The use and cost of health services prior to death: a comparison of the Medicare-only and the Medicare-Medicaid elderly populations.

Lack of data has limited research into the high cost and ethical dilemmas associated with care of the dying elderly. This study is based on a five-year, person-specific file of Medicare and Medicaid use and cost data for residents of Monroe County, New York, over the age of 65. It examines and compares utilization and expenditure patterns of the Medicare-only and the Medicare-Medicaid (dually eligible) decedents in 1988. Examination of reimbursement for nonacute services, not covered by Medicare, reveals that services for the "older old" may be less costly immediately prior to death than for younger decedents. However, when expenses in the year prior to the year of death are also counted, services for the dually eligible, older old decedents appear to be neither more nor less costly than for younger decedents. Distribution of expenses does, however, vary considerably with age. The younger decedents, aged 65 to 74, use 55 percent of their medical resources on hospital care, paid for by Medicare; the older old use 26 percent for hospital services and pay 67 percent for supportive care, reimbursed by Medicaid. The study suggests that medical intervention associated with dying is utilized more often and at a higher cost by younger decedents.


The organized American medical profession's response to financial conflicts of interest: 1890-1992.

Recent issues of financial conflicts of interest for physicians are illuminated by the history of similar issues from 1890 to 1992 and the response of the organized medical profession to activities like payment of commissions, or "fee splitting," physical ownership of medical facilities, and dispensing of drugs. The medical profession tried to confront these and related conflicts early in the century, in some cases by adopting ethical codes, but was unable to enforce standards of conduct. Since the 1950s, substitutes for fee splitting emerged and were tolerated by the profession. Moreover, its stance became weaker as it abandoned its earlier clear ethical prohibitions and relied instead on subjective standards and, more recently, on disclosure to patients. The medical profession still favors voluntary codes of ethics, setting aspirational goals, promoting a patient-centered ethos, and relying on the good will of individual doctors--tactics for dealing with financial conflicts of interest that this review of recent history suggests are inadequate. Instead, public intervention is required to address these problems.


Searching for answers. Using computers to find the literature you need for patient care.

The medical journal literature is extensive and contains information that can help physicians care for patients. Minnesota physicians are able to tap into a variety of resources to help them locate relevant articles. Literature searches may be run by librarians in a local hospital library, a larger health sciences library, or via various professional associations. Physicians interested in conducting their own searches are able to use local systems or dial into national ones. Innovations in computers will allow easier and faster access to the medical literature in the future.


The effects of childhood trauma upon adult recovery from injury and illness.

Many patients with chronic injury or illness suffer from childhood trauma syndrome. As a result, they do not take proper care of themselves and are at risk of developing long-term disabilities. Physicians who understand the dynamics of CTS can help these patients regain control over their own bodies and health. By building therapeutic relationships with these patients, physicians can restore trust and help them lead happier, healthier lives.


Emergency medicine. The rural challenge.

Overcrowding, stress-related job burnout for emergency room physicians and other personnel, violence spilling over into the hospital with the surge in gang activity--it's the stuff of movies and, increasingly, real life in urban emergency departments, even in Minnesota. The trials and tribulations of providing emergency medical care in our metropolitan areas are well documented, both in professional journals and the conventional media. It's dramatic--lives hang in the balance--and it's important to everyone, since those who will one day require emergency medical care must entrust their well-being to this system. But it's only part of the story.


Lead levels among pregnant women in Hennepin County.

The purpose of this study was to determine the blood lead levels (Pb-B) of urban pregnant women with low incomes and/or living in areas with heavily traveled roads, dilapidated housing, and industrial plants. We measured blood lead in 1,055 pregnant Minneapolis-area women at entry to prenatal care and in one-third of the sample during the second half of pregnancy. The mean Pb-B level of the first sample (n = 1,055) was 1.83 +/- 1.83 micrograms/dL; of the second sample (n = 375), 1.99 +/- 1.92 micrograms/dL. Only one woman had a Pb-B level greater than 12.0 micrograms/dL, which was the result of occupational exposure. The low lead levels found in this study indicate that it is not necessary to routinely screen pregnant women for elevated Pb-B levels in our geographic area. Rather, women should be screened via an environmental questionnaire to ascertain the risk of lead exposure.


Mountain sickness.

This article outlines the fundamental physiology and pathology of high altitude. It covers the latest findings from the unique experiment Operation Everest II in which eight persons were gradually decompressed to the "summit" of Mt. Everest and extensive physiologic studies done. The article contains an examination of the various high altitude pathologies, including acute mountain sickness, high altitude pulmonary edema, high altitude cerebral edema, and high altitude retinal hemorrhage. Information on prevention and current approaches to treatment conclude the article.


Neurologic injury from undersea diving.

Underwater diving may cause several unique neurologic injuries because of exposure to rapid changes in pressure and volume. DCS, which results from extended deep dives and too rapid ascent, is a systemic disease that frequently causes spinal cord injury but may involve other organs as well. AGE results from pulmonary overpressure on ascent, with extravasation of air into the arterial system, and causes stroke-like brain injury. Both conditions are of sudden onset, progress rapidly, and require urgent attention. Definitive treatment includes administration of oxygen and recompression in a chamber. Permanent neurologic injury may result.


Lightning and electrical injuries.

Lightning and electrical injuries are similar in that both produce immediate tissue injury from burn and trauma induced by fall and both can arrest the heart and respiratory center. Immediate support of circulation and respiration is life-saving. Subsequently the nervous system may show signs of injury, and seizures, cerebral edema, and muscle and nerve lesions should be handled as the indications arise. Prevention of the injury is more effective than any postinjury treatment. Outdoors hikers and campers must take shelter to minimize their exposure; indoors properly installed equipment and attention to the relation of the equipment user to the electrical ground are the key elements in avoiding electrocution.


Neurologist as expert witness.

Prevailing liberal rules of evidence permit qualified medical and scientific experts to offer opinions designed to help courts decide issues to which their expertise relates. The opinions can be based on direct examinations, review of data assembled by others and data or inferences of a type relied on by other experts in the field. Application of these rules is illustrated through analysis of expert testimony in litigation involving a neurologic syndrome allegedly caused by an immunization and in a case involving controversy over the extent and outcome of major brain injury. Concerns about misuse of expert medical and scientific testimony in litigation are addressed. The article closes with a consideration of approaches designed to improve the reliability of expert testimony.


The postconcussion syndrome and the sequelae of mild head injury.

The postconcussion syndrome refers to a large number of symptoms and signs that may occur alone or in combination following usually mild head injury. The most common complaints are headaches, dizziness, fatigue, irritability, anxiety, insomnia, loss of consciousness and memory, and noise sensitivity. Mild head injury is a major public health concern because the annual incidence is about 150 per 100,000 population, accounting for 75% or more of all head injuries. The postconcussion syndrome has been recognized for at least the last few hundred years and has been the subject of intense controversy for more than 100 years. The Hollywood head injury myth has been an important contributor to persisting skepticism and might be countered by educational efforts and counter-examples from boxing. The organicity of the postconcussion syndrome has now become well documented. Abnormalities following mild head injury have been reported in neuropathologic, neurophysiologic, neuroimaging, and neuropsychologic studies. There are multiple sequelae of mild head injury, including headaches of multiple types, cranial nerve symptoms and signs, psychologic and somatic complaints, and cognitive impairment. Rare sequelae include hematomas, seizures, transient global amnesia, tremor, and dystonia. Neuroimaging and physiologic and psychologic testing should be used judiciously based on the problems of the particular patient rather than in a cookbook fashion. Prognostic studies clearly substantiate the existence of a postconcussion syndrome. Manifestations of the postconcussion syndrome are common, with resolution in most patients by 3 to 6 months after the injury. Persistent symptoms and cognitive deficits are present in a distinct minority of patients for additional months or years. Risk factors for persisting sequelae include age over 40 years; lower educational, intellectual, and socioeconomic level; female gender; alcohol abuse; prior head injury; and multiple trauma. Although a small minority are malingerers, frauds, or have compensation neurosis, most patients have genuine complaints. Contrary to a popular perception, most patients with litigation or compensation claims are not cured by a verdict. Treatment is individualized depending on the specific complaints of the patient. Although a variety of medication and psychologic treatments are currently available, ongoing basic and clinical research of all aspects of mild head injury are crucial to provide more efficacious treatment in the future.


Posttraumatic cranial neuropathies.

Injury to cranial nerves is a common sequela of blunt head trauma. The olfactory, facial, and audiovestibular nerves are damaged most often, followed by the optic and ocular motor nerves. The trigeminal and lower cranial nerves are rarely involved. Chances of recovery are greatest for the facial nerve, intermediate for the ocular motor nerves, and least likely for the olfactory, optic, and audiovestibular nerves. Treatment is usually symptomatic, although steroids or surgical decompression of the optic and facial nerves can lead to dramatic results in selected patients.


Posttraumatic epilepsy.

The risk for development of posttraumatic epilepsy is generally related to the severity of injury or the brain trauma dose. This article discusses mechanisms and biomechanical effects of brain injury. Genetic factors, prevention and prophylaxis, and the use of antiepileptic drugs are discussed also.


Cognitive impairment following closed head injury.

Cognitive impairments are usually the most disabling sequelae of CHI. The earliest stage of recovery from moderate to severe closed head injury is a period of PTA that typically includes memory loss for events preceding and surrounding the injury and memory loss for events occurring since the injury. Following resolution of PTA, deficits may be present in a number of cognitive domains. Memory and attention/information processing speed and efficiency are typically the cognitive domains most severely affected by head injury. Intellectual, language, and perceptual skills tend to be relatively preserved. Neurologic variables such as pupillary reactivity and worst GCS score are prognostic of cognitive impairment at 1 to 2 years postinjury. Following mild head injury, impairments of memory and information processing may be apparent within the first week of recovery. These deficits usually resolve in 1 to 3 months, although chronic complaints persist in a minority of individuals. The long-term cognitive effects of CHI are typically more severe for younger children than older children. Neuropsychologic assessment provides an objective way to measure the presence and severity of cognitive impairment.


Psychiatric aspects of the neurology of trauma.

This article discusses posttraumatic stress disorders, their relationship to conversion and dissociation responses, and the role of neurologic examination in diagnosis. The concentration camp syndrome (CSS) and its organic and psychological components, the emotional impact of physical illness, and psychiatric syndromes related to brain lesions are presented also.


