Increase In Rate Of Synthesis Research Articles

Promoter-independent synthesis of chemically modified RNA by human DNA polymerase θ variants.

Synthetic RNA oligonucleotides composed of canonical and modified ribonucleotides are highly effective for RNA antisense therapeutics and RNA-based genome engineering applications utilizing CRISPR-Cas9. Yet, synthesis of synthetic RNA using phosphoramidite chemistry is highly inefficient and expensive relative to DNA oligonucleotides, especially for relatively long RNA oligonucleotides. Thus, new biotechnologies are needed to significantly reduce costs, while increasing synthesis rates and yields of synthetic RNA. Here, we engineer human DNA polymerase theta (Polθ) variants and demonstrate their ability to synthesize long (95-200 nt) RNA oligonucleotides with canonical ribonucleotides and ribonucleotide analogs commonly used for stabilizing RNA for therapeutic and genome engineering applications. In contrast to natural promoter-dependent RNA polymerases, Polθ variants synthesize RNA by initiating from DNA or RNA primers, which enables the production of RNA without short abortive byproducts. Remarkably, Polθ variants show the lower capacity to misincorporate ribonucleotides compared to T7 RNA polymerase. Automation of this enzymatic RNA synthesis technology can potentially increase yields while reducing costs of synthetic RNA oligonucleotide production.

The role of the platelet pool of Plasminogen Activator Inhibitor-1 in well-controlled type 2 diabetes patients.

BackgroundThe main inhibitor of the fibrinolytic system, Plasminogen Activator Inhibitor -1 (PAI-1), irreversibly binds tissue-type Plasminogen Activator (t-PA) and thereby inhibits the protective action of tPA against thrombus formation. Elevated levels of plasma PAI-1 are associated with an increased risk of cardiovascular events and are observed in subjects with type 2 diabetes (T2D) and obesity. Platelets contain the majority of PAI-1 present in blood and exhibit the ability to synthesis active PAI-1. Diabetic platelets are known to be hyper-reactive and larger in size; however, whether these features affect their contribution to the elevated levels of plasma PAI-1 in T2D is not established.ObjectivesTo characterize the PAI-1 antigen content and the mRNA expression in platelets from T2D subjects compared to obese and lean control subjects, in order to elucidate the role of platelet PAI-1 in T2D.MethodsNine subjects with T2D and obesity were recruited from Primary Care Centers together with 15 healthy control subjects (8 lean subjects and 7 with obesity). PAI-1 antigen levels in plasma, serum and platelets were determined by ELISA, and PAI-1 mRNA expression was analyzed by qPCR.ResultsThere was no significant difference in PAI-1 mRNA expression or PAI-1 antigen in platelets in T2D subject in comparison to obese and lean control subjects. An elevated level of plasma PAI-1 was seen in both T2D and obese subjects. PAI-1 gene expression was significantly higher in both obese groups compared to lean.ConclusionSimilar levels of protein and mRNA expression of PAI-1 in platelets from T2D, obese and lean subjects indicate a limited role of platelets for the elevated plasma PAI-1 levels. However, an increased synthesis rate of mRNA transcripts in platelets from T2D and an increased release of PAI-1 could also result in similar mRNA and protein levels. Hence, synthesis and release rates of PAI-1 from platelets in T2D and obesity need to be investigated to further elucidate the role of platelets in obesity and T2D.

Impaired Proteostasis, not Protein Synthesis, Limits Recovery of Aged Skeletal Muscle After Disuse Atrophy

