Polycystic ovarian syndrome: signs and feedback effects of hyperandrogenism and insulin resistance.

Polycystic ovary syndrome (PCOS) is a disease whose diagnosis is based on the detection of hyperandrogenism (HA) and ovulatory dysfunction. Women with PCOS frequently develop insulin resistance (IR), which generates a metabolic condition that involves a decrease in the action of insulin at the cellular level and is linked to compensatory hyperinsulinemia (HI). In PCOS, the ovary remains sensitive to the action of insulin. Additionally, it has been observed that the main effect of insulin in the ovary is the stimulation of androgen synthesis, resulting in HA, one of the fundamental characteristics of the PCOS. In this sense, the excess of androgens favors the development of IR, thus perpetuating the cycle of IR-HI-HA, and therefore PCOS. Moreover, mitochondrial dysfunction is present in PCOS patients and is a common feature in both IR and HA. This review places electron transfer as a key element in HA and IR development, with emphasis on the relationship between androgen biosynthesis and mitochondrial function. Indeed, metformin has been involved in repair mitochondrial dysfunction, decrease of oxidative stress, reduction of androgens levels and the enhancing of insulin sensitivity. Therefore, we propose that treatment with metformin could decrease HI and consequently HA, restoring, at least in part, the metabolic and hormonal disorders of PCOS.

Contribution of the mitogen-activated protein kinase Hog1 to the halotolerance of the marine yeast Debaryomyces hansenii.

Halotolerant species are adapted to dealing continually with hyperosmotic environments, having evolved strategies that are uncommon in other organisms. The HOG pathway is the master system that regulates the cellular adaptation under these conditions; nevertheless, apart from the importance of Debaryomyces hansenii as an organism representative of the halotolerant class, its HOG1 pathway has been poorly studied, due to the difficulty of applying conventional recombinant DNA technology. Here we describe for the first time the phenotypic characterisation of a null HOG1 mutant of D. hansenii. Dhhog1Δ strain was found moderately resistant to 1 M NaCl and sensitive to higher concentrations. Under hyperosmotic shock, DhHog1 fully upregulated transcription of DhSTL1 and partially upregulated that of DhGPD1. High osmotic stress lead to long-term inner glycerol accumulation that was partially dependent on DhHog1. These observations indicated that the HOG pathway is required for survival under high external osmolarity but dispensable under low and mid-osmotic conditions. It was also found that DhHog1 can regulate response to alkali stress during hyperosmotic conditions and that it plays a role in oxidative and endoplasmic reticulum stress. Taken together, these results provide new insight into the contribution of this MAPK in halotolerance of this yeast.

Rapamycin induces morphological and physiological changes without increase in lipid content in Ustilago maydis.

The evolutionarily conserved serine/threonine kinase TOR recruits different subunits to assemble the Target of Rapamycin Complex 1 (TORC1), which is inhibited by rapamycin and regulates ribosome biogenesis, autophagy, and lipid metabolism by regulating the expression of lipogenic genes. In addition, TORC1 participates in the cell cycle, increasing the length of the G2 phase. In the present work, we investigated the effect of rapamycin on cell growth, cell morphology and neutral lipid metabolism in the phytopathogenic fungus Ustilago maydis. Inhibition of TORC1 by rapamycin induced the formation of septa that separate the nuclei that were formed after mitosis. Regarding neutral lipid metabolism, a higher accumulation of triacylglycerols was not detected, but the cells did contain large lipid bodies, which suggests that small lipid bodies became fused into big lipid droplets. Vacuoles showed a similar behavior as the lipid bodies, and double labeling with Blue-CMAC and BODIPY indicates that vacuoles and lipid bodies were independent organelles. The results suggest that TORC1 has a role in cell morphology, lipid metabolism, and vacuolar physiology in U. maydis.

Tolerance to Oxidative Stress in Budding Yeast by Heterologous Expression of Catalases A and T from Debaryomyces hansenii.

