Mitochondrial DNA sequence variation and genetic stock structure of Atlantic cod (Gadus morhua) from bay and offshore locations on the Newfoundland continental shelf

Bay cod, Atlantic cod (Gadus morhua) that over-winter in the deep-water bays of northeastern Newfoundland, have historically been regarded as distinct in migration and spawning behaviour from offshore (Grand Bank) cod stocks. To investigate their genetic relationships, we determined the DNA sequence of a 307-base-pair portion of the mitochondrial cytochrome b gene for 236 adult cod taken from the waters off northeastern Newfoundland, including fish found over-wintering and spawning in Trinity Bay. Although 17 genotypes were found, a single common genotype occurs at a frequency of greater than 80% in all samples, and no alternative genotype occurs at a frequency of greater than 3%. Genotype proportions did not differ significantly among samples. Measures of genetic subdivision among sampling locations are nil. Cod over-wintering in Trinity Bay are not genetically distinct from offshore cod. In combination with tagging and physiological studies, these data suggest that there is sufficient movement of cod between bay and offshore locations to prevent the development or maintenance of independent inshore stocks. Adult cod that over-winter in Trinity Bay appear to represent an assemblage of temporarily nonmigratory fish that have become physiologically acclimated to cold-water inshore environments. The pattern of genetic variation in northern cod suggests a recent population structure characterized by extensive movement of contemporary individuals superimposed on an older structure characterized by a bottleneck in the population size of cod in the north-western Atlantic.     

Conformational analysis of the tumor-associated carbohydrate antigen 19-9 and its Lea blood group antigen component as related to the specificity of monoclonal antibody CO19-9

A structural comparison between the synthetic, tumor-associated 19-9 tetrasaccharide, NeuAc alpha 2----3Gal beta 1----3GlcNAc(4----1 alpha Fuc)-O(CH2)8CO2CH3 and its Lea blood group antigen component, Gal beta 1----3GlcNAc(4----1 alpha Fuc)-O(CH2)8CO2CH3 was carried out by two-dimensional 1H NMR spectroscopy and hard-sphere energy calculations. Significant chemical shift differences between the two molecules were detected only for protons at or near the linkage site of NeuAc to the Lea trisaccharide core. Coupling constants for the ring protons of both molecules did not suggest major deviation from the 4C1 chair conformation for Gal and GlcNAc, the 1C4 conformation for Fuc, or the 2C5 conformation for NeuAc. Two-dimensional nuclear Overhauser enhancement experiments revealed through-space, inter-proton interactions that corresponded to some extent with those predicted by diffraction data and hard-sphere energy minimization programs for both saccharides. However, a significant number of interactions did not obey the distance dependence predicted from a rigid structure model. These data suggest that, while the average conformation of the 19-9 antigen's Lea core may be invariant to NeuAc alpha 2----3Gal linkage, the dynamics of the Lea trisaccharide are altered upon sialylation. Data also indicate that the terminal NeuAc linkage is more flexible than the inter-residue bonds of the core trisacharide. This analysis, in combination with the fact that the monoclonal anti-19-9 antibody CO 19-9 does not cross-react with the Lea antigen, provides evidence in favor of NeuAc as an epitope-creating unit involved directly at the antibody binding site. However, given the possible role of variable dynamics in epitope formation, these results do not preclude crucial roles in antibody recognition for regions on the 19-9 antigen that are distanced from NeuAc.     

Rational Design of a Fibroblast Growth Factor 21-Based Clinical Candidate,LY2405319

Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7–14 days resulted in a 25–50% lowering of plasma glucose coupled with a 10–30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans.     