Older individuals undergo more frequent inpatient hospitalizations and bedrest compared to younger persons, causing rapid losses of muscle mass and strength. Evidence suggests that only aged muscle fails to completely recover following disuse despite aged and adult muscle having similar levels of muscle loss. Current efforts to improve muscle recovery in older individuals are commonly aimed at increasing myofibrillar protein synthesis via mTOR stimulation despite recent evidence demonstrating that old muscle has chronically elevated levels of mTORC1 activity. We hypothesized that old muscle fails to fully recover muscle mass due to impaired proteostasis and not due limitations in protein synthesis.MethodsAdult (10 month) and old (30 month) F344BN rats were hindlimb unloaded (HU) for 14‐days to induce atrophy, followed by subsequent reloading (RE) to study muscle regrowth. During RE the rats were labeled with deuterium oxide (D2O) for 7, 15, 30, 45 or 60 days (n=4‐6 per timepoint per group) to determine bulk and individual protein synthesis and RNA synthesis. We also assessed muscle mass, fiber size by immunohistochemistry, mTORC1 signaling by western blot, and aggregate formation by tissue fractionation.ResultsAdult and old gastrocnemius (GA) muscle had significant losses of muscle mass (359 mg ±55 and 724 mg ±60 loss respectively,p<0.05) and fiber size (1296 um2±202 and 1480 um2 ±142 loss respectively, p<0.01) with HU. While adult muscle fully recovered GA mass and fiber size by day 15, old GA muscle did not fully recover during the entire 60 days of RE (18% CSA and 11% mass loss still present). Despite limited mass and fiber size recovery in old muscle, GA myofibrillar protein synthesis rates (1.45 ±0.18 vs 2.28 ±0.24, FSR%, p<0.05) and mTORC1‐related signaling were higher in old muscle compared to adult. Additionally, GA RNA synthesis rates (2.79 ±0.139 vs 5.18 ±0.684, p<0.05) and RNA concentration (183 ng ±3.6 vs 249 ng ±13.9, p<0.05), which are markers of translational capacity, were also elevated in old muscle relative to adult during RE. Old muscle had higher levels of insoluble protein aggregates, a marker of impaired proteostasis, during RE compared to adult (1.2 ±0.09 vs 1.5 ±012 fold increase, p<0.05). Lastly, we assessed individual protein synthesis rates of the whole muscle proteome and discovered that old GA muscle had a larger number of proteins with increased synthesis rates compared to adult. In conclusion, these data strongly suggest that limitations in old muscle to recover after disuse are not due to limitations in protein synthesis but are instead due to impaired proteostasis with age. Therefore, understanding how proteostasis responds during these periods surrounding unloading in old muscle are critical to improve muscle recovery after disuse.

Frequency of mispackaging of Prochlorococcus DNA by cyanophage

Prochlorococcus cells are the numerically dominant phototrophs in the open ocean. Cyanophages that infect them are a notable fraction of the total viral population in the euphotic zone, and, as vehicles of horizontal gene transfer, appear to drive their evolution. Here we examine the propensity of three cyanophages—a podovirus, a siphovirus, and a myovirus—to mispackage host DNA in their capsids while infecting Prochlorococcus, the first step in phage-mediated horizontal gene transfer. We find the mispackaging frequencies are distinctly different among the three phages. Myoviruses mispackage host DNA at low and seemingly fixed frequencies, while podo- and siphoviruses vary in their mispackaging frequencies by orders of magnitude depending on growth light intensity. We link this difference to the concentration of intracellular reactive oxygen species and protein synthesis rates, both parameters increasing in response to higher light intensity. Based on our findings, we propose a model of mispackaging frequency determined by the imbalance between the production of capsids and the number of phage genome copies during infection: when protein synthesis rate increase to levels that the phage cannot regulate, they lead to an accumulation of empty capsids, in turn triggering more frequent host DNA mispackaging errors.

Re-Differentiation Capacity of Human Chondrocytes in Vitro Following Electrical Stimulation with Capacitively Coupled Fields.

Treatment of cartilage lesions remains a clinical challenge. Therefore, biophysical stimuli like electric fields seem to be a promising tool for chondrocytic differentiation and treatment of cartilage lesions. In this in vitro study, we evaluated the effects of low intensity capacitively coupled electric fields with an alternating voltage of 100 mVRMS (corresponds to 5.2 × 10−5 mV/cm) or 1 VRMS (corresponds to 5.2 × 10−4 mV/cm) with 1 kHz, on human chondrocytes derived from osteoarthritic (OA) and non-degenerative hyaline cartilage. A reduction of metabolic activity after electrical stimulation was more pronounced in non-degenerative cells. In contrast, DNA contents in OA cells were significantly decreased after electrical stimulation. A difference between 100 mVRMS and 1 VRMS was not detected. However, a voltage-dependent influence on gene and protein expression was observed. Both cell types showed increased synthesis rates of collagen (Col) II, glycosaminoglycans (GAG), and Col I protein following stimulation with 100 mVRMS, whereas this increase was clearly higher in OA cells. Our results demonstrated the sensitization of chondrocytes by alternating electric fields, especially at 100 mVRMS, which has an impact on chondrocytic differentiation capacity. However, analysis of further electrical stimulation parameters should be done to induce optimal hyaline characteristics of ex vivo expanded human chondrocytes.

