Dynamic protein methylation in chromatin biology.

Post-translational modification of chromatin is emerging as an increasingly important regulator of chromosomal processes. In particular, histone lysine and arginine methylation play important roles in regulating transcription, maintaining genomic integrity, and contributing to epigenetic memory. Recently, the use of new approaches to analyse histone methylation, the generation of genetic model systems, and the ability to interrogate genome wide histone modification profiles has aided in defining how histone methylation contributes to these processes. Here we focus on the recent advances in our understanding of the histone methylation system and examine how dynamic histone methylation contributes to normal cellular function in mammals.

Molecular cloning, expression analysis and assignment of the porcine tumor necrosis factor superfamily member 10 gene (TNFSF10) to SSC13q34-->q36 by fluorescence in situ hybridization and radiation hybrid mapping.

We have cloned the complete coding region of the porcine TNFSF10 gene. The porcine TNFSF10 cDNA has an ORF of 870 nucleotides and shares 85% identity with human TNFSF10, and 75% and 72% identity with rat and mouse Tnfsf10 coding sequences, respectively. The deduced porcine TNFSF10 protein consists of 289 amino acids with the calculated molecular mass of 33.5 kDa and a predicted pI of 8.15. The amino acid sequence similarities correspond to 86, 72 and 70% when compared with human, rat and mouse sequences, respectively. Northern blot analysis detected TNFSF10-specific transcripts (approximately 1.7 kb) in various organs of a 10-week-old pig, suggesting ubiquitous expression. Real-time RT-PCR studies of various organs from fetal (days 73 and 98) and postnatal stages (two weeks, eight months) demonstrated developmental and tissue-specific regulation of TNFSF10 mRNA abundance. The chromosomal location of the porcine TNFSF10 gene was determined by FISH of a specific BAC clone to metaphase chromosomes. This TNFSF10 BAC clone has been assigned to SSC13q34-->q36. Additionally, the localization of the TNFSF10 gene was verified by RH mapping on the porcine IMpRH panel.

[Invasive techniques in emergency medicine. II. Preclinical thorax drainage--indications and technique]. / Invasive Techniken in der Notfallmedizin. II. Präklinische Thoraxdrainage--Indikationen und Technik.

The preclinical tension pneumothorax which even without technical support is easily recognizable, requires immediate decompression. However, there are a number of patients with thoracic injuries such as serial rib fractures or palpable skin emphysema which--in combination with a ventilator--may necessitate the insertion of a thoracic tube. In the preclinical setting this procedure usually only takes place in the ventilated patient. With patients who are respiratorily compensated and are breathing spontaneously, careful control and a conservative approach is advised even if pneumothorax is suspected.

Mapping of a minimal apolipoprotein(a) interaction motif conserved in fibrin(ogen) beta - and gamma -chains.

Lipoprotein(a) (Lp(a)) is a major independent risk factor for atherothrombotic disease in humans. The physiological function(s) of Lp(a) as well as the precise mechanism(s) by which high plasma levels of Lp(a) increase risk are unknown. Binding of apolipoprotein(a) (apo(a)) to fibrin(ogen) and other components of the blood clotting cascade has been demonstrated in vitro, but the domains in fibrin(ogen) critical for interaction are undefined. We used apo(a) kringle IV subtypes to screen a human liver cDNA library by the yeast GAL4 two-hybrid interaction trap system. Among positive clones that emerged from the screen, clones were identified as fibrinogen beta- and gamma-chains. Peptide-based pull-down experiments confirmed that the emerging peptide motif, conserved in the carboxyl-terminal globular domains of the fibrinogen beta and gamma modules specifically interacts with apo(a)/Lp(a) in human plasma as well as in cell culture supernatants of HepG2 and Chinese hamster ovary cells, ectopically expressing apo(a)/Lp(a). The influence of lysine in the fibrinogen peptides and of lysine binding sites in apo(a) for the interaction was evaluated by binding experiments with apo(a) mutants and a mutated fibrin(ogen) peptid. This confirmed the lysine binding sites in kringle IV type 10 of apo(a) as the major fibrin(ogen) binding site but also demonstrated lysine-independent interactions.