The function of catalases A and T from the budding yeast Saccharomyces cerevisiae (ScCta1 and ScCtt1) is to decompose hydrogen peroxide (H2O2) to mitigate oxidative stress. Catalase orthologs are widely found in yeast, suggesting that scavenging H2O2 is crucial to avoid the oxidative damage caused by reactive oxygen species (ROS). However, the function of catalase orthologs has not yet been experimentally characterized in vivo. Here, we heterologously expressed Debaryomyces hansenii DhCTA1 and DhCTT1 genes, encoding ScCta1 and ScCtt1 orthologs, respectively, in a S. cerevisiae acatalasemic strain (cta1Δ ctt1Δ). We performed a physiological analysis evaluating growth, catalase activity, and H2O2 tolerance of the strains grown with glucose or ethanol as carbon source, as well as under NaCl stress. We found that both genes complement the catalase function in S. cerevisiae. Particularly, the strain harboring DhCTT1 showed improved growth when ethanol was used as carbon source both in the absence or presence of salt stress. This phenotype is attributed to the high catalase activity of DhCtt1 detected at the exponential growth phase, which prevents intracellular ROS accumulation and confers oxidative stress resistance.

TRPV4 Regulates Tight Junctions and Affects Differentiation in a Cell Culture Model of the Corneal Epithelium.

TRPV4 (transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat, or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca2+ imaging experiments showed that TRPV4 activation with GSK1016790A produced an influx of calcium that was blunted by the specific TRPV4 blocker RN-1734. We analyzed the involvement of TRPV4 in RCE1(5T5) epithelial differentiation by measuring the development of transepithelial electrical resistance (TER) as an indicator of the tight junction (TJ) assembly. We showed that TRPV4 activity was necessary to establish the TJ. In differentiated epithelia, activation of TRPV4 increases the TER and the accumulation of claudin-4 in cell-cell contacts. Epidermal Growth Factor (EGF) up-regulates the TER of corneal epithelial cultures, and we show here that TRPV4 activation mimicked this EGF effect. Conversely, TRPV4 inhibition or knock down by specific shRNA prevented the increase in TER. Moreover, TRPP2, an EGF-activated channel that forms heteromeric complexes with TRPV4, is also concentrated in the outmost cell layer of differentiated RCE1(5T5) sheets. This suggests that the EGF regulation of the TJ may involve a heterotetrameric TRPV4-TRPP2 channel. These results demonstrated TRPV4 activity was necessary for the correct establishment of TJ in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. J. Cell. Physiol. 232: 1794-1807, 2017. © 2016 Wiley Periodicals, Inc.

Diversification of Paralogous α-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae.

Production of α-isopropylmalate (α-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeast Saccharomyces cerevisiae has two paralogous genes, LEU4 and LEU9, that encode α-IPM synthase (α-IPMS) isozymes. Little is known about the biochemical differences between these two α-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. The leu4Δ mutant, which expresses only the feedback-resistant Leu9 homodimer, grows slowly with either glucose or ethanol and accumulates elevated pools of leucine; this phenotype is alleviated by the addition of leucine. Transformation of the leu4Δ mutant with a centromeric plasmid carrying LEU4 restored the wild-type phenotype. Bimolecular fluorescent complementation analysis showed that Leu4-Leu9 heterodimeric isozymes are formed in vivo. Purification and kinetic analysis showed that the hetero-oligomeric isozyme has a distinct leucine sensitivity behavior. Determination of α-IPMS activity in ethanol-grown cultures showed that α-IPM biosynthesis and growth under these respiratory conditions depend on the feedback-sensitive Leu4 homodimer. We conclude that retention and further diversification of two yeast α-IPMSs have resulted in a specific regulatory system that controls the leucine-α-IPM biosynthetic pathway by selective feedback sensitivity of homomeric and heterodimeric isoforms.

Nitric oxide signalling pathway in Duchenne muscular dystrophy mice: up-regulation of L-arginine transporters.

