On the variability of “flexion creases” in the distal plantar areas of newborns

The plantae of 83 male and 95 female healthy, full-term Caucasian newborns from Austria (gestational age 235–301 days) were photographed and the flexion creases in the distal part were examined. Analysis of the creases was done under four different aspects, the main evaluation based on a scheme of nine creases introduced by Schenk and Patzer (1959). Two transversal (No. 1 and No. 9) and three longitudinal (Nos. 3, 6, and 7) creases are always or predominantly present, a finding corresponding with the data of Schenk on 52 male and 48 female newborns from Germany. In addition, the nine plantar creases were evaluated according to six degrees of expression, and some typical crease combinations are shown. Bilateral and bisexual similarity prevails. The results of this study might serve investigations of different populations and of medical disorders.     

The F0 complex of the ATP synthase of Escherichia coli contains a proton pathway with large proton polarizability caused by collective proton fluctuation.

The F0 complex of the Escherichia coli ATP synthase embedded into cardiolipin liposomes was studied by FT-IR spectroscopy. For comparison, respective studies were performed with dried F0 liposomes and with F0 liposomes treated with N,N'-dicyclohexyl-carbodiimide (DCCD), which binds to Asp-61 of subunit c. Furthermore, the effect of H2O-->D2O exchange on the infrared spectrum was investigated. With F0 liposomes an infrared continuum is observed beginning at about 3000 cm-1 and extending toward smaller wavenumbers. In the DCCD-treated sample, this continuum is no longer observed. It vanishes also with drying of the liposomes. After H2O-->D2O exchange, this infrared continuum begins at about 2350 cm-1 and is less intense. All of these results demonstrate that a proton pathway in native F0 is present, in which the protons are shifted in a hydrogen-bonded chain with large proton polarizability due to collective proton tunneling. With the D2O-hydrated system, deuteron polarizability due to collective deuteron motion is observed, but the polarizability due to collective deuteron motion is smaller. Such pathways are very efficient, because they conduct protons or deuterons within picoseconds. These pathways lose their polarizability if the F0 complex is blocked by DCCD or if the liposomes are dried. On the basis of our results on the proton polarizability of hydrogen bonds and hydrogen-bonded systems and on the basis of structural data from the literature, the nature of the proton pathway of the F0 complex of E. coli is discussed.     

Diagnostic Tests for Primary Renal Hypouricemia

Primary renal hypouricemia is a genetic disorder characterized by defective renal uric acid (UA) reabsorption with complications such as nephrolithiasis and exercise-induced acute renal failure. The known causes are: defects in the SLC22A12 gene, encoding the human urate transporter 1 (hURAT1), and also impairment of voltage urate transporter (URATv1), encoded by SLC2A9 (GLUT9) gene. Diagnosis is based on hypouricemia (<119 μmol/L) and increased fractional excretion of UA (>10%). To date, the cases with mutations in hURAT1 gene have been reported in East Asia only. More than 100 Japanese patients have been described. Hypouricemia is sometimes overlooked; therefore, we have set up the flowchart for this disorder. The patients were selected for molecular analysis from 620 Czech hypouricemic patients. Secondary causes of hyperuricosuric hypouricemia were excluded. The estimations of (1) serum UA, (2) excretion fraction of UA, and (3) analysis of hURAT1 and URATv1 genes follow. Three transitions and one deletion (four times) in SLC22A12 gene and one nucleotide insertion in SLC2A9 gene in seven Czech patients were found. Three patients had acute renal failure and urate nephrolithiasis. In addition, five nonsynonymous sequence variants and three nonsynonymous sequence variants in SLC2A9 gene were found in two UK patients suffering from acute renal failure. Our finding of the defects in SLC22A12 and SLC2A9 genes gives further evidence of the causative genes of primary renal hypouricemia and supports their important role in regulation of serum urate levels in humans.     