Molecular characterisation of congenital glaucoma in a consanguineous Canadian community: a step towards preventing glaucoma related blindness.

Glaucoma is a leading cause of irreversible blindness in Canada. Congenital glaucoma usually manifests during the first years of life and is characterised by severe visual loss and autosomal recessive inheritance. Two disease loci, on chromosomes 1p36 and 2p21, have been associated with various forms of congenital glaucoma. A branch of a large six generation family from a consanguineous Amish community in south western Ontario was affected with congenital glaucoma and was studied by linkage and mutational analysis to identify the glaucoma related genetic defects. Linkage analysis using the MLINK component of the LINKAGE package (v 5.1) showed evidence of linkage to the 2p21 region (Zmax=3.34, theta=0, D2S1348 and D2S1346). Mutational analysis of the primary candidate gene, CYP1B1, was done by direct cycle sequencing, dideoxy fingerprinting analysis, and fragment analysis. Two different disease causing mutations in exon 3, 1410del13 and 1505G-->A, both segregated with the disease phenotype. The two different combinations of these alleles appeared to result in a variable expressivity of the phenotype. The compound heterozygote appeared to have a milder phenotype when compared to the homozygotes for the 13 bp deletion. The congenital glaucoma phenotype for this large inbred Amish family is the result of mutations in CYP1B1 (2p21). The molecular information derived from this study will be used to help identify carriers of the CYP1B1 mutation in this community and optimise the management of those at risk of developing glaucoma.

Characterization of an overexpressed spindle protein during a baculovirus infection.

The nucleopolyhedrovirus CfDEFNPV contains a gene encoding a viral protein, which accumulates as bipyramidal inclusion bodies (spindles) in the cytoplasm of infected cells. The spindles appear as early as 24 h postinfection, approximately 1 day earlier than viral occlusion bodies (OBs). Purification and characterization of the spindle protein was complicated by the fact that the OBs copurified with the spindles. We therefore modified CfDEFNPV by replacing the polyhedrin gene (plh) with a cassette containing the green fluorescent protein (GFP) gene. The recombinant virus did not produce OBs; however, the synthesis and morphogenesis of the spindles were not altered. When analyzed by SDS-PAGE, the spindles produced a 50-kDa protein, which was termed spindlin. Tunicamycin inhibition and endoglycosidase studies showed that spindlin was glycosylated. The N-terminus of spindlin was sequenced and its gene (gp50) was located on the viral genome. The gene was cloned and sequenced. Homologs of gp50 were found in several baculoviruses as well as in entomopoxviruses (EPV). In the latter virus, the homologous gene is that of fusolin, which also encodes a protein that forms spindle-shaped inclusion bodies in the cytoplasm of infected cells. Immunoblot analysis indicated that spindlin and fusolin were not serologically related, even though they share conserved polypeptide domains. Sequence analysis showed that gp50 of CfDEFNPV contains two late promoter motifs (TTAAG) in its 5' flanking region. Both were used, but the proximal motif (-14 to -18 nt relative to the ATG) was the primary sequence from which most of the mRNA was initiated. When gp50 was cloned in a heterologous baculovirus expression system, spindlin was synthesized, although the spindles were irregular in shape. This suggested that the spindle structure may be species-specific or it may require more than one gene product for its morphogenesis.

Genetic heterogeneity of the Coppock-like cataract: a mutation in CRYBB2 on chromosome 22q11.2.

