Amino acid racemization and the preservation of ancient DNA.

The extent of racemization of aspartic acid, alanine, and leucine provides criteria for assessing whether ancient tissue samples contain endogenous DNA. In samples in which the D/L ratio of aspartic acid exceeds 0.08, ancient DNA sequences could not be retrieved. Paleontological finds from which DNA sequences purportedly millions of years old have been reported show extensive racemization, and the amino acids present are mainly contaminates. An exception is the amino acids in some insects preserved in amber.

Growth factor-functionalized silk membranes support wound healing in vitro.

Chronic wounds represent a serious problem in daily medical routine requiring improved wound care. Silk of the domesticated silkworm (Bombyx mori) has been used to form a variety of biomaterials for medical applications. We genetically engineered B. mori to produce silk functionalized with growth factors to promote wound healing in vitro. In this study FGF-, EGF-, KGF-, PDGF- or VEGF-functionalized silk membranes were compared to native B. mori silk membranes without growth factors for their ability to support wound healing in vitro. All silk membranes were cytocompatible and supported macrophage secretion of neutrophil recruiting factor CXCL1 and monocyte chemoattractant protein 1 (MCP-1). VEGF-functionalized silk significantly outperformed other growth factor-functionalized silk membranes, but not native silk in angiogenesis assays. In addition, EGF- and VEGF-functionalized silk membranes slightly enhanced macrophage adhesion compared to silk without growth factors. In wound healing assays in vitro (reduction of wound lesion), dermal equivalents showed a higher wound healing capacity when covered with EGF-, FGF- or VEGF-functionalized silk membranes compared to native, KGF- or PDGF-functionalized silk membranes. Keratinocyte migration and growth is overstimulated by KGF- and VEGF-functionalized silk membranes. In conclusion, growth factor-functionalized silk membranes prepared from genetically engineered silk worm glands are promising wound dressings for future wound healing therapies.

Telomerase-dependent repeat divergence at the 3' ends of yeast telomeres.

Yeast telomeres consist of approximately 300 nt of degenerate repeats with the consensus sequence G(2-3)(TG)(1-6). We developed a method for the amplification of a genetically marked telomere by PCR, allowing precise length and sequence determination of the G-rich strand including the 3' terminus. We examined wild-type cells, telomerase RNA deficient cells and a strain deleted for YKU70, which encodes for a protein involved in telomere maintenance and DNA double strand break repair. The 3' end of the G-rich strand was found to be at a variable position within the telomeric repeat. No preference for either thymine or guanine as the 3' base was detected. Comparison of telomere sequences from clonal populations revealed that telomeres consist of a centromere-proximal region of stable sequence and a distal region with differing degenerate repeats. In wild-type as well as yku70-Delta cells, variation in the degenerate telomeric repeats was detected starting 40-100 nt from the 3' end. Sequence divergence was abolished after deletion of the telomerase RNA gene. Thus, this region defines the domain where telomere shortening and telomerase-mediated extension occurs. Since this domain is much larger than the number of nucleo-tides lost per generation in the absence of telomerase, we propose that telomerase does not extend a given telomere in every cell cycle.

Time-dependent changes of plasma ribonuclease activity in female Wistar rats under combination therapy of ovarian carcinoma.

Determinations of plasma ribonuclease were recommended in 1977 by Sheid et al. for laboratory diagnosis of ovarian carcinoma. We investigated the turnover of transfer ribonucleic acid (tRNA) as the substrate of the enzyme in healthy female inbred rats under the multistep-massive dose therapy of ovarian carcinoma usual at the Department of Gynecology, University of Giessen. Whereas mitopodozide, percutaneous irradiation of the abdomen and teniposide did not have any detectable influence on the enzyme activity, the enzyme was strongly suppressed by the other therapy steps: minima of tRNA turnover were observed on the 2nd day with adriamycine, on the 4th day with methotrexate and methotrexate/folinic acid, on the 6th day with cyclophosphamide and fractionated percutaneous irradiation with cyclophosphamide medication in the intervening periods. According to the results, a plasma ribonuclease determination to check the result of therapy should only be performed at an interval of 2 weeks after each therapy step.

Instructions for motor learning: differential effects of internal versus external focus of attention.

