Measurement of 7-dehydrocholesterol and cholesterol in hair can be used in the diagnosis of Smith-Lemli-Opitz syndrome

7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50–4000 ng/mg and 30–6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Geographic isolation and evolution of Mediterranean endemic Cyclamen: insights from chloroplast trnL (UAA) intron sequence variation

 In this study we construct a phylogenetic hypothesis for the relatedness among disjunct subspecies of Cyclamen repandum and its two allopatric congeners, C. creticum and C. balearicum in order to examine the evolutionary divergence of currently isolated populations across the western Mediterranean. The most parsimonious phylogenetic tree obtained from sequencing the cpDNA trnL (UAA) intron suggests a major phylogeographic divide in southern Greece between two clades. The first clade comprises samples of C. repandum subsp. peloponnesiacum (from the Peloponnese) and C. creticum (from Crete). The second comprises samples of C. repandum subsp. repandum (from Croatia, Italy, southern France, Corsica, Sardinia and Sicily), C. repandum subsp. rhodense (from Rhodes and Kos) and C. balearicum (from the Balearic Islands and southern France). These data suggest that C. creticum has evolved in allopatry from C. repandum subsp. peloponnesiacum and that C. balearicum and C. repandum ssp. rhodense have diverged from C. repandum subsp. repandum at its western and eastern distribution limits. At one small site on Corsica, a population of C. repandum may have introgressed with relictual populations of C. balearicum. These divergence patterns illustrate how a phylogenetic perspective can be used to better understand the evolution of endemism in the Mediterranean flora. Received February 19, 2001 Accepted August 22, 2001     

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

Assessment and comparison of neural morphology through metrical feature extraction and analysis in neuron and neuron–glia cultures

The morphology of dissociated single cerebellar Purkinje cells obtained from wild-type P1 CD1 mice was assessed in the absence and in the presence of glia. A dedicated noninvasive technique based on optical microscopy was developed. Image processing algorithms were implemented to extract metrical features characterizing cell structure and dendritic arborization. The morphological features were analyzed in order to identify quantitative differences in Purkinje cell morphology due to interactions with astrocytes.     

Retrograde Traffic Out of the Yeast Vacuole to the TGN Occurs via the Prevacuolar/Endosomal Compartment

A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid–containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.     

Historical and recent evidence for close relationships among Rickettsia parkeri,R. conorii,R. africae,and R. sibirica: Implications for rickettsial taxonomy

Rickettsia parkeri, a member of the spotted fever group rickettsias, was first described in 1939 and was thought to be non‐pathogenic until recently, when it was found to cause a spotted fever‐like illness in humans and areas of necrosis (eschars) at the sites of tick bites. Accordingly, there is currently much interest in this emerging pathogen. In this study, all published articles concerning R. parkeri were reviewed and analyzed for evidence of relatedness among this agent and other spotted fever group (SFG) rickettsiae which also produce similar clinical syndromes and/or eschars, including R. conorii, R. africae, and R. sibirica. A synthesis of the historical (antigenic) and recent (molecular) data supporting a phylogenetic sub‐grouping of these SFG organisms is presented and comments are offered about the taxonomy of rickettsial organisms in general, and R. parkeri in particular.     

Mechanisms and development of self–other distinction in dyads and groups

This opinion piece offers a commentary on the four papers that address the theme of the development of self and other understanding with a view to highlighting the important contribution of developmental research to understanding of mechanisms of social cognition. We discuss potential mechanisms linking self–other distinction and empathy, implications for grouping motor, affective and cognitive domains under a single mechanism, applications of these accounts for joint action and finally consider self–other distinction in group versus dyadic settings.     

The control of flower initiation by gibberellin in Helianthus annuus L. (sunflower), a non-photoperiodic plant

The involvement of gibberellins in the control of flowering of sunflower was studied by direct application of GA₃ to the apex of the plants, analysis of the endogenous levels of gibberellin-like substances at different plant ages, and indirectly by the application of paclobutrazol, an inhibitor of gibberellin synthesis. GA₃ speeded-up flower initiation and floral apex development. The time of GA₃ application was more critical than the amount of GA₃ applied. The endogenous levels of gibberellin-like compounds increased significantly by day 15 after sowing. The application of paclobutrazol markedly delayed floral initiation and this effect was also depedent on plant age. Both GA₃ and paclobutrazol had their greatest effects between 10 and 20 days after sowing suggesting that an increase in gibberellins in that time period plays a role in floral initiation.     

Trace elements in rock salt and their bioavailability estimated from solubility in acid

In this study, 18 partly commercially available samples of rock salt from Austria, Germany, Pakistan, Poland, Switzerland, and Ukraine were investigated with respect to their content of trace elements using instrumental neutron activation analysis. Elements detected were Al, Ba, Br, Ca, Ce, Cl, Co, Cr, Cs, Eu, Fe, Hf, La, Mn, Na, Rb, Sb, Sc, Sm, Sr, Ta, Tb, Th, and Zn, some of them only in individual cases. An estimation of the bioavailability of these trace elements was performed by dissolving an equivalent of the sodium chloride samples in diluted hydrochloric acid (simulating stomach acid), filtering off the insoluble components, and analyzing the evaporated filtrate. It could be shown that in most cases bioactive trace elements like Fe can be found in rock salt in the form of almost insoluble compounds and are therefore not significantly bioavailable, whereas thorium, for example, was partly bioavailable in two cases. A significant contribution to the recommended daily intake of metal trace elements by using rock salt for nutrition can be excluded.