Natural genetic engineering of the bacterial genome.

The term 'natural genetic engineering' means viewing genetic change as a coordinated cell biological process, the reorganization of discrete genomic modules, resulting in the formation of new DNA structures. Examples of natural genetic engineering continue to accumulate, and the concept can be used to integrate observations which demonstrate the similarity between in vitro genetic engineering and the action of in vivo agents of genetic change.

Genome organization, natural genetic engineering and adaptive mutation.

Bacterial evolution is considered in the light of molecular discoveries about genome organization, biochemical mechanisms of genetic change, and cellular control networks. Prokaryotic genetic determinants are organized as modular composites of coding sequences and protein-factor binding sites joined together during evolution. Studies of genetic change have revealed the existence of biochemical functions capable of restructuring the bacterial genome at various levels and joining together different sequence elements. These natural genetic engineering systems can be subject to regulation by signal transduction networks conveying information about the extracellular and intracellular environments. Mu-mediated araB-lacZ coding sequence fusions provide one example of adaptive mutation (increased formation of useful mutations under selection) and illustrate how physiological regulation can modulate the activity of a natural genetic engineering system under specific conditions.

Bacteria are small but not stupid: cognition, natural genetic engineering and socio-bacteriology.

Forty years' experience as a bacterial geneticist has taught me that bacteria possess many cognitive, computational and evolutionary capabilities unimaginable in the first six decades of the twentieth century. Analysis of cellular processes such as metabolism, regulation of protein synthesis, and DNA repair established that bacteria continually monitor their external and internal environments and compute functional outputs based on information provided by their sensory apparatus. Studies of genetic recombination, lysogeny, antibiotic resistance and my own work on transposable elements revealed multiple widespread bacterial systems for mobilizing and engineering DNA molecules. Examination of colony development and organization led me to appreciate how extensive multicellular collaboration is among the majority of bacterial species. Contemporary research in many laboratories on cell-cell signaling, symbiosis and pathogenesis show that bacteria utilise sophisticated mechanisms for intercellular communication and even have the ability to commandeer the basic cell biology of 'higher' plants and animals to meet their own needs. This remarkable series of observations requires us to revise basic ideas about biological information processing and recognise that even the smallest cells are sentient beings.

Cigar smoking in men and risk of death from tobacco-related cancers.

BACKGROUND: Cigar consumption in the United States has increased dramatically since 1993, yet there are limited prospective data on the risk of cancer associated with cigar smoking. We examined the association between cigar smoking and death from tobacco-related cancers in a large, prospective cohort of U. S. men. METHODS: We used Cox proportional hazards models to analyze the relationship between cigar smoking at baseline in 1982 and mortality from cancers of the lung, oral cavity/pharynx, larynx, esophagus, bladder, and pancreas over 12 years of follow-up of the American Cancer Society's Cancer Prevention Study II cohort. A total of 137 243 men were included in the final analysis. Women were not included because we had no data on their cigar use. We excluded men who ever smoked cigarettes or pipes and adjusted all rate ratio (RR) estimates for age, alcohol use, and use of snuff or chewing tobacco. RESULTS: Current cigar smoking at baseline, as compared with never smoking, was associated with an increased risk of death from cancers of the lung (RR = 5.1; 95% confidence interval [CI] = 4.0-6.6), oral cavity/pharynx (RR = 4.0 [95% CI = 1.5-10.3]), larynx (RR = 10.3 [95% CI = 2.6-41.0]), and esophagus (RR = 1.8; 95% CI = 0.9-3.7). Although current cigar smokers overall did not appear to be at an increased risk of death from cancer of the pancreas (RR = 1.3; 95% CI = 0.9-1.9) or bladder (RR = 1.0; 95% CI = 0.4-2.3), there was an increased risk for current cigar smokers who reported that they inhaled the smoke (for pancreas, RR = 2.7; 95% CI = 1.5-4.8; for bladder, RR = 3.6; 95% CI = 1.3-9.9). CONCLUSIONS: Results from this large prospective study support a strong association between cigar smoking and mortality from several types of cancer.

Cloning and expression of IDDM-specific human autoantigens.

A DNA cloning approach was taken to identify islet cell protein antigens that are recognized specifically by insulin-dependent diabetes mellitus (IDDM) sera. A human islet cDNA library was generated and screened with diabetic sera. In this article, identification of two clones is described. Proteins expressed by these lambda phages appeared to react specifically with newly diagnosed diabetic sera. Islet cell antibody 12 (ICA12) was tested by Western blotting. ICA512 was not reactive with sera in the Western format but was specifically immunoprecipitated by diabetic sera from an Escherichia coli extract.