Trauma and movement disorders.

Movement disorders have been linked to trauma; however, these cases are uncommon and best substantiated if the movement disorder occurs in close temporal association with the trauma, occurs in context of other clinical signs of neurologic damage, and if evidence of CNS lesions on neuroimaging scans is evident. The pathophysiology of movement disorders relates to basal ganglia dysfunction, and traumatic lesions of peripheral nervous system structures or cortical regions could theoretically alter basal ganglia function indirectly and influence movement disorders. Parkinson's disease, the prototype of movement disorders, does not appear to be caused by trauma.


Reflex sympathetic dystrophy and causalgia.

Converging lines of research suggest that RSD is a form of reflex neurogenic inflammation. Early the sympathetic nervous system appears to have an initiating and sustaining role. In later states of the illness, structural changes at the DH level seem to correlate with centralization of the process. Neuropeptides and EAAs released from thin or unmyelinated nociceptive afferents may produce both the inflammation and the suspected central sensitization of DH neurons seen in later stages of the disease. Effective treatment will follow a clearer understanding of the physiology of each stage of the disease.


Some observations on whiplash injuries.

Motor vehicle accidents with a whiplash mechanism of injury are one of the most common causes of neck injuries, with an incidence of perhaps 1 million per year in the United States. Proper adjustment of head restraints can reduce the incidence of neck pain in rear-end collisions by 24%. Persistent neck pain is more common in women by a ratio of 70:30. Whiplash injuries usually result in neck pain owing to myofascial trauma, which has been documented in both animal and human studies. Headaches, reported in 82% of patients acutely, are usually of the muscle contraction type, often associated with greater occipital neuralgia and less often temporomandibular joint syndrome. Occasionally migraine headaches can be precipitated. Dizziness often occurs and can result from vestibular, central, and cervical injury. More than one third of patients acutely complain of paresthesias, which frequently are caused by trigger points and thoracic outlet syndrome and less commonly by cervical radiculopathy. Some studies have indicated that a postconcussion syndrome can develop from a whiplash injury. Interscapular and low back pain are other frequent complaints. Although most patients recover within 3 months after the accident, persistent neck pain and headaches after 2 years are reported by more than 30% and 10% of patients. Risk factors for a less favorable recovery include older age, the presence of interscapular or upper back pain, occipital headache, multiple symptoms or paresthesias at presentation, reduced range of movement of the cervical spine, the presence of an objective neurologic deficit, preexisting degenerative osteoarthritic changes; and the upper middle occupational category. There is only a minimal association of a poor prognosis with the speed or severity of the collision and the extent of vehicle damage. Whiplash injuries result in long-term disability with upward of 6% of patients not returning to work after 1 year. Although litigation is very common and always raises questions of secondary gain in patients with persistent symptoms, most patients are not cured by a verdict. Acute treatment of neck pain consists of ice for 24 hours followed by heat applications, pain pills, NSAIDs, and muscle relaxants. Trigger point injections can be beneficial in both the acute and the persistent phases. Use of cervical collars should probably be kept to a minimum during the first 2 to 3 weeks after the injury and then avoided. Early passive mobilization and range of motion exercises may accelerate recovery. Physical therapy and transcutaneous nerve stimulators may be helpful in reducing pain and improving movement.(ABSTRACT TRUNCATED AT 400 WORDS)


Neurology of microgravity and space travel.

Exposure to microgravity and space travel produce several neurologic changes, including SAS, ataxia, postural disturbances, perceptual illusions, neuromuscular weakness, and fatigue. Inflight SAS, perceptual illusions, and ocular changes are of more importance. After landing, however, ataxia, perceptual illusions, neuromuscular weakness, and fatigue play greater roles in astronaut health and readaptation to a terrestrial environment. Cardiovascular adjustments to microgravity, bone demineralization, and possible decompression sickness and excessive radiation exposure contribute further to medical problems of astronauts in space. A better understanding of the mechanisms by which microgravity adversely affects the nervous system and more effective treatments will provide healthier, happier, and longer stays in space on the space station Freedom and during the mission to Mars.


The management of acute diarrhea in children: oral rehydration, maintenance, and nutritional therapy. Centers for Disease Control and Prevention.

Worldwide, diarrhea remains one of the most common illnesses among children. In the United States, children < 5 years of age experience > 20 million episodes of diarrhea each year, leading to several million doctor visits, 200,000 hospitalizations, and approximately 400 deaths. Much of this morbidity is due to the dehydration associated with acute watery diarrhea. Consequently, the proper management of children with acute diarrhea is important for all practitioners as well as for parents of small children. The development of oral therapy for the rehydration and maintenance of children with dehydrating diarrhea has become the worldwide mainstay of national diarrheal control programs. More recently, proper nutrition for children with diarrhea is viewed as an important adjunct to therapy, whereas antibiotics and other drugs play only a limited role. Intravenous therapy remains essential for diarrheal episodes associated with severe dehydration. This document reviews the proper management of diarrhea among children. Particular attention is given to the use of oral therapy for rehydration and maintenance therapy for the dehydrated child and nutritional management. In the United States, the improved management of children with diarrhea could lead to a noticeable decrease in the number of children who are hospitalized or die as a result of diarrheal illness. This report contains recommendations prepared by the Centers for Disease Control and Prevention (CDC), with input from a panel of pediatric and diarrheal management experts, which are consistent with recommendations endorsed by the American Academy of Pediatrics.


Incidence of treatment for end-stage renal disease attributed to diabetes mellitus--United States, 1980-1989.

End-stage renal disease (ESRD) is defined as renal insufficiency requiring dialysis or kidney transplantation for survival. In the United States, diabetes mellitus is the major cause of ESRD. This report summarizes trends during the 1980s in the incidence of treatment for ESRD attributable to diabetes mellitus (ESRD-DM).


Hospitalizations for diabetic ketoacidosis--Washington State, 1987-1989.

Diabetic ketoacidosis (DKA) is an acute metabolic complication of diabetes mellitus that can be life threatening. Although DKA is often preventable, approximately 84,000 DKA-associated hospitalizations and 1800 DKA-associated deaths occurred in the United States during 1988. The Washington Department of Health (WDH) monitors DKA-associated hospitalizations to assist its chronic disease programs in preventing DKA-associated hospitalizations and deaths. This report summarizes surveillance of DKA hospitalizations among Washington state residents from 1987 through 1989.


Incidence of treatment for end-stage renal disease attributed to diabetes mellitus, by race/ethnicity--Colorado, 1982-1989.

Diabetes mellitus (DM) is the principal known cause of end-stage renal disease (ESRD) (i.e., renal insufficiency requiring kidney dialysis or transplantation for survival) and accounts for one third of all incident cases of treated ESRD in the United States. Rates for initiating diabetes-related ESRD (ESRD-DM) treatment have been characterized for blacks and whites but have not been well characterized for Hispanics. To describe ESRD-DM treatment by race/ethnicity in Colorado for improved program planning for intervention services, the Colorado Diabetes Surveillance Project of the Colorado Department of Health investigated differences in the rates of initiating ESRD-DM treatment for non-Hispanic white, Hispanic, and black Colorado residents with diabetes. This report describes trends in the incidence of ESRD-DM treatment by race/ethnicity among persons with diabetes in Colorado from 1982 through 1989.


Cigarette smoking among Southeast Asian immigrants--Washington state, 1989.

Since 1975, approximately one million Southeast Asians have immigrated to the United States. In general, the efforts of local public health agencies to meet the needs of these immigrants have focused on identifying and treating acute and chronic diseases rather than identifying and modifying health-risk behaviors (e.g., smoking) among these immigrants. However, efforts to determine the prevalence of smoking suggest that smoking rates are high, especially among men of Southeast Asian origin. During 1989, to characterize cigarette smoking among Southeast Asian immigrants, the Seattle-King County (Washington) Health Department surveyed newly arriving Southeast Asian immigrants who intended to reside in the county regarding their health problems and health-risk behaviors. This report summarizes survey findings regarding their smoking habits.


Cigarette smoking among American Indians and Alaskan Natives--Behavioral Risk Factor Surveillance System, 1987-1991.

Cardiovascular disease and cancer are two of the leading causes of premature death among American Indians and Alaskan Natives. Although cigarette smoking contributes to these diseases, cigarette smoking behaviors among American Indians and Alaskan Natives have not been well characterized nationally. To better assess the impact of smoking on these populations, CDC analyzed data obtained from the Behavioral Risk Factor Surveillance System (BRFSS) during 1987-1991. This report summarizes the findings from this study.


HIV instruction and selected HIV-risk behaviors among high school students--United States, 1989-1991.

Efforts to prevent human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) in the United States must be targeted toward persons of all age groups at risk, including adolescents. Many high school students have reported engaging in behaviors that increase their risk for HIV infection (1,2). During April and May of 1989, 1990, and 1991, CDC conducted three national school-based surveys among high school students that addressed, in part, HIV-risk behavior and school-based instruction. This report summarizes findings from these surveys.


AIDS Community Demonstration Projects: implementation of volunteer networks for HIV-prevention programs--selected sites, 1991-1992.

States and cities have effectively used community-level intervention projects to reduce cigarette smoking and other risk behaviors associated with chronic disease (1-3); similar strategies have been introduced to prevent human immunodeficiency virus (HIV) infection in men who have sex with men and who identify themselves as homosexual or bisexual (4,5). Many of these projects used community volunteers to deliver interventions (2,4,5). An important concern for community-level intervention projects that rely on volunteers is the need to address the recruitment and retention of these volunteers. CDC AIDS Community Demonstration Projects have developed and maintained volunteer networks among hard-to-reach populations for HIV prevention. This report summarizes methods used by demonstration projects in five cities to develop and maintain volunteer networks during 1991-1992.


HIV infection and AIDS--Georgia, 1991.

Public health surveillance efforts for acquired immunodeficiency syndrome (AIDS) in the United States have documented an increasing proportion of cases among persons who reside outside the largest metropolitan areas (1,2). These findings, coupled with results of human immunodeficiency virus (HIV) prevalence studies, have led to the development of HIV-related prevention and treatment services in smaller cities and rural areas. This report presents results of HIV-infection and AIDS surveillance in Georgia (1990 population: 6.5 million) for 1991 and compares these findings for urban and rural areas.


Trends in years of potential life lost before age 65 among whites and blacks--United States, 1979-1989.

