Mechanics Reveals the Biological Trigger in Wrinkly Fingers.

Fingertips wrinkle due to long exposure to water. The biological reason for this morphological change is unclear and still not fully understood. There are two main hypotheses for the underlying mechanism of fingertip wrinkling: the 'shrink' model (in which the wrinkling is driven by the contraction of the lower layers of skin, associated with the shrinking of the underlying vasculature), and the 'swell' model (in which the wrinkling is driven by the swelling of the upper layers of the skin, associated with osmosis). In reality, contraction of the lower layers of the skin and swelling of the upper layers will happen simultaneously. However, the relative importance of these two mechanisms to drive fingertip wrinkling also remains unclear. Simulating the swelling in the upper layers of skin alone, which is associated with neurological disorders, we found that wrinkles appeared above an increase of volume of [Formula: see text] Therefore, the upper layers can not exceed this swelling level in order to not contradict in vivo observations in patients with such neurological disorders. Simulating the contraction of the lower layers of the skin alone, we found that the volume have to decrease a [Formula: see text] to observe wrinkles. Furthermore, we found that the combined effect of both mechanisms leads to pronounced wrinkles even at low levels of swelling and contraction when individually they do not. This latter results indicates that the collaborative effect of both hypothesis are needed to induce wrinkles in the fingertips. Our results demonstrate how models from continuum mechanics can be successfully applied to testing hypotheses for the mechanisms that underly fingertip wrinkling, and how these effects can be quantified.

Atomic Force Microscopy Study of Protein-Protein Interactions in the Cytochrome CYP11A1 (P450scc)-Containing Steroid Hydroxylase System.

Atomic force microscopy (AFM) and photon correlation spectroscopy (PCS) were used for monitoring of the procedure for cytochrome CYP11A1 monomerization in solution without phospholipids. It was shown that the incubation of 100 µM CYP11A1 with 12% Emulgen 913 in 50 mM KP, pH 7.4, for 10 min at T = 22°C leads to dissociation of hemoprotein aggregates to monomers with the monomerization degree of (82 ± 4)%. Following the monomerization procedure, CYP11A1 remained functionally active. AFM was employed to detect and visualize the isolated proteins as well as complexes formed between the components of the cytochrome CYP11A1-dependent steroid hydroxylase system. Both Ad and AdR were present in solution as monomers. The typical heights of the monomeric AdR, Ad and CYP11A1 images were measured by AFM and were found to correspond to the sizes 1.6 ± 0.2 nm, 1.0 ± 0.2 nm and 1.8 ± 0.2 nm, respectively. The binary Ad/AdR and AdR/CYP11A1mon complexes with the heights 2.2 ± 0.2 nm and 2.8 ± 0.2 nm, respectively, were registered by use of AFM. The Ad/CYP11A1mon complex formation reaction was kinetically characterized based on optical biosensor data. In addition, the ternary AdR/Ad/CYP11A1 complexes with a typical height of 4 ± 1 nm were AFM registered.

Analysis of the interaction of a hybrid system consisting of bovine adrenodoxin reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119.

The mitochondrial steroid-hydroxylating system in vertebrates and the NADPH producing electron transfer chain in photosynthetic organisms contain structurally and functionally similar components. Examination of a potential hybrid reconstitution of the electron transfer chain between different components of both systems could help to improve our knowledge on protein-protein interaction and subsequent electron transfer. Here we analyzed the interaction between bovine adrenodoxin reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119. Optical biosensor as well as steady state and fast kinetic experiments showed their ability to form distinct productive complexes. Compared with the corresponding physiological systems the electron transfer is rather slow, probably due to the lack of specificity at the interaction surface.

Hypothalamic actions of neuromedin U.

