Cholesterol efflux from larval Manduca sexta fat body in vitro: high-density lipophorin as the acceptor

The objective of this study was to characterize the transfer of cholesterol from Manduca sexta larvae fat body to high-density lipophorin. [³H]-Cholesterol-labeled fat body was incubated with lipophorin under different conditions and cholesterol transfer was determined. Transfer rate exhibited a hyperbolic dependency on lipophorin concentration with an apparent K_m of 3.6 mg/ml, which is consistent with either an aqueous diffusion mechanism of cholesterol transfer or a receptor-mediated process. Several results, including the high K_m, the high activation energy, and the lack of Ca²⁺ dependence favor aqueous diffusion model. In addition, anti-lipid transfer particle antibodies had only a small inhibitory effect, suggesting it is not involved in cholesterol transfer. However, the transfer was inhibited in the presence of suramin, which would be consistent with a receptor-mediated process. The effects of suramin may be complex because it can change membrane properties when bound to the lipophorin receptor and affect the rate of cholesterol desorption. The preponderance of data suggests that the export of cholesterol from fat body to lipophorin follows a simple aqueous diffusion pathway. Although we cannot completely exclude some contribution from a receptor-mediated pathway, it seems that if such a pathway were present, it represents a minor route.     

Missense mutations in GJB2 encoding connexin-26 cause the ectodermal dysplasia keratitis-ichthyosis-deafness syndrome

Keratitis-ichthyosis-deafness syndrome (KID) is a rare ectodermal dysplasia characterized by vascularizing keratitis, profound sensorineural hearing loss (SNHL), and progressive erythrokeratoderma, a clinical triad that indicates a failure in development and differentiation of multiple stratifying epithelia. Here, we provide compelling evidence that KID is caused by heterozygous missense mutations in the connexin-26 gene, GJB2. In each of 10 patients with KID, we identified a point mutation leading to substitution of conserved residues in the cytoplasmic amino terminus or first extracellular domain of Cx26. One of these mutations was detected in six unrelated sporadic case subjects and also segregated in one family with vertical transmission of KID. These results indicate the presence of a common, recurrent mutation and establish its autosomal dominant nature. Cx26 and the closely related Cx30 showed differential expression in epidermal, adnexal, and corneal epithelia but were not significantly altered in lesional skin. However, mutant Cx26 was incapable of inducing intercellular coupling in vitro, which indicates its functional impairment. Our data reveal striking genotype-phenotype correlations and demonstrate that dominant GJB2 mutations can disturb the gap junction system of one or several ectodermal epithelia, thereby producing multiple phenotypes: nonsyndromic SNHL, syndromic SNHL with palmoplantar keratoderma, and KID. Decreased host defense and increased carcinogenic potential in KID illustrate that gap junction communication plays not only a crucial role in epithelial homeostasis and differentiation but also in immune response and epidermal carcinogenesis.     

Beta-cyclodextrin facilitates cholesterol efflux from larval Manduca sexta fat body and midgut in vitro

The ability of 2-hydroxypropyl-β-cyclodextrin (HPβCD) and methyl-β-cyclodextrin (MβCD) to promote cholesterol efflux from [³H]cholesterol-labeled larval Manduca sexta fat body and midgut was tested. In fat body, both β-cyclodextrins induced a two-phase efflux of cholesterol. The first rapid phase depended on cyclodextrin concentration and was more rapid for MβCD than for HPβCD. The second, slower, phase was independent of cyclodextrin concentration and type. In midgut, only the concentration-dependent phase was observed; the rate constants are approximately 85% slower than for fat body. In both cases, a low activation energy for transfer was observed, consistent with a collision mechanism where cyclodextrin interacts directly with cholesterol in plasma membrane to affect transfer. In fat body, the second slower phase is suggestive of a second pool of exchangeable cholesterol and most likely represents transfer of cholesterol from internal membranes or different lateral domains of the plasma membrane. The lack of this second phase in midgut suggests that midgut has only a single pool of exchangeable cholesterol. Although the rates are somewhat different, the overall kinetic pattern for cyclodextrin-mediated cholesterol transfer in insect fat body closely resembles that for vertebrate cells, while the single pool behavior of the midgut is not found in vertebrate cells.     

