Extent Of Synthesis Research Articles

Biomimetic synthesis of iron oxide nanoparticles from Bacillus megaterium to be used in hyperthermia therapy

The suitable structural characteristics of magnetic nanoparticles have resulted in their widespread use in magnetic hyperthermia therapy. Moreover, they are considered a proper and operational choice for pharmaceutical nanocarriers. Using the biomimetic method, we were able to produce iron oxide magnetic nanoparticles from the bacterial source of PTCC1250, Bacillus megaterium, for therangostic diagnosis systems and targeted drug delivery. Some of the benefits of this method include mitigated environmental and biological dangers, low toxicity, high biocompatibility, cheap and short-term mass production possibilities in each synthesis round compared to other biological sources, simple equipment required for the synthesis; and the possibility of industrial-scale production. Bacillus megaterium is a magnetotactic bacteria (MTB) that has a magnetosome organelle capable of orienting based on external magnetic fields, caused by the mineralization of magnetic nanocrystals. Utilizing this capability and adding an iron nitrate solution to the bacterial suspension, we synthesized iron oxide nanoparticles. The extent of synthesis was measured using UV–visible spectrophotometry. The morphology was evaluated using FESEM. The crystallized structure was characterized using RAMAN and XRD. The size and distribution of the nanoparticles were assessed using DLS. The surface charge of the nanoparticles was measured using zeta potential. The synthesis of iron oxide nanoparticles was confirmed using FT-IR, and the magnetic property was measured using VSM. This study is continued to identify industrial and clinical applications.

In vitro synthesis of noradrenaline in ganglia and salivary glands after in vivo preganglionic stimulation.

Abstract The extent of synthesis of [14C]noradrenaline from [14C]tyrosine and from [14C]dopamine was assessed in slices of superior cervical ganglia, representing cell bodies, and in submaxillary salivary glands, representing terminals of noradrenergic neurons of the cat. Immediately and 6 h after preganglionic stimulation for 3 h the rate of synthesis of noradrenaline from tyrosine and dopamine was not altered in ganglia. In salivary glands, however, synthesis of noradrenaline from both tyrosine and dopamine was increased at both times. These results suggest that acute periods of increased neural activity results in the acceleration of noradrenaline synthesis in the terminals but not in the cell bodies of noradrenergic neurons.

Probing Mechanism for the Enhancement of Uptake of Fatty Acid into Cells by the Membrane Protein CD36

CD36 is a membrane protein found in various cell types including adipocytes and endothelial cells. Physiological studies have suggested that CD36 is associated with insulin-resistant diabetes, however, the mechanism remains poorly understood. It is well recognized as a receptor for multiple ligands such as collagen and thrombospondin 1, and possibly acts as a catalyst to enhance the rate of transmembrane movement of fatty acid. To separate the two independent events transport across the plasma membrane and the subsequent intracellular metabolism, we have applied biophysical approaches and metabolic analyses to cells in vitro. Our results using cultured cells (HEK293 cells overexpressing CD36 and 3T3-L1 adipocyte cells) as well as mice adipocyte cells isolated from a CD36 null mouse showed that fatty acids diffuse through the plasma membrane rapidly with or without CD36. In HEK 298 cells, which normally synthesize triglycerides very slowly and to a limited extent, expression of CD36 enhanced the rate and extent of synthesis. Even in the presence of CD36, incorporation into triglycerides is a much slower process (min) relative to the transmembrane movement (sec), indicating that the rate-limiting step of the regulation of fatty acid uptake by CD36 is intracellular metabolism. Lastly, by PCR array analysis of 84 proteins, we have identified several enzymes involved in human fatty acid metabolism with gene expression levels altered by overexpression of CD36. Taken together, our results showed that CD36 increases fatty acid uptake by enhancing triglycerides synthesis rather than acting as a membrane transporter, but as yet by unidentified molecular mechanisms.

Peptide bond synthesis: function of the efp gene product.

