Derivatives of Aminoquinones with N-protected Amino Acids

The synthesis of derivatives of aminoquinones with N-protected amino acids is reported here. 2-Amino-1,4-benzoquinone and 2-amino-1,4-naphthoquinone, prepared by the azide method in yields of 60 and 95% respectively, were coupled with N-Boc-protected amino acids including glycine, serine, proline and tyrosine, to give the correspondening derivatives. N,N'-Diisopropylcarbodiimide/1-hydroxybenzotriazole or N,N'-dicyclohexylcarbodiimide/HOBt used as coupling reagents provided the expected products in satisfactory yields and purities as supported by TLC, HPLC and spectral analysis.     

Scavenger receptor class B type I is expressed in cultured keratinocytes and epidermis. Regulation in response to changes in cholesterol homeostasis and barrier requirements.

Cholesterol is a key lipid in the stratum corneum, where it is critical for permeability barrier homeostasis. The epidermis is an active site of cholesterol synthesis, but inhibition of epidermal cholesterol synthesis with topically applied statins only modestly affects epidermal permeability barrier function, suggesting a possible compensatory role for extraepidermal cholesterol. Scavenger receptor class B type I (SR-BI) is a recently described cell surface receptor for high density lipoproteins (HDL) that mediates the selective uptake of cholesterol esters from circulating HDL. In the present study, we demonstrate that SR-BI is present in cultured human keratinocytes and that calcium-induced differentiation markedly decreases SR-BI levels. Additionally, the cell association of [(3)H]cholesterol-labeled HDL decreased in differentiated versus undifferentiated keratinocytes. Furthermore, the inhibition of cholesterol synthesis with simvastatin resulted in a 3-4-fold increase in both SR-BI mRNA and protein levels, whereas conversely, addition of 25-hydroxycholesterol suppressed SR-BI levels by approximately 50%. SR-BI mRNA is also expressed in murine epidermis, increasing by 50% in parallel with cholesterol requirements following acute barrier disruption. Because the increase is completely blocked by occlusion with a vapor-impermeable membrane, changes in epidermal SR-BI expression are regulated specifically by barrier requirements. Lastly, using immunofluorescence we demonstrated that SR-BI is present in human epidermis predominantly in the basal layer and increases following barrier disruption. In summary, the present study demonstrates first that SR-BI is expressed in keratinocytes and regulated by cellular cholesterol requirements, suggesting that it plays a role in keratinocyte cholesterol homeostasis. Second, the increase in SR-BI following barrier disruption suggests that SR-BI expression increases to facilitate cholesterol uptake leading to barrier restoration.     

Characterization of Microbial Activities and U Reduction in a Shallow Aquifer Contaminated by Uranium Mill Tailings

A characterization of the Shiprock, NM, uranium mill tailing site focused on the geochemical and microbiological factors governing in-situ uranium-redox reactions. Groundwater and aqueous extracts of sediment samples contained a wide concentration range of sulfate, nitrate, and U(VI) with median values of 21.2 mM, 16.1 µM, and 2.7 µM, respectively. Iron(III) was not detected in groundwater, but a median value of 0.3 mM in sediment extracts was measured. Bacterial diversity down gradient from the disposal pile reflected the predominant geochemistry with relatively high numbers of sulfate- and nitrate-reducing microorganisms, and smaller numbers of acetogenic, methanogenic, nitrate-dependent Fe(II)-oxidizing, Fe(III)-reducing, and sulfide-oxidizing bacteria. In aquifer slurry incubations, nitrate reduction was always preferred and had a negative impact on sulfate-, Fe(III)-, and U-reduction rates. We also found that sulfate-reduction rates decreased sharply in the presence of clay, while Fe(III)-reduction increased with no clear impact on U reduction. In the absence of clay, iron and sulfate reduction correlated with concentrations of Fe(III) and sulfate, respectively. Rates of U(VI) loss did not correlate with the concentration of any electron acceptor. With the exception of Fe(III), electron donor amendment was largely unsuccessful in stimulating electron acceptor loss over a 2-week incubation period, suggesting that endogenous forms of organic matter were sufficient to support microbial activity. Our findings suggest that efforts to accelerate biological U reduction should initially focus on stimulating nitrate removal.     

