Decoupling of DNA excision repair and RNA transcription in translocation breaksite regions of plasmacytoma-susceptible BALB/cAnPt mice

BACKGROUND: Reduction of potential pathogens by selective intestinal decontamination has been proposed to improve intensive care. Despite large scientific interest in this method, little is known about its benefit in homogeneous trauma populations. METHODS: In a prospective, controlled study, we enrolled non-infected trauma patients (age over 18 years, mechanical ventilation > or = 48 hours, intensive care for more than 3 days) who primarily were admitted to our university medical center. We randomized patients to be treated with two different topical regimens (polymyxin, tobramycin, and amphotericin (PTA) or polymyxin, ciprofloxin, amphotericin (PCA)) or the carrier only (placebo), administered four times daily both to the oropharynx and to the gastrointestinal tract. All patients received intravenous ciprofloxacin (200 mg, bd) for 4 days. FINDINGS: Of 357 enrolled patients, 310 (age 38.0 +/- 16.5 years, Injury Severity Score 35.2 +/- 12.7) met all inclusion criteria. Selective decontamination successfully reduced intestinal bacterial colonization. However, we did not identify significant differences between groups regarding pneumonia (PTA 47.5%, PCA 39.0%, placebo 45.3%), sepsis (PTA 47.5%, PCA 37.8%, placebo 42.6%), multiple organ failure (PTA 56.3%; PCA 52.4%, placebo 58.1%), and death (PTA 11.3%, PCA 12.2%, placebo 10.8%). Total costs per patient were highest with the PTA regimen. CONCLUSIONS: We found no benefit of selective decontamination in trauma patients. Apparently, bacterial overgrowth in the intestinal tract is not the sole link between trauma, sepsis, and organ failure.     

Single-stage repair of aortic arch obstruction and associated intracardiac defects with pulmonary homograft patch aortoplasty

PURPOSE: To investigate the involvement of the cornea during endotoxin-induced uveitis (EIU) in the rat and the effect of Ngamma-nitro-L-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor, administered by iontophoresis. METHODS: EIU was induced in Lewis rats that were killed at 8 and 16 hours after lipopolysaccharide (LPS) injection. The severity of uveitis was evaluated clinically at 16 hours, and nitrite levels were evaluated in the aqueous humor at 8 hours. Corneal thickness was measured, 16 hours after LPS injection, on histologic sections using an image analyzer. Transmission electron microscopy (TEM) was used for fine analysis of the cornea. Transcorneoscleral iontophoresis of L-NAME (100 mM) was performed either at LPS injection or at 1 and 2 hours after LPS injection. RESULTS: At 16 hours after LPS injection, mean corneal thickness was 153.7+/-5.58 microm in the group of rats injected with LPS (n=8) compared with 126.89+/-11.11 microm in the saline-injected rats (n=8) (P < 0.01). TEM showed stromal edema and signs of damage in the endothelial and epithelial layers. In the group of rats treated by three successive iontophoreses of L-NAME (n=8), corneal thickness was 125.24+/-10.36 microm compared with 146.76+/-7.52 microm in the group of rats treated with iontophoresis of saline (n=8), (P=0.015). TEM observation showed a reduction of stromal edema and a normal endothelium. Nitrite levels in the aqueous humor were significantly reduced at 8 hours by L-NAME treatment (P=0.03). No effect on corneal edema was observed after a single iontophoresis of L-NAME at LPS injection (P=0.19). Iontophoresis of saline by itself induced no change in corneal thickness nor in TEM structure analysis compared with normal rats. CONCLUSIONS: Corneal edema is observed during EIU. This edema is significantly reduced by three successive iontophoreses of L-NAME, which partially inhibited the inflammation. A role of nitric oxide in the corneal endothelium functions may explain the antiedematous effect of L-NAME.     

