Cervical wear pathobiology by robot-simulated 3-year toothbrushing - New methodological approach.

OBJECTIVES: An ex-vivo study was aimed at (i) programming clinically validated robot three-year random toothbrushing, (ii) evaluating cervical macro- and microwear patterns on all tooth groups of different functional age, (iii) documenting and codificating wear related morphological features at the cemento-enamel junction in young teeth and on roots in older teeth. DESIGN: Following ethical approval random toothbrushing (44 strokes per tooth horizontally, rotating, vertically; 2x/d) with manual toothbrushes and low-abrasive dentifrice was performed in an artificial oral cavity with brushing-force 3.5 N on 14 extracted human teeth. Morphological features were examined by SEM at baseline and after simulated 3 years using the replication technique. 3D-SEM analyses were carried out with a four-quadrant back scattered electron detector. Wilcoxon-Mann-Whitney-test was used for statistical analyses. RESULTS: 3-year random toothbrushing with horizontal, rotating and vertical brushing movements revealed morphological features classified as four enamel patterns, one dentin pattern and three cervical patterns. Negative impacts were enamel, cementum and dentin loss. Positive impact on oral health was removing dental calculus and straightening cervical traumatic and iatrogenic damages. The volume loss varied from x̅=34.25nl to x̅=87.75nl. Wear extended apically from 100 to 1500 micrometres. CONCLUSION: Robot simulated toothbrushing in an artificial oral cavity, with subsequent SEM and 3D-SEM assessment, elucidated both negative and oral health-contributing micromorphology patterns of cervical wear after simulated 3-year random toothbrushing. Cervical macro- and microwear of cementum revealed, for the first time, what we describe as overhanging enamel peninsulas and enamel islands on roots in young teeth, but no enamel islands on roots from older teeth after root cementum loss. In contrast, many older teeth exhibited enamel peninsulas.

Visualizing nuclei in skin cryosections: viable options to 4'6-diamidino-2-phenylindol for confocal laser microscopy.

BACKGROUND: Visualization of nuclei in skin (cryo-) sections is essential for both, rapid overview and reliable orientation within skin samples. Therefore, nuclear staining is a very common counterstain for immunohistochemical studies of human skin as this nuclear staining precisely depicts the cellular distribution within the epidermis. Moreover, it clearly shows the epidermal-dermal border as well as the transition zone between the living and the cornified layers of the epidermis. For standard epifluorescence microscopy, 4'6-diamidino-2-phenylindol (DAPI) is commonly used. For confocal laser scanning microscopy (CLSM), however, DAPI is often not suitable because its excitation maximum is in the ultraviolet (UV) range (Ex(max) 359 nm) when bound to DNA, and UV lasers and the corresponding optics are not part of CLSM standard configuration. METHODS: In order to find an adequate DAPI substitute that is excitable with standard visible light lasers, the following nuclear stains were tested: LOLOt-1 iodide (Ex(max) 565 nm), TOTO s -3 iodide (Ex(max) 642 nm), LO-PROt-1 iodide (Ex(max) 567 nm), SYTO s 84 (Ex(max) 567 nm), SYTO s 85 (Ex(max) 567 nm), SYTOX s Green (Ex(max) 488 nm) and SYTOX s Orange (Ex(max) 547 nm), Propidium iodide (Ex(max) 535 nm). Besides optimal concentration and incubation time, following criteria were also evaluated: photobleaching, background, e.g. cytoplasmic staining of RNA, and sensitivity to different fixation conditions (unfixed, IEM fixation, PLP fixation and PFA fixation). RESULTS: According to these criteria Sytox s Green showed the best overall staining score and can be used for variously fixed skin samples and shows a distinct and stable green nuclear fluorescence.

A short history of sweat gland biology.

The axilla, especially its microflora and axillary sweat glands as well as their secretions, is the main target of cosmetic compositions such as deodorants or antiperspirants. There are three types of sweat glands present in the axillary skin, namely apocrine, eccrine and apoeccrine sweat glands. Here, we provide an overview of the morphological, structural and functional characteristics of the different gland types and present techniques that allow their clear distinction. Moreover, we describe different forms of perspiration as physical reactions to external and internal stimuli.

Are sweat glands an alternate penetration pathway? Understanding the morphological complexity of the axillary sweat gland apparatus.

