[Birth anomalies of coronary arteries. Responsibility in myocardial ischemia].

Anomalous origin of the left or right coronary artery from the contralateral sinus of Valsalva with coursing between the aorta and the pulmonary trunk way cause angina, myocardial infarction or sudden death. This anomaly should be suspected especially when ischemic symptoms occur in young patients without risk factor for atherosclerosis. We believe that surgical operation after demonstration of myocardial ischemia is indicated to prevent severe myocardial ischemia or sudden death.


[Aging of the arterial system].

The high-pressure systemic arterial network plays a major physiological role as it buffers the impact of the blood flow wave generated by the left ventricle and transforms it into a more continuous flow in the peripheral arterioles. In ageing processes unrelated to hypertensive or vascular disease the structural properties of the arterial network are significantly altered. The collagen, elastin and smooth muscle contents of the tunica media and the geometrical arrangement of arterial wall components are constantly modified according to the subject's age. These morphometric changes are responsible for changes in the mechanical properties of the arterial walls leading to rigidity. An increase in caliber of the arterial lumen has been found in all studies; it partially compensates for the effects of arterial wall rigidity on vascular compliance, thereby limiting the haemodynamic and functional changes that occur in blood circulation.


[Effects of aging on arterial function in man].

The structure of arteries changes with age. Gradual disappearance of elastic fibres facilitates dilatation of the arterial lumen and increase in length of the vessels. Simultaneously, the arterial wall becomes thicker, but it is often difficult to distinguish the changes due to ageing from those caused by atherosclerosis. These structural alterations result in a greater rigidity of the arteries, which can now be evaluated by non-invasive segmental methods. This shows that ageing does not have the same functional effects on wide-caliber elastic arteries and on middle-sized muscular arteries. The increase of systolic arterial pressure observed in the elderly is a consequence of structural and functional changes in the arterial system.


[Aging of heart function in animals].

Most of the laboratory animals studied for cardiovascular ageing are rodents. Anatomical studies have demonstrated an increase in heart weight and volume, hypovascularization of the myocardium and, more recently, hypertrophy of myocytes. Studies on the mechanical properties of rat papillary muscle have shown that ageing is associated with reduction of myocardial distensibility, whereas the habitual parameters of contractility (Tmax and Vmax) are unchanged in terms of baseline values. On the other hand, mechanical responses to stimulation of beta-adrenoceptors are reduced. The essential change in myocardial contraction in ageing rats is its chronology: ageing is attended by a longer duration of both contraction and relaxation. Biochemical studies of old rats' myocardium have yielded a fundamental information: in this species ageing is accompanied by a change in synthesized myosin subtype. This change is identical to that observed in experimental systolic overload; it is also adaptative, as it enables oxygen consumption to be reduced during a constant work, and it explains the modifications observed in the chronology of a contraction. Studies on the "pump" function of old rats' myocardium have confirmed the appearance with age of another adaptative change: isolated hearts of old animals have a greater output in a rigid circuit while the hearts of adult rats have their output in a more compliant circuit. Altogether, experimental studies on cardiac ageing lead to the conclusion that the changes observed are similar to those due to systolic overload, the origin of which is a loss of arterial compliance. These changes are adaptative in that they reduce oxygen consumption by a myocardium with systolic overload, but the resulting changes in contraction chronology are such that the aged heart cannot deviate from a mean rate without notably widening the differential blood pressure. Thus, experimental studies of myocardial ageing have presented ageing as a contradiction between ideal adaptation to a chronic stress and reduced possibilities of adaptation to acute aggressions.


[Functional coupling of the left ventriculo-arterial system and aging in man].

In a haemodynamic and angiographic study at rest conducted in 25 men aged from 22 to 68 years who had no more than another risk factor for arterial disease and showed no evidence of cardiac or arterial disease several parameters were measured or calculated. Firstly, a global index of arterial system function (Ea) and its various factors: Ea = ESP/SV [ESP: left ventricular end-systolic pressure; SV: systolic volume]; Ea = (HR x TSR)+Ea' [HR: heart rate/min; TSR: total systemic resistance] where Ea' = (ESP - AoP/SV) [AoP: mean aortic pressure]. Secondly, the parameters concerning the left ventricle were the mass (m) and the m/EDV ratio [EDV: end-diastolic volume] and indices of the left ventricular systolic and diastolic functions, such as ELV = ESP/ESV [ESV: end-systolic volume], kp: the volumic distensibility module of the left ventricular chamber; relations EF - o ES [EF: ejection fraction; o ES: end-systolic constraint], and kp - m/EDV as indice of left ventricular muscle distensibility. In parallel with the subjects' age, Ea increased by joint augmentation of TSR and Ea' while m, m/EDV, ELV and kp also significantly increased. The inotropic quality of the left ventricular muscle and its intrinsic distensibility were found to be decreased in a few subjects aged over 45. Ea/ELV (reverse of ejection fraction -1) tended to increase (ELV relatively less than Ea), but this increase was not significant (P = 0.10). These results show that in the ageing man the improvement observed in the ejection fraction of the left ventricular pump corresponds roughly to the degradation of the arterial system transfer function, and the arterial system-left ventricle coupling, evaluated by the Ea/ELV ratio, is maintained (better in fact than in arterial hypertension and heart failure). This improvement is achieved by increases of m and, chiefly, m/EDV which compensate for both the increase of Ea and the relative decline of left ventricular muscle contractile quality. There is a disorder of the left ventricular pump diastolic function which is due to geometric changes in the chamber and to changes in the intrinsic distensibility of the left ventricular muscle.


[Cerebral blood flow. Changes in regulation with aging].

In subjects without cardiovascular or neuronal diseases ageing does not seem to be accompanied by a significant fall in global cerebral blood flow at rest. Yet non-negligible changes in cerebral blood flow rate can be demonstrated by haemodynamic or metabolic stimuli. Several intra- and extracranial vascular mechanisms may be involved in these changes. During ageing, a decrease of carotid artery compliance due to parietal calcium deposition might be an important factor in the disturbance of cerebral blood flow regulatory mechanisms.


[Cerebral and cardiovascular aging and brain energy metabolism. Studies with positron-emission tomography in man].

Positron emission tomography (PET) is the only method that measures concomitantly in the same cerebral region the main haemodynamic (blood flow, blood volume) and metabolic (oxygen and glucose consumption) parameters, thereby providing essential data concerning the supply of substrates by circulation and their principally neuronal/synaptic use by the cells. In "healthy" (i.e. normal) ageing, there is a progressive decrease of oxygen and glucose consumption--with subsequent reduction of cerebral blood flow (CBF)--which predominates in the neocortex and suggests a dysfunction of neuronal circuits perhaps responsible for some age-related changes in cognitive functions. The metabolic depression in the neocortex seems to be accentuated during the more "common" ageing which may include cerebrovascular risk factors, lesions of the white matter in varying numbers and/or degradation of cognitive functions. Strategically located brain lesions of vascular origin (e.g. damaging of the thalamic nuclei), may produce a thalamo-cortical disconnection which induces a diffuse cortical hypometabolism associated with severe cognitive disorders. In degenerative dementias (particularly Alzheimer's disease), which include cortical and subcortical neuronal lesions (e.g. those involving the cholinergic system), there is a hypometabolism preferentially affecting the associative cortex. The cumulative effects on cortical functioning of multiple vascular lesions, superimposed on degenerative neuron loss, might account for age-related cognitive disorders in "normal" ageing as well as in the so-called "vascular" or "mixed" dementias.


[Normal and pathological renal aging in animals].

Ageing of the kidneys has long been associated with a fall in the number of functioning nephrons resulting in a reduction of renal blood flow and glomerular filtration. This narrow concept of age-related changes in renal function has been developed chiefly during the last few years by Brenner et al. on the basis of experimental studies conducted on rodents. According to these authors, the size and frequency of segmental and focal lesions of glomerulosclerosis increase regularly with age, and in its final phase this pathology results in occlusion of glomerular capillaries. Renal ageing, therefore, can be assimilated to the nephron reduction models obtained by surgical ablation. The hypothesis that hypofiltration in certain nephrons is compensated by hyperfiltration in healthy glomerulis, leading to a vicious circle of self-destruction, was then applied to both ageing and experimental renal impairment: the smaller the number of nephrons, the greater the filtration achieved by the remaining nephrons, a process that accelerates the probability of their destruction. Conversely, any attempt to reduce intracapillary pressure or glomerular filtration slows down the progression of renal failure. This hypothesis is supported by experiments showing that reduction of protein intake or chronic inhibition of angiotensin I-converting enzyme activity are truly capable of limiting the progression of glomerulosclerosis induced in rats by partial renal mass ablation. Similarly, prolonged food restriction increases the life expectancy of rodents and almost totally prevents the occurrence of glomerulosclerosis. The experimental finding that degenerative renal lesions do not necessarily develop with age raises the problem of normal and pathological ageing. With an adequate choice of rats' food, strain and sanitary surroundings it is possible to obtain very old animals devoid of occluded glomerular capillaries and loss of nephron. What about the functional and structural changes due to ageing and not to pathology? This question has given rise to numerous studies which concluded, on the whole, that there exists a normal ageing of the kidneys without loss of nephron and that ageing is expressed by the fact that the kidneys have difficulties in adjusting themselves to disturbances in the inner environment. As regards renal functional reserve, response to the antidiuretic hormone in case of water restriction, or stimulation of the renin-angiotensin system in response to decrease of sodium intake, it is clear that the renal cells responsible for glomerular filtration, tubular transport or synthesis and release of peptidic hormones exhibit functional alterations that are age-related. The cellular and molecular mechanisms underlying these physiological changes are little known.(ABSTRACT TRUNCATED AT 400 WORDS)


[Renal aging in man].

