Specification translation process

Designed experiments are a key tool for performing this specification translation process and helping to establish such controls. However, designed experiments are not the only tool required to accomplish this task. We will also explore other tools, such as tolerance analysis, robust design, capability studies, and Failure Modes and Effects Analysis (FMEA), to see how to combine these tools into an effective system for vahdation. 

Designed experiments can play a key role in this process. They can be used to establish operating windows for attribute characteristics. However, their most important use is as part of the specification translation process. Designed experiments must be carefully integrated into the overall process. This process requires five items ... 

Biosynthesis. The biosynthesis of neuropeptides is much more complex and involves the multistep process of transcription of specific mRNA from specific genes, formation of a high molecular weight protein product by translation, post-translational processing of the protein precursor to allow for... 

In order to measure the exact amount of a specific protein (analyte) by IHC signal intensity, a critical requirement is the availability of a standard reference material (present in a known amount by weight) that can be used to calibrate the assay (IHC stain). It is then possible to determine the amount of test analyte (protein) by a translation process from the intensity of IHC signals. In this respect it is helpful to consider the IHC stain as a tissue based ELISA assay (Enzyme Linked ImmunoSorbent Assay), noting that ELISA is used in the clinical laboratory as a standard quantitative method for measuring protein by weight in fluids, by reference to a calibrating reference standard. 

FIGURE 1 8-5 Tissue-specific processing of the pro-opiomelanocortin (POMC) precursor yields a wide array of bioactive peptide products. Processing of the POMC precursor varies in various tissues. In anterior pituitary, adrenocorticotropic hormone (ACTH (1-39)) and P-1 ipo tropin (P-LPH) are the primary products of post-translational processing. Arcuate neurons produce the potent opiate P-endorphin (P-endo (1-31)) as well as ACTIK1 -13) NIT,. Intermediate pituitary produces a-melanocyte-stimulating hormone (aMSH), acetylated P endof 1 31) and P-endo(l-27). NTS, nucleus tractus solitarius. 

The Ras proteins are synthesized as biologically inactive, cytosolic precursor proteins. They are then modified by several post-translational processing steps at the carboxyl terminal end and thereby converted into biologically active proteins localized at the plasma membrane. The cysteine of the C-terminal CAAX sequence (C is cysteine, A is generally an aliphatic amino acid, and X is methionine, serine, alanine, or glutamine) is first enzymatically S-farnesylated the AAX part is then cleaved off by a specific protease, and the free C-terminal cysteine is finally converted into a methyl ester (Scheme 1). 

A further difference in CB between normal and malignant tissues is that in the latter, a greater proportion of CB is found on the cell membrane (K2). This preferential binding to the cell membrane in malignant tumors may be due to defects in glycosylation during post-translational processing of CB (PI). Whether CB is bound to a specific receptor, like that described earlier for uPA, is presently unclear. 

A set of coding rules are in action as in the translation process. First, a set of three adjacent nucleotides compose the code for each amino acid. A single amino acid can have several triplet codes or codons. Since there are four different nucleotides (or four different bases) in DNA and RNA, there exist 4 = 64 trinucleotide combinations. For instance, using U as a symbol for uracil, which is present in RNA, the triplet or code or codon UUU is specific for phenylalanine. 

Data from in vitro activity assays with these purified recombinant proteins can typically be interpreted much more easily than data obtained from experiments with crude or partially purified protein extracts, because (1) there will be no competing proteins with similar activity present in the assay, and (2) there will no enzymes present that convert the product generated by the enzyme of interest, and hence reduce the effective product concentration. A potential downside of the use of recombinant protein over crude extracts is the fact that critical co-factors that will ensure proper activity may not be present in the purified protein fraction. If that is the case, the researcher will have to empirically determine which co-factor and at what concentration needs to be included in the assay. Another consideration is that the native protein may have undergone post-translational processing, such as acetylation, glycosylation, myristoylation, etc. These modifications may not occur or may not occur properly when the protein is expressed in bacterial, fungal or insect cells. Assuming that these potential problems do not occur or can be dealt with, the availability of pure recombinant protein will enable the determination of substrate specificity, as well as kinetic experiments in which the rate of conversion is measured as a function of time and/or substrate concentration. 

Within the amino acid sequence there may be specific sequences which act as signals for the post-translational processing of the protein (e.g. glycosylation or proteolytic processing see Topic H5). 

The question of the biosynthesis of GSHPx has been the subject of much research and it is now known with certainty that the formation of the seleno-cysteinyl residue depends on the existence of a gene that specifies a unique UGA codon that codes specifically for selenocysteine the complimentary tRNA binds serine to which is then added the selenyl moiety to form selenocysteine which is added to the nascent protein by the normal translational process [13], This mechanism has also been demonstrated for the biosynthesis of other seleno-enzymes in several bacterial species [14-16]. 

The levels of particular enzymes (and indeed of specific proteins in general) is determined by the balance of protein degradation versus the specific expression of the protein (through the process of specific gene transcription, translation and post-translational processing of the protein). Genes can either be constitutively expressed (in which case they are normally always being transcribed) or are inducible, that is, specific transcription factors are activated... 

Direct protein sequencing, by the methods discussed above (Sects. 5.1.5 and 5.1.6), is the preferred route to primary structure. It is now widely accepted that an indirect approach, via the sequence of cloned cDNA is also appropriate, and possibly easier. It does need to be established, however, that the protein primary structure is not affected by tissue specific mRNA processing (splicing or editing) or by post-translational modification such as N- or C-terminal processing or protein splicing. 

The biogenesis of both acetylcholine receptor and chromaffin granules share several common properties. The specific polypeptides are synthesized and transported into the membrane by a vectorial translation process. The specific proteins are sorted out by the Golgi apparatus and eventually fuse with the plasma membrane via the secretory pathway. Yet the acetylcholine receptor functions on the plasma membrane, and therefore it should stay on this membrane for a long time (2-7 days). On the other hand, the function of chromaffin granules is to store neurotransmitters. Therefore they stay most of their lifetime inside the cell and their fusion with the plasma membrane is temporary. Soon after the secretion process, the constituents of the chromaffin granule membrane must be removed from the plasma membrane by endocytosis. 

TRANSLATIONAL CONTROL Eukaryotic cells can respond to various stimuli (e.g., heat shock, viral infections, and cell cycle phase changes) by selectively altering protein synthesis. The covalent modification of several translation factors (nonribosomal proteins that assist in the translation process) has been observed to alter the overall protein synthesis rate and/or enhance the translation of specific mRNAs. For example, the phosphorylation of the protein eIF-2 affects the rate of hemoglobin synthesis in rabbit reticulocytes (immature red blood cells). 

In addition to the ribosomal subunits, mRNA, and aminoacyl-tRNAs, translation requires an energy source (GTP) and a wide variety of protein factors. These factors perform several roles. Some have catalytic functions others stabilize specific structures that form during translation. Translation factors are classified according to the phase of the translation process that they affect, that is, initiation, elongation, or termination. The major differences between prokaryotic and eukaryotic translation appear to be due largely to the identity and functioning of these protein factors. 

What are the three phases of protein synthesis Describe the principal events in each phase. What specific roles do translation factors play in both prokaryotic and eukaryotic translation processes ... 

The implementation of human-like post-translational processing in specific tissues... 

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