To build an effective barrier against the penetration of extrinsic agents is one of the skin's main functions. The barrier properties of the stratum corneum and the epidermis have been subject to extensive studies in the past while the role of skin appendages as possible pathways of penetration are only rarely described. In order to study the possible penetration barriers in these complex appendages, a careful investigation of their morphology and ultrastructure has to be done. Studying the morphology of axillary skin appendages requires clear-cut criteria for the differentiation between eccrine, apocrine and apoeccrine glands. Therefore we studied the distribution of proteins described to be specific for either eccrine or apocrine glands (CD15, CD44, S-100 and milk fat globulin) on axillary skin samples from healthy young adults by immunofluorescence. Additionally, we examined the distribution of cytoskeletal proteins such as cytokeratins (1/10/11, 14, 18) and F-actin. For a more detailed understanding of the possible versatile barrier elements of the axillary sweat glands, we studied the distribution of tight-junction-associated proteins (occludin, claudin 1, claudin 4). The coils and the dermal duct may provide an active barrier built of tight junctions as occludin and claudin 4 are co-localized. However, the intra-epidermal duct did not show any co-localization of the investigated proteins. By combining morphological features as revealed by F-actin staining and the distribution of the above-mentioned proteins, immunocytochemical typing of eccrine and apocrine glands becomes possible. With this tool, we could also confirm the existence of apoeccrine glands and locate them in their 'natural environment'.

[Taxanes in the chemotherapy of hormone-refractory prostate carcinoma]. / Taxane in der Chemotherapie des hormonrefraktären Prostatakarzinoms.

Prostate cancer represents one of the most prevalent malignancies in men. Standard therapy of metastatic prostate cancer consists of androgen deprivation, which is a palliative therapy yielding a clinical response of limited duration. In hormone-refractory prostate cancer (HRPC), response to chemotherapy with regimens available until about ten years ago has been disappointing. Nowadays, due to increasing life expectancy and earlier diagnosis and therapy of prostate cancer, more patients with hormone-refractory disease are still in relatively good overall condition. With the taxanes, much more effective cytostatic substances for chemotherapy of HRPC are available today. Using modern taxane-based chemotherapy, effective palliation of pain can be achieved in 50-70% of patients with HRPC, while retaining an acceptable quality of life. There is also evidence for improved overall survival after taxane-based chemotherapy, although this remains to be proven by ongoing studies. This article presents an overview of current studies investigating the outcome after taxane-based chemotherapy, as well as new therapeutic approaches in combination with docetaxel.

Diagnosis of haploidy and triploidy based on measurement of gene copy number by real-time PCR.

We report the development of a method for diagnosis of heterozygous deletions or duplications based on measurement of gene copy number. The method involves amplifications of a test locus with unknown copy number and a reference locus with known copy number using real-time PCR. Progress of the PCR reactions is monitored using fluorigenic probes and a real-time fluorescence detection system. For each reaction, the number of cycles is measured at which a defined threshold fluorescence emission is reached. Using standard curves, the copy number of the test DNA relative to a common standard DNA is determined for each locus. From the ratio of the relative copy numbers, the genomic copy number of the test locus is determined. In order to demonstrate the accuracy and reliability of the method for genetic testing, we analyzed 43 patients with hereditary neuropathy with liability to pressure palsies (HNPP), containing a heterozygous deletion of a 1.5 Mb region on chromosome 17p11.2-p12, eight patients with Charcot-Marie-Tooth disease, containing a heterozygous duplication of the same genomic region, and 50 normal control individuals. As a test locus we analyzed the PMP22 gene located within the 1.5 Mb region. The genomic copy number of the test locus was precisely measured, and the presence or absence of the genomic deletion or duplication was unambiguously diagnosed in all individuals.

MHC expression in fragment and full-thickness allogeneic embryonic retinal transplants.

