Differential recognition of tyrosine-based basolateral signals by AP-1B subunit mu1B in polarized epithelial cells

To investigate the importance of tyrosine recognition by the AP-1B clathrin adaptor subunit mu1B for basolateral sorting of integral membrane proteins in polarized epithelial cells, we have produced and characterized a mutant form of mu1B. The mutant (M-mu1B) contains alanine substitutions of each of the four conserved residues, which in the AP-2 adaptor subunit micro2 are critical for interacting with tyrosine-based endocytosis signals. We show M-mu1B is defective for tyrosine binding in vitro, but is nevertheless incorporated into AP-1 complexes in transfected cells. Using LLC-PK1 cells expressing either wild type or M-mu1B, we find that there is inefficient basolateral expression of membrane proteins whose basolateral targeting signals share critical tyrosines with signals for endocytosis. In contrast, membrane proteins whose basolateral targeting signals are distinct from their endocytosis signals (transferrin and low-density lipoprotein receptors) accumulate at the basolateral domain normally, although in a manner that is strictly dependent on mu1B or M-mu1B expression. Our results suggest that mu1B interacts with different classes of basolateral targeting signals in distinct ways.     

Predicting demographic group structures based on DNA sequence data

The ability to infer relationships between groups of sequences, either by searching for their evolutionary history or by comparing their sequence similarity, can be a crucial step in hypothesis testing. Interpreting relationships of human immunodeficiency virus type 1 (HIV-1) sequences can be challenging because of their rapidly evolving genomes, but it may also lead to a better understanding of the underlying biology. Several studies have focused on the evolution of HIV-1, but there is little information to link sequence similarities and evolutionary histories of HIV-1 to the epidemiological information of the infected individual. Our goal was to correlate patterns of HIV-1 genetic diversity with epidemiological information, including risk and demographic factors. These correlations were then used to predict epidemiological information through analyzing short stretches of HIV-1 sequence. Using standard phylogenetic and phenetic techniques on 100 HIV-1 subtype B sequences, we were able to show some correlation between the viral sequences and the geographic area of infection and the risk of men who engage in sex with men. To help identify more subtle relationships between the viral sequences, the method of multidimensional scaling (MDS) was performed. That method identified statistically significant correlations between the viral sequences and the risk factors of men who engage in sex with men and individuals who engage in sex with injection drug users or use injection drugs themselves. Using tree construction, MDS, and newly developed likelihood assignment methods on the original 100 samples we sequenced, and also on a set of blinded samples, we were able to predict demographic/risk group membership at a rate statistically better than by chance alone. Such methods may make it possible to identify viral variants belonging to specific demographic groups by examining only a small portion of the HIV-1 genome. Such predictions of demographic epidemiology based on sequence information may become valuable in assigning different treatment regimens to infected individuals.     

Effect of linker sequence on the stability of circularly permuted variants of ribonuclease T1

Circularly permuted variants of ribonuclease T1 were constructed with a library of residues covalently linking the original amino and carboxyl terminal ends of the wild-type protein. The library of linking peptides consisted of three amino acids containing any combination of proline, aspartate, asparagine, serine, threonine, tyrosine, alanine, and histidine. Forty two unique linker sequences were isolated and 10 of these mutants were further characterized with regard to catalytic activity and overall thermodynamic stability. The 10 mutants with the different linking sequences (HPD, TPH, DTD, TPD, PYH, PAT, PHP, DSS, SPP, and TPS), in addition to GGG and GPG, were 4.0-6.2 kcal/mol less stable than the wild-type ribonuclease T1. However, these circular permuted variants were only 0.4-2.6 kcal/mol less stable than the direct parent protein that is missing the disulfide bond connecting residues 2 and 10. The most stable linking peptide was HPD.     

Tumor-derived cytokines dysregulate macrophage interferon-gamma responsiveness and interferon regulatory factor-8 expression

Tumors can evade immune responses through suppressor signals that dysregulate host effector cell function. In this study we demonstrate that tumor-derived suppressor molecules impede host antitumor immune activity through dysregulation of multiple macrophage (Mphi) pathways, including suppressed production of cytotoxic and immunostimulatory agents and impaired expression of the interferon regulatory factor-8 (IRF-8) protein, a critical transducer of interferon-gamma-mediated activation pathways. The tumor-derived immunosuppressive cytokines interleukin-10 and transforming growth factor-beta(1) constrain IRF-8 production by normal Mphis, regardless of priming, and IRF-8 is also dysregulated in primary Mphis from tumor-burdened hosts. Collectively, these data describe a new mechanism by which tumors disrupt immune function and suggest that abrogation of tumor-derived immunoregulatory factors in situ can restore immune function and enhance antitumor efficacy.     

The molecular machinery for lysosome biogenesis

The lysosome serves as a site for delivery of materials targeted for removal from the eukaryotic cell. The mechanisms underlying the biogenesis of this organelle are currently the subject of renewed interest due to advances in our understanding of the protein sorting machinery. Genetic model systems such as yeast and Drosophila have been instrumental in identifying both protein and lipid components of this machinery. Importantly, many of these components, as well as the processes in which they are involved, are proving conserved in mammals. Other recently identified components, however, appear to be unique to higher eukaryotes. BioEssays 23:333-343, 2001. Published 2001 John Wiley & Sons, Inc.     

