Stiffening of human skin fibroblasts with age

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ∼60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.     

Molecular investigation of the parental origin of a de novo unbalanced translocation 13/18

We report a patient with a de novo translocation 13/18, identified by high-resolution banding. The breakpoints were ascertained by fluorescence in situ hybridisation with whole chromosome 13 and 18 paints. Short tandem repeat typing demonstrated the aberration to be of combined maternal/paternal origin and thereby confirmed its de novo and postzygotic formation. Thus, a gonadal mosaic in one of the parents resulting in a higher recurrence risk could be excluded. Received: 1 September 1996 / Revised: 3 November 1996     

Molecular Insights into Variable Electron Transfer in Amphibian Cryptochrome

Cryptochrome proteins are activated by the absorption of blue light, leading to the formation of radical pairs through electron transfer in the active site. Recent experimental studies have shown that once some of the amino acid residues in the active site of Xenopus laevis cryptochrome DASH are mutated, radical-pair formation is still observed. In this study, we computationally investigate electron-transfer pathways in the X. laevis cryptochrome DASH by extensively equilibrating a previously established homology model using molecular dynamics simulations and then mutating key amino acids involved in the electron transfer. The electron-transfer pathways are then probed by using tight-binding density-functional theory. We report the alternative electron-transfer pathways resolved at the molecular level and, through comparison of amino acid sequences for cryptochromes from different species, we demonstrate that one of these alternative electron-transfer pathways could be general for all cryptochrome DASH proteins.     

Tetrasomy 18p de novo: Identification by FISH with conventional and microdissection probes and analysis of parental origin and formation by short sequence repeat typing

We report a de novo supernumerary isochromosome 18p in a child with tetrasomy 18p, analyzed by a straightforward combination of cytogenetic and molecular cytogenetic methods. The diagnostic procedure consisted of standard banding techniques and fluorescence in situ hybridization (FISH) with centromere and library DNA probes for chromosome 18, and 18p-specific FISH probes prepared by chromosome microdissection and in vitro amplification. The maternal origin as well as the most probable cell stages of formation of the supernumerary isochromosome were determined by typing of short sequence repeats (SSRs). The pattern of allelic distribution suggests a nondisjunction during meiosis followed by a centromeric misdivision in an early postzygotic mitosis as the most probable mode of isochromosome 18p formation. The combination of the applied methods represents a powerful tool to investigate the nature and the origin of de novo marker chromosomes. Received: 28 August 1995 / Revised: 3 November 1995; 20 December 1995     

A high-performance liquid chromatographic method for the determination of pseudouridine and uric acid in native human urine and ultrafiltrated serum

A simple and reliable HPLC method for quantitative determination of pseudouridine and uric acid in human urine and serum using a cation-exchange resin is described. This method is straightforward (12 runs of urine samples per day since the sample is only diluted into buffer and then chromatographed), sensitive, and highly reproducible. The column is stable over long periods (3 months of uninterrupted use at a time; it is thereafter easily restored to the original state). Mean excretion values for pseudouridine (in μmol/mmol creatinine) are 26.4 ± 3.1 (17 female adults), 23.8 ± 2.5 (12 male adults), 164.7 ± 32.2 (37 male preterm infants); mean values for uric acid (μmol/mmol creatinine) are, respectively, 310.3 ± 90.5, 278.2 ± 56.1, and 1108 ± 314. Human serum is deproteinized by pressure ultrafiltration in microcollodion bags with a nominal exclusion molecular weight of 12,400 and then put directly onto the HPLC column. The complete procedure takes 4 h.     