Desmoglein 4 in hair follicle differentiation and epidermal adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris

Cell adhesion and communication are interdependent aspects of cell behavior that are critical for morphogenesis and tissue architecture. In the skin, epidermal adhesion is mediated in part by specialized cell-cell junctions known as desmosomes, which are characterized by the presence of desmosomal cadherins, known as desmogleins and desmocollins. We identified a cadherin family member, desmoglein 4, which is expressed in the suprabasal epidermis and hair follicle. The essential role of desmoglein 4 in skin was established by identifying mutations in families with inherited hypotrichosis, as well as in the lanceolate hair mouse. We also show that DSG4 is an autoantigen in pemphigus vulgaris. Characterization of the phenotype of naturally occurring mutant mice revealed disruption of desmosomal adhesion and perturbations in keratinocyte behavior. We provide evidence that desmoglein 4 is a key mediator of keratinocyte cell adhesion in the hair follicle, where it coordinates the transition from proliferation to differentiation.     

Altered antigenicity is seen in the lipopolysaccharide profile of non-serotypeable Actinobacillus actinomycetemcomitans strains

Non-serotypeable Actinobacillus actinomycetemcomitans strains may be derived from the serotypeable ones. In the present study, we compared the outer membrane proteins (OMPs) and lipopolysaccharides (LPSs) of serotypeable and non-serotypeable A. actinomycetemcomitans strains (n=24) of the same genotype in the same subject (n=6) to find out if alterations on the cell-surface contribute to the non-serotypeability. Serotypeable and non-serotypeable A. actinomycetemcomitans strains showed great similarity in the OMP patterns both within and between subjects. Using serotype-specific antisera, clear immunoblotting LPS profiles in the O-antigenic region were seen in serotype b and c strains but not in non-serotypeable strains from the same subjects. The results suggest that changes in LPS lead to the altered antigenicity of non-serotypeable A. actinomycetemcomitans strains.     

Environmental Variation Generates Environmental Opportunist Pathogen Outbreaks

Many socio-economically important pathogens persist and grow in the outside host environment and opportunistically invade host individuals. The environmental growth and opportunistic nature of these pathogens has received only little attention in epidemiology. Environmental reservoirs are, however, an important source of novel diseases. Thus, attempts to control these diseases require different approaches than in traditional epidemiology focusing on obligatory parasites. Conditions in the outside-host environment are prone to fluctuate over time. This variation is a potentially important driver of epidemiological dynamics and affect the evolution of novel diseases. Using a modelling approach combining the traditional SIRS models to environmental opportunist pathogens and environmental variability, we show that epidemiological dynamics of opportunist diseases are profoundly driven by the quality of environmental variability, such as the long-term predictability and magnitude of fluctuations. When comparing periodic and stochastic environmental factors, for a given variance, stochastic variation is more likely to cause outbreaks than periodic variation. This is due to the extreme values being further away from the mean. Moreover, the effects of variability depend on the underlying biology of the epidemiological system, and which part of the system is being affected. Variation in host susceptibility leads to more severe pathogen outbreaks than variation in pathogen growth rate in the environment. Positive correlation in variation on both targets can cancel the effect of variation altogether. Moreover, the severity of outbreaks is significantly reduced by increase in the duration of immunity. Uncovering these issues helps in understanding and controlling diseases caused by environmental pathogens.     