The effects of different types of instructions on complex motor skill learning were examined. The instructions were related either to the participant's own body movements (internal focus) or to the effects of those movements on the apparatus (external focus). The hypothesis tested was that external-focus instructions would be more beneficial for learning than internal-focus instructions. In Experiment 1, the participants (N = 33) performed slalom-type movements on a ski-simulator. The instructions referred to the way in which force should be exerted on the platform that the participant was standing on. The instructions given 1 group of participants referred to the performers' feet (internal focus), whereas the instructions given another group referred to the wheels of the platform, which were located directly under the feet (external focus). The control group was given no focus instructions. All participants practiced the task on 2 consecutive days and performed a retention test on Day 3. Compared with the effects of internal-focus instructions and no instructions, the external-focus instructions enhanced learning. Internal-focus instruction was not more effective than no instructions. In Experiment 2, an attempt was made to replicate the differential effects of external-versus internal-focus instructions with a different task (balancing on a stabilometer). Consistent with Experiment 1, instructing learners (N = 16) to focus on 2 markers on the platform of the stabilometer (external focus) led to more effective learning than instructing them to focus on their feet (internal focus), as measured by a retention test after 2 days of practice. Practical and theoretical implications of those results are discussed.

[Ultrasound control of thermal tissue therapy]. / Ultraschall-Kontrolle Thermischer Tumor Therapien.

As alternatives to surgical resection and/or supportive to radio- or chemo-therapy of tumors and metastases minimal invasive interstitial thermal treatment procedures by which the tissue is heated up locally to temperatures up to 100 degrees C are used. However beside nuclear magnetic resonance tomography there is no economical, by routine applicable procedure for non invasive therapy control at present disposal. In this work the possibility of non invasive control of thermal therapies by means of temporal and spectral analysis of radio frequency ultrasound signals are evaluated. Two different ultrasonic procedures, the first beeing based on the analysis of local modifications in the time of flight of the ultrasound signal for determination of the temperature distribution in the tissue, the second beeing based on the physical attenuation characteristics of biological tissue and their dependence on the tissue structure are proposed and evaluated for therapy control. With in vitro experiments the possibilities and limitations of both procedures and preliminary results of a prototype control system are demonstrated.

[Ultrasound hard tissue detection for registration in orthopedics and traumatology]. / Ultraschall-Hartgewebedetektion zur Registrierung in der Orthopädie und Traumatologie.

The application of x-ray diagnostics for intraoperative navigation and for registration of hard tissue structures is restrained due to high radiation loads and the not given real time ability. Previous approaches with conventional ultrasonic imaging are only interactively applicable due to the high information content of soft tissue optimised B-mode-images. A pure image processing does not allow an automatic identification of individual structures, so that this must be done by the physician. To decrease the high expense of time, this report presents a concept for an adapted chain of rf-signal processing as well as an ultrasonic system for the contrast-enhanced representation of tissue borders. The system permits an exact measurement of the body geometry, which is demonstrated by determining the position of the pelvis entry plane.

Ancient DNA.

The investigation of molecular traits for evolutionary studies is a valuable alternative to the classic morphological approach. Since the mid-1980s molecular evolution has extended its field of investigation into the past. This has become possible with development of the polymerase chain reaction, which allows amplification of, and hence the study of, DNA from individuals long since dead. Many ancient, mostly extinct, animals and plants have been studied in order to determine their phylogenetic relationships with extant species. The biochemical properties of ancient DNA are also under investigation. The modifications that this molecule undergoes over long periods of time are of great interest as attempts are made to retrieve DNA from increasingly older remains.

[Alterations of the plasma ribonuclease activity in female wistar rats under the conditions of combined therapy of the ovary carcinoma (author's transl)]. / Veränderungen der Plasmaribonuklease-Aktivität bei weiblichen Wistar-Ratten unter den Bedingungen der Kombinationstherapie des Ovarialkarzinoms.

The authors have analysed the influence exercised by the combined therapy of the ovary carcinoma on the plasma ribonuclease activity in Wistar rats. The medicaments "Proresid", "VM-26", and "Methotrexat" together with a percutaneous irradiation of the abdomen did not alterate perceptibly the enzyme activity during the experiment, but there was a considerable reduction of the enzyme activity after an application of "Endoxan", "Adriblastin" and "Methotrexat" with subsequent administration of "Leukovorin". The most violent ribonuclease suppression, however, was obtained by an intraperitoneal instillation of radiogold and a fractioned percutaneous irradiation of the abdomen with intercalated administration of "Endoxan".

[Determination of the plasma ribonuclease activity as a diagnostic aid in the diagnosis of ovarian cancer. Results of a double blind study (author's transl)]. / Bestimmung der Plasmaribonukleaseaktivität als diagnostisches Hilfsmittel zur Erfassung von Ovarialkarzinomen - Ergebnisse einer Doppelblindstudie.

