Different glycoforms of prostate-specific membrane antigen are intracellularly transported through their association with distinct detergent-resistant membranes

Hormone-refractory prostate carcinomas as well as the neovasculature of different tumours express high levels of PSMA (prostate-specific membrane antigen). PSMA is a type II-transmembrane glycoprotein and a potential tumour marker for both diagnosis and passive immunotherapy. Here, we report on the association of PSMA with DRMs (detergent-resistant membranes) at different stages of the protein maturation pathway in human prostate carcinoma LNCaP cells. At least three PSMA glycoforms were biochemically identified based on their extractability behaviour in different non-ionic detergents. In particular, one precursor glycoform of PSMA is associated with Tween 20-insoluble DRMs, whereas the complex glycosylated protein segregates into membrane structures that are insoluble in Lubrol WX and display a different lipid composition. Association of PSMA with these membranes occurs in the Golgi compartment together with the acquisition of a native conformation. PSMA homodimers reach the plasma membrane of LNCaP cells in Lubrol WX-insoluble lipid/protein complexes. At the steady state, the majority of PSMA remains within these membrane microdomains at the cell surface. We conclude that the intracellular transport of PSMA occurs through populations of DRMs distinct for each biosynthetic form and cellular compartment.     

Combined Effects of Turbulence and Different Predation Regimes on Zooplankton in Highly Colored Water—Implications for Environmental Change in Lakes

In aquatic ecosystems, predation is affected both by turbulence and visibility, but the combined effects are poorly known. Both factors are changing in lakes in the Northern Hemisphere; the average levels of turbulence are predicted to increase due to increasing wind activities, while water transparency is decreasing, e.g., due to variations in precipitation, and sediment resuspension. We explored experimentally how turbulence influenced the effects of planktivorous fish and invertebrate predators on zooplankton when it was combined with low visibility caused by high levels of water color. The study was conducted as a factorial design in 24 outdoor ponds, using the natural zooplankton community as a prey population. Perch and roach were used as vertebrate predators and Chaoborus flavicans larvae as invertebrate predators. In addition to calm conditions, the turbulent dissipation rate used in the experiments was 10⁻⁶ m² s⁻³, and the water color was 140 mg Pt L⁻¹. The results demonstrated that in a system dominated by invertebrates, predation pressure on cladocerans increased considerably under intermediate turbulence. Under calm conditions, chaoborids caused only a minor reduction in the crustacean biomass. The effect of fish predation on cladocerans was slightly reduced by turbulence, while predation on cyclopoids was strongly enhanced. Surprisingly, under turbulent conditions fish reduced cyclopoid biomass, whereas in calm water it increased in the presence of fish. We thus concluded that turbulence affects fish selectivity. The results suggested that in dystrophic invertebrate-dominated lakes, turbulence may severely affect the abundance of cladocerans. In fish-dominated dystrophic lakes, on the other hand, turbulence-induced changes in planktivory may considerably affect copepods instead of cladocerans. In lakes inhabited by both invertebrates and fish, the response of top-down regulation to turbulence resembles that in fish-dominated systems, due to intraguild predation. The changes in planktivorous predation induced by abiotic factors may possibly cascade to primary producers.     

Protein interactions within the N-end rule ubiquitin ligation pathway

Rate studies have been employed as a reporter function to probe protein-protein interactions within a biochemically defined reconstituted N-end rule ubiquitin ligation pathway. The concentration dependence for E1-catalyzed HsUbc2b/E2(14kb) transthiolation is hyperbolic and yields K(m) values of 102 +/- 13 nm and 123 +/- 19 nm for high affinity binding to rabbit and human E1/Uba1 orthologs. Competitive inhibition by the inactive substrate and product analogs HsUbc2bC88A (K(i) = 104 +/- 15 nm) and HsUbc2bC88S-ubiquitin oxyester (K(i) = 169 +/- 17 nm), respectively, indicates that the ubiquitin moiety contributes little to E1 binding. Under conditions of rate-limiting E3alpha-catalyzed conjugation to human alpha-lactalbumin, HsUbc2b-ubiquitin thiolester exhibits a K(i) of 54 +/- 18 nm and is competitively inhibited by the substrate analog HsUbc2bC88S-ubiquitin oxyester (K(i) = 66 +/- 29 nm). In contrast, the ligase product analog HsUbc2bC88A exhibits a K(i) of 440 +/- 55 nm with respect to the wild type HsUbc2b-ubiquitin thiolester, demonstrating that ubiquitin binding contributes to the ability of E3alpha to discriminate between substrate and product E2. A survey of E1 and E2 isoform distribution in selected cell lines demonstrates that Ubc2 isoforms are the predominant intracellular ubiquitin carrier protein. Intracellular levels of E1 and Ubc2 are micromolar and approximately equal based on in vitro quantitation by stoichiometric (125)I-ubiquitin thiolester formation. Comparison of intracellular E1 and Ubc2 pools with the corresponding ubiquitin pools reveals that most of the free ubiquitin in cells is present as thiolesters to the components of the conjugation pathways. The present data represent the first comprehensive analysis of protein interactions within a ubiquitin ligation pathway.     

