Seasonal changes in the diel vertical migration of walleye pollock (<Emphasis Type="Italic">Theragra chalcogramma</Emphasis>) in the northern Gulf of Alaska

Walleye pollock (Theragra chalcogramma) perform diel vertical migration (DVM) as juveniles, but have an increasing tendency to be associated with the bottom with age. We studied the DVM of a local population of adult pollock in the northern Gulf of Alaska in August and November 2003. There was no relationship between the depth of pollock and the isolume (line of equal light intensity) necessary for visual foraging in August. Pollock passed through the thermocline at this time. In November there was a significant relationship between pollock biomass above/below the 200 m isobath and the isolume necessary for visual foraging. It is hypothesized that in August pollock ignore the isolume and thermocline, simply tracking the movements of their prey (euphausiids) to feed upon them near the surface at night. In November, relatively denser pollock shoals migrate up and down with the isolume necessary for visual foraging to feed on decapods.     

Human poly(ADP-ribose) glycohydrolase is expressed in alternative splice variants yielding isoforms that localize to different cell compartments

Poly(ADP-ribose) glycohydrolase (PARG) is the only protein known to catalyze hydrolysis of ADP-ribose (ADPR) polymers to free ADP-ribose. While numerous genes encode different poly(ADP-ribose) polymerases (PARPs) that all synthesize ADP-ribose polymer, only a single gene coding for PARG has been detected in mammalian cells. Here, we describe two splice variants of human PARG mRNA, which lead to expression of PARG isoforms of 102 kDa (hPARG102) and 99 kDa (hPARG99) in addition to the full-length PARG protein (hPARG111). These splice variants differ from hPARG111 by the lack of exon 1 (hPARG102) or exons 1 and 2 (hPARG99). They are generated by the utilization of ambiguous splice donor sites in the PARG gene 5' untranslated region. The hPARG111 isoform localizes to the nucleus, whereas hPARG102 and hPARG99 are cytoplasmic proteins. The nuclear targeting of hPARG111 is due to a nuclear localization signal (NLS) in exon 1 that was mapped to the amino acids (aa) (10)CTKRPRW(16). Immunocytochemistry, immunoblotting, and PARG enzyme activity measurements show that the cytoplasmic isoforms of PARG account for most of the PARG activity in cells in the absence and presence of genotoxic stress. The predominantly cytoplasmic location of cellular PARG is intriguing as most known cellular PARPs have a nuclear localization.     

FeNO structure in distal pocket mutants of myoglobin based on resonance Raman spectroscopy

FeNO vibrational frequencies were investigated for a series of myoglobin mutants using isotope-edited resonance Raman spectra of (15/14)NO adducts, which reveal the FeNO and NO stretching modes. The latter give rise to doublet bands, as a result of Fermi resonances with coincident porphyrin vibrations; these doublets were analyzed by curve-fitting to obtain the nuNO frequencies. Variations in nuNO among the mutants correlate with the reported nuCO variations for the CO adducts of the same mutants. The correlation has a slope near unity, indicating equal sensitivity of the NO and CO bonds to polar influences in the heme pocket. A few mutants deviate from the correlation, indicating that distal interactions differ for the NO and CO adducts, probably because of the differing distal residue geometries. In contrast to the strong and consistent nuFeC/nuCO correlation found for the CO adducts, nuFeN correlates only weakly with nuNO, and the slope of the correlation depends on which residue is being mutated. This variability is suggested to arise from steric interactions, which change the FeNO angle and therefore alter the Fe-NO and N-O bond orders. This effect is modeled with Density Functional Theory (DFT) and is rationalized on the basis of a valence isomer bonding model. The FeNO unit, which is naturally bent, is a more sensitive reporter of steric interactions than the FeCO unit, which is naturally linear. An important additional factor is the strength of the bond to the proximal ligand, which modulates the valence isomer equilibrium. The FeNO unit is bent more strongly in MbNO than in protein-free heme-NO complexes because of a combination of a strengthened proximal bond and distal interactions.     

Structural plasticity and noncovalent substrate binding in the GroEL apical domain. A study using electrospay ionization mass spectrometry and fluorescence binding studies

Advances in understanding how GroEL binds to non-native proteins are reported. Conformational flexibility in the GroEL apical domain, which could account for the variety of substrates that GroEL binds, is illustrated by comparison of several independent crystallographic structures of apical domain constructs that show conformational plasticity in helices H and I. Additionally, ESI-MS indicates that apical domain constructs have co-populated conformations at neutral pH. To assess the ability of different apical domain conformers to bind co-chaperone and substrate, model peptides corresponding to the mobile loop of GroES and to helix D from rhodanese were studied. Analysis of apical domain-peptide complexes by ESI-MS indicates that only the folded or partially folded apical domain conformations form complexes that survive gas phase conditions. Fluorescence binding studies show that the apical domain can fully bind both peptides independently. No competition for binding was observed, suggesting the peptides have distinct apical domain-binding sites. Blocking the GroES-apical domain-binding site in GroEL rendered the chaperonin inactive in binding GroES and in assisting the folding of denatured rhodanese, but still capable of binding non-native proteins, supporting the conclusion that GroES and substrate proteins have, at least partially, distinct binding sites even in the intact GroEL tetradecamer.     

