¿Sigue siendo útil la microscopia de campo oscuro en el diagnóstico de la sífilis primaria en el siglo XXI? / Is dark-field microscopy still useful for the primary syphilis diagnosis in the 21ST century?

IntroducciónLa serología luética en la sífilis primaria puede ser negativa los primeros 5-15 días. El objetivo de este trabajo fue evaluar los beneficios de incluir la microscopia de campo oscuro (MCO) en el algoritmo diagnóstico de la sífilis primaria.MetodologíaSe incluyó a todos los pacientes que acudieron a una clínica de infecciones de transmisión sexual de la Comunidad de Madrid entre 2015 y 2019 que presentaban una úlcera genital sospechosa de sífilis primaria. Se les realizó MCO y serología (EIA/TPPA/RPR).ResultadosDe las 806 muestras, el 53,2% (429) fueron positivas para MCO. De los 429, el 48% presentaba screening serológico negativo (EIA/RPR) y de ellos en el 77,6% el TPPA fue positivo.ConclusionesLa MCO permite un diagnóstico de sífilis primaria precoz, incluso sin confirmación serológica. Si no se dispone de técnicas directas, en primoinfección, la TPPA es de gran ayuda en el diagnóstico.

IntroductionSerological test for primary syphilis could be negative the first 5-15 days. The aim of this study was to evaluate the benefit of including dark field microscopy (DFM) in the diagnosis algorythm for primary syphilis.Materials/methodsPatients attended to a sexual transmission diseases clinic of Madrid, from 2015 to 2019, for a genital ulcer with clinical suspicion of primary syphilis. They were tested for DMF and serological test (EIA/TPPA/RPR).ResultsOver the total amount of samples (806), 53.2% (429) were positive for DFM. Thus, the 48% of the 429 patients had negative serological test (EIA/RPR) of which the 77.6% were positive at TPPA.ConclusionsDFM allows primary syphilis early diagnosis, even without serological test. If no direct detection methods are available, for patients without history of syphilis, TPPA could help to diagnose primary syphilis.

Unraveling RNA dynamical behavior of TPP riboswitches: a comparison between Escherichia coli and Arabidopsis thaliana.

Riboswitches are RNA sensors that affect post-transcriptional processes through their ability to bind to small molecules. Thiamine pyrophosphate (TPP) riboswitch class is the most widespread riboswitch occurring in all three domains of life. Even though it controls different genes involved in the synthesis or transport of thiamine and its phosphorylated derivatives in bacteria, archaea, fungi, and plants, the TPP aptamer has a conserved structure. In this study, we aimed at understanding differences in the structural dynamics of TPP riboswitches from Escherichia coli and Arabidopsis thaliana, based on their crystallographic structures (TPPswec and TPPswat, respectively) and dynamics in aqueous solution, both in apo and holo states. A combination of Molecular Dynamics Simulations and Network Analysis empowered to find out slight differences in the dynamical behavior of TPP riboswitches, although relevant for their dynamics in bacteria and plants species. Our results suggest that distinct interactions in the microenvironment surrounding nucleotide U36 of TPPswec (and U35 in TPPswat) are related to different responses to TPP. The network analysis showed that minor structural differences in the aptamer enable enhanced intramolecular communication in the presence of TPP in TPPswec, but not in TPPswat. TPP riboswitches of plants present subtler and slower regulation mechanisms than bacteria do.

Integrative method for three-dimensional imaging of the entire Golgi apparatus by combining thiamine pyrophosphatase cytochemistry and array tomography using backscattered electron-mode scanning electron microscopy.

Thiamine pyrophosphatase (TPPase) cytochemistry is an established method for specific labeling of the trans-Golgi cisterns in tissue sections. Herein, we combined this enzyme cytochemical method with array tomography using scanning electron microscopy (SEM), a new imaging technique based on collection of backscattered electron (BSE) images of consecutive resin-embedded sections on glass slides, to detect the entire three-dimensional (3D) organization of the Golgi apparatus with sufficient spatial resolution. As the signal intensity of BSE depends on the atomic number of the materials, lead precipitates confined to the trans-Golgi cisterns after TPPase cytochemistry were clearly observed by BSE-mode SEM. The mild fixative used for TPPase cytochemistry also enabled accurate identification of target gonadotropes in the composite pituitary tissue by immunocytochemical staining. By 3D reconstruction of the entire trans-Golgi cisterns based on serial ultrathin section images of tissues after TPPase cytochemistry, we detected ultrastructural differences in the 3D configuration of the Golgi apparatus between cerebellar Purkinje cells and pituitary gonadotropes. The appropriate combination of enzyme cytochemistry and/or immunostaining with array tomography will further clarify the relationship between the organization and functional states of the Golgi apparatus.