PURPOSE: To identify the genetic defect for the Coppock-like cataract (CCL) affecting a Swiss family, which defect was unlinked to the chromosome 2q33-35 CCL locus. METHODS: A large family was characterized for linkage analysis by slit lamp examination or by the review of drawings made before cataract extraction. The affection status was attributed before genotyping, and the genotyping was masked to the affection status. Two-point and multipoint linkage analyses were performed using the MLINK and the LINKMAP components of the LINKAGE program package (ver. 5.1), respectively. Mutational analysis of candidate genes was performed by a combination of direct cycle sequencing and an amplification refractory mutation system assay. RESULTS: Ten individuals were affected with the CCL phenotype. The disease was autosomal dominant and appeared to be fully penetrant. A new CCL locus was identified on chromosome 22q11.2 within a 11.67-cM interval (maximum lod score [Zmax] = 4.14; theta = 0). Mutational analysis of the CRYBB2 candidate gene identified a disease-causing mutation in exon 6. This sequence change was identical with that previously described to be associated with the cerulean cataract, a clinically distinct entity. CONCLUSIONS: The CCL phenotype is genetically heterogeneous with a second gene on chromosome 22q11.2, CRYBB2. The CCL and the cerulean cataract are two distinct clinical entities associated with the same genetic defect. This work provides evidence for a modifier factor that influences cataract formation and that remains to be identified.

Technical aspects of telepathology with emphasis on future development.

Pathology undergoes presently changes due to new developments in diagnostic opportunities and cost saving efforts in health care. Out of the wide field of telepathology the paper selects three prototype applications: telepathology in teleeducation, expert advice for preselected details of a slide and finally telepathology for remote diagnosis. The most challenging field for remote diagnosis is the application in the frozen section scenario. The paper starts with the mental experiment to map conventional procedures to counterparts in telepathology. Technical opportunities and economical restrictions of telepathology equipment are discussed with respect to the components: electronic camera, display devices, haptic sensors and displays, available telecommunication channels and telepathology software. As an example and for illustration of the state of the art for an advanced telemicroscopy system able to perform remote frozen section diagnosis, the HISTKOM equipment is presented in more details. The section concerning future developments regards the aspects of the acceptance by tentative users, legal aspects, costs and affordability of equipment, the market for equipment components and the adequate telecommunication services. Further is regarded the mutual influence of properties of existing systems and application experiences gained with them on the next generation of equipment and application software. Conclusions and references close the paper.

European field tests with HISTKOM telepathology equipment.

HISTKOM telemicroscopy equipment for telepathology is designed for the most challenging application in telepathology: intraoperational frozen section diagnosis. Adapted to this application, it is also excellently suited for all other telepathology modes requesting less sophisticated equipment. The technical concept and user interface are oriented to routine daily pathology. HISTKOM underwent heavy field-tests at several locations. The field-tests designs and the results of five of these are reported in this paper. Telepathology will exploit its advantages in networks hosting participants requesting and offering services. The solution of the interoperability problem caused by different equipment from different suppliers within such a network will be a major task, the solution for which is in progress. The new generation of HISTKOM equipment and software is designed in a modularized concept, allowing the integration of various hardware components from different manufacturers; thus special configurations can be realized easily. HISTKOM is offered as complete turnkey system, but can also be installed in yet existing configurations of the customer if they meet specifications.

Centerfire frangible ammunition: wounding potential and other forensic concerns.

Recently developed frangible ammunition of copper particulate construction in .38 Special, 9 mm, and .223 calibers was evaluated for wounding performance by firing into pigs' heads. The ability to match fired bullets with the corresponding gun was also examined. Results showed that wounds caused by 9-mm and .38 Special frangible bullets were comparable in severity to those caused by regular service ammunition of the same caliber. The recovered 9-mm and .38 Special bullets demonstrated class characteristics but not the individual rifling marks necessary for bullet-to-gun matching. High-velocity .223-caliber rifle bullets fragmented extensively within target tissues, causing severe wounding. Radiologic examination of resulting wounds showed images strikingly similar to the lead "snowstorm" picture caused by high-velocity hunting ammunition.

[Ultrasound diagnosis of acute rupture of the anterior cruciate ligament. An experimental and clinical study]. / Sonographische Diagnostik der frischen Rupturen des vorderen Kreuzbands. Eine experimentelle und klinische Studie.