How repeated retroelements format genome function.

Genomes operate as sophisticated information storage systems. Generic repeated signals in the DNA format expression of coding sequence files and organize additional functions essential for genome replication and accurate transmission to progeny cells. Retroelements comprise a major fraction of many genomes and contain a surprising diversity of functional signals. In this article, we summarize some features of the taxonomic distribution of retroelements, especially mammalian SINEs, tabulate functional roles documented for different classes of retroelements, and discuss their potential roles as genome organizers. In particular, the fact that certain retroelements serve as boundaries for heterochromatin domains and provide a significant fraction of scaffolding/matrix attachment regions (S/MARs) suggests that the reversed transcribed component of the genome plays a major architectonic role in higher order physical structuring. Employing an information science model, the "functionalist" perspective on repetitive DNA leads to new ways of thinking about the systemic organization of cellular genomes and provides several novel possibilities involving retroelements in evolutionarily significant genome reorganization.

Action of a transposable element in coding sequence fusions.

The original Casadaban technique for isolating fused cistrons encoding hybrid beta-galactosidase proteins used a Mucts62 prophage to align the upstream coding sequence and lacZ prior to selection. Kinetic analysis of araB-lacZ fusion colony emergence indicated that the required DNA rearrangements were regulated and responsive to conditions on selection plates. This has been cited as an example of "directed mutation." Here we show genetically that the MuA and integration host factor (IHF) transposition functions are involved in the formation of hybrid araB-lacZ cistrons and propose a molecular model for how fusions can form from the initial strand-transfer complex. These results confirm earlier indications of direct Mu involvement in the fusion process. The proposed model explains how rearranged Mu sequences come to be found as interdomain linkers in certain hybrid cistrons and indicates that the fusion process involves a spatially and temporally coordinated sequence of biochemical reactions.

Insulin independence of unstable diabetic patient after single living donor islet transplantation.

BACKGROUND: Current success in islet transplantation will lead to a donor shortage. Living donor islet transplantation could be an alternative approach to expand the potential donor pool. In this study we describe the first successful living donor islet transplantation for unstable diabetes, performed at Kyoto University Hospital on January 19, 2005. METHODS: The donor was a healthy 56-year-old woman and mother of the recipient. The recipient was a 27-year-old woman with insulin-dependent diabetes since the age of 15 years. She experienced frequent hypoglycemic unawareness episodes. Her blood glucose concentration was difficult to control and C-peptide level was negative after glucagon stimulation. She needed an average 28 of units of insulin per day. The donor underwent a distal pancreatectomy and islets were isolated from the resected pancreas graft. The total islet yield was 408,114 islet equivalents and isolated islets were immediately transplanted into the recipient's liver. RESULTS: After transplant, the blood glucose level of the recipient was tightly controlled without hypoglycemic episodes. She was discharged on day 37 with a normal oral glucose tolerance test (OGTT). The recipient remained insulin-independent for >3 months, since day 22 posttransplant. The donor's postoperative clinical course was uneventful. She was discharged on postoperative day 18 and returned to her job within 1 month. CONCLUSIONS: We report the first successful living donor islet transplantation for the treatment of unstable diabetes. We believe that living donor islet transplantation may become an option in the treatment of insulin-dependent diabetes.

Long-term efficacy and safety of acarbose in the treatment of obese subjects with non-insulin-dependent diabetes mellitus.

BACKGROUND: Acarbose delays the release of glucose from complex carbohydrates and disaccharides by inhibiting intestinal alpha-glucosidases, attenuating postprandial increments in blood glucose and insulin. This multicenter double-blind study compared the efficacy and safety of acarbose with placebo in the treatment of obese subjects with non-insulin-dependent diabetes mellitus (NIDDM) managed by diet. METHODS: Two hundred twelve obese subjects with NIDDM who had not received any diabetic medication for at least 12 weeks were randomized to receive acarbose or placebo. The subjects were stratified by fasting glucose level above or below 11.1 mmol/L (200 mg/dL). Based on the subject's therapeutic response and tolerance, the acarbose dosage was titrated from 50 to 300 mg three times per day. This 36-week study consisted of a 6-week pretreatment period, a 24-week double-blind treatment period, and a 6-week posttreatment period. RESULTS: Ninety-one subjects given acarbose and 98 subjects who received placebo were evaluable for efficacy. During a standard meal tolerance test at the double-blind end point, the differences between treatment groups in mean change from baseline were as follows: 0.9 mmol/L (16 mg/dL) for fasting plasma glucose level, approximately 2.8 mmol/L (50 mg/dL) for postprandial plasma glucose level, and 0.59% (P < .0001) for hemoglobin A1c concentration (for all three measurements, values decreased in the acarbose group and increased in the placebo group). CONCLUSIONS: Acarbose improved both fasting and postprandial hyperglycemia and improved overall glycemic control as measured by the hemoglobin A1c level. These findings suggest a beneficial role for acarbose in combination with diet in the treatment of obese subjects with NIDDM.

