Conductance Ratios and Cellular Identity

Recent experimental evidence suggests that coordinated expression of ion channels plays a role in constraining neuronal electrical activity. In particular, each neuronal cell type of the crustacean stomatogastric ganglion exhibits a unique set of positive linear correlations between ionic membrane conductances. These data suggest a causal relationship between expressed conductance correlations and features of cellular identity, namely electrical activity type. To test this idea, we used an existing database of conductance-based model neurons. We partitioned this database based on various measures of intrinsic activity, to approximate distinctions between biological cell types. We then tested individual conductance pairs for linear dependence to identify correlations. Contrary to experimental evidence, in which all conductance correlations are positive, 32% of correlations seen in this database were negative relationships. In addition, 80% of correlations seen here involved at least one calcium conductance, which have been difficult to measure experimentally. Similar to experimental results, each activity type investigated had a unique combination of correlated conductances. Finally, we found that populations of models that conform to a specific conductance correlation have a higher likelihood of exhibiting a particular feature of electrical activity. We conclude that regulating conductance ratios can support proper electrical activity of a wide range of cell types, particularly when the identity of the cell is well-defined by one or two features of its activity. Furthermore, we predict that previously unseen negative correlations and correlations involving calcium conductances are biologically plausible.     

Effects of inhibition of N-linked glycosylation by tunicamycin on nucleoside transport polypeptides of L1210 leukemia cells

Membrane polypeptides (relative mass (Mr) 48,000--55,000) associated with the equilibrative transport of nucleosides were identified in cultured murine leukemia (L1210/C2) cells by site-specific photolabeling with [3H]nitrobenzylthioinosine ([3H]NBMPR). Growth of cells in the presence of tunicamycin resulted in the gradual conversion of 3H-labeled polypeptides to a form that migrated more rapidly (Mr 42,000--47,000) during sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis. When plasma membrane fractions were photolabeled and incubated with O-glycanase or endoglycosidase F, the [3H]NBMPR-labeled polypeptides migrated in SDS-polyacrylamide gels with the same mobility as native NBMPR-binding polypeptides, whereas incubation with either N-glycanase or trifluoromethane sulfonic acid converted [3H]NBMPR-labeled polypeptides to the more rapidly migrating form (Mr 41,000--48,000). These observations are consistent with the presence of N-linked oligosaccharides of the complex type on the NBMPR-binding polypeptides of L1210/C2 cells. Tunicamycin exposures that reduced incorporation of [3H]mannose into plasma membrane fractions by greater than 95% had little, if any, effect on either the affinity (Kd values, 0.1-0.2 nM) or abundance (Bmax values, 200,000--220,000 sites/cell) of NBMPR-binding sites, whereas uridine transport kinetics at 37 degrees C were altered in a complex way. Thus, although N-linked glycosylation is not required for insertion of the NBMPR-binding protein into the plasma membrane or for interaction of NBMPR with the high-affinity binding sites, it is important for function of at least one of the three nucleoside transporters expressed by L1210/C2 cells.     

Redundancy of signal and anchor functions in the NH2-terminal uncharged region of influenza virus neuraminidase, a class II membrane glycoprotein

Class II membrane glycoproteins share a common topology of the NH2 terminus inside and the COOH terminus outside the cell. Their transport to the cell surface is initiated by the function of a single hydrophobic domain near the NH2 terminus. This functional domain serves both as an uncleaved signal sequence and as a transmembrane anchor. We examined the signal and anchor functions of influenza virus neuraminidase, a prototype class II membrane glycoprotein, by deletion analysis of its long, uncharged amino-terminal region. The results presented here show that the entire stretch of 29 uncharged amino acids (7 to 35) is not required for either a signal sequence or an anchor sequence function. On the basis of translocation and membrane stability data for different mutants, we suggest that the first 20 amino acid residues (7 to 27) are likely to provide the hydrophobic core for these functions and that within this putative subdomain some sequences are more efficient than the other sequences in providing a translocation function. Finally, it appears that neuraminidase and its mutant proteins are translocated with the proper orientation, regardless of the characteristics of the flanking sequences.     

Structure and orientation of expressed bovine coronavirus hemagglutinin-esterase protein.

The sequence of the hemagglutinin-esterase (HE) gene for the Mebus strain of bovine coronavirus was obtained from cDNA clones, and its deduced product is a 47,700-kilodalton apoprotein of 424 amino acids. Expression of the HE protein in vitro in the presence of microsomes revealed N-terminal signal peptide cleavage and C-terminal anchorage but not disulfide-linked dimerization. Dimerization was observed only after expression in vivo, during which HE was also transported to the cell surface.     

Production of a foreign protein product with genetically modified plant cells.