The central nervous system and gut peptide neuromedin U (NMU) inhibits feeding after intracerebroventricular injection. This study explored the hypothalamic actions of NMU on feeding and the hypothalamo-pituitary-adrenal axis. Intraparaventricular nucleus (intra-PVN) NMU dose-dependently inhibited food intake, with a minimum effective dose of 0.1 nmol and a robust effect at 0.3 nmol. Feeding inhibition was mapped by NMU injection into eight hypothalamic areas. NMU (0.3 nmol) inhibited food intake in the PVN (0-1 h, 59 +/- 6.9% of the control value; P < 0.001) and arcuate nucleus (0-1 h, 76 +/- 10.4% of the control value; P < 0.05). Intra-PVN NMU markedly increased grooming and locomotor behavior and dose-dependently increased plasma ACTH (0.3 nmol NMU, 24.8 +/- 1.9 pg/ml; saline, 11.4 +/- 1.0; P < 0.001) and corticosterone (0.3 nmol NMU, 275.4 +/- 40.5 ng/ml; saline, 129.4 +/- 25.0; P < 0.01). Using hypothalamic explants in vitro, NMU stimulated CRH (100 nM NMU, 5.9 +/- 0.95 pmol/explant; basal, 3.8 +/- 0.39; P < 0.01) and arginine vasopressin release (100 nM NMU, 124.5 +/- 21.8 fmol/explant; basal, 74.5 +/- 7.6; P < 0.01). Leptin stimulated NMU release (141.9 +/- 20.4 fmol/explant; basal, 92.9 +/- 9.4; P < 0.01). Thus, we describe a novel role for NMU in the PVN to stimulate the hypothalamo-pituitary-adrenal axis and locomotor and grooming behavior and to inhibit feeding.

Two parameters improve efficiency of mitochondrial uptake of adenylate kinase: decreased folding velocity and increased propensity of N-terminal alpha-helix formation.

The long isoform of eukaryotic adenylate kinase has a dual subcellular location in the cytoplasm and in the mitochondrial intermembrane space. Protein sequences and modifications are identical in both locations. In yeast, the bulk of the major form of adenylate kinase (Aky2p) is in the cytoplasm and, in the steady state, only 5-8% is sorted to the mitochondrial intermembrane space. Since the reasons for exclusion from mitochondrial import are unclear, we have constructed aky2 mutants with elevated mitochondrial uptake efficiency of Aky2p in vivo and in vitro. We have analyzed the effect of the mutations on secondary structure prediction in silico and have tested folding velocity and folding stability. One type of mutants displayed decreased proteolytic stability and retarded renaturation kinetics after chaotropic denaturation implying that deterioration of folding leads to prolonged presentation of target information to mitochondrial import receptors, thereby effecting improved uptake. In a second type of mutants, increased import efficiency was correlated with an increased probability of formation of an alpha-helix with increased amphipathic moment at the N-terminus suggesting that targeting interactions with mitochondrial import receptors had been improved at the level of binding affinity.

Evidence of an orexigenic role for cocaine- and amphetamine-regulated transcript after administration into discrete hypothalamic nuclei.

Cocaine- and amphetamine-regulated transcript is expressed in hypothalamic regions involved in the central control of food intake. Previous data have implicated cocaine- and amphetamine-regulated transcript as an anorectic peptide. We studied the effect of the active fragment of cocaine- and amphetamine-regulated transcript, cocaine- and amphetamine-regulated transcript-(55-102), on feeding when injected into discrete nuclei of the hypothalamus. Cocaine- and amphetamine-regulated transcript-(55-102) (0.04 nmol) elicited a delayed, but significant, increase in feeding in 24-h fasted rats after injection into the ventromedial nucleus (1-2 h, 261 +/- 60% of control; P < 0.05) and arcuate nucleus (1-2 h, 225 +/- 38% of control; P < 0.05) of the hypothalamus. Administration of a higher dose of cocaine- and amphetamine-regulated transcript-(55-102) (0.2 nmol) elicited a significant increase in feeding after injection into the ventromedial nucleus (1-2 h, 1253 +/- 179% of control; P < 0.001), arcuate nucleus (1-2 h, 265 +/- 43% of control; P < 0.05), paraventricular nucleus (2-4 h food intake, 186 +/- 29% of control; P < 0.05), lateral hypothalamic area (2-4 h, 280 +/- 34% of control; P < 0.001), anterior hypothalamic area (2-4 h, 252 +/- 42% of control; P < 0.01), dorsomedial nucleus (2-4 h, 368 +/- 29% of control;P < 0.001) and supraoptic nucleus (2-4 h, 212 +/- 34% of control; P < 0.05) of the hypothalamus. Administration of cocaine- and amphetamine-regulated transcript-(55-102) into the third ventricle of the hypothalamus resulted in an inhibition in feeding [0-4 h (0.4 nmol), 33 +/- 13% control; P < 0.001], but was associated with marked abnormalities in behavior, which may have interfered with feeding. These behavioral abnormalities were not observed after the administration of cocaine- and amphetamine-regulated transcript-(55-102) directly into the arcuate nucleus. These data suggest that cocaine- and amphetamine-regulated transcript may play an orexigenic role in the hypothalamic feeding circuitry.

