Enrichment of grape berries and tomato fruit with health-promoting tartaric acid by expression of the Vitis vinifera transketolase VvTK2 gene.

Tartaric acid (TA) is a major non-fermentable plant soluble acid that abundantly occur in grapes and wines, imparting low pH and tart flavour to berries thereby regulating numerous quality attributes of wine, such as flavour, microbial stability, and aging potential. Evaluation of acidity in mature fruits of 21 wine grape (Vitis vinifera) varieties revealed significant variation between 'Beichun' and 'Gewürztraminer', which was correlated with TA content. RNA-seq analysis of fruits from the two cultivars at different developmental stages revealed that a transketolase gene, VvTK2, was significantly dominantly expressed in the high TA phenotype 'Beichun' variety. Subcellular localization assay showed that VvTK2 protein was located in the chloroplast. Virus-induced VvTK2 gene silencing significantly decreased the expression of 2-keto-L-gulonic acid reductase (Vv2-KGR) as well as L-idonate dehydrogenase (VvL-IdnDH3) and inhibited TA accumulation, while its transient over-expression in grape showed the opposite results. Heterologous VvTK2 over-expression in tomato demonstrated its obvious capacity to induce TA synthesis. Overall, these results highlights a novel role of VvTK2 in modulating TA biosynthesis, which could be an excellent strategy for future genetic improvement of grape flavour.

TKTL1 as a Prognostic Marker in Pancreatic Ductal Adenocarcinoma and Its Correlation with FDG-PET-CT.

INTRODUCTION: Glucose metabolism in cancer cells differs from noncancerous cells. The expression of transketolase-like protein 1 (TKTL1), a key enzyme in the glucose metabolism of cancer cells, predicts poor prognosis in several cancer types. We studied TKTL1 as a prognostic tool and whether TKTL1 expression correlates with 18F-FDG-PET-CT among patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: This retrospective study examined two PDAC patient cohorts: 168 patients operated on at Helsinki University Hospital between 2001 and 2011, and 20 patients with FDG-PET-CT results available from the Auria Biobank. We used immunohistochemistry for TKTL1 expression, combining results with clinicopathological data. RESULTS: Five-year disease-specific survival (DSS) was slightly but not significantly better in patients with a high versus low TKTL1 expression, with DSS of 28.0 versus 17.3%, respectively (p = 0.123). TKTL1 served as a marker of a better prognosis in patients over 65 years old (p = 0.012) and among those with TNM class M1 (p = 0.018), stage IV disease (p = 0.027), or perivascular invasion (p = 0.008). CONCLUSIONS: Our study shows that TKTL1 cannot be used as a prognostic factor in PDAC with the exception of elderly patients and those with advanced disease. The correlation of TKTL1 with 18F-FDG-PET-CT requires further study in a larger patient cohort.

Erythrocyte transketolase deficiency in patients suffering from Crohn's disease.

OBJECTIVE: Aim of the study was to assess the possible vitamin B1 deficiency in relation to the exacerbation of Crohn's disease (CD) in adult patients. PATIENTS AND METHODS: Forty-nine Crohn's disease (CD) patients with different disease activity (The Crohn's Disease Activity Index-CDAI) were included in the study. Anthropometrical and biochemical parameters, i.e., high sensitive C-reactive protein, were assessed. The spectrophotometric method was used to measure the transketolase activity (TK) in erythrocytes. The normalized transketolase activity ratio (NTKZ) and the percentage of activation with thiamine pyrophosphate (%TPP) were also evaluated. RESULTS: The mean values of BMI were close to cut-off: 18.5 kg/m2, indicating a poor nutritional status in CD patients. The patients with moderate-to-severe active CD had a statistically significant higher value of CDAI and hsCRP concentrations compared to those being in the asymptomatic remission or at the mildly active stage of the disease. The level of NTKZ and %TPP were statistically different between the analyzed groups, showing the deficit of vitamin B1 in the group of moderate-to-severe active CD patients (Mean ± SD; NTKZ: 1.99 ± 0.87 vs. 1.54 ± 0.62 U/g Hb; % of TPP: 0.15 ± 0.78 vs. 54.90 ± 38.80). CONCLUSIONS: Vitamin B1 deficiency is part of the Crohn's disease manifestation in moderate-to-severe active patients.

Identification of transketolase as a target of PARIS in substantia nigra.