Barium-Promoted Ruthenium Catalysts on Yittria-Stabilized Zirconia Supports for Ammonia Synthesis

The cost-effective, small-scale, distributed synthesis of ammonia depends on effective catalysts and processes that operate under modest elevated pressure (i.e., p < 20 bar) conditions. The present paper considers Ru as the active catalyst supported on yittria-stabilized zirconia (YSZ). The addition of alkali and alkaline-earth metal promoters is found to increase synthesis rates by an order of magnitude. The rate enhancement is largely insensitive to the promoter concentration, with Cs outperforming Ba and K by a factor of 2. However, Ba is found to be stable whereas Cs degrades more rapidly, which is attributed to the low melting point of its oxide. At 400 °C and 1.0 MPa, the specific synthesis rate over Ba–Ru is measured to be approximately 1410 mmol gRu–1 h–1, higher than the most active oxide-supported Ru catalysts reported in the literature. The rate becomes inhibited by H2 absorption at low temperature (below 350 °C), but lower H2/N2 ratios enable the rate to remain comparable to what is observed i...

Synthesis of MnO2 Nanoparticles in the Presence and Absence of Ultrasonic Irradiation

Previous studies show that ultrasound has a critical role in the synthesis of nanoparticles. In this paper, we compare the synthesis of MnO2 nanoparticles in the presence and absence of ultrasonic waves. The reaction efficiency is measured by the back titration method. The purity, phase, and morphology of the prepared samples are analyzed by X-ray diffraction (XRD) and field-emission scanning electron microscopy (FESEM). The surface area and total pore volume are measured by Brunauer–Emmett–Teller (BET) analysis. The results showed that the reaction efficiency reached 95% in the presence of ultrasonic waves during only 20 min, while it needed more than 6 h to reach such efficiency in the absence of ultrasonic waves. The XRD analysis and FESEM images showed that both samples were MnO2 nanoparticles and their phase was α. The increased synthesis rate is due to the production of free radicals during water sonolysis. Nitrogen adsorption–desorption isotherms displayed mesoporous nanoparticles with a pore size in the range of 2–10 nm for both samples. The surface area was 28.092 m2/g for samples under ultrasonic irradiation and 77.533 m2/g for other samples. A decrease of 2.75 times in the surface area was demonstrated for samples under ultrasonic irradiation. Also, the total pore volume of samples under ultrasonic irradiation was 1.85 times less than that of other samples. The reduction in the surface area and total pore volume is due to the role of the shock wave phenomenon in ultrasound irradiation. The shock wave increases the collision among nanoparticles and the adhesion among them. Based on the analysis of FESEM images, a new index is introduced describing the cohesiveness of the samples. This measure is compatible with the results of the BET analysis and shows increased adhesion among nanoparticles.

Design and optimisation of a microwave reactor for kilo-scale polymer synthesis

Current industrial production of polymer resins is generally undertaken in large multi-tonne stirred tank reactors. These are characterised by relatively slow heating and cooling cycles, resulting in long vessel cycle times and extended production campaigns. In this work we present a design for a hybrid microwave/oil jacket proof of concept system capable of producing up to 4.1 kg of polymer resin per batch. By exploiting rapid volumetric heating effects of microwave energy at 2.45 GHz, we have optimised the synthetic regime, such that a 3.7 kg batch of polyester resin pre-polymer can be made in only 8 h 20 min, with higher molecular weight (Mn 2100) compared to the conventional process taking 22 h 15 min (Mn 1200), yielding an increase in synthesis rate of at least 265%. The increase in polymer molecular weight also suggests a higher conversion was achieved over a shorter time scale.

The role of amino acid metabolism during abiotic stress release.