DMD (Duchenne muscular dystrophy) is an incurable rapidly worsening neuromuscular degenerative disease caused by the absence of dystrophin. In skeletal muscle a lack of dystrophin disrupts the recruitment of neuronal NOS (nitric oxide synthase) to the sarcolemma thus affecting NO (nitric oxide) production. Utrophin is a dystrophin homologue, the expression of which is greatly up-regulated in the sarcolemma of dystrophin-negative fibres from mdx mice, a mouse model of DMD. Although cardiomyopathy is an important cause of death, little is known about the NO signalling pathway in the cardiac muscle of DMD patients. Thus we used cardiomyocytes and hearts from two month-old mdx and mdx:utrophin-/- (double knockout) mice (mdx:utr) to study key steps in NO signalling: L-arginine transporters, NOS and sGC (soluble guanylyl cyclase). nNOS did not co-localize with dystrophin or utrophin to the cardiomyocyte membrane. Despite this nNOS activity was markedly decreased in both mdx and mdx:utr mice, whereas nNOS expression was only decreased in mdx:utr mouse hearts, suggesting that utrophin up-regulation in cardiomyocytes maintains nNOS levels, but not function. sGC protein levels and activity remained at control levels. Unexpectedly, L-arginine transporter expression and function were significantly increased, suggesting a novel biochemical compensatory mechanism of the NO pathway and a potential entry site for therapeutics.

Increased sarcolipin expression and decreased sarco(endo)plasmic reticulum Ca2+ uptake in skeletal muscles of mouse models of Duchenne muscular dystrophy.

Abnormal intracellular Ca(2+) handling is an important factor in the progressive functional decline of dystrophic muscle. In the present study, we investigated the function of sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase (SERCA) in various dystrophic muscles of mouse models of Duchenne muscular dystrophy. Our studies show that the protein expression of sarcolipin, a key regulator of the SERCA pump is abnormally high and correlates with decreased maximum velocity of SR Ca(2+) uptake in the soleus, diaphragm and quadriceps of mild (mdx) and severe (mdx:utr-/-) dystrophic mice. These changes are more pronounced in the muscles of mdx:utr-/- mice. We also found increased expression of SERCA2a and calsequestrin specifically in the dystrophic quadriceps. Immunostaining analysis further showed that SERCA2a expression is associated both with fibers expressing slow-type myosin and regenerating fibers expressing embryonic myosin. Together, our data suggest that sarcolipin upregulation is a common secondary alteration in all dystrophic muscles and contributes to the abnormal elevation of intracellular Ca(2+) concentration via SERCA inhibition.

Seasonally increasing parasite load is associated with microbiome dysbiosis in wild bumblebees.

The microbiome is increasingly recognized for its complex relationship with host fitness. Bumblebees are host to a characteristic gut microbiome community that is derived and reinforced through social contact between individuals. The bumblebee microbiome is species-poor, and primarily composed from a small number of core taxa that are associated with the greater tribe of corbiculate bees. Experimental findings support a role for the core bumblebee microbiome in resistance to severe infections by a common trypanosomal parasite, Crithidia bombi. However, most studies have been small in scale, often considering just one or two bumblebee species, or making use of commercially-reared bees. To better understand the microbiome diversity of wild populations, we have deeply sampled field populations of ten sympatric species found throughout central and down east Maine in a three-year microbiome field survey. We have used 16S amplicon sequencing to produce microbiome community profiles, and qPCR to screen samples for infections by Crithidia bombi. The breadth of our dataset has enabled us to test for seasonal and interspecific trends in the microbiome community. Controlling for these external sources of variation, we have identified microbial factors associated with infection and parasite load that support the role of the core microbiome in resistance to severe infection.

The Nß motif of NaTrxh directs secretion as an endoplasmic reticulum transit peptide and variations might result in different cellular targeting.

Soluble secretory proteins with a signal peptide reach the extracellular space through the endoplasmic reticulum-Golgi conventional pathway. During translation, the signal peptide is recognised by the signal recognition particle and results in a co-translational translocation to the endoplasmic reticulum to continue the secretory pathway. However, soluble secretory proteins lacking a signal peptide are also abundant, and several unconventional (endoplasmic reticulum/Golgi independent) pathways have been proposed and some demonstrated. This work describes new features of the secretion signal called Nß, originally identified in NaTrxh, a plant extracellular thioredoxin, that does not possess an orthodox signal peptide. We provide evidence that other proteins, including thioredoxins type h, with similar sequences are also signal peptide-lacking secretory proteins. To be a secretion signal, positions 5, 8 and 9 must contain neutral residues in plant proteins-a negative residue in position 8 is suggested in animal proteins-to maintain the Nß motif negatively charged and a hydrophilic profile. Moreover, our results suggest that the NaTrxh translocation to the endoplasmic reticulum occurs as a post-translational event. Finally, the Nß motif sequence at the N- or C-terminus could be a feature that may help to predict protein localisation, mainly in plant and animal proteins.