Renal morphological and functional changes occur in the elderly. Between the ages of 50 and 80 years the renal mass diminishes by a mean of 20 percent, to the expense of the renal cortex. Glomerular sclerosis results in a gradual fall in the number of functioning glomeruli. Simultaneously, a decrease of about 7 percent in glomerular filtration rate per decade after the age of 40 years and a 10 percent decrease in renal plasma flow per decade, and therefore an increase in filtration fraction, are observed. These haemodynamic abnormalities are accompanied by changes in tubular function, including a delay in renal adjustment to sodium overload, and above all to sodium restriction, together with a decrease in concentrating and, to a lesser extent, diluting capacity. In actual fact, the intensity of glomerular sclerosis varies considerably from one subject to another, and the decrease in glomerular filtration rate is far from being constant. These changes are perhaps not ineluctably associated with aging; they might result from pathological processes that have gone unnoticed.


[Cardiovascular pathology related to aging. Role of prevention].

During the last 50 years the increase of life expectancy has been dramatic in all Western countries. In France, the life expectancy in 1988 was 80.6 years for women and 72.3 years for men. In 1980, the life expectancy at the age of 60 was more than 20 years in women and 16 years in men. Side by side with this increase of life expectancy there has been a change in the structure of the causes of death. Cardiovascular diseases accounted for 15.6 percent of all causes in 1930, 31 percent in 1950 and 37.4 percent in 1980. Cardiovascular diseases therefore rank very high among the causes of mortality, and one of the principal reasons for this is ageing of the population. Studying the epidemiology of cardiovascular ageing is tantamount to determining the part played by prevention in these diseases. There are wide fluctuations in cardiovascular mortality rate according to some demographic variables: men are more affected than women, the poorer subjects more than the richer subjects, and people living in the North of France more than those living in the South. These disparities are real, irrespective of age, and they can be explained, at least partly, by differences in the prevalence of well-known risk factors. Cardiovascular ageing plays a role, probably important, in the genesis of physical and mental disabilities, but markers that would measure them precisely are lacking. The development of such indicators is a major line of research which should make it possible to evaluate the prospective results of a policy of prevention in a segment of the population where people aged 60 or more accounted for 19.1 percent in 1990 and will account for 25 percent in the year 2020.


[Prevention of pathological aging. The arterial hypertension example].

Studies conducted during the last decade have made it increasingly easier to distinguish between normal and pathological ageing. Previous studies on general populations had established a dogma: most of the major body functions, such as cardiac output, glomerular clearance, muscular strength, visual acuity and so forth, were supposed to decline with age. However, in recent studies on the effects of age on cardiac output all subjects with heart disease were excluded and as a result the negative correlation found between cardiac output and age disappeared, which shows that a phenomenon attributed to ageing was in fact caused by disease. This does not mean that there is no such thing as fundamental ageing, and indeed the mechanisms that maintain cardiac output are different in young and elderly subjects. These data are of more than theoretical interest, since it may be presumed that prevention, the instruments of which are already available in some fields, will modify the profile of ageing. The risk of arterial hypertension is not restricted to the classical cardiovascular diseases: it extends to other diseases, such as vascular dementia, which are potent factors of pathological ageing. Most of the controlled trials carried out in arterial hypertension have demonstrated that treating certain types of hypertension significantly reduces the morbidity and/or mortality of cardiovascular diseases. The specific properties of new antihypertensive drugs, such as angiotensin-converting enzyme inhibitors, open stimulating vistas on arterial ageing.


Breast and cervical cancer screening in older women: the San Diego Medicare Preventive Health Project.

BACKGROUND. This study presents rates and correlates of cancer screening, including mammography, clinical breast exam, breast self-examination, and Pap tests, in older women. Age was the predictor of main interest. RESULTS. Among a sample of female Medicare beneficiaries ages 65 and older, who were members of a health maintenance organization, there was a significant, inverse relationship between age and mammography adherence, while controlling for health status, physician utilization, education, and income. The proportions of subjects ages 65-74 and 75+ never having had a mammogram were 17 and 32%, respectively, and the proportions having had annual mammograms were 40 and 28%, respectively. Pap test frequency showed a trend toward an inverse relationship with age, controlling for demographic and health-related variables. In the youngest age group (65-69), 52% had annual Pap tests, whereas in the oldest group (80+), only 36% had annual Pap tests. Age did not predict frequency of clinical breast exam, and the relationship of age to breast self-examination was inconclusive. The role of the physician in promoting cancer screening is discussed.


Breast cancer screening attitudes and behaviors of rural and urban women.

This study was carried out to assess the breast cancer knowledge, attitudes, and awareness of women age 40 to 74 in Alberta, a Canadian province of 2.4 million people. This analysis compares the attributes of 538 rural women, defined as those living between 1 and 3 hr drive from the major cities in Alberta, and 735 urban women who lived in one of these two cities. Rural women were found to have the same basic knowledge of breast cancer or perceptions of barriers to mammography, but had more negative attitudes about breast cancer itself. Despite their similar access to physician care, they were less likely to have had a recent clinical breast examination or mammogram (P less than 0.001). These differences remained when adjustment was made for demographic background variables; the adjusted prevalence rate ratio for a screening mammogram in the past 2 years was 0.52 (95% C.I., 0.43, 0.64), and for intention to have a mammogram in the next 2 years, 0.75 (0.63, 0.90). The results suggest particular program delivery strategies when planning for provision of breast screening information and service to the large subgroup of rural women.


Impact of a clinical preventive medicine curriculum for primary care faculty: results of a dissemination model.

This study was designed to test a dissemination model for providing clinical preventive medicine (CPM) training to general internal medicine faculty across the United States. The model incorporated direct instruction of a few faculty as seminar facilitators who, in turn, taught a CPM curriculum to their faculty colleagues, who then could teach it to housestaff and students. The CPM curriculum consisted of six seminars that focused primarily on the risk factors for chronic diseases and on behavior change methods for modifying smoking, diet, and exercise. Faculty who participated in the seminars had significant pre- to post-test increase in knowledge and reported self-efficacy to implement CPM strategies with patients, as well as changes in CPM clinical practices. These faculty, in turn, successfully disseminated CPM information to their housestaff, who also had increases in self-efficacy and changed clinical practices regarding CPM topics. The successful implementation of the dissemination model attests to its viability as a mechanism for disseminating CPM curricula and increasing the emphasis on CMP issues in both clinical teaching and clinical encounters with patients.


A comparison of knowledge of medical students and practicing primary care physicians about cardiovascular risk assessment and intervention.

Heart attack and stroke are still prevalent causes of death and disability in the U.S. adult population (1, 2). Studies (3-9) have shown that modification of hypertension, smoking, and hypercholesterolemia can reduce risks for atherosclerosis and subsequent cardiovascular events. Therefore, it is important that physicians be skilled in assessing and modifying patients' overall cardiovascular risk. This study compares acquired knowledge of second-year medical students about cardiovascular risk assessment with knowledge in a selected group of practicing primary care physicians, who are members of the medical school's clinical faculty, using a new experimental testing technique called the tailored response test (TRT). Students performed a structured cardiovascular risk intervention on a patient in primary care clinics. Their acquired knowledge was then tested using the TRT, which contained 43 discrete judgments about a clinical case. Test scores of students and faculty were compared. Both students and faculty demonstrated knowledge about the most important risk factors, appropriate screening tools, and interventions. However, the selected physicians did not demonstrate knowledge of certain important risk assessment and intervention recommendations, based on national standards. Only 38% of faculty and 27% of students were aware that a "fasting" serum cholesterol is not needed for screening, 30% of faculty believed that if cholesterol was over 300 they would "probably prescribe medicine" before other intervention strategies were tried, and 32% of faculty and 30% of students would order a screening chest X-ray, which is incorrect in the case history. The TRT, in contrast to self-report surveys, demonstrates that important cardiovascular risk assessment and intervention knowledge, with implications for cost effectiveness in health care delivery, has not penetrated to a selected group of physicians who are members of the medical school's clinical faculty and therefore serve as role models for medical students. This is disturbing, in light of current emphases on cost effectiveness in health care. Greater undergraduate curricula and CME emphasis on cardiovascular preventive practice is needed, such that almost 100% of students and faculty demonstrate knowledge, and practice, of preventive medicine according to national standards. In turn, groups developing national standards are enjoined to design and implement effective approaches for disseminating these recommendations.


Results from a statewide approach to adolescent tobacco use prevention.

The 1985 Minnesota Legislature established guidelines for school-based tobacco-use prevention programming and provided financial incentives to school districts to encourage them to adopt a broad range of preventive measures. The Minnesota-Wisconsin Adolescent Tobacco-Use Research Project was funded by the National Cancer Institute in 1986 to evaluate the Minnesota initiative through two parallel studies. The Four Group Comparison Study was a prospective study of 48 school "units" which were randomly assigned to one of four conditions in 1987. Baseline observations were taken in the sixth grade in 1987, interventions were delivered in the seventh grade, and follow-up observations were taken in the seventh, eighth, and ninth grades. The Four Group Comparison Study was designed to evaluate the three middle-school interventions that were most widely adopted by Minnesota school districts as a result of the 1985 legislation. The Two State Comparison Study was a serial cross-sectional study of representative districts in Minnesota and Wisconsin. Annual surveys of ninth graders were conducted from 1986-1990. The Two State Comparison Study was designed to determine whether tobacco-use patterns changed in Minnesota relative to Wisconsin following the Minnesota legislation. The prospective study indicated that none of the interventions was more effective in reducing adolescent tobacco use compared with a randomized control group. The serial cross-sectional study revealed that there was a modest net decline in Minnesota relative to Wisconsin from 1986 to 1990, but that it was within the range of chance variation. Taken together, these results indicate that this legislative initiative was insufficient to reduce adolescent tobacco use statewide during the 5-year study period. Together with results from other recent studies, they suggest that even more intensive efforts may be required to effect widespread reductions in adolescent tobacco use.


Calcium intake in youth: sex, age, and racial differences in NHANES II.