The efp gene encodes a protein that is essential for the growth and for the viability of Escherichia coli cells. Interruption of this gene results in cell death due to a defect in protein synthesis. We report here that the EFP protein, encoded by the efp gene, is required for in vitro reconstitution of polypeptide synthesis in a system programmed by a native template which contains each of the purified initiation factors, IF1, IF2, IF3; the elongation factors, EFTu, EFTs and EFG, and a protein called W that is required to eject tRNAs from ribosomes. The EFP protein is required for enhancing the rate and the extent of synthesis in the presence of all of the above factors. The EFP protein stimulates synthesis of poly(Phe) programmed with poly(rU) only if N-acetyl Phe-tRNA initiates the reactions under conditions that foster the dissociation of the 70S ribosome. Study of the ability of the ribosome to synthesize a number of fMet-initiated dipeptides from CCA amino acyl acceptors suggests that EFP acts to promote synthesis with acceptors that are poor donors for the the reconstituted peptidyl transferase.

Synthesis and phosphorylation of androgen receptor of the mouse brain cortex and their regulation by sex steroids during aging.

To examine the synthesis and phosphorylation of androgen receptor (AR) and their regulation by sex steroids, adult (24 weeks) and old (65 weeks) male and female mice were gonadectomized and administered with testosterone and estradiol. AR amount, synthesis and phosphorylation were measured in the brain cortex by immunoblotting and immunoprecipitation using antibody raised against rat AR transactivation domain (TAD) which was expressed in E. coli as a fusion protein. We found that the amount of AR was high in adult and declined in old mice of both sexes. Administration of testosterone and estradiol significantly down-regulated the level of AR in old male and adult female. Similarly, the rate of AR synthesis also declined with age. Exogenous treatment of gonadectomized mice with testosterone and estradiol reduced the extent of synthesis significantly in all groups except in old female. No sex-dependent variation was noticed either in the level or synthesis of AR. In contrast, the extent of phosphorylation was higher in old mice of both sexes as compared to their adult counterparts. Testosterone and estradiol supplementation resulted in remarkable increase in AR phosphorylation in all groups. Thus it is evident from our findings that the amount and synthesis of AR decrease but phosphorylation of AR increases in the brain cortex with advancing age of mice and they are regulated by testosterone and estradiol in age- and sex-specific manner.

Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: A different perspective

Marine and terrestrial photosynthetic and chemoautotrophic microorganisms assimilate considerable amounts of carbon dioxide. Like green plastids, the predominant means by which this process occurs is via the Calvin-Benson-Bassham reductive pentose phosphate pathway, where ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a paramount role. Recent findings indicate that this enzyme is subject to diverse means of control, including specific and elaborate means to guarantee its high rate and extent of synthesis. In addition, powerful and specific means to regulate Rubisco activity is a characteristic feature of many microbial systems. In many respects, the diverse properties of microbial Rubisco enzymes suggest interesting strategies to elucidate the molecular basis of CO2/O2 specificity, the ‘holy grail’ of Rubisco biochemistry. These systems thus provide, as the title suggests, ‘different perspectives’ to this fundamental problem. These include vast possibilities for imaginative biological selection using metabolically versatile organisms with well-defined genetic transfer capabilities to solve important issues of Rubisco specificity and molecular control. This review considers the major issues of Rubisco biochemistry and regulation in photosynthetic microoganisms including proteobacteria, cyanobacteria, marine nongreen algae, as well as other interesting prokaryotic and eukaryotic microbial systems recently shown to possess this enzyme.

Expression of the alternatively spliced EIIIB segment of fibronectin.

Previous descriptions of the expression and distribution of the alternatively spliced EIIIB segment of fibronectin (FN) relied upon an antibody which, on subsequent testing, was shown not to recognize this segment directly. This raises concerns regarding the reliability of all such previous descriptions. In order to prepare reagents directly reactive with this segment, we raised polyclonal antibodies to two different bacterial fusion proteins containing intact EIIIB segments, and to a synthetic 36 amino acid peptide from the center of the EIIIB segment. Antibodies raised to each of these three immunogens recognized fusion proteins containing the EIIIB segment, but failed to recognize full length EIIIB+ FNs produced by mammalian cells, suggesting that oligosaccharide linked to Asn1359 within the EIIIB segment, or potentially to other residues in FN, might interfere with antibody recognition of this segment. Consistent with this hypothesis, N-deglycosylation of recombinant full and partial length EIIIB+ FNs permitted their specific recognition by the anti-fusion protein (but not anti-peptide) antibodies. Using anti-fusion protein antibodies coupled with deglycosylation procedures, we provide a series of new results relevant to the functions of the EIIIB segment: 1) Endogenously synthesized EIIIB+ FN is incorporated into the extracellular matrix of cultured fibroblasts, where it appears by immunofluorescence microscopy and radioimmunoprecipitation analyses to have a distribution very similar to both EIIIA+ forms and the total pool of FNs. 2) No reproducible changes can be shown to occur in the extent of synthesis or matrix incorporation of EIIIB+ FNs upon cellular transformation. 3) Low levels of EIIIB+ FN are normally present in blood plasma and consequently also in purified preparations of plasma FN. 4) EIIIB+ FN is present in blood platelets, where it constitutes a minor fraction of total platelet FN, yet is greater than 4-fold enriched relative to plasma FN. 5) EIIIB+ FN is synthesized by first passage cultured endothelial cells, suggesting that the endothelium could constitute a source for this FN isoform in the circulating blood. The antibodies and methods used in this study constitute the first direct assays of EIIIB+ FN protein expression and are applicable to a variety of species.