Systematic profiling of subtelomeric silencing factors in budding yeast.

Subtelomeric gene silencing is the negative transcriptional regulation of genes located close to telomeres. This phenomenon occurs in a variety of eukaryotes with salient physiological implications, such as cell adherence, virulence, immune-system escape, and ageing. The process has been widely studied in the budding yeast Saccharomyces cerevisiae, where genes involved in this process have been identified mostly on a gene-by-gene basis. Here, we introduce a quantitative approach to study gene silencing, that couples the classical URA3 reporter with GFP monitoring, amenable to high-throughput flow cytometry analysis. This dual silencing reporter was integrated into several subtelomeric loci in the genome, where it showed a gradual range of silencing effects. By crossing strains with this dual reporter at the COS12 and YFR057W subtelomeric query loci with gene-deletion mutants, we carried out a large-scale forward screen for potential silencing factors. The approach was replicable and allowed accurate detection of expression changes. Results of our comprehensive screen suggest that the main players influencing subtelomeric silencing were previously known, but additional potential factors underlying chromatin conformation are involved. We validate and report the novel silencing factor LGE1, a protein with unknown molecular function required for histone H2B ubiquitination. Our strategy can be readily combined with other reporters and gene perturbation collections, making it a versatile tool to study gene silencing at a genome-wide scale.

Neo-functionalization in Saccharomyces cerevisiae: a novel Nrg1-Rtg3 chimeric transcriptional modulator is essential to maintain mitochondrial DNA integrity.

In Saccharomyces cerevisiae, the transcriptional repressor Nrg1 (Negative Regulator of Glucose-repressed genes) and the ß-Zip transcription factor Rtg3 (ReTroGrade regulation) mediate glucose repression and signalling from the mitochondria to the nucleus, respectively. Here, we show a novel function of these two proteins, in which alanine promotes the formation of a chimeric Nrg1/Rtg3 regulator that represses the ALT2 gene (encoding an alanine transaminase paralog of unknown function). An NRG1/NRG2 paralogous pair, resulting from a post-wide genome small-scale duplication event, is present in the Saccharomyces genus. Neo-functionalization of only one paralog resulted in the ability of Nrg1 to interact with Rtg3. Both nrg1Δ and rtg3Δ single mutant strains were unable to use ethanol and showed a typical petite (small) phenotype on glucose. Neither of the wild-type genes complemented the petite phenotype, suggesting irreversible mitochondrial DNA damage in these mutants. Neither nrg1Δ nor rtg3Δ mutant strains expressed genes encoded by any of the five polycistronic units transcribed from mitochondrial DNA in S. cerevisiae. This, and the direct measurement of the mitochondrial DNA gene complement, confirmed that irreversible damage of the mitochondrial DNA occurred in both mutant strains, which is consistent with the essential role of the chimeric Nrg1/Rtg3 regulator in mitochondrial DNA maintenance.

Entropic intermediates and hidden rate-limiting steps in seemingly concerted cycloadditions. Observation, prediction, and origin of an isotope effect on recrossing.

An unusual intramolecular kinetic isotope effect (KIE) in the reaction of dichloroketene with cis-2-butene does not fit with a simple asynchronous cycloaddition transition state, but it can be predicted from trajectory studies on a bifurcating energy surface. The origin of the KIE is related to a high propensity for transition state recrossing in this system, with heavier masses recrossing less. The KIE can also be predicted by a statistical model that treats the cycloaddition as a stepwise mechanism, the rate-limiting second step being associated with an entropic barrier for formation of the second carbon-carbon bond. The relevance of this stepwise mechanism to other asynchronous but seemingly concerted cycloadditions is suggested by examination of organocatalytic Diels-Alder reactions.

Yeasts Inhabiting Extreme Environments and Their Biotechnological Applications.