A t(3;8) chromosomal translocation associated with hepatitis B virus intergration involves the carboxypeptidase N locus

Integrated hepatitis B virus (HBV) DNA is found in the great majority of human hepatocellular carcinomas, suggesting that these viral integrations may be implicated in liver oncogenesis. Besides the insertional mutagenesis characterized in a few selected cases and the contribution of viral transactivators to cell transformation to malignancy, HBV has been shown to generate gross chromosomal rearrangements potentially involved in carcinogenesis. Here, we report a t(3;8) chromosomal translocation present in a hepatocellular carcinoma developed in noncirrhotic liver tissue. One side of the translocation, in 8p23, is shown to be in the vicinity of the carboxypeptidase N gene, a locus that is heavily transcribed in liver tissue and frequently deleted in hepatocellular carcinomas and other epithelial tumors. The other side of the translocation, in 3q27-29, is widely implicated in several types of translocations occurring in different malignancies, such as large-cell lymphomas. The present data strongly support a model in which HBV-induced chromosomal rearrangements play a key role during multistep liver oncogenesis.     

Diagnosis of genetic defects by chromosomal analysis

Of 901 karyotypes performed over a period of 4 years, genetic anomalies were detected in 162 cases. Down's syndrome (trisomy 21) was the most common (168.8%) genetic disorder followed by Turner's syndrome, Philadelphia chromosome, Klinefelter's syndrome, Edward's syndrome (trisomy 18) and Patau's syndrome (trisomy 13). All the three trisomies were detected very early in life. Mean age at the time of diagnosis for Turner's syndrome was 13.3 years, allowing a timely hormone replacement therapy to improve secondary sexual characters. Patients with Klinefelter's syndrome were diagnosed late (mean age 23.6 years), which greatly reduced their chances of an effective therapy to improve the clinical and social outcome.     

The contribution of deficient DNA repair to chromosomal radiosensitivity of CHO cells after G2 irradiation

Neuroendocrine tumors, particularly those of gastrointestinal tract origin, have a predisposition for metastasizing to the liver. In such patients, the clinical course is often dominated by the hepatic disease, either because of hormone secretion or because of tumor bulk. Because the liver has a dual vascular supply and hepatic metastases derive the majority of blood from the hepatic artery, the regional delivery of chemotherapy can offer pharmacokinetic advantages over systemic administration. The hepatic artery is also a nonsurgical avenue for inducing selective metastasis ischemia by the embolization of tumor vessels. The combination of these two therapies, or chemoembolization, may provide additive benefits. Such an approach has been demonstrated to reduce tumor bulk, reduce hormone levels, and palliate the symptoms of many patients with liver-dominant neuroendocrine metastases. Embolization or chemoembolization is an appropriate modality for some patients with neuroendocrine tumors.     

Balanced reciprocal translocation mosaicism associated with an abnormal phenotype

Balanced reciprocal translocation mosaicism is rarely reported in humans. Only two previous cases have been associated with an abnormal phenotype. We report on a third case of apparently balanced reciprocal translocation mosaicism associated with an abnormal phenotype, largely different from those reported previously. Since low levels of mosaicism may not be detected in routine cytogenetic analyses, balanced reciprocal translocation mosaicism may be associated with an abnormal phenotype more often than has been recognized to date.     

Association of a host DNA structure with retroviral integration sites in chromosomal DNA

Integration of retroviral genomes is a site-specific process with respect to the virus but not the host genome. Numerous chromosomal sites and various sequences can be used as targets. Nevertheless, preferential regions and integration patterns have been observed. Using a functional assay, we investigated if host structural DNA elements could be associated with retroviral integration sites. The results were that 9 of 10 distinct retroviral integration events occurred in close proximity of structural elements behaving like intrinsically bent DNA.     