Interaction of proliferating cell nuclear antigen with yeast DNA polymerase delta.

Previously, we described the purification of the catalytic core subunit of yeast DNA polymerase delta from Escherichia coli carrying the yeast POL3 gene cloned in a vector that allowed efficient expression. The basic catalytic properties of the single subunit enzyme are virtually identical to the heterodimeric core enzyme purified from yeast cells (Brown, W. C., Duncan, J. A., and Campbell, J. L. (1993) J. Biol. Chem. 268, 982-990). In this work, we investigate the ability of yeast proliferating cell nuclear antigen (PCNA) to stimulate processive synthesis by the bacterially produced, single-subunit DNA polymerase delta. Yeast PCNA was found to stimulate the full-length single-subunit yeast DNA polymerase delta and to increase its processivity. A truncated version of DNA polymerase delta, from which the NH2-terminal 220 amino acids had been removed, was prepared and similarly investigated. While the catalytic properties of the truncated protein were nearly identical to those of the full-length enzyme, neither the extent of synthesis nor processivity was increased in the presence of PCNA. Thus, we conclude that the single-subunit DNA polymerase can associate productively with PCNA in the absence of other proteins and that the NH2-terminal domain of the catalytic subunit must be intact for this interaction.

Phosphate limitation affects global gene expression inthiobacillus ferrooxidans

Abstract During some biogeochemical processes mediated by bacteria, essential nutrients such as phosphate can become limiting for the microorganisms, affecting their biological activity. We have analyzed changes in total protein synthesis of Thiobacillus ferrooxidans when it was subjected to phosphate starvation. By two‐dimensional polyacrylamide gel electrophoresis, at least 25 proteins changed in extent of synthesis under the limiting conditions. Several of these proteins could be traced to the surface of the bacteria by total cell labeling in the presence of 125I and analysis of outer membrane. The observed changes reflect the appearance of an environmental phosphate scavenging system in T. ferrooxidans.

Constraints on prostaglandin biosynthesis in tissues.

The formation of prostaglandins by prostaglandin H synthase can be limited by the availability of the fatty acid substrate or the hydroperoxide activator and also by a self-catalyzed inactivation associated with the oxygenation reaction. Each pmol of synthase appeared able to form only about 1300 pmol of prostaglandin from arachidonate before it was inactivated. This extent of synthesis was not diminished when substrate fatty acid was complexed with cytosolic proteins even though the velocity of the oxygenation reaction was greatly decreased by the lower availability of substrate acid. When the availability of hydroperoxide activator was decreased by added glutathione peroxidase, the extent of oxygenation per mol of synthase was decreased irrespective of the amount of cytosolic protein present. Approximately 65% of the total prostaglandin synthesis by homogenates was suppressed with a glutathione peroxidase to prostaglandin H synthase ratio of about 90. The remaining prostaglandin synthetic activity was more resistant, being completely suppressed only when the ratio of peroxidase to synthase exceeded 750. The overall ratio of glutathione peroxidase (peroxide-removing) capacity to prostaglandin synthetic (peroxide-forming) capacity in selected tissues ranged from over 1800 in rat liver to less than 30 in leukocytes. A comparison between the daily urinary output of prostaglandin metabolites and tissue prostaglandin synthetic capacity suggested that prostaglandin H synthase inactivation along with glutathione peroxidase suppression of the extent of prostaglandin synthase may be important in limiting prostaglandin biosynthesis within cells.

