Pathogeny and immunological control of toxoplasmosis. 1. Toxoplasma gondii is a ubiquitous, obligate intracellular parasite of worldwide distribution. In humans, the parasite exists in two forms: the tachyzoite is the rapidly multiplying stage of the parasite which actively invades host cells and represents the principal pathogenic form at the acute phase of the disease; the bradyzoite is the form which multiplies slowly in host cells, resulting in the formation of cysts which persist in tissues. Several antigenic components have been identified, some of which are characteristic for each parasitic stage; particularly, in tachyzoites, the 30 kDa membrane protein represents up to 5% of the total protein content. 2. Toxoplasma infection in humans is usually asymptomatic because of effective immunity involving antibodies, T cells and cytokines. Activated macrophages, CD4 and CD8 lymphocytes and cytokines, such IFN gamma, play a major role in the control of acute infection and the maintenance of infection at the chronic stage. The alteration of immune functions, as observed in congenitally infected children and in HIV-infected patients, may induce the recrudescence of previously latent toxoplasmosis, in relation to disruption of the cyst form of the parasite. The resulting reactivation is responsible for life-threatening infections which are frequently manifested as toxoplasmic encephalitis. 3. In this review, the parasite and immunological factors participating in the pathogenesis of the lesions associated with acute, chronic and reactivated toxoplasmosis are presented.


DNA alkylation by carbon-centered radicals. 1. Over the last two decades, the prevalent view in chemical carcinogenesis has been that most free radicals do not bind to DNA. Recent studies, however, are demonstrating formation of adducts between DNA and free radicals such as hydroxyl radicals and aromatic cation radicals. 2. Within this context, we discuss the recent work from our group demonstrating DNA alkylation by carbon-centered radicals formed during biotransformation of genotoxic hydrazine derivatives both in vitro and in vivo. 3. The mutagenic potential of the identified methyl radical adduct, C8-methylguanine, is discussed, and other possible biological sources of carbon-centered radicals are presented.


Effects of cyclosporin A immunosuppression on pancreaticoduodenal transplantation in alloxan-diabetic rats. 1. Forty-five outbred Wistar rats were randomly assigned to three experimental groups: GI, 10 non-diabetic control rats; GII, 10 alloxan-diabetic control rats; GIII, 25 alloxan-diabetic rats which received pancreaticoduodenal transplantation (PDT) from normal Wistar donor rats and were immunosuppressed with cyclosporin A (Cy-A), 10 mg kg body weight-1 day-1, administered intraperitoneally for 30 days. 2. In parallel, 15 alloxan-diabetic inbred Wistar rats received isogeneic PDT from normal Wistar donor rats. 3. Cy-A prevented graft rejection in the 15 surviving animals in group III. These observations were confirmed by clinical and biochemical parameters (body weight, urine output, water and food intake, blood and urinary glucose and plasma insulin) and by histology and immunohistochemistry of the pancreas. 4. However, Cy-A was associated with 60% of the infectious complications in transplanted rats leading to 40% mortality. Pulmonary infections were the main cause of death. There were no side effects of immunosuppression on the pancreas. Infections were not significant in inbred rats submitted to PDT.


Effect of aging on the glutaminase activity of neoplastic and immune tissues. 1. The effect of age and Walker 256 tumor on maximal phosphate-dependent glutaminase activity of rat immune tissue was determined. Glutaminase is a key enzyme in the metabolism of glutamine, an important fuel for normal and neoplastic cells. 2. Maximal activity of phosphate-dependent glutaminase was measured in immune tissues and tumors of Walker 256 tumor-bearing young (28 days old), mature (3 months old) and aged (15 months old) Wistar rats. The following tissues were examined: thymus, spleen, mesenteric lymph nodes and tumor. 3. Tumor implantation for 14 days reduced glutaminase activity in the thymus and mesenteric lymph nodes. Tumor glutaminase activity was lowest in aged rats and highest in the mature group. 4. Comparison of glutaminase activity in immune and tumor tissues suggested the flux of glutamine between these tissues in the 3 groups. Glutaminase activity was 2.8-fold higher in immune tissues in aged rats (2.58 +/- 0.35 vs 0.93 +/- 0.16 mumol min-1 g tissue wet weight-1, mean +/- SEM, 5 rats), and 1.9- (4.14 +/- 0.47 vs 8.36 +/- 1.29 mumol min-1 g tissue wet weight-1, mean +/- SEM, 5 rats) and 2.5-fold increased (2.41 +/- 0.20 vs 5.92 +/- 0.22 mumol min-1 g tissue wet weight-1, mean +/- SEM, 5 rats) in tumor tissue in the mature and young groups, respectively. These results suggest the deviation of glutamine flux from defense cells to the neoplastic tissue in tumor-bearing young and mature rats and may partially explain the slow cancer growth in elderly patients.