Highly Diverse Cyanobactins in Strains of the Genus Anabaena

Cyanobactins are small, cyclic peptides recently found in cyanobacteria. They are formed through proteolytic cleavage and posttranslational modification of short precursor proteins and exhibit antitumor, cytotoxic, or multi-drug-reversing activities. Using genome project data, bioinformatics, stable isotope labeling, and mass spectrometry, we discovered novel cyclic peptides, anacyclamides, in 27 Anabaena strains. The lengths of the anacylamides varied greatly, from 7 to 20 amino acids. Pronounced sequence variation was also detected, and only one amino acid, proline, was present in all anacyclamides. The anacyclamides identified included unmodified proteinogenic or prenylated amino acids. We identified an 11-kb gene cluster in the genome of Anabaena sp. 90, and heterologous expression in Escherichia coli confirmed that this cluster was responsible for anacyclamide production. The discovery of anacyclamides greatly increases the structural diversity of cyanobactins.Cyanobacteria are a prolific source of secondary metabolites with potential as drug leads or useful probes for cell biology studies (23). They include biomedically interesting compounds, such as the anticancer drug lead cryptophycin (15), and environmentally problematic hepatotoxic peptides, such as microcystins and nodularins produced by bloom-forming cyanobacteria (23). Many of these compounds contain nonproteinogenic amino acids and modified peptides and are produced by nonribosomal peptide synthesis (23, 26).The cyanobactins are a new group of cyclic peptides recently found in cyanobacteria (4). They are assembled through posttranslational proteolytic cleavage and head-to-tail macrocyclization of short precursor proteins. The cyanobactins are low-molecular-weight cyclic peptides that contain heterocyclized amino acids and can be prenylated or contain d-amino acids (3, 4). The cyanobactins that contain heterocyclized amino acids include patellamides, ulithiacyclamides, trichamide, tenuecyclamides, trunkamides, patellins, and microcyclamides and are synthesized in this manner (3, 4, 20, 24, 28). They possess antitumor, cytotoxic, and multi-drug-reversing activities and have potential as drug leads (4, 18, 20).Cyanobactins containing heterocyclized amino acids are found in a variety of cyanobacteria (4). A recent study demonstrated that the cyanobactin biosynthetic pathway is prevalent in planktonic bloom-forming cyanobacteria (14). However, the products of these gene clusters encoding new cyanobactins are unknown. Here we report discovery of a novel family of low-molecular-weight cyanobactins and show that these compounds are common in strains of the genus Anabaena. These anacyclamides exhibit pronounced length and sequence variation and contain unmodified or prenylated amino acids.     

Quantitative real-time PCR detection of toxic Nodularia cyanobacteria in the Baltic Sea

A specific quantitative real-time PCR (qPCR) method was developed for the quantification of hepatotoxin nodularin-producing Nodularia, one of the main bloom-forming cyanobacteria in the Baltic Sea. Specific PCR primers were designed for subunit F of the nodularin synthetase gene (ndaF), which encodes the NdaF subunit of the nodularin synthetase gene complex needed for nodularin production. The qPCR method was applied to water samples (a total of 120 samples) collected from the Baltic Sea in July 2004. As few as 30 ndaF gene copies ml(-1) of seawater could be detected, and thus, the method was very sensitive. The ndaF gene copy numbers and nodularin concentrations were shown to correlate in the Baltic seawater, indicating the constant production of nodularin by Nodularia. This qPCR method for the ndaF gene can be used for detailed studies of Nodularia blooms and their formation. ndaF gene copies and nodularin were detected mostly in the surface water but also in deeper water layers (down to 30 m). Toxic Nodularia blooms are not only horizontally but also vertically widely distributed, and thus, the Baltic fauna is extensively exposed to nodularin.     

Mice deficient in involucrin, envoplakin, and periplakin have a defective epidermal barrier

The cornified envelope is assembled from transglutaminase cross-linked proteins and lipids in the outermost epidermal layers and is essential for skin barrier function. Involucrin, envoplakin, and periplakin form the protein scaffold on which the envelope assembles. To examine their combined function, we generated mice deficient in all three genes. The triple knockouts have delayed embryonic barrier formation and postnatal hyperkeratosis (abnormal accumulation of cornified cells) resulting from impaired desquamation. Cornified envelopes form but are ultrastructurally abnormal, with reduced lipid content and decreased mechanical integrity. Expression of proteases is reduced and the protease inhibitor, serpina1b, is highly upregulated, resulting in defective filaggrin processing and delayed degradation of desmoglein 1 and corneodesmosin. There is infiltration of CD4⁺ T cells and a reduction in resident γδ⁺ T cells, reminiscent of atopic dermatitis. Thus, combined loss of the cornified envelope proteins not only impairs the epidermal barrier, but also changes the composition of T cell subpopulations in the skin.