Role of vacuolar transporter proteins in plant secondary metabolism: Catharanthus roseus cell culture

Here the current status of knowledge on some well-characterized transporters located in the vacuolar membrane is reviewed. As different cellular compartments and even different cells may be involved in certain steps of a biosynthetic pathway, the regulation of the flux is not only dependent on structural genes encoding enzymes catabolizing certain steps but also transport has a major regulatory function. The aim of the present review is to give an overview of the present knowledge of transport of secondary metabolites in plants, and to use this information in the context of our knowledge about Catharanthus roseus alkaloid biosynthesis. This should lead to further insight in the possible role of various transporters in the regulation of the biosynthesis of these alkaloids.     

Schiff bases of tryptamine as potent inhibitors of nucleoside triphosphate diphosphohydrolases (NTPDases): Structure-activity relationship

Overexpression of NTPDases leads to a number of pathological situations such as thrombosis, and cancer. Thus, effective inhibitors are required to combat these pathological situations. Different classes of NTPDase inhibitors are reported so far including nucleotides and their derivatives, sulfonated dyes such as reactive blue 2, suramin and its derivatives, and polyoxomatalates (POMs). Suramin is a well-known and potent NTPDase inhibitor, nonetheless, a range of side effects are also associated with it. Reactive blue 2 also had non-specific side effects that become apparent at high concentrations. In addition, most of the NTPDase inhibitors are high molecular weight compounds, always required tedious chemical steps to synthesize. Hence, there is still need to explore novel, low molecular weight, easy to synthesize, and potent NTPDase inhibitors.Keeping in mind the known NTPDase inhibitors with imine functionality and nitrogen heterocycles, Schiff bases of tryptamine, 1–26, were synthesized and characterized by spectroscopic techniques such as EI-MS, HREI-MS, ¹H-, and ¹³C NMR. All the synthetic compounds were evaluated for the inhibitory avidity against activities of three major isoforms of NTPDases: NTPDase-1, NTPDase-3, and NTPDase-8. Cumulatively, eighteen compounds were found to show potent inhibition (Ki = 0.0200–0.350 μM) of NTPDase-1, twelve (Ki = 0.071–1.060 μM) of NTPDase-3, and fifteen compounds inhibited (Ki = 0.0700–4.03 μM) NTPDase-8 activity. As a comparison, the Kis of the standard inhibitor suramin were 1.260 ± 0.007, 6.39 ± 0.89 and 1.180 ± 0.002 μM, respectively. Kinetic studies were performed on lead compounds (6, 5, and 21) with human (h-) NTPDase-1, -3, and -8, and Lineweaver-Burk plot analysis showed that they were all competitive inhibitors. In silico study was conducted on compound 6 that showed the highest level of inhibition of NTPDase-1 to understand the binding mode in the active site of the enzyme.     

Ruminal transport and metabolism of short-chain fatty acids (SCFA) in vitro: effect of SCFA chain length and pH

The unidirectional transport and metabolism of 14C-labeled acetate, propionate and butyrate across the isolated bovine rumen epithelium was measured in vitro by the Ussing chamber technique. There was a significant, but relatively small, net secretion of acetate and propionate, and a large and significant net absorption of butyrate. The results demonstrate that the mucosal-serosal (MS) pathway for short-chain fatty acids (SCFA) is different from the serosal-mucosal (SM) pathway, and that butyrate is treated differently from acetate and propionate by the epithelium. The results support that the main route for epithelial SCFA transport is transcellular. The correlation between SCFA lipophility and the flux rate was positive but weak at both pH 7.3 and 6.0. Decreasing pH increased all SCFA fluxes significantly, but not proportionally to the increase of protonized SCFA in the bathing solution. There was a significant and apparently non-competitive interaction between the transport of acetate, propionate and butyrate. It seems that mediated transport mechanisms must be involved in epithelial SCFA transport in the bovine rumen, but the data do not exclude that passive diffusion could account for a significant part of the flux. The metabolism of SCFA in the Ussing chamber system was considerable, and there was a clear preference for excretion of CO2 from this metabolism to the mucosal side, while side preference for non-CO2 metabolite excretion was not studied. Of the propionate and butyrate transported in the MS direction, 78 and 95% was metabolised, while only 37 and 38% was metabolised in the SM direction (acetate metabolism could not be measured). There was, however, no simple relation between the degree of metabolism and the transport rate or the transport asymmetry of the SCFA.     

