Targeted deletion of the integrin beta4 signaling domain suppresses laminin-5-dependent nuclear entry of mitogen-activated protein kinases and NF-kappaB, causing defects in epidermal growth and migration

The alpha6beta4 integrin-a laminin-5 receptor-mediates assembly of hemidesmosomes and recruitment of Shc and phosphoinositide 3-kinase through the unique cytoplasmic extension of beta4. Mice carrying a targeted deletion of the signaling domain of beta4 develop normally and do not display signs of skin fragility. The epidermis of these mice contains well-structured hemidesmosomes and adheres stably to the basement membrane. However, it is hypoplastic due to reduced proliferation of basal keratinocytes and undergoes wound repair at a reduced rate. Keratinocytes from beta4 mutant mice undergo extensive spreading but fail to proliferate and migrate in response to epidermal growth factor (EGF) on laminin-5. EGF causes significant phosphorylation of extracellular signal-regulated kinase (ERK) and Jun N-terminal protein kinase (JNK) and phosphorylation and degradation of IkappaB in beta4 mutant cells adhering to laminin-5. Unexpectedly, however, ERK, JNK, and NF-kappaB remain in the cytoplasm in beta4 mutant cells on laminin-5, whereas they enter effectively into the nucleus in the same cells on fibronectin or in wild-type cells on both matrix proteins. Inhibitor studies indicate that alpha6beta4 promotes keratinocyte proliferation and migration through its effect on NF-kappaB and P-JNK. These findings provide evidence that beta4 signaling promotes epidermal growth and wound healing through a previously unrecognized effect on nuclear translocation of NF-kappaB and mitogen-activated protein kinases.     

Blood group antigens: molecules seeking a function?

The blood group antigens have been dismissed by some researchers as merely ‘icing on the cake’ of glycoprotein structures. The fact that there are no lethal mutations and individuals have been described lacking ABO, H and Lewis antigens seems to lend weight to the argument. This paper reviews the research which suggests that these antigens do indeed have function and argues that blood group antigens play important roles in modulation of protein activity, infection and cancer. It explores the evidence and poses questions as to the relevance and implications of the results. This revised version was published online in November 2006 with corrections to the Cover Date.     

Characterization of the Particles of Purified κ-Casein: Trypsin as a Probe of Surface-Accessible Residues

κ-Casein as purified from bovine milk exhibits a rather unique disulfide bonding pattern as revealed by SDS–PAGE. The disulfide-bonded caseins present range from dimer to octamer and above and preparations contain about 10% monomer. All of these heterogeneous polymers, however, self-associate into nearly spherical particles with an average diameter of 13 nm at pH 8.0, as revealed by negatively stained transmission electron micrographs and dynamic light scattering. The weight-average molecular weight of the aggregates at pH 8.0, as judged by analytical ultracentrifugation, is 648,000. Trypsin digestion at pH 8.0 was used to probe the surface groups of the κ-casein A polymers. The reaction with trypsin was rapid and the peptides liberated were identified by separation with reverse-phase HPLC, amino acid analysis, and protein sequencing. The most rapidly released peptides (t _1/2 < 30 sec) were from cleavage at Arg 97 and Lys residues 111 and 112. These results suggest a surface orientation for these residues, and the data are in accord with earlier proposed 3D predictive models for κ-casein. It is speculated that Arg 97, together with adjacent His residues (98 and 100) and Lys residues 111 and 112, form two positively charged clusters on the surface of the otherwise negatively charged casein. These clusters bracket the neutral chymosin cleavage site (whose hydrolysis triggers a well-known digestive process) and so these clusters may facilitate docking of the substrate caseins with chymosin.     

Diets in Transition: Hunter-Gatherer to Station Diet and Station Diet to the Self-Select Store Diet

The transition of Australian Aborigines from a diet based on hunting and gathering to an essentially Western diet has been proceeding for almost 200 years, but in some regions was greatly delayed. In 1966/1967 G ould (1980) studied operational aspects of hunting-gathering in desert regions of northwestern Australia and recorded sufficient quantitative and species-specific data to allow definition of the diet over 5 months. By 1966, many Aboriginal people in that region had been displaced from their hunting grounds and were living in camps on cattle stations or in missions. Aboriginal diet on cattle stations in the north-westwas studied in 1951 (Commonwealth Departmentof Health) and shown to consist chiefly of fresh meat, wheaten flour, and sugar with small amounts of vegetables and dairy products. With the granting of citizenship in 1967, most Aborigines were dismissed from cattle stations and were moved first to town camps and later formed remote Aboriginal communities. Studies in the 1980s showed that the self-selected diet in such communities reflected the station diet to a greater extent than the traditional diet. Quantitative presentation of the above three diets, in terms both of foods and of major nutrients, show that many of the dietary inadequacies of the station diet identified in 1951 still persisted in self-selected Aboriginal diets in the 1980s. A comparison of the three diets with a modern recommended dietsupports the nutritional adequacy of the hunter-gatherer diet. Traditional cultural values assigned to food preferences continued to influence food choices in all three diets.     