Plant responses to abiotic stress include various modifications in amino acid metabolism. By using a hydroponic culture system, we systematically investigate modification in amino acid profiles and the proteome of Arabidopsis thaliana leaves during initial recovery from low water potential or high salinity. Both treatments elicited oxidative stress leading to a biphasic stress response during recovery. Degradation of highly abundant proteins such as subunits of photosystems and ribosomes contributed to an accumulation of free amino acids. Catabolic pathways for several low abundant amino acids were induced indicating their usage as an alternative respiratory substrate to compensate for the decreased photosynthesis. Our results demonstrate that rapid detoxification of potentially detrimental amino acids such as Lys is a priority during the initial stress recovery period. The content of Pro, which acts as a compatible osmolyte during stress, was adjusted by balancing its synthesis and catabolism both of which were induced both during and after stress treatments. The production of amino acid derived secondary metabolites was up-regulated specifically during the recovery period, and our dataset also indicates increased synthesis rates of the precursor amino acids. Overall, our results support a tight relationship between amino acid metabolism and stress responses.

Increasing Protein Production Rates Can Decrease the Rate at Which Functional Protein Is Produced and Their Steady-State Levels.

The rate at which soluble, functional protein is produced by the ribosome has recently been found to vary in complex and unexplained ways as various translation-associated rates are altered through synonymous codon substitutions. To understand this phenomenon, here, we combine a well-established ribosome-traffic model with a master-equation model of cotranslational domain folding to explore the scenarios that are possible for the protein production rate, J, and the functional-nascent protein production rate, F, as the rates of various translation processes are altered for five different E. coli proteins. We find that while J monotonically increases as the rates of translation-initiation, -elongation, and -termination increase, F can either increase or decrease. We show that F's nonmonotonic behavior arises within the model from two opposing trends: the tendency for increased translation rates to produce more total protein but less cotranslationally folded protein. We further demonstrate that under certain conditions these nonmonotonic changes in F can result in nonmonotonic variations in post-translational, steady-state levels of functional protein. These results provide a potential explanation for recent experimental observations in which the specific activity of enzymatic proteins decreased with increased synthesis rates. Additionally our model has the potential to be used to rationally design transcripts to maximize the production of functional nascent protein by simultaneously optimizing translation initiation, elongation, and termination rates.

An in-silico insight into the substrate binding characteristics of the active site of amorpha-4, 11-diene synthase, a key enzyme in artemisinin biosynthesis.

The enzyme amorphadiene synthase (ADS) conducts the first committed step in the biosynthetic conversion of the substrate farnesyl pyrophosphate (FPP) to artemisinin, which is a highly effective natural product against multidrug-resistant strains of malaria. Due to the either low abundance or low turn-over rate of the enzyme, obtaining artemisinin from both natural and synthetic sources is costly and laborious. In this in silico study, we strived to elucidate the substrate binding site specificities of the ADS, with the rational that unraveling enzyme features paves the way for enzyme engineering to increase synthesis rate. A homology model of the ADS from Artemisia annua L. was constructed based on the available crystal structure of the 5-epiaristolochene synthase (TEAS) and further analyzed with molecular dynamic simulations to determine residues forming the substrate recognition pocket. We also investigated the structural aspects of Mg2+ binding. Results revealed DDYTD and NDLMT as metal-binding motifs in the putative active site gorge, which is composed of the D and H helixes and one loop region (aa519-532). Moreover, several representative residues including Tyr519, Asp444, Trp271, Asn443, Thr399, Arg262, Val292, Gly400 and Leu405, determine the FPP binding mode and its fate in terms of stereochemistry as well as the enzyme fidelity for the specific end product. These findings lead to inferences concerning key components of the ADS catalytic cavity, and provide evidence for the spatial localization of the FPP and Mg2+. Such detailed understanding will probably help to design an improved enzyme.