Enhanced differentiation of splenic plasma cells but diminished long-lived high-affinity bone marrow plasma cells in aged mice

In the present work, we have dissected the mechanisms responsible for the impaired humoral responses in aging. We found that there was a substantially higher level of Ab-forming cells in the spleens of aged mice than that of young controls. However, the number of high-affinity, class-switched Ab-forming cells was severely decreased in the spleen of aged mice. The accumulation of low-affinity IgM Ab-forming cells in the spleens of aged animals was not due to a deficiency in isotype switching because the number of total IgG1 splenic plasma cells was not significantly reduced. Remarkably, plasma cells of both low and high affinity were significantly diminished in the bone marrow of aged mice compared with that of young mice. The results from reconstitution experiments showed that aged bone marrow was less supportive for plasma cells derived from young splenic B cells. These findings suggest that humoral immune deficiency in aging results from at least two mechanisms: the inability to generate sufficient numbers of high-affinity Ab-forming cells, which is a result of diminished germinal center reaction, and the defective bone marrow environment that has diminished ability to support the selection and survival of long-term Ab-forming cells.     

Genetic correlations between nicotine reinforcement‐related behaviors and propensity toward high or low alcohol preference in two replicate mouse lines

Common genetic factors may contribute to the high comorbidity between tobacco smoking and alcohol use disorder. Here, we assessed behavioral and biological effects of nicotine in replicate mouse lines selectively bred for high (HAP2/3) or low alcohol preference (LAP2/3). In Experiment 1, free‐choice (FC) oral nicotine and quinine intake were assessed in HAP2/3 and LAP2/3 mice. Effects of nicotinic acetylcholine receptor blockade by mecamylamine on nicotine intake in HAP2 mice were also examined. In Experiment 2, HAP2/3 and LAP2/3 mice were tested for differences in sensitivity to nicotine‐induced taste conditioning. In Experiment 3, the effects of a single nicotine injection on nucleus accumbens (NAc) and dorsal striatum monoamine levels in HAP2/3 and LAP2/3 mice were tested. In Experiment 1, HAP2/3 mice showed greater nicotine intake and intake ratio than LAP2/3 mice. There were no line differences in quinine intake. Mecamylamine reduced nicotine intake and intake ratio in HAP2 mice. In Experiment 2, HAP2/3 mice showed weaker nicotine‐induced conditioned taste aversion (CTA) compared with LAP2/3 mice. In Experiment 3, nicotine treatment increased NAc dopamine turnover across both HAP2/3 and LAP2/3 mouse lines. These results show that there is a positive genetic correlation between oral alcohol intake (high alcohol intake/preference selection phenotype) and oral nicotine intake and a negative genetic correlation between oral alcohol intake and sensitivity to nicotine‐induced CTA.     

The NMDA receptor NR1 C1 region bound to calmodulin: structural insights into functional differences between homologous domains

Calmodulin (CaM) regulates tetrameric N-methyl-D-aspartate receptors (NMDARs) by binding tightly to the C0 and C1 regions of its NR1 subunit. A crystal structure (2HQW; 1.96 A) of calcium-saturated CaM bound to NR1C1 (peptide spanning 875-898) showed that NR1 S890, whose phosphorylation regulates membrane localization, was solvent protected, whereas the endoplasmic reticulum retention motif was solvent exposed. NR1 F880 filled the CaM C-domain pocket, whereas T886 was closest to the N-domain pocket. This 1-7 pattern was most similar to that in the CaM-MARCKS complex. Comparison of CaM-ligand wrap-around conformations identified a core tetrad of CaM C-domain residues (FLMM(C)) that contacted all ligands consistently. An identical tetrad of N-domain residues (FLMM(N)) made variable sets of contacts with ligands. This CaM-NR1C1 structure provides a foundation for designing mutants to test the role of CaM in NR1 trafficking as well as insights into how the homologous CaM domains have different roles in molecular recognition.     

Analysis of helicase activity and substrate specificity of Drosophila RECQ5

RecQ5 is one of five RecQ helicase homologs identified in humans. Three of the human RecQ homologs (BLM, WRN and RTS) have been linked to autosomal recessive human genetic disorders (Bloom syndrome, Werner syndrome and Rothmund–Thomson syndrome, respectively) that display increased genomic instability and cause elevated levels of cancers in addition to other symptoms. To understand the role of RecQ helicases in maintaining genomic stability, the WRN, BLM and Escherichia coli RecQ helicases have been characterized in terms of their DNA substrate specificity. However, little is known about other members of the RecQ family. Here we show that Drosophila RECQ5 helicase is a structure-specific DNA helicase like the other RecQ helicases biochemically characterized so far, although the substrate specificity is not identical to that of WRN and BLM helicases. Drosophila RECQ5 helicase is capable of unwinding 3′ Flap, three-way junction, fork and three-strand junction substrates at lower protein concentrations compared to 5′ Flap, 12 nt bubble and synthetic Holliday junction structures, which can be unwound efficiently by WRN and BLM.