Role of mitochondrial DNA variants and copy number in diabetic atherogenesis.

Hyperglycemia-induced reactive oxygen species production can cause diabetes and its complications, including atherosclerosis. The role of mitochondrial DNA variants and mitochondrial copy number in the pathogenesis of diabetic atherogenesis is not well understood. We examined 36 diabetic patients who had undergone amputation for diabetic foot and seven non-diabetic patients who had undergone amputation after traumatic injury. Mitochondrial DNA was extracted and used for sequencing. Single nucleotide polymorphisms (SNPs) relative to the Cambridge reference sequence were analyzed. Mitochondrial DNA copy number was quantified by real-time PCR. Twenty-one novel variants were detected in 29 diabetic patients with arterial stenosis; six of the variants were heteroplasmic, and most occurred in highly evolutionarily conserved residues. These variants were more prevalent in patients with arterial stenosis than in those without stenosis. The novel variants included four in complex I (ND1: C3477A/C, A3523A/G; ND5: C13028A/C, C13060A/C), one in complex IV (COX1: T6090A/T), and one in rRNA (12srRNA: G857G/T). Compared with non-diabetic patients, the diabetic patients had significantly less mitochondrial DNA. Furthermore, among diabetic patients with arterial stenosis, there was a significant positive correlation between mitochondrial DNA copy number and the number of total SNPs. In conclusion, we identified six novel heteroplasmic mitochondrial DNA variants among diabetic patients with arterial stenosis, and we found that diabetic atherogenesis is associated with decreased amounts of mitochondrial DNA.

Analysis of a 69-kb contiguous genomic sequence at a putative tumor suppressor gene locus on human chromosome 6q27.

Multiple neoplasias including B-cell non-Hodgkin's lymphoma, breast carcinoma, and ovarian carcinoma, have been associated with frequent deletions of the distal region on the long arm of human chromosome 6, suggesting the presence of one or more tumor suppressor gene(s) at this locus. Loss of heterozygosity analysis of breast and ovarian tumors has further restricted the minimal region of loss within 6q27. To further characterize this genomic region for gene content including putative tumor suppressor genes as well as other elements that may contribute to tumorigenesis, a 68940-bp contiguous sequence, encompassing markers D6S193 and D6S297, was generated by random shotgun sequencing of a cosmid, P1, and PAC contig. In addition, exon trapping was performed utilizing a subset of these clones. Sixteen trapped exons, ranging in size from 44 to 399 bp, span this approximately 69-kb region. Many other putative exons have been identified computationally. Further analysis has identified 13 potential promoters and 13 putative polyadenylation sites in the region. Northern analysis identified a transcript mapping within this interval that is expressed in ovarian, breast, and lymphoid-derived tumor cell lines. Consideration of these data, together with the demonstration of several regions of high CpG content, suggests the possibility of several genes at this locus.

Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8).

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G + C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor.

Gastrointestinal transit in cirrhotic patients: effect of hepatic encephalopathy and its treatment.

Chronic hepatic encephalopathy is highly responsive to changes in diet, to antibiotic therapy and to ingestion of nondigestible disaccharides. The precise pathophysiology of chronic hepatic encephalopathy in individual cases is highly variable, although ammonia toxicity and production of neurotransmitterlike substances in the gut have been proposed to contribute to the overall syndrome of chronic hepatic encephalopathy. The support for this hypothesis is based on the empiric observation that reduction in protein intake, a catharsis or both are effective treatments for chronic hepatic encephalopathy. This study was performed to evaluate the effect of mild subclinical and low-grade (grade 0 to 1) chronic hepatic encephalopathy on gastric emptying and oral-cecal transit times. Thirty patients were studied. Ten had no evidence of chronic hepatic encephalopathy, as determined with a battery of neuropsychiatric studies (group 1); 10 had subclinical hepatic encephalopathy, as judged on the basis of abnormal neuropsychiatric test performance but normal neurological examination (group 2); and 10 had grade 1 hepatic encephalopathy. Each underwent a liquid gastric emptying study and a lactulose oral-cecal transit time study. No significant differences between groups were evident in the results of the gastric emptying studies. In contrast, the time required for a lactulose load to reach the cecum was significantly greater in the patients with hepatic encephalopathy (p < 0.01) and increased as a function of the hepatic encephalopathy grade.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytomegalovirus infection and gastric emptying.