Equivalent genetic roles for bmp7/snailhouse and bmp2b/swirl in dorsoventral pattern formation

A bone morphogenetic protein (BMP) signaling pathway acts in the establishment of the dorsoventral axis of the vertebrate embryo. Here we demonstrate the genetic requirement for two different Bmp ligand subclass genes for dorsoventral pattern formation of the zebrafish embryo. From the relative efficiencies observed in Bmp ligand rescue experiments, conserved chromosomal synteny, and isolation of the zebrafish bmp7 gene, we determined that the strongly dorsalized snailhouse mutant phenotype is caused by a mutation in the bmp7 gene. We show that the original snailhouse allele is a hypomorphic mutation and we identify a snailhouse/bmp7 null mutant. We demonstrate that the snailhouse/bmp7 null mutant phenotype is identical to the presumptive null mutant phenotype of the strongest dorsalized zebrafish mutant swirl/bmp2b, revealing equivalent genetic roles for these two Bmp ligands. Double mutant snailhouse/bmp7; swirl/bmp2b embryos do not exhibit additional or stronger dorsalized phenotypes, indicating that these Bmp ligands do not function redundantly in early embryonic development. Furthermore, overexpression experiments reveal that Bmp2b and Bmp7 synergize in the ventralization of wild-type embryos through a cell-autonomous mechanism, suggesting that Bmp2b/Bmp7 heterodimers may act in vivo to specify ventral cell fates in the zebrafish embryo.     

Membrane topology of the Mep/Amt family of ammonium transporters

The Mep/Amt proteins constitute a new family of transport proteins that are ubiquitous in nature. Members from bacteria, yeast and plants have been identified experimentally as high-affinity ammonium transporters. We have determined the topology of AmtB, a Mep/Amt protein from Escherichia coli, as a representative protein for the complete family. This was established using a minimal set of AmtB-PhoA fusion proteins with a complementary set of AmtB-LacZ fusions. These data, accompanied by an in silico analysis, indicate that the majority of the Mep/Amt proteins contain 11 membrane-spanning helices, with the N-terminus on the exterior face of the membrane and the C-terminus on the interior. A small subset, including E. coli AmtB, probably have an additional twelfth membrane-spanning region at the N-terminus. Addition of PhoA or LacZ alpha-peptide to the C-terminus of E. coli AmtB resulted in complete loss of transport activity, as judged by measurements of [14C]-methylammonium uptake. This C-terminal region, along with four membrane-spanning helices, contains multiple residues that are conserved within the Mep/Amt protein family. Structural modelling of the E. coli AmtB protein suggests a number of secondary structural features that might contribute to function, including a putative ammonium binding site on the periplasmic face of the membrane at residue Asp-182. The implications of these results are discussed in relation to the structure and function of the related human Rhesus proteins.     

An in vitro approach for the characterization of the cycling B cell response

Summary Because isolation of sufficient numbers of cycling, germinal center B cells from mice for biochemical characterization of BCR-derived signals can be problematic, we have designed an experimental approach for generating large numbers of cycling B cells for further study. In the experiments reported here, small, resting B cells were polyclonally stimulated with lipopolysaccharide (LPS), and cycling B cells isolated as two bands on three-step Percoll gradients. Cycling B cells isolated at Days 2, 4, or 6 of preactivation showed an increased expression of Fas receptor and peanut agglutinin binding, with a concomitant decrease in sIgD positivity. These cells phenotypically resembled extrafollicular or early germinal center B cells. These cycling B cells were used to study the functional consequences of differential signaling through the BCR. Strong cross-linking of BCR, by restimulation of cycling normal B cells with either immobilized or soluble F(ab’)₂ anti-μ and cycling hen egg lysozyme (HEL) transgenic B cells with either soluble or immobilized HEL, extended cellular proliferation by 2–3 d. In contrast, cycling B cells either restimulated with soluble, whole anti-μ (to mimic binding of soluble immune complexes) or cultured in the absence of restimulation (to mimic cycling B cells not competitive for antigen) resulted in the rapid exit of the cells from cycle. This system will enable the molecular and biochemical characterization of signal delivery to cycling B cells.     

p21WAF1/CIP1 RNA Expression in Highly HIV-1 Exposed,Uninfected Individuals

Some individuals remain HIV-1 antibody and PCR negative after repeated exposures to the virus, and are referred to as HIV-exposed seronegatives (HESN). However, the causes of resistance to HIV-1 infection in cases other than those with a homozygous CCR5Δ32 deletion are unclear. We hypothesized that human p21^WAF1/CIP1 (a cyclin-dependent kinase inhibitor) could play a role in resistance to HIV-1 infection in HESN, as p21 expression has been associated with suppression of HIV-1 in elite controllers and reported to block HIV-1 integration in cell culture. We measured p21 RNA expression in PBMC from 40 HESN and 40 low exposure HIV-1 seroconverters (LESC) prior to their infection using a real-time PCR assay. Comparing the 20 HESN with the highest exposure risk (median = 111 partners/2.5 years prior to the 20 LESC with the lowest exposure risk (median = 1 partner/2.5 years prior), p21 expression trended higher in HESN in only one of two experiments (P = 0.11 vs. P = 0.80). Additionally, comparison of p21 expression in the top 40 HESN (median = 73 partners/year) and lowest 40 LESC (median = 2 partners/year) showed no difference between the groups (P = 0.84). There was a weak linear trend between risk of infection after exposure and increasing p21 gene expression (R² = 0.02, P = 0.12), but again only in one experiment. Hence, if p21 expression contributes to the resistance to viral infection in HESN, it likely plays a minor role evident only in those with extremely high levels of exposure to HIV-1.