Long-chain polyunsaturated fatty acids in maternal and infant nutrition

Homo sapiens has evolved on a diet rich in alpha-linolenic acid and long chain polyunsaturated fatty acids (LCP). We have, however, gradually changed our diet from about 10,000 years ago and accelerated this change from about 100 to 200 years ago. The many dietary changes, including lower intake of omega3-fatty acids, are related to 'typically Western' diseases. After a brief introduction in essential fatty acids (EFA), LCP and their functions, this contribution discusses our present low status of notably LCPomega3 in the context of our rapidly changing diet within an evolutionary short time frame. It then focuses on the consequences in pregnancy, lactation and neonatal nutrition, as illustrated by some recent data from our group. We discuss the concept of a 'relative' EFA/LCP deficiency in the fetus as the outcome of high transplacental glucose flux. This flux may in the fetus augment de novo synthesis of fatty acids, which not only dilutes transplacentally transported EFA/LCP, but also causes competition of de novo synthesized oleic acid with linoleic acid for delta-6 desaturation. Such conditions were encountered by us in mothers with high body mass indices, diabetes mellitus and preeclampsia. The unifying factor might be compromised glucose homeostasis. In search of the milk arachidonic acid (AA) and docosahexaenoic acid (DHA) contents of our African ancestors, we investigated women in Tanzania with high intakes of freshwater fish as only animal lipid source. These women had milk AA and DHA contents that were well above present recommendations for infant formulae. Both studies stimulate rethinking of 'optimal homeostasis'. Subtle signs of dysbalanced maternal glucose homeostasis may be important and observations from current Western societies may not provide us with an adequate basis for dietary recommendations.     

Wooden objects from Ohalo II (23,000 cal BP), Jordan Valley, Israel

Eight wooden objects were found at Ohalo II, a submerged and well-preserved site in the Sea of Galilee, Israel. The fisher-hunter-gatherers' site has been radiometrically dated to 22,500-23,500 (cal BP) with 45 assays read by four laboratories. The wooden objects were found on brush-hut floors. They include a bark plank with polish and use signs, pencil-shaped specimens with longitudinal shavings, and other types that may have been decorative or symbolic. One incised wooden object is identical in size and incision pattern to a gazelle bone implement found in a grave, behind a human skull. The recovered wooden objects are not directly related to hunting, gathering, or fishing, and frustratingly, there are no remains of bows, arrows, spears, handles, or other such items. Nonetheless, the objects present a wide repertoire in terms of size, shape, and possible function. The new finds add to the growing body of evidence concerning the use of perishable materials during the Upper Paleolithic.     

Engineering novel Lec1 glycosylation mutants in CHO-DUKX cells: molecular insights and effector modulation of N-acetylglucosaminyltransferase I

Many secreted or cell surface proteins are post-translationally modified by carbohydrate chains which are a primary source of heterogeneity. The Lec1 mutant, which is defective in Golgi N-acetylglucosaminyltransferase I (GnTI) activity, produces relatively homogeneous Man(5) GlcNAc(2) glycan modifications, and is widely used for various applications. To facilitate the investigation of GnTI, its Man5 glycan endproduct, and the impact of Man5 on effector function, the present study has established several novel Lec1 mutants in dhfr(-) CHO-DUKX cells through chemical mutagenesis and lectin selection. A total of nine clonal lines exhibiting the Lec1-phenotype are characterized, six of which harbor non-sense mutations leading to a truncated GnTI, and three (R415K, D291N, and P138L) of which are novel loss-of-function sense mutations. Analysis of the rabbit GnTI structure (Unligil et al., 2000) indicates that D291 is the proposed catalytic base and R415 is a crucial residue in forming the substrate binding pocket, whereas P138 is key to maintaining two β strands in proximity to the substrate binding pocket. Computational modeling reveals that the oligomannose glycan backbone of a glycoprotein (the acceptor substrate) fits nicely into the unoccupied channel of the substrate binding pocket partly through hydrogen bonding with R415 and D291. This finding is consistent with the ordered sequential Bi Bi kinetic mechanism suggested for GnTI, in which binding of UDP-GlcNAc (the donor substrate)/Mn(2+) induces conformational changes that promote acceptor binding. When an anti-human CD20 antibody protein is stably expressed in one CHO-DUKX-Lec1 line, it is confirmed that N-glycans are predominantly Man(5) GlcNAc(2) and they do not contain an α1,6-fucose linked to the innermost GlcNAc. Furthermore, this Man(5) GlcNAc(2) modified antibody exhibits a significantly increased ADCC activity than the wild-type protein, while displaying a lower CDC activity. The data support the hypothesis that modulating GnTI activity can influence antibody effector functions for proteins with an IgG1 immunoglobulin Fc domain.     