Data from the NHANES II survey were used to determine whether the calcium intake of children ages 3-18 met the minimum recommended daily allowance. In addition, the impact of age, race, sex, and socioeconomic status on calcium intake and the density of calcium intake was explored. The results indicated significant differences between groups of children based on age, sex, and race (P less than 0.05). Males across all age groups (3-5, 6-7, 8-10, 11-12, 13-18) had a higher calcium intake than females. In addition, calcium intake levels among males always met or exceeded the minimum RDA. Levels among all blacks started below the minimum RDA in the age group 3-5. During the adolescent years, calcium intake again fell below the RDA for black males and for females of both races, with the calcium intake of the adolescent females resembling that of the group ages 3-5. In a multiple regression model sex, age, race, and total energy intake predicted 49% (P less than 0.0001) of the variance in calcium intake. Socioeconomic status (using parents' reported income) was not a significant predictor. Another regression model was used to examine the relationship among age, race, sex, and socioeconomic status and its effect on the density of calcium intake (mg/kcal). While the overall model was significant (P less than 0.0001) it accounted for only 3.5% of the variance. Age, sex, and race of the child significantly predicted the density of calcium (P less than 0.05); however, parents' gross income did not (P less than 0.81), indicating that income level does not contribute to explaining these differences.


Elderly health beliefs, attitudes, and maintenance.

Why older persons engage in varying amounts of health maintenance activity is becoming both an increasingly important policy issue and a topic of interest to health services researchers. Such activity may help the elderly to delay the onset of the health-related problems associated with aging, maintain if not improve their functional abilities, and perhaps improve their quality of life. Using a conceptual model largely based upon the health belief model, this study sought to examine predictors of variability of health maintenance activity among older persons. The project included cross-sectional data drawn from the first phase of a multiyear panel study of elderly community residents. Results of ordinary least-squares and logistic regression analyses of seven types of health maintenance activity suggest that health beliefs are an important consideration but that other variables, namely, type of insurance plan and select sociodemographic factors, also had significant impacts. Another consistent finding was that each of the types of health maintenance activity was associated with different types of predictor variables. These findings suggest that in order for levels of health maintenance activity to be increased, intervention programs need to be targeted toward specific types of health beliefs and need to take into account the importance of social differences.


Environmental effects on chromosomes in oncology and radiology department personnel.

Long-term, low levels of antineoplastic drugs and ionizing radiation in the work environment may have genotoxic effects on chromosomes in personnel. The effects of ionizing radiation is acknowledged in occupational medicine and therefore various measures have been undertaken to prevent such effects. However, there are no protective measures for workers exposed to antineoplastic drugs. In this study metaphase chromosome preparations were analyzed in 126 nurses and radiology technicians. They were divided into three equal groups. The first group consisted of shift nurses in an oncology department handling antineoplastic drugs. The second group consisted of radiology technicians in a radiology department and the third group acted as controls. Structural chromosomal aberrations were increased among those nurses exposed to antineoplastic drugs in comparison with technicians (P less than 0.05) and controls (P less than 0.01), respectively.


Anticarcinogenic effects of (-)-epigallocatechin gallate.

Our research objective is to develop nontoxic cancer chemopreventive agents and to apply these agents in treating humans. We are identifying agents that inhibit the process of tumor promotion in two-stage carcinogenesis experiments on mouse skin. We review (a) the inhibitory effect of penta-O-galloyl-beta-D-glucose (5GG) on tumor promotion by teleocidin, one of the 12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters (5GG is structurally similar to (-)-epigallocatechin gallate (EGCG) and is isolated from hydrolyzed tannic acid); (b) the inhibitory effects of EGCG, the main constituent of Japanese green tea, on tumor promotion with two tumor promoters, teleocidin and okadaic acid, a non-TPA-type tumor promoter; (c) the mechanisms of action of EGCG, a single application of which reduced the specific binding of [3H]TPA and [3H]okadaic acid to a particulate fraction of mouse skin; and (d) the anticarcinogenic effects of EGCG on duodenal carcinogenesis induced by N-ethyl-N'-nitro-N-nitrosoguanidine in male C57BL/6 mice. EGCG is a nontoxic compound. We believe that the main constituent of Japanese green tea, EGCG, is a practical cancer chemopreventive agent available in everyday life.


Chemopreventive effects of green tea components on hepatic carcinogenesis.

Catechin components of green tea have been shown to possess anticarcinogenic properties possible related to their antioxidant activity. In the present study, a catechin containing green tea extract (GTE) was examined for its effect on three previously defined properties of liver tumor promoters, induction of cytolethality, inhibition of gap junctional intercellular communication, and induction of cell proliferation. Hepatocytes from male B6C3F1 mice were isolated and placed in primary culture. The effects of GTE of oxygen free radical-induced cytolethality was examined by coincubating GTE with the oxygen radical generating compounds paraquat, glucose oxidase (GO), and xanthine oxidase (XO). GTE prevented the induction of hepatocyte cytolethality by GO, XO, and paraquat in a dose-responsive manner. Similarly, GTE prevented the inhibition of gap junctional-mediated intercellular communication (measured by lucifer yellow dye coupling) by phenobarbital, lindane, and paraquat in a dose-dependent manner. The effect of GTE on hepatocyte DNA synthesis was examined in male mice containing preneoplastic liver lesions induced by diethylnitrosamine. GTE significantly decreased the labeling index in hepatic preneoplastic foci from animals treated with phenobarbital for 7 days. These studies suggest that the previous reported anticarcinogenic activity of green tea may be related to its effect on the tumor promotion stage of the cancer process.


Antioxidative effect of polyphenol extract prepared from various Chinese teas.

METHODS. Twelve different types of Chinese teas, including green, semifermented, and black tea, were studied for their antioxidant activities and active components. Compositions of (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, and gallic acid were identified by fast atom bombardment-mass spectrometry and high-performance liquid chromatography-mass spectrometry and quantified by high-performance liquid chromatography. Antioxidant activities in lard were measured by the Rancimat method. RESULTS. The results showed that both yields of polyphenol extract and antioxidant activities varied with different tea processing methods. It was found that (-)-epigallocatechin gallate, (-)-epigallocatechin, and (-)-epicatechin gallate inhibited soybean lipoxygenase at the IC50 values ranging from 10 to 20 microM.


Green tea consumption and serum lipid profiles: a cross-sectional study in northern Kyushu, Japan.

METHODS. The relation between green tea consumption and serum lipid concentrations was examined using cross-sectional data on 1,306 males who received the retirement health examination at the Self-Defense Forces Fukuoka Hospital between October 1986 and December 1988. RESULTS. After adjustment for rank, smoking, alcohol use, physical activity, and body mass index, serum total cholesterol levels were found to be inversely related to the consumption of green tea while no association was noted with serum triglycerides and high-density lipoprotein cholesterol. Adjusted mean concentrations of total cholesterol were 8 mg/dl lower in men drinking nine cups or more per day than in those consuming zero to two cups per day. Serum cholesterol levels were inversely associated with traditional Japanese dietary habits (intake of rice and soy bean paste soup) and positively associated with Westernized habits. Additional adjustment for these dietary variables did not alter the inverse relation between green tea and total cholesterol.


Association of serum lipoproteins and health-related habits with coffee and tea consumption in free-living subjects examined in the Israeli CORDIS Study.

A positive association between coffee consumption and serum cholesterol levels has been demonstrated and recent results indicate that this may be due to a lipid-rich fraction present mainly in boiled coffee. The possible effects of tea consumption are less clear, although evidence has been presented which suggests that tea drinkers have lower cholesterol levels. The associations between serum lipids and lipoproteins and coffee and tea consumption were examined in the Israel CORDIS study. Employees of 21 factories were screened for cardiovascular disease risk factors between 1985 and 1987. Detailed data on coffee and tea consumption and serum lipids and lipoproteins were available for 3,858 men and 1,511 women. Consumption of five or more cups of coffee per day was reported by 10.1% of men and 8.7% of women, whereas only 3.4% of men and 2.2% of women consumed similar quantities of tea. After controlling for a number of potential confounders, coffee consumption (particularly "mud" coffee) was strongly and positively associated with higher serum total and low-density lipoprotein cholesterol levels in both sexes (consumers of five or more cups per day had cholesterol levels up to 18 mg/dl higher than abstainers) and with high-density lipoprotein cholesterol in women. There was a nonsignificant negative association between tea consumption and serum cholesterol (cholesterol levels about 5 mg/dl more in abstainers than in those consuming five or more cups per day). In general, more negative health-related habits were reported with increased coffee consumption, whereas this relationship was not found for tea drinking. The relatively small percentage of heavy tea drinkers limits the conclusions that one can draw from this study and controlled trials are needed to evaluate possible lipid-lowering effects of tea consumption.


Tea consumption. relationship to cholesterol, blood pressure, and coronary and total mortality.

The relation of tea to cholesterol, systolic blood pressure, and mortality from coronary heart disease and all causes was studied in 9,856 men and 10,233 women without history of cardiovascular disease or diabetes. All men and women 35-49 years of age from the county of Oppland (Norway) were invited to participate; the attendance rate was 90%. Mean serum cholesterol decreased with increasing tea consumption, the linear trend coefficient corresponded to a difference of 0.24 mmol/liter (9.3 mg/dl) in men and 0.15 mmol/liter (5.8 mg/dl) in women between drinkers of less than one cup and those of five or more cups/day, when other risk factors were taken into account. Systolic blood pressure was inversely related to tea with a difference between the same two tea groups of 2.1 mm in men and 3.5 mm in women. Altogether 396 men and 237 women died from all causes, and of these 141 and 18, respectively, died from coronary heart disease during the 12-year follow-up period. The mortality rate was higher (not statistically significant) among persons drinking no tea or less than one cup compared with persons drinking one or more cups/day. This applies to men and women and to coronary heart disease and all-cause mortality. For men, the relative risk (one or more versus less than one cup) for coronary death from Cox regression was 0.64 (95% CI:0.38, 1.07).


[Video bronchoscopy--the electronic view of the bronchus].

The optical resolution in the fibrescopes commonly in use is limited by the number and length of glass fibres used in the endoscopes. In the videoscope--i.e. in the direct videoscopy--the endoscopic picture is transformed already in the tip of the endoscope into electronic signals which can be guided through electrical wire nearly free from losses. This results in an enormous improvement of the image quality. Some problems arise from the adjustment of the light intensity yielding too much brightness in the short-distance and darkness in the depth. Furthermore, the work channel of the prototype we used is still too small. The investigation with this new technique requires an increased coordinative effort between the handling of the instrument and the picture on the monitor; but with some practice one becomes familiar with it. The pictures of the bronchial wall are extremely brilliant. Unusual structures of bronchial mucosa can be analyzed. Microscopic endoscopy seems to be only a short step away. Digital processing of the electronic image offers extraordinary perspectives for the future.


