Pyruvate shuttle in muscle cells: high-affinity pyruvate transport sites insensitive to trans-lactate efflux

The specificity of the transport mechanisms for pyruvate and lactate and their sensitivity to inhibitors were studied in L6 skeletal muscle cells. Trans- and cis-lactate effects on pyruvate transport kinetic parameters were examined. Pyruvate and lactate were transported by a multisite carrier system, i.e., by two families of sites, one with low affinity and high capacity (type I sites) and the other with high affinity and low capacity (type II). The multisite character of transport kinetics was not modified by either hydroxycinnamic acid (CIN) or p-chloromercuribenzylsulfonic acid (PCMBS), which exert different types of inhibition. The transport efficiency (TE) ratios of maximal velocity to the trans-activation dissociation constant (Kt) showed that lactate and pyruvate were preferentially transported by types I and II sites, respectively. The cis-lactate effect was observed with high Ki values for both sites. The trans-lactate effect on pyruvate transport occurred only on type I sites and exhibited an asymmetric interaction pattern (Kt of inward lactate > Kt of outward lactate). The inability of lactate to trans-stimulate type II sites suggests that intracellular lactate cannot recruit these sites. The high-affinity type II sites act as a specific pyruvate shuttle and constitute an essential relay for the intracellular lactate shuttle.     

Relations Between the Stage of Cell Maturation and Lactate Transporter Activities in Rat Neonatal Muscle Cells in Culture

Lactate transport was investigated in newborn rat muscle cells in culture. The aim was to study the lactate transport function at two stages of cell differentiation in culture: (i) during the proliferative phase characterized by myoblasts and myotubes (MyB/MyT2) obtained after 2–3 seedings, (ii) when myotubes (MyT1) grow old in culture after 8–9 seedings. In both developmental stages MyB/MyT2, lactate was carried following a saturable and sigmoidal velocity curve: the Hill and the Scatchard plot analyses confirmed an allosteric or multisite mechanism of lactate transport with two classes of carriers: one of low and one of high affinity i.e., 8.6 and 0.95 mm, respectively, which are associated with high and low transport capacities (V _m ) i.e., 9.1 and 0.67 nm/min/mg, respectively. With MyT1, the velocity curve of lactate transport presented a hyperbolic profile, and the Hill plot analysis gave a Hill number near one suggesting that for cell aging in culture the decrease in cooperativity shows that lactate transport essentially occurs through the low affinity transport system. Inhibitor effects also contributed to evidence for at least two systems of transport. Results obtained from primary cells give evidence for the early activity of lactate transport system at the Myb/MyT2 stage and its evolution during cell aging in culture (MyT1). Sarcolemmal lactate transport in primary cultures of myocytes is accomplished by multiple carriers, neither of which are MCT1 or MCT2 as confirmed by immunoblots. Received: 31 March 1999/Revised: 22 September 1999     

Assessment of Phytochemical Analysis,Nutritional Composition and Antimicrobial Activity of<i> Moringa oleifera</i>

Moringa oleifera is a miracle plant rich in nutrients, antioxidants, and antibiotic properties. Present study was designed to evaluate various biochemical attributes of leaves and flowers of M. oleifera. Plant parts (leaves, flowers) of M. oleifera, collected from different roadsides of Multan district, Punjab, Pakistan, were used as experimental material. Result indicates that alkaloids, saponin, carbohydrates, fats, and protein had a high value in the aqueous extract of both leaves and flowers of M. oleifera. Whereas phenol content was high in methanolic leaves extract and the phenol contents were high in aqueous extract of flowers. The extract yield of M. oleifera leaves and flowers both showed a higher percentage in aqueous extract (57.5%), followed by methanol extract and lowest in ethyl acetate extract. Flavonoids contents were higher in ethyl acetate extract of leaves (33.67%) and aqueous extract of flowers (53.71%). While crude fiber was high in methanolic extract of leaves (12.40%) and in flowers crude fiber was high in ethyl acetate extract (15.86%). The moisture contents were higher in leaves (8.87%) than flowers (7.3%) and similarly, ash percentage in flowers (52.60%) than leaves (41.84%). Ethyl acetate extracts of M. oleifera leaves show antibacterial activity against Pseudomonas aeruginosa while methanolic extract of M. oleifera flowers shows antibacterial activity against Xanthomonas sp. Maximum growth inhibits show in all extracts of leaves against Aspergillus flavus, F. oxysporum, and P. glabrum except for the concentrated aqueous extract of leaves. While in flowers maximum growth inhibits all extracts against P. glabrum, A. niger, and A. flavus except the diluted ethyl acetate extract. Phytochemicals present in different parts of moringa have significant edible and commercial potential. Moringa extracts exhibited significant antimicrobial activity, therefore have applications in pharmaceuticals.     