Two newly identified exons in human GRM1 express a novel splice variant of metabotropic glutamate 1 receptor

To date, five human metabotropic glutamate (mGlu) 1 receptor splice variants (1a, 1b, 1d, 1f, and 1g) have been described, all of which involve alternative C-terminal splicing. mGlu1a receptor contains a long C-terminal domain (341 amino acids), which has been shown to scaffold with several proteins and contribute to the structure of the post-synaptic density. However, several shorter mGlu1 receptor splice variants lack the sequence required for these interactions, and no major functional differences between these short splice variants have been described. By using RT-PCR we have shown that two human melanoma cell lines express both mGlu1a and mGlu1b receptors. In addition, using 3′RACE, we identified three previously unknown mGlu1 receptor mRNAs. Two differ in the length of their 3′ untranslated region (UTR), and encode the same predicted protein as mGlu1g receptor—the shortest of all mGlu1 receptor splice variants. The third mRNA, named mGlu1h, encodes a predicted C-terminal splice variant of 10 additional amino acids. mGlu1h mRNA was observed in two different melanoma cell lines and is overexpressed, compared with melanoma precursor cells, melanocytes. Most importantly, this new splice variant, mGlu1h receptor, is encoded by two previously unidentified exons located within the human GRM1 gene. Additionally, these new exons are found exclusively within the GRM1 genes of higher primates and are highly conserved. Therefore, we hypothesize that mGlu1h receptors play a distinct role in primate glutamatergic signaling.     

Stimulation of the gastrin-cholecystokinin(B) receptor promotes branching morphogenesis in gastric AGS cells

Epithelial organization is maintained by cell proliferation, migration, and differentiation. In the case of the gastric epithelium, at least some of these events are regulated by the hormone gastrin. In addition, gastric epithelial cells are organized into characteristic tubular structures (the gastric glands), but the cellular mechanisms regulating the organization of tubular structures (sometimes called branching morphogenesis) are uncertain. In the present study, we examined the role of the gastrin-cholecystokinin(B) receptor in promoting branching morphogenesis of gastric epithelial cells. When gastric cancer AGS-G(R) cells were cultured on plastic, gastrin and PMA stimulated cell adhesion, formation of lamellipodia, and extension of long processes in part by activation of protein kinase C (PKC) and phosphatidylinositol (PI)-3 kinase. Branching morphogenesis was not observed in these circumstances. However, when cells were cultured on artificial basement membrane, the same stimuli increased the formation of organized multicellular arrays, exhibiting branching morphogenesis. These effects were reversed by inhibitors of PKC but not of PI-3 kinase. We conclude that, in the presence of basement membrane, activation of PKC by gastrin stimulates branching morphogenesis.     

Calf blood flow during prolonged tilt in idiopathic dilated cardiomyopathy and after cardiac transplantation

In severe congestive heart failure (CHF), abnormal reflex control of calf blood flow during brief head-up tilt that appears to normalize after transplantation (HTX) may be present during prolonged observation also. Therefore, we studied the effect of prolonged (30 min) 50 degrees head-up tilt on calf skeletal muscle blood flow measured by the local (133)Xe washout method in CHF and after HTX and in patients with the presence vs. absence of native right atrium (+PNA and -PNA, respectively). During brief head-up tilt, skeletal muscle blood flow increased 13 +/- 42% in 9 severe CHF patients in contrast to a -28 +/- 22% decrease (P < 0.01) in 11 control subjects, -24 +/- 30% decrease in 15 moderate CHF patients (P < 0.05), -25 +/- 14% decrease in 12 patients with recent HTX (P < 0.01), and -21 +/- 24% decrease in 8 patients with distant HTX (P = 0.06). However, during sustained tilt, blood flow declined to similar levels of that in the other groups in severe CHF. HTX -PNA vs. +PNA showed blunted skeletal muscle vasomotor control (P < 0.05) and a higher systolic blood pressure (139 +/- 14 vs. 125 +/- 15 mmHg, P < 0.05) and heart rate (92 +/- 10 vs. 83 +/- 8 beats/min, P < 0.05). Thus paradox vasodilatation of calf skeletal muscle in severe CHF is present only during brief but not prolonged tilt. This may be one explanation of the rare presence of orthostatic intolerance in CHF and implies only a minor possible role for the abnormality in edema pathogenesis. Removal of all right atrium in HTX has an important hemodynamic impact that may possibly affect later clinical outcome.