Recently, PARIS (ZNF746) is introduced as authentic substrate of parkin and transcriptionally represses PGC-1α by binding to insulin responsive sequences (IRSs) in the promoter of PGC-1α. The overexpression of PARIS selectively leads to the loss of dopaminergic neurons (DN) and mitochondrial abnormalities in the substantia nigra (SN) of Parkinson's disease (PD) models. To identify PARIS target molecules altered in SN region-specific manner, LC-MS/MS-based quantitative proteomic analysis is employed to investigate proteomic alteration in the cortex, striatum, and SN of AAV-PARIS injected mice. Herein, we find that the protein and mRNA of transketolase (TKT), a key enzyme in pentose phosphate pathway (PPP) of glucose metabolism, is exclusively decreased in the SN of AAV-PARIS mice. PARIS overexpression suppresses TKT transcription via IRS-like motif in the TKT promoter. Moreover, the reduction of TKT by PARIS is found in primary DN but not in cortical neurons, suggesting that PARIS-medicated TKT suppression is cell type-dependent. Interestingly, we observe the reduced level of TKT in the SN of PD patients but not in the cortex. These findings indicate that TKT might be a SN-specific target of PARIS, providing new clues to understand the mechanism underlying selective DNs death in PD.

Implantação de um método para determinação da atividade da transcetolase eritrocitária para avaliação indireta da tiamina / Implementation of a method for determining the erythrocyte transketolase activity for indirect evaluation of thiamin

O teste de ativação da transcetolase eritrocitária (TK-E) pelo pirofosfato de tiamina (TPP) exógeno é um método indireto para mensurar a tiamina (vitamina B1). A diminuição da atividade da transcetolase eritrocitária e o aumento da estimulação in vitro com o TPP maior do que 17 % indicam deficiência de tiamina. Este é um método plausível, pois são nos eritrócitos que estão concentradas a maior parte desta vitamina. Em virtude de surtos de beribéri que tem ocorrido no Brasil desde 2006, o Instituto Adolfo Lutz (IAL), como Laboratório Central de Saúde Pública, propôs a implantação desse método para auxiliar na investigação de novos surtos ou de casos isolados. Foram avaliados o teste de precisão, a linearidade, a estabilidade do hemolisado e da amostra, e estimados os limites de detecção e de quantificação. A atividade da TK-E sem ativação pelo TPP foi de 0,732 UI/gHb e com ativação foi de 0,827 UI/gHb. Todos os resultados dos parâmetros avaliados neste estudo apresentaram-se dentro dos critérios de aceitabilidade garantindo-se a confiabilidade do método. Fica, assim, disponível mais um ensaio bioquímico para a Rede Pública de Saúde, mas ainda necessário definir os valores de referência para estabelecer os limites clínicos da deficiência de tiamina.

Erythrocyte transketolase activation test (TK-E) by exogenous thiamine pyrophosphate (TPP) is an indirect method to measure thiamine (vitamin B1). The decrease in the erythrocyte transketolase activity and the increase of in vitro stimulation with TPP greater than 17 % indicate thiamine deficiency. It is a reasonable method as the major portion of this vitamin are concentrated in erithrocytes. Due to the beriberi outbreaks that have occurred in Brazil since 2006, the Adolfo Lutz Institute (IAL), as a Central Public Health Laboratory, proposed the implementation of this method to give support to the investigation on the new outbreaks or isolated cases. The evaluated parameters were precision, linearity, hemolysate and sample stability, and the limits of detection and quantification were estimated. The TK-E activity without activation by TPP was 0.732 UI/gHb, and with activation was 0.827 UI/gHb. All of the results obtained from the evaluated parameters showed to be within the eligibility criteria, ensuring the reliability of the proposed methods. Thus, this method showed to be adequate as biochemical assay for the Public Health Network. However, there is a need to define the reference values to establish the clinical limits of thiamine deficiency.

Electrochemical detection of transketolase activity using a tyrosinase biosensor.

This paper proposes a new concept of transketolase (TK) activity profiling. A tyrosinase (PPO) biosensor, based on the immobilization of this enzyme in a Mg(2)Al-Cl layered double hydroxide, was developed for the amperometric detection of N-acetyl-l-tyrosine ethyl ester monohydrate (N-Ac-Tyr-OEt) at -0.2V. This compound was released during an enzymatic reaction catalyzed by TK with N-acetyl-O-(2R, 3S, 5-trihydroxy-4-oxopentyl)-l-tyrosine ethyl ester used as donor substrate. This tyrosinase biosensor was optimized for the detection of TK activity, including PPO optimum substrate concentration, electrolyte nature, pH, and influence of bovine serum albumin (BSA). It was found that N-Ac-Tyr-OEt release is dependent on TK concentration (U/mL) in the electrolyte medium. These results demonstrate the sensitivity and specificity of the tyrosinase biosensor designed for in vitro detection of TK activity, which is known to be involved in several diseases.