In an experimental study on 20 cadaver knee joints we found examined the ventral translation of the tibial head, which is otherwise shown by the Lachmann test, by dorsal sonography. These examinations were performed in knees in which arthroscopical examination had confirmed intact ACL. After endoscopical partial and complete rupture of the ACL the examinations were repeated. Radiological examination was performed as a reference method. In a sonographically standardized clinical study of 243 traumatized knee joints the ventral translation of the tibial head was evaluated. In all knee joints examined an arthroscopy or arthrotomy was performed. In 56% a rupture of the ACL was verified. There was a positive correlation between translation of the tibial head by 3 mm or more compared with the non-injured side and a rupture of the ACL (sensitivity 0.95; specificity 0.98). The dynamic sonographic technique is a simple and highly sensitive specific diagnostic method that can be used to quantify the results found on clinical examination.

Hydrosalpinx formation and its regeneration after microsurgical reconstruction--a functional and morphological study on rabbits.

The development of mechanically induced hydrosalpinx and the subsequent regeneration of the uterine tube following correction were investigated with reference to morphological and functional parameters in 33 rabbits. Mechanically induced hydrosalpinx leads to reduced fertility. The characteristic morphological alterations are flattening of the folds, deciliation, reduced secretory activity and general de-differentiation of the epithelial cells. The intensity of these changes peaked as little as 4 weeks after induction of the hydrosalpinx. The deciliation is generally patchy. The division into different fields, islet formation and celestial chart phenomena reflect different degrees of vascularization and/or oxygenation. These alterations were completely reversible within 8 weeks after reconstruction. In isolated cases only the formation of polypose stumps was seen, which were, however, covered with a normal epithelial surface. The discrepancy between the reduced tubal function following reconstruction for hydrosalpinx and the complete regeneration of the tubal mucosa suggests that other regulatory mechanisms more important for the function of the uterine tube are impaired.

[Healing of tubal anastomoses--microsurgery vs. fibrin gluing: morphologic aspects]. / Die Heilung von Tubenanastomosen--Mikrochirurgie vs. Fibrinklebung: morphologische Aspekte.

The healing of anastomoses either by microsurgical suture technique or by fibrin sealant technique has been examined in an experimental morphological study. With view to morphological criteria the healing of tubal anastomoses after one month is completed. Afterwards only little areas of regeneration could be found in the region of the anastomosis. These statements are valid for both types of anastomoses. In single cases a more progressive regeneration of the mucosa could be demonstrated. Using fibrin glue a more pronounced fibrosis could not be seen.

Fibrin glue for anastomosis of the fallopian tube--morphology.

In a morphological study fibrin-glued tubal anastomoses were compared with classical anastomoses using microsutures. In the isthmic-isthmic anastomoses, whether they were glued or sutured, the opposition of the folds and the continuity of the tubal wall were good. Scanning electron microscope studies of Fallopian tubes subjected to anastomosis using fibrin glue or microsurgical sutures showed a break in the fold structure regardless of the technique used, with formation of polyp-like and tuborous structures in some of the anastomoses in the ampullary region. In one case of an ampullar-ampullar anastomosis with fibrin glue, formation of a fistula was observed and in another case of the same type of anastomosis a hydrosalpinx developed as the consequence of intratubal adhesions and stenosis. In glued anastomoses in particular, intraluminal fibrin deposits were observed. Otherwise our scanning and transmission electron microscope investigations of the region of the anastomoses revealed a normal cell picture with abundant cilia-bearing, structurally unremarkable cells. Fibrin glueing can thus be regarded as a possible alternative to the conventional microsurgical suturing technique for the construction of anastomoses in the isthmic segment of the tube. In the case of wide-lumen ampullary anastomoses, however, the danger of fistula formation, dehiscence, development of intraluminal adhesions and stenosis must be regarded as increased. Fibrin glueing also does not appear to be appropriate for anastomoses requiring approximation of differing luminal widths.