Effect of acarbose on biochemical responses and clinical symptoms in dumping syndrome

A dose of 50 mg of acarbose was administered with a standard breakfast to 13 subjects with dumping syndrome. Significant attenuation of hyperglycaemia (p less than 0.01) was observed, and rises in plasma gastric inhibitory polypeptide, insulin and enteroglycagon were reduced (p less than 0.05). Plasma levels of neurotensin, vasoactive intestinal polypeptide and somatostatin were not affected. Dumping score was reduced, but this did not achieve statistical significance. In a longer-term study, 9 patients took acarbose, 50 mg t.i.d., for 1 month. No significant reduction in the number or severity of dumping attacks was observed, but a majority expressed a preference for the drug and some individuals experienced a marked improvement of symptoms.

Triose phosphate isomerase from the blood fluke Schistosoma mansoni:Biochemical characterisation of a potential drug and vaccine target

The glycolytic enzyme triose phosphate isomerase from Schistosoma mansoni is a potential target for drugs and vaccines. Molecular modelling of the enzyme predicted that a Ser-Ala-Asp motif which is believed to be a helminth-specific epitope is exposed. The enzyme is dimeric (as judged by gel filtration and cross-linking), resistant to proteolysis and highly stable to thermal denaturation (melting temperature of 82.0°C). The steady-state kinetic parameters are high (Km for dihydroxyacetone phosphate is 0.51mM; Km for glyceraldehyde 3-phosphate is 1.1mM; kcat for dihydroxyacetone phosphate is 7800s(-1) and kcat for glyceraldehyde 3-phosphate is 6.9s(-1)).

Biochemical characterisation of triose phosphate isomerase from the liver fluke Fasciola hepatica

Triose phosphate isomerase (TPI) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, a reaction in the glycolytic pathway. TPI from the common liver fluke, Fasciola hepatica, has been cloned, sequenced and recombinantly expressed in Escherichia coli. The protein has a monomeric molecular mass of approximately 28 kDa. Crosslinking and gel filtration experiments demonstrated that the enzyme exists predominantly as a dimer in solution. F. hepatica TPI is predicted to have a ß-barrel structure and key active site residues (Lys-14, His-95 and Glu-165) are conserved. The enzyme shows remarkable stability to both proteolytic degradation and thermal denaturation. The melting temperature, estimated by thermal scanning fluorimetry, was 67 °C and this temperature was increased in the presence of either dihydroxyacetone phosphate or glyceraldehyde 3-phosphate. Kinetic studies showed that F. hepatica TPI demonstrates Michaelis-Menten kinetics in both directions, with Km values for dihydroxyacetone phosphate and glyceraldehyde 3-phosphate of 2.3 mM and 0.66 mM respectively. Turnover numbers were estimated at 25,000 s(-1) for the conversion of dihydroxyacetone phosphate and 1900 s(-1) for the conversion of glyceraldehyde 3-phosphate. Phosphoenolpyruvate acts as a weak inhibitor of the enzyme. F. hepatica TPI has many features in common with mammalian TPI enzymes (e.g. ß-barrel structure, homodimeric nature, high stability and rapid kinetic turnover). Nevertheless, recent successful identification of specific inhibitors of TPI from other parasites, suggests that small differences in structure and biochemical properties could be exploited in the development of novel, species-specific inhibitors.

Biochemical and physiochemical characteristics of a pre-B cell stimulating factor produced by the plasmacytoma cell line LICR LON HMY2

We have characterized the pre-B cell colony stimulating activity (pre-B cell CSA) from LICR LON HMY2 conditioned medium (CM) by a variety of biochemical techniques. Pre-B cell CSA was found to be associated with a heat stable glycoprotein which has an isoelectric point of 8.3 and a mol. wt, as determined by polyacrylamide gel electrophoresis, of 28-32 kD. The relationship of this activity to previously described factors acting on cells of the B cell lineage is discussed.

Prostate stereotactic ablative body radiotherapy using a standard linear accelerator:toxicity, biochemical, and pathological outcomes

Biological dose escalation through stereotactic ablative radiotherapy (SABR) holds promise of improved patient convenience, system capacity and tumor control with decreased cost and side effects. The objectives are to report the toxicities, biochemical and pathologic outcomes of this prospective study.

Biochemical characterisation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the liver fluke, Fasciola hepatica

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses one of the two steps in glycolysis which generate the reduced coenzyme NADH. This reaction precedes the two ATP generating steps. Thus, inhibition of GAPDH will lead to substantially reduced energy generation. Consequently, there has been considerable interest in developing GAPDH inhibitors as anti-cancer and anti-parasitic agents. Here, we describe the biochemical characterisation of GAPDH from the common liver fluke Fasciola hepatica (FhGAPDH). The primary sequence of FhGAPDH is similar to that from other trematodes and the predicted structure shows high similarity to those from other animals including the mammalian hosts. FhGAPDH lacks a binding pocket which has been exploited in the design of novel antitrypanosomal compounds. The protein can be expressed in, and purified from Escherichia coli; the recombinant protein was active and showed no cooperativity towards glyceraldehyde 3-phosphate as a substrate. In the absence of ligands, FhGAPDH was a mixture of homodimers and tetramers, as judged by protein-protein crosslinking and analytical gel filtration. The addition of either NAD(+) or glyceraldehyde 3-phosphate shifted this equilibrium towards a compact dimer. Thermal scanning fluorimetry demonstrated that this form was considerably more stable than the unliganded one. These responses to ligand binding differ from those seen in mammalian enzymes. These differences could be exploited in the discovery of reagents which selectively disrupt the function of FhGAPDH.