Lactate-dehydrogenase 5 is overexpressed in non-small cell lung cancer and correlates with the expression of the transketolase-like protein 1.

AIMS: As one of the five lactate dehydrogenase (LDH) isoenzymes, LDH5 has the highest efficiency to catalyze pyruvate transformation to lactate. LDH5 overexpression in cancer cells induces an upregulated glycolytic metabolism and reduced dependence on the presence of oxygen. Here we analyzed LDH5 protein expression in a well characterized large cohort of primary lung cancers in correlation to clinico-pathological data and its possible impact on patient survival. METHODS: Primary lung cancers (n = 269) and non neoplastic lung tissue (n = 35) were tested for LDH5 expression by immunohistochemistry using a polyclonal LDH5 antibody (ab53010). The results of LDH5 expression were correlated to clinico-pathological data as well as to patient's survival. In addition, the results of the previously tested transketolase like 1 protein (TKTL1) expression were correlated to LDH5 expression. RESULTS: 89.5% (n = 238) of NSCLC revealed LDH5 expression whereas LDH5 expression was not detected in non neoplastic lung tissues (n = 34) (p < 0.0001). LDH5 overexpression was associated with histological type (adenocarcinoma = 57%, squamous cell carcinoma = 45%, large cell carcinoma = 46%, p = 0.006). No significant correlation could be detected with regard to TNM-stage, grading or survival. A two sided correlation between the expression of TKTL1 and LDH5 could be shown (p = 0.002) within the overall cohort as well as for each grading and pN group. A significant correlation between LDH5 and TKTL1 within each histologic tumortype could not be revealed. CONCLUSIONS: LDH5 is overexpressed in NSCLC and could hence serve as an additional marker for malignancy. Furthermore, LDH5 correlates positively with the prognostic marker TKTL1. Our results confirm a close link between the two metabolic enzymes and indicate an alteration in the glucose metabolism in the process of malignant transformation.

Molecular-assisted immunohistochemical optimization.

Immunohistochemistry (IHC) is an essential tool in diagnostic surgical pathology, allowing analysis of protein subcellular localization. The use of IHC by different laboratories has lead to inconsistencies in published literature for several antibodies, due to either interpretative (inter-observer variation) or technical reasons. These disparities have major implications in both clinical and research settings. In this study, we report our experience conducting an IHC optimization of antibodies against five proteins previously identified by proteomic analysis to be breast cancer biomarkers, namely 6PGL (PGLS), CAZ2 (CAPZA2), PA2G4 (EBP1) PSD2 and TKT. Large variations in the immunolocalizations and intensities were observed when manipulating the antigen retrieval method and primary antibody incubation concentration. However, the use of an independent molecular analysis method provided a clear indication in choosing the appropriate biologically and functionally relevant "staining pattern". Without this latter step, each of these contradictory results would have been a priori "technically acceptable" and would have led to different biological and functional interpretations of these proteins and potentially different applications in a routine pathology setting. Thus, we conclude that full validation of immunohistochemical protocols for scientific and clinical use will require the incorporation of biological knowledge of the biomarker and the disease in question.

Subcellular localization and enzymatic properties of differentially expressed transketolase genes isolated from the desiccation tolerant resurrection plant Craterostigma plantagineum.

The desiccation tolerant resurrection plant Craterostigma plantagineum encodes three classes of transketolase transcripts, which are distinguished by their gene structures and their expression patterns. One class, represented by tkt3, is constitutively expressed and two classes, represented by tkt7 and tkt10, are upregulated upon rehydration of desiccated C. plantagineum plants. The objective of this work was to characterize the differentially expressed transketolase isoforms with respect to subcellular localization and enzymatic activity. Using GFP fusion constructs and enzymatic activity assays, we demonstrate that C. plantagineum has novel forms of transketolase which localize not to the chloroplast, but mainly to the cytoplasm and which are distinct in the enzymatic properties from the transketolase enzymes active in the Calvin cycle or oxidative pentose phosphate pathway. A transketolase preparation from rehydrated leaves was able to synthesize the unusual C8 carbon sugar octulose when glucose-6-phosphate and hydroxy-pyruvate were used as acceptor and donor molecules in in vitro assays. This suggests that a transketolase catalyzed reaction is likely to be involved in the octulose biosynthesis in C. plantagineum.