Molecular cloning and characterization of the mouse histone deacetylase 1 gene: integration of a retrovirus in 129SV mice

Reversible histone acetylation plays an important role for chromatin structure and gene expression. The acetylation state of core histones is controlled by histone acetyltransferases and histone deacetylases. Here we report the cloning and characterization of the mouse histone deacetylase 1 (HDAC1) gene. The mouse genome contains several HDAC1-related structures representing the HDAC1 gene and at least three pseudogenes. The HDAC1 gene comprises 14 exons ranging from 49 to 539 bp. Interestingly the murine HDAC1 gene strongly resembles the previously published mouse HDAC2 gene (Zeng et al., J. Biol. Chem. 273 (1998) 28921-28930). The sizes of ten of the 14 exons are identical for both genes and the splicing sites for 11 introns align in identical positions suggesting a gene duplication event. The HDAC1 gene is located only 128 bp downstream from the MARCKS-related protein (MRP) gene in a tail-to-tail orientation. The murine MRP gene was previously mapped to a conserved gene cluster on chromosome 4 sharing linkage homology to human chromosome 1p32-36. The genes for HDAC1 and MRP are co-expressed in a variety of cell types. In the genome of 129SV mice the largest intervening sequence of the HDAC1 gene, intron 3, harbors a complete copy of the endogenous retrovirus MuERV-L. In contrast the HDAC1 gene in other mouse strains such as C57B16, C3H/An and C-RY lacks the retrovirus. Our study provides useful tools for future targeted gene disruption studies.     

Blowflies Calliphora vicina and Lucilia sericata as passive vectors of Mycobacterium avium subsp. avium, M. a. paratuberculosis and M. a. hominissuis

Mycobacterium avium subsp. paratuberculosis (Actinomycetales: Mycobacteriaceae) isolates of identical restriction fragment length polymorphism (RFLP) type B-C1 were isolated from: intestinal mucosa of two cows showing clinical signs of paratuberculosis, a specimen of the blowfly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) captured while perched on these cattle intestines in a waste container at the site of the slaughter, and the blowflies C. vicina and Lucilia caesar Linnaeus captured the next day at the same site when no infected cattle with paratuberculosis were slaughtered. Subsequently, second-stage larvae of the blowflies C. vicina and Lucilia sericata (Meigen) were experimentally infected by feeding them liver from hens with avian tuberculosis caused by M. a. avium (serotype 1, genotype IS901+ and IS1245+) and small cuts of pork meat contaminated with M. a. hominissuis (serotype 8, genotype IS901- and IS1245+). Mycobacterium a. avium of identical serotype, genotype and RFLP type F-C3 was isolated from C. vicina larvae on days 4 and 11 post infection (p.i.) and from L. sericata larvae on day 4 p.i. Identical RFLP type B-C1 of M. a. paratuberculosis was isolated from adult C. vicina fed with artificially contaminated saccharose solution on day 2 p. i. Investigation of M. a. paratuberculosis distribution inside the adult C. vicina showed that the majority of Colony Forming Units (CFU) were isolated from the abdomen and head, fewer from the thorax and wings and none from the legs. Larvae and adults may participate in spreading causal agents of mycobacterial infections and this fact should be considered during sanitation of infected herds and in slaughterhouses when materials from animals affected by mycobacterial infections are processed.     

Rapid purification and crystal structure analysis of a small protein carrying two terminal affinity tags

Small peptide tags are often fused to proteins to allow their affinity purification in high-throughput structure analysis schemes. To assess the compatibility of small peptide tags with protein crystallization and to examine if the tags alter the three-dimensional structure, the N-terminus of the chicken alpha-spectrin SH3 domain was labeled with a His6 tag and the C-terminus with a StrepII tag. The resulting protein, His6-SH3-StrepII, consists of 83 amino-acid residues, 23 of which originate from the tags. His6-SH3-StrepII is readily purified by dual affinity chromatography, has very similar biophysical characteristics as the untagged protein domain and crystallizes readily from a number of sparse-matrix screen conditions. The crystal structure analysis at 2.3 A resolution proves native-like structure of His6-SH3-StrepII and shows the entire His6 tag and part of the StrepII tag to be disordered in the crystal. Obviously, the fused affinity tags did not interfere with crystallization and structure analysis and did not change the protein structure. From the extreme case of His6-SH3-StrepII, where affinity tags represent 27% of the total fusion protein mass, we extrapolate that protein constructs with N- and C-terminal peptide tags may lend themselves to biophysical and structural investigations in high-throughput regimes.     

