DNA methylation in the promoter region of the p16 (CDKN2/MTS-1/INK4A) gene in human breast tumours

The p16 (CDKN2/MTS-1/INK4A) gene is one of several tumour-suppressor genes that have been shown to be inactivated by DNA methylation in various human cancers including breast tumours. We have used bisulphite genomic sequencing to examine the detailed sequence specificity of DNA methylation in the CpG island promoter/exon 1 region in the p16 gene in DNA from a series of human breast cancer specimens and normal human breast tissue (from reductive mammaplasty). The p16 region examined was unmethylated in the four normal human breast specimens and in four out of nine breast tumours. In the other five independent breast tumour specimens, a uniform pattern of DNA methylation was observed. Of the nine major sites of DNA methylation in the amplified region from these tumour DNAs, four were in non-CG sequences. This unusual concentration of non-CG methylation sites was not a general phenomenon present throughout the genome of these tumour cells because the methylated CpG island regions of interspersed L1 repeats had a pattern of (almost exclusively) CG methylation similar to that found in normal breast tissue DNA and in DNA from tumours with unmethylated p16 genes. These data suggest that DNA methylation of the p16 gene in some breast tumours could be the result of an active process that generates a discrete methylation pattern and, hence, could ultimately be amenable to therapeutic manipulation.     

Membrane-type 1 matrix metalloprotease (MT1-MMP) enables invasive migration of glioma cells in central nervous system white matter

Invasive glioma cells migrate preferentially along central nervous system (CNS) white matter fiber tracts irrespective of the fact that CNS myelin contains proteins that inhibit cell migration and neurite outgrowth. Previous work has demonstrated that to migrate on a myelin substrate and to overcome its inhibitory effect, rat C6 and human glioblastoma cells require a membrane-bound metalloproteolytic activity (C6-MP) which shares several biochemical and pharmacological characteristics with MT1-MMP. We show now that MT1-MMP is expressed on the surface of rat C6 glioblastoma cells and is coenriched with C6-MP activity. Immunodepletion of C6-MP activity is achieved with an anti-MT1-MMP antibody. These data suggest that MT1-MMP and the C6-MP are closely related or identical. When mouse 3T3 fibroblasts were transfected with MT1-MMP they acquired the ability to spread and migrate on the nonpermissive myelin substrate and to infiltrate into adult rat optic nerve explants. MT1-MMP-transfected fibroblasts and C6 glioma cells were able to digest bNI-220, one of the most potent CNS myelin inhibitory proteins. Plasma membranes of both MT1-MMP-transfected fibroblasts and C6 glioma cells inactivated inhibitory myelin extracts, and this activity was sensitive to the same protease inhibitors. Interestingly, pretreatment of CNS myelin with gelatinase A/MMP-2 could not inactivate its inhibitory property. These data imply an important role of MT1-MMP in spreading and migration of glioma cells on white matter constituents in vitro and point to a function of MT1-MMP in the invasive behavior of malignant gliomas in the CNS in vivo.     

Analysis of germline mutation spectra at the Huntington's disease locus supports a mitotic mutation mechanism

Trinucleotide repeat disease alleles can undergo 'dynamic' mutations in which repeat number may change when a gene is transmitted from parent to offspring. By typing >3500 sperm, we determined the size distribution of Huntington's disease (HD) germline mutations produced by 26 individuals from the Venezuelan cohort with CAG/CTG repeat numbers ranging from 37 to 62. Both the mutation frequency and mean change in allele size increased with increasing somatic repeat number. The mutation frequencies averaged 82% and, for individuals with at least 50 repeats, 98%. The extraordinarily high mutation frequency levels are most consistent with a mutation process that occurs throughout germline mitotic divisions, rather than resulting from a single meiotic event. In several cases, the mean change in repeat number differed significantly among individuals with similar somatic allele sizes. This individual variation could not be attributed to age in a simple way or to ' cis ' sequences, suggesting the influence of genetic background or other factors. A familial effect is suggested in one family where both the father and son gave highly unusual spectra compared with other individuals matched for age and repeat number. A statistical model based on incomplete processing of Okazaki fragments during DNA replication was found to provide an excellent fit to the data but variation in parameter values among individuals suggests that the molecular mechanism might be more complex.     

A detailed structural description of Escherichia coli succinyl-CoA synthetase

Succinyl-CoA synthetase (SCS) carries out the substrate-level phosphorylation of GDP or ADP in the citric acid cycle. A molecular model of the enzyme from Escherichia coli, crystallized in the presence of CoA, has been refined against data collected to 2.3 A resolution. The crystals are of space group P4322, having unit cell dimensions a=b=98.68 A, c=403.76 A and the data set includes the data measured from 23 crystals. E. coli SCS is an (alphabeta)2-tetramer; there are two copies of each subunit in the asymmetric unit of the crystals. The crystal packing leaves two choices for which pair of alphabeta-dimers form the physiologically relevant tetramer. The copies of the alphabeta-dimer are similar, each having one active site where the phosphorylated histidine residue and the thiol group of CoA are found. CoA is bound in an extended conformation to the nucleotide-binding motif in the N-terminal domain of the alpha-subunit. The phosphoryl group of the phosphorylated histidine residue is positioned at the amino termini of two alpha-helices, one from the C-terminal domain of the alpha-subunit and the other from the C-terminal domain of the beta-subunit. These two domains have similar topologies, despite only 14 % sequence identity. By analogy to other nucleotide-binding proteins, the binding site for the nucleotide may reside in the N-terminal domain of the beta-subunit. If this is so, the catalytic histidine residue would have to move about 35 A to react with the nucleotide.     