Control of dehydrodiferulate cross-linking in pectins from sugar-beet tissues

Pectins were extracted from roots, petioles and leaves of sugar beet, and cross-linked using hydrogen peroxide and peroxidase. The effects on dehydrodiferulate formation were monitored by HPLC and TLC. Dehydrodimers were formed in different proportions to those found in vivo. There was a net loss of around 50% of the phenolic groups (monomers plus dimers) during dimerisation. Gel filtration showed that root and petiole pectin, but not leaf pectin, increased in molecular weight during cross-linking. The effects of varying the cross-linking conditions were investigated, and it was found that hydrogen peroxide concentration was the most important factor in controlling both the type and amount of dehydrodiferulate formed.     

A Novel Familial Mutation in the PCSK1 Gene That Alters the Oxyanion Hole Residue of Proprotein Convertase 1/3 and Impairs Its Enzymatic Activity

Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, encoding the neuroendocrine convertase 1 precursor (PC1/3) which was recently reported as a cause of Congenital Diarrhea Disorder (CDD). The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred.     

Clonal Relationship among Atypical Enteropathogenic Escherichia coli Strains Isolated from Different Animal Species and Humans

Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.Enteropathogenic Escherichia coli (EPEC) strains are among the major causes of infantile diarrhea in developing countries (71) and can be classified as typical and atypical, depending on the presence or absence of the E. coli adherence factor plasmid (pEAF), respectively (39).The pathogenesis of EPEC resides in the ability to cause the attaching and effacing (A/E) lesion in the gut mucosa of human or animal hosts, leading to diarrheal illness (40). The genes responsible for the A/E lesion formation are located in a chromosomal pathogenicity island of ∼35 kb, known as the locus of enterocyte effacement (LEE) (23, 47). LEE encodes an adhesin called intimin (38), its translocated receptor (Tir) (42), components of a type III secretion system (36), and effector molecules, named E. coli-secreted proteins (Esp proteins) (41). These virulence factors have a crucial role in A/E lesion formation, and their detection in EPEC strains is an indicator of their potential to produce these lesions (19, 56).Atypical EPEC strains have been associated with diarrhea outbreaks in developed countries (31, 73, 77) and with sporadic cases of diarrhea in developing and developed countries (1, 12, 26, 52, 55). At present, the prevalence of atypical EPEC is higher than that of typical EPEC in several countries (1, 12, 26, 52, 55, 65).Different from the situation in developed countries, where atypical EPEC outbreaks and sporadic infections are associated with children and adults, atypical EPEC infection in Brazil is mainly associated with children''s illnesses (32, 71).Typical EPEC strains are rarely isolated from animals, and humans are the major natural reservoir for these pathogens (14, 32, 53, 71). In contrast, atypical EPEC strains are present in both healthy and diseased animals (dog, monkey, cats, and bovines) and humans (4, 6, 18, 28, 71). Some studies have associated pets and farm and wild animals as reservoirs and infection sources of atypical EPEC strains for humans (32). However, these studies did not compare atypical EPEC strains isolated from humans and animals by gold-standard molecular methods like multilocus sequence typing (MLST) or pulsed-field gel electrophoresis (PFGE) (15, 35, 43, 53). For this reason, there are some doubts about whether atypical EPEC strains isolated from animals represent risks for human health and whether animals really play the role of reservoirs of atypical EPEC.The aim of this study was to compare atypical EPEC strains isolated from humans and different animals, including pets (cats and dogs), farm animals (bovines, ovines, and rabbits), and wild animals (monkeys), by molecular phylogenetic techniques to verify the role of animals as reservoirs of and sources of infection with atypical EPEC in humans.     