Testicular effects of monensin, a golgi interfering agent in male rats.

OBJECTIVE: The present study was undertaken to explore the effects of monensin, a potent Golgi disturbing agent on male fertility. METHODS: Male Wistar rats were administered monensin at the dose levels of 2.5, 5, and 10 mg/kg b wt. Animals were sacrificed after 67 days of the treatment. The activities of lactate dehydrogenase (LDH), ATPase, acid phosphatase and thiamine pyrophosphatase (TPPase) were measured in the testis. Cytochemical assay of Golgi body marker enzyme, thiamine pyrophosphatase was also performed. Ultrastructural changes in testis were studied by Transmission electron microscopy. Sperm number and motility were also examined. RESULTS AND DISCUSSION: The alterations in the activities of above mentioned enzymes indicate the pronounced effect of the drug on the functioning of spermatogenic cells. The findings from electron microscopy such as membrane disruption, swelling and disintegration of Golgi apparatus strongly suggest the interference of monensin with the functioning of Golgi apparatus in the spermatogenic cells. Data from the sperm number and motility as well as the fertility studies and the resulted litter size further points towards the antifertility effects of monensin in male rats. CONCLUSION: The findings from the present study strongly indicated the effects of monensin on the testis, involving alterations in key enzyme activities and changes at the ultrastructural level.

[Novel targets for antibiotics discovery: riboswitches].

Riboswitches are cis-acting domains located in mRNA sequences that could regulate gene expression by sensing small molecules without employing protein. Most known riboswitches in bacteria have naturally evolved to bind essential metabolite ligands and are involved in the regulation of critical genes that are responsible for the biosynthesis or transport of the cognate ligand. The riboswitch-mediated gene expression could be repressed by metabolite analogs, which caused bacterial growth inhibition or even death. A number of leading compounds targeting riboswitches have been discovered. A promising avenue for the development of new class of riboswitch-based antibiotics has been opened. Herein we reviewed the current findings of riboswitches that served as targets for antibacterial drug development and the underlying mechanisms. The development of high-throughput methods and rational drug design for riboswitch-specific drug discovery are relevant challenges are discussed. summarized.

Novel targets for antibiotics discovery: riboswitches / 药学学报

Riboswitches are cis-acting domains located in mRNA sequences that could regulate gene expression by sensing small molecules without employing protein. Most known riboswitches in bacteria have naturally evolved to bind essential metabolite ligands and are involved in the regulation of critical genes that are responsible for the biosynthesis or transport of the cognate ligand. The riboswitch-mediated gene expression could be repressed by metabolite analogs, which caused bacterial growth inhibition or even death. A number of leading compounds targeting riboswitches have been discovered. A promising avenue for the development of new class of riboswitch-based antibiotics has been opened. Herein we reviewed the current findings of riboswitches that served as targets for antibacterial drug development and the underlying mechanisms. The development of high-throughput methods and rational drug design for riboswitch-specific drug discovery are relevant challenges are discussed. summarized.

Tripeptidil peptidasa 1 en pacientes con ceroidolipofuscinosis neuronal infantil tardía / Tripeptidyl peptidase 1 in patients with late infantile neuronal ceroid lipofuscinosis