Gastrointestinal infection due to cytomegalovirus occurs frequently in liver transplant recipients. Upper gastrointestinal cytomegalovirus infection is associated with subjective complaints of nausea, a sense of abdominal fullness, and occasionally emesis and/or dysphagia. In order to determine whether these symptoms reflect a disruption of the normal motility of the stomach, the following study was performed. Eleven individuals who were evaluated for liver transplantation were prospectively recruited and studied as follows: (1) upper gastrointestinal endoscopy with biopsy of the gastric antral mucosa; (2) viral culture of the gastric mucosa; (3) a histologic examination of the gastric mucosa; and (4) a radionuclide gastric emptying study was obtained before and 4-8 weeks following successful liver transplantation. Prior to liver transplantation, none had symptoms of nausea, vomiting, or epigastric fullness. All were culture-negative for cytomegalovirus. All had endoscopic and histologic evidence of portal hypertensive gastropathy but none had antral erosions or ulcers. All demonstrated normal gastric emptying of a liquid meal. Following liver transplantation, 6 remained free of gastric cytomegalovirus while 5 developed a culture-confirmed gastric cytomegalovirus infection. Those that developed a gastric cytomegalovirus infection also had more gastric symptoms, and more gastric histologic abnormalities. Moreover, those with a gastric cytomegalovirus infection demonstrated enhanced gastric retention of a liquid meal (P less than 0.01).

The ICT in situ experimental model in dental research.

The intra-oral cariogenicity test (ICT) in situ experimental model was introduced in 1964 for the study of caries on sample enamel in the human mouth. Slabs of human or bovine enamel are mounted with a Dacron gauze cover in the acrylic flanges of prosthetic appliances. The extent of enamel demineralization or remineralization of lesions is assessed from surface microhardness measurements and microradiography of the enamel sections. The ICT model offers the potential of studying various parameters related to caries. This publication presents a typical ICT study comparing the cariogenicity of 10% sucrose solutions containing 1, 3, 10, and 30 ppm F with that of the control solution, 10% sucrose (without fluoride). The treatments were applied to the ICT as 10-minute extra-oral immersions. The results indicate: (1) a strong effect of F in decreasing demineralization of sound enamel and increasing remineralization of pre-softened enamel in the ICT; (2) pronounced resistance to a subsequent in vitro acid test; (3) pronounced F incorporation into pre-softened enamel; and (4) a characteristic acid-resistant zone, as seen in microradiographs, associated with exposure to F. This in situ model enables one to study experimental caries with repeated testing of enamel that follows the development and/or the regression of subsurface enamel lesions, F incorporation, increased acid resistance, cariogenicity of substrates, and other parameters of caries that can be assessed under standard conditions of tooth substrate and microbial sheltering.

Gastrointestinal tract hemorrhage. The value of a nasogastric aspirate.

A bloody nasogastric aspirate is believed to imply active upper gastrointestinal tract bleeding, while a nonbloody yellow-green nasogastric aspirate that contains duodenal secretions suggests the absence of bleeding proximal to the ligament of Treitz. To validate these beliefs, physicians were asked to predict the presence of active gastrointestinal tract bleeding and whether bile was present in a nasogastric aspirate obtained immediately before endoscopy in 73 episodes of bleeding in 62 patients. A relationship was found between the physician's assessment of the presence of active bleeding demonstrated endoscopically and the appearance of the nasogastric aspirate. However, the sensitivity and specificity were low (79% and 55%, respectively). No association between the assessment of bile in the nasogastric aspirate and the presence of bile acids was demonstrated. These data do not support the placement of a nasogastric tube to determine whether or not a patient is bleeding, the location of the bleeding, and whether endoscopy should be performed.

Isolation of a genomic sublibrary enriched for glucocorticoid-regulated genes.