A double-blind placebo-controlled trial evaluating the safety and efficacy of acarbose for the treatment of patients with insulin-requiring type II diabetes.

OBJECTIVE: To determine whether a forced titration of acarbose (from 50 to 300 mg three times daily) administered over a 24-week period, in conjunction with diet and insulin therapy, improves glycemic control and reduces daily insulin requirements in insulin-requiring type II diabetes. RESEARCH DESIGN AND METHODS: This multicenter, randomized, double-blind, placebo-controlled trial was 36 weeks in duration. The trial consisted of a 6-week pretreatment period, a 24-week double-blind treatment period, and a 6-week post-treatment follow-up period. The primary efficacy variables were the mean change from baseline in HbA1c levels and the mean percentage change from baseline in total daily insulin dose. RESULTS: Treatment with acarbose was associated with significant reductions in HbA1c levels of 0.40% (P = 0.0001) and in total daily insulin dose of 8.3% (P = 0.0015). There were also significant reductions in all plasma glucose variables measured, including a 0.9 mmol/l reduction in fasting glucose (P = 0.0440), a 2.6 mmol/l reduction in glucose Cmax (P = 0.0001) and a 270 mmol.min-1.l-1 reduction in glucose area under the curve (P = 0.0002). Although acarbose treatment was associated with a greater incidence of adverse events than was placebo treatment, primarily flatulence and diarrhea, these events did not generally prevent patients from completing the study. CONCLUSIONS: The results of this study suggest that acarbose is a safe and effective adjunct to diet and insulin therapy for the management of insulin-requiring type II diabetes.

Reduction of glycosylated hemoglobin and postprandial hyperglycemia by acarbose in patients with NIDDM. A placebo-controlled dose-comparison study.

OBJECTIVE: To compare the safety and efficacy of three doses of acarbose (100, 200, and 300 mg three times daily) with placebo for the treatment of non-insulin-dependent diabetes mellitus (NIDDM) in patients maintained on dietary therapy alone. RESEARCH DESIGN AND METHODS: This multicenter double-blind placebo-controlled trial was 22 weeks in duration. The trial consisted of a 2-week screening period, a 4-week placebo run-in period, and a 16-week double-blind treatment period. The primary measure of drug efficacy was the mean change from baseline in HbA1c levels. Additional efficacy variables included the mean change from baseline in fasting and postprandial plasma glucose and serum insulin levels. RESULTS: After 16 weeks of treatment, acarbose-treated patients had statistically significant reductions in mean HbA1c levels of 0.78, 0.73, and 1.10% (relative to placebo) in the 100-, 200-, and 300-mg t.i.d. groups, respectively. Significant reductions in fasting and postprandial plasma glucose levels, glucose area under the time-concentration curve, and maximum glucose concentration were also observed in acarbose-treated patients. Although there were no statistically significant differences among the 100-, 200-, and 300-mg treatment groups, there was a trend toward a dose-response relationship for most plasma glucose variables that were measured. Gastrointestinal side effects (e.g., abdominal pain, flatulence, and diarrhea) and serum transaminase elevations (e.g., aspartate aminotransferase [AST] and alanine aminotransferase [ALT] were more frequently reported in the acarbose-treated patients than in the placebo-treated control patients. Transaminase elevations occurred only at the 200-, and 300-mg dosages and were readily reversible on discontinuation of treatment. CONCLUSIONS: Acarbose at doses of 100, 200, and 300 mg administered three times daily for 16 weeks significantly reduced HbA1c levels and postprandial hyperglycemia. Treatment with acarbose is a safe and effective adjunct to dietary therapy for the treatment of NIDDM.

Nasopharyngeal cancer in a low-risk population: defining risk factors by histological type.