Family studies in scleroderma (systemic sclerosis) demonstrating an HLA-linked increased chromosomal breakage rate in cultured lymphocytes

Summary An increased chromosomal breakage rate (ICBR) was found in 27 of 28 patients with scleroderma (systemic sclerosis, SS)-5 with the syndrome including calcinosis cutis, Raynaud phenomenon, esophagus hypomotility, sclerodactyly and telangiectasia (CREST), 4 incomplete CREST, 1 overlapping syndrome, 18 progressive systemic sclerosis (PSS). Not only the patients, but also about half of their first-degree relatives showed an increased chromosomal breakage rate (more than 5 breaks per 100 metaphases). This character segregated as a dominant marker in nine families of scleroderma patients. In the six informative of the nine families, the ICBR trait showed close linkage with the HLA region on chromosome 6 (total lod score 5.5 at =0). In these families, ICBR was predominantly observed in linkage with HLA haplotype A1, Cw7, B8, C4AQ0B1, DR3 which is frequently observed in autoimmune diseases. The nature of the agent inducing chromosomal breakage in cultured lymphocytes of some, but not all family members of scleroderma patients remains to be clarified.Presented in part at the 7th International Congress of Human Genetics, Berlin 1986, and the 9th International Workshop on Human Gene Mapping, Paris 1987     

Ultrastrukturveränderungen motorischer Vorderhornzellen des Kaninchens unter abgestufter Ischämie

Zusammenfassung Die motorischen Vorderhornzellen im Rückenmark des Kaninchens wurden im Lumbaibereich nach abgestufter temporärer Ischämie elektronenmikroskopisch untersucht. Die cytoplasmatischen Strukturen und Organellen dieser Zellen werden unterschiedlich stark durch die Zirkulationsunterbrechung beeinflußt; besonders eindrucksvoll sind die Veränderungen der Mitochondrien und des endoplasmatischen Retikulums. Vor allem zeigen die Mitochondrien mit zunehmender Ischämiedauer ganz charakteristische, reproduzierbare Strukturveränderungen. Sowohl an den Mitochondrien als auch am endoplasmatischen Retikulum sind bereits nach einer Blockade der Blutzufuhr von 5 min deutliche Folgen der Ischämie zu erkennen (Schwellung, Wasseraufnahme). Zentral gelegene Vorderhornzellen sind durchgehend stärker von der Zirkulationsunterbrechung betroffen als peripher gelegene Zellen. Innerhalb einer Zelle finden sich nach längerer Ischämiedauer nebeneinander Gruppen stark geschwollener und weniger geschädigter Mitochondrien. Die durch Ischämie an den Mitochondrien und am endoplasmatischen Retikulum hervorgerufenen Schwellungen und Membranveränderungen sind als Folge einer Störung des Energie- und Wasserhaushaltes der Zelle aufzufassen.

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Ultrastructural alterations of spinal motoneurons during gradually different ischemias in the rabbit

Summary Spinal ventral horn motoneurons from the lumbar region in the rabbit were examined in the electron microscope during different states of temporary ischemia. Ischemia caused variable effects upon the motoneuron cytoplasmic structures. Alterations were particularly evident in mitochondria and endoplasmic reticulum; a swelling and water uptake by these organelles were observed already after a 5-minute blockade of blood supply. After prolonged ischemia, reproducible structural changes and membrane alterations were especially evident in mitochondria; within a cell, the swollen mitochondria occurred next to less damaged mitochondria. Centrally located ventral horn cells were affected more than peripherally located cells. The structural alterations of the mitochondria and the endoplasmic reticulum are interpreted to be caused by a disturbance of cellular energy and water metabolism.

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Herrn Prof. Dr. Dr. h. c. H. Spatz gewidmet.

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Herrn Prof. Dr. A. Oksche danke ich für einen Arbeitsplatz an der Elektronenmikroskopischen Abteilung des Anatomischen Institutes, Lehrstuhl I.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Die Wanderung der Pigmentgranula in der Epidermis beim physiologischen Farbwechsel von Carausius morosus

Carausius morosus is one of the few insects exhibiting physiological colour change in the epidermal cells. The distribution of ommochromes, carotinoids, and pterindines is analysed in light and dark adapted animals.In light adapted animals the ommochrome granules are concentrated at the proximal cell membrane. During dark adaption they move to the distal cell membrane, dispersing there over the whole cell forming a shield of dark pigment. The carotinoid granules behave in a similar way. The rod shaped pteridine granules are concentrated in the distal half of the epidermis. They show no daytime dependent movements. However, they take part in the physiological colour change indirectly. Apparently, biliverdin is not attached to granules but dissolved in the epidermal cells.