Functional and structural genomics using PEDANT.

MOTIVATION: Enormous demand for fast and accurate analysis of biological sequences is fuelled by the pace of genome analysis efforts. There is also an acute need in reliable up-to-date genomic databases integrating both functional and structural information. Here we describe the current status of the PEDANT software system for high-throughput analysis of large biological sequence sets and the genome analysis server associated with it. RESULTS: The principal features of PEDANT are: (i) completely automatic processing of data using a wide range of bioinformatics methods, (ii) manual refinement of annotation, (iii) automatic and manual assignment of gene products to a number of functional and structural categories, (iv) extensive hyperlinked protein reports, and (v) advanced DNA and protein viewers. The system is easily extensible and allows to include custom methods, databases, and categories with minimal or no programming effort. PEDANT is actively used as a collaborative environment to support several on-going genome sequencing projects. The main purpose of the PEDANT genome database is to quickly disseminate well-organized information on completely sequenced and unfinished genomes. It currently includes 80 genomic sequences and in many cases serves as the only source of exhaustive information on a given genome. The database also acts as a vehicle for a number of research projects in bioinformatics. Using SQL queries, it is possible to correlate a large variety of pre-computed properties of gene products encoded in complete genomes with each other and compare them with data sets of special scientific interest. In particular, the availability of structural predictions for over 300 000 genomic proteins makes PEDANT the most extensive structural genomics resource available on the web.

Implant-supported denture in a patient with Huntington's disease: interdisciplinary aspects.

Patients with extrapyramidal diseases often cannot maintain independent, efficient oral hygiene due to restricted motor ability of the upper extremities and lack of coordination. The hermetic closure of the mouth and lips, and the associated ability to keep liquid and toothpaste in the mouth, can become so weak that effective oral hygiene cannot be maintained. Over a period of many years, this illness leads to loss of teeth and the need for complete prosthodontic care. Dyskinesia and hyperkinesia of the tongue and the peri-oral musculature, combined with xerostomia and pooling of saliva, make it impossible for the patient to wear a conventional complete denture, despite an anatomically-adequate bearing area. In such cases, an implant-supported prosthesis is a better therapeutic measure, although some aspects of oral hygiene must initially be disregarded. Two ITI implants were inserted into the anterior mandibular region of a patient with Huntington's chorea, because a complete denture could not be retained on the alveolar ridge, despite adequate vestibule depth, due to tongue dyskinesia. A bar joint was used to anchor this mucosal-borne denture. This implant-supported complete denture led to a clear improvement in the patient's chewing function when observed over a period of a year.

Integrative analysis of protein interaction data.

We have developed a method for the integrative analysis of protein interaction data. It comprises clustering, visualization and data integration components. The method is generally applicable for all sequenced organisms. Here, we describe in detail the combination of protein interaction data in the yeast Saccharomyces cerevisiae with the functional classification of all yeast proteins. We evaluate the utility of the method by comparison with experimental data and deduce hypotheses about the functional role of so far uncharacterized proteins. Further applications of the integrative analysis method are discussed. The method presented here is powerful and flexible. We show that it is capable of mining large-scale data sets.

Pulp reactions to different preparation techniques on teeth exhibiting periodontal disease.

