Replication timing of human telomeric DNA and other repetitive sequences analyzed by fluorescence in situ hybridization and flow cytometry.

The replication timing of telomeres seems to differ between species. Yeast telomeres are late replicating, whereas limited data from very few human cell lines have indicated telomere replication throughout S phase. In the present study a series of permanent cell lines and patient samples was investigated using a flow cytometric approach for telomere length determination based on in situ hybridization using peptide nucleic acid probes and DNA staining. This method permits selective analysis of cells in specific phases of the cell cycle without perturbation of the cell cycle machinery. The timing of replication of telomeric C(3)TA(2) and T(2)AG(3) repeats was found to differ between individual samples and could precede or be concomitant with the replication of bulk DNA. Replication of the T(2)AG(3) strand seemed to occur somewhat later than that of the C(3)TA(2) strand in some samples. (GTG)(n) and other repetitive sequences generally showed a replication pattern similar to that of the bulk of DNA with slightly individual differences, whereas centromeric DNA repeats consistently replicated within a short time frame in late S phase. The apparent variability in replication timing seen for telomeric DNA might suggest individual differences in firing of replication origins.

Defective satellite cells in congenital myotonic dystrophy.

In this study we have developed an in vitro cell culture system which displays the majority of the defects previously described for congenital myotonic dystrophy (CDM) muscle in vivo. Human satellite cells were isolated from the quadriceps muscles of three CDM fetuses with different clinical severity. By Southern blot analysis all three cultures were found to have approximately 2300 CTG repeats. This CTG expansion was found to progressively increase in size during the proliferative life span, confirming an instability of this triplet in skeletal muscle cells. The CDM myoblasts and myotubes also showed abnormal retention of mutant RNA in nuclear foci, as well as modifications in their myogenic program. The proliferative capacity of the CDM myoblasts was reduced and a delay in fusion, differentiation and maturation was observed in the CDM cultures compared with unaffected myoblast cultures. The clinical severity and delayed maturation observed in the CDM fetuses were closely reflected by the phenotypic modifications observed in vitro. Since the culture conditions were the same, this suggests that the defects we have described are intrinsic to the program expressed by the myoblasts in the absence of any trophic factors. Altogether, our results demonstrate that satellite cells are defective in CDM and are probably implicated in the delay in maturation and muscle atrophy that has been described previously in CDM fetuses.

Decreased levels of myotonic dystrophy protein kinase (DMPK) and delayed differentiation in human myotonic dystrophy myoblasts.

Muscle cell cultures derived from a myotonic dystrophy (DM1) fetus were established in order to determine on the one hand, whether the differentiation of DM1 myoblasts is altered and, on the other hand, whether the levels of myotonic dystrophy protein kinase (DMPK) protein is decreased in DM1 muscle cells. DM1 myoblasts isolated from a quadriceps of a 12-weeks old fetus proliferate at a similar rate as normal myoblasts isolated from a quadriceps of an unaffected 15-weeks old fetus but their maturation is altered as shown by the decreased levels in slow myosin heavy chain protein. In contrast, no change was observed in the expression of vimentin, myogenin and embryonic myosin heavy chain. The levels of DMPK transcripts sharply increased during myoblast differentiation and the mutant DMPK transcripts are retained in discrete foci in the nuclei of muscle cells. The levels of 85-kDa DMPK protein was reduced by about 50% in DM1 cells compared with normal cells. Our study demonstrates that delay in DM1 myoblast maturation is associated with nuclear retention of mutant DMPK transcripts and decreased levels of DMPK protein.

Multicolor fluorescence in situ hybridization with peptide nucleic acid probes for enumeration of specific chromosomes in human cells.

In previous studies, we showed that peptide nucleic acid (PNA) probes have significant advantages over conventional synthetic RNA or DNA probes in FISH procedures for detecting telomeric and trinucleotide repeat sequences. Here, we report that directly labeled PNA probes recognizing chromosome-specific repeat sequences are also powerful tools for detecting and enumerating specific chromosomes in interphase and metaphase cells. This is illustrated by multicolor FISH experiments with cells from normal individuals and patients with numerical sex chromosome aberrations.

Effect of feeding protein deficient diet on phospholipid turnover and protein kinase C mediated protein phosphorylation in rat brain.