[Negative cortisone attitude in patients with bronchial asthma].

Patients with bronchial asthma often exhibit irrational fears of cortisone medication beyond justified worries about side-effects. A negative cortisone image, which means overemphasizing the damaging and threatening aspects of cortisone, often underlies non-compliant illness behaviour. In the present study, cortisone image was investigated in 54 patients with bronchial asthma before and after participation in the Düsseldorf Asthma treatment and teaching programme (ATTP) and one year later. By participating in ATTP, a significant reduction of the negative cortisone image and thus a more realistic view of cortisone could be achieved in most patients. This effect was also evident at the follow-up examination one year later. On the other hand, persistence of a negative cortisone image in a subgroup of patients turned out to be a potent predictor of later non-compliant illness behaviour. Psychological implications of a persistent negative cortisone image are discussed.


[Comparison of six peak flow meters for patients].

Five meters of six models of peak flowmeters have been checked by 40 measurements each in series with a pneumotachograph. There were no substantial differences between the five meters of one type, but the various models showed a different scatter, partly due to differences in the applied flow pattern. We prefer those peak flowmeters with less influence of the flow pattern.


[Case report: endoscopic resection of a 5 cm intrathoracic lipoma].

Technical possibilities of operative thoracoscopy are demonstrated on the occasion of the presentation of an endoscopic resection of a benign intrathoracic tumour. Thoracic video-endoscopy is definitely a useful constituent of the treatment of defined pleural or pulmonary processes, since it can be used in addition to biopsy (which serves to assure the correct diagnosis) without rendering the diagnostic procedure more complicated to any significant extent.


[Social significance of research studies in the fields of phthisiology and pulmonology in Uzbekistan].

The direction of research work in phthisiology and pulmonology is determined by the epidemiologic situation for tuberculosis and CNPD in the republic. The results of a complex research work in phthisiology and pulmonology carried out in the rural areas of Uzbekistan, and in the cotton--growing regions in particular, are given. Practical significance of this work is stressed, which comprises direct inspection of the tuberculosis infection foci and subjects in contact and subsequent accomplishment of preventive and health--improvement measures, comprehensive screening of the rural population for tuberculosis and CNPD detection, conduction of chemotherapy and pathogenetic and surgical treatment of tuberculosis patients. The results of research into phthisiology and pulmonology will make the basis of the national program with respect to tuberculosis in Uzbekistan.


[Use of computer technology for teaching phthisiology and pulmonology at medical institutes].

The experience of computer technology application at the Phthisiology Department of the Kazan Medical Institute over the last 5 years was generalized. Four classes of the teaching programs were described: program control of the accumulated experience, diagnostic search, a mathematical model of an emergency situation and expert medical systems. Preference is given to the latter two types of programs. It is emphasized that application of the IBM-compatible systems as the most prevalent in clinical practice is promising. Description of the specific programs is presented.


[Characteristics of diagnosis, clinical aspects and clinical course of tuberculosis in adolescents with concomitant diseases].

The authors present the data on special features of the course of tuberculosis in adolescents having other diverse conditions such as nonspecific pulmonary diseases and congenital abnormalities, prior acute respiratory diseases and concurrent iron deficiency anemia. The combination of tuberculosis with other diseases results in a longer course of tuberculosis with slow clinical and X-ray dynamics and in a number of cases (++prior acute respiratory diseases) in bouts of the specific process. The favourable outcomes of the disease are determined by the correct combined treatment of tuberculosis and concurrent diseases. When tuberculosis runs along with congenital abnormalities the effectiveness of treatment is achieved by surgical methods after specific chemotherapy.


[Variants of the involution of pulmonary tuberculosis].

The results of a many years' clinico-dispensary followup of 1597 patients with focal, infiltrative and disseminated tuberculosis and pulmonary tuberculomas were analysed. Current complex therapy of patients made it possible to distinguish 3 basic variants of the process involution: I--rapid resolution, II--at first rapid and then slow resolution and III-slow resolution. The main factor responsible for one or the other involution variant was a morphologic substrate of the process at the moment of its detection and primarily its recent character and the presence of extensive old specific background in the zone of a repeated outburst. The quality of treatment, including use of rifampicin, was of the decisive importance only in the presence of a recent process and the absence of such a background. Other factors including the patients' age, clinical form, phase and dissemination of the process were not crucial. Recent tuberculosis chemotherapy promoted the acceleration of reparative processes in the lung tissue in the presence of fresh changes.


[Optimal effectiveness of complex treatment of patients with chronic obstructive bronchitis by intravascular laser irradiation of blood].

Results of combined treatment of 100 patients with chronic obstructive bronchitis are presented in the paper. Intravascular laser irradiation of blood had a favourable effect on the clinical disease course, normalized parameters of lipid peroxidation, produced a marked immune stimulating and anti-inflammatory action, controlled the blood coagulation system and improved rheological blood properties. Intravascular laser irradiation of blood included in the complex of therapeutic measures increased the efficiency of therapy in patients with chronic obstructive bronchitis by 12.0 +/- 2.6%.


[Possibilities of intravenous use of helium-neon laser in the treatment of experimental tuberculosis of animals].

The study presents experimental finding of 40 mongrel dogs whose intravascular blood was irradiated with laser as a supplement to the multimodality treatment of respiratory tuberculosis. Earlier disappearance of intoxication symptoms and reduced terms of destruction cavity decrease and closure, as roentgenologically evidenced, was achieved. The influence of this treatment on certain lipid peroxidation parameters, hemocoagulation, immunity status and bacteriostatic blood activity were found. There were no side effects during treatment.


[Antioxidant treatment of pulmonary tuberculosis in patients with chronic alcoholism].

Study of the state of the antioxidant system (AOS) and lipid peroxidation (LPO), according to the data on the alpha-tocopherol content, diene conjugates and acid erythrocyte hemolysis in 69 patients suffering from pulmonary tuberculosis concurrent with chronic alcoholism, has revealed significant differences as against the findings related to healthy subjects (36), to those with stage 11 alcoholism (31) and pulmonary tuberculosis patients (51). Favourable changes in the AOS and LPO during complex treatment that includes antioxidant agents were less manifested in patients with combined pathology and appeared at later terms than in tuberculosis patients. The antioxidant therapy of tuberculosis in chronic alcoholics should take a prolonged time, at least 4 months, since the first favourable changes in the AOS and LPO parameters appear after 4 months, while normalization does not set in even after 6 months.


[Combined chemotherapy of toxic hepatitis in patients with pulmonary tuberculosis].

The follow-up involved patients with infiltrative pulmonary tuberculosis who developed toxic medicamentous hepatitis associated with antituberculosis drugs. They were treated by the method of combined pharmacotherapy which comprised sodium nucleinate (0.5 g 4 times daily), splenin (2 ml twice a day) and quercetin. The given combination of drugs rapidly improved clinical parameters and normalized immunologic tests.


[Functional morphology of the lungs in patients with tuberculosis and diabetes mellitus].

The lungs of 20 patients (between the ages of 14 and 46 years) with tuberculosis and type I diabetes mellitus were subjected to a morphological examination with the help of a light and electron microscope. Thirteen patients were operated on for fibrous-cavernous tuberculosis and 7 for pulmonary tuberculoma. A duration of the concurrent diseases was 3 to 10 years (5.5 years, on average). All patients had a serious form of diabetes mellitus. The combined affection was characterized by a progressive course of tuberculosis with involvement of bronchi in the process. Specific features of tissue reactions pertinent to this combined pathology were revealed which comprised defective defense mechanisms (alveolar macrophages, type II alveolocytes and fibroblasts in the form of their dystrophy) generalized affection of pulmonary vessels, intensive fibre formation and disorganization of the forming connective tissue, which has a bearing on the development of the pathological process.


[Population migration and tuberculosis].

Characteristic features of a migratory process of tuberculosis patients in the Republic of Moldova were subjected to study. Two tendencies have been revealed: growth of a number of the arriving subjects and deterioration of the structure of pulmonary tuberculosis among them. Patients who arrived from the places of confinement where morbidity persists on a high level constitute a particular hazard concerning tuberculosis dissemination. Certain organization measures have been taken. The epidemiologic situation for tuberculosis in the penitentiary-labour establishments at the republican Ministry of Internal Affairs was subjected to a comprehensive analysis with subsequent discussion of the results at a meeting of the staff of the Ministry of Public Health; instruction and plan of measures to be taken have been compiled by both ministries; a permanent board has been instituted for rendering help to medical workers of the penitentiary establishments; all law-protective organs have been involved in tuberculosis control; a specialized institution has been set up with a hospital for 200 beds intended for skilled examination and treatment of patients. As a result, the index of tuberculosis morbidity in the republican penitentiary-labour establishments reduced by more than half to promote an improvement of the epidemiologic situation in the republic.


[Morphological characteristics of reparative processes in tuberculosis and the effect of pesticides].

The paper deals with the results of a comparative morphologic, toxicologic and biochemical study of the character of reparative processes in destructive forms of pulmonary tuberculosis, specific features of non-coordination of collagen metabolism depending on the cumulation of organochlorine and organophosphorus pesticides and in the absence of their residual quantities in the patient's body on admission to a hospital and during current antibacterial therapy. The resected and removed lungs and blood of 1063 patients were used for the study. Specific features of a tuberculosis course, the duration and effectiveness of antibacterial therapy, the character of reparative processes, characteristic features of collagen metabolism and collagenosis, the character and dissemination of residual changes due to therapy were established in the compared groups of patients.


[Estimation of the degree of risk of reactivation of residual tuberculous changes based on the status of mycobacterial population].