Introducción: Las ceroidolipofuscinosis neuronales (CLN) representan un grupo de enfermedades lisosomales hereditarias de herencia autosómica recesiva, de presentación más frecuente durante la niñez, caracterizadas neuropatológicamente por acumulación de lipopigmentos autofluorescentes en los lisosomas de neuronas y otras células. Clínicamente se presentan con pérdida de las habilidades psicomotoras adquiridas, incoordinación motora, ataxia, pérdida de la visión, cambios de conducta, convulsiones de difícil tratamiento asociadas a mioclonías y una corta expectativa de vida. En la actualidad, se conocen 10 formas genéticamente distintas de esta enfermedad, entre ellas la forma infantil tardía donde las manifestaciones clínicas aparecen entre el segundo y cuarto año de vida. El gen responsable de la enfermedad es el TPP1 ubicado en 11p15 y codifica la enzima tripeptidil peptidasa 1. Pacientes y métodos: Se estandarizó la técnica para el diagnóstico enzimático de la ceroidolipofuscinosis neuronal infantil tardía a través de sangre seca en papel de filtro en 76 individuos sanos en edad preescolar y adulta de población venezolana. La actividad enzimática de la TPP1 fue determinada en 9 pacientes con diagnóstico clínico de ceroidolipofuscinosis infantil tardía 2 (CLN2). Resultados: Seis pacientes mostraron valores de actividad muy por debajo del rango establecido (0,11-0,45 nmol/mancha) para los controles sanos en edad preescolar, confirmando el diagnostico enzimático. Tres de los 14 padres estudiados presentaron valores en el rango de heterocigotos. Conclusiones: El diagnóstico enzimático de CLN2 a través de la determinación de la actividad enzimática de la enzima TPP1 mediante la técnica de sangre seca en papel de filtro permite un diagnóstico rápido, sencillo, económico y confiable(AU)

Introduction: Neuronal ceroid lipofuscinoses are a group of inherited autosomal recessive lysosomal diseases, most commonly found in infancy. These are neuropathologically characterised by accumulation of an autofluorescent lipopigment in neurons and other cells. This condition is clinically characterised by loss of motor and cognitive skills, lack of motor coordination, ataxia, progressive visual impairment, behavioural changes; seizures of difficult to manage seizures, particularly myoclonic, and premature death. Ten clinical forms have been described, one of which is late infantile where clinical signs begin between two and four years. The gene responsible for this disease is located at 11p15 locus, and the enzyme encoded by this gene is the tripeptidyl peptidase 1. Patients and methods: We standardised the technique for the enzymatic diagnosis of late infantile neuronal ceroid lipofuscinoses from dried blood on filter paper card in 76 healthy individuals adults and children in order to establish a normal range in the Venezuelan population. The tripeptidyl peptidase activity was also determined in 9 patients with a clinical diagnosis of late infantile neuronal ceroid lipofuscinoses. Results: Six of the samples showed activity lower than the lowest control value (0.11 to 0.45 nmol/spot) from healthy controls of infantile age, confirming the enzymatic diagnosis. Three of the 14 parent samples analysed showed values in the heterozygote ranges. Conclusions: The enzymatic diagnosis of late infantile neuronal ceroid lipofuscinoses from dried blood on filter paper card is a rapid, easier, less expensive and accurate molecular diagnosis tool(AU)

Identification and characterization of oxalate oxidoreductase, a novel thiamine pyrophosphate-dependent 2-oxoacid oxidoreductase that enables anaerobic growth on oxalate.

Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO(2) fixation for heterotrophic or autotrophic growth. This paper describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. Exposure to oxalate leads to the induction of three proteins that are subunits of OOR, which oxidizes oxalate coupled to the production of two electrons and CO(2) or bicarbonate. Like other members of the 2-oxoacid:ferredoxin oxidoreductase family, OOR contains thiamine pyrophosphate and three [Fe(4)S(4)] clusters. However, unlike previously characterized members of this family, OOR does not use coenzyme A as a substrate. Oxalate is oxidized with a k(cat) of 0.09 s(-1) and a K(m) of 58 µM at pH 8. OOR also oxidizes a few other 2-oxoacids (which do not induce OOR) also without any requirement for CoA. The enzyme transfers its reducing equivalents to a broad range of electron acceptors, including ferredoxin and the nickel-dependent carbon monoxide dehydrogenase. In conjunction with the well characterized Wood-Ljungdahl pathway, OOR should be sufficient for oxalate metabolism by M. thermoacetica, and it constitutes a novel pathway for oxalate metabolism.

Determination of the genetic, molecular, and biochemical basis of the Arabidopsis thaliana thiamin auxotroph th1.