Multiple drugs and multiple targets: An analysis of the electrostatic determinants of binding between non‐nucleoside HIV‐1 reverse transcriptase inhibitors and variants of HIV‐1 RT

We present a systematic, computational analysis of the electrostatic component of binding of three HIV‐1 RT inhibitors—nevirapine (NVP), efavirenz (EFV), and the recently approved rilpivirine (RPV)—to wild‐type (WT) and mutant variants of RT. Electrostatic charge optimization was applied to determine how suited each molecule's charge distribution is for binding WT and individual mutants of HIV‐1 RT. Although the charge distributions of NVP and EFV are rather far from being optimal for tight binding, RPVs charge distribution is close to the theoretical, optimal charge distribution for binding WT HIV‐1 RT, although slight changes in charge can dramatically impact binding energetics. Moreover, toward the L100I/K103N double mutant, RPVs charge distribution is quite far from optimal. We also determine the contributions of chemical moieties on each molecule toward the electrostatic component of binding and show that different regions of a drug molecule may be used for recognition by different RT variants. The electrostatic contributions of certain RT residues toward drug binding are also computed to highlight critical residues for each interaction. Finally, the charge distribution of RPV is optimized to promiscuously bind to three RT variants rather than to each one in turn, with the resulting charge distribution being a compromise between the optimal charge distributions to each individual variant. Taken together, this work demonstrates that even in a binding site considered quite hydrophobic, electrostatics play a subtle yet varying role that must be considered in designing next‐generation molecules that recognize rapidly mutating targets. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Complex interactions between GSK3 and aPKC in Drosophila embryonic epithelial morphogenesis

Generally, epithelial cells must organize in three dimensions to form functional tissue sheets. Here we investigate one such sheet, the Drosophila embryonic epidermis, and the morphogenetic processes organizing cells within it. We report that epidermal morphogenesis requires the proper distribution of the apical polarity determinant aPKC. Specifically, we find roles for the kinases GSK3 and aPKC in cellular alignment, asymmetric protein distribution, and adhesion during the development of this polarized tissue. Finally, we propose a model explaining how regulation of aPKC protein levels can reorganize both adhesion and the cytoskeleton.     

Absolute quantification of human tear lactoferrin using multiple reaction monitoring technique with stable-isotopic labeling

The mass spectrometry technique of multiple reaction monitoring (MRM) was used to quantify and compare the expression level of lactoferrin in tear films among control, prostate cancer (CaP), and benign prostate hyperplasia (BPH) groups. Tear samples from 14 men with CaP, 15 men with BPH, and 14 controls were analyzed in the study. Collected tears (2 μl) of each sample were digested with trypsin overnight at 37 °C without any pretreatment, and tear lactoferrin was quantified using a lactoferrin-specific peptide, VPSHAVVAR, both using natural/light and isotopic-labeled/heavy peptides with MRM. The average tear lactoferrin concentration was 1.01 ± 0.07 μg/μl in control samples, 0.96 ± 0.07 μg/μl in the BPH group, and 0.98 ± 0.07 μg/μl in the CaP group. Our study is the first to quantify tear proteins using a total of 43 individual (non-pooled) tear samples and showed that direct digestion of tear samples is suitable for MRM studies. The calculated average lactoferrin concentration in the control group matched that in the published range of human tear lactoferrin concentration measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the lactoferrin was stably expressed across all of the samples, with no significant differences being observed among the control, BPH, and CaP groups.     

Cutting edge: CD4+CD25+ regulatory T cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis

Autoreactive CD4(+) T cells exist in normal individuals and retain the capacity to initiate autoimmune disease. The current study investigates the role of CD4(+)CD25(+) T-regulatory (T(R)) cells during autoimmune disease using the CD4(+) T cell-dependent myelin oligodendrocyte glycoprotein (MOG)-specific experimental autoimmune encephalomyelitis model of multiple sclerosis. In vitro, T(R) cells effectively inhibited both the proliferation of and cytokine production by MOG(35-55)-specific Th1 cells. In vivo, adoptive transfer of T(R) cells conferred significant protection from clinical experimental autoimmune encephalomyelitis which was associated with normal activation of autoreactive Th1 cells, but an increased frequency of MOG(35-55)-specific Th2 cells and decreased CNS infiltration. Lastly, transferred T(R) cells displayed an enhanced ability to traffic to the peripheral lymph nodes and expressed increased levels of the adhesion molecules ICAM-1 and P-selectin that may promote functional interactions with target T cells. Collectively, these findings suggest that T(R) cells contribute notably to the endogenous mechanisms that regulate actively induced autoimmune disease.