To evaluate the histopathological outcome of two preparation techniques (featheredge preparation/shoulder preparation) on teeth exhibiting pulp reactions due to age and periodontal disease, 11 teeth were prepared for full veneer crowns. Laboratory made resin crowns were fixed with a zinc phosphate cement for a period of 90 days. After extraction, adjacent pulpal areas were histopathologically rated according to the BRD criteria comprising the parameters (i) Bacterial invasion, (ii) Regenerative parameters, (iii) Degenerative parameters. Degenerative reactions were more correlated with tooth preparation than with advanced periodontal disease. The severity of endondontal reactions depends more on remaining dentin thickness than on the type of preparation.

Overview of the yeast genome.

The collaboration of more than 600 scientists from over 100 laboratories to sequence the Saccharomyces cerevisiae genome was the largest decentralised experiment in modern molecular biology and resulted in a unique data resource representing the first complete set of genes from a eukaryotic organism. 12 million bases were sequenced in a truly international effort involving European, US, Canadian and Japanese laboratories. While the yeast genome represents only a small fraction of the information in today's public sequence databases, the complete, ordered and non-redundant sequence provides an invaluable resource for the detailed analysis of cellular gene function and genome architecture. In terms of throughput, completeness and information content, yeast has always been the lead eukaryotic organism in genomics; it is still the largest genome to be completely sequenced.

Heme binding to a conserved Cys-Pro-Val motif is crucial for the catalytic function of mitochondrial heme lyases.

Covalent attachment of heme to the apoforms of mitochondrial cytochromes c and c1 requires the activity of cytochrome c heme lyase (CCHL) and cytochrome c1 heme lyase (CC1HL), respectively. The two enzymes differ in their cytochrome specificity, but they are related in sequence, and both contain conserved Cys-Pro-Val (CPV) motifs. By using various in vitro assays we investigated whether heme can bind directly to heme lyases and whether the CPV motif may be involved in heme binding. Heme stabilized CC1HL, as a model protein, in a folded, protease-resistant conformation, stimulated the refolding of CC1HL after urea denaturation, and inhibited the import of the CC1HL precursor into mitochondria. These effects were not observed with a point mutant, CC1HLSPV, in which cysteine was replaced by serine, and with CC1HLDeltaCPV, in which the motif was deleted. These results show that heme lyases can bind heme directly, and they identify the CPV sequence as a structural element important for this interaction. The phenotype of a yeast mutant expressing CC1HLSPV is in good agreement with such a role of the CPV motif. The mutant cells accumulate the heme-free intermediate form of cytochrome c1 and display a severe deficiency in the holo form. We suggest that the CPV motif forms a crucial part of the substrate binding site for heme.

The role of androgen receptor gene mutations in male breast carcinoma.

Certain germline mutations (607Arg-Gln, 608Arg-Lys) in the androgen receptor gene have been associated with the occurrence of breast cancer in males suffering from partial androgen insensitivity. To assess whether somatic mutations in this gene could be detected in breast carcinoma, archival tumor tissue of males without clinical evidence of androgen insensitivity was screened for point mutations in the androgen receptor gene. DNA was retrieved by chloroform-phenol extraction from formalin-fixed, paraffin-embedded tissues. Exons 2-8 of the androgen receptor gene, encoding the DNA- and hormone-binding regions of the receptor, were amplified by polymerase chain reaction and subjected to nonisotopic single strand conformation assay (SSCA) to screen for point mutations. In the tumor DNA, no variations suggestive of mutations were encountered on SSCA. However, in a control patient with partial androgen insensitivity and predominantly female phenotype, the germline mutation 607Arg-Gln was identified in blood leukocyte DNA. Our results indicate that somatic mutations of the androgen receptor are not required for the development of male breast cancer. This, however, does not exclude an increased risk of breast carcinoma in patients with androgen insensitivity.

Nonisotopic single strand conformation analysis of the 5 alpha-reductase type 2 gene for the diagnosis of 5 alpha-reductase deficiency.