Feeding of protein deficient diet is known to alter the transmembrane signalling in brain of rat by reducing total protein kinase C (PKC) activity. Phospholipid metabolism regulates the activation of PKC through generation of second messengers and the extent of PKC activation accordingly influences the magnitude of phosphorylation of its endogenous substrate proteins. Thus it was speculated that ingestion of protein deficient diet may modify the turnover rate of membrane phospholipids and magnitude of phosphorylation of endogenous substrate proteins of PKC. The experiments were conducted on rats fed on three different types of laboratory prepared diets viz. casein (20% casein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threonine) for 28 days. The metabolism of phosphoinositides (PIs) and phosphatidyl choline (PC) was studied by equilibrium labeling with [3H] myo inositol and [14C methyl] choline chloride respectively. The phosphorylation of endogenous substrate proteins of PKC was studied by using 32P-gamma-ATP followed by SDS-PAGE and autoradiography. The results suggest that in deficient group, there is an increased incorporation of [3H] myo inositol in PIs and inositol phosphate pool in comparison to the casein group. The phosphatidyl inositol (PI) turnover reduced, although there was a marginal increase in the phosphatidyl inositol monophosphate (PIP) and phosphatidyl inositol bis phosphate (PIP2). Supplementation of diet showed a reversal of the pattern towards control to a considerable extent. In the deficient group, PC metabolism showed an increased incorporation of [14C methyl] choline in choline phospholipids but decreased incorporation in phosphoryl choline in comparison with the casein group. The increase in total PC contents was significant but marginal in residue contents. The turnover rate of PC increased only marginally and that of residue declined. Supplementation of diet reduced the total contents of PC and residue, but the turnover rate of PC and residue remained still higher. Phosphorylation of endogenous proteins showed four different proteins of 78, 46, 33 and 16 kDa to be the substrates of PKC in casein group. In deficient group, phosphorylation of these proteins increased markedly while supplementation of diet had a reversing effect rendering the values to be intermediate between casein and the supplemented group. The changes in phospholipid metabolism and in phosphorylation of endogenous substrate proteins of PKC suggest that dietary protein deficiency causes alterations in transmembrane signalling mechanism in rat brain. These effects are partially reversed by improving the quality of proteins in the diet.

CT portography by direct intrasplenic contrast injection: a new technique.

BACKGROUND: The evaluation of percutaneous contrast injection into splenic parenchyma as an alternative technique for computed tomographic (CT) portography in the preoperative assessment of primary hepatobiliary tumors. METHODS: Thirty-two patients underwent a nonenhanced CT scan of the liver, after which a 19-gauge, 10-cm-long needle was introduced into the splenic parenchyma under CT guidance. One hundred forty milliliters of contrast medium (200 mgI/mL; 28 g/I) were injected through this needle: first, a 20-mL bolus (in 5 s) and then 2 mL/s for 60 s. At the end of the bolus injection (5 s), 8-mm-thick contiguous axial scans of the liver were obtained. RESULTS: The success rate of the procedure was 93.7% (30/32; two technical failures). The average time required for the entire study was 13 min and 50 s (range = 7 min 53 s to 25 min 17 s). Hepatic parenchymal enhancement was good in 24/30 (80%), moderate in 3/30 (10%), and unsatisfactory in caudal sections of the liver in 3/30 (10%). Artifactual perfusion defects were seen in 4/30 (13%) due to inadvertant injection of small quantities of air. Intrasplenic subcapsular contrast accumulation occurred in 56.2% (18/32; minimal 15, moderate 3), extrasplenic contrast leakage in 12. 5% (4/32), and left shoulder pain in 18.7% (6/32). No major complications were observed. CONCLUSIONS: Direct intrasplenic contrast injection for CT portography is a simple, effective, and safe technique with a high success rate and requires significantly less time and lower doses of contrast medium; it also eliminates angiography, indwelling arterial catheters, and patient transfers from angiography to the CT area.

Amiodarone-induced pulmonary toxicity in an adolescent.