2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate kinase/thiamin monophosphate pyrophosphorylase (HMPPK/TMPPase) is a key enzyme involved in thiamin biosynthesis. A candidate HMPPK/TMPPase gene identified in the Arabidopsis genome complemented the thiamin auxotrophy of the th1 mutant, thus proving that the th1 locus corresponds to the structural gene for the HMPPK/TMPPase. Sequence comparisons between the wild-type HMPPK/TMPPase gene and the th1-201 mutant allele identified a single point mutation that caused the substitution of a phenylalanine for a conserved serine residue in the HMPPK domain. Functional analyses of the mutant HMPPK/TMPPase in Escherichia coli revealed that the amino acid substitution in the HMPPK domain of mutant enzyme resulted in a conformational change that severely compromised both activities of the bifunctional enzyme. Studies were also performed to identify the chloroplast as the specific subcellular locale of the Arabidopsis HMPPK/TMPPase.

Effect of monensin on the enzymes of oxidative stress, thiamine pyrophosphatase and DNA integrity in rat testicular cells in vitro.

Monensin, a sodium specific ionophore was evaluated for its in vitro effects on rat testis by studying changes at biochemical parameters as well as at the DNA level. It was observed that monensin produced marked alterations in the activities of various enzymes associated with the testicular functions. The significant inhibition of different enzymes of oxidative defense system points toward the generation of reactive oxygen species (ROS) by monensin treatment. The significant depletion of reduced glutathione and elevation in the level of lipid peroxidation further support the above findings. The significant inhibition of the activities of lactate dehydrogenase and adenosine triphosphatase shows the interference of monensin with the normal energy supply in spermatogenesis. Moreover, the significant increase in the activities of acid phosphatase and thiamine pyrophosphatase demonstrates the interference of monensin with the Golgi-lysosomal complex of the rat testis. Induced DNA fragmentation indicates towards the impact of monensin on the DNA integrity and apoptosis. Further studies are needed to understand the important molecular mechanisms responsible for these effects.

Mutational analysis of ThiH, a member of the radical S-adenosylmethionine (AdoMet) protein superfamily.

Thiamine pyrophosphate (TPP) is an essential cofactor for all forms of life. In Salmonella enterica, the thiH gene product is required for the synthesis of the 4-methyl-5-beta hydroxyethyl-thiazole monophosphate moiety of TPP. ThiH is a member of the radical S-adenosylmethionine (AdoMet) superfamily of proteins that is characterized by the presence of oxygen labile [Fe-S] clusters. Lack of an in vitro activity assay for ThiH has hampered the analysis of this interesting enzyme. We circumvented this problem by using an in vivo activity assay for ThiH. Random and directed mutagenesis of the thiH gene was performed. Analysis of auxotrophic thiH mutants defined two classes, those that required thiazole to make TPP (null mutants) and those with thiamine auxotrophy that was corrected by either L-tyrosine or thiazole (ThiH* mutants). Increased levels of AdoMet also corrected the thiamine requirement of members of the latter class. Residues required for in vivo function were identified and are discussed in the context of structures available for AdoMet enzymes.

In vitro cytochemical studies of the golgi apparatus of pancreatic acinar cells exposed to drugs that inhibit the transport of membranes and proteins.

In a preceding article, we described alterations occurring in rat pancreas acinar cells at successive post-mortem (PM) intervals. In ultra-thin sections from samples obtained from 0.5, 1, 2, 4, 8 and 12 h, we observed in the Golgi apparatus the appearance of an anomalous membrane bound structure. Such structures are formed by tubules and vesicles that we have called tubular vesicular structure (TVS), and they are frequently located in the position corresponding to the 4th cisterna of the Golgian cisternal pile. Lobules of rat pancreas, incubated in vitro with metabolic inhibitors (such as antimycin A, sodium fluoride, sodium azide and potassium cyanide), were processed in order to be compared with the PM samples of the rat acinar cells. In sliced pieces of lobules, acid phosphatase (AcPase) and tiaminopirophosphatase (TPPase) activity were evaluated. Except for the potassium cyanide treatment, we frequently observed the TVS located at the position corresponding to the 4th cisternae (similar to those observed in the PM acinar cells). These TVS's are predominantly TPPase positive. Based on this result and the fact that the TVS's are surrounded by a membrane (as confirmed by the freeze-fracture replica results) with no structural elements inside, they seem not to correspond to autophagosomes. The TVS's, observed either at PM consecutive times or incubated with metabolic inhibitors, seem to be structures formed in response to ATP deprivation. In 0,5 h PM cells and in cells incubated for 30 and 60 min with metabolic inhibitors, the subcellular structures reacted for AcPase in the rigid lamellae, CV and lysosomes.