The determination of the plasmaribonucleases was reported by Sheid and co-workers in 1977 for the diagnosis of carcinoma of the ovary. The authors investigated the accuracy of this test with a modified method in a double blind study encompassing healthy controls. Blood samples from 62 patients with ovarian cancer were tested. The results were accurate in 56 cases. In 6 cases the test results were doubtful. The difference in the ribonuclease activity was significant between patients with ovarian cancer and healthy patients or patients in remission from ovarian cancer. The results were not dependant upon the FIGO staging of the disease. Our results encourage the recommendation to use this test as a diagnostic aid in carcinoma of the ovary.

Ancient DNA: methodological challenges.

The study of ancient DNA offers the possibility of following genetic change over time. However, the field is plagued by a problem which is unique in molecular biology--the difficulty of verifying results by reproduction. Some of the reasons for this are technical and derive from the low copy number and damaged state of ancient DNA molecules. Other reasons are the unique nature of many of the objects from which DNA is extracted. We describe methodological approaches with which these problems can be alleviated in order to ensure that results are scientific in the sense that they can be reproduced by others.

Molecular phylogeny of the extinct ground sloth Mylodon darwinii.

DNA was extracted from the remains of 35 ground sloths from various parts of North and South America. Two specimens of Mylodon darwinii, a species that went extinct at the end of the last glaciation, yielded amplifiable DNA. However, of the total DNA extracted, only approximately 1/1000 originated from the sloth, whereas a substantial part of the remainder was of bacterial and fungal origin. In spite of this, > 1100 bp of sloth mitochondrial rDNA sequences could be reconstructed from short amplification products. Phylogenetic analyses using homologous sequences from all extant edentate groups suggest that Mylodon darwinii was more closely related to the two-toed than the three-toed sloths and, thus, that an arboreal life-style has evolved at least twice among sloths. The divergence of Mylodon and the two-toed sloth furthermore allows a date for the radiation of armadillos, anteaters, and sloths to be estimated. This result shows that the edentates differ from other mammalian orders in that they contain lineages that diverged before the end of the Cretaceous Period.

DNA damage and DNA sequence retrieval from ancient tissues.

Gas chromatography/mass spectrometry (GC/MS) was used to determine the amounts of eight oxidative base modifications in DNA extracted from 11 specimens of bones and soft tissues, ranging in age from 40 to >50 000 years. Among the compounds assayed hydantoin derivatives of pyrimidines were quantitatively dominant. From five of the specimens endogenous ancient DNA sequences could be amplified by PCR. The DNA from these specimens contained substantially lower amounts of hydantoins than the six specimens from which no DNA could be amplified. Other types of damage, e.g. oxidation products of purines, did not correlate with the inability to retrieve DNA sequences. Furthermore, all samples with low amounts of damage and from which DNA could be amplified stemmed from regions where low temperatures have prevailed throughout the burial period of the specimens.

A human DNA editing enzyme homologous to the Escherichia coli DnaQ/MutD protein.

Mammalian DNA polymerases alpha and beta lack 3' exonuclease activity and are unable to edit errors after DNA synthesis. However, editing exonucleases can be functions of separate polypeptides. We isolated a widely distributed DNA-specific 3' exonuclease from rabbit liver nuclei, sequenced tryptic peptides by mass spectrometry, and identified the corresponding human open reading frame. The protein expressed from the cloned human sequence exhibits 3' exonuclease activity. The human clone shares sequence homology with the editing function of the Escherichia coli DNA polymerase III holoenzyme, i.e., the DnaQ/MutD protein, and weakly with the editing 3' exonuclease domain of eukaryotic DNA polymerase epsilon. The gene maps to human chromosome 3p21.2-21.3. In a reconstituted human DNA repair system containing DNA polymerase beta and DNA ligase III-XRCC1, accurate rejoining of a 3' mismatched base residue at a single-strand break is dependent on addition of the exonuclease.

Base excision repair of oxidative DNA damage activated by XPG protein.

Oxidized pyrimidines in DNA are removed by a distinct base excision repair pathway initiated by the DNA glycosylase--AP lyase hNth1 in human cells. We have reconstituted this single-residue replacement pathway with recombinant proteins, including the AP endonuclease HAP1/APE, DNA polymerase beta, and DNA ligase III-XRCC1 heterodimer. With these proteins, the nucleotide excision repair enzyme XPG serves as a cofactor for the efficient function of hNth1. XPG protein promotes binding of hNth1 to damaged DNA. The stimulation of hNth1 activity is retained in XPG catalytic site mutants inactive in nucleotide excision repair. The data support the model that development of Cockayne syndrome in XP-G patients is related to inefficient excision of endogenous oxidative DNA damage.