We report amiodarone-induced pulmonary toxicity in an 18-year-old boy who had undergone corrective surgery for tetralogy of Fallot 4 years earlier, and was treated with amiodarone because of recurrent malignant postoperative ventricular tachyarrhythmias. Toxicity was recognized on the basis of clinical features, chest X-ray, high-resolution contrast enhanced computerized tomography, and resolution of the findings subsequent to withdrawal of amiodarone and treatment with steroids. Pulmonary toxicity due to amiodarone, as far as we know, has not been reported in children and young adults, and its occurence even in young adults requires wider appreciation.

Obstructed infracardiac total anomalous pulmonary venous connection in an adult.

Adult survival in patients with uncorrected obstructed infracardiac total anomalous pulmonary venous drainage has not been reported. A 17-year-old man who presented to us with features of severe pulmonary arterial hypertension was diagnosed to have obstructed pulmonary venous drainage to the hepatic vein. Presence of a large ventricular septal defect in this patient may have contributed favorably to survival.

Results of renal angioplasty in nonspecific aortoarteritis (Takayasu disease).

PURPOSE: To evaluate the long-term results of percutaneous transluminal renal angioplasty (PTRA) in the management of renovascular hypertension caused by nonspecific aortoarteritis (Takayasu disease). MATERIALS AND METHODS: The results of 96 stenoses in 66 patients were retrospectively studied. The indications for PTRA included hypertension uncontrolled by single-drug therapy, evidence of greater than 70% diameter stenosis in the renal artery with a peak systolic gradient of greater than 20 mm Hg, and clinically inactive disease. RESULTS: Technical success was obtained in 91 (95%) stenoses in 62 patients. Clinical success was seen in 59 (89%) and included "cure" in 14 and "improvement" in 45 patients. The stenosis decreased from 88% +/- 6% (range, 70%-100%) to 11% +/- 12% (range, 0%-40%), systolic pressure gradient decreased from 95 mm Hg +/- 22 (range, 30-140 mm Hg) to 9 mm Hg +/- 8 (range, 0-30 mm Hg), blood pressure improved from 181 +/- 16 (range, 150-220)/115 +/- 10 (range, 90-146) to 136 +/- 25 (range, 130-210)/86 +/- 16 (range, 80-130) mm Hg, and the drug requirement decreased from 3.9 +/- .6 (range, 2-5) to 1.1 +/- .9 (range, 0-3) (P value for all < .001). Complications included transient intrarenal arterial spasm in three patients, groin hematoma in two patients, and ipsilateral renal vein injury in one patient. At 22 months +/- 17 (range, 4-84 months) follow-up, the restenosis rate, as determined by recurrence of hypertension and angiographic demonstration of restenosis, was 16%. CONCLUSION: Despite some technical problems, PTRA is safe and effective in treating renovascular hypertension caused by nonspecific aortoarteritis. The complication rate is low.

Congenital H-type urethroanal fistula.

A case of congenital urethroanal fistula with a normal anterior urethra in a male child is reported. The fistula was demonstrated between the prostatic urethra and anorectum. This anomaly is usually associated with an atretic anterior urethra and has been variously described as a variant of a urethral duplication by some authors, and of an anorectal malformation (ARM) by others. We conclude that its rightful classification is as a variant of ARM in which the fistula is a result of persistence of the cloacal duct and corresponds to the anorecto-vestibular fistula with a normal anus (perineal canal) in a female.

Localization of trinucleotide repeat sequences in myotonic dystrophy cells using a single fluorochrome-labeled PNA probe.

A labeled peptide nucleic acid (PNA) antisense probe was used to study the spatial distribution of triplet repeats (CTG) in human myotonic dystrophy (DM) cells by high-resolution fluorescence in situ hybridization (FISH). It was found that transcripts containing triplet repeats were present as a number of discrete foci in the DM nuclei. Greater numbers of foci were visible with the PNA probe than a comparable DNA probe. The PNA probe was also used to visualize the triplet-repeat expansion within the DM gene located on chromosome 19. Using the intensity of the expanded triplet-repeat on the chromosomes as a reference, it was estimated there were between 15-230 RNA molecules in each focus observed in DM nuclei.

Morphological mural changes in the aorta in non-specific aortoarteritis (Takayasu's arteritis): assessment by intravascular ultrasound imaging.