Glucocorticoids regulate gene expression by causing the glucocorticoid receptor to bind to an enhancer-like DNA element termed the glucocorticoid regulatory element (GRE). The resultant effect on transcription of specific genes causes a cascade of intracellular events that determines the growth or differentiated function of the target tissue. Although virtually all animal tissues respond to glucocorticoids, it has proven difficult to elucidate the molecular events which underlie physiologically important glucocorticoid effects such as lymphocyte death or poor wound healing. In this paper, a tryptic fragment of the glucocorticoid receptor (17K-GR) is shown to bind selectively to DNA containing a GRE. When a mixture of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) region and plasmid vector DNA was extracted using the intact glucocorticoid receptor or the 17K-GR, the 17K-GR retained a greater proportion of LTR vs. plasmid DNA. The 17K-GR-LTR complex was also more resistant to salt extraction. Extraction of Bam HI-digested mouse genomic DNA resulted in enrichment of the pro-opiomelanocortin (POMC) gene 5' fragment (which contains a GRE) vs. the 3' fragment which does not. A mouse genomic phage library was enriched for GRE-containing sequences by extraction using the 17K-GR. The frequency of POMC-positive plaques was determined to gauge enrichment of down-regulated genes, and the frequency of phosphoenolpyruvate carboxy-kinase-positive plaques was determined to gauge enrichment of up-regulated genes. The frequencies obtained (1.2 x 10(-3) and 3.5 x 10(-3), respectively) indicated that a family of glucocorticoid-regulated genes totaling approximately 300 had been isolated in a genomic sublibrary.

Does primary sclerosing cholangitis occurring in association with inflammatory bowel disease differ from that occurring in the absence of inflammatory bowel disease? A study of sixty-six subjects.

Primary sclerosing cholangitis often occurs in association with inflammatory bowel disease, particularly ulcerative colitis but also Crohn's disease of the colon either with or without terminal ileal disease. Little data exist as to the effect of inflammatory bowel disease on the presenting symptoms, radiological features, response to liver transplantation, and potential risk of bile duct carcinoma in individuals with primary sclerosing cholangitis. In an effort to answer these questions, 66 patients with primary sclerosing cholangitis were studied. The definitive diagnosis of primary sclerosing cholangitis in each was accomplished using cholangiography, which in each case demonstrated characteristic beading, ectasia and stricturing of the intrahepatic and extrahepatic bile ducts. Inflammatory bowel disease was present in 47 (71.2%) patients. Thirty nine (59.1%) had ulcerative colitis; their mean age was 42.5 +/- 11.6 yr (mean +/- SD), and the male/female ratio was 2.9:1. In addition, eight patients (12.1%) had Crohn's colitis; their mean age was 40.5 +/- 6.5 yr, and the male/female ratio of this group was 1:1. Nineteen patients (28.8%) had primary sclerosing cholangitis without any inflammatory bowel disease; their mean age was 42.0 +/- 12.1 yr, and the male/female ratio in this group was 0.72:1. Seventy-two percent of the patients without inflammatory bowel disease had either jaundice, pruritus or fatigue at presentation compared with 41% of the patients with inflammatory bowel disease (p less than 0.05). In contrast, abnormal liver function tests were more common as the first manifestation of liver disease in the latter group (38% vs. 11%; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

T-lymphocyte subsets in gut and blood of liver transplant recipients with and without cytomegalovirus gastroenteritis.