Yeasts are microscopic fungi inhabiting all Earth environments, including those inhospitable for most life forms, considered extreme environments. According to their habitats, yeasts could be extremotolerant or extremophiles. Some are polyextremophiles, depending on their growth capacity, tolerance, and survival in the face of their habitat's physical and chemical constitution. The extreme yeasts are relevant for the industrial production of value-added compounds, such as biofuels, lipids, carotenoids, recombinant proteins, enzymes, among others. This review calls attention to the importance of yeasts inhabiting extreme environments, including metabolic and adaptive aspects to tolerate conditions of cold, heat, water availability, pH, salinity, osmolarity, UV radiation, and metal toxicity, which are relevant for biotechnological applications. We explore the habitats of extreme yeasts, highlighting key species, physiology, adaptations, and molecular identification. Finally, we summarize several findings related to the industrially-important extremophilic yeasts and describe current trends in biotechnological applications that will impact the bioeconomy.

SARS-CoV-2 Variants and Clinical Outcomes: A Systematic Review.

BACKGROUND: From the start of the COVID-19 pandemic, new SARS-CoV-2 variants have emerged that potentially affect transmissibility, severity, and immune evasion in infected individuals. In the present systematic review, the impact of different SARS-CoV-2 variants on clinical outcomes is analyzed. METHODS: A systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020. Two databases (PubMed and ScienceDirect) were searched for original articles published from 1 January 2020 to 23 November 2021. The articles that met the selection criteria were appraised according to the Newcastle-Ottawa Quality Assessment Scale. RESULTS: Thirty-three articles were included, involving a total of 253,209 patients and 188,944 partial or complete SARS-CoV-2 sequences. The most reported SARS-CoV-2 variants showed changes in the spike protein, N protein, RdRp and NSP3. In 28 scenarios, SARS-CoV-2 variants were found to be associated with a mild to severe or even fatal clinical outcome, 15 articles reported such association to be statistically significant. Adjustments in eight of them were made for age, sex and other covariates. CONCLUSIONS: SARS-CoV-2 variants can potentially have an impact on clinical outcomes; future studies focused on this topic should consider several covariates that influence the clinical course of the disease.

Impaired Carbohydrate Metabolism and Excess of Lipid Accumulation in Offspring of Hyperandrogenic Mice.

Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder of unknown etiology. Hyperandrogenism (HA) is the main diagnostic criteria for PCOS, in addition to being a risk factor for developing several disorders throughout the patient's life, including pregnancy. However, the impact on offspring is little known. Therefore, the aim of this work was to evaluate the effect of maternal HA on glucose metabolism and hepatic lipid accumulation in adult offspring. We used Balb/c mice treated with dehydroepiandrosterone (DHEA) for 20 consecutive days. The ovary of DHEA-treated mice showed hemorrhagic bodies, an increased number of atretic follicles, and greater expression of genes related to meiotic cell cycle and DNA repair. The DHEA offspring (O-DHEA) had low birth weight, and some pups showed malformations. However, O-DHEA individuals gained weight rapidly, and the differences between them and the control group became significantly greater in adulthood. Moreover, O-DHEA presented higher serum glucose after a 6 h fast and a larger area under glucose, insulin, and pyruvate tolerance test curves. Oil Red O staining showed a more significant accumulation of fat in the liver but no changes in serum cholesterol and triacylglycerol levels. In summary, our results show that HA, induced by DHEA, affects gene expression in oocyte, which in turn generates defects in embryonic development, insulin resistance, and alteration in hepatic gluconeogenesis and lipid metabolism in O-DHEA, thereby increasing the risk of developing metabolic diseases.

Alternative CUG Codon Usage in the Halotolerant Yeast Debaryomyces hansenii: Gene Expression Profiles Provide New Insights into Ambiguous Translation.