In Fission Yeast the Constriction Rate is not Set by the Cytokinetic Ring, but by the Septum Growth Machinery

Constriction of the actomyosin contractile ring is often thought to drive division of the cell into two and to set the constriction rate. We measured a tension in the cytokinetic ring of fission yeast, where ring constriction is accompanied by septation, the growth of new cell wall in the wake of the constricting ring. Using a cell wall modulus ∼ 30 MPa, we conclude ring tension produces negligible septum strains ∼ 0.01% for a ring of radius and width ∼0.1 μm. Thus the inward septum growth rate determines the constriction rate, not the ring tension.Instead, ring tension maintains the circular shape of the septum, ensuring proper closure, using a mechanism that exploits the mechanosensitivity of septum growth. Hypothesizing mechanonsensitivity of the septum growth machinery, a mathematical model showed that ring tension suppresses septum edge roughness. Measurements of septum edge roughness in live cells agreed with model predictions, implying a mechanosensitivity per beta glucan synthase (Bgs) complex, the relative synthesis rate increase per force. Estimating the density of active Bgs complexes, a ring at constriction onset () therefore exerts centripetal force per Bgs, giving a small rate of synthesis increase. A rigorous calculation, from our mathematical model, shows little effect of tension on the constriction rate throughout constriction: ring radius versus time is almost linear with a small downward curvature.Thus, ring tension has little effect on the constriction rate, which is set by the intrinsic rate of synthesis by the cell's septum synthesis machinery.

Increased synthesis of folate transporters regulates folate transport in conditions of ethanol exposure and folate deficiency.

Excessive alcohol consumption and dietary folate inadequacy are the main contributors leading to folate deficiency (FD). The present study was planned to study regulation of folate transport in conditions of FD and ethanol exposure in human embryonic kidney cell line. Also, the reversible nature of effects mediated by ethanol exposure and FD was determined by folate repletion and ethanol removal. For ethanol treatment, HEK293 cells were grown in medium containing 100mM ethanol, and after treatment, one group of cells was shifted on medium that was free from ethanol. For FD treatment, cells were grown in folate-deficient medium followed by shifting of one group of cells on folate containing medium. FD as well as ethanol exposure resulted in an increase in folate uptake which was due to an increase in expression of folate transporters, i.e., reduced folate carrier, proton-coupled folate transporter, and folate receptor, both at the mRNA and protein level. The effects mediated by ethanol exposure and FD were reversible on removal of treatment. Promoter region methylation of folate transporters remained unaffected after FD and ethanol exposure. As far as transcription rate of folate transporters is concerned, an increase in rate of synthesis was observed in both ethanol exposure and FD conditions. Additionally, mRNA life of folate transporters was observed to be reduced by FD. An increased expression of folate transporters under ethanol exposure and FD conditions can be attributed to enhanced rate of synthesis of folate transporters.

High-Throughput Contact Flow Lithography.

High-throughput fabrication of graphically encoded hydrogel microparticles is achieved by combining flow contact lithography in a multichannel microfluidic device and a high capacity 25 mm LED UV source. Production rates of chemically homogeneous particles are improved by two orders of magnitude. Additionally, the custom-built contact lithography instrument provides an affordable solution for patterning complex microstructures on surfaces.

Abstract 249: High Salt and Aldosterone Increase (pro)renin Receptor Expression Through Enac α Activation in Neuronal Cells

Activation of the brain renin-angiotensin-aldosterone system plays an essential role in hyperactivity of the sympathetic nervous system during salt sensitive hypertension. We previously reported an up-regulation of (pro)renin receptor (PRR) expression levels in DOCA-salt hypertensive mice brain. However, the mechanism by which PRR is regulated in DOCA-salt hypertension remains unknown. We hypothesize that aldosterone increases PRR expression by activation of epithelial sodium channel (ENaC). To test this hypothesis, we used neuro-2A cell line from brain hypothalamus which expresses all ENaC α, β, and γ subunits, as well as mineralocorticoid receptor (MR). Cells were incubated in normal salt (NS, 142 mM) or high salt (HS, 160 mM) culture medium for 1, 3, or 5 days, with or without the addition of aldosterone (1μM) for 2 hours. Interestingly, the combination of HS (5 days) with aldosterone significantly increased PRR mRNA levels (fold change) (3.28 ± 0.11) compared to NS (1.00 ± 0.01), HS (1.23 ± 0.01), or aldosterone (1.40 ± 0.01) alone in Neuro-2A cells. The PRR expression levels (1.253 ± 0.006; 1.89 ± 0.002) were significantly attenuated by ENaC inhibition (amiloride, 10μM) or MR inhibition (eplernone, 10μM) respectively (P&lt;0.05), suggesting that ENaC and MR activation mediates the up-regulation of PRR expression in neuronal cells. Furthermore, HS increased ENaC α (2.42 ± 0.01), while decreased ENaC β (0.05 ± 0.01), and ENaC γ (0.03 ± 0.01) mRNA expression levels compared to NS treatment (1.00 ±0.01) in Neuro-2A cells suggesting an increase in synthesis rate of α subunit following HS treatment. In summary, HS and aldosterone increase PRR expression levels via activation of ENaC. ENaC α may be the regulating subunit in the assembly of functional ENaC in neuronal cells. We conclude that regulation of PRR expression by high salt and aldosterone may be the critical step to activate angiotensinergic sympatho-excitatory pathways leading to salt sensitive hypertension.