Cytochemical study of the involvement of cell organelles in formation and accumulation of fibrillar amyloid in the pancreas of NORbeta transgenic mice.

Phosphatase ultrastructural cytochemistry was used to evaluate the participation of cytoplasmic organelles in the accumulation of fibrillar amyloid beta (Abeta) in exocrine acinar cells and in macrophages of the pancreas of transgenic mice overexpressing a carboxy-terminal fragment of Abeta protein precursor (ABPP). Nucleoside diphosphatase (NDPase) and glucose-6-phosphatase (G6Pase) were used as cytochemical markers of the endoplasmic reticulum (ER), thiamine pyrophosphatase (TPPase) as a marker of the Golgi apparatus (GA), and acid phosphatase (AcPase) as a marker of lysosomes. Monoclonal antibody 4G8 raised against the 17-24 aa sequence of human Abeta protein was used for immunogold localization of fibrillar Abeta. The results of this study indicate that the formation of Abeta in acinar cells occurs directly in the vacuolar areas of the rough ER (RER) without evident participation of the elements of the GA, whereas an intimate structural relation with primary lysosomes suggests their role in modification or digestion of the deposited amyloid. In macrophages, fibrillar amyloid was present in numerous cytoplasmic vacuoles located frequently in close proximity to flattened saccules of the ER. This structural pattern revealed similarity to that observed previously in microglial cells producing fibrillar PrP amyloid in scrapie-infected mice and Abeta in brains of human elderly patients and in Alzheimer's type brain pathology.

Hormonal control of enzyme activity during the plasma membrane transformation of uterine epithelial cells.

Ultrastructural and light microscopic catalytic histochemical methods were used to study the distribution and changes in distribution of four phosphatase enzymes; alkaline phosphatase, 5'-nucleotidase, thiamine pyrophosphatase and adenosine triphosphatase in uterine epithelial cells in response to the ovarian hormones, oestrogen, progesterone or a combination of both used in different regimes on ovariectomised rats. Reaction product for all four enzymes was clearly localised in the epithelial cells, especially with oestrogen priming. However, the four enzymes showed markedly different patterns of organisation of reaction product in response to other hormonal treatments. Our findings clearly show that the expression of these enzymes is under ovarian hormonal control. However, while all of the enzymes are upregulated by oestrogen, the response to progesterone is variable, which can upregulate or downregulate different enzymes. The findings are particularly obvious at the electron microscopic level on the apical plasma membrane of the uterine epithelial cells, which was the main focus of our study.

Nuclear changes and acrosome formation during spermiogenesis in Euchistus heros (Hemiptera: Pentatomidae).

Ultrastructural and cytochemical studies were carried out on nuclear changes and acrosome formation during the spermiogenesis of the phytophagous bug Euchistus heros. The development of the nucleus involves changes in the shape and in degree of chromatin condensation: initially it is dispersed and with a low-electron density, then assumes a fibrillar arrangement and finally compacts in an electron-dense material. The acrosome is formed by the Golgi complex and presents unusual morphological features during its development. The reaction product of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase activities were detected during various stages of acrosome development. In contrast, residues of alpha-N-acetylgalactosamine and basic proteins were only reported in the intermediate and late stages of the differentiation process, respectively.

Autophagy-related vacuoles in mouse gallbladder epithelium.

The mouse gallbladder epithelial cells contain very heterogeneous vacuolar population. In an attempt to classify these vacuoles we identified NADPase and TPPase activity as well as the location of HRP which is used as the endocytotic marker. The results of the present study show that the vacuoles can be classified into three categories: (1) the vacuoles predominantly containing loose membrane coils related to the nascent autophagic vacuoles, (2) vacuoles containing densely packed membranes and exhibiting a positive HRP reaction, indicating the convergence of endocytotic and autophagic pathway, and (3) vacuoles composed of degraded membrane structures and containing the reaction product of NADPase activity, showing that the fusion of the lysosomes with the autophagosome-endosome took place. The highly developed cis, medial and trans Golgi compartments reflect the biosynthetic and endocytotic activity of the gallbladder epithelium.

Cytoskeleton mediating transport between the ER system and the Golgi apparatus in the green alga Scenedesmus acutus.