In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans

Deinococcus radiodurans R1 and other members of this genus share extraordinary resistance to the lethal and mutagenic effects of ionizing radiation. We have recently identified a RecA homolog in strain R1 and have shown that mutation of the corresponding gene causes marked radiosensitivity. We show here that following high-level exposure to gamma irradiation (1.75 megarads, the dose required to yield 37% of CFU for plateau-phase wild-type R1), the wild-type strain repairs > 150 double-strand breaks per chromosome, whereas a recA-defective mutant (rec30) repairs very few or none. A heterologous Escherichia coli-D. radiodurans shuttle plasmid (pMD68) was constructed and found to be retained in surviving D. radiodurans R1 and rec30 following any radiation exposure up to the highest dose tested, 3 megarads. Plasmid repair was monitored in vivo following irradiation with 1.75 megarads in both R1/pMD68 and rec30/pMD68. Immediately after irradiation, plasmids from both strains contained numerous breaks and failed to transform E. coli. While irradiation with 1.75 megarads was lethal to rec30 cultures, a small amount of supercoiled plasmid was regenerated, but it lacked the ability to transform E. coli. In contrast, wild-type cultures showed a cell division arrest of about 10 h, followed by exponential growth. Supercoiled plasmid was regenerated at normal levels, and it readily transformed E. coli. These studies show that D. radiodurans retains a heterologous plasmid following irradiation and repairs it with the same high efficiency as its chromosomal DNA, while the repair defect in rec30 prevents repair of the plasmid. Taken together, the results of this study suggest that plasmid DNA damaged in vivo in D. radiodurans is repaired by recA-dependent mechanisms similar to those employed in the repair of chromosomal DNA.     

8-hydroxyguanine levels in nuclear DNA and its repair activity in rat organs associated with age

8-Hydroxyguanine (8-OH-Gua) is one of the most abundant types of oxidative DNA damage. The levels of 8-OH-Gua, and its repair activity, were quantified in 3-week-, 5-month-, and 30-month-old male Sprague-Dawley rat organs such as liver, kidney, spleen, lung, small intestine, and brain. The levels of 8-OH-Gua were significantly higher in the 5-month-old rat kidney and brain and 30-month-old rat spleen when compared to that of the 3-week-old rats. However, no significant differences were found in the organs between 5- and 30-month-old rats that were due to the aging process. The repair activity levels of kidney, spleen, and lung were higher than those of liver, small intestine, and brain. This pattern was consistent for the three age stages.     

Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer

The human DNA mismatch repair gene homologue hMSH2, on chromosome 2p is involved in hereditary non-polyposis colon cancer (HNPCC). On the basis of linkage data, a second HNPCC locus was assigned to chromosome 3p21-23 (ref. 3). Here we report that a human gene encoding a protein, hMLH1 (human MutL homologue), homologous to the bacterial DNA mismatch repair protein MutL, is located on human chromosome 3p21.3-23. We propose that hMLH1 is the HNPCC gene located on 3p because of the similarity of the hMLH1 gene product to the yeast DNA mismatch repair protein, MLH1, the coincident location of the hMLH1 gene and the HNPCC locus on chromosome 3, and hMLH1 missense mutations in affected individuals from a chromosome 3-linked HNPCC family.     

A new non-random chromosomal translocation t(3;6)(q27;p21.3) associated with BCL6 rearrangement in two patients with non-Hodgkin's lymphoma

We describe two cases of non-Hodgkin's lymphoma associated with t(3;6)(q27;p21.3) and BCL6 rearrangement. The first case was in a 78-year old woman, whose performance status (PS) was 1, the serum lactate dehydrogenase (LDH) level was elevated, and the Ann Arbor stage was IIIA with no extra nodal lymphomatous site. The pathological diagnosis from a biopsy of the inguinal lymph node was 'malignant lymphoma (ML), follicular, small cleaved' according to the Working Formulation. Complete remission was achieved. Although she had relapse in 1992, remission was obtained again. The second case was in a 62-year old man, whose PS was 1, the serum LDH was normal, and Ann Arbor stage was IVA with the involvement of the small intestine. Histological diagnosis of the cervical lymph node was 'ML, diffuse, large cell'. Complete remission was obtained without relapse. The 3q27 translocations, found in 20-30% of non-Hodgkin's lymphoma, are unique in having multiple chromosomal translocation partners. Chromosome band 6p21.3 is one of these partner sites that may be the site of a novel gene. The two cases presented here show that this translocation is a non-random chromosomal change involving 3q27 and BCL6. Since t(3;6) was the sole karyotypic abnormality in one case, this translocation may play a role in lymphomagenesis.     