The use of a real‐time luciferase assay to quantify gene expression dynamics in the living yeast cell

A destabilized version of firefly luciferase was used in living yeast cells as a real-time reporter for gene expression. This highly sensitive and non-invasive system can be simultaneously used upon many different experimental conditions in small culture aliquots. This allows the dose-response behaviour of gene expression driven by any yeast promoter to be reported and can be used to quantify important parameters, such as the threshold, sensitivity, response time, maximal activity and synthesis rate for a given stimulus. We applied the luciferase assay to the nutrient-regulated GAL1 promoter and the stress-responsive GRE2 promoter. We find that luciferase expression driven by the GAL1 promoter responds dynamically to growing galactose concentrations, with increasing synthesis rates determined by the light increment in the initial linear phase of activation. In the case of the GRE2 promoter, we demonstrate that the very short-lived version of luciferase used here is an excellent tool to quantitatively describe transient transcriptional activation. The luciferase expression controlled by the GRE2 promoter responds dynamically to a gradual increase of osmotic or oxidative stress stimuli, which is mainly based on the progressive increase of the time the promoter remains active. Finally, we determined the dose-response behaviour of a single transcription factor binding site in a synthetic promoter context, using the stress response element (STRE) as an example. Taken together, the luciferase assay described here is an attractive tool to rapidly and precisely determine and compare kinetic parameters of gene expression.

Fe 2+ binds iron responsive element-RNA, selectively changing protein-binding affinities and regulating mRNA repression and activation

Iron increases synthesis rates of proteins encoded in iron-responsive element (IRE)-mRNAs; metabolic iron ("free," "labile") is Fe(2+). The noncoding IRE-RNA structure, approximately 30nt, folds into a stem loop to control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. IRE-RNA riboregulators bind specifically to iron-regulatory proteins (IRP) proteins, inhibiting ribosome binding. Deletion of the IRE-RNA from an mRNA decreases both IRP binding and IRP-independent protein synthesis, indicating effects of other "factors." Current models of IRE-mRNA regulation, emphasizing iron-dependent degradation/modification of IRP, lack answers about how iron increases IRE-RNA/IRP protein dissociation or how IRE-RNA, after IRP dissociation, influences protein synthesis rates. However, we observed Fe(2+) (anaerobic) or Mn(2+) selectively increase the IRE-RNA/IRP K(D). Here we show: (i) Fe(2+) binds to the IRE-RNA, altering its conformation (by 2-aminopurine fluorescence and ethidium bromide displacement); (ii) metal ions increase translation of IRE-mRNA in vitro; (iii) eukaryotic initiation factor (eIF)4F binds specifically with high affinity to IRE-RNA; (iv) Fe(2+) increased eIF4F/IRE-RNA binding, which outcompetes IRP binding; (v) exogenous eIF4F rescued metal-dependent IRE-RNA translation in eIF4F-depeleted extracts. The regulation by metabolic iron binding to IRE-RNA to decrease inhibitor protein (IRP) binding and increase activator protein (eIF4F) binding identifies IRE-RNA as a riboregulator.