Renal sodium conservation during starvation in rats. The natriuresis of fasting has been well characterized in man and rabbits but not in rats. The daily effects of fasting on glomerular filtration rate (GFR) and urinary sodium and potassium excretion were evaluated in Munich-Wistar rats (260-310 g) submitted to prolonged starvation (2-8 days). Rats do not present the natriuresis of fasting. Sodium excretion was reduced since the first few hours (0-4 h) of starvation. Antinatriuresis was abrupt during the early periods (1st and 2nd days) and stabilized at very low levels. During the early phase (4 days), sodium retention occurred due to both reduced glomerular filtration and increased tubular reabsorption. However, during the late phase (after the 4th day), antinatriuresis was mainly induced by the elevation in tubular reabsorption, since a normalization of GFR was observed. Thus, these homeostatic mechanisms permit adequate renal sodium conservation during starvation in rats.


Angiotensin converting activity assessed in vivo is increased in hereditary hypertensive rats. 1. The angiotensin converting enzyme (ACE) activity of spontaneously hypertensive (SHR) and spontaneously hypertensive stroke-prone (SHRSP) rats was compared to the ACE activity of normotensive Wistar-Kyoto rats (WKY). 2. ACE activity was assessed indirectly in conscious unrestrained rats using the equipressor response end point to simultaneously calculate the extent of conversion of angiotensin I (AI) to angiotensin II (AII) and the pulmonary degradation of bradykinin (BK). 3. The pulmonary degradation of BK was significantly elevated (99.4%) in SHR rats whereas the elevation was not significant in SHRSP rats (99.2%) compared to WKY rats, even though the pulmonary inactivation of BK in WKY rats was higher (98.6%) than in normotensive Wistar rats (95.6% and 97.5%) previously studied. 4. Blood pressure responsiveness to intra-aortically injected BK (bolus injection and infusion) was markedly increased in SHR and SHRSP rats with no change in reactivity to sodium nitroprusside. 5. Conversion of AI to AII assessed by the equipressor doses of the hormones which produced a 20-mmHg rise in blood pressure was markedly elevated in SHR (86 +/- 4%) and SHRSP (80 +/- 7%) rats when compared to WKY rats (38 +/- 4%). 6. The marked increase in conversion of AI to AII in hypertensive animals, accompanied by an increased pulmonary degradation of BK in SHR rats, suggests that ACE activity is increased in conscious SHR and SHRSP rats and may participate in the genesis of hypertension in this model of genetic hypertension.