Gel-forming capsular polysaccharide of fast-growing rhizobia: occurrence and rheological properties

In addition to the excretion of soluble acidic polysaccharides many fast-growing rhizobia deposit insoluble neutral capsular polysaccharide (CPS), which is composed of d-mannose, d-galactose, and d-glucose in the ratios 1:4:1. CPS was found to occur in all strains of Rhizobium leguminosarum and R. trifolii. Synthesis takes place in the stationary phase of growth, but the extent of synthesis differs widely for individual strains. CPS was not found in the species R. phaseoli and R. meliloti. CPS can be extracted from the cell pellet with N NAOH and the so obtained material is notable for its gelling character. It is insoluble in cold water and dissolves in hot water to a clear solution. On cooling to room temperature the solution solidifies to a resilient gel at a setting point of 40–45° C, and remelts on heating at 50–55° C. Gel strength of CPS in 500 g/cm2 for a 1% suspension.

In vitro synthesis of genome length complementary RNA of vesicular stomatitis virus in the presence of inosine 5′-triphosphate

The virion-associated RNA polymerase of vesicular stomatitis virus (VSV) synthesizes RNA in vitro when GTP is replaced by inosine 5′-triphosphate (ITP). The synthesis is optimal at an ITP concentration of 200 μ M and the extent of synthesis is between 15 to 20% compared to normal transcription in the presence of GTP. Analyses of the RNA products revealed that approximately 10% of the product RNA represented plus-strand complement of the genome RNA. Defective interfering particles of VSV were also capable of synthesizing complementary RNA in the presence of ITP, in addition to 46-base RNA, although in lesser amount (2%). Since I substitution facilitates read-through of the genome RNA, these results suggest that interaction of the product RNA with the genome template or ITP-mediated modification of a viral protein may be involved in the readthrough process in vitro.

Vitamin A deprivation selectively lowers uridine nucleotide pools in cultured sertoli cells.

The effects of retinoid addition of vitamin A-depleted (UV-irradiated) culture medium on uridine metabolism in cultured Sertoli cells have been studied. After vitamin A depletion, a consistent 2- to 4-fold enhancement of [3H]uridine incorporation into RNA was observed. Several lines of evidence indicate that this enhancement is the result of an increase in the specific activity of the uridine-labeled precursors of RNA. Although vitamin A depletion did not affect either uridine uptake or alter cellular RNA content, a 5-fold increase in the specific activity of UMP was found in vitamin A-depleted cells. This increase results because the cellular content of uracil nucleosides plus nucleotides is selectively lowered in vitamin A-depleted cells. The decreased content of uridine derivatives could be accounted for by a 45-57% decrease in the activity of glutamine-dependent carbamylphosphate synthetase in vitamin A-depleted cells. The effects of vitamin A deprivation on uridine incorporation, as well as carbamylphosphate synthetase activity, could be completely restored to or above control values by supplementing vitamin A-depleted cell culture medium with either retinol or retinoic acid. This effect of vitamin A depletion appears to be highly specific. Under the same conditions, no gross alteration in either the pattern or extent of synthesis of cellular or secreted proteins, glycoproteins, glycosaminoglycans, and lipids was observed. In addition, vitamin A depletion/repletion had no effect on the growth rate or morphology of the cells.

Deoxyribonucleic Acid Synthesis in Saccharomyces cerevisiae Cells Permeabilized with Ether