The halotolerant yeast Debaryomyces hansenii belongs to the CTG-Ser1 clade of fungal species that use the CUG codon to translate as leucine or serine. The ambiguous decoding of the CUG codon is relevant for expanding protein diversity, but little is known about the role of leucine-serine ambiguity in cellular adaptations to extreme environments. Here, we examine sequences and structures of tRNACAG from the CTG-Ser1 clade yeasts, finding that D. hansenii conserves the elements to translate ambiguously. Then, we show that D. hansenii has tolerance to conditions of salinity, acidity, alkalinity, and oxidative stress associated with phenotypic and ultrastructural changes. In these conditions, we found differential expression in both the logarithmic and stationary growth phases of tRNASer, tRNALeu, tRNACAG, LeuRS, and SerRS genes that could be involved in the adaptive process of this yeast. Finally, we compare the proteomic isoelectric points and hydropathy profiles, detecting that the most important variations among the physicochemical characteristics of D. hansenii proteins are in their hydrophobic and hydrophilic interactions with the medium. We propose that the ambiguous translation, i.e., leucylation or serynation, on translation of the CUG-encoded residues, could be linked to adaptation processes in extreme environments.

Parasitemia and Differential Tissue Tropism in Mice Infected with Trypanosoma cruzi Isolates Obtained from Meccus phyllosoma in the State of Oaxaca, Mexico.

Trypanosoma cruzi is a parasite transmitted by the feces of triatomines. Many triatomine species are found in Mexico, and various T. cruzi variants have been isolated from these species, each showing very different virulence and cell tropism. The isolates were obtained from Meccus phyllosoma specimens in three localities in the state of Oaxaca, Mexico: Tehuantitla, Vixhana, and Guichivere. The virulence of each isolate was assessed by quantifying parasitemia, survival, and histopathologic findings. The lineage of each isolate was identified using the mini-exon gene. The expression of the tssa gene during infection was detected in the heart, esophagus, gastrocnemius, and brain. Our results show that the maximum post-infection parasitemia was higher for the Tehuantitla isolate. On genotyping, all isolates were identified as T. cruzi I. The amastigotes in the heart and gastrocnemius were verified for all isolates, but in the brain only for Tehuantitla and Vixhana. The tssa expression allowed us to detect T. cruzi isolates, for Tehuantitla, predominantly in the heart. For Vixhana, a higher tssa expression was detected in gastrocnemius, and for Guichivere, it was higher in the esophagus. Results show that virulence, tropism, and tssa expression can vary, even when the isolates are derived from the same vector species, in the same region, and at similar altitudes.

Molecular Epidemiology Surveillance of SARS-CoV-2: Mutations and Genetic Diversity One Year after Emerging.

In December 2019, the first cases of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were identified in the city of Wuhan, China. Since then, it has spread worldwide with new mutations being reported. The aim of the present study was to monitor the changes in genetic diversity and track non-synonymous substitutions (dN) that could be implicated in the fitness of SARS-CoV-2 and its spread in different regions between December 2019 and November 2020. We analyzed 2213 complete genomes from six geographical regions worldwide, which were downloaded from GenBank and GISAID databases. Although SARS-CoV-2 presented low genetic diversity, there has been an increase over time, with the presence of several hotspot mutations throughout its genome. We identified seven frequent mutations that resulted in dN substitutions. Two of them, C14408T>P323L and A23403G>D614G, located in the nsp12 and Spike protein, respectively, emerged early in the pandemic and showed a considerable increase in frequency over time. Two other mutations, A1163T>I120F in nsp2 and G22992A>S477N in the Spike protein, emerged recently and have spread in Oceania and Europe. There were associations of P323L, D614G, R203K and G204R substitutions with disease severity. Continuous molecular surveillance of SARS-CoV-2 will be necessary to detect and describe the transmission dynamics of new variants of the virus with clinical relevance. This information is important to improve programs to control the virus.

Isotope effect, mechanism, and origin of catalysis in the decarboxylation of mandelylthiamin.

The mechanism of decarboxylations in water and the catalysis of mandelylthiamin (MTh) decarboxylation by pyridinium ions is explored. It has been recently proposed that a decarboxylation step forming an intermediate molecule/CO(2) pair is reversible and that pyridinium ions catalyze the MTh decarboxylation by trapping the intermediate, preventing reversion to MTh. A calculation of the barrier for the back reaction goes against this proposal, as the diffusional separation of CO(2) would be on the order of 10,000 times faster. A comparison of predicted and experimental isotope effects for a series of decarboxylations including the MTh reaction shows in each case the absence of significant reversibility of the decarboxylation step. An alternative origin of the pyridinium catalysis of decarboxylation is proposed, based on the formal binding of the pyridinium ions to the decarboxylation transition state.