Endopeptidase and carboxypeptidase activities in human urine which hydrolyze bradykinin. 1. We have fractionated the bradykinin inactivating activity of human urine by stepwise elution chromatography on DEAE-cellulose and recovered 95% of the inactivating activity and 29% of the protein (absorbance at A280 nm). 2. Seven of nine fractions which presented activity were also tested for angiotensin I and II inactivating activity, angiotensin converting activity and for the hydrolysis of hippuryl-His-Leu and hippuryl-Arg. Sites of hydrolysis in bradykinin were determined by HPLC of the hydrolysates and fragments were compared with authentic peptides. 3. Cleavage sites demonstrated for Fractions A through G were: Phe8-Arg9 (A and B), Phe5-Ser6 (C and F), Pro7-Phe8 (D), Gly4-Phe5 and Pro7-Phe8 (E) and Pro3-Gly4 (G). 4. The relative molecular weight of the bradykininase activity present in each fraction, determined by gel filtration, was: 16 kDa (A), 70 kDa (B), 60 kDa (C), 88 kDa (D), 230 kDa (E), 45 kDa (F) and 49 kDa (G). 5. Bradykinin inactivating activity was inhibited 50-100% by 3 mMEDTA (A, B, D, E and G), 1 mMM 2-mercaptoethanol (A, B, C and G), 0.1 microM Hg2+ (A, C and G), 0.1 mM PMSF (C and F), 1 mM TPCK (C and F), 1 mM Zn2+ (C), 60 microM BPP5a and 40 microM BPP9a (D), 0.1 microM phosphoramidon (E) and 3 mM sodium p-hydroxymercuribenzoate (G). 6. The properties of some of these bradykinin inactivating activities correspond to enzymes previously described in urine and tissues: carboxypeptidases (Fractions A and B), angiotensin I converting enzyme (Fraction D), neutral endopeptidase (Fraction E). However, the chymotrypsin-like activity of Fractions C and F and the prolylendopeptidase activity of Fraction G have not been described before in urine and they are being purified in order to obtain a more accurate characterization.


Insulin autoantibodies in first degree relatives of type I diabetic patients. 1. Insulin autoantibodies (IAA) of first-degree relatives of type I diabetic patients and recent-onset type I diabetics were measured by radioimmunoassay. A cut-off of 60 nU/ml was established on the basis of the values of normal control individuals. The intra-assay coefficient of variation was 9.2% for a moderately positive serum (1908 +/- 176 nU/ml (mean +/- SD), N = 7; range, 1708 to 2158 nU/ml). The interassay coefficient of variation was 23.8% for a negative (normal control) serum (28.1 +/- 6.7 nU/ml, N = 6; range, 22 to 39 nU/ml) and 14.5% in a highly positive serum (6185 +/- 899 nU/ml, N = 7; range, 5053 to 7009 nU/ml). 2. Insulin autoantibody levels (mean +/- SEM) were 19.3 +/- 2.8 nU/ml (range, -19 to 40 nU/ml) in 25 controls, 24.8 +/- 3.4 nU/ml (range, -17 to 59 nU/ml) in 41 type II diabetic patients, 18.5 +/- 2.4 nU/ml (range, -58 to 268 nU/ml) in 171 first-degree relatives of type I diabetic patients and 208.9 +/- 87.0 nU/ml (range, 10 to 1101 nU/ml) in 16 recent-onset type I diabetic patients. IAA levels were significantly higher in the last group compared with the other groups (P < 0.01). 3. None of the controls or type II diabetics exceeded the upper limit of normality. In contrast, 9 of 171 (5.3%) first-degree relatives and 9 of 16 (56.0%) recent-onset type I diabetic patients had IAA levels above the 60 nU/ml cut-off point.(ABSTRACT TRUNCATED AT 250 WORDS)


Serum levels of tumor necrosis factor in insulin-dependent diabetic patients. We determined the serum concentrations of tumor necrosis factor (TNF) in 15 nondiabetic healthy subjects and in 36 insulin-dependent (type I) diabetic outpatients. The mean (+/- SD) annual fasting plasma glucose, urine glucose and HbA1 levels of the diabetic group were 179 +/- 71 mg/dl, 13.0 +/- 13.2 g/day and 12.3 +/- 1.3%, respectively. The mean serum TNF concentration measured by immunoradiometric assay of the diabetic group (8.6 +/- 1.9 pg/ml) was significantly higher than healthy controls (6.9 +/- 0.9 pg/ml). Within the diabetic group, there was no correlation between serum TNF levels and either duration of diabetes or indices of metabolic control. However, serum TNF levels progressively increased from the well to the poorly controlled diabetic groups: 8.1 +/- 1.5 (G), 8.2 +/- 1.4 (F) and 9.4 +/- 2.4 (P) pg/ml, respectively, which parallel levels of HbA1 (%): 8.4 +/- 2.4, 11.7 +/- 1.8 and 14.6 +/- 1.2, respectively. Serum TNF levels of the diabetic patients with chronic complications (N = 7, 9.5 +/- 2.3 pg/ml) and without complications (N = 29, 8.4 +/- 1.7 pg/ml) were statistically higher than control subjects. The progressive increase of the serum TNF levels from the well to the poorly controlled diabetic groups suggests a relationship between levels of this cytokine and protein glycosylation.