A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes

This paper outlines a simple method of estimating both the Ca-buffering properties of the cytoplasm and the time-course of changes of sarcoplasmic reticulum (s.r.) Ca concentration during systole. The experiments were performed on voltage-clamped ferret single ventricular myocytes loaded with the free acid of fluo-3 through a patch pipette. The application of caffeine (10 mM) resulted in a Na-Ca exchange current and a transient increase of the free intracellular Ca concentration ([Ca2+]i). The time-course of change of total Ca in the cell was obtained by integrating the current and this was compared with the measurements of [Ca2+]i to obtain a buffering curve. This could be fit with a maximum capacity for the intrinsic buffers of 114+/-18 micromol l-1 and Kd of 0.59+/-0.17 microM (n=8). During the systolic rise of [Ca2+]i, the measured changes of [Ca2+]i and the buffering curve were used to calculate the magnitude and time-course of the change of total cytoplasmic Ca and thence of both s.r. Ca content and Ca release flux. This method provides a simple and reversible mechanism to measure Ca buffering and the time-course of both total cytoplasmic and s.r. Ca.     

Effects of test conditions on the outcome of place conditioning with morphine and naltrexone in mice

BACKGROUND: Although it is widely proposed that surgeons, before introducing a novel laparoscopic technique in man, should practice in an appropriate animal model for acquisition of the necessary technical skills, the effectiveness of those hands-on training courses are rarely documented. METHODS: In 1995 we have organized eight hands-on training courses for laparoscopic anterior interbody spine fusion in an in vivo porcine model. A total of 72 colleagues from 50 different centers of 12 countries participated, including orthopedic, trauma, visceral, neuro-, and vascular surgeons. Quality and effectiveness of the course were evaluated by a questionnaire after a 1.5- to 2.5-year period. RESULTS: During this time, 42.2% of the participating centers had applied the new technique successfully in man. Centers which participated in the course with a team that included a skilled laparoscopic surgeon and an orthopedic or trauma surgeon introduced the technique more frequently to clinical practice (57.9%) than those represented by only one participant (30. 8%). Moreover, there was a tendency toward a more frequent introduction of the technique to clinical practice in centers associated with university hospitals (57.1% vs. 29.2%), indicating the requirement of a particular infrastructure for this complex interdisciplinary procedure. Almost all participants (98.3%) agreed that for novel surgical techniques requiring advanced technical skills, there should first be training in a large animal model before the technique is applied in man. CONCLUSIONS: Complex laparoscopic procedures (i.e., laparoscopic spine surgery) can be successfully learned by in vivo hands-on training courses. We propose that for refinements and modifications of the technique (e.g. , the lumboscopic approach), there should also first be training in a large animal model before these are applied in man.     

Functional analysis of human MutSalpha and MutSbeta complexes in yeast

Mismatch repair (MMR) is initiated when a heterodimer of hMSH2*hMSH6 or hMSH2*hMSH3 binds to mismatches. Here we perform functional analyses of these human protein complexes in yeast. We use a sensitive genetic system wherein the rate of single-base deletions in a homopolymeric run in the LYS2 gene is 10 000-fold higher in an msh2 mutant than in a wild-type strain. Expression of the human proteins alone or in combination does not reduce the mutation rate of the msh2 strain, and expression of the individual human proteins does not increase the low mutation rate of a wild-type strain. However, co-expression of hMSH2 and hMSH6 in wild-type yeast increases the mutation rate 4000-fold, while co-expression of hMSH2 and hMSH3 elevates the rate 5-fold. Analysis of cell extracts indicates that the proteins are expressed and bind to mismatched DNA. The results suggest that hMutSalpha and hMutSbeta complexes form, bind to and prevent correction of replication slippage errors in yeast. Expression of hMSH6 with hMSH2 containing a proline substituted for a conserved Arg524 eliminates the mutator effect and reduces mismatch binding. The analogous mutation in humans is associated with microsatellite instability, defective MMR and cancer, illustrating the utility of the yeast system for studying human disease alleles.     

Toxin involvement in staphylococcal scalded skin syndrome

The production of staphylococcal exfoliative toxin A (ETA) and toxin B (ETB), toxic shock syndrome toxin (TSST-1), and enterotoxins A-E was analyzed in 60 Staphylococcus aureus strains isolated from children with scalded skin syndrome (15 with generalized exfoliative syndrome, 28 with bullous impetigo, and 17 with staphylococcal scarlet fever). All strains isolated from patients with generalized exfoliative syndrome or bullous impetigo produced ETA and/or ETB and caused a Nikolsky's sign when injected subcutaneously into newborn mice. In contrast, exfoliative toxin was detected in an S. aureus strain from only one of 17 case of staphylococcal scarlet fever; the 16 other S. aureus strains produced TSST-1 and/or an enterotoxin. In conclusion, enterotoxins or TSST-1 are more frequently associated with staphylococcal scarlet fever than are exfoliative toxins. Hence staphylococcal scarlet fever may well represent an abortive form of toxic shock syndrome rather than a milder form of staphylococcal scalded skin syndrome.