The reduction of preventable deaths among minority populations in the United States is a national health objective for the year 2000 (1). One measure used to assess progress toward this objective is years of potential life lost before age 65 (YPLL-65), which measures the impact of deaths occurring in years preceding 65 years of age and emphasizes the effects of deaths among younger persons (2). This report compares trends in YPLL-65 among U.S. whites and blacks from 1979 through 1989.


Factors potentially associated with reductions in alcohol-related traffic fatalities--United States, 1990 and 1991.

Traffic crashes are the single greatest cause of death among persons aged 5-32 years in the United States (1); almost half of all traffic fatalities are alcohol-related (1,2). An estimated 40% of persons in the United States may be involved in an alcohol-related traffic crash sometime during their lives (1). In 1991, the number of alcohol-related traffic fatalities (ARTFs) declined almost 10% when compared with 1990 (3), and the total number of deaths during 1991 (19,900) is the lowest since more complete alcohol-related fatal crash data became available in 1982. This report summarizes data from the National Highway Traffic Safety Administration's (NHTSA) Fatal Accident Reporting System on trends in ARTFs in the United States from 1982 through 1991 and presents information regarding several factors potentially related to the decline in fatalities during 1991.


Heterosexual transmission of HIV--Puerto Rico, 1981-1991.

Puerto Rico has the second highest overall rate of acquired immunodeficiency syndrome (AIDS) cases among states and territories of the United States and the second highest rate of cases among women (1,2). Although heterosexual transmission of human immunodeficiency virus (HIV) among persons reported with AIDS has increased throughout the United States--accounting for 8% of all U.S. AIDS cases diagnosed in 1991 (2,3)--the proportion of cases attributed to heterosexual transmission is highest in Puerto Rico (18%). This report summarizes data collected through the Puerto Rico AIDS Surveillance Program to characterize AIDS cases associated with heterosexual transmission during 1981-1991.


Cardiovascular disease control efforts among primary-care physicians--Missouri, 1990.

Nearly half (43%) of all deaths in the United States each year are related to cardiovascular diseases (CVD), including coronary heart disease and cerebrovascular disease (1). CVD is linked to certain risk factors and behaviors, including high blood pressure, elevated total serum cholesterol, cigarette smoking, and physical inactivity (2). Because approximately 70% of the population has at least one contact with a physician each year (3), primary-care physicians are central to health promotion and disease-prevention efforts and early detection of CVD. In May and August 1990, the Missouri Department of Health (MDH) conducted a survey of practicing Missouri physicians to characterize their efforts to identify and control CVD risk factors among their patients.


Abortion surveillance--United States, 1989.

Since 1980, the number of legal induced abortions reported to CDC has remained stable, varying each year by < 5%. In 1989, 1,396,658 abortions were reported--a 1.9% increase from 1988. The abortion ratio for 1989 was 346 legal induced abortions/1,000 live births, and the abortion rate was 24/1,000 women ages 15-44 years. The abortion ratio was highest for black women and women of other minority racial groups and for women < 15 years of age. Overall, women undergoing abortions tended to be young, white, and unmarried; to have had no previous live births; and to be having the procedure for the first time. Approximately half of all abortions were performed before the eighth week of gestation, and 87% were before the thirteenth week of gestation. Younger women tended to obtain abortions later in pregnancy than older women. This report also includes newly reported abortion-related deaths for 1986 and 1987, as well as an update on abortion-related deaths for the period 1978-1985. Ten deaths in 1986 and six deaths in 1987 were associated with legal induced abortion. The case-fatality rate in 1986 was 0.8 abortion-related deaths/100,000 legal induced abortions and 0.4/100,000 in 1987.


Influenza surveillance--United States, 1991-92.

During the 1991-92 influenza season, sustained regional influenza activity began to be reported by state and territorial epidemiologists in the United States in mid-October 1991. Sustained reporting of widespread influenza activity began in early November 1991, 5-10 weeks earlier than in any of the previous nine influenza seasons. Influenza caused substantial morbidity among school-age children and excess mortality among the elderly. Regional outbreaks of influenza ended 2-6 weeks earlier than in the previous nine influenza seasons, based on the last sustained state and territorial epidemiologists' reports. Nationally, > 99% of isolates were influenza A. Influenza A(H3N2) predominated in all regions of the country, but isolation of influenza A(H1N1) increased proportionally as the season progressed. Isolation of influenza B (< 1% of total isolates) clustered after February. The majority of isolates characterized were antigenically similar to components in the 1991-92 influenza vaccine. However, an influenza A(H1N1) strain that had undergone antigenic drift was detected in many regions of the country; this strain will be included in the 1992-93 influenza vaccine.


[Innovative elastomeric bio-adhesive (pup-201) synthesized by urethane-prepolymer].

Properties of PUP-201 for the application to vital organs were estimated. Basic properties of PUP-201 were compared with two kinds of commercial fibrin and synthetic adhesives. Its initial and wet adhesive strength was 40 and 150 times stronger than those of fibrin adhesives respectively. Its curing time was 50 seconds, and elongating rate was 150% nearly as same as that of the intestine. This adhesive had not antimicrobial activities. The strength of adhesive bonded pig skin in shear by tension loading by way of JIS K6850 recorded 2.0-2.7kg/cm2 five minutes after, and 2.0-2.8kg/cm2 after 24 hours. The results of the same test in small intestine defined 0.4-1.0kg/cm2 in sero-sero attachment. There was no toxicity on the pig skin. Practical examinations for application to hemostasis on the section of dog liver and sealing of the lung section were performed successfully. PUP-201 was demonstrated excellently useful material for the adhesive, hemostasis and sealant of vital tissue.


[In vivo effect of interleukin-2 (IL-2) on immunopotentiation in irradiated mice].

This study's purpose was to evaluate the effect of IL-2 on the recovery from immunosuppressive state in vivo. Young adult mice (ddY) were rendered immunodeficient by whole body irradiation (300 rad). IL-2 (20000 u/head/day) was administered intraperitoneally for 5 days after irradiation. In the experiment (I) immunological parameters were investigated. In the IL-2-treated mice peripheral lymphocytes and neutrophils were increased, but there was no change in the T and B lymphocyte ratio. The proliferative responses of spleen cells to mitogens were improved in the IL-2-treated mice. A significant expansion of macrophages in the intraperitoneal cavity was demonstrated. In the experiment (II) irradiated mice were given peritonitis by 1 puncture with a 23 gauge needle through their ligated cecum. This peritonitis model was followed for 2 weeks after ligation and puncture of the cecum. In the mice with IL-2 pretreatment and antibiotic therapy, the survival rate was significantly improved, compared with mice that only received antibiotic therapy. Administration of carrageenan and anti-T cell monoclonal antibodies significantly decreased the survival rate. These results suggested macrophages as well as functional T cells were necessary to decrease the mortality rate. In conclusion, IL-2 may have a remarkably protective effect against infection in immunodeficient state.


[Motility and mucosal hemodynamics of the lower esophagus after transabdominal esophageal transection for esophageal varices].

Lower esophageal motility and mucosal hemodynamics were investigated in 20 patients who underwent transabdominal esophageal transection for esophageal varices (ET), to evaluate their association with reflux esophagitis and variceal recurrence. In the manometric study with microtransducer catheter, maximum swallowing pressure in the lower esophagus of the patients was significantly lower than that of the healthy controls (20 cases) (26.1 +/- 20.5mmHg vs. 80.0 +/- 10.0mmHg: p < 0.01), while high pressure zone pressure did not differ between the two groups. In comparison between patients with and without esophagitis (E(+) and E(-)), maximum swallowing pressure of E(+) was statistically lower than that of E(-) (12.4 +/- 18.7mmHg vs. 31.0 +/- 19.1mmHg: p < 0.05). In the hemodynamic study by reflectance spectrophotometry, the index of esophageal mucosal blood volume (IHb) and the index of oxygen saturation of hemoglobin (ISo2) of E(+) and E(-) were no different from those in the patients with non-operated esophageal varices (10 cases). Although there was no correlation between the recurrence of RC-sign and mucosal microcirculation, the patients with larger varices tended to have a higher IHb and a patients with F1-varices had significantly lower ISo2 than the patients without varices. This study indicated that the poor clearance ability after ET may lead to reflux esophagitis and the patients with variceal recurrence had the congested mucosal microcirculation, compared to those without variceal recurrence.


[Histopathological features of the lymph node metastases in patients with thoracic esophageal cancer].

Histopathological features of the lymph node involvement were studied in 104 patients with thoracic esophageal cancer who underwent subtotal esophagectomy combined with extended radical lymph adenectomy in cervicothoracoabdominal region. Metastatic involvement was found in a total number of 503 lymph nodes from 73 patients by histologic examination. The mean of long and short diameter was found to be less than 5mm in 125 (24.9%) of these 503 nodes. The involved area on the section was less than one third in 149 nodes (29.6%), and was significantly smaller in mediastinal lymph nodes than those in cervical or abdominal ones. Sixty-seven (13.3%) of 503 nodes were partially invaded by micrometastasis of 1mm or less in diameter. Micrometastasis also more frequently occurred in mediastinal nodes with a statistically significant difference. Extranodal proliferation (ENP) of cancer cells was found in 106 nodes (21.1%), and extranodal lymphatic and/or blood vessel invasion (ENly, v) was also recognized in 60 nodes (11.9%). Micrometastasis and ENP with or without ENly, v were found in 24 (32.9%) and 29 (39.7%) of 73 patients with positive lymph node metastasis, respectively. Postoperative survival rate in patients with micrometastasis and/or ENP with or without ENly, v was inferior to that in patients with neither of them.


[Decision analysis in therapeutic strategies for small bowel obstructions].

Clinical decision analysis was applied in therapeutic decision-making regarding small bowel obstructions. For strangulation obstruction, the three strategies of immediate surgery, observation of clinical course, and decision according to ultrasonographic findings were analyzed. When mortality rate, morbidity rate, or duration of hospital stay was used as the utility value, decision on the basis of ultrasonographic findings was selected as the most effective strategy, while the strategy of immediate surgery was selected when the rate of intestinal necrosis was used as the utility value. For adhesive obstructions, the three strategies of immediate surgery, conservative treatment with long tube decompression, and long tube decompression with enteroclysis (infusion contrast radiography) were analyzed. The strategy of enteroclysis was selected when morbidity rate and duration of hospital stay were used as utility values, while immediate surgery was selected when recurrence rate of adhesive obstruction was used as the utility value. For obstruction caused by intraabdominal recurrent cancer, operative treatment and conservative treatment were analyzed. The strategy of surgery was selected when rate of obstruction resolved and number of months of survival were used as utility values. Clinical decision analysis is valuable in quantitatively assessing individual variables affecting therapeutic decision-making.