Structural and mutational studies of the catalytic domain of colicin E5: a tRNA-specific ribonuclease

Colicin E5 specifically cleaves four tRNAs in Escherichia coli that contain the modified nucleotide queuosine (Q) at the wobble position, thereby preventing protein synthesis and ultimately resulting in cell death. Here, the crystal structure of the catalytic domain of colicin E5 (E5-CRD) from E. coli was determined at 1.5 A resolution. Unexpectedly, E5-CRD adopts a core folding with a four-stranded beta-sheet packed against an alpha-helix, seen in the well-studied ribonuclease T1 despite a lack of sequence similarity. Beyond the core catalytic domain, an N-terminal helix, a C-terminal beta-strand and loop, and an extended internal loop constitute an RNA binding cleft. Mutational analysis identified five amino acids that were important for tRNA substrate binding and cleavage by E5-CRD. The structure, together with the mutational study, allows us to propose a model of colicin E5-tRNA interactions, suggesting the molecular basis of tRNA substrate recognition and the mechanism of tRNA cleavage by colicin E5.     

A septin GTPase scaffold of dynein–dynactin motors triggers retrograde lysosome transport

The metabolic and signaling functions of lysosomes depend on their intracellular positioning and trafficking, but the underlying mechanisms are little understood. Here, we have discovered a novel septin GTPase–based mechanism for retrograde lysosome transport. We found that septin 9 (SEPT9) associates with lysosomes, promoting the perinuclear localization of lysosomes in a Rab7-independent manner. SEPT9 targeting to mitochondria and peroxisomes is sufficient to recruit dynein and cause perinuclear clustering. We show that SEPT9 interacts with both dynein and dynactin through its GTPase domain and N-terminal extension, respectively. Strikingly, SEPT9 associates preferentially with the dynein intermediate chain (DIC) in its GDP-bound state, which favors dimerization and assembly into septin multimers. In response to oxidative cell stress induced by arsenite, SEPT9 localization to lysosomes is enhanced, promoting the perinuclear clustering of lysosomes. We posit that septins function as GDP-activated scaffolds for the cooperative assembly of dynein–dynactin, providing an alternative mechanism of retrograde lysosome transport at steady state and during cellular adaptation to stress.     

T-cell recognition of HLA-DQ2-bound gluten peptides can be influenced by an N-terminal proline at p-1

Recent research has implicated a large number of gluten-derived peptides in the pathogenesis of celiac disease, a preponderantly HLA-DQ2-associated disorder. Current evidence indicates that the core of some of those peptides is ten amino acids long, while HLA class II normally accommodates nine amino acids in the binding groove. We have now investigated this in detail, using gluten-specific T-cell clones, HLA-DQ2-specific peptide-binding assays and molecular modelling. T-cell recognition of both a -gliadin peptide and a low-molecular-weight glutenin peptide was found to be strictly dependent on a ten-amino acids-long peptide. Subsequent peptide-binding studies indicated that the glutenin peptide bound in a conventional p1/p9 register, with an additional proline at p-1. Testing of substitution analogues demonstrated that the nature of the amino acid at p-1 strongly influenced T-cell recognition of the peptide. Moreover, molecular modelling confirmed that the glutenin peptide binds in a p1/p9 register, and that the proline at p-1 points upward towards the T-cell receptor. Database searches indicate that a large number of potential T-cell stimulatory gluten peptides with an additional proline at relative position p-1 exist, suggesting that the recognition of other gluten peptides may depend on this proline as well. This knowledge may be of importance for the identification of additional T-cell stimulatory gluten peptides and the design of a peptide-based, tolerance-inducing therapy.     

An integrated genetic and cytogenetic map for the Mediterranean fruit fly, <Emphasis Type="Italic">Ceratitis capitata</Emphasis>, based on microsatellite and morphological markers

A genetic map based on microsatellite polymorphisms and visible mutations of the Mediterranean fruit fly (medfly), Ceratitis capitata is presented. Genotyping was performed on single flies from several backcross families. The map is composed of 67 microsatellites and 16 visible markers distributed over four linkage groups. Fluorescence in situ hybridization of selected microsatellite markers on salivary gland polytene chromosomes allowed the alignment of these groups to the second, fourth, fifth and sixth chromosome. None of the markers tested showed segregation either with the X or the third chromosome. However, this map constitutes a substantial starting point for a detailed genetic map of C. capitata. The construction of an integrated map covering the whole genome should greatly facilitate genetic studies and future genome sequence projects of the species.