FTIR spectroscopy of complexes formed between metarhodopsin II and C-terminal peptides from the G-protein alpha- and gamma-subunits

Metarhodopsin II (MII) provides the active conformation of rhodopsin for interaction with the G-protein, Gt. Fourier transform infrared spectra from samples prepared by centrifugation reflect the pH dependent equilibrium between MII and inactive metarhodopsin I. C-terminal synthetic peptides (Gtalpha(340-350) and Gtgamma(60-71)farnesyl) stabilize MII. We find that both peptides cause similar spectral changes not seen with control peptides (Gtalpha (K341R, L349A) and non-farnesylated Gtgamma). The spectra reflect all the protonation dependent bands normally observed when MII is formed at acidic pH. Beside the protonation dependent bands, additional features, similar with both peptides, appear in the amide I and II regions.     

Modelling the optimal timing in metabolic pathway activation—Use of Pontryagin's Maximum Principle and role of the Golden section

The time course of enzyme concentrations in metabolic pathways can be predicted on the basis of the optimality criterion of minimizing the time period in which an essential product is generated. This criterion is in line with the widely accepted view that high fitness requires high pathway flux. Here, based on Pontryagin's Maximum Principle, a method is developed to solve the corresponding constrained optimal control problem in an almost exclusively analytical way and, thus, to calculate optimal enzyme profiles, when linear, irreversible rate laws are assumed. Three different problem formulations are considered and the corresponding optimization results are derived. Besides the minimization of transition time, we consider an operation time in which 90% of the substrate has been converted into product. In that case, only the enzyme at the lower end of the pathway rather than all enzymes are active in the last phase. In all cases, biphasic or multiphasic time courses are obtained. The biological meaning of the results in terms of a consecutive just-in-time expression of metabolic genes is discussed. For the special case of two-enzyme systems, the role of the Golden section in the solution is outlined.     

Induction of Hsp22 (HspB8) by estrogen and the metalloestrogen cadmium in estrogen receptor-positive breast cancer cells

Estrogen (E2) plays a critical role in the etiology and progression of human breast cancer. The estrogenic response is complex and not completely understood, including in terms of the involved responsive genes. Here we show that Hsp22 (synonyms: HspB8, E2lG1, H11), a member of the small heat shock protein (sHSP) superfamily, was induced by E2 in estrogen receptor-positive MCF-7 breast cancer cells, resulting in an elevated Hsp22 protein level, whereas it was not induced in estrogen receptor-negative MDA-MB-231 cells. This induction was prevented by the pure anti-estrogen ICI182780 (faslodex, fulvestrant), whereas tamoxifen, a substance with mixed estrogenic and antiestrogenic properties, had no major inhibitory effect on this induction, nor did it induce Hsp22 on its own. Cadmium (Cd) is an environmental pollutant with estrogenic properties (metalloestrogen) that has been implicated in breast cancer. Treatment of MCF-7 cells with Cd also resulted in induction of Hsp22, and this induction was also inhibited by ICI182780. In live MCF-7 cells, Hsp22 interacted at the level of dimers with Hsp27, a related sHSP, as was shown by quantitative fluorescence resonance energy transfer measurements. In cytosolic extracts of MCF-7 cells, most of the E2- and Cd-induced Hsp22 was incorporated into high-molecular mass complexes. In part, Hsp22 and Hsp27 were components of distinct populations of these complexes. Finally, candidate elements in the Hsp22 promoter were identified by sequence analysis that could account for the induction of Hsp22 by E2 and Cd. Taken together, Hsp22 induction represents a new aspect of the estrogenic response with potential significance for the biology of estrogen receptor-positive breast cancer cells.     

The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin

Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (G_t). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.