Pathways of Ca²⁺ entry and cytoskeletal damage following eccentric contractions in mouse skeletal muscle

Muscles that are stretched during contraction (eccentric contractions) show deficits in force production and a variety of structural changes, including loss of antibody staining of cytoskeletal proteins. Extracellular Ca(2+) entry and activation of calpains have been proposed as mechanisms involved in these changes. The present study used isolated mouse extensor digitorum longus (EDL) muscles subjected to 10 eccentric contractions and monitored force production, immunostaining of cytoskeletal proteins, and resting stiffness. Possible pathways for Ca(2+) entry were tested with streptomycin (200 μM), a blocker of stretch-activated channels, and with muscles from mice deficient in the transient receptor potential canonical 1 gene (TRPC1 KO), a candidate gene for stretch-activated channels. At 30 min after the eccentric contractions, the isometric force was decreased to 75 ± 3% of initial control and this force loss was reduced by streptomycin but not in the TRPC1 KO. Desmin, titin, and dystrophin all showed patchy loss of immunostaining 30 min after the eccentric contractions, which was substantially reduced by streptomycin and in the TRPC1 KO muscles. Muscles showed a reduction of resting stiffness following eccentric contractions, and this reduction was eliminated by streptomycin and absent in the TRPC1 KO muscles. Calpain activation was determined by the appearance of a lower molecular weight autolysis product and μ-calpain was activated at 30 min, whereas the muscle-specific calpain-3 was not. To test whether the loss of stiffness was caused by titin cleavage, protein gels were used but no significant titin cleavage was detected. These results suggest that Ca(2+) entry following eccentric contractions is through a stretch-activated channel that is blocked by streptomycin and encoded or modulated by TRPC1.     

Electrical stimulation influences satellite cell proliferation and apoptosis in unloading-induced muscle atrophy in mice

Muscle atrophy caused by disuse is accompanied by adverse physiological and functional consequences. Satellite cells are the primary source of skeletal muscle regeneration. Satellite cell dysfunction, as a result of impaired proliferative potential and/or increased apoptosis, is thought to be one of the causes contributing to the decreased muscle regeneration capacity in atrophy. We have previously shown that electrical stimulation improved satellite cell dysfunction. Here we test whether electrical stimulation can also enhance satellite cell proliferative potential as well as suppress apoptotic cell death in disuse-induced muscle atrophy. Eight-week-old male BALB/c mice were subjected to a 14-day hindlimb unloading procedure. During that period, one limb (HU-ES) received electrical stimulation (frequency: 20 Hz; duration: 3 h, twice daily) while the contralateral limb served as control (HU). Immunohistochemistry and western blotting techniques were used to characterize specific proteins in cell proliferation and apoptosis. The HU-ES soleus muscles showed significant improvement in muscle mass, cross-sectional area, and peak tetanic force relative to the HU limb (p<0.05). The satellite cell proliferative activity as detected within the BrdU+/Pax7+ population was significantly higher (p<0.05). The apoptotic myonuclei (detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and the apoptotic satellite cells (detected by cleaved Poly [ADP-ribose] polymerase co-labeled with Pax7) were reduced (p<0.05) in the HU-ES limb. Furthermore the apoptosis-inducing factor and cleaved caspase-3 were down-regulated while the anti-apoptotic Bcl-2 protein was up-regulated (p<0.05), in the HU-ES limb. These findings suggest that the electrical stimulation paradigm provides an effective stimulus to rescue the loss of myonuclei and satellite cells in disuse muscle atrophy, thus maintaining a viable satellite cell pool for subsequent muscle regeneration. Optimization of stimulation parameters may enhance the outcome of the intervention.     

Renaturation, purification and characterization of streptokinase expressed as inclusion body in recombinant E. coli

Recombinant protein purification is facilitated using high expression systems which produce larger quantities of streptokinase protein as inclusion bodies. As the accumulation of active streptokinase is toxic to the host cells, we have optimized the conditions to achieve large amounts of streptokinase in the form of inclusion bodies. The solubility and yield of pure protein are highly dependent on various combinations of chemical additives, ionic and non-ionic detergents and salts, with solubilizing agents followed by refolding of denatured protein into active form. As the extraction of the purified streptokinase from inclusion bodies requires denaturation and a subsequent refolding step, careful balancing steps were needed to develop under different controlled conditions. Here the purified fragments of refolded proteins were screened to select the conditions that yield the active streptokinase having native conformation. The maximum specific activity of the purified streptokinase was achieved by these methods. The refolded recombinant streptokinase was analyzed by RP-HPLC showing a purity of 99%. Size exclusion chromatography profile shows that there are minimal aggregates in the active streptokinase protein and the percentage of renaturation is around 99%.     

Involvement of a Stat3 binding site in inflammation-induced enteric apelin expression

Apelin is the endogenous ligand for the APJ receptor; both are expressed in the gastrointestinal tract. Experimental colitis in rodents and inflammatory bowel disease in humans are associated with increased intestinal apelin production. Our aim was to use LPS and proinflammatory cytokine-treated (IL-6 and IFN-gamma) rodents or enteric cells to identify signaling mechanisms underlying inflammation-induced enteric apelin expression. LPS, IL-6, or IFN-gamma treatment of rodents increased enteric apelin expression. Pharmacological blockade of Jak/Stat signaling or IL-6 antibody administration inhibited elevations in enteric apelin expression. Transient transfection experiments showed that LPS, IL-6, or IFN-gamma increased apelin expression by stimulation of apelin promoter activity, and blockade of Jak/Stat signaling abolished elevations in apelin promoter activity. A chromatin immunoprecipitation assay showed that IL-6 induced binding of phospho-Stat3 to a putative Stat3 site in the apelin promoter; mutation of this site abrogated the LPS-induced elevation in apelin promoter activity. Together, our findings indicate that binding of phospho-Stat3 to the apelin promoter is the final step underlying proinflammatory cytokine-induced enteric apelin expression during intestinal inflammation.