A new erythrose 4-phosphate dehydrogenase coupled assay for transketolase.

The standard assay for transketolase (E.C 2.2.1.1) has depended upon the use of D-xylulose 5-phosphate as the ketose donor substrate since the production of D-glyceraldehyde 3-phosphate can be readily coupled to a reaction that consumes NADH allowing the reaction to be followed spectrophotometrically. Unfortunately, commercial supplies of D-xylulose 5-phosphate recently became unavailable. In this article we describe the coupling of a transketolase reaction (using Leishmania mexicana transketolase) that converts D-fructose 6-phosphate to D-erythrose 4-phosphate. D-Erythrose 4-phosphate can then be converted to 4-phosphate D-erythronate using erythrose-4-phosphate dehydrogenase (E.C 1.2.1.72), a reaction that reduces NAD+ to NADH and can be easily followed spectrophotometrically. D-Ribose 5-phosphate and D-glyceraldehyde 3-phosphate can both be used as ketol acceptor substrates in the reaction although D-ribose 5-phosphate is also a substrate for the coupling enzyme.

A novel assay system for the measurement of transketolase activity using xylulokinase from Saccharomyces cerevisiae.

The conventional method of transketolase (TKT) activity assay uses ribose 5-phosphate and xylulose 5-phosphate as substrates. However, a new method of TKT assay is currently required since xylulose 5-phosphate is no longer commercially available and is difficult to synthesize chemically. Although there are effective assays for TKT using non-natural substrates, these are inadequate for evaluating changes in enzyme activity and affinity toward real substrates. As a solution to such problems, we describe a novel assay system using xylulokinase (XK) from Saccharomyces cerevisiae. As for this purpose, the XK was overexpressed in E. coli, separated and purified in a single step, added to induce a reaction that generated xylulose 5-phosphate, which was integrated into the conventional TKT assay. The new coupling assay gave reproducible results with E. coli TKT and had a detection limit up to 5 x 10(-4)unit/mg protein. A reliable result was also achieved for the incorporation of XK and TKT into a single reaction.

Optimisation and evaluation of a generic microplate-based HPLC screen for transketolase activity.

A microplate-based HPLC assay for transketolase is described for rapidly determining substrate and product concentration suitable for optimisation of biocatalytic process conditions and screening directed evolution libraries. Transketolase catalyses the enantioselective carbon-carbon bond formation of chiral keto-diol products. The assay was used to determine dissociation constants for the two cofactors required by transketolase with 5-11% error. The preparation of samples by microplate-based fermentation, cell lysis, addition of cofactor, addition of substrates was also evaluated and optimised for increased transketolase activity. The whole process enables 3-fold improved enzyme variants to be identified from a single measurement.

Transketolase protein TKTL1 overexpression: A potential biomarker and therapeutic target in breast cancer.

Malignant tumors degrade glucose to lactate even in the presence of oxygen via the pentose phosphate pathway (ppp). The non-oxidative part of the ppp is controlled by thiamine-dependant transketolase enzyme reactions. Overexpression of the transketolase-like-1-gene (TKTL1) in urothelial and colorectal cancer is associated with poor patient outcome. We analyzed the expression of the TKTL1 protein in a retrospective institution-based patient cohort with invasive breast cancer by immunohistochemical analysis of 124 paraffin-embedded breast cancer tissues. Our study revealed TKTL1 expression in 86% of breast cancer specimens with 45% showing high expression levels. In contrast, only 29% of corresponding non-neoplastic breast tissues were TKTL1 immunopositive, including 9% with high expression levels. High expression levels of TKTL1 correlated significantly to Her2/neu overexpression (p=0.015). However, TKTL1 expression failed to reach statistical significance for other common prognostic parameters. In contrast to recent data for e.g. colorectal cancer TKTL1 overexpression did not correlate to patient outcome and survival. However, in the context of novel insights into TKTL1-related tumor metabolism and the high proportion of TKTL1 overexpressing breast cancers, this enzyme represents a potential candidate for targeted inhibition of tumor growth in this tumor entity.

Avitaminosis B1, actividad de la transcetolasa y efecto TPP eritrocitarios / No disponible

No disponible

Aldehyde dehydrogenase (ALDH) 3A1 expression by the human keratocyte and its repair phenotypes.