In the green alga Scenedesmus acutus, Golgi bodies are located near the nucleus and supplied with transition vesicles that bud from the outer nuclear envelope membrane. Using this alga, we have shown previously that thiamine pyrophosphatase (TPPase), a marker enzyme of Golgi bodies, migrates in vesicles from the Golgi bodies to the ER via the nuclear envelope in the presence of BFA (Noguchi et al., Protoplasma 201, 202-212, 1998). In this study we demonstrate that both cytochalasin B and oryzalin (microtubule-disrupting agent) inhibit the BFA-induced migration of TPPase from Golgi bodies to the nuclear envelope. However, only actin filaments--not microtubules--can be detected between the nuclear envelope and the Golgi bodies in both BFA-treated and untreated cells. These observations suggest that actin filaments mediate the BFA-induced retrograde transport of vesicles. This mechanism differs from that found in mammalian cells, in which microtubules mediate BFA-induced retrograde transport by the elongation of membrane tubules from the Golgi cisternae. We also discuss the non-participation of the cytoskeleton in anterograde transport from the nuclear envelope to the Golgi bodies.

The role of the Golgi apparatus during terminal differentiation of mouse urothelial surface cells.

The development of the Golgi apparatus in the surface cells of mouse urinary bladder during embryonic development was investigated by electronmicroscopic cytochemistry. The distributions of NADPase and TPPase activities were studied in the urinary bladder during day 15 to day 18 of gestation. At the early embryonic stage, the products of the NADPase and TPPase reactions were visible exclusively in 1 to 2 medial and/or trans Golgi saccules. The strongest increment of NADPase and TPPase positive Golgi cisternae was detected at day 17 when the activity of the urothelial cells was very prominent. At this age, NADPase activity was detected also in lysosomes and on the apical surface of the urothelial cells. The highest distribution pattern of NADPase and TPPase activities observed at this stage rapidly decreases at day 18 of fetal life. The results suggest that the organization of the Golgi apparatus reflected the intensity of the processes occuring in the urothelial cells during gestation.

Ultrastructural and cytochemical studies of the spermatozoa of Acrosternum aseadum (Hemiptera: Pentatomidae) after copulation.

Beside the morphofunctional modifications undergone during spermiogenesis, the spermatozoon could undergo other modifications after copulation. Since no structural modification occurs in the spermatozoon of Acrosternum aseadum after copulation, we used cytochemical studies to show the enzymatic activities variations of acid phosphatase, thiamine pyrophosphatase, glucose-6-phosphatase and cytochrome C oxidase, when the spermatozoon passes through the spermatheca. The enzymatic activity, few hours after copulation, is strong and specifically located. However, 40 h after copulation, there is considerable loss of enzymatic activity, with the exception of thiamine pyrophosphatase, which shows the same activity. This result indicates that the spermatozoon of A. aseadum undergoes physiological modifications when passing through the spermatheca and that these modifications may be involved with survival in this organ as well as with the fertilization process.

Molecular cloning and expression of a mouse thiamin pyrophosphokinase cDNA.

Thiamin pyrophosphokinase (EC 2.7.6.2) catalyzes the pyrophosphorylation of thiamin with adenosine 5'-triphosphate to form thiamin pyrophosphate. A mouse thiamin pyrophosphokinase cDNA clone (mTPK1) was isolated using a combination of mouse expressed sequence tag database analysis, a two-step polymerase chain reaction procedure, and functional complementation screening with a Saccharomyces cerevisiae thiamin pyrophosphokinase-deficient mutant (thi80). The predicted protein contained 243 amino acid residues with a calculated molecular weight of 27,068. When the intact mTPK1 open reading frame was expressed as a glutathione S-transferase fusion protein in Escherichia coli lacking thiamin pyrophosphokinase, marked enzyme activity was detected in the bacterial cells. The corresponding 2.5-kilobase pair mRNA was expressed in a tissue-dependent manner and was found at relatively high levels in the kidney and liver, indicating that the mode of expression of mTPK1 genes differs with cell type. The expression of mTPK1 genes in cultured mouse neuroblastoma and normal liver cells was unaffected by the thiamin concentration in the medium (10 microM versus 3.0 nM). This is the first report on identification of the primary sequence for mammalian thiamin pyrophosphokinase.