Endogenous C-terminal fragments of beta-amyloid precursor protein from Xenopus laevis skin exudate

Using a monoclonal antibody against the entire C-terminal end of human APP₆₉₅ (643–695 sequence) and a monoclonal antibody directed against human β[1–40] amyloid peptide (βA), we show the existence of endogenous peptides proteolytically derived from APP in skin exudate of the non transgenic Xenopus laevis frog. The majority of the immunoreactivity is found associated with a 30 kDa molecular species. Biochemical fractionation followed by mass spectrometry identification allowed us to assign this molecular species to C-terminal APP fragments containing all or part of βA. According to the nature of N- and C-terminal amino acids we identified endogenous β-, γ-, ε-secretase-like activities, caspase-like activity and numerous endogenous cleavage sites within the β-amyloid sequence at same sites as those observed in human βA sequence. All these homologies with human indicate that X. laevis skin exudate is a good natural model to study βA metabolism. In this way, interestingly, we identified endogenous cleavages at prohormone convertase-like sites not yet described at the same sites in human. Finally, all identified peptide fragments were stably associated with a 20.2 kDa protein. These new observed features suggest new research pathways concerning human βA metabolism and carriage of hydrophobic peptide fragments issued from APP processing.     

From protein catalogues towards targeted proteomics approaches in cereal grains

Due to their importance for human nutrition, the protein content of cereal grains has been a subject of intense study for over a century and cereal grains were not surprisingly one of the earliest subjects for 2D-gel-based proteome analysis. Over the last two decades, countless cereal grain proteomes, mostly derived using 2D-gel based technologies, have been described and hundreds of proteins identified. However, very little is still known about post-translational modifications, subcellular proteomes, and protein-protein interactions in cereal grains. Development of techniques for improved extraction, separation and identification of proteins and peptides is facilitating functional proteomics and analysis of sub-proteomes from small amounts of starting material, such as seed tissues. The combination of proteomics with structural and functional analysis is increasingly applied to target subsets of proteins. These “next-generation” proteomics studies will vastly increase our depth of knowledge about the processes controlling cereal grain development, nutritional and processing characteristics.     

OsSIZ1 Regulates the Vegetative Growth and Reproductive Development in Rice

SAP and MIZ (SIZ) is a small ubiquitin-related modifier (SUMO) E3 ligase that facilitates conjugation of SUMO to protein substrates. Although there have been a number of reports about the functions of SIZ1 in Arabidopsis in the regulation of diverse life processes, no information regarding the role of SIZ in other plants is available yet. In this work, two homologous genes from rice (Oryza sativa) were isolated and designated as OsSIZ1 and OsSIZ2 based on amino acid sequence homology to AtSIZ1 and their phylogenetic relationship. The function in the vegetative growth and reproductive development in rice was investigated using OsSIZ1 mutants containing a T-DNA insertion. The results showed that the mutant Ossiz1 exhibited the significant changes in several growth and developmental parameters, including primary root length, adventitious root number, plant height, leaf and panicle length, flower formation, and seed-setting rate compared with wild type. Taking together these results indicate that OsSIZ1 plays an important role in regulating growth and development in rice.     