The effects of orthotopic liver transplantation (OLTx) and cytomegalovirus (CMV) gastroenteritis on the type of mononuclear cells within the upper gastrointestinal tract were determined. Nineteen liver transplant recipients were studied both before and after transplantation. Each underwent a pan-upper gastrointestinal endoscopy with biopsy of the antrum and duodenum before and four weeks following liver transplantation. A panel of monoclonal antibodies prepared against HLA-DR, NK, IL-2R, T11, T4, T8, and B1 cell surface antigens was used to examine the tissues. Before OLTx, none of the 19 subjects studied had clinical or histologic evidence for CMV gastroenteritis. Following OLTx, five of the 19 subjects had CMV gastroenteritis. The number of HLA-DR positive staining lymphocytes present in biopsies obtained post-OLTx was significantly greater (P less than 0.005) than those present in biopsies obtained pre-OLTx regardless of the presence or absence of CMV gastroenteritis. No difference in the intensity of HLA-DR antigen expression between pre- and post-OLTx biopsies and those with and without CMV gastroenteritis was evident. No difference in the number of natural killer (NK) cells and the number of cells expressing the interleukin-2 receptor (IL-2R) was evident between biopsies obtained pre- and post-OLTx. In contrast, the number of T lymphocytes bearing the T11, T4, and T8 markers and the calculated T4/T8 ratio differed between biopsies obtained pre- and post-OLTx and between those positive for CMV gastroenteritis post-OLTx and those without evidence for CMV gastroenteritis either before or after OLTx, although these changes were not consistent throughout the gastrointestinal tract.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of midazolam for percutaneous liver biopsy.

The standard procedure for percutaneous liver biopsy (PLB) involves only the use of local anesthesia. However, at times, a PLB can be frightening and uncomfortable. Such experiences often limit the willingness of patients to undergo subsequent follow-up biopsies. To investigate the ability of midazolam, a new water-soluble benzodiazepine preparation, noted for its potency, rapid onset of action, and amnestic qualities, to enhance patient acceptability of a follow-up liver biopsy, a "sedative dose" of midazolam (2 mg) or saline was administered immediately prior to and following a percutaneous liver biopsy. The initial dose was used to sedate the subject while not impairing patient cooperation during the biopsy procedure; the second dose was used to induce amnesia for the biopsy procedure. The next morning patient recollection for the preceding biopsy procedure and their willingness to undergo a future PLB were assessed using a questionnaire. Forty-one patients (ages 18-78) were randomized to receive either midazolam (N = 21) or saline/placebo N = 20) treatment. All PLBs were obtained with a Trucut needle. All subjects were given 2-5 cc of 2% xylocaine local anesthetic at the biopsy site. The questionnaire utilized evaluated patient experience of the procedure with respect to their recall, level of anxiety during the procedure, and willingness to undergo a repeat procedure. The data obtained revealed that those receiving midazolam admitted to experiencing less discomfort during the biopsy procedure (P less than 0.04) and had less memory for the procedure (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

T-cell populations in liver and renal transplant recipients with infectious esophagitis.

Individuals undergoing hepatic and renal transplants are susceptible to infections of the gastrointestinal tract, particularly the esophagus. The most common responsible agents are Candida and herpes simplex virus (HSV) with cytomegalovirus (CMV), Aspergillus, and other agents being regarded as unusual pathogens even in this unique population. Altered T-cell populations have been associated with CMV colitis in healthy homosexuals and in individuals with the acquired immunodeficiency syndrome (AIDS). Similarly, individuals with Epstein-Barr virus infections have altered T-cell populations. Whether these infections alter T-cell populations in infected individuals or the abnormalities in T-cell subpopulations occur first and enhance the likelihood of an infection in susceptible populations is as yet unknown. In this study peripheral blood T-cell populations in individuals before (19 patients) and after (47 patients) liver transplantation and after receiving a renal allograft (21 patients) were compared. Those individual having any symptoms related to esophageal disease underwent upper gastrointestinal endoscopy combined with mucosal biopsies, brushings, and cultures and were subdivided into those with and without infectious esophagitis. CMV esophagitis was found to be associated with an arithmetically decreased T-cell helper/suppressor (H/S) ratio principally due to an increase in the suppressor cell number. Such a reduction in the TH/S ratio and in the number of circulating suppressor cells was not found in esophagitis due to either HSV or Candida and was unrelated to the serum cyclosporine level or prednisone dosage prescribed.(ABSTRACT TRUNCATED AT 250 WORDS)

Infectious esophagitis following liver and renal transplantation.