5 alpha-Reductase deficiency is a rare autosomal recessive disorder of defective virilization in karyotypic males due to reduced conversion of testosterone to dihydrotestosterone. The gene encoding the affected 5 alpha-reductase type 2 enzyme has recently been cloned, and mutations within the coding region have been discovered as the cause of this disease. We address the possibility of a rapid nonradioactive molecular genetic screening technique for initial diagnosis and report different point mutations in this gene in eight unrelated patients with clinical features of 5 alpha-reductase deficiency. For molecular genetic analysis, DNA from peripheral blood leukocytes was studied. The coding region of the 5 alpha-reductase type 2 gene was characterized by exon-specific PCR amplification, nonradioactive single strand conformation analysis, and direct sequencing. In seven patients, homozygous point mutations were identified (Leu55-Gln, delta Met157, Gly196-Ser, Arg227-Gln, Ala228-Thr, and His231-Arg). One individual was a compound heterozygote carrier of two mutations (Ile112-Asn and Gln126-Arg). We conclude that molecular genetic characterization of point mutations in the 5 alpha-reductase type 2 gene may be used as an additional valuable procedure for the diagnosis of this disorder.

Biogenesis of mitochondrial heme lyases in yeast. Import and folding in the intermembrane space.

Heme lyases are components of the mitochondrial intermembrane space facilitating the covalent attachment of heme to the apoforms of c-type cytochromes. The precursors of heme lyases are synthesized in the cytosol without the typical N-terminal mitochondrial targeting signal. Here, we have analyzed the mode of import and folding of the two heme lyases of the yeast Saccharomyces cerevisiae, namely of cytochrome c heme lyase and of cytochrome c1 heme lyase. For transport into mitochondria, both proteins use the general protein import machinery of the outer membrane. Import occurred independently of a membrane potential, delta psi, across the inner membrane and ATP in the matrix space, suggesting that the inner membrane is not required for transport along this direct sorting pathway. The presence of a large folded domain in heme lyases was utilized to study their folding in the intermembrane space. Formation of this domain occurred at the same rate as import, indicating that heme lyases fold either during or immediately after their transfer across the membrane. Folding was not affected by depletion of ATP and delta psi or by inhibitors of peptidylprolyl cis-trans isomerases, i.e. it does not involve homologs of known folding factors (like Hsp60 and Hsp70). The energy derived from folding cannot be regarded as a major driving force for import, since the folded domain could be imported into mitochondria with the same efficiency as the intact protein. We conclude that protein folding in the intermembrane space obeys principles different from those established for other subcellular compartments.

Mutation of a highly conserved base in the yeast mitochondrial 21S rRNA restricts ribosomal frameshifting.

A mutation shown to cause resistance to chloramphenicol in Saccharomyces cerevisiae was mapped to the central loop in domain V of the yeast mitochondrial 21S rRNA. The mutant 21S rRNA has a base pair exchange from U2677 (corresponding to U2504 in Escherichia coli) to C2677, which significantly reduces rightward frameshifting at a UU UUU UCC A site in a +1 U mutant. There is evidence to suggest that this reduction also applies to leftward frameshifting at the same site in a -1 U mutant. The mutation did not increase the rate of misreading of a number of mitochondrial missense, nonsense or frameshift (of both signs) mutations, and did not adversely affect the synthesis of wild-type mitochondrial gene products. It is suggested here that ribosomes bearing either the C2677 mutation or its wild-type allele may behave identically during normal decoding and only differ at sites where a ribosomal stall, by permitting non-standard decoding, differentially affects the normal interaction of tRNAs with the chloramphenicol resistant domain V. Chloramphenicol-resistant mutations mapping at two other sites in domain V are described. These mutations had no effect on frameshifting.

Detection of point mutations in the androgen receptor gene using non-isotopic single strand conformation polymorphism analysis. German Collaborative Intersex Study Group.