[Lymphoid infiltration of colon cancer and clinical significance of the skin test].

It was reported that lymphoid infiltration (LI) of colon cancer tended to be found more commonly in patients with colonic cancer in an early stage than in an advanced stage. This time, the correlation among LI of colon cancer, cell-mediated immunity and the cumulative 5-year survival rate was studied in 124 patients with resected colon cancer. The results were as follows: 1) The cumulative 5-year survival rate in patients with positive LI was higher than that in the patients with negative LI. 2) There was correlation between the degree of LI of the cancer and Su-PS skin reaction. 3) Postoperative cell-mediated immunity was reduced in the cases of poor prognosis. Therefore we insist on paying attention to immunochemotherapy for 24 months after surgery.


[Immunohistochemical detection of p53 in colorectal cancer and its relationship to prognosis].

The expression of p53 in colorectal tumors was studied immunohistochemically by monoclonal antibody (PAb1801). No nuclear staining was evident in the tumor cells of colorectal adenomas. p53 immunoreactivity was found in 59 (61.5%) of 96 colorectal cancers. There was no significant correlation between the p53 immunoreactivity and histologic type, tumor size, invasion of bowel wall, lymphatic invasion, venous invasion, lymph node metastasis, peritoneal dissemination, or liver metastasis. However, the p53 negative tumors showed a recurrence rate of 3.3%, while for the p53 positive tumors a recurrence rate of 20.9%. p53 negative tumors were associated with favorable prognosis, whereas those with p53 positive tumors were related to poor prognosis. DNA polymerase alpha positive cells rate in p53 positive tumors was significantly higher than in p53 negative tumors. The results suggested that p53 immunoreactivity might possibly be a useful prognostic marker of colorectal cancers.


[The effect of hyperthermia against pancreatic cancer cell--various examinations including flow cytometric bromodeoxyuridine/DNA analysis].

We investigated the effect of hyperthermia against pancreatic cancer cell in various aspects including of cytokinetics. The hyperthermia above 43 degrees C was thought to have strong effect for the studies on surviving cell number and morphological change of Capan-2 and RWP-1 cells after heating treatment. The cell inoculated to the nude mice subcutaneously after heating treatment at 43 degrees C was recognized the tumor forming at 3 weeks later, however, the growth speed was slower than that of control to which untreated Capan-2 cells were inoculated. As the case of 44 degrees C, the tumor forming was not recognized at all. The cytokinetics was measured by flow cytometric bromodeoxyuridine (BrdUrd)/DNA analysis. Up to 43 degrees C in accordance with increase in the heating temperature, the accumulation in G2M phase increased, and the amount of BrdUrd incorporated 5 phase decreased. At the temperature above 44 degrees C, BrdUrd was hardly incorporated into S phase cells and the cell cycle was almost stopped. These results suggested the usefulness of hyperthermia and strong effect of heating treatment above 44 degrees C against pancreatic cancer cell.


[Preoperative diagnosis of internal mammary node metastases in patients with breast cancer by using ultrasonography].

US findings of 81 patients with breast cancer were analyzed respectively to evaluate the usefulness of US in the preoperative diagnosis of internal mammary mode metastases. The internal mammary area (I.M.A.) in which internal mammary nodes lie was shown as a hypoechoic stripe in a sagittal scan along the sternum and as a triangular or spindle-shaped hypoechoic area in a transverse scan in the intercostal space. The US features of each I.M.A. in the first three intercostal spaces were divided into three patterns: Pattern-A wad defined as widening of the ipsilateral side of the I.M.A. compared with the contralateral side; Pattern-C was defined as normal at both sides of the I.M.A. Pathological examination confirmed the node metastases in 85.7% of intercostal scans showing Pattern-A and in 35% of intercostal scans showing Pattern-B. Only one of 189 intercostal scans showing Pattern-C had nodal metastases. The overall accuracy was 88.1% in 243 intercostal scans and 90.1% in 81 patients. We conclude that US is useful in the detection of internal mammary node metastases in patients with breast cancer.


[Coronary artery bypass surgery in the elderly].

In recent years it has become apparent that the number of persons 70 years of age or older is increasing. Between June, 1980, and December, 1990, 75 patients 70 years of age and older (range, 70-79 years; mean 73.1) underwent isolated coronary artery bypass grafting (CABG). During this same interval, 507 patients less than 70 years old underwent isolated CABG. Preoperative status in the elderly showed a significantly greater prevalence of women, urgent or emergent operation, triple vessel disease and diabetes. Operative mortality was higher among the elderly compared with the younger patients (p < 0.05). The increased frequency of urgent or emergent operation in the elderly was associated with postoperative complications. Major complications related CABG in the elderly were serious cerebrovascular accidents and low cardiac output syndrome. In order to improve the results of CABG in the elderly, it is essential to perform the elective operation following careful evaluation of both cardiac function and central nervous system.


[Experimental study of the prostacyclin production of autogeneous vein graft].

In order to investigate the blood compatibility of autogenous vein graft (AVG), changes in prostacyclin (PGI2) production following harvesting and arterial implantation were studied experimentally. Basal PGI2 production, which means the value obtained without stimulation by arachidonic acid (AA), decreased significantly 60 minutes after harvesting. At this time, no morphological changes in the endothelial cells were observed. Under stimulation by AA, a significant decrease in the PGI2 production of the specimens was seen 120 minutes after harvesting. Despite severe morphological changes in endothelial cells, the PGI2 production of vein grafts was maintained until this time. The AVG in the canine abdominal aorta was completely reendothelialized 1 week after implantation, but another 2 weeks were needed to restore PGI2 production. It is concluded from these results: 1. It is rational to implant an AVG within 60 minutes after harvesting from the viewpoints of morphology and PGI2 production of the endothelial cells. 2. PGI2 production from sources other than endothelial cells is possible and may contribute to the compatibility of the AVG in the early period after implantation. 3. AVG in the aorta could restore PGI2 production 3 weeks after implantation.


[Oxidized cellulose occlusion of a peripheral bronchial fistula communicating to the left subphrenic abscess].

A 52-year-old man was complicated with a left subphrenic abscess after total pancreatectomy and gastrectomy for advanced pancreatic cancer. A left subphrenic silicon tube penetrated the diaphragm and the bottom of the left lung as well, causing a bronchial fistula with bilateral aspiration pneumonia. Then bronchoscopically, the fistula was successfully treated by packing a few pieces of oxidized cellulose into the affected bronchus. One month later the patient died of sepsis due to multiple liver abscess. On autopsy, the bronchial fistula and any active inflammation were not recognized in the left lower lung area.


[The comparison of postoperative multiple organ failure with arterial disease to that with gastrointestinal cancer].

The clinical features of 22 postoperative multiple organ failure (MOF) patients, comprised of 8 with arterial disease (A-MOF) and 14 with gastrointestinal cancer (G-MOF), were investigated. Differences in the operative time, blood loss, and mortality were not significant. The initial organ impaired was the lungs in 78.6% of G-MOF patients and the heart or kidneys in all A-MOF patients. Infection developed in over 80% of both groups. In many A-MOF patients, the pneumonia or septicemia developed secondary to organ failure, while intraabdominal infection triggered respiratory failure in many G-MOF patients. Our organisms in infected specimens and their antibiotic sensitivities was valuable for the early administration of effective antibiotics. Upper gastrointestinal tract bleeding was important in the prognosis of both groups and occurred more frequently in A-MOF than in G-MOF patients. Consumption coagulopathy in A-MOF patients and DIC induced by infection in G-MOF patients mainly caused such bleeding. Preoperative administration of heparin was effective in improving coagulopathy. Furthermore, measurement of intramural pH with tonometer in the stomach and gastric irrigation with oxygenated perfluorochemicals were effective in the prediction and prevention of gastrointestinal bleeding.


[Principles of lymphadenectomy for gastric cancer according to depth of wall invasion, tumor site and regional lymphatic flow].

The principles of lymphadenectomy for gastric cancer are discussed based on the 1010 gastric cancer patients who underwent gastrectomy with curative intent between 1970 and 1990. In 147 of these patients, regional lymphatic flow was examined by injecting activated carbon particles CH40. 1) One hundred and ninety patients (19.8%) with cancer invasion confined to the mucosa had lymph node metastases limited to the perigastric nodes (n1). 2) Two hundred and five patients (20.3%) had cancer invasion to the submucosa. For 99.0% of them the lymph node metastases were limited to compartment II (n2). 3) Three hundred and twenty-two patients (31.9%), with cancer invasion to the muscle layer or serosa and limited to the upper or middle third of the stomach, had lymph node metastases in compartment II and No. 12 (n 2 + No. 12). 4) Two hundred and ninety-three patients (29.0%) with cancer invasion to the muscle layer or serosa and limited to lower third of the stomach or to its extension, had lymph nodes metastases in compartment III (n3). 5) Consequent to observations on the regional lymph flow of the stomach by CH40, we now perform paraaortic lymph node dissection, when gastric cancer patients with serosal invasion have metastases lymph nodes No. 2, 7, 8a, 9, 11, 12 or No. 14V.


[Relationship between prolonged life span and changes of serum IAP and albumin induced by the therapy of lentinan plus tegafur in inoperable and recurrent gastric cancer].

Relationship between prolonged life span and changes of serum IAP and albumin induced by the therapy of lentinan plus tegafur were analysed on 43 cases with inoperable and recurrent gastric cancer. Antitumor effect was observed only in one case (2%). Other clinical effects such as improvement of performance status (PS), appetite or pain were observed in 18 cases (42%). Decrease of serum IAP was observed in 25 cases (58%) and increase of albumin was observed in 20 cases (47%). The changes of these two factors seemed to be reversely correlated. Among 30 cases which didn't show decrease of albumin, we found no increase of serum-IAP in 22 cases (72%). In the cases which showed decrease of serum IAP from abnormally high level (more than 500 mu/ml) and increase of albumin from abnormally low level (less than 3.5g/dl), prolonged life span was observed by comparison with the other cases. The cases with any clinical effect contained 78% of the cases without increase of serum IAP, and 72% of the cases without decrease of albumin. These results suggested that life prolongation effect or improvement of clinical symptoms by our therapy was closely related to the change of these serum factors.