Upper gastrointestinal endoscopy was performed for the evaluation of infectious esophagitis in 19 consecutive subjects evaluated prospectively before orthotopic liver transplantation (OLTx), in a separate group of 27 subjects post-OLTx, and in 21 subjects following orthotopic renal transplantation (ORTx). None of the pre-OLTx patients had evidence of infectious esophagitis, whereas 11% of the post-OLTx and 24% of the post-ORTx patients had esophageal infections. Candida esophagitis was found only in the post-ORTx patients, whereas cytomegalovirus and herpes simplex viral esophagitis were found in both the post-ORTx and post-OLTx patients. Dysphagia was associated with evidence of herpes simplex virus infection (P less than 0.001) and epigastric pain was associated with Candida infection (P less than 0.001). No association between the administration of prednisone or the blood level of cyclosporine A and esophagitis was found. Finally, the use of standard low-dose mycostatin prophylaxis was not effective for prevention of Candida esophagitis. Nonetheless the use of higher doses of mycostatin was therapeutic.

The natural course of hepatitis B surface antigen-positive chronic active hepatitis in Taiwan.

A prospective study on the natural course of hepatitis B surface antigen (HBsAg)-positive chronic active hepatitis (CAH) in 84 patients was conducted at the Taiwan Veterans General Hospital in Taipei. On presentation, 36% of the patients had jaundice and 94% had elevated serum transaminase activity in addition to clinical symptoms. Of the 84 patients with HBsAg-positive CAH, 76 were followed for an average of 46 months, and at the time of the last visit 46% either had evidence of progressive liver disease or had died. Of the 15 deaths, nine were due to complications of liver disease and six were due to primary hepatocellular carcinoma (PHC), alpha-Fetoprotein levels of greater than 400 ng/ml were found only in patients in whom PHC was detected. The course of HBsAg-positive CAH in this predominantly male Asian population was progressive, and deaths occurred either by liver failure, by bleeding esophageal varices, or by progression to PHC.

Hepatitis B viral markers in patients with primary hepatocellular carcinoma in Taiwan.

The presence of hepatitis B viral markers in patients with primary hepatocellular carcinoma (PHC) was studied retrospectively at the Taiwan Veterans General Hospital in Taipei, Taiwan. Serum samples from 102 PHC patients and from 100 control individuals were tested for hepatitis B surface antigen (HBsAg), antibody to HBsAg (anti-HBs), antibody to hepatitis B core antigen (anti-HBc), hepatitis Be antigen (HBeAg), and antibody to HBeAg (anti-HBe). Of the 102 PHC patients, 72 (71%) were positive for HBsAg. Nine (9%) additional patients were positive for anti-HBc alone in high titer, 19 (19%) had both anti-HBc and anti-HBs, and 9 (9%) had HBsAg, anti-HBc, and anti-HBs. In the 100 controls, 12 (12%) were HBsAg-positive, whereas 22 (22%) had anti-HBc alone and 50 (50%) had both anti-HBc and anti-HBs. Only 4 (4%) controls and no PHC patients had anti-HBs alone. Of the HBsAg-positive patients with PHC, 17 (29%) had HBeAg and 36 (61%) had anti-HBe. The alpha-fetoprotein (AFP) levels above 400 ng/ml were found in 44% of the PHC patients. Values of AFP above 1 x 10(5) ng/ml were more frequently detected in PHC patients who were HBsAg-positive. Categorization of the geographic origins of the families whose members had PHC revealed that most families had originated from southern China. This study confirms that hepatitis B viral markers are frequently present in Chinese patients with PHC.

A sequence-specific endonuclease (Xmn I) from Xanthomonas manihotis.

A type II restriction endonuclease Xmn I with a novel site specificity has been isolated from Xanthomonas manihotis. Xmn I does not cleave SV40 DNA, but cleaves phi X174 DNA into three fragments, which constitute 76.61%, 18.08% and 5.31% of the total length of 5386 base pairs, and cleaves pBR322 DNA into two fragments of 55.71% and 44.29% of the entire 4362 base pairs. The nucleotide sequences around the cleavage sites made by Xmn I are not exactly homologous, but they have a common sequence of 5' GAANNNNTTC 3' according to a simple computer program analysis on nucleotide sequences of phi X174 DNA, pBR322 DNA and SV40 DNA. The results suggest that the cleavage site of Xmn I is located within its recognition sequence of 5' GAANNNNTTC 3'.