Nasopharyngeal cancer (NPC) is a major public health problem in parts of Southeast Asia and North Africa, but is rare among whites and blacks. Although infection with the EBV and genetic susceptibility appear to play large roles in high-incidence populations, migrant studies suggest that environmental factors may also be important. Aside from the high risks associated with ingestion of salted fish, surprisingly few other risk factors have been established from studies in endemic areas. We studied a low-incidence population to determine whether tobacco use, alcohol consumption, and certain medical conditions and treatments are related to NPC and to examine variations in risk by histology. We reasoned that new relationships might be best identified in the absence of strong causal pathways, such as intake of preserved foods and genetic susceptibility. A population-based case-control study was conducted from 1987 to 1993 at five cancer registries in the United States: western Washington, metropolitan Detroit, Connecticut, Iowa, and Utah. Controls were identified by random digit dialing and frequency matched to the gender and age distribution of cases at each registry. Telephone interviews were completed by 231 cases and 246 controls. We observed a strong dose-response relationship between cigarette smoking and risk of differentiated squamous cell carcinoma (test for trend, P < .001). The highest risk [odds ratio (OR), 6.5; 95% confidence interval (CI), 2.0-21.3] occurred among current smokers with a history of more than 60 pack-years. In contrast, there was no evidence that undifferentiated or nonkeratinizing carcinomas were associated with cigarette smoking. Similarly, a significant increase in risk was observed for the heaviest alcohol consumers (21 or more drinks/week) only for differentiated squamous cell carcinomas (OR, 2.9; 95% CI, 1.2-6.9). The associations with cigarettes and alcohol appeared to be stronger among persons 50 years or older. There was a suggestion that diagnosis with infectious mononucleosis (a marker of late infection with EBV) is linked with decreased NPC risk (OR, 0.4; 95% CI, 0.1-1.1). This report indicates that over two-thirds of differentiated squamous cell NPC cases arising in older persons in the United States can be accounted for by cigarettes and alcohol, but leaves unexplained cases arising in the young and carcinomas of undifferentiated or nonkeratinizing histology. Future studies of NPC need to take into account histology and age in evaluating these and other environmental and genetic risk factors.

Multicenter, placebo-controlled trial comparing acarbose (BAY g 5421) with placebo, tolbutamide, and tolbutamide-plus-acarbose in non-insulin-dependent diabetes mellitus.

BACKGROUND: Acarbose delays release of glucose from complex carbohydrates and disaccharides by inhibiting intestinal alpha-glucosidases, thereby attenuating postprandial increments in blood glucose and insulin. This multicenter, double-blind, placebo-controlled study compared the efficacy and safety of diet alone, acarbose, tolbutamide, and acarbose-plus-tolbutamide in non-insulin-dependent diabetes mellitus (NIDDM) patients. PATIENTS AND METHODS: A total of 290 patients with NIDDM and fasting plasma glucose levels of at least 140 mg/dL were randomized to receive treatment TID with acarbose 200 mg, tolbutamide 250 to 1,000 mg, a combination of both drugs, or placebo. A 6-week run-in period was followed by double-blind treatment for 24 weeks, then a 6-week follow-up period. RESULTS: All active treatments were superior (P < 0.05) to placebo in reducing postprandial hyperglycemia and HbA1c levels. The ranking in order of efficacy was: acarbose-plus-tolbutamide, tolbutamide, acarbose, and placebo. The postprandial reductions in glucose were approximately 85 mg/dL for acarbose-plus-tolbutamide, 71 mg/dL for tolbutamide, 56 mg/dL for acarbose, and 13 mg/dL for placebo. Tolbutamide was associated with increases in body weight and postprandial insulin levels when taken alone, but these were ameliorated when tolbutamide was taken in combination with acarbose. Acarbose alone or in combination with tolbutamide caused significantly more gastrointestinal adverse events (mainly flatulence and soft stools or diarrhea) than tolbutamide or placebo, but these were generally well tolerated. Clinically significant elevations in hepatic transaminase levels occurred in 3 patients in the acarbose group and 2 in the acarbose-plus-tolbutamide group. Transaminase levels returned to normal when therapy was discontinued. CONCLUSIONS: Acarbose was effective and well tolerated in the treatment of NIDDM. Control of glycemia was significantly better with acarbose compared with diet alone. Acarbose-plus-tolbutamide was superior to tolbutamide alone.

Efficacy of lamivudine in chronic hepatitis B patients with active viral replication and decompensated cirrhosis undergoing liver transplantation.