Transparency is essential for normal corneal function. Recent studies suggest that corneal cells express high levels of so-called corneal crystallins, such as aldehyde dehydrogenase (ALDH) and transketolase (TKT) that contribute to maintaining cellular transparency. Stromal injury leads to the appearance of repair phenotype keratocytes, the corneal fibroblast and myofibroblast. Previous studies on keratocytes from species such as bovine and rabbit indicate that the transformation from the normal to repair phenotype is accompanied by a loss of corneal crystallin expression, which may be associated with loss of cellular transparency. Here we investigated if a similar loss occurs with human keratocyte repair phenotypes. Human corneal epithelial cells were collected by scraping and keratocytes were isolated by collagenase digestion from cadaveric corneas. The cells were either processed immediately (freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. RT-PCR, western blotting and immunolabeling were used to detect mRNA and protein expression of ALDH isozymes and TKT. ALDH enzyme activity was also quantitated and immunolabeling was performed to determine the expression of ALDH3A1 in human corneal tissue sections from normal and diseased corneas. Human corneal keratocytes isolated from three donors expressed ALDH1A1 and ALDH3A1 mRNA, and one donor also expressed ALDH2 and TKT. Corneal epithelial cells expressed ALDH1A1, ALDH2, ALDH3A1 and TKT. Compared to normal keratocytes, corneal fibroblast expression of ALDH3A1 mRNA was reduced by 27% (n=5). ALDH3A1 protein expression as detected by western blotting was markedly reduced in passage zero fibroblasts and undetectable in higher passages (n=3). TKT protein expression was reduced in fibroblasts compared to keratocytes (n=2). ALDH3A1 enzyme activity was not detectable in corneal fibroblasts (n=6) but was readily detected in corneal epithelial cells (0.29+/-0.1U/mg protein, n=4) and keratocytes (0.05+/-0.009U/mg protein, n=7). ALDH3A1 expression was also reduced in corneal fibroblasts and myofibroblasts as determined by immunolabeling of the cells in culture (n=3) and in diseased corneal tissues in situ (n=2). We conclude that expression of the crystallin ALDH3A1 is decreased in repair phenotype human keratocytes, compared to normal human keratocytes. Extrapolating from studies of bovine and rabbit, the reduced expression of ALDH3A1 may contribute to the loss of corneal transparency experienced by human patients after injury and refractive surgeries.

Thiamine deficiency results in downregulation of the GLAST glutamate transporter in cultured astrocytes.

Pyrithiamine-induced thiamine deficiency (TD) is a well-established model of Wernicke's encephalopathy in which a glutamate-mediated excitotoxic mechanism may play an important role in determining selective vulnerability. In order to examine this possibility, cultured astrocytes were exposed to TD and effects on glutamate transport and metabolic function were studied. TD led to decreases in cellular levels of thiamine and thiamine diphosphate (TDP) after 24 h of treatment and decreased activities of the TDP-dependent enzymes alpha-ketoglutarate dehydrogenase and transketolase after 4 and 7 days, respectively. TD treatment for 10 days led to a reversible decrease in the uptake of [(3)H]-D-aspartate, a nonmetabolizable analogue of glutamate. Kinetic analysis revealed that the uptake inhibition was caused by a 47% decrease in the V(max) for uptake of [(3)H]-D-aspartate, with no change in the K(m) value. Immunoblotting showed that this decrease in uptake was due to an 81% downregulation of the astrocyte-specific GLAST glutamate transporter. Loss of uptake activity and GLAST protein were blocked by treatment with the protein kinase C inhibitor H7, while exposure to DCG IV, a group II metabotropic glutamate receptor (mGluR) agonist, resulted in improvement of [(3)H]-D-aspartate uptake and a partial reversal of transporter downregulation. These results are consistent with our recent in vivo findings of a loss of astrocytic glutamate transporters in TD and provide evidence that TD conditions may increase phosphorylation of GLAST, contributing to its downregulation. In addition, manipulation of group II mGluR activity may provide an important strategy in the treatment of this disorder.

Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization-time of flight-mass spectrometry: II. Biological applications.

A novel disposable high-density matrix assisted laser desorption/ionization (MALDI) target plate made either from polymethylmethacrylate (PMMA) or polycarbonate (PC) is presented where thousands (1,200-1,600) of samples can be deposited and subsequently analyzed by MALDI-time of flight (TOF) mass spectrometry. Good reproducibility was obtained across the plate regardless of position on the target plate with a relative standard deviation (RSD) on the peak intensity of typically 30% calculated from data generated by analysis of a 10 nm peptide mixture of angiotensin I, II, III and bradykinin. The nanovial array format combined with microdispensing technology makes it possible to carry out in-vial chemistry on deposited samples. This is demonstrated by the analysis of peptides from beta-casein and subsequent in-vial dephosphorylation of its phosphopeptides at 10 fmol levels by microdispensing of alkaline phosphatase, into the nanovial. The mass spectra obtained from these polymeric targets provides can also be used in high sensitivity applications as shown by peptide mass fingerprinting of human fibroblast proteins separated by two-dimensional gel electrophoresis.

