Re: Pic Serial Communication Assembly Code
Cherrie Patete
I need to use 8086 assembly language to connect with arduino in my assignment. The thing i want to do is when I run the assembly program, the buzzer will sound. I run the assembly program using DOSBox.
Above is my assembly program code. When I run the assembly code, it supposed to send 'H' to com1, and the buzzer will sound. However, there is nothing happen when I run the assembly program. What is the problem?
LianChyn:
I need to use 8086 assembly language to connect with arduino in my assignment. The thing i want to do is when I run the assembly program, the buzzer will sound. I run the assembly program using DOSBox.
Before we get too far debugging your programs, please explain how you have the Arduino connected to the PC. Are you using the USB port on the PC to connect to the Arduino. OR are you using a serial RS232 port on the PC? If the latter, do you have a RS232 to TTL adapter making the connection?
You have posted a piece of assembler code in your Original Post. Are you really suggesting that that is all that is needed to open the serial port, set the baud rate and send a character? Seems very unlikely to me.
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Elucidating the molecular mechanisms that guide collagen assembly is central to our understanding of collagen biosynthesis. In humans, there are 28 known collagen types, but >45 genetically distinctive collagen strands1. The excess of collagen strands relative to collagen types means that some collagens form 2:1 or even 1:1:1 heterotrimers. For example, type-II and type-III collagen are homotrimeric; the most abundant collagen, type-I, is typically a 2:1 heterotrimer of two Colα1(I) chains and one Colα2(I) chain (Fig. 1a); and collagen type-V can form a 1:1:1 heterotrimer of Colα1(V), Colα2(V), and Colα3(V)1. Homotrimeric versus heterotrimeric triple helices have different stability, altered propensities for extracellular supramolecular assembly, and customized functions and binding partners7. The unique features and functionalities of collagen heterotrimers have rendered formation of defined synthetic collagen heterotrimers a major goal of peptide and protein engineers8,9,10,11,12,13,14,15. However, attaining and maintaining defined triple-helix compositions and proper register in short collagen-like peptides capable of biomimetic supramolecular assembly continues to present significant challenges.
C-Pro domain-mediated assembly of collagen type-I. a Schematic representation of collagen-I assembly. Two strands of Colα1(I) and one strand of Colα2(I) typically assemble into heterotrimers, a process that is initiated by the respective C-Pro domains. Colα1(I) is also known to form homotrimers, whereas Colα2(I) does not homotrimerize and only forms heterotrimers. The crystal structure of a homotrimeric C-Pro domain is used here for illustration purposes (PDBID 5K31), with each collagen-I C-Pro domain differentially colored to demonstrate possible assembly schematics16. b Alignment of the Colα1(I) and Colα2(I) C-Pro domains highlights high sequence similarity. The cysteine network is numbered from C1 to C8 in the N-terminal to C-terminal direction, with each cysteine residue colored in red
We began by aligning collagen C-Pro domains from distantly related species across the animal kingdom to track the appearance of each factor that has been proposed to play a key role in collagen assembly through evolutionary history. Our goal was to identify a conserved factor that emerged near the same point as collagen-I heterotrimers, which likely evolved no later than the last common ancestor of all chordates (see Fig. 2b for a cladogram). Previous modeling based on the crystal structure of C-Proα1(I) suggested that Lys1248 and Glu1249 (numbering based on the full-length procollagen α1(I) sequence) in the chain recognition sequence of human C-Proα2(I) may form heterotrimer-stabilizing salt bridges with the adjacent C-Proα1(I) in a collagen-I heterotrimer16. However, Lys1248 probably did not fix until osteichthyes, and a negatively charged amino acid at position 1249 probably did not evolve until tetrapods (see Fig. 2b and Supplementary Fig. 1). Moreover, only Asp1347, but not Asp1344, the salt bridge-forming amino acids in C-Proα1(I), is present in sharks, one of the early-diverging groups that later lost bone (Supplementary Fig. 1). Therefore, Asp1344 probably was absent in the last common ancestor of gnathostomes and did not fix until osteichthyes. Other amino acids in C-Proα2(I), including Arg1165 and Lys1366, have also been proposed to form salt bridges with the chain recognition sequence in C-Proα1(I)16, but evidence that Arg1165 and Lys1366 impact homotrimerization of C-Proα2(I) is lacking. Beyond these specific amino acids, the chain recognition sequence itself likely did not appear until gnathostomes (Supplementary Fig. 1). Thus, this sequence appears to be a recently evolved vertebrate mechanism of chain selection that developed only when the problem of type-specific collagen assembly became significantly complicated by the presence of many collagen types in a single organism. An alternative explanation is clearly required to account for the homotrimerizing versus heterotrimerizing propensities of collagen strands.
