properties of stem cells and thus may be able to support the generation of both differentiated progeny cells as well as their own continued replenishment throughout adult life.

scholarly journals Сhanges in intracellular calcium concentration and mitochondrial potential in primary cultures of rat brain cells in acute mechanical injury

2018 ◽  
pp. 124-128
Author(s):  
И.А. Красильникова ◽  
З.В. Бакаева ◽  
В.Г. Пинелис ◽  
О.Ю. Лисина ◽  
А.М. Сурин
Keyword(s):  
Calcium Concentration ◽  
Primary Cultures ◽  
Mechanical Injury ◽  
Ionotropic Glutamate Receptors ◽  
Mitochondrial Potential ◽  
Microscopy Technique ◽  
Nmda Channels ◽  
Old Rats

Актуальность. Моделирование in vitro травматического повреждения мозга помогает выяснить патологические механизмы, ответственные за гибель клеток или их последующую дисфункцию в деталях, труднодостижимых in vivo. Цель. Определить изменения внутриклеточной концентрации свободного Са2+ ([Ca2+]i) и митохондриального потенциала (m) в первичной нейроглиальной культуре непосредственно в момент нанесения механической травмы. Методы и материалы. Методом флуоресцентной микроскопии отслеживали изменения [Ca2+]i) и m в первичной нейроглиальной культуре из коры головного мозга 1-2-дневных крыс. Возраст культуры в момент измерений 11-14 дней. Результаты. Обнаружено, что нейротравма вызывает скачок [Ca2+]i и совпадающее с ним по времени резкое падение m. Эти изменения затрагивали клетки, расположенные не далее 100мкм от границы травмы. Блокирование ионотропных глутаматных рецепторов NMDA-типа с помощью МК-801 снижало в 8,5 раз долю нейронов, имевших высокий подъем [Ca2+]i. Выводы. Поступления Са2+ в клетки при механическом повреждении первичной нейроглиальной культуры происходит преимущественно по NMDA-каналам и отчасти, вероятно, по АТФ-активируемым каналам. Background. In vitro modeling of traumatic brain injury helps clarifying pathological mechanisms responsible for cell death or their subsequent dysfunction in detail, which is difficult to accomplish in vivo. Aim. To determine changes in intracellular free Ca2+ concentration ([Ca2+]i) and mitochondrial potential (m) in a primary neuroglial culture during infliction of a mechanical injury (scratch). Methods and materials. Changes in [Ca2+]i and m in the primary neuroglial culture from the cerebral cortex of 1-2 day old rats were monitored using a fluorescence microscopy technique. Measurements were performed in 11-14-day old cultures. Results. Neurotrauma resulted in a sharp increase in [Ca2+]i and a synchronous profound drop of m. These changes affected cells located not farther than 100 µm from the boundary of the injury. Inhibition of NMDA-type ionotropic glutamate receptors with MK-801 reduced by approximately 8.5 times the proportion of neurons, which indicated a high [Ca2+]i rise. Conclusion. Са2+ influx into cells during mechanical injury of the primary neuroglial culture occurs predominantly through NMDA-channels and perhaps partially through ATP-activated channels.

scholarly journals The N-terminus of hepcidin is essential for its interaction with ferroportin: structure-function study

Blood ◽  
2006 ◽  
Vol 107 (1) ◽  
pp. 328-333 ◽  
Author(s):  
Elizabeta Nemeth ◽  
Gloria C. Preza ◽  
Chun-Ling Jung ◽  
Jerry Kaplan ◽  
Alan J. Waring ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Progressive Decrease ◽  
Cellular Iron ◽  
Gfp Fusion Protein ◽  
N Terminus ◽  
A Cell ◽  
Terminal Amino ◽  
Peptide Treatment

Abstract Hepcidin is the principal iron-regulatory hormone. It acts by binding to the iron exporter ferroportin, inducing its internalization and degradation, thereby blocking cellular iron efflux. The bioactive 25 amino acid (aa) peptide has a hairpin structure stabilized by 4 disulfide bonds. We synthesized a series of hepcidin derivatives and determined their bioactivity in a cell line expressing ferroportin-GFP fusion protein, by measuring the degradation of ferroportin-GFP and the accumulation of ferritin after peptide treatment. Bioactivity was also assayed in mice by the induction of hypoferremia. Serial deletion of N-terminal amino acids caused progressive decrease in activity which was completely lost when 5 N-terminal aa's were deleted. Synthetic 3-aa and 6-aa N-terminal peptides alone, however, did not internalize ferroportin and did not interfere with ferroportin internalization by native hepcidin. Deletion of 2 C-terminal aa's did not affect peptide activity. Removal of individual disulfide bonds by pairwise substitution of cysteines with alanines also did not affect peptide activity in vitro. However, these peptides were less active in vivo, likely because of their decreased stability in circulation. G71D and K83R, substitutions previously described in humans, did not affect hepcidin activity. Apart from the essential nature of the N-terminus, hepcidin structure appears permissive for mutations.

Biosynthesis of Nitrogenous Phospholipids in Spinach Leaves

1974 ◽  
Vol 52 (6) ◽  
pp. 469-482 ◽  
Author(s):  
M. O. Marshall ◽  
M. Kates
Keyword(s):  
Microsomal Fraction ◽  
Enzyme System ◽  
Supernatant Fraction ◽  
Exchange Reactions ◽  
Methylation Pathway ◽  
Ethanolamine Kinase ◽  
Spinach Leaves

Pathways for biosynthesis of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), in spinach leaves have been studied both in vivo (whole leaves and leaf slices) and in vitro (cell-free leaf fractions). Biosynthesis of PS was shown to occur by the action of a particle-bound CDP-diglyceride: serine phosphatidyltransferase, and PE by the action of a PS-decarboxylase localized in the 100 000 × g supernatant fraction. PE was also formed by the operation of the CDP-ethanolamine:diglyceride phosphorylethanolamine transferase, localized in the microsomal fraction. The presence of ethanolamine kinase required for formation of phosphorylethanolamine was demonstrated in vitro, but not the presence of CTP:phosphorylethanolamine cytidyltransferase; however, the latter is presumed present on the basis of in vivo results. Operation of the methylation pathway for biosynthesis of PC was established in vivo, and direct methylation of phosphatidyl-N-methylethanolamine to phosphatidyl-N,N-dimethylethanolamine (PE-diMe) and of PE-diME to PC by S-adenosylmethionine was demonstrated with a particulate enzyme system localized in the microsomal fraction; direct methylation of PE itself could not be shown in this system. PC was also synthesized by the CDP-choline:diglyceride phosphorylcholine transferase system localized in the microsomal fraction. Synthesis of PE and PC by Ca2+-stimulated exchange reactions with ethanolamine and choline, respectively, could be demonstrated, but at low rates. However, no synthesis of PS by exchange reactions with serine could be detected.

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