[Experimental study on catheter-bypass between superior mesenteric and hepatic hilar portal veins (SMV-HPV bypass)].

Hepatic ischemia is a consequence of concomitant resection of the portal vein and the hepatic artery when it is necessitated in hepatobiliary or pancreatic surgery. We developed an antithrombogenic catheter with a balloon and experimentally evaluated the safety of construction of SMV-HPV bypass using this catheter to simultaneously prevent intestinal vascular congestion and hepatic ischemia. The main portal vein and the hepatic artery were blocked and SMV-HPV shunting was made in five dogs (bypass group); the main portal vein and the hepatic artery were blocked and the SMV was shunted to the femoral vein in others (ischemia group). In the ischemia group, 3 of the 5 dogs died within 5 days, but all animals of the bypass group survived for a long period. In the bypass group, the changes of adenine nucleotide levels, energy charge ratio, liver function, blood coagulation, and fibrinolysis were mild and within physiologic ranges, and none of changes of the hepatic tissue were observed histologically. This bypass procedure is simple and safe to simultaneously prevent hepatic ischemia and intestinal vascular congestion in cases requiring concomitant resection and reconstruction of the portal vein and the hepatic artery.


[Establishment of an experimental model with a high frequency of liver metastasis and recurrence from gastric VX2 cancer: histological analysis of the developmental process of primary and metastatic cancer lesions].

An experimental model with a high frequency of liver metastases and recurrence was established by the non-resection and resection of gastric cancer lesions induced with implanting VX2 cancer cells into the stomach of 35 rabbits. The frequency of liver metastases was 0% on Days 7 and 14, 40% on Day 21 and 60% on Day 28 in the non-resection group. In the resection group, primary lesions were resected on Days 7, 10 and 14, and the metastases were found in all the animals 14 days after the resection on Day 14, though they did not occur in every animal 18 and 21 days after the resection on Days 10 and 7. The metastatic lesions were found in the peri-lobular area, accompanied by cancer emboli in the interlobular veins. Vascular invasion was found in almost all (90%) the primary lesions of animals with liver metastases or recurrence. These results suggest that hepatic micrometastases occur between 10 and 14 days after implantation, and that vascular invasion plays an important role in the formation and extension of liver metastases or recurrence. They also suggest that this model is utilized as a useful tool for studying many aspects of liver metastases or recurrence in gastric cancer.


[Investigation of lipid peroxidation in regenerating rat liver].

The purpose of this study was to estimate the effects of lipid peroxidation in regenerating rat liver. Partial 70% hepatectomy was performed in rat according to Higgings and Anderson. EPC (alpha Toc: Ascorbic acid = 6:4, radical scavenger) was injected intravenously (5mg/kg weight) one hour before operation. Lipid peroxidation in regenerating liver reached a peak at 24 hours after operation. The administration of EPC markedly suppressed the increase of lipid peroxide in the plasma and remnant liver and that of GPT after hepatectomy, with subsequent good liver regeneration ratio. Moreover, the pretreatment with EPC remarkably elevated the activity of thymidine kinase (index of DAN synthesis). The EPC administration had not notable effects on the level of plasma ketone body ratio in animal but pathologically caused early advent of glycogen granule in the remnant liver tissue after partial hepatectomy, which reflected restoration of mitochondrial energy level. The results of the present study suggest that scavenger may be useful not only for impairment of liver dysfunction but also for recovery of mitochondrial energy level and DNA synthesis after liver resection.


[Experimental study of hepatic inflow occlusion in extrahepatic obstructive jaundice--effects on systemic and hepatic hemodynamics and oxygen metabolism].

The effect of clamping both the hepatic artery and portal vein for 20 min in extrahepatic obstructive jaundice on systemic and hepatic hemodynamics and oxygen metabolism were studied in dogs, and the following results was obtained. 1) Dogs with obstructive jaundice were in a hyperdynamic state before hepatic inflow occlusion, showing an increased demand for oxygen in the liver. 2) After declamping, cardiac index and systemic oxygen delivery were decreased in normal dogs, but a further decrease was found in dogs with jaundice. 3) After declamping, hepatic arterial blood flow was increased in normal dogs. Portal blood flow was decreased just after declamping, but was soon restored. In dogs with jaundice, hepatic arterial blood flow increased to a lesser extent, and recovery of portal blood flow was delayed. 4) Hepatic oxygen consumption and oxygen extraction ratio were already increased markedly just after declamping in normal dogs, whereas the degree of the increase in these parameters was much smaller in dogs with jaundice. Thus, hepatic inflow occlusion in extrahepatic obstructive jaundice induces evident disorder of hepatic oxygen metabolism, and is suggested to be dangerous.


[A study of phagocytic cell function in aspiration pneumonia induced by carrageenan].

To estimate the changes in phagocytic cell function associated with aspiration pneumonia, induced in mice by intra-tracheal injection of carrageenan. In inflammatory sites, the dominant increased cell component was polymorphonuclear leukocytes (PMNs). Intracellular 2'-7'-dichlorofluorescein (DCFH) oxidation of PMNs infiltrated into the lung tissue of the carrageenan-treated mice revealed a lower level of activity than that of a control group throughout a 120-hour period. The DCFH oxidative activity of alveolar macrophages (AMs) was lower in the broncho-alveolar lavage fluid and lung tissue of the carrageenan-treated mice than the control group. A decrease in the DCFH oxidative activity of AMs was observed after treatment with KCN or deferoxamine in the control group. In contrast, activity in the carrageenan-treated group was not decreased by KCN. Moreover, increased expression of Ia and F4/80 antigens was detected in the AMs of the carrageenan-treated group. These results show that increasing numbers of PMNs have a major role as phagocytic cells in inflammatory lung tissue, whereas AMs are reduced to their oxidative product and change into antigen-presenting cells, revealing a high ratio of Ia expression. The decrease in DCFH oxidation in the AMs of the carrageenan-treated mice may have been due to decreased mitochondrial oxidative metabolism.


[A case of minimal-sized adenocarcinoma in a fistula-in-ano].

The patient was 47 year-old man with a simple intermuscular fistula-in-ano. Fistulectomy revealed that there was a small mucinous adenocarcinoma. The tumor was small and localized in the wall of the fistula and the muscular invasion was not recognized. The mucin contained a little O-acylated sainomucin, and the origin of the tumor was considered as anal gland cell. This case appears as one evidence that the cancer in fistula-in-ano originates from the anal gland cell in the fistula.


Purification and properties of the RuvA and RuvB proteins of Escherichia coli.

The RuvA and RuvB proteins of Escherichia coli play important roles in the post-replicational repair of damaged DNA, genetic recombination and cell division. In this paper, we describe the construction of over expression vectors for RuvA and RuvB and detail simple purification schemes for each protein. The purified 22 kDa RuvA polypeptide forms a tetrameric protein (M(r) ca. 100,000) as observed by gel filtration. The tetramer is stabilised by strong disulphide bridges that resist denaturation during SDS-PAGE (in the absence of boiling and beta-mercaptoethanol). In contrast, purified RuvB polypeptides (37 kDa) weakly associate to form a dimeric protein (M(r) ca. 85,000). At low protein concentrations, the RuvB dimer dissociates into monomers. The multimeric forms of each protein may be covalently linked by the bifunctional cross-linking reagent dimethyl suberimidate. Addition of purified RuvA and RuvB to a RecA-mediated recombination reaction was found to stimulate the rate of strand exchange leading to the rapid formation of heteroduplex DNA.


GEBF-I in Drosophila species and hybrids: the co-evolution of an enhancer and its cognate factor.

The activation of the Drosophila melanogaster salivary gland secretion protein gene Sgs-3 is marked by important changes in chromatin structure in the distal regulatory region at -600 bp from the Sgs-3 start site. A stage- and tissue-specific glue enhancer binding factor, GEBF-I, binds in vitro to sequences from this region. Previous studies have revealed considerable variation in the DNA sequences of comparable regions in the related Drosophila species, D. simulans, D. erecta and D. yakuba. We detected GEBF-I-like proteins in these species, which appear to evolve as rapidly as the corresponding DNA sequences, and studied in detail the binding characteristics of the GEBF-I proteins of the two most closely related species, D. melanogaster and D. simulans. In crosses between these species, certain strains produce hybrid larvae which, unexpectedly, synthesised a single intermediate form of the protein. This suggests that the factor is subject to species-specific post-transcriptional modifications. In these hybrid larvae, which carry one D. melanogaster and one D. simulans Sgs-3 gene, the hybrid GEBF-I protein appears equally effective in the induction of both target genes.


Functional conservation between Schizosaccharomyces pombe ste8 and Saccharomyces cerevisiae STE11 protein kinases in yeast signal transduction.

In fission yeast (Schizosaccharomyces pombe), the mat1-Pm gene, which is required for entry into meiosis, is expressed in response to a pheromone signal. Cells carrying a mutation in the ste8 gene are unable to induce transcription of mat1-Pm in response to pheromone, suggesting that the ste8 gene product functions in the signal transduction pathway. The ste8+ gene encodes a 659 amino acid putative protein kinase, which is identical to the previously identified byr2 suppressor of the ras1 defect. Furthermore, ste8+ is highly homologous to the Saccharomyces cerevisiae STE11 gene, which functions in signal transduction in budding yeast. Expression of the S. cerevisiae STE11 gene in S. pombe ste8 mutants restores the ability to transcribe mat1-Pm in response to pheromone. Also, such cells become capable of conjugation and sporulation. When mat1-Pm is artifically expressed from a heterologous promoter, ste8 mutant cells will enter meiosis. This demonstrates that the meiotic defect of ste8 mutants is due to the absence of the mat1-Pm gene product.


FinOP repression of the F plasmid involves extension of the half-life of FinP antisense RNA by FinO.

The transfer operon of the F plasmid is positively regulated by the traJ gene product, expression of which, in turn, is regulated by both an antisense RNA, FinP, and the FinO protein (the FinOP system). A finP- F plasmid, pSFL20, was constructed by site-directed mutagenesis and was found to produce wild-type levels of pili encoded by the transfer operon. Transcription of the traJ gene was decreased by a factor of 3-5 fold in the presence of FinOP with no accumulation of a stable RNA duplex between the FinP RNA and the portion of the traJ mRNA which is complementary to finP. Stabilization of FinP RNA by FinO occurs in the absence of traJ transcripts, suggesting that FinO may interact directly with FinP to prevent its degradation.