BACKGROUND: The study was carried out to evaluate the expression of major histocompatibility complex (MHC) molecules in retinal transplants with different tissue integrity. METHODS: Twelve adult rabbits received an allogeneic subretinal neuroretinal transplant, in the form of either fragmented embryonic cells or a complete full-thickness embryonic retina. A controlled transvitreal approach was used for both transplantation types. The grafts were examined histologically after 31 or 49 days with hematoxylin and eosin staining and immunohistochemical analysis of MHC class I and class II expression. RESULTS: All five fragment transplants developed into rosettes. Two of them displayed MHC class I-labeled cells, and four MHC class II-labeled cells. The cells were concentrated on the scleral side of the graft, and there was also a marked increase of labeled cells in the choroid. MHC labeling was often associated with defects in the retinal pigment epithelium. Six of the seven full-thickness grafts displayed a laminated morphology with well-developed retinal layers. The seventh consisted of rosettes. None of these grafts displayed MHC class I- or class II-labeled cells. CONCLUSIONS: The findings suggest that host immune response against fragmented and intact neuroretinal grafts is different, indicating tissue integrity as one factor affecting graft-host immune interactions. The absence of immune response in full-thickness grafts is encouraging and important in the struggle to find therapies for retinal degenerative disease.

Beta-thalassemia in the German population: mediterranean, Asian and novel mutations. Mutations in brief no.228. Online.

The beta-thalassemia mutations of 13 unrelated heterozygous Germans who remained unidentified in a previous study of 40 subjects were investigated at the DNA level. Two Mediterranean, one Asian and three novel mutations (CD6 -G, CDs 108 /112-12nt, CDs 130/131 + GCCT) were identified. Altogether, in 30 of the 35 subjects (86%) in which a mutation in the beta-globin gene was identified, the mutation was of Mediterranean origin. The geographical distribution suggests recent migration from the Mediterranean region as cause of the high proportion of frequent Mediterranean beta-thalassemia mutations in the German population. Our results support the notion that the majority of beta-thalassemia genes in the western and central European population are of Mediterranean origin.

Molecular diagnostics of 15 hemophilia A patients: characterization of eight novel mutations in the factor VIII gene, two of which result in exon skipping.

The X-linked bleeding disorder hemophilia A is caused by mutations in the coagulation factor VIII gene. A high frequency of de novo mutations and the large size of this gene complicate the molecular diagnostic of hemophilia A. Characterization of mutations, however, may help identify amino acids or regions with essential functional or structural properties and thereby clarify the mechanism of pathogenesis. In the present study, we describe the identification of 15 mutations in the factor VIII gene, of which eight are novel. Among the patients with severe hemophilia A, two splice mutations (IVS5-3 and IVS19-2), a 4-bp deletion ((TACA) at codon 1215, and a missense mutation G1850V have been characterized. The missense mutations G479R, R531C, V537D, N2129S and I2190N were found for five patients with a moderate course of hemophilia A disease. A silent mutation resulting in activation of a cryptic acceptor splice site within exon 11 and four other missense mutations Y114C, R1689H, R2150H (2x), M2164V have been identified for six patients with mild hemophilia A.

Urodynamic effects of oral oxybutynin chloride in children with myelomeningocele and detrusor hyperreflexia.

OBJECTIVES: To investigate the effects of oral oxybutynin chloride (OC) on standard urodynamic measures in children with myelomeningocele (MMC) and detrusor hyperreflexia. METHODS: Forty-one MMC children with detrusor hyperreflexia (19 boys and 22 girls, aged 2 months to 15 years; mean 4.9 years) were evaluated urodynamically before and within 3 months after initiation of oral OC therapy (0.2 to 0.3 mg/kg/day). Therapy with oral OC was always combined with clean intermittent catheterization (CIC). RESULTS: Oral OC treatment caused an increase in bladder capacity from 141 +/- 96 to 197 +/- 99 mL (+ 40%; P < 0.01), a decrease in detrusor pressure at maximal capacity from 45 +/- 32 to 28 +/- 23 cm H2O (-38%; P < 0.01), and an increase in detrusor compliance from 6.5 +/- 5.6 to 16.8 +/- 13.7 mL/cm H2O (+ 158%; P < 0.01). Improvement in urodynamic measures and continence were correlated. After a follow-up of at least 2 years, effective protection of renal function was achieved in 38 of the 41 children (93%) with conservative therapy alone. Adverse effects resulted in discontinuation of oral OC treatment in only 2 cases. CONCLUSIONS: Treatment with oral OC and CIC is effective and safe in children with MMC and detrusor hyperreflexia and should be initiated early when indicated by urodynamic findings.

Different ocular abnormalities in individuals of a three-generation family caused by a new nonsense mutation in the PST domain of the PAX6 gene. Mutations in brief no. 189. Online.