The study was undertaken to define the role of L-forms of M. tuberculosis having different qualitative and ++quantitative characteristics in the development of recurrences of a tuberculosis process in subjects with residual tuberculous changes in the lungs. The microbiological and ++clinico-roentgenological+ examination included 2412 subjects who were in VIIA and VIIB Dispensary Groups. These subjects were found to have a considerable reservoir of tuberculosis infection: the causative agent of tuberculosis in the bacterial and L-forms was detected in 214 (9%) of the examinees. Detection of M. tuberculosis L-forms tended to reversion in a pathological material of subjects with residual tuberculous changes in the lungs is a major predictors of high potential risk of tuberculosis process reactivation.


[Indicators of iron metabolism in adolescents with tuberculosis].

Iron metabolic parameters were studied in a sample of 25 adolescent patients with tuberculosis who had been followed up. ++Criteria were examined for assessing the data obtained which differ from those found in iron-deficiency anemia. A number of parameters (hemoglobin, total serum iron-binding capacity, erythrocytic ferritin) have been proposed, which should be considered in detecting iron-deficiency anemia in a tuberculosis process.


[Analysis of the quality of clinical diagnosis based on autopsy data].

The material is based on the autopsy findings collected over 15 years (1975-1989). Findings related to 748 fatal outcomes were subjected to analysis in which 90 (12%) cases were misdiagnosed regarding the principal disease. A percentage of diagnostic errors with respect to tuberculosis was 1.8 on the whole and in the diagnosis of clinical forms and phases it was considerably higher-15.9 and 19.4, respectively. Hyperdiagnosis was typical of disseminated tuberculosis and caseous pneumonia, while hypodiagnosis--of primary, fibrocavernous tuberculosis and post-tuberculous changes. A percentage of hypodiagnosis in nontuberculous pathology varied from 12.5 to 50. Dynamic observation by the five-year plans demonstrated an increase in the percentage of misdiagnosis of the concurrent pathology and its decrease when the phase of a specific process was established. Purely medical errors accounted for 75% of misdiagnosis.


[An optimal method of pharmacological prevention of tuberculosis among adolescents].

The paper presents the findings of a comparative study of the efficacy of chemoprophylaxis which was conducted by the everyday (1 course) and intermittent (2 courses) method in adolescents aged 12-17 years, depending on the presence of aggravating factors. Examination covered 150 adolescents from the VI registration group. Following chemoprophylaxis there was a drop in sensitivity to tuberculin in 95.5 +/- 0.8% of group 1 (main) and in 78.5 +/- 1.1% of group 2 cases during which a medium size of an infiltrate in group 1 reduced by 2.8 mm more than that in group 2 (p less than 0.001). After the end of an observation period, 92.4 +/- 0.8% of subjects of group 1 and 79.4 +/- 0.5% of subjects of group 2 were struck off the register from the VIA registration group; the respective figures for the VIB registration group were 73.2 +/- 0.2% and 58.6 +/- 3%. The epidemiologic and economic effect of chemoprophylaxis conducted by 2 courses was 2 and 1.5 times higher than that in the traditional method.


[Effectiveness of BCG vaccination in children in relation to genetic and social factors].

The social status, genetic parameters, somatic and infectious diseases have been studied in BCG--vaccinated children who presented a postvaccinal cicatrix and in those in whom it did not form. Children having a postvaccinal cicatrix were found to belong mainly to the muscular type of constitution, while those without it--to the thoracic type. It was shown that a one--time positive tuberculin reaction to the Mantoux test with 2 TU (infiltrate of 6-7 mm) is associated with somatic and infectious pathology. This reaction can be considered to be para--allergy within 4 months after the disease.


Inhibitory effect of Trypanosoma brucei brucei on Glossina morsitans midgut trypsin in vitro.

The ability of Trypanosoma brucei brucei to inhibit trypsin or trypsin-like enzymes in crude midgut homogenates of Glossina morsitans morsitans was studied in vitro. The isolated parasites caused a concentration-dependent decrease in midgut trypsin activity. Furthermore, trypanosomes lysed by repeated freeze-thawing had a similar effect on trypsin activity. In both cases, the inhibition by either intact or lysed parasites was partial as revealed by Dixon plots. Similarly, trypanosome membrane proteins stoichiometrically inhibited trypsin activity, suggesting that the enzyme interacts specifically with a moiety on the parasite surface. The Km and Ki values obtained in this case were 35 microM and 0.18 mg/ml, respectively. These results suggest that one of the ways in which trypanosomes overcome the hostile tsetse-fly midgut barrier involves the inhibition of enzyme activity.


Effect of buparvaquone on the expression of interleukin 2 receptors in Theileria annulata-infected cells.

Theileria annulata-infected cells were cultured in the presence or absence of human recombinant interleukin 2 (hrIL-2). This growth factor proved to be capable of enhancing the growth of the infected cells: its effect was marked, particularly when the cells were seeded at low densities, and it varied from cell line to cell line. The infected cells produced a factor that possessed the biological activities of IL-2, since their supernatants could enhance the proliferation of concanvalin A-stimulated (Con A) blasts. The reactivity of the parasitized cells to hrIL-2 was abolished following their treatment with the antitheilerial drug buparvaquone. In addition, the drug inhibited the binding of 125I-IL-2 to T. annulata-infected cells but failed to suppress its binding to Con A blasts. Northern blot analysis revealed that the drug had no effect on the expression of the alpha chain of the IL-2 receptor (IL-2R). Therefore, it is possible that buparvaquone interferes with the expression of the beta chain of the IL-2R. The role of IL-2 and the IL2R in the permanent proliferation of T. annulata-infected cells is discussed.


Echinococcus multilocularis: immunological study on the "Em2-positive" laminated layer during in vitro and in vivo post-oncospheral and larval development.

Echinococcus multilocularis oncospheres, primary vesicular cysts, and protoscolices were assessed in vitro and in vivo for their potential to synthesize a PAS-positive laminated layer containing monoclonal antibody (mAb) G11-binding Em2 antigen. The presence of Em2 antigen in developed oncospheres and cysts was subsequently correlated to the potential of in vivo development into a secondary metacestode in recipient host mice, which also responded by anti-Em2 serum antibody formation. In contrast, protoscolices failed to develop the "Em2-positive" layer in vitro under the selected experimental conditions. The failure to develop subsequently in vivo into a secondary metacestode was underlined by a lack of anti-Em2 serum antibody formation by the hosts. We furthermore developed a technique to obtain E. multilocularis clones by inoculating single oncospheres into recipient mice.


Use of a recombinant Dictyocaulus viviparus antigen in an enzyme-linked immunosorbent assay for immunodiagnosis of bovine dictyocaulosis.

A specific recombinant antigen was evaluated for its immunodiagnostic potential in an enzyme-linked immunosorbent assay (ELISA). The antigen was used as a glutathione S-transferase (GST) fusion protein (DvGST3-14) or as a pure recombinant parasite protein (Dv3-14). A total of 55 sera from cattle experimentally infected with Dictyocaulus viviparus, 24 sera from naturally infected cattle and 25 sera from helminth-free cattle were examined. ELISA results obtained for the sera from experimental infections showed a calculated specificity of greater than 99% for both antigen preparations and a sensitivity of 93% (DvGST3-14) and 91% (Dv3-14). For field sera from natural infections, the specificity was calculated to be 90% (DvGST3-14) and greater than 99% (Dv3-14) and the sensitivity, 85% (DvGST3-14) and 70% (Dv3-14).


Detection of Echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs, dingoes and foxes.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Echinococcus coproantigens in fecal samples from dogs, dingoes or foxes infected with either E. granulosus or E. multilocularis. The ELISA was based on protein-A-purified polyclonal antibodies [anti-E. granulosus excretory/secretory (E/S) antigens]. The specificity of the assay as determined in 155 samples derived from carnivores that were free of helminth infection (n = 37) or infected with non-Echinococcus cestodes (n = 76) or with various nematodes (n = 42) was found to be 98% overall. The diagnostic sensitivity was strongly dependent on the homologous worm burden. All 13 samples from foxes harboring greater than 1,000 E. multilocularis worms and 13 of 15 (87%) samples from dogs or dingoes containing greater than 200 E. granulosus worms were ELISA-positive, whereas 34 of 46 samples from foxes harboring less than 1,000 E. multilocularis and 9 of 10 samples from dogs or dingoes bearing less than 200 E. granulosus tested negative. Experimental prepatent infections of dogs with E. granulosus revealed positive ELISA reactions within the prepatent period (10-20 days post-infection) for six animals bearing greater than 1,000 E. granulosus each; a low worm burden (less than 1,000 tapeworms/animal) resulted in ELISA positivity in only 2 of 3 animals at 30 days post-infection at the earliest. All five dogs that had been experimentally infected with E. multilocularis tested positive in the coproantigen ELISA as early as on day 5 post-infection.


Neoglycoproteins as tools for the detection of carbohydrate-specific receptors on the cell surface of Leishmania.

Promastigote and amastigote forms of human pathogenic Leishmania from the Old and New World, including promastigotes of L. enrietti, were tested with neoglycoproteins to ascertain the existence of endogenous lectins. These tools expose the chemically coupled sugar that is attached to the inert carrier as a potential ligand for the binding reaction. Agglutination tests demonstrated that the promastigotes of human Leishmania reacted only with the neoglycoproteins N-acetyl-D-galactosamine-para-aminophenyl-bovine serum albumin (gal-NAc-BSA) and N-acetyl-D-glucosamine-para-amino-phenyl-bovine serum albumin (glcNAc-BSA), whereas the amastigote forms failed to react with the neoglycoproteins. In contrast, the promastigotes of L. enriettii were agglutinated by the neoglycoprotein D-mannose-bovine serum albumin (man-BSA). The agglutination reactions could be inhibited by the homologous sugars N-acetyl-beta-D-glucosamine, N-acetyl-beta-D-galactosamine, and alpha-D-mannose. Fluorescence tests yielded the same results. The incubation of the promastigotes with ethylenedinitrolotetraacetic acid (EDTA) prevented their reaction with the neoglycoproteins, whereas the addition of calcium restored it. This result demonstrates that Leishmania express calcium-dependent lectins that are accessible on their surface.


Cellular responses to culture-derived soluble exoantigens of Trypanosoma lewisi.