The two common polymorphic forms of human NRH-quinone oxidoreductase 2 (NQO2) have different biochemical properties

There are two common forms of NRH-quinone oxidoreductase 2 (NQO2) in the human population resulting from SNP rs1143684. One has phenylalanine at position 47 (NQO2-F47) and the other leucine (NQO2-L47). Using recombinant proteins, we show that these variants have similar steady state kinetic parameters, although NQO2-L47 has a slightly lower specificity constant. NQO2-L47 is less stable towards proteolytic digestion and thermal denaturation than NQO2-F47. Both forms are inhibited by resveratrol, but NQO2-F47 shows negative cooperativity with this inhibitor. Thus these data demonstrate, for the first time, clear biochemical differences between the variants which help explain previous biomedical and epidemiological findings. © 2014 Federation of European Biochemical Societies.

Comparative biochemical analysis of three members of the Schistosoma mansoni TAL family: Differences in ion and drug binding properties

The tegumental allergen-like (TAL) proteins from Schistosoma mansoni are part of a family of calcium binding proteins found only in parasitic flatworms. These proteins have attracted interest as potential drug or vaccine targets, yet comparatively little is known about their biochemistry. Here, we compared the biochemical properties of three members of this family: SmTAL1 (Sm22.6), SmTAL2 (Sm21.7) and SmTAL3 (Sm20.8). Molecular modelling suggested that, despite similarities in domain organisation, there are differences in the three proteins’ structures. SmTAL1 was predicted to have two functional calcium binding sites and SmTAL2 was predicted to have one. Despite the presence of two EF-hand-like structures in SmTAL3, neither was predicted to be functional. These predictions were confirmed by native gel electrophoresis, intrinsic fluorescence and differential scanning fluorimetry: both SmTAL1 and SmTAL2 are able to bind calcium ions reversibly, but SmTAL3 is not. SmTAL1 is also able to interact with manganese, strontium, iron(II) and nickel ions. SmTAL2 has a different ion binding profile interacting with cadmium, manganese, magnesium, strontium and barium ions in addition to calcium. All three proteins form dimers and, in contrast to some Fasciola hepatica proteins from the same family; dimerization is not affected by calcium ions. SmTAL1 interacts with the anti-schistosomal drug praziquantel and the calmodulin antagonists trifluoperazine, chlorpromazine and W7. SmTAL2 interacts only with W7. SmTAL3 interacts with the aforementioned calmodulin antagonists and thiamylal, but not praziquantel. Overall, these data suggest that the proteins have different biochemical properties and thus, most likely, different in vivo functions.

Nestor-Guillermo Progeria Syndrome: a biochemical insight into Barrier-to-Autointegration Factor 1, alanine 12 threonine mutation

Background: Premature aging syndromes recapitulate many aspects of natural aging and provide an insight into this phenomenon at a molecular and cellular level. The progeria syndromes appear to cause rapid aging through disruption of normal nuclear structure. Recently, a coding mutation (c.34G > A [p.A12T]) in the Barrier to Autointegration Factor 1 (BANF1) gene was identified as the genetic basis of Nestor-Guillermo Progeria syndrome (NGPS). This mutation was described to cause instability in the BANF1 protein, causing a disruption of the nuclear envelope structure.

Results: Here we demonstrate that the BANF1 A12T protein is indeed correctly folded, stable and that the observed phenotype, is likely due to the disruption of the DNA binding surface of the A12T mutant. We demonstrate, using biochemical assays, that the BANF1 A12T protein is impaired in its ability to bind DNA while its interaction with nuclear envelope proteins is unperturbed. Consistent with this, we demonstrate that ectopic expression of the mutant protein induces the NGPS cellular phenotype, while the protein localizes normally to the nuclear envelope.

Conclusions: Our study clarifies the role of the A12T mutation in NGPS patients, which will be of importance for understanding the development of the disease.

UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors

Leloir pathway enzyme uridine diphosphate (UDP)-galactose 4'-epimerase from the common liver fluke Fasciola hepatica (FhGALE) was identified and characterized. The enzyme can be expressed in, and purified from, Escherichia coli. The recombinant enzyme is active: the K(m) (470 μM) is higher than the corresponding human enzyme (HsGALE), whereas the k(cat) (2.3 s(-1)) is substantially lower. FhGALE binds NAD(+) and has shown to be dimeric by analytical gel filtration. Like the human and yeast GALEs, FhGALE is stabilized by the substrate UDP-galactose. Molecular modelling predicted that FhGALE adopts a similar overall fold to HsGALE and that tyrosine 155 is likely to be the catalytically critical residue in the active site. In silico screening of the National Cancer Institute Developmental Therapeutics Program library identified 40 potential inhibitors of FhGALE which were tested in vitro. Of these, 6 showed concentration-dependent inhibition of FhGALE, some with nanomolar IC50 values. Two inhibitors (5-fluoroorotate and N-[(benzyloxy)carbonyl]leucyltryptophan) demonstrated selectivity for FhGALE over HsGALE. These compounds also thermally destabilized FhGALE in a concentration-dependent manner. Interestingly, the selectivity of 5-fluoroorotate was not shown by orotic acid, which differs in structure by 1 fluorine atom. These results demonstrate that, despite the structural and biochemical similarities of FhGALE and HsGALE, it is possible to discover compounds which preferentially inhibit FhGALE.