Programmed death-1 targeting can promote allograft survival

The recently identified CD28 homolog and costimulatory molecule programmed death-1 (PD-1) and its ligands, PD-L1 and PD-L2, which are homologs of B7, constitute an inhibitory regulatory pathway of potential therapeutic use in immune-mediated diseases. We examined the expression and functions of PD-1 and its ligands in experimental cardiac allograft rejection. In initial studies, we found that most normal tissues and cardiac isografts had minimal expression of PD-1, PD-L1, or PD-L2, but intragraft induction of all three molecules occurred during development of cardiac allograft rejection. Intragraft expression of all three genes was maintained despite therapy with cyclosporin A or rapamycin, but was prevented in the early posttransplant period by costimulation blockade using CD154 or anti-inducible costimulator mAb. We prepared PD-L1.Ig and PD-L2.Ig fusion proteins and showed that each bound to activated PD-1(+) T cells and inhibited T cell functions in vitro, thereby allowing us to test the effects of PD-1 targeting on allograft survival in vivo. Neither agent alone modulated allograft rejection in wild-type recipients. However, use of PD-L1.Ig administration in CD28(-/-) recipients, or in conjunction with immunosuppression in fully MHC-disparate combinations, markedly prolonged cardiac allograft survival, in some cases causing permanent engraftment, and was accompanied by reduced intragraft expression of IFN-gamma and IFN-gamma-induced chemokines. PD-L1.Ig use also prevented development of transplant arteriosclerosis post-CD154 mAb therapy. These data show that when combined with limited immunosuppression, or in the context of submaximal TCR or costimulatory signals, targeting of PD-1 can block allograft rejection and modulate T and B cell-dependent pathologic immune responses in vivo.     

Metallothionein and zinc homeostasis during tumor progression

Zinc homeostasis was studied during the induction, growth, and methotrexate (MTX) treatment of Dark Agouti rat mammary adenocarcinomas (DAMA). A progressive fall in plasma Zn concentration (pZn), significant at a tumor burden of less than 1% body weight (bw), was sustained during tumor enlargement to give a 54% reduction in pZn at 16.3% bw (n=6/group). The hypozincemia was attributed to the increasing Zn demand for tumor growth. Zn content of the 16.3% bw tumors equaled that of muscle (normally 60% of total body Zn). Tumor metallothionein (tMT) was sufficient to bind <3% of total tumor Zn, and hepatic MT (hMT) remained at basal concentrations during early tumor growth, doubling only in the presence of significant necrosis in large tumors. Methotrexate (MTX, 0.5 mg/Kg im x 2 d) at respective tumor burdens of 5 and 10% bw (n=9, 10/group) gave 2 therapeutic effects, dependent on tumor size: 1.5% bw tumors in 7 rats remained close to their original size until experiment end when pZn, hMT, and tMT were typical of 5% bw untreated tumors. 2. Tumors in 5 rats given MTX at 10% bw had marked subcapsular necrosis and regression to a size similar to those in group 1; pZn returned toward normal, whereas hMT was 6 times its 5% bw counterpart. Host weight loss was significantly reduced, as were tumor-associated changes in plasma glucose and calcium. In summary, neither tMT nor hMT appears to play a role in the hypozincemia that follows DAMA Zn sequestration and growth. Critically timed MTX can result in tumor regression and return of plasma Zn, Ca, and glucose toward normal. This is associated with an increase in hMT and reduction in host weight loss, suggesting a flow of Zn from the resorbing tumor to the host, enabling the synthesis of hMT and retention of host structural proteins.     

CHARACTERIZATION OF THE BASIS FOR DIFFERENCES IN SERUM DBH ACTIVITY IN IMMATURE AND ADULT RATS BY USE OF HOMOLOGOUS ANTIBODY

Abstract– Rat serum dopamine-β-hydroxylase (DBH) activity decreased 5-7-fold between 15 and 60 days of age. Immunoprecipitation performed with homologous antibody (guinea-pig anti-rat adrenal DBH) showed that during this time period the quantity of antibody necessary to precipitate 50% of the enzymatic activity (AD₅₀) decreased 5-fold from 0.25 to 0.05 μl/ml. The biochemical properties of rat serum DBH at 15 and 60 days of age were compared to test the hypothesis that there might be different biochemical forms of the enzyme in the blood of immature and adult rats. Thermal stability, apparent K_m for tyramine, electrophoretic mobility, pH optima and elution profile on gel filtratioh chromatography were all found to be similar for rat serum DBH at both ages. On the basis of homospecific activity and multiple similarities in biochemical characteristics, it appears that differences in serum activity at the two ages reflect differences in the steady-state levels of enzyme. To determine the turnover of serum DBH in the two age groups, the recovery of enzyme activity was monitored after acute clearance of the circulating pool of DBH by treatment with the homologous antiserum. Immunotitration of DBH activity in vivo indicated that the total pool of serum enzyme was 4-fold greater in the mature rat than in 4-day-olds. After treatment of adult rats with 2μl of homologous antiserum, serum DBH activity was reduced by 85% with a half-life of recovery of 3.0 ± 0.6 days; the estimated fractional rate of degradation was 0.23 ± 0.06 day^?1 and the rate of entrance was 2.3 ± 0.2 units/ml/day. After treatment of 4-day-old rats with 1 μl of homologous antiserum, serum DBH activity was reduced by 95% with a half-life of recovery of 3.3 ± 0.5 days: the estimated average fractional rate of degradation was 0.22 ± 0.06 day^?1 and the average rate of entrance was 10.7 ± 1.6 units/ml/day. Thus, the several-fold difference in steady-state levels of serum DBH in rat pups as compared to adult rats appears to be due to greatly increased rates of entrance of the enzyme in the immature rats.