Soluble exoantigens of Trypanosoma lewisi were obtained from short-culture systems of dividing epimastigotes and non-dividing trypomastigotes incubated in phosphate-buffered saline glucose solution (PBSG) for 3 h at 37 degrees C. Suppression of normal rat-spleen cell responses to mitogen concanavalin A (Con A) occurred at high exoantigens concentrations. The epimastigote-derived exoantigens were more suppressive than the trypomastigote-derived exoantigens. The suppression of Con A stimulation was ablated by the addition of exogenous interleukin-2 (IL-2) or washing of spleen cells that had been incubated with suppressing concentrations of the epimastigote exoantigens for 24 h prior to Con A stimulation. These results strongly suggest that exoantigens of T. lewisi play an important immunoregulatory role during the course of infection and that they achieve this by inhibiting the production of IL-2 by T-helper cells.


Immunoreactivity to two specific regions of chromogranin A in the nervous system of Ascaris suum: an immunocytochemical study.

Antisera to a highly conserved region of chromogranin A (sequence KELTAE) and to a hexapeptide (sequence KGQELE) adjacent to the putative C-terminus of pancreastatin, a peptide whose sequence is found within the chromogranin A molecule, have been used to examine the localisation of immunoreactivity (IR) to these peptides in Ascaris suum. IR to both peptides was found in the nerve rings and nerve cords. In addition, KGQELE-IR was also observed in the pharyngeal neurones and in a network of fibres on the surface of the female gonoduct. The staining was specific in that it could be abolished by preincubation of the antisera with the appropriate antigen. The two antisera appeared to be staining different subsets of neurones, suggesting that (at least) two peptides were being recognised. The wide-spread distribution of IR to both peptides throughout the nervous system of the parasite suggests that the peptides carrying the epitopes recognised by the antisera are of fundamental importance to the functioning of the parasite's nervous system.


Adherence between Entamoeba histolytica trophozoites and undifferentiated or DMSO-induced HL-60 cells.

Differentiation of promyelocytic HL-60 cells along the pathway toward granulocytes using dimethyl-sulfoxide (DMSO) led to increased expression of the adhesion molecules CD11b (Mac-1), CD11c (p150,95), and CD35 (CR1). Undifferentiated and differentiated HL-60 cells adhered similarly to Entamoeba histolytica trophozoites, and monoclonal antibodies against adhesion molecules CD11b and CD11c did not inhibit adherence. We therefore suggest that CD11b and CD11c are not involved in the adherence between polymorphonuclear granulocytes and E. histolytica trophozoites.


Distribution of a fluorescent ivermectin probe, bodipy ivermectin, in tissues of the nematode parasite Ascaris suum.

A fluorescent derivative of the anthelmintic ivermectin (4''-5,7-dimethyl bodipy proprionylivermectin, referred to hereafter as bodipy ivermectin) was synthesized for an investigation of the distribution of avermectins. Injected into adult Ascaris suum at doses greater than 0.1 micron per worm, it produced a dose-dependent immobilization. Fluorescent microscopy of frozen sections revealed the distribution of the probe in the whole nematode. Staining of collagenase-isolated muscle cells was studied using bath application of bodipy ivermectin. The trypan-blue quenching technique showed that the ivermectin probe was located in the outer monolayer of the muscle membrane. The cytoplasm was not stained. The interpretation of these observations is discussed in view of the known lipophilic nature of avermectins. Staining of the muscle membrane and nerve cord is consistent with the view that avermectins act at these sites. The significance of the hypodermal and lateral line staining is also discussed.


In vitro effects of milbemycin oxime: mechanism of action against Angiostrongylus cantonensis and Dirofilaria immitis.

The neuropharmacological mechanisms underlying the action of milbemycin oxime on the motility of Angiostrongylus cantonensis and Dirofilaria immitis were examined in vitro. In A. cantonensis, milbemycin oxime caused inhibitory effects at low concentrations of > or = 10(-9) g/ml, and paralysis was elicited at 10(-8) - 10(-6) g/ml. The paralysis was antagonized by picrotoxin and bicuculline but not by dibenamine. In addition, stimulatory effects were observed when the antibiotic was used at higher concentrations of 3-5 x 10(-6) g/ml, and the action was antagonized by strychnine. Both effects were also observed in the preparation contracted by eserine or pyrantel. When D. immitis was treated with milbemycin oxime at concentrations of 10(-7) and 3-5 x 10(-6) g/ml, only slight inhibitory and stimulatory effects, respectively, were observed. These effects were partially antagonized by picrotoxin and strychnine, respectively. These results suggest that the inhibitory and stimulatory actions of milbemycin oxime are caused through gabergic and cholinergic mechanisms in A. cantonensis and D. immitis.


Fasciola hepatica: early lesions in the viscera of seven pulmonate species after penetration of the miracidium.

Early lesions were studied in four viscera of seven pulmonate species on the 2nd day after miracidial exposure. The albumen gland, the digestive gland, and the gonad of infected snails demonstrated not only normal tissue but also the presence of two stages of epithelial necrosis. Multifocal necrosis predominated, with varying percentages of frequency depending on the snail species and stage of life; generalized necrosis was more seldom observed. In the kidney, epithelial necrosis was encountered more frequently than intralamellar deposits.


Quantitative analysis of protein far UV circular dichroism spectra by neural networks.

A new method based on neural network theory is presented to analyze and quantify the information content of far UV circular dichroism spectra. Using a backpropagation network model with a single hidden layer between input and output, it was possible to deduce five different secondary structure fractions (helix, parallel and antiparallel beta-sheet, beta-turn and random coil) with satisfactory correlations between calculated and measured secondary structure data. We demonstrate that for each wavelength interval a specific network is suitable. The remaining discrepancy between the secondary structure data from neural network prediction and crystallography may be attributed to errors in the determination of protein concentration and random noise in the CD signal, as indicated by simulations.


The alpha/beta hydrolase fold.

We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.


The functions of tryptophan residues in membrane proteins.

Membrane proteins have a significantly higher Trp content than do soluble proteins. This is especially true for the M and L subunits of the photosynthetic reaction center from purple bacteria. The Trp residues are not uniformly distributed through the membrane but are concentrated at the periplasmic side of the complex. In addition, Trp residues are not randomly aligned. Within the protein subunits, many form hydrogen bonds with carbonyl oxygens of the main chain, thereby stabilizing the protein. On the surface of the molecule, they are correctly positioned to form hydrogen bonds with the lipid head groups while their hydrophobic rings are immersed in the lipid part of the bilayer. These observations suggest that Trp residues are involved in the translocation of protein through the membrane and that following translocation, Trp residues serve as anchors on the periplasmic side of the membrane.


Calculations of antibody-antigen interactions: microscopic and semi-microscopic evaluation of the free energies of binding of phosphorylcholine analogs to McPC603.

The study of antibody-antigen interactions should greatly benefit from the development of quantitative models for the evaluation of binding free energies in proteins. The present work addresses this challenge by considering the test case of the binding free energies of phosphorylcholine analogs to the murine myeloma protein McPC603. This includes the evaluation of the differential binding energy as well as the absolute binding energies and their corresponding electrostatic contributions. Four different approaches are examined: the Protein Dipoles Langevin Dipoles (PDLD) method, the semi-microscopic PDLD (PDLD/S) method, a free energy perturbation (FEP) method based on an adiabatic charging procedure and a linear response approximation that accelerates the FEP calculation. The PDLD electrostatic calculations are augmented by estimates of the relevant hydrophobic and steric contributions. The determination of the hydrophobic energy involves an approach which considers the modification of the effective surface area of the solute by local field effects. The steric contributions are analyzed in terms of the corresponding reorganization energies. This treatment, which considers the protein as a harmonic system, views the steric forces as the restoring forces for the electrostatic interactions. The FEP method is found to give unreliable results with regular cut-off radii and starts to give quantitative results only in very expensive treatment with very large cut-off radii. The PDLD and PDLD/S methods are much faster than the FEP approach and give reasonable results for both the relative and absolute binding energies. The speed and simplicity of the PDLD/S method make it an effective strategy for interactive docking studies and indeed such an option is incorporated in the program MOLARIS. A component analysis of the different energy contributions of the FEP treatment and a similar PDLD analysis indicate that electrostatic effects provide the largest contribution to the differential binding energy, while the hydrophobic and steric contributions are much smaller. This finding lends further support to the idea that electrostatic interactions play a major role in determining the antigen specificity of McPC603.


A functional antibody mutant with an insertion in the framework region 3 loop of the VH domain: implications for antibody engineering.

We have studied the effects of a four residue insertion into the FR3 loop of the heavy chain variable region from the anti-NP antibody B1-8. The insertion mutant is obtained as secreted antibody without major defects in biosynthesis, indicating that antibody variable domains can accommodate length variation not only in complementarity determining regions (CDRs), but also in framework region (FR) loops. The B1-8 antigen binding site is not affected by the change in a neighbouring loop. FR3 insertions represent a new method of antibody engineering with a potential to obtain strong antigen binding by designing additional antigen contacting residues.


Fluorescent properties of the Escherichia coli D-xylose isomerase active site.

The consequences of active site mutations of the Escherichia coli D-xylose isomerase (E.C. 5.3.1.5) on substrate binding were examined by fluorescence spectroscopy. Site-directed mutagenesis of conserved tryptophan residues in the E. coli enzyme (Trp49 and Trp188) reveals that fluorescence quenching of these residues occurs during the binding of xylose by the wild-type enzyme. The fluorescent properties of additional active site substitutions at His101 were also examined. Substitutions of His101 which inactivate the enzyme were shown to have altered spectral characteristics, which preclude detection of substrate binding. In the case of H101S, a mutant protein with measurable isomerizing activity, substrate binding with novel fluorescent properties was observed, possibly the bound pyranose form of xylose under steady-state conditions.


Phe496 and Leu497 are essential for receptor binding and cytotoxic action of the murine interleukin-4 receptor targeted fusion toxin DAB389-mIL-4.