OBJECTIVE: Non-specific aortoarteritis (Takayasu's arteritis) is a panarteritis of unknown aetiology which primarily involves the vessel walls. The imaging morphology of wall abnormalities has been infrequently studied and their intravascular ultrasound appearance is not reported in the literature. METHOD: We studied this morphology by intravascular ultrasound in nine patients in whom the diagnosis of Takayasu's arteritis was made by clinical and angiographic criteria. Intravascular ultrasound was performed by the transfemoral route. Images of the aorta were obtained in each patient. Qualitative and quantitative analysis was performed. RESULTS: All procedures were successful, without complication. Typical findings included thickening and altered echogenicity of the media, adventitia and peri-arterial tissues. The inner echogenic layer was thin. Pliability of aortic walls was lost in stenotic segments. Aortic calcification was seen in one patient. The aortic wall thickness was 0.17-0.58 cm. Angiograms showed skip areas of aortic involvement. Intravascular ultrasound showed wall changes even in the skip areas which were normal at angiography. CONCLUSION: Intravascular ultrasound shows changes in the aortic wall morphology in non-specific aortoarteritis. Our findings suggest that this disease involves contiguous aortic segments, producing wall and luminal changes in some areas and only wall changes in intervening areas.

Sorting of beta-actin mRNA and protein to neurites and growth cones in culture.

The transport of mRNAs into developing dendrites and axons may be a basic mechanism to localize cytoskeletal proteins to growth cones and influence microfilament organization. Using isoform-specific antibodies and probes for in situ hybridization, we observed distinct localization patterns for beta- and gamma-actin within cultured cerebrocortical neurons. beta-Actin protein was highly enriched within growth cones and filopodia, in contrast to gamma-actin protein, which was distributed uniformly throughout the cell. beta-Actin protein also was shown to be peripherally localized after transfection of beta-actin cDNA bearing an epitope tag. beta-Actin mRNAs were localized more frequently to neuronal processes and growth cones, unlike gamma-actin mRNAs, which were restricted to the cell body. The rapid localization of beta-actin mRNA, but not gamma-actin mRNA, into processes and growth cones could be induced by dibutyryl cAMP treatment. Using high-resolution in situ hybridization and image-processing methods, we showed that the distribution of beta-actin mRNA within growth cones was statistically nonrandom and demonstrated an association with microtubules. beta-Actin mRNAs were detected within minor neurites, axonal processes, and growth cones in the form of spatially distinct granules that colocalized with translational components. Ultrastructural analysis revealed polyribosomes within growth cones that colocalized with cytoskeletal filaments. The transport of beta-actin mRNA into developing neurites may be a sequence-specific mechanism to synthesize cytoskeletal proteins directly within processes and growth cones and would provide an additional means to deliver cytoskeletal proteins over long distances.

Recurrent pseudoaneurysm of the left ventricle with subcutaneous herniation into the chest wall. A case report.

Pseudoaneurysm of the left ventricle is rare, and recurrence is extremely rare. We report the case of a 62-year-old man who presented at our hospital with a painless pulsatile swelling in the left breast. He had undergone coronary artery bypass grafting and left-ventricular aneurysmectomy 14 years earlier. On investigation, the swelling was diagnosed to be a pseudoaneurysm of the left ventricle with subcutaneous herniation. The extreme rarity of this condition prompted us to report the case. The investigative techniques and the surgical strategy are discussed.

Expansion of a CUG trinucleotide repeat in the 3' untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts.

Expansion of a CTG trinucleotide repeat in the 3' untranslated region (UTR) of DMPK, the gene encoding myotonic dystrophy protein kinase, induces the dominantly inherited neuromuscular disorder myotonic dystrophy (DM). Transcripts containing the expanded trinucleotide are abundant in differentiated cultured myoblasts, and they are spliced and polyadenylylated normally. However, mutant transcripts never reach the cytoplasm in these nonmitotic cells; instead, they form stable clusters that are tightly linked to the nuclear matrix, which can prevent effective biochemical purification of these transcripts. In DM patients, reduced DMPK protein levels, consequent to nuclear retention of mutant transcripts, are probably a cause of disease development. Formation of nuclear foci is a novel mechanism for preventing transcript export and effecting a loss of gene function.