Cells of Saccharomyces cerevisiae permeabilized by treatment with ether take up and incorporate exogenous deoxynucleoside triphosphate into deoxyribonucleic acid (DNA). With rho(+) strains, more than 95% of the product was mitochondrial DNA (mtDNA). This report characterizes ether-permeabilized yeast cells and describes studies on the mechanism of mtDNA synthesis with this system. The initial rate of in vitro mtDNA synthesis with one strain (X2180-1Brho(+)) was close to the rate of mtDNA replication in vivo. The extent of synthesis after 45 min was sufficient for the duplication of about 25% of the total mtDNA in the cells. The incorporated radioactivity resulting from in vitro DNA synthesis appeared in fragments that were an average of 30% mitochondrial genome size. Density-labeling experiments showed that continuous strands of at least 7 kilobases after denaturation, and up to 25 kilobase pairs before denaturation, were synthesized by this system. Pulse-chase experiments demonstrated that a large proportion of DNA product after short labeling times appeared in 0.25-kilobase fragments (after denaturation), which served as precursors of high-molecular-weight DNA. It is not yet clear whether the short pieces participate in a mechanism of discontinuous replication similar to that of bacterial and animal cell chromosomal DNA or whether they are related to the rapidly turning over, short initiation sequence of animal cell mtDNA. In rho(0) strains, which lack mtDNA, the initial rate of nuclear DNA synthesis in vitro was 1 to 2% of the average in vivo rate. With temperature-sensitive DNA replication mutants (cdc8), the synthesis of nuclear DNA was temperature sensitive in vitro as well, and in vitro DNA synthesis was blocked in an initiation mutant (cdc7) that was shifted to the restrictive temperature before the ether treatment.

Deoxyribonucleic acid synthesis in isolated nuclei from baby-hamster kidney cells (BHK-21/C13). Characterization of the system

A comparative study of some commonly employed laboratory procedures for studying DNA synthesis in isolated nuclei was carried out. Nuclei isolated from baby-hamster kidney (BHK-21/C13) cells synthesize DNA for 30-60min at 37 degrees C in a reaction requiring uni- and bi-valent cations, ATP and all four deoxyribonucleoside 5'-triphosphates. The addition of either ribonucleotides or cytosol from S-phase cells had no effect, but DNA synthesis was stimulated by some dextrans (mol.wt. 5x10(6)). The extent of synthesis was influenced by apparently minor variations in experimental conditions. For example, DNA synthesis by nuclei in Tris/HCl, pH7.5, was only 50% of that observed in Hepes/NaOH, pH7.5; the presence of detergents Triton X-100, Triton N-101, Nonidet P-40, Brij 58 and Tween 80 in the incubation medium altered the amount of synthesis to different extents. Although most detergents inhibited synthesis, a stimulation occurred with Tween 80 (150% of controls). These effects were reversed on washing the nuclei, except that of Brij 58, which inhibited DNA synthesis by 90-95% irreversibly. Anomalous sucrose-density-gradient sedimentation behaviour of the DNA, and of precursor [(3)H]-dTTP, was observed when nuclei were lysed with solutions of sodium dodecyl sulphate/Mg(2+) or with Sarkosyl/Mg(2+), but consistent results, showing that the DNA synthesized in vitro sedimented exclusively at about 4S, were obtained when nuclei were lysed with sodium dodecyl sulphate (without Mg(2+))/EDTA, digested with proteinase K and heated at 100 degrees C with 11% (v/v) formaldehyde to prevent macromolecular association. These results, coupled with density-labelling studies with bromodeoxyuridine and CsCl-density-gradient analysis, showed that DNA synthesis in these nuclei was replicative and was restricted to a covalent extension of Okazaki pieces previously initiated in vivo. No new initiations were observed, and the DNA was not ligated into larger molecules. The cessation of DNA synthesis after about 60 min was due to the complete utilization of available primer/template DNA.

Distribution of 7-methylguanine and of replication sites in the different kinetic classes of DNA from rats treated with dimethylnitrosamine.

The distribution of 7-methylguanine in the families of repetitive and unique sequences of rat liver chromatin DNA has been studied using the technique of DNA-DNA reassociation. Rats were injected with di[14C]methylnitrosamine and chromatin DNA was prepared 3 h later. The distribution of 7-methylguanine was found to be random between these classes of DNA. We have also studied chromatin DNA from rats treated with unlabelled DMN plus [3H]thymidine in this way, in order to find if DMN affects DNA synthesis within any one kinetic class. Our results suggest that there is no difference in the extent of synthesis between these classes.

Quantitation of vitellogenin messenger RNA in the liver of male Xenopus toads during primary and secondary stimulation by estrogen.