Point mutations in the androgen receptor gene cause androgen insensitivity syndromes, clinically characterized by masculinization defects in karyotypic males due to endorgan resistance to androgenic steroids. Characterization of these mutations with single strand conformation polymorphism analysis utilizing radioactive PCR can serve as a diagnostic tool for molecular subclassification of these syndromes. It is the basis for genetic counseling and for therapeutic decisions. Here we report an improved non-radioactive single strand polymorphism analysis for rapid detection of androgen receptor gene mutations in affected individuals. In addition to previously reported mutations, 10 patients with clinical features of androgen resistance were studied. DNA was isolated from peripheral blood leucocytes and exons 1 to 8 of the coding region of the androgen receptor gene amplified by PCR. Amplification products were denatured and run on non-denaturing gels. These were subjected to fixation and silver staining. Variations were directly sequenced. In all patients a different point mutation in one of the exons was detected. While one insertion mutation was found in a patient with complete androgen insensitivity, all other mutations cause amino acid substitutions. These data suggest that the described non-radioactive single strand polymorphism analysis is a useful tool for the characterization of androgen receptor gene mutations. The omission of radioisotopes is advantageous in a clinical setting. The mutations described emphasize the clinical and molecular heterogeneity of this disease.

Expression of the Saccharomyces cerevisiae CYT2 gene, encoding cytochrome c1 heme lyase.

In this paper we examine the expression of the Saccharomyces cerevisiae CYT2 gene, which encodes cytochrome c1 heme lyase. This enzyme is required for covalent attachment of heme to apocytochrome c1, a subunit of the mitochondrial respiratory chain. Transcription of the 1-kb CYT2 mRNA initiates at four prominent sites at a distance of 52-225 bp in front of the AUG start codon. The level of CYT2 mRNA is not influenced by the presence or absence of oxygen or of heme, but it is subject to carbon-source control. The concentration of the CYT2 mRNA is significantly reduced in glucose-grown cells as compared to cells grown under non-repressing conditions. Neither the HAPp activator proteins nor MIG1p, a repressor protein involved in glucose repression, seem to mediate this effect.

Molecular cloning and characterization of the Saccharomyces cerevisiae CYT2 gene encoding cytochrome-c1-heme lyase.

Cytochrome c1, a subunit of the mitochondrial ubiquinol--cytochrome-c reductase, is synthesized on cytosolic ribosomes as a precursor protein of 37 kDa. Maturation to the mature 31-kDa form involves two proteolytic processing steps of the amino-terminal presequence. After removal of the amino-terminal part by the matrix-localized processing peptidase, the carboxy-terminal part of the presequence is cleaved off by an unknown intermembrane space protease. This step depends on covalent linkage of heme to the apoprotein. At least two complementation groups (I and II) can be distinguished among mutants of the yeast Saccharomyces cerevisiae, which are defective in this second proteolytic processing, i.e. they accumulate the intermediate-sized form of cytochrome c1 instead of the mature form. Recently, it was shown that complementation group II defines the structural gene for cytochrome c1 [Sadler, I., Suda, K., Schatz, G., Kaudewitz, F. & Haid, A., (1984) EMBO J. 3, 2137-2143]. We report on the molecular cloning and characterization of the CYT2 gene representing complementation group I. It maps on chromosome XI and encodes a mitochondrial protein of about 26 kDa. Extensive similarity to Neurospora crassa and S. cerevisiae cytochrome-c--heme lyase, as well as the phenotype of cyt2 mutants, strongly suggest that we have identified the gene for cytochrome-c1--heme lyase.

Expression of yeast cytochrome c1 is controlled at the transcriptional level by glucose, oxygen and haem.

The nuclear gene for cytochrome c1 in Saccharomyces cerevisiae (CYT1) was localized on chromosome XV. Its upstream region was identified by functional complementation. Fusion to the lacZ reporter gene on a CEN plasmid allowed study of the effect of carbon sources and of specific deletion mutations on expression of the gene in yeast transformants. Detailed promoter analysis combined with expression studies in recipient strains defective in regulatory genes identified cis-acting sites and transcription factors involved in the regulated expression of the cytochrome c1 gene. These analyses showed that, in the presence of glucose, transcription of CYT1 is positively controlled by oxygen, presumably through the haem signal, and mediated by the HAP1-encoded transactivator. It is additionally regulated by the HAP2/3/4 complex which mediates gene activation mainly under glucose-free conditions. Basal transcription is, in part, effected by CPF1, a centromere and promoter-binding factor.