DAB389-mIL-4 is a murine interleukin-4 (mIL-4) diphtheria toxin-related fusion protein which has been shown to be selectively toxic to cells expressing the mIL-4 receptor. In this report, we have used site-directed and in-frame deletion mutagenesis to study the role of the putative C-terminal alpha-helix (helix E) of the mIL-4 component of DAB389-mIL-4 in the intoxication process. We demonstrate that deletion of the C-terminal 15 amino acids of the fusion toxin leads to loss of cytotoxicity. The substitution of Phe496 with either Pro, Ala or Tyr, results in a greater than 20-fold decrease in cytotoxic activity of the respective mutant fusion toxins. In addition, substitution of Leu497 with either Ala or Glu results in a similar loss of cytotoxic activity. All of these mutant forms of the mIL-4 fusion toxin demonstrate a significant decrease in binding affinity (Ki) to the mIL-4 receptor in a competitive radioligand binding assay. In marked contrast, however, the substitution of Asp495 with Asn results in a 4-fold increase in cytotoxic potency and binding affinity to mIL-4 receptor bearing cells in vitro.


A point mutation that decreases the thermal stability of human interferon gamma.

We have identified a mutation of human gamma-interferon (IFN gamma) causing a temperature-sensitive phenotype. We used a randomized oligonucleotide to mutagenize a synthetic human IFN gamma gene, then screened the resulting mutants produced in Escherichia coli for proteins with altered biological activity. One mutant protein selected for detailed characterization exhibited less than 0.3% of the specific biological activity of native IFN gamma in an antiviral activity assay performed at 37 degrees C. However, the protein bound the human IFN gamma receptor with native efficiency at 4 degrees C. Sequencing the plasmid DNA encoding this protein showed that the mutation changed the lysine residue at amino acid 43 to glutamic acid (IFN gamma/K43E). Site-specific mutagenesis at amino acid 43 showed that this protein's phenotype resulted from positioning a negative charge at position 43. Structural characterization of IFN gamma/K43E using CD demonstrated that the protein had native conformation at 25 degrees C, but assumed an altered conformation at 37 degrees C. IFN gamma/K43E in this altered conformation bound poorly to the IFN gamma receptor at 37 degrees C, providing a rationale for the mutant's decreased antiviral activity.


A point mutation of human interferon gamma abolishes receptor recognition.

We identified a single amino acid mutation that abolished the bioactivity of human IFN gamma. The mutation was identified by screening a mutagenized IFN gamma expression library for molecules with altered biological activity. The mutant protein was expressed at high levels in Escherichia coli, and remained soluble upon purification. However, the protein was completely inactive in all IFN gamma assays investigated, exhibiting less than 0.0006% of the specific activity of native IFN gamma antiviral activity. Sequencing the plasmid DNA encoding this mutant protein showed that the histidine at position 111 of native human IFN gamma is changed to aspartic acid (IFN gamma/H111D). Other mutations at this site showed that only hydrophobic amino acids at position 111 maintain significant, though low, biological activity. Structural characterization of the IFN gamma/H111D protein by NMR as well as CD spectroscopy demonstrated that the protein has limited conformational differences from native IFN gamma. Models of the X-ray crystal structure of human IFN gamma [Ealick, P.E., W.J. Cook, S. Vijay-Kumar, M. Carson, T.L. Nagabhushan, P.P. Trotta and C.E. Bugg (1991) Science, 252, 698-702] suggest that this histidine residue is located at a severe 55 degrees bend in the C-terminal F helix. We conclude that H111 lies within or affects the receptor binding domain of human IFN gamma.


Involvement of residues 296-299 in the enzymatic activity of tissue-type plasminogen activator.

The tetra-alanine substitution variant KHRR 296-299 AAAA of tissue-type plasminogen activator (t-PA) was previously shown to have enhanced fibrin specificity and enhanced activity in the presence of fibrin compared with the wild-type form of the molecule. The structural requirements for these alterations in enzymatic activity were investigated by constructing several amino acid substitution variants at each of the positions from 296 to 299 and evaluating their activities under a variety of conditions. Effects on plasminogen activator activity were common among the point mutants at positions 296-299; nearly all had a phenotype similar to the KHRR 296-299 AAAA variant. The greatest effects on enzymatic function were found with multiple substitution variants, but some single charge reversals and proline substitutions had substantial effects. The enhanced fibrin specificity of KHRR 296-299 AAAA t-PA results in less fibrinogenolysis than seen with wild-type t-PA. Approximately four times greater concentration of KHRR 296-299 AAAA compared with wild-type t-PA was required to consume 50% of the fibrinogen in human plasma.


A new protein conjugate that replaces the use of secondary antibodies engineered from the two staphylococcal enzymes protein A and 6-phospho-beta-galactosidase.

The lacG gene encoding the 6-phospho-beta-galactosidase (E.C.3.2.1.85) of Staphylococcus aureus was fused to the protein A gene in the plasmid pRIT2T. Escherichia coli cells containing this plasmid produce a fusion protein with both IgG binding and 6-phospho-beta-galactosidase activities after heat induction. The recombinant gene was overexpressed and the hybrid protein was purified to homogeneity in high yield. The chimeric protein was shown to have almost identical enzymatic characteristics to pure 6-phospho-beta-galactosidase. This result leads to the conclusion that a free N-terminus of the 6-phospho-beta-galactosidase is not required for biological activity. The hybrid protein of protein A and 6-phospho-beta-galactosidase was used as an enzyme conjugate in enzyme-linked immunosorbent assays (ELISA). The experiments presented demonstrate that the 6-phospho-beta-galactosidase is a suitable fusion partner in various diagnostic applications where an unique biological activity is required.


Insertion of a disulfide-containing neurotoxin into E. coli alkaline phosphatase: the hybrid retains both biological activities.

We have inserted a disulfide-containing snake neurotoxin into the N-terminal end of Escherichia coli alkaline phosphatase, between residues +6 and +7 of the mature enzyme. For this purpose, we have designed a cloning and expression vector which allows insertion of foreign DNA between the corresponding codons, and visual selection of the desired recombinant clones upon recovery of phosphatase activity. The hybrid protein is exported to the bacterial periplasm, the alkaline phosphatase signal peptide is correctly processed, and both domains are functionally conformed. The phosphatase domain displays catalytic activity, and the inserted toxin is able to bind to its biological target, the nicotinic acetylcholine receptor. The hybrid molecule is remarkably stable and resistant to proteolysis. Crude periplasmic extract containing the hybrid can be used as a tracer-containing reagent in competitive enzymo-immuno and enzymo-receptor assays. We propose to use the system described in this paper for fast preparation of properly folded disulfide-containing enzymatic probes.


An SS1-SS2 beta-barrel structure for the voltage-activated potassium channel.

To examine the feasibility of a beta structure for the pore-lining region of the voltage-gated potassium channel, we have characterized a family of 12 antiparallel beta-barrels. Each is comprised of four identical pairs of beta-strands organized with approximate 4-fold symmetry about a channel axis. The C- and N-termini of the beta-strand pairs are assumed to be at the extracellular end of the channel, and each pair is connected by a hairpin turn at the intracellular end of the channel. The models differ in the residues located in the hairpin turn and in the orientation of the two strands of each pair in the barrel, i.e. whether the C-terminus of a pair is clockwise (CW) or counterclockwise (CCW) from the N-terminus when the channel is viewed from outside the cell. Following known structure precedents and potential energy predictions, the barrel is assumed to be right-twisting in all cases. All models have crowded layers of inward-projecting aromatic side-chains near the center of the channel which could regulate channel selectivity. The models with an odd number of amino acids in the hairpin turn have the advantage of predicting that F433 points into the barrel, but the disadvantage that V438 does not. Of these models, two of the models are most consistent with the external tetraethylammonium (TEA) block data, and of those, one (T439 CCW 3:5) is most consistent with the internal TEA block data.


Main structural and functional features of the basic cytosolic bovine 21 kDa protein delineated through hydrophobic cluster analysis and molecular modelling.

A 21 kDa protein purified from bovine brain cytosol was previously described as a hydrophobic ligand binding protein; however, its accurate biological function remained still uncertain. In order to get further information about its potential biological role, an extended prediction of its secondary and three dimensional structures was undertaken. We describe here a process which permitted us to discover a structural homology between the 21 kDa protein and the N-domain of yeast phosphoglycerate kinase (PGK). This process is based on comparing the 21 kDa protein with all the proteins presenting a slight homology, by using the Hydrophobic Cluster Analysis (HCA) method. According to the observed similarity between the N-domain of yeast PGK and the 21 kDa protein, we built a model which was shown to possess a potential binding site for nucleotides. Moreover, the model obtained presents three-dimensional (3D) structure similarity with adenylate kinase. These results suggest two main hypotheses: (i) the 21 kDa protein may belong to the kinase family; (ii) the binding of a nucleotide could imply a modification of the 3D structure of the 21 kDa protein that can promote the transfer of hydrophobic ligands to the plasma membrane. Meanwhile, verification of these hypotheses has been in part performed experimentally: the 21 kDa protein binds MgATP as well as, to a lesser extent, phosphoglycerate.


Towards an automatic method of predicting protein structure by homology: an evaluation of suboptimal sequence alignments.

A major problem in predicting protein structure by homology modelling is that the sequence alignment from which the model is built may not be the best one in terms of the correct equivalencing of residues assessed by structural or functional criteria. A useful strategy is to generate and examine a number of suboptimal alignments as better alignments can often be found away from the optimal. A procedure to filter rapidly suboptimal alignments based on measurement of core volumes and packing pair potentials is investigated. The approach is benchmarked on three pairs of sequences which are non-trivial to align correctly, namely two immunoglobulin domains, plastocyanin with azurin and two distant globin sequences. It is shown to be useful to reduce a large ensemble of possible alignments down to a few which correspond more closely to the correct (structure based) alignment.


Computational complexity of a problem in molecular structure prediction.