Clinical utility of a 22-kDa growth hormone-specific assay. Human growth hormone (hGH) circulates in different molecular forms, with the 22-kDa monomer being the predominant one and the 20-kDa variant corresponding to 5 to 15% of the serum hGH on a weight basis. Using monoclonal antibodies with different specificities we developed two immunoenzymometric assays, one with 22 + 20-kDa specificity and the other specific only for the 22-kDa form. Both assays used microtiter plates as solid phase and streptavidin-peroxidase for color development; intra-assay CV was less than 10% in the range of 1 to 100 mIU/l for the 22 + 20-kDa assay and in the range of 3 to 100 for the 22-kDa assay, with an inter-assay CV of less than 14% for both assays; sensitivity was 0.2 mIU/l for the 22 + 20-kDa assay and 0.5 mIU/l for the 22-kDa assay. The two assays were compared by measuring 200 serum samples with detectable hGH levels by both assays. Higher values were obtained with the 22 + 20-kDa assay (62.1 +/- 5.9 vs 59.2 +/- 6.1 mIU/l, mean +/- SD) with a correlation coefficient (r) of 0.99. In no clinical condition (28 patients with growth retardation and 14 acromegalics) did the two assays give discrepant values. We conclude that there was no practical advantage in using an assay with specificity restricted to the 22-kDa form for measuring hGH in clinical serum samples.


A method for screening drugs against the liver stages of malaria using Plasmodium gallinaceum and Aedes mosquitos. 1. The radical cure of human malaria caused by Plasmodium vivax requires two drugs, i.e., a blood schizontocide such as chloroquine to clear the circulating parasites, and primaquine aimed at the liver stages (hyponozoites) responsible for the late relapses of this parasite. Primaquine is unique as a radical curative drug but is highly toxic. The only useful model currently available for screening drugs to replace primaquine is Plasmodium cynomolgi-induced malaria in Rhesus monkeys. Because of the limited availability and cost of these animals, the development of non-primate models for such screening would be of considerable value. 2. We used a drug-screening assay for the liver stage malaria parasite based on the ability of such drugs to stop development of gametocytes in the mosquito vector. The inhibition of the sporogonic cycle of malaria in the mosquito by primaquine (15 mg/kg) was confirmed here and used for re-evaluation of the gametocyte method. 3. We observed that the level of parasitemia in the untreated control chicken used to infect mosquitos was a crucial factor affecting the subsequent development of sporogony. Thus, parasitemia was carefully controlled in the studies involving oocyst development. Parasitemias lower than 6% at the beginning of the experiment and increasing were found to be most appropriate for the production of the infectious gametocytes during a period of 8 h.(ABSTRACT TRUNCATED AT 250 WORDS)


Pressor effects of acetylcholine microinjected into forebrain nuclei of unanesthetized rats. 1. The injection of 13.5-54 nmol/500 nl of acetylcholine (ACH) into different brain areas of unanesthetized freely-moving 200-250 g male Wistar rats caused only pressor responses. 2. In the prosencephalon, the lateral septal area was the site at which ACH was more effective, whereas injections into surrounding areas, such as the accumbens/bed nucleus striae terminalis, the medial septal area or the lateral ventricle were less effective. No blood pressure effects were observed after injection into the anterior amygdala. 3. In the diencephalon, the ventromedial hypothalamic nucleus was the most sensitive site, whereas injection of ACH into surrounding areas, such as the posterior and lateral hypothalamic or the dorsal and ventral premammillary nuclei was less effective. 4. At all sites tested, the local pretreatment with 138-276 nmol atropine abolished the pressor response to ACH, suggesting a mediation through muscarinic receptors. 5. The sites of injection were confirmed histologically. 6. The present data indicate the existence of a cholinergic-sensitive site involved in the control of blood pressure at the level of the lateral septal area