Serum lactate dehydrogenase isoenzyme 1 activity in patients with testicular germ cell tumors correlates with the total number of copies of the short arm of chromosome 12 in the tumor.

The aim of our study was to assess the relationship between the serum lactate dehydrogenase isoenzyme 1 (S-LDH-1) activity in patients with testicular germ cell tumors and the number of copies of the short arm of chromosome 12 (12p) present in tumor. Twenty-seven adult patients with measurable tumor lesions were studied. Twenty-five had three or more copies of chromosome 12 per cell in the tumors. Nineteen had one or more copies of a specific chromosomal abnormality, an isochromosome of the short arm of chromosome 12, i(12p). Fourteen had increased S-LDH-1 levels. S-LDH-1 activity correlated significantly with the product of total tumor volume and the total number of copies of the short arm of chromosome 12 present per cell (total tumor 12p). We conclude that the total number of copies of the short arm of chromosome 12 in the tumors is most probably a factor contributing to the LDH-1 activity released from the tumors.


Cloning and molecular characterization of the secY genes from Bacillus licheniformis and Staphylococcus carnosus: comparative analysis of nine members of the SecY family.

SecY is a central component of the export machinery that mediates the translocation of secretory proteins across the plasma membrane of Escherichia coli. We have cloned and sequenced the secY genes from Bacillus licheniformis and Staphylococcus carnosus. The deduced amino acid sequences are highly homologous to those of other known SecY polypeptides, all having the potential to form 10 transmembrane segments. Comparative analysis of 9 SecY polypeptides, derived from different bacteria, revealed that 14 amino acid positions (2.7%) are identical in all SecY proteins and 89 (16.9%) show conservative changes. Clusters of conserved amino acid residues were found in 4 of the 10 transmembrane segments and 2 of the 6 cytoplasmic domains. It is suggested that the conserved regions might be involved in the translocation activity of SecY or might be required for the correct interaction of SecY with other components of the secretion apparatus.


Characterization of a chromosomally encoded, non-PTS metabolic pathway for sucrose utilization in Escherichia coli EC3132.

A wild-type isolate, EC3132, of Escherichia coli, that is able to grow on sucrose was isolated and its csc genes (mnemonic for chromosomally coded sucrose genes) transferred to strains of E. coli K12. EC3132 and all sucrose-positive exconjugants and transductants invariably showed a D-serine deaminase (Dsd)-negative phenotype. The csc locus maps adjacent to dsdA, the structural gene for the D-serine deaminase, and contains an inducible regulon, controlled by a sucrose-specific repressor CscR, together with structural genes for a sucrose hydrolase (invertase) CscA, for a D-fructokinase CscK, and for a transport system CscB. Based on DNA sequencing studies, this last codes for a hydrophobic protein of 415 amino acids. CscB is closely related to the beta-galactoside transport system LacY (31.2% identical residues) and a raffinose transport system RafB (32.3% identical residues) of the enteric bacteria, both of the proton symport type. A two-dimensional model common to the three transport proteins, which is based on the integrated consensus sequence, will be discussed.


Differential manifestation of seed mortality induced by seed-specific expression of the gene for diphtheria toxin A chain in Arabidopsis and tobacco.

A pea vicilin promoter-diphtheria toxin A (DTx-A) chain gene fusion was introduced into Arabidopsis and tobacco. The chimeric Dtx-A gene behaves as a dominant, seed-lethal, Mendelian factor, and the segregation ratios are consistent with the numbers of integrated copies as revealed by Southern blotting. Germination deficiency results from distinct developmental abnormalities, thus allowing genetic dissection of seed development. The endosperm is affected first in both species. In Arabidopsis, full cellularization of the initially syncytial endosperm does not take place, which results in shrinkage and a shriveled appearance of the mature dry seed. The embryo, which appears structurally normal and lacks visible lesions, ceases to develop at the partially recurved cotyledon stage and does not use the remaining endosperm. In tobacco, peripheral degeneration and premature termination of cellular endosperm development occurs at the cotyledon initiation stage. Lesions appear in the cotyledons at the advanced cotyledon stage, but the embryo continues to grow and attains nearly the same size and level of differentiation as mature wild-type embryos before degeneration and intracellular disintegration take place throughout. Accumulation of protein bodies and other cytoplasmic inclusions is very limited and occurs only in few cells. The timing and distribution of lesions follow a pattern typical for accumulation of protein bodies in wild-type seeds. These observations are consistent with expression of the vicilin promoter in the enlargement phase of cell differentiation. A novel tissue interaction arises, when the embryo uses up all the arrested endosperm: the embryo proves to be capable of absorbing the parenchyma layers of the integument, which are normally obliterated by, and incorporated into, the endosperm.(ABSTRACT TRUNCATED AT 250 WORDS)


Induction of a Streptomyces cacaoi beta-lactamase gene cloned in S. lividans.

The previously cloned class A beta-lactamase gene (bla) of Streptomyces cacaoi was shown to be inducible by beta-lactam compounds in the host organism S. lividans. A regulatory region of 2.75 kb was identified and the nucleotide sequence determined. It contained four open reading frames (ORFs) of which only two were complete and required for induction. ORF1-ORF2 exerted a positive regulatory effect on the expression of bla. Inactivation of ORF1 or of ORF2 resulted not only in the loss of induction, but also in a 30- to 60-fold decrease in the basal (non-induced) level of beta-lactamase production. ORF1 codes for a DNA-binding protein related to the AmpR repressor/activator, which controls the expression of ampC (class C beta-lactamase) genes in several Enterobacteria.


Modular structure of the FixL protein of Rhizobium meliloti.

FixL protein of Rhizobium meliloti is a haemo-protein kinase which activates the transcription of nifA and fixK genes via the transcriptional activator protein FixJ under microaerobic conditions. FixL and FixJ proteins belong to the family of two-component regulatory systems for which primary sequence data predicts a modular structure. We showed, using Escherichia coli as heterologous host, that FixL indeed has a modular structure. The amino-terminal hydrophobic domain is dispensable for the oxygen-regulated activity of FixL in vivo. The central cytoplasmic non-conserved domain is necessary for the oxygen-sensing function of FixL whereas it is not necessary for the activation of FixJ by FixL. We propose that, under aerobic conditions, the central domain represses the activating function associated with the carboxy-terminal conserved domain.


The region essential for efficient autonomous replication of pSa in Escherichia coli.

Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.


The PYR1 gene of the plant pathogenic fungus Colletotrichum graminicola: selection by intraspecific complementation and sequence analysis.

A spontaneous uridine-requiring auxotroph of Colletotrichum graminicola was recovered by selection for resistance to 5-fluoro-orotic acid. The auxotroph lacked orotate phosphoribosyl transferase (OPRTase) and was complemented with a clone from a cosmid library of C. graminicola DNA. A 3.1 kb HindIII-SalI fragment was subcloned from the cosmid and it could efficiently transform the auxotrophic strain to uridine prototrophy and integrate by site-specific recombination. This DNA fragment contains an open reading frame that is similar to OPRTase genes of the fungi Sordaria macrospora, Trichoderma reesei, Podospora anserina, and Saccharomyces cerevisiae. Based on the sequence similarities and the ability to restore uridine prototrophy, we conclude that the fragment contains the C. graminicola gene for OPRTase, which we have named PYR1. Our results demonstrate that cloning by complementation is feasible in C. graminicola, that the gene for OPRTase from C. graminicola can be useful as a selectable marker in transformation of the fungus, and that the OPRTase gene product is similar to OPRTase from other fungi.


Analysis of the site of action of the amdR product for regulation of the amdS gene of Aspergillus nidulans.

The amdR gene of Aspergillus nidulans regulates a number of structural genes in response to omega amino acid inducers. The site of action of the amdR product on expression of the amdS gene was investigated by studying the effects of changes in the 5' region of amdS, generated in vitro, on the induction, and on responses of an amdS-lacZ fusion gene to an amdRc allele. A sequence was identified that is sufficient for amdR regulation and that shows identity with sequences involved in amdR regulation of the gatA and lam genes. This sequence includes a CCAAT sequence and it was shown that this sequence is an important element in setting the basal level of amdS expression.


Suppression of the growth and export defects of an Escherichia coli secA(Ts) mutant by a gene cloned from Bacillus subtilis.

A gene library of Bacillus subtilis chromosomal DNA was screened for genes capable of reverting the growth defects of the Escherichia coli secA51(Ts) mutant at 42 degrees C. A B. subtilis gene, designated csaA, was found to phenotypically suppress not only the growth defects of the E. coli mutant, but also to relieve the detrimental accumulation of precursors of exported proteins. The csaA gene encoded a protein of 15 kDa (137 amino acids) and was likely to be the distalmost member of an operon. No similarity to csaA was found among DNA or protein sequences deposited in databases. In contrast to other homologous or heterologous suppressors of the E. coli secA51(Ts) mutation, the csaA gene did not exert pleiotropic effects on either the E. coli secY24(Ts) or lep9(Ts) mutations. However, it restored the ability of a SecB-deficient mutant to grow on complex medium. It is proposed that CsaA serves as a molecular chaperone for exported proteins or alternatively acts by stabilizing the SecA protein.


Effect of donor copy number on the rate of gene conversion in the yeast Saccharomyces cerevisiae.

Nonreciprocal recombination (gene conversion) between homologous sequences at nonhomologous locations in the genome occurs readily in the yeast Saccharomyces cerevisiae. In order to test whether the rate of gene conversion is dependent on the number of homologous copies available in the cell to act as donors of information, the level of conversion of a defined allele was measured in strains carrying plasmids containing homologous sequences. The level of recombination was elevated in a strain carrying multiple copies of the plasmid, compared with the same strain carrying a single copy of the homologous sequences either on a plasmid or integrated in the genome. Thus, the level of conversion is proportional to the number of copies of donor sequences present in the cell. We discuss these results within the framework of currently favoured models of recombination.


Adipocyte fatty acid-binding protein: regulation of gene expression in vivo and in vitro by an insulin-sensitizing agent.