Abstract From livers of estrogen-stimulated female Xenopus toads, large quantities of estrogen-induced, poly(A)-containing RNA could be isolated, showing the same characteristics as vitellogenin mRNA obtained from hormone-treated males. Using cDNA hybridization, vitellogenin mRNA was monitored in the cytoplasmic poly(A)-containing RNA of the liver of male toads during 13 days of primary and the initial phase of secondary stimulation with estrogen. During primary stimulation, low amounts of vitellogenin mRNA, not exceeding 0.18% of the cytoplasmic poly(A)-containing RNA, were first detected after 12 hr of hormone treatment, and vitellogenin mRNA was found to increase on the average to 34% of the cytoplasmic poly(A)-containing RNA on the seventh day of hormone treatment. After 3 days of primary stimulation, accumulation of vitellogenin mRNA leveled off, showing no significant increase in the cytoplasm up to 13 days of hormone treatment. As judged from incorporation of 32 PO 4 into blood plasma proteins of males during primary stimulation, vitellogenin was first detected after 1 day, and its synthesis was found to increase dramatically until the thirteenth day of hormone treatment. This implies that there is a coincidence between appearance and extent of synthesis of vitellogenin and the abundance of vitellogenin mRNA in the cytoplasm, but there is evidence that during later phase of primary stimulation (day 3–13), the increase in synthesis of vitellogenin cannot be attributed anymore to a significant accumulation of vitellogenin mRNA. In male Xenopus, estrogen-induced synthesis of vitellogenin is no more detectable 41 days after hormone injection, and the concentration of vitellogenin mRNA was found to be

Synthesis of ribosomal proteins L7L12 in relaxed and stringent strains of Escherichia coli.

The control of the synthesis of ribosomal proteins L7L12 (which lack histidine) was examined during growth and histidine starvation of stringent and relaxed histidine mutants. Since no ribosomes are synthesized during starvation, these proteins, in both the stringent and relaxed organisms, accumulated in the supernatant and were shown to possess both biological and physical characteristics typical of normal L7L12. However, the rate and extent of synthesis of these proteins during starvation is greater in the relaxed strain than in the stringent. These data suggest that the regulation of the synthesis of these proteins, similar to that of ribosomal RNA is regulated by the stringent control system. It was also shown that during normal growth of both organisms, L7L12 is also found in the supernatant as well as on the ribosomes. The L7L12 found in the supernatant under these conditions, however, appears to be different than ribosomal L7L12.

Synthesis and release of polyribophosphate by Haemophilus influenzae type b in vitro.

Polyribophosphate, the capsular polysaccharide antigen of Haemophilus influenzae type b, can be assayed in crude bacterial extracts by a combination of immunoprecipitation, hot-acid extraction, and colorimetry. In vitro, the antigen is synthesized during growth and, in well-buffered media, in early stationary phase as well. It is shed from the cell throughout the course of synthesis, and at 37 C almost all the extractable antigen has been released spontaneously within several hours after synthesis. Various strains are similar in extent of synthesis and gross composition of the antigen; however, strains differ in the rate of release during exponential phase. The antigen is released as (or as part of) a very large structure, although under certain culture conditions much smaller antigenic units are found.

Proteins of bacteriophage phi6.

We investigated the protein composition of the lipid-containing bacteriophage phi 6. We also studied the synthesis of phage-specific proteins in the host bacterium Pseudomonas phaseolicola HB10Y. The virion was found to contain 10 proteins of the following molecular weights: P1, 93,000; P2, 88,000; P3, 84,000; P4, 36,800; P5, 24,000; P6, 21,000; P7, 19,900; P8, 10,500; P9, 8,700; and P10, less than 6,000. Proteins P3, P9, and P10 were completely extracted from the virion with 1% Triton X-100. Protein P6 was partially extracted. Proteins P8 and P9 were purified by column chromatography. The amino acid composition of P9 was determined and was found to lack methionine. Labeling of viral proteins with [35S]methionine in infected cells indicated that proteins P5, P9, P10, and P11 lacked methionine. Treatment of host cells with UV light before infection allowed the synthesis of P1, P2, P4, and P7; however, the extent of viral protein synthesis fell off exponentially with increasing delay time between irradiation and infection. Treatment of host cells with rifampin during infection allowed preferential synthesis of viral proteins, but the extent of synthesis also fell off exponentially with increasing delay time between the addition of rifampin and the addition of radioactive amino acids. All of the virion proteins were seen in gels prepared from rifampin-treated infected cells. In addition, two proteins, P11 and P12, were observed; their molecular weights were 25,200 and 20,100, respectively. Proteins P1, P2, P4, and P7 were synthesized early, whereas the rest began to increase at 45 min post-infection.