Assembly of DNA with histones and nonhistone chromosomal proteins in vitro

We have examined, by protein binding assays, thermal denaturation, and circular dichroism, the possible effects of histones on nonhistone chromosomal protein (NHCP) interactions with DNA. For these studies, we have fractionated mouse Krebs II chromosomal proteins into three discrete fractions: Mo, 5 M urea-soluble NHCP; M1, 5 M urea-1 M NaCl-soluble NHCP from 5 M urea-extracted chromatin; and M3, 5 M urea-3 M NaCl-soluble chromosomal proteins from 5 M urea-1 M NaCl-extracted chromatin. These fractions contain heterogeneous populations of NHCP, and were found to differentially affect histone binding to DNA by methods of reconstitution, or by direct binding of M0, M1, or M3 to urea-salt reconstituted DNA with histones. M0 was found to exert a unique effect on the thermal denaturation and circular dichroic spectra of DNA-histone complexes. M0 from Krebs II chromatin was also found to complete for DNA sites in the presence of M0 from mouse liver chromatin. In addition, in 5 M urea, pH 8.0, histone binding to DNA reached saturation at 1.85 mg/mg of DNA, higher than the in vivo ratio of 1.00 mg/mg of DNA. Saturation of histone binding to DNA occurred only in the presence of 5 M urea, resulting in a reduction of nonspecific histone-histone interactions on DNA.     

Minimally invasive repair of atrial septal defects

BACKGROUND: Minimally invasive pediatric cardiac surgical techniques continue to evolve and remain challenged by technologic advances in percutaneous devices developed to treat congenital heart disease exclusive of cardiopulmonary bypass. Public tenacity for "incisionless" operations, however, must remain balanced scrupulously against the collective safety of the surgical procedure. METHODS: Twenty-three pediatric patients underwent repair of atrial septal defects through a partial sternal split and a limited skin incision (5 to 7 cm) at our institution between July 1995 and October 1996. RESULTS: The average age of the patients was 6 years and 2 months (range, 19 months to 15 years) and the average weight was 23.3 kg (range, 11.3 to 61.7 kg). The average bypass time was 35 minutes (range, 19 to 81 minutes). Fourteen patients had a single dose of blood cardioplegia administered, whereas 9 had ventricular fibrillation electrically induced. Twenty-two patients had ostium secundum defects and 1 had a sinus venosus defect. The average length of the hospital stay was 3.6 days (range, 3 to 6 days). There were no operative or late deaths. CONCLUSIONS: Modifications of this technique continue to evolve as an effective cosmetic alternative to submammary and thoracotomy approaches. Advantages of this modification include excellent cosmetic results in all age groups and the concomitant security and familiarity of mediastinal access and full sternotomy when required.     

Increased nuchal translucency as a marker for fetal chromosomal defects

BACKGROUND: Screening for trisomy 21 (Down's syndrome) by measuring maternal serum alpha-fetoprotein, chorionic gonadotropin, and estriol concentrations and then performing chorionic-villus sampling or amniocentesis identifies approximately 60 percent of fetuses with this disorder. We used ultrasonography to detect increased nuchal translucency and cystic hygroma, which are characteristic features of fetuses with chromosomal defects. METHODS: We performed transvaginal ultrasonography in 10,010 unselected adolescents and women less than 40 years of age with live singleton fetuses at 10 to 15.9 weeks of gestation. Increased fetal nuchal translucency was defined as an area of translucency at least 3 mm in width, and cystic hygromas were defined as septated, fluid-filled sacs in the nuchal region. Subjects whose fetuses had these findings were offered fetal karyotyping. Information on pregnancies, deliveries, and neonates was subsequently obtained from hospital records and national birth and malformation registries. RESULTS: Nuchal translucency or cystic hygroma was seen in 76 fetuses (0.8 percent), of which 18 (24 percent) had an abnormal karyotype. The sensitivity for trisomies 21, 18, and 13 combined was 62 percent (13 of 21 fetuses), and the sensitivity for trisomy 21 alone was 54 percent (7 of 13 fetuses). CONCLUSIONS: The use of transvaginal ultrasonography to detect increased nuchal translucency and cystic hygroma is a sensitive test for fetal aneuploidy. It can be done earlier in pregnancy than serum screening, and it decreases the subsequent need for chorionic-villus sampling or amniocentesis.     