Acute treatment with fluvoxamine elevates rat brain serotonin synthesis in some terminal regions: An autoradiographic study

A considerable body of evidence indicates the involvement of the neurotransmitter serotonin (5-HT) in the pathogenesis and treatment of depression. The acute effect of fluvoxamine, on 5-HT synthesis rates was investigated in rat brain regions, using α-(14)C-methyl-L-tryptophan as a tracer. Fluvoxamine (25 mg/kg) and saline (control) were injected intraperitoneally, one hour before the injection of the tracer (30 μCi). There was no significant effect of fluvoxamine on plasma free tryptophan. After Benjamini-Hochberg False Discovery Rate correction, a significant decrease in the 5-HT synthesis rate in the fluvoxamine treated rats, was found in the raphe magnus (-32%), but not in the median (-14%) and dorsal (-3%) raphe nuclei. In the regions with serotonergic axon terminals, significant increases in synthesis rates were observed in the dorsal (+41%) and ventral (+43%) hippocampus, visual (+38%), auditory (+65%) and parietal (+37%) cortex, and the substantia nigra pars compacta (+56%). There were no significant changes in the 5-HT synthesis rates in the median (+11%) and lateral (+24%) part of the caudate-putamen, nucleus accumbens (+5%), VTA (+16%) or frontal cortex (+ 6%). The data show that the acute administration of fluvoxamine affects 5-HT synthesis rates in a regionally specific pattern, with a general elevation of the synthesis in the terminal regions and a reduction in some cell body structures. The reasons for the regional specific effect of fluvoxamine on 5-HT synthesis are unclear, but may be mediated by the presynaptic serotonergic autoreceptors.

Effect of chronic protein ingestion on tyrosine and tryptophan levels and catecholamine and serotonin synthesis in rat brain

ObjectivesPrevious studies have shown that brain tyrosine (TYR) levels and catecholamine synthesis rate increase in rats as chronic dietary protein content increases from 2 to 10% (% weight). A single protein, casein, was examined. The present study explores how TYR levels and catecholamine synthesis (and tryptophan (TRP) levels and serotonin synthesis) change when different proteins are ingested chronically over the same range of dietary protein contents.MethodsMale rats ingested for 8 days diets contain 2 or 10% protein (zein, gluten, casein, soy protein, or alpha-lactalbumin). On the last day, they were killed 2.5 hours into the dark period, 30 minutes after receiving an injection of m-hydroxybenzylhydrazine, an inhibitor of aromatic l-amino acid decarboxylase. Brain samples were analyzed for amino acids, including 5-hydroxytryptophan (index of serotonin synthesis rate) and dihydroxyphenylalanine (index of catecholamine synthesis rate), by HPLC-electrochemical detection.ResultsTYR levels and catecholamine synthesis rate in brain were unaffected by the particular protein ingested. However, TRP levels and serotonin synthesis rate varied markedly, depending on the protein ingested, with effects being most prominent in the 10% protein groups. The effect of dietary protein on brain TRP correlated very highly with its effect on serotonin synthesis.DiscussionThe results indicate that the protein ingested can chronically modify TRP levels and serotonin synthesis in brain, but not TYR levels or catecholamine synthesis, with effects most distinct at an adequate level of protein intake (10%).

Translation Initiation: A Regulatory Role for Poly(A) Tracts in Front of the AUG Codon in Saccharomyces cerevisiae

The 5′-UTR serves as the loading dock for ribosomes during translation initiation and is the key site for translation regulation. Many genes in the yeast Saccharomyces cerevisiae contain poly(A) tracts in their 5′-UTRs. We studied these pre-AUG poly(A) tracts in a set of 3274 recently identified 5′-UTRs in the yeast to characterize their effect on in vivo protein abundance, ribosomal density, and protein synthesis rate in the yeast. The protein abundance and the protein synthesis rate increase with the length of the poly(A), but exhibit a dramatic decrease when the poly(A) length is ≥12. The ribosomal density also reaches the lowest level when the poly(A) length is ≥12. This supports the hypothesis that a pre-AUG poly(A) tract can bind to translation initiation factors to enhance translation initiation, but a long (≥12) pre-AUG poly(A) tract will bind to Pab1p, whose binding size is 12 consecutive A residues in yeast, resulting in repression of translation. The hypothesis explains why a long pre-AUG poly(A) leads to more efficient translation initiation than a short one when PABP is absent, and why pre-AUG poly(A) is short in the early genes but long in the late genes of vaccinia virus.