PAX6 is a candidate gene for familial aniridia. We have carried out a mutational analysis of the PAX6 gene in a three-generation family from Germany, containing 5 individuals affected with ocular abnormalities. In all affected individuals, a heterozygous mutation was detected in the PAX6 gene, exchanging tyrosine 369 by a stop codon. The mutation is located in the 3' moiety of the PST domain, at the C terminus of the PAX6 protein. In the affected family members, the same heterozygous mutation leads to distinct phenotypes of varying severity. Most notably, no aniridia was observed in one of the family members carrying the mutation, although other ocular abnormalities (underdeveloped iris and cataracts) were present. We discuss the possibility that small C terminal truncations of the PAX6 protein might lead to less severe or more divergent phenotypes than trancations at internal positions.

Isolation of human and mouse HMG2a cDNAs: evidence for an HMG2a-specific 3' untranslated region.

We have isolated cDNAs of the human gene for high mobility group protein HMG2a, using the method of direct cDNA selection. The gene maps to chromosome band Xq28, and is located within 40 kb from marker DXS1684, at a distance of 5.4 Mb from the telomere. The deduced human HMG2a protein sequence has a length of 199 amino acids and is 97% identical to the sequence of chicken HMG2a. The 3' untranslated regions of the HMG2a gene in both species are highly homologous (87% identical nucleotides), and are even more conserved than the coding sequences (84% identical nucleotides). In addition, a partial cDNA sequence of the putative HMG2a gene from mouse was identified. The 3' untranslated regions from human and mouse are 90% identical. We conclude that the 3' untranslated sequences have been under strong selective pressure during evolution. Whereas expression of the chicken HMG2a gene has previously been demonstrated in liver of newly hatched chicken, the human HMG2a gene is transcribed mainly in placenta.

A gene in human chromosome band Xq28 (GABRE) defines a putative new subunit class of the GABAA neurotransmitter receptor.

We have isolated and sequenced a novel human gene (GABRE) of the GABAA neurotransmitter receptor family. A cDNA sequence of the gene coding for a 506 amino acid protein was identified, representing a member of a putative new class (epsilon) of the GABAA receptor. The gene is transcribed at least at low level in several different tissues, with the highest levels being detected in adult heart and placenta. Alternative splicing of GABRE transcripts isolated from different tissues was observed at multiple positions of the gene, yielding an unusually complex variety of cDNA variants. The structure of the 5' region of most cDNAs is compatible with expression of protein sequence epsilon only in adult brain, whereas in other tissues, the majority of transcripts code for truncated protein sequences. The GABRE gene extends over 14 kb and is clustered together with the alpha 3 and the putative beta 4 GABAA receptor subunit genes in an approximately 0.8-Mb interval in chromosome band Xq28, located in the candidate regions of two different neurologic diseases. Based on features of conservation of protein sequences, gene structure, and genomic organization of GABAA receptor gene clusters, we propose that the epsilon and gamma subunit genes have a common ancestor and that GABAA receptor gene clusters in the human genome have diverged by multiple duplication events of an ancestral gene cluster containing one each alpha, beta, and gamma/epsilon precursor gene.

The melanoma antigen gene (MAGE) family is clustered in the chromosomal band Xq28.

The melanoma antigen gene (MAGE) family comprises 12 known genes, of which 6 are expressed in tumors. In the course of a systematic analysis of transcripts in Xq28, we have identified cDNAs related to different MAGE genes. Analysis of cell hybrids, ordered YACs, and cosmids showed that all MAGE genes are located in Xq28 and are clustered in three main intervals within 3.5 Mb. The six genes expressed in tumors are contained in the two intervals closest to the telomere and are highly homologous to each other. Analysis of different species suggests that human MAGE sequences are conserved in primates, but less well conserved in other vertebrate species.

A YAC clone map spanning 7.5 megabases of human chromosome band Xq28.