Comparative study of the repair kinetics of chromosomal aberrations and DNA strand breaks in proliferating and quiescent CHO cells

Repair kinetics observable at the level of exchange-type chromosomal aberrations (dicentric chromosomes), using fractionation and delayed-plating techniques, have been compared with repair kinetics of radiation-induced DNA double-strand breaks, measured with PFGE, and with repair kinetics of all strand breaks, measured with the alkali-unwinding technique. Only data from quiescent or proliferating CHO K1 cells obtained in the same laboratory were used. We determined repair kinetics in terms of the time constant tau (equal to half-time/log(e)2). The repair kinetics (tau approximately 11-14 min) observed in the split-dose formation of dicentric chromosomes agrees with fast repair kinetics of double-strand breaks (tau approximately 11-13 min), thus permitting us to identify the latter as the 'primary lesions' whose pairwise interaction leads to the beta D2 yield term of the aberrations. The repair kinetics observed for dicentric chromosomes formed under delayed-plating conditions (tau approximately 75 min), which mainly affects the alpha D yield term, is attributed to an intermediate interchromosomal product temporarily existing in the course of aberration formation; it is suggested that this product is mechanistically correlated with the slow repair kinetics of 'clustered damage' to DNA seen with the applied molecular methods (tau approximately 90 min).     

Hypersensitivity of ataxia telangiectasia fibroblasts to ionizing radiation is associated with a repair deficiency of DNA double-strand breaks

Amphiregulin (Ar) is an EGF receptor ligand that functions to modulate the growth of both normal and malignant epithelial cells. We asked whether mouse preimplantation embryos express Ar, and if so, what the function of Ar is during preimplantation development. We used RT-PCR to show expression of Ar mRNA in mouse blastocysts, and using a polyclonal anti-Ar antibody and indirect immunofluorescence, we detected the presence of Ar protein in morula- and blastocyst-stage embryos. Ar protein was present in both the cytoplasm and nucleus in both morulae- and blastocyst-stage embryos, which is similar to Ar distribution in other cell types. Embryos cultured in Ar developed into blastocysts more quickly and also exhibited increased cell numbers compared to control embryos. In addition, 4-cell stage embryos cultured in an antisense Ar phosphorothioate-modified oligodeoxynucleotide (S-oligo) for 48 hr exhibited slower rates of blastocyst formation and reduced embryo cell numbers compared to embryos exposed to a random control S-oligo. TGF-alpha significantly improved blastocyst formation, but not cell numbers, for embryos cultured in the antisense Ar S-oligo. From these observations, we propose that Ar may function as an autocrine growth factor for mouse preimplantation embryos by promoting blastocyst formation and embryo cell number. We also propose that blastocyst formation is stimulated by Ar and TGF-alpha, while Ar appears to exert a greater stimulatory effect on cell proliferation than does TGF-alpha in these embryos.     

Elemental diet-induced bacterial translocation associated with systemic and intestinal immune suppression

In a study of 1,002 consecutive Malaysian male newborns, 48 (4.8%) were found to have undescended testes (UDT). The rate and laterality of the UDT were associated with lower birth weight (P < 0.001) and prematurity (P < 0.001). Boys with UDT were also more likely to have other congenital abnormalities of the external genitalia, the commonest being hydrocele. No correlation between UDT and maternal age, birth order, social class, or mode of delivery was demonstrated in this study. Although 26/34 (76.5%) of UDT achieved full spontaneous descent by 1 year of age, 1.1% of all infants whose testes remained undescended required regular long-term follow-up with surgical referral and correction at an appropriate time. A premature infant with UDT is more likely to achieve full testicular descent at 1 year of age than a term infant.     

DNA replication, repair, and repair tests

The rate of inhibition and recovery of DNA synthesis can be used in a rapid assay system to detect genotoxic potentials of chemicals. Also, the observation that an agent stimulates DNA repair in a test system indicates its ability to cause damage in DNA. Different experimental approaches to the study of repair synthesis are discussed.