Differential expression of cytosolic proteins in the rat kidney cortex and medulla: preliminary proteomics.

The rodent kidney is a target of many xenobiotics and is typified by regionally specific structure and function. This renders distinct regions of the kidney differentially susceptible to toxic exposure and effect. To characterize these differences at the proteome level, protein patterns from male rat kidney cortex and medulla cytosols were examined by two-dimensional electrophoresis (2-DE) and image analysis and prominent proteins identified immunologically or by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and electrospray/ionization-tandem mass spectrometry (ESI-MS/MS) sequence tag identification. An average of 727 protein spots were resolved and matched to the cortex cytosol reference pattern, and 716 in the medulla. Of this total, 127 proteins were found to differ in abundance (86 higher in cortex; 41 higher in medulla) (P < 0.001). Of those proteins that were detectable in both cortex and medulla, the abundance of 97 differed significantly while 30 proteins were found to be unique to one region or the other (26 in cortex, 4 in medulla). Twenty protein spots were identified and their regional differences are discussed. These results both confirm and expand our understanding of the molecular heterogeneity characterizing structurally and functionally distinct regions of the kidney and serve as a useful foundation for future nephrotoxicologic studies.

Transketolase is a major protein in the mouse cornea.

Earlier experiments in this laboratory identified a highly expressed 65-68-kDa protein in both mouse and human corneas (Cuthbertson, R. A. , Tomarev, S. I., and Piatigorsky J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4004-4008). Here, we demonstrate that this protein is transketolase (TKT; EC 2.2.1.1), an enzyme in the nonoxidative branch of the pentose-phosphate pathway, based on peptide and cDNA isolation and sequence analysis of mouse cornea protein and RNA samples, respectively. While expressed at low levels in a number of tissues, the 2.1-kilobase TKT mRNA was expressed at a 50-fold higher level in the adult mouse cornea. The area of most abundant expression was localized to the cornea epithelial cell layer by in situ hybridization. Western blot analysis confirmed TKT protein abundance in the cornea and indicated that TKT may comprise as much as 10% of the total soluble protein of the adult mouse cornea. Soluble cornea extracts exhibited a correspondingly high level of TKT enzymatic activity. TKT expression increased progressively through cornea maturation, as shown by Northern blot, in situ hybridization, Western blot, and enzymatic analyses. TKT mRNA and protein were expressed at low levels in the cornea prior to eye opening, while markedly increased levels were observed after eye opening. Taken together, these observations suggest that TKT may be a cornea enzyme-crystallin, and suggest that the crystallin paradigm and concept of gene sharing, once thought to be restricted to the lens, apply to other transparent ocular tissues.

Characteristic transketolase alterations in dermal fibroblasts of Alzheimer patients are modulated by culture conditions.

Cultured Alzheimer fibroblasts were found to present peculiar alterations of transketolase (TK) ascribed to enhanced proteolytic activities in these cells and tentatively proposed as a marker of the disease. TK abnormalities, consisting of enzyme forms (alkaline bands) with unusually high alkaline pI, were investigated with respect to the mechanism of their generation and modulation by culture conditions. Alzheimer fibroblasts propagated at different pH, within a range of 7.3-7.8, exhibited TK abnormalities whose expression correlated directly with increases in medium pH values. Alterations were mostly evident in cells grown at 5% CO2 saturation in the atmosphere and with 3.7 g/liter NaHCO3 in the medium to yield an initial pH of about 7.75. Alkaline bands were not detected in either Alzheimer fibroblasts incubated at 10% CO2 or in control cells under any of the other conditions tested. Changes in initial medium pH also affected the morphology of fibroblasts, which shifted from a relatively large to an elongated shape as the medium pH decreased. The formation of alkaline bands was abolished by the addition of E-64, a known cysteine protease inhibitor, to cells just prior to extraction. On the contrary, Alzheimer fibroblasts cultured for 2 days in the presence of the inhibitor maintained the typically altered TK pattern. The establishment of conditions suitable for the expression of TK alterations in Alzheimer fibroblasts might be of help for diagnostic purposes and provide information on still elusive pathogenetic mechanisms of the disease.