Proteome reference map of Lactobacillus acidophilus NCFM and quantitative proteomics towards understanding the prebiotic action of lactitol

Lactobacillus acidophilus NCFM is a probiotic bacterium adapted to survive in the gastrointestinal tract and with potential health benefits to the host. Lactitol is a synthetic sugar alcohol used as a sugar replacement in low calorie foods and selectively stimulating growth of L. acidophilus NCFM. In the present study the whole-cell extract proteome of L. acidophilus NCFM grown on glucose until late exponential phase was resolved by 2-DE (pH 3-7). A total of 275 unique proteins assigned to various physiological processes were identified from 650 spots. Differential 2-DE (DIGE) (pH 4-7) of L. acidophilus NCFM grown on glucose and lactitol, revealed 68 spots with modified relative intensity. Thirty-two unique proteins were identified in 41 of these spots changing 1.6-12.7-fold in relative abundance by adaptation of L. acidophilus NCFM to growth on lactitol. These proteins included β-galactosidase small subunit, galactokinase, galactose-1-phosphate uridylyltransferase and UDP-glucose-4-epimerase, which all are potentially involved in lactitol metabolism. This first comprehensive proteome analysis of L. acidophilus NCFM provides insights into protein abundance changes elicited by the prebiotic lactitol.     

Pulmonary Aspergillosis and Aflatoxins in Chronic Lung Diseases

Recent studies have clearly defined the vaginopathic Candida albicans strains that cause severe vulvovaginal candidiasis (VVC). Therefore, genotyping C. albicans isolates may predict the success of and assist in choosing the appropriate antifungal therapy. The purpose of this study was to compare the genotypes of C. albicans isolates causing VVC with those found in asymptomatic healthy pregnant and non-pregnant women in Adana, Turkey, as well as the antifungal susceptibility profiles of these isolates. A total of 216 independent C. albicans isolates were genotyped by allelic combination based on the microsatellite marker analysis of one such microsatellite, present in the promoter region of the elongation factor 3-encoding gene (CEF3) of C. albicans. The susceptibility testing profiles of all of the isolates against five antifungals and boric acid were obtained retrospectively from our laboratory records. We identified 20 genotypes on the basis of different allelic combinations at the CEF3 locus with a discriminatory power of 0.85. Genotypes 136–144 and 126–135 were present in 50 % of the isolates. No differences existed in the genotypic profiles of fungal isolates between pregnant and non-pregnant women. Remarkably, we did not find a single vaginopathic genotype. All of the isolates were susceptible to amphotericin B and 5-fluorocytosine, and the fluconazole and ketoconazole resistance rates were 0.9 and 3.7 %, respectively. Therefore, we did not find any correlation between genotype, severity of VVC, and antifungal resistance (P > 0.05). Even so, additional molecular data may provide new insights into the management of VVC.     

5T4 Interacts with TIP-2/GIPC, a PDZ Protein, with Implications for Metastasis

Overexpression of the 5T4 transmembrane glycoprotein can have marked effects on both the actin cytoskeleton and cell migration. Using a yeast two-hybrid approach, we describe a novel interaction between 5T4 and TIP-2/GIPC, a cytoplasmic interacting protein containing a PDZ domain. The cytoplasmic tail of 5T4 contains a class I PDZ-binding motif (Ser-Asp-Val) and we demonstrate that this region, in particular the terminal valine, is required for 5T4 interaction with TIP-2/GIPC. HeLa cells expressing hemagglutinin-tagged TIP-2/GIPC (HA-TIP-2/GIPC) have an altered distribution of endogenous 5T4, which colocalizes with HA-TIP-2/GIPC, thus supporting an interaction. Furthermore, TIP-2/GIPC can be coimmunoprecipitated with 5T4 from HeLa cell lysates. Identification of the 5T4 and TIP-2/GIPC interaction provides the first link between 5T4 and the actin cytoskeleton. Since other proteins, like 5T4, associate with TIP-2/GIPC and are linked with cancer, we explore the possibility that TIP-2/GIPC may be a common factor involved in the cancer process.