Pioglitazone, a thiazolidinedione, is a novel antidiabetic compound that can lower blood glucose in diabetic rodents by increasing insulin sensitivity in target tissues. We have previously demonstrated that pioglitazone can enhance the insulin- or insulin-like growth factor-1-regulated differentiation of 3T3-L1 cells, a cell line that undergoes morphological and biochemical differentiation to mature adipocytes [Mol. Pharmacol. 41:393-398 (1992)]. In this study, we have examined the effect of pioglitazone on the expression of the adipocyte fatty acid-binding protein (aFABP) in ob/ob mice and 3T3-L1 cells. Administration of the drug to mice was observed to cause a dose-dependent increase in aFABP mRNA expression in epididymal fat, which was correlated with a decrease in blood glucose and insulin levels. Treatment of 3T3-L1 cells with pioglitazone enhanced aFABP expression in a time-dependent fashion. To explore a possible direct effect of pioglitazone on aFABP expression, a chimeric gene was constructed containing the aFABP promoter fused upstream of the bacterial reporter gene for chloramphenicol acetyltransferase. After transfection into 3T3-L1 cells and selection of stable transformants, regulation of the chimeric gene was studied. Pioglitazone, in combination with insulin or insulin-like growth factor-1, was observed to elicit a dose-dependent increase in expression, indicating a role for pioglitazone in regulating transcription of the aFABP gene. Several thiazolidinedione analogs were tested for their ability to induce the expression of the chimeric gene, and it was found that activity in this assay paralleled the structure-activity relationships observed for enhancement of 3T3-L1 cell differentiation. These observations on control of aFABP gene expression by pioglitazone suggest possible mechanisms by which cellular sensitivity to insulin may be regulated.


Cloning and expression of a high affinity taurine transporter from rat brain.

A cDNA clone (designated rB16a) encoding a taurine transporter has been isolated from rat brain, and its functional properties have been examined in mammalian cells. The nucleotide sequence of the clone predicts a 621-amino acid protein with significant homology to other neurotransmitter transporters. Hydropathy analysis reveals stretches of hydrophobic amino acids indicative of 12 transmembrane domains, similar to those proposed for other cloned neurotransmitter transporters. The transporter displays high affinity for taurine (Km approximately 40 microM) and is dependent on external sodium and chloride for transport activity. Specific transport is sensitive to inhibition by beta-alanine and gamma-aminobutyric acid, similar to taurine transporters in vivo. Localization studies demonstrate that the transporter mRNA is located in both the brain and peripheral tissues. The structural similarity of the taurine transporter to neurotransmitter transporters is consistent with a neuromodulatory role for taurine in the nervous system.


Equilibrium and kinetic studies of the interactions of salmeterol with membrane bilayers.

The interaction of salmeterol with model membranes has been studied with regard to equilibrium and kinetic behavior, including determination of the membrane-based partition coefficient, the rate of dissociation of salmeterol from membranes, and the rate of association. These data were obtained in various membrane preparations and under various conditions (e.g., temperature, cholesterol content). The compound is very lipophilic, compared with other beta 2 agonists such as salbutamol, and has a rapid association rate and a moderate dissociation rate. The equilibrium data support the assertion that the salmeterol action measured in perfused tissue involves an exo-site for nonspecific binding that may be identified with or related to the lipid bilayer. The kinetic data in unilamellar and multilamellar liposomes of synthetic lipids further suggest that the approach to the exo-site and the active site may involve components in the native system other than the lipid bilayer in which the beta 2 receptor is located. These additional components may explain the slow onset and the extraordinarily long duration of action.


Mono- and diglucuronide formation from chrysene and benzo(a)pyrene phenols by 3-methylcholanthrene-inducible phenol UDP-glucuronosyltransferase (UGT1A1).

Mono- and diphenols of chrysene and benzo(a)pyrene are suspected substrates of a 3-methylcholanthrene (MC)-inducible phenol UDP-glucuronosyltransferase (UGT1A1). Mono- and diglucuronide formation from these compounds was studied in two systems, (a) livers of MC-treated rats (homologous expression) and (b) a Chinese hamster lung fibroblast cell line (V79) containing rat UGT1A1 cDNA and stably expressing this isozyme (heterologous expression). In liver microsomes of MC-treated rats, glucuronidation of 6-hydroxychrysene was stimulated 11-fold by MC treatment. With 3,6-dihydroxychrysene, formation of its 3-hydroxy-6-monoglucuronide and of the diglucuronide was increased 24- and 310-fold, respectively. Induction factors obtained for monoglucuronide formation (but not for diglucuronide formation) were in line with published data on the increase of immunodetectable UGT1A1 protein and of its mRNA. It is suggested that the high induction factors for diglucuronide formation are the result of a combination of the induction of UGT1A1 and facilitated interaction of neighboring UGT1A1 molecules in endoplasmic reticulum membranes. Glucuronidation of 6-hydroxychrysene, 3-hydroxybenzo(a)pyrene, and 3,6-dihydroxybenzo(a)pyrene to their mono- and diglucuronides was clearly detectable in V79 cells expressing UGT1A1. However, conjugation of 3,6-dihydroxychrysene to its monoglucuronides was low and diglucuronide formation was not detectable, suggesting that UGT isozymes other than UGT1A1 are responsible for these reactions.


Identification of receptor domains that modify ligand binding to 5-hydroxytryptamine2 and 5-hydroxytryptamine1c serotonin receptors.

Serotonin [5-hydroxytryptamine (5-HT)] receptors are distinguished pharmacologically by their characteristic affinities for agonists and antagonists. Two serotonin receptors, the 5-HT2 and 5-HT1c, share a number of pharmacologic and structural properties while differing in their affinities for certain agonists and antagonists. To identify regions of the 5-HT2 and 5-HT1c receptors important for specifying their unique pharmacology, we constructed six chimeric 5-HT2/5-HT1c receptors in which domains of each receptor were exchanged. The abilities of several drugs to inhibit [3H]mesulergine bound to the chimeric and parent receptors transiently expressed in COS-7 cells were then examined. For spiperone and haloperidol (both butyrophenones), chimeras that exchanged transmembrane (TM) domains I and II or TMs I-III had the greatest effects on binding affinity. The binding affinity of cinanserin (a cinnamanilide) was significantly changed in all the chimeras studied. In contrast, the binding of ketanserin (a 4-fluorobenzoylpiperidine) was strongly influenced by chimeras that exchanged TMs I-III (but not I and II) and by chimeras that exchanged intracellular loop 3 to TM VII. 5-HT binding affinity was greatly altered for chimeras that exchanged domains of intracellular loop 3 to TM VII, with minor effects being noted for chimeras that exchanged TMs I and II and I-III. The affinities of the nonselective drugs mesulergine, mianserin, and m-chlorophenylpiperazine were relatively unaffected when domains of the 5-HT2 and 5-HT1c receptors were exchanged. Taken together, these results imply that structurally diverse 5-HT2 antagonists utilize distinct regions of the 5-HT2 receptor for high affinity binding.


Two allosteric modulators interact at a common site on cardiac muscarinic receptors.

The abilities of gallamine, obidoxime, tetrahydroaminoacridine (THA), and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) to alter the rate of dissociation of N-[3H]methylscopolamine from rat cardiac muscarinic receptors were investigated. All four ligands monotonically slowed the dissociation, with the order of potency gallamine > TMB-8 > THA > obidoxime. There was a dramatic difference in the efficacy of these allosteric modulators. Gallamine, TMB-8, and THA slowed the dissociation of N-methylscopolamine by > 90% at maximally effective concentrations, whereas obidoxime was capable of slowing it by only about 50%. In a manner analogous to the action of a partial agonist, obidoxime was able to partially reverse the effects of the other three modulators. Furthermore, the concentration-dependent effects of combinations of obidoxime and gallamine were in good agreement with the model of competitive interaction between these two ligands. These results provide the first evidence that two muscarinic allosteric modulators interact competitively at a well defined site.


Chronic lithium treatment inhibits basal and agonist-stimulated responses in rat cerebral cortex and GH3 pituitary cells.

Li+ is used clinically in the management of bipolar-disordered (manic-depressive) illness, but the mechanism of its clinical efficacy remains unclear. Li+ inhibits the metabolism of certain inositol phosphates, leading to a decreased cycling of inositol that may be sufficient to reduce phosphoinositide metabolism. We have tested this hypothesis in slices of rat cerebral cortex and in rat pituitary GH3 cells grown in the presence of low extracellular inositol. We show that basal and stimulated mass levels of inositol-1,4,5-trisphosphate were reduced in rat cerebral cortex and in GH3 cells after chronic, but not acute, treatment with a therapeutic concentration of Li+. In GH3 cells chronic treatment with Li+ also decreased basal levels of intracellular Ca2+ and secretion of prolactin, effects that were prevented by the presence of myo-inositol. Agonist-stimulated mobilization of Ca2+ and prolactin release were also reduced in Li(+)-treated cells. These findings show that chronic perturbation of the phosphoinositide pathway by Li+ is sufficient to reduce basal and agonist-stimulated cellular responses, an action that may underlie its effectiveness in the alleviation of affective disorders.


Effect of extracellular pH on the potency of N-methyl-D-aspartic acid receptor competitive antagonists.

Structure-activity analysis reveals that acidic alpha-amino acids containing an omega-PO3H2 group are more potent antagonists at N-methyl-D-aspartate (NMDA) receptors than are analogs with omega-COOH or omega-tetrazole groups. At physiological values of extra-cellular pH the omega-PO3H2 group is only partially deprotonated and the corresponding antagonists exist as ions with one or two negative charges. In contrast, competitive antagonists with omega-COOH and omega-tetrazole groups are fully ionized at physiological pH but carry only a single negative charge. Dose-inhibition analysis was performed with (2R)-AP7 and its piperidine derivative LY 257883 to determine whether ionization of the omega-PO3H2 group influences NMDA receptor antagonist potency; these experiments revealed a > 3-fold increase in potency on raising of the extracellular pH from 7.3 to pH 8.2, consistent with the increase in the relative concentration of the ionic form of the antagonist in which the omega-PO3H2 group contains two negative charges. Experiments with the omega-COOH-containing analog of LY 257883 and with SDZ EAB 515, an omega-PO3H2-containing antagonist of novel structure, revealed only 1.5- and 1.3-fold increases in potency, respectively, over the same pH range. Analysis of the kinetics of block of NMDA-activated currents resulting from rapid application of LY 257883 suggests that the increase in potency on raising of the extracellular pH results largely from an increase in the antagonist association rate constant but also from a small decrease in the dissociation rate constant. Together, these results suggest that the fully ionized forms of the R-enantiomers of AP7 and LY 257883 act as the active antagonist species at NMDA receptors.