The computational task of protein structure prediction is believed to require exponential time, but previous arguments as to its intractability have taken into account only the size of a protein's conformational space. Such arguments do not rule out the possible existence of an algorithm, more selective than exhaustive search, that is efficient and exact. (An efficient algorithm is one that is guaranteed, for all possible inputs, to run in time bounded by a function polynomial in the problem size. An intractable problem is one for which no efficient algorithm exists.) Questions regarding the possible intractability of problems are often best answered using the theory of NP-completeness. In this treatment we show the NP-hardness of two typical mathematical statements of empirical potential energy function minimization of macromolecules. Unless all NP-complete problems can be solved efficiently, these results imply that a function minimization algorithm can be efficient for protein structure prediction only if it exploits protein-specific properties that prohibit the simple geometric constructions that we use in our proofs. Analysis of further mathematical statements of molecular structure prediction could constitute a systematic methodology for identifying sources of complexity in protein folding, and for guiding development of predictive algorithms.


Design, synthesis and structure of an amphipathic peptide with pH-inducible haemolytic activity.

A synthetic, 26-residue peptide having a strong helix forming potential in the protonated state was designed to interact with lipid bilayers in a pH-dependent way. On the basis of this concept a cluster of four glutamic acid residues was inserted in the central region of the amphipathic peptide to promote helix destabilization by mutual charge repulsion at neutral pH. Protonation of these residues might then bring about both a pH-mediated change in hydrophobicity and conformation forming a membrane-active amphiphilic helix. The sequence GLGTLLTLLEFLLEELLEFLKRKRQQamide produced by the design strategy induced pH-triggered lysis of human erythrocytes. A molecular model correlating the lytic activity to the formation of transmembrane pores which were detected by electron microscopy in erythrocyte membranes is discussed. Circular dichroism studies indicated a self-association of the monomeric random coil form with increasing peptide concentration leading to the apparent induction of strong alpha-helix formation (approximately 100% helicity) in the fully aggregated state. However, no pH-dependent helix-random coil transition was observed, implying that interhelical hydrophobic and ionic interactions not only govern the self-association but also decisively influence the conformational stability of the peptide.


Molecular modelling and site-directed mutagenesis on a bovine anti-testosterone monoclonal antibody.

A three-dimensional (3D) molecular model of the antigen-combining site of a bovine anti-testosterone monoclonal antibody has been constructed. In the model, the CDRs, and a single heavy chain framework region residue (Trp47), associate to form a hydrophobic cavity large enough to accommodate a single molecule of testosterone. Tyr97 of CDR-H3 lies at the bottom of the cavity with its hydroxyl group exposed to solvent. Using the model and data from binding studies, we predicted that the cavity forms the antibody's paratope and on binding testosterone a hydrogen bond is formed between Tyr97 of CDR-H3 and the hydroxyl group on the D-ring of testosterone. This prediction has subsequently been tested by site-directed mutagenesis. An antibody with phenylalanine in place of tyrosine at position 97 in CDR-H3 has its affinity reduced by approximately 800 fold. The reduction in binding energy associated with the reduced affinity has been calculated to be 3.9 kcal/mol which is within the range (0.5-4.0 kcal/mol) expected for the loss of a single hydrogen bond. The model has been used to suggest ways of increasing the antibody's affinity for testosterone.


Interchain cysteine bridges control entry of progesterone to the central cavity of the uteroglobin dimer.

The progesterone-binding protein uteroglobin has been expressed in Escherichia coli in an unfused, soluble form. Like mature uteroglobin from rabbit endometrium (UG), the E.coli produced uteroglobin (UG1) dimerizes in vitro, forms an antiparallel dimer with Cys3-Cys69' and Cys69-Cys3' disulfide bonds and binds progesterone under reducing conditions. In order to analyze the dimerization and the reduction dependence of progesterone binding in more detail, we separately replaced cysteine 3 and cysteine 69 by serines. Under reducing conditions, both uteroglobin variants (UG1-3Ser and UG1-69Ser) bind progesterone with the same affinity as the wild-type suggesting that both cysteine residues are not directly involved in progesterone binding. In contrast to the wild-type protein, both cysteine variants also bind progesterone with high affinity in the absence of reducing agents. In addition, UG1-3Ser and UG1-69Ser both form covalently linked homodimers. Thus, unnatural Cys69-69' and Cys3-3' disulfide bonds exist in UG1-3Ser and UG1-69Ser, respectively. These data together with computer models based on X-ray diffraction data strongly support the idea that progesterone reaches its binding site located in an internal hydrophobic cavity via a hydrophobic tunnel along helices 1 and 4. Under non-reducing conditions the tunnel is closed by two disulfide bridges (Cys3-Cys69' and Cys69-Cys3') that lie in the most flexible region of the dimer. Reduction or replacement of a cysteine residue enables conformational changes that open the channel allowing progesterone to enter.


Cellulose-binding domains: potential for purification of complex proteins.

The endoglucanase CenA and the exoglucanase Cex from Cellulomonas fimi each contain a discrete cellulose-binding domain (CBD), at the amino-terminus or carboxyl-terminus respectively. The gene fragment encoding the CBD can be fused to the gene of a protein of interest. Using this approach hybrid proteins can be engineered which bind reversibly to cellulose and exhibit the biological activity of the protein partner. Alkaline phosphatase (PhoA) from Escherichia coli, and a beta-glucosidase (Abg) from an Agrobacterium sp. are dimeric proteins. The fusion polypeptides CenA-PhoA and Abg-CBC(Cex) are sensitive to proteolysis at the junctions between the fusion partners. Proteolysis results in a mixture of homo- and heterodimers; these bind to cellulose if one or both of the monomers carry a CBD, e.g. CenA-PhoA/CenA-PhoA and CenA-PhoA/PhoA. CBD fusion polypeptides could be used in this way to purify polypeptides which associate with the fusion partner.


Expression of a synthetic gene coding for the amino acid sequence of Clostridium pasteurianum rubredoxin.

A synthetic gene based on the published amino acid sequence for Clostridium pasteurianum rubredoxin was constructed, cloned in Escherichia coli 71/18 and expressed using the T7 RNA polymerase/promoter system in E. coli HMS273. UV/visible spectroscopy and metal analyses indicated that the as-isolated synthetic gene product is a mixture of holo-(i.e. iron-containing) rubredoxin and zinc-substituted rubredoxin, with the latter amounting to approximately 70% of the total rubredoxin. The UV/visible absorption and resonance Raman spectra of the cloned holorubredoxin are characteristic of the native rubredoxin-type iron site. N-terminal amino acid sequencing suggests that the gene product consists of at least three polypeptide species with the initial sequences (approximate relative abundances): Met-Met-Lys-... (63%), blocked (30%) and Met-Lys-... (7%). The blocked portion presumably consists of a mixture of nMet-Met-Lys-... and nMet-Lys-..., where nMet represents an amino-blocked methionine residue.


Monte Carlo minimization with thermalization for global optimization of polypeptide conformations in cartesian coordinate space.

A new minimization procedure for the global optimization in cartesian coordinate space of the conformational energy of a polypeptide chain is presented. The Metropolis Monte Carlo minimization is thereby supplemented by a thermalization process, which is initiated whenever a structure becomes trapped in an area containing closely located local minima in the conformational space. The method has been applied to the endogenous opioid pentapeptide methionine enkephalin. Five among 13 different starting conformations led to the same apparent global minimum of an in-house developed energy function, a type II' reverse turn, the central residues of which are Gly-3-Phe-4. A comparison between the ECEPP/2 global minimum conformation of methionine enkephalin and the apparent one achieved by the present method shows that minimum-energy conformations having a certain similarity can be generated by relatively different force fields.


Overproduction, crystallization, and preliminary X-ray diffraction studies of the major cold shock protein from Bacillus subtilis, CspB.

The major cold shock protein from Bacillus subtilis (CspB) was overexpressed using the bacteriophage T7 RNA polymerase/promoter system and purified to apparent homogeneity from recombinant Escherichia coli cells. CspB was crystallized in two different forms using vapor diffusion methods. The first crystal form obtained with ammonium sulfate as precipitant belongs to the trigonal crystal system, space group P3(1)21 (P3(2)21) with unit cell dimensions a = b = 59.1 A and c = 46.4 A. The second crystal form is tetragonal, space group P4(1)2(1)2 (P4(3)2(1)2) with unit cell dimensions a = b = 56.9 A and c = 53.0 A. These crystals grow with polyethylene glycol 4000 as precipitant.


A systematic search for protein signature sequences.

Signature sequences are contiguous patterns of amino acids 10-50 residues long that are associated with a particular structure or function in proteins. These may be of three types (by our nomenclature): superfamily signatures, remnant homologies, and motifs. We have performed a systematic search through a database of protein sequences to automatically and preferentially find remnant homologies and motifs. This was accomplished in three steps: 1. We generated a nonredundant sequence database. 2. We used BLAST3 (Altschul and Lipman, Proc. Natl. Acad. Sci. U.S.A. 87:5509-5513, 1990) to generate local pairwise and triplet sequence alignments for every protein in the database vs. every other. 3. We selected "interesting" alignments and grouped them into clusters. We find that most of the clusters contain segments from proteins which share a common structure or function. Many of them correspond to signatures previously noted in the literature. We discuss three previously recognized motifs in detail (FAD/NAD-binding, ATP/GTP-binding, and cytochrome b5-like domains) to demonstrate how the alignments generated by our procedure are consistent with previous work and make structural and functional sense. We also discuss two signatures (for N-acetyltransferases and glycerol-phosphate binding) which to our knowledge have not been previously recognized.


Modeling of substrate and inhibitor binding to phospholipase A2.

Molecular graphics and molecular mechanics techniques have been used to study the mode of ligand binding and mechanism of action of the enzyme phospholipase A2. A substrate-enzyme complex was constructed based on the crystal structure of the apoenzyme. The complex was minimized to relieve initial strain, and the structural and energetic features of the resultant complex analyzed in detail, at the molecular and residue level. The minimized complex was then used as a basis for examining the action of the enzyme on modified substrates, binding of inhibitors to the enzyme, and possible reaction intermediate complexes. The model is compatible with the suggested mechanism of hydrolysis and with experimental data about stereoselectivity, efficiency of hydrolysis of modified substrates, and inhibitor potency. In conclusion, the model can be used as a tool in evaluating new ligands as possible substrates and in the rational design of inhibitors, for the therapeutic treatment of diseases such as rheumatoid arthritis, atherosclerosis, and asthma.