Contribution of serum albumin to the transport of orally administered L-tryptophan into liver of rats with L-tryptophan depletion

Summary The role of serum albumin in the transport of orally administered L-tryptophan (Trp) into rat tissues was examined using analbuminemic and Sprague-Dawley (SD) rats with and without a-methyl-DL-tryptophan (AMT)-induced Trp depletion. Trp was orally administered to rats 16h after AMT or 0.85% NaCl administration, when liver tryptophan 2,3-dioxygenase and protein synthetic activities in AMT-treated rats were similar to those of 0.85% NaCl-treated rats. After oral Trp administration, regardless of the presence or absence of Trp depletion, free serum Trp concentrations were similar in the analbuminemic and SD rats, while total serum Trp concentrations were lower in analbuminemic rats than in SD rats. Although liver, brain, and muscle Trp concentrations after oral Trp administration under Trp depletion were lower in analbuminemic rats than in SD rats, the ratio of the liver Trp concentration in analbuminemic rats to that in SD rats was smaller than that of the brain or muscle Trp concentration. These results suggest that variations in serum albumin levels could affect the transport of orally administered Trp into the liver of rats with Trp depletion.     

Effects of Methylphenidate Analogues on Phenethylamine Substrates for the Striatal Dopamine Transporter

Methylphenidate (MPD) was found to inhibit competitively the striatal dopamine transporter (DAT) and bind at sites on the DAT in common with both cocaine (a non-substrate site ligand) and amphetamine (a substrate site ligand). Some methylphenidate analogues modified on the aromatic ring and/or at the nitrogen were tested to determine whether the profile of inhibition could be altered. None was found to stimulate the release of dopamine in the time frame (< or = 60 s) of the experiments conducted, and each of the analogues tested was found to noncompetitively inhibit the transport of dopamine. It was found that halogenating the aromatic ring with chlorine (threo-3,4-dichloromethylphenidate hydrochloride; compound 1) increased the affinity of MPD to inhibit the transport of dopamine. A derivative of MPD with simultaneous, single methyl group substitutions on the phenyl ring and at the nitrogen (threo-N-methyl-4-methylphenidate hydrochloride; compound 2) bound at a site in common with MPD. A benzyl group positioned at the nitrogen (threo-N-benzylmethylphenidate hydrochloride; compound 3) imparted properties to the inhibitor in which binding at substrate and non-substrate sites could be distinguished. This analogue bound at a mutually interacting site with that of methylphenidate and had a K(int) value of 4.29 microM. Furthermore, the N-substituted analogues (compounds 2 and 3), although clearly inhibitors of dopamine transport, were found to attenuate dramatically the inhibition of dopamine transport by amphetamine, suggesting that the development of an antagonist for substrate analogue drugs of abuse may be possible.     

Expression, Isolation, and Crystallization of the Catalytic Domain of CopB, a Putative Copper Transporting ATPase from the Thermoacidophilic Archaeon Sulfolobus solfataricus

The P-type CPX-ATPases are responsible for the transport of heavy metal ions in archaea, bacteria, and eukaryotes. We have chosen one of the two CPX-ATPases of the thermophile Sulfolobus solfataricus, CopB (= SSO2896) for the investigation of the molecular mechanism of this integral membrane protein. We recombinately expressed three different soluble domains of this protein (named CopB-A, CopB-B, and CopB-C) in Escherichia coli and purified them to homogeneity. 3D crystals of CopB-B, the 29 kDa catalytic ATP binding/phosphorylation domain were produced, which diffracted to a resolution of 2.2 A. CopB-B has heavy metal stimulated phosphatase activity, which was half maximal in the presence of 80 microM Cu2+. The protein forms a phosphorylated intermediate with the substrate gamma-(32P)-ATP. No specific activation of the polypeptide was observed, when CopB-B phosphatase activity was tested in the presence of the purified CopB-C and CopB-A proteins, which provide the cation binding and the phosphatase domains. We conclude that CopB is a putatively copper translocating ATPase, in which structural elements integrally located in the membrane are required for full, coordinated activation of the catalytic ATP binding domain.     

The cytoplasmic male-sterile type and normal type mitochondrial genomes of sugar beet share the same complement of genes of known function but differ in the content of expressed ORFs

The complete nucleotide sequence (501,020 bp) of the mitochondrial genome from cytoplasmic male-sterile (CMS) sugar beet was determined. This enabled us to compare the sequence with that previously published for the mitochondrial genome of normal, male-fertile sugar beet. The comparison revealed that the two genomes have the same complement of genes of known function. The rRNA and tRNA genes encoded in the CMS mitochondrial genome share 100% sequence identity with their respective counterparts in the normal genome. We found a total of 24 single nucleotide substitutions in 11 protein genes encoded by the CMS mitochondrial genome. However, none of these seems to be responsible for male sterility. In addition, several other ORFs were found to be actively transcribed in sugar beet mitochondria. Among these, Norf246 was observed to be present in the normal mitochondrial genome but absent from the CMS genome. However, it seems unlikely that the loss of Norf246 is causally related to the expression of CMS, because previous studies on mitochondrial translation products failed to detect the product of this ORF. Conversely, the CMS genome contains four transcribed ORFs (Satp6presequence, Scox2-2 , Sorf324 and Sorf119) which are missing from the normal genome. These ORFs, which are potential candidates for CMS genes, were shown to be generated by mitochondrial genome rearrangements.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. Hagemann