Determinants of antifolate cytotoxicity: folylpolyglutamate synthetase activity during cellular proliferation and development.

Previous studies have documented the metabolism of a broad range of folate antimetabolites to polyglutamate derivatives by the enzyme folylpoly-gamma-glutamate synthetase (FPGS). The activity of the more recently developed classes of antifolates directed against thymidylate synthase and de novo purine synthesis is sufficiently dependent on polyglutamation that these compounds should be specifically cytotoxic to any normal or malignant proliferating cell expressing this enzyme. We have studied the patterns of expression of FPGS in mammalian cells and tissues during rapid growth, growth arrest, differentiation, and embryonic development. During embryogenesis in the rat, FPGS levels in liver and brain were higher during the period of proliferative activity and then dropped to a level characteristic of the adult organs. However, the levels in liver were substantially higher than those in brain at any given time. This pattern was mimicked in mouse C3H 10T1/2 embryo fibroblast cells, in which FPGS activity decreased after cessation of growth but then remained at a lower steady state level during an extended period of postconfluent culture. Enzyme activity also dropped after the differentiation of human HL-60 promyelocytic leukemia cells. In a human homolog of these experimental systems, FPGS levels were below the limits of detection in circulating mature human hematopoietic cells of the granulocytic, lymphoblastic, and erythrocytic lineages. In striking contrast, substantial levels of FPGS were found in circulating lymphoblasts from eight patients with acute lymphoblastic leukemia. The levels of FPGS found in these transformed stem cells would help to explain the sensitivity of many acute lymphoblastic leukemias to folate antimetabolites. We concluded that expression of FPGS is regulated by at least two mechanisms, one of which is linked to proliferation and the other of which controls enzyme levels after differentiation and is tissue specific.


Regiochemical differences in cytochrome P450 isozymes responsible for the oxidation of methylenedioxyphenyl groups by rabbit liver.

The cytochrome P450 isozymes catalyzing the oxidation of the methylenedioxyphenyl compounds methylenedioxybenzene (MDB) and methylenedioxyamphetamine (MDA) have been investigated in rabbit liver preparations. The aromatic ring in MDB undergoes both demethylenation to catechol and aromatic hydroxylation to sesamol, whereas that in MDA undergoes only demethylenation to dihydroxyamphetamine. Formation of catechol and sesamol from MDB in microsomal incubation mixtures was enhanced about 5- and 3-fold, respectively, by pretreatment of the rabbits with phenobarbital, which induced CYP2B4 and CYP4B1. The cytochrome P450 isozyme responsible for aromatic hydroxylation of MDB was induced by beta-naphthoflavone and was inhibited by alpha-naphthoflavone. Microsomal demethylenation of MDA was minimally sensitive to pretreatment of the rabbits with phenobarbital, beta-naphthoflavone, pyrazole, or rifampicin. However, MDA competitively inhibited the N-demethylation of erythromycin. Antibodies against CYP2B4, but not those against CYP4B1, caused a marked inhibition of the demethylenation and aromatic hydroxylation of MDB. Antibodies against CYP2C3 did not inhibit the demethylenation of MDA, nor did substrates or inhibitors of the CYP2D family except for bufuralol. MDB and MDA were both capable of forming metabolic intermediate complexes, and the rate of complex formation was accelerated by phenobarbital induction. Reconstitution experiments with CYP2B4 suggested that phenobarbital-inducible complex formation from MDA was not due to the carbene pathway involving the methylenedioxy group but was due to oxidation of the amino group. These results indicate that CYP2B4 oxidizes different regions of methylenedioxyphenyl compounds depending on their structure. MDB undergoes oxidation at the methylenedioxy group (major) and the benzene ring (minor). MDA is oxidized at the alkylamino side chain at the nitrogen and alpha-carbon. The results suggested that one or more constitutive isoforms (probably unknown) of cytochrome P450 present in rabbit liver microsomes are primarily responsible for MDA demethylenation but that CYP3A6 contributes slightly.


Cellular pool of transient ferric iron, chelatable by deferoxamine and distinct from ferritin, that is involved in oxidative cell injury.

A cellular pool of transient ferric iron that is chelatable by deferoxamine, distinct from ferritin, and required for oxidative cell injury has been identified in cultured rat hepatocytes labeled with 59FeCl3. Pretreatment of hepatocytes with deferoxamine depleted the cellular pool of chelatable iron and protected the cells from an oxidative injury. Incubation of deferoxamine-pretreated hepatocytes in serum-free medium restored both the chelatable iron pool and the susceptibility to oxidative injury. Furthermore, inhibition of protein degradation with chymostatin prevented the restoration of both the chelatable pool and susceptibility to oxidative injury. The deferoxamine-chelatable iron pool was distinguished kinetically and immunochemically from the larger cellular pool of ferritin iron. The labeled iron in the deferoxamine-chelatable pool was transient, unlike either the total cellular uptake of 59Fe or its incorporation into ferritin, both of which increased with time of labeling. With pulse-chase labeling, the percentage of the total uptake of 59Fe that was represented by the deferoxamine-chelatable pool decreased. At the same time, the percentage represented by radioactivity immunoprecipitable as ferritin increased. Furthermore, immunoprecipitation of ferritin from the labeled lysates enriched the resulting immunosupernatants in deferoxamine-chelatable iron. The degree of enrichment for chelatable iron correlated with the percentage of the cellular label that was immunoprecipitable as ferritin. The deferoxamine-chelatable iron appears to represent a metabolically common pool of iron that is rapidly in transit through the cell. Extracellular iron entering the pool can be utilized for heme synthesis or stored in ferritin, whereas protein degradation releases storage iron into this pool.


Hypersensitivity to acetaldehyde-protein adducts.

Acetaldehyde, the first product in the metabolism of ethanol, is known to condense with plasma proteins, forming stable adducts. We have previously shown that these adducts can be recognized as foreign by the immune system. In the present study the existence of type I hypersensitivity-mediating antibodies against these adducts was investigated in humans and in animals. Immunization of mice with acetaldehyde-protein condensates, followed by adoptive transfer of splenocytes, led to the production of IgE anti-acetaldehyde adducts. A monoclonal IgE antibody was obtained by the hybridization technique. This antibody recognized acetaldehyde adducts, independently of the carrier protein used, indicating that the acetaldehyde moiety behaves as a hapten. The affinity of the antibody for the acetaldehyde adduct of polylysine was 7 orders of magnitude higher than that for polylysine. Passive immunization by intradermal or intravenous administration of this monoclonal antibody to rats rendered the animals hypersensitive to acetaldehyde-protein conjugates, as shown by marked anaphylaxis. A study was conducted to determine the existence of naturally occurring hypersensitivity reactions to alcohol in > 1000 non-Oriental individuals. A prevalence of severe hypersensitivity reactions of 0.46% was found. The reactions were severe enough to deter these individuals from consuming all types of alcoholic beverages. Individuals presenting such reactions had significantly elevated levels of circulating anti-acetaldehyde-protein IgE antibodies.


Correlations between the rate constant of singlet oxygen quenching by imidazole derivatives and anti-inflammatory activity in rats.

The second-order rate constants, k delta, for quenching of molecular singlet oxygen O2 (1 delta g) by nonsteroidal anti-inflammatory imidazole drugs have been determined using time-resolved phosphorescence detection of singlet oxygen. A linear correlation was observed between log k delta (ranging from 7.90 to 8.50) and the anti-inflammatory activity of these compounds (ranging from ED50 = 15 to 300 mg/kg), as measured in rats by Jørgensen and Dyrsting [United States Patent 4,424,229 (1984)]. The correlation between this physico-chemical parameter measured in vitro and a biological activity measured in vivo might be useful in screening other types of candidate anti-inflammatory drugs. The rate constant (k delta) can be considered as a quantitative expression of the electron-donating power of the imidazole drug, as suggested by a correlation of log k deta (ranging from 6.02 to 7.45) with Hammett substituent parameters observed in the case of 2-substituted imidazoles.


Epidermal growth factor activates phospholipase C in rat hepatocytes via a different mechanism from that in A431 or rat1hER cells.

Epidermal growth factor (EGF) can stimulate inositol lipid hydrolysis in rat hepatocytes and can accelerate GTP/GDP exchange in hepatic membranes. Both of these responses can be abolished by pretreatment with pertussis toxin, suggesting that EGF may regulate phospholipase C (PLC) activity via a guanine nucleotide-binding regulatory protein (G protein) in liver cells. In contrast, in A431 human epidermoid carcinoma cells EGF can induce a rapid phosphorylation of PLC-gamma on tyrosine residues that increases the activity of immunoprecipitated PLC-gamma, suggesting that tyrosine phosphorylation of PLC-gamma may be the mechanism for EGF-stimulated inositol trisphosphate production in these cells. To determine the importance of the phosphorylation of PLC-gamma on tyrosine residues in a system where the EGF receptor apparently couples to a G protein, the effect of EGF on tyrosine phosphorylation of PLC-gamma was examined in rat hepatocytes. PLC-gamma was immunoprecipitated from cell lysates with a PLC-gamma antiserum and its tyrosine phosphorylation state was determined using both Western blot analysis with phosphotyrosine antibodies and direct measurement of phosphorylated amino acids. The results were compared with analogous experiments performed with A431 cells and another cultured cell line expressing high levels of human EGF receptors, Rat1hER fibroblasts. Although the amount of PLC-gamma in rat hepatocytes is similar to that in A431 cells and slightly higher than that in Rat1hER cells, EGF causes a barely detectable increase in the phosphorylation of PLC-gamma on tyrosine in hepatocytes, whereas it stimulates a significant degree of phosphorylation of PLC-gamma on tyrosine in Rat1hER or A431 cells. Pretreatment of hepatocytes with pertussis toxin abolishes the ability of EGF to activate PLC, as determined by an increase in intracellular Ca2+, but has no effect on the small amount of phosphate incorporated into tyrosine residues on the PLC-gamma protein, demonstrating that this low level of PLC-gamma phosphorylation does not correlate with changes in PLC activity. The data suggest that phosphorylation of PLC-gamma on tyrosine is not important for EGF-enhanced PLC activity in hepatocytes. This conclusion implies that EGF may use a mechanism to regulate PLC activity in hepatocytes that is different from that used in cultured cells expressing high levels of EGF receptors.