Liver transplantation for endstage hepatitis B virus (HBV) infection has been associated with survival inferior to that of liver transplantation in other chronic liver diseases due to HBV reinfection of the graft. Lamivudine is a new nucleoside analog with potent antiviral effects against hepatitis B. Our aim was to test its efficacy when used pre- and posttransplantation in HBV-DNA positive patients with endstage liver disease. Patients received oral lamivudine 100 mg daily both pretransplant and posttransplant. Viral serology, serum and tissue HBV-DNA and liver histology were assessed sequentially. Five consecutive patients with endstage hepatitis B were entered into the trial. Serum HBV-DNA was cleared pretransplant in all patients. Three of four transplanted patients cleared HBeAg and HBsAg postoperatively, whereas all four became negative for serum HBV-DNA (dot-blot and PCR). Liver biopsies were negative for HBV-DNA by PCR in 3 of 4 cases. Lymphocytes were negative for HBV-DNA by PCR in all cases. With follow-up of 3, 14, 16, and 26 months, two patients have normal liver enzymes and normal liver histology and two have developed recurrent hepatitis B. No significant side effects were seen. This pilot study shows that lamivudine can effectively inhibit hepatitis B virus in cirrhotic patients pretransplant and posttransplant. A lamivudine resistant mutant developed in two patients. Transplant recipients with actively replicating HBV related cirrhosis may achieve a good outcome after liver transplantation using lamivudine, but viral resistance is likely to be a significant problem.

Long-term outcome of living related liver transplantation for patients with intrapulmonary shunting and strategy for complications.

BACKGROUND: In 320 living related liver transplantation performed between June 1990 and September 1997, there were 21 living related liver transplantation for patients with intrapulmonary shunting, manifested by digital clubbing, cyanosis, and dyspnea. We report the long-term outcome for more 6 months and our strategy to overcome complications in these recipients. PATIENTS: A total of 21 patients (age range 2-33 years, 19 children and 2 adults, 6 males and 15 females) were classified into three grades according to shunt ratio calculated by TcMAA pulmonary scintigraphy; 5 in mild group (shunt ratio: less than 20%), 6 in moderated group (20%-40%), and 10 in severe group (more than 40%). The original underlying liver disease was biliary atresia in all patients. RESULTS: Spearmen's correlation coefficient rank test revealed that shunt ratio correlated significantly with PaO2 in room air (P=0.0001), PaO2 in 100% oxygen (P=0.0004), hematocrit (P=0.0276), and period of dyspnea before transplantation (P=0.023). COMPLICATIONS: Wound infection occurred in 80, 66, and 80%, and bile leakage in 20, 0, 40% in mild, moderate, and severe group, respectively. Patients who had portal vein thrombosis, and intracranial complication were classified as severe group and the incidence was 20 and 20%, respectively. The patient actuarial one year survival was 80, 66.7, and 48%, in mild, moderate, and severe group, respectively, although there was no significant difference. All patients who survived improved hepatopulmonary syndrome and the length of period required for the resolution was significantly correlated to the preoperative shunt ratio (P=0.023). COMMENTS: Patients with severe shunting are susceptible to wound infection and bile leak. The trend of higher incidence of portal thrombosis and intracranial complications in the severe group was closely related high hematocrit. Secure surgical technique to reduce bile leak and delayed primary wound closure to reduce wound infection were found to be effective. Anticoagulant therapy by infusing heparin through the portal vein followed by coumadin could prevent fatal portal vein thrombosis without counter risk of fatal cerebral hemorrhage.

Genome system architecture and natural genetic engineering in evolution.

Molecular genetics teaches three lessons relevant to the nature of genetic change during evolution: (1) Genomes are organized as hierarchies of composite systems (multidomain protein-coding sequences; functional loci made up of regulatory, coding, processing, and intervening sequences; and multilocus regulons and replicons) interconnected and organized into specific "system architectures" by repetitive DNA elements. (2) Genetic change often occurs via natural genetic engineering systems (cellular biochemical functions, such as recombination complexes, topoisomerases, and mobile elements, capable of altering DNA sequence information and joining together different genomic components). (3) The activity of natural genetic systems is regulated by cellular control circuits with respect to the timing, activity levels, and specificities of DNA rearrangements (e.g., adaptive mutation, Ty element mobility, and P factor insertions). These three lessons provide plausible molecular explanations for the episodic, multiple, nonrandom DNA rearrangements needed to account for the evolution of novel genomic system architectures and complex multilocus adaptations. This molecular genetic perspective places evolutionary change in the biologically responsive context of cellular biochemistry.