In contrast to the relatively recent evolution of the chain recognition sequence, analysis of distantly related collagens reveals that the distinctive cysteine substitution patterns of Colα1(I) and Colα2(I) C-Pro domains is much more highly conserved (C1 through C4 shown in Fig. 2c; see also the full alignment shown in Supplementary Fig. 1)24. Indeed, the pattern of one strand containing eight cysteine residues and the other containing seven cysteine residues emerged along with the chordates, which were likely the earliest organisms to display collagen-I heterotrimers, and has been maintained since that time throughout distantly related groups of chordates.
The strong conservation of the cysteine pattern (Fig. 2c) across chordates, a group of animals with highly divergent body plans, appears to provide compelling support for the hypothesis that the presence or absence of specific Cys residues critically regulates the ability of collagen-I strands to homotrimerize versus heterotrimerize. Therefore, we were motivated to revisit the conclusion from prior research that re-introduction of C2 in C-Proα2(I) does not allow the protein to stably homotrimerize21,23.
We began by creating plasmids for expression of hemagglutinin (HA)-tagged C-Proα1(I) and FLAG-tagged C-Proα2(I) in human cells. The distinctive antibody epitopes were included to simplify differential detection by immunoblotting. We incorporated a preprotrypsin signal sequence to target the proteins to the ER for folding and subsequent secretion. We found that, when expressed alone in human embryonic kidney 293 (HEK293) cells, both C-Proα1(I) (as previously observed16) and C-Proα2(I) were robustly secreted. Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblot analysis of the media under non-reducing conditions demonstrated the expected assembly patterns for these proteins. C-Proα1(I) migrated as a stable, disulfide-linked homotrimer (Fig. 3a), whereas C-Proα2(I) migrated as a monomer. Both C-Proα1(I) and C-Proα2(I) migrated as monomers under reducing SDS-PAGE conditions (Fig. 3a). These results recapitulate the known ability of full-length Colα1(I) to form disulfide-linked homotrimers and the known inability of full-length Colα2(I) to do the same. Furthermore, co-expression of C-Proα1(I) and C-Proα2(I) rescued monomeric C-Proα2(I) into a disulfide-linked heterotrimer with C-Proα1(I) (Fig. 3a; shown by the yellow overlap on a non-reducing gel upon co-expression). Thus, consistent with prior work16, these biochemically amenable constructs provide a valid model system to examine the molecular code for collagen assembly.
The data in Fig. 3 show that the presence or absence of C2 in the collagen-I C-Pro domain is a defining feature controlling the ability to homotrimerize in a disulfide-dependent manner, consistent with the phylogenetic analyses in Fig. 2. Critically, however, results derived from SDS-PAGE gels do not address the innate ability of these C-Pro domains to trimerize independent of disulfide bond formation, as the analyses are necessarily performed under denaturing instead of native conditions. Furthermore, our observations in Fig. 3 conflict with prior work on the C2 variant of a Colα2(I) mini-gene consisting of the N-Pro domain, a short triple-helical domain, and the C-Pro domain21,23. In those prior studies, disulfide-linked homotrimers were not observed for S2C C-Proα2(I) on a non-reducing SDS-PAGE gel. Moreover, the short triple-helical domain was reported to be sensitive to proteolysis, suggesting that a triple helix was not formed23. Notably, the model proteins in those early studies were expressed in a cell-free, rabbit reticulocyte expression system, either in the presence or absence of canine pancreatic microsomes. The final step before inducing expression of a protein of interest in such a system is treatment with EDTA or EGTA to complex Ca2+, thereby inactivating the Ca2+-dependent nuclease used to degrade endogenous mRNAs25. Examination of the crystal structures of C-Pro homotrimers suggests that the resulting Ca2+ depletion during synthesis and folding may be confounding, as Ca2+ ions are bound at the interfaces between individual subunits of the trimer (Fig. 4a)16,17.
To address this conflict and better understand the innate trimerization propensities of collagen-I C-Pro domains, we purified milligram quantities of His-tagged versions of all four C-Proα(I) variants, cleaving the His tag to yield unmodified C-Pro domains in the final purification step (see Supplementary Fig. 2). We note that liquid chromatography-mass spectrometry analysis of both the purified C-Proα1(I) and S2C C-Proα2(I) homotrimers revealed the expected disulfide linkages based on the existing crystal structures of homotrimeric C-Pro domains16,17 upon proteolysis of the oxidized and denatured samples, suggesting that the purified homotrimers are both properly folded (Supplementary Tables 1 and 2).
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