Xq28 has been of special interest in human genetics because a large number of diseases map to this region. As a step in the molecular analysis of the as yet uncloned disease genes, and as a test for the detailed analysis of larger regions of the genome, we have constructed YAC clone contigs covering the 7.5 Mb region between IDS to the telomere on the long arm of the human X chromosome. Contigs were assembled and verified by an integrated hybridization-based strategy. Data was combined from the physical map, from YAC and cosmid mapping experiments, and from the localization of specific transcripts in the region. Two gaps in the YAC map of 250 and 100 kb were covered in part by the aid of cosmid clones, but small gaps of 50 kb each remain. The cloned region is expected to contain yet unidentified genes for at least ten genetic diseases. The construction of ordered YAC clone contigs of Xq28 represents an important step in the molecular identification of these genes, and the further analysis of one of the genetically most interesting regions of the human genome.

Reversed ratio of color-specific cones in rabbit retinal cell transplants.

Recently, we have reported on the emergence of various retinal cell types in embryonic rabbit retina transplanted to adult rabbits. When comparing the relative numbers of the spectrally different cone types in the transplants to those in the host or age-matched control retinas, a surprising shift was observed. While in the normal rabbit retina the middle-wavelength-sensitive (M) cones are considerably more abundant than the short-wave-sensitive (S) cones, the S/M cone ratio was found to be the opposite in the graft. The number of rosettes containing only S-cones in high density was found to be considerably higher than that of M-cone rich rosettes. The number of S-cones also exceeded that of the M-cones in each rosette that contained both cell types. Our results were obtained from the systematic immunocytochemical analysis of 15 different transplants derived from transplantations of embryonic rabbit retinas into adult hosts of the same species. The emergence and proportion of the two cone types were followed between 14 and 63 days after transplantation (between 29 and 78 postconceptional days of the donor tissue). Sections from various parts of the transplants were reacted with the monoclonal antibodies COS-1 and OS-2, specific for the middle- and short-wavelength-sensitive cones, respectively. The explanation for the reverse cone ratio in these transplants is not known yet, however, the observed phenomenon may indicate differences between the specification of the two basic cone types.

[Bacteria-reducing properties of copper-containing and non-copper-containing materials. II. Relationship between microbiocide effect of copper-containing materials and copper ion concentration after contamination with moist and dry hands]. / Bakterienreduzierende Eigenschaften kupferhaltiger und nicht kupferhaltiger Materialien. II. Mitteilung: Beziehung zwischen mikrobizider Wirkung kupferhaltiger Materialien und Kupferionen-Konzentration bei Kontamination mit feuchten und trockenen Händen.

The bactericide quality of different materials containing copper (copper, brass, brass-sheet) was tested and particularly the effect of humidity and copper-concentration was examined. In first examinations (I. communication) (19) the materials were contaminated by hands, which were dipped into a suspension of different microorganisms (Micrococcus luteus, Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa). The survival rate of the microorganisms on the surfaces was determined after defined exposure times (0, 5, 30, 60, 120 min). The following ways of contaminating the test materials were chosen: single contamination with wet hands, repeated contamination with wet and dry hands. In this examination the results were tested for statistical significance and in parallel experiments the copper-concentration on the surfaces of the materials was determined. Materials containing copper contaminated with wet hands show a significant bactericide effect after an exposure time of 120 min. The bactericide effect on Micrococcus luteus and Pseudomonas aeruginosa is inhibited after contamination with dry hands. This results can not be explained by a different copper-concentration on the surfaces of the materials, because a statistically significant difference of copper-ion concentrations between contamination with wet and dry hands could not be obtained.

Porcine (GT)n sequences: structure and association with dispersed and tandem repeats.

Loci containing (GT)n repeats were isolated from three different plasmid libraries with inserts of porcine genomic DNA between 140 and 200, 200 and 300, and 350 and 400 bp. Sequencing showed that the average repeat length and the fraction of perfect repeats were increased in the libraries containing longer inserts (> or = 200 bp). The polymorphism of (GT)n loci containing at least 10 repeat units was analyzed using the polymerase chain reaction and an automated DNA sequencer. Nearly all tested loci are polymorphic and can therefore be used as marker loci for gene mapping and for other applications. The (GT)n loci were categorized into three classes: (1) loci containing the (GT)n repeats associated with a SINE element, (2) loci containing the (GT)n repeats associated with one or more other simple repeats, and (3) loci containing (GT)n as the only detected repetitive element. At most loci of the first class, the (GT)n repeat was in a fixed configuration adjacent to the 3' end of the SINE. The findings support the notion of clustering of different repeat types in the mammalian genome.