Plant cells (Nicotiana tabacum) were genetically engineered to produce a foreign protein, chloramphenicol acetyltransferase (CAT), and the CAT production from suspension cultures was investigated. Suspension cultures were grown in a shake flask, a stirred fermenter, and a bubble-column fermenter. The CAT production was growth related and the maximum activity was reached during the early stationary phase. A 41-day, semicontinuous stirred fermenter run, consisting of five sequential batch runs, demonstrated long-term CAT production. Continuous CAT production was also accomplished in a bubble-column fermenter at a medium flow rate of 3.1 ml h-1, which was equivalent to a dilution rate of 0.25 day-1.     

Change in climatically suitable breeding distributions reduces hybridization potential between Vermivora warblers

Aim

Climate change is affecting the distribution of species and subsequent biotic interactions, including hybridization potential. The imperiled Golden-winged Warbler (GWWA) competes and hybridizes with the Blue-winged Warbler (BWWA), which may threaten the persistence of GWWA due to introgression. We examined how climate change is likely to alter the breeding distributions and potential for hybridization between GWWA and BWWA.

Location

North America.

Methods

We used GWWA and BWWA occurrence data to model climatically suitable conditions under historical and future climate scenarios. Models were parameterized with 13 bioclimatic variables and 3 topographic variables. Using ensemble modeling, we estimated historical and modern distributions, as well as a projected distribution under six future climate scenarios. We quantified breeding distribution area, the position of and amount of overlap between GWWA and BWWA distributions under each climate scenario. We summarized the top explanatory variables in our model to predict environmental parameters of the distributions under future climate scenarios relative to historical climate.

Results

GWWA and BWWA distributions are projected to substantially change under future climate scenarios. GWWA are projected to undergo the greatest change; the area of climatically suitable breeding season conditions is expected to shift north to northwest; and range contraction is predicted in five out of six future climate scenarios. Climatically suitable conditions for BWWA decreased in four of the six future climate scenarios, while the distribution is projected to shift east. A reduction in overlapping distributions for GWWA and BWWA is projected under all six future climate scenarios.

Main Conclusions

Climate change is expected to substantially alter the area of climatically suitable conditions for GWWA and BWWA, with the southern portion of the current breeding ranges likely to become climatically unsuitable. However, interactions between BWWA and GWWA are expected to decline with the decrease in overlapping habitat, which may reduce the risk of genetic introgression.     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.     

Protein interactions during coronavirus assembly.

Coronaviruses assemble and obtain their envelope at membranes of the intermediate compartment between the endoplasmic reticulum and Golgi complex. Like other enveloped viruses, coronavirus assembly is presumably dependent on protein localization and protein-protein as well as protein-RNA interactions. We have used the bovine coronavirus (BCV) as a model to study interactions between the viral proteins in virus-infected cells that are important for coronavirus assembly. BCV is a prototype for the coronaviruses that express an additional major structural protein, the hemagglutinin esterase (HE), in addition to the spike (S) glycoprotein, membrane (M) glycoprotein, and nucleocapsid (N) protein. Complexes consisting of the M, S, and HE proteins were detected in virus-infected cells by coimmunoprecipitations. Kinetic analyses demonstrated that S protein and HE each quickly formed a complex with M protein after synthesis, whereas heterocomplexes consisting of all three proteins formed more slowly. The kinetics of HE biosynthesis revealed that the half-life of oligomerization was approximately 30 min, which correlated with the appearance of complexes consisting of M, HE, and S proteins, suggesting that oligomerization and/or conformational changes may be important for the S-M-HE protein complexes to form. Only HE dimers were found associated with the heterocomplexes consisting of all three proteins. S-M-HE protein complexes were detected prior to processing of the oligosaccharide chains on HE, indicating that these protein complexes formed in a premedial Golgi compartment before trimming of sugar chains. Transient coexpressions and double-labeling immunofluorescence demonstrated that HE and S proteins colocalized with M protein. This was further supported by coimmunoprecipitation of specific HE-M and S-M protein complexes from transfected cells, indicating that these proteins can form complexes in the absence of other viral proteins.     

Mapping of antigenic determinants of human apolipoprotein B using monoclonal antibodies against low density lipoproteins

The reactivity of a series of monoclonal antibodies directed against human low density lipoproteins (LDL) has been tested with hepatic and intestinal apolipoprotein B (apo-B) termed B-100 and B-48, respectively (Kane, J. P., Hardman, D. A., and Paulus, H. E. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2465-2469). Whereas those antibodies that have been previously shown to recognize determinants close to the LDL receptor recognition site reacted only with B-100, two antibodies specific for other regions of apo-B reacted with both B-100 and B-48. Therefore, it is probable that sequence homologies exist between the two proteins and it must be considered that all or parts of the B-48 sequence may be contained within that of B-100. The specificity of the reaction of these antibodies with proteins designated B-74 and B-26 supports the concept that they represent complementary fragments of B-100. The present results have been incorporated in a theoretical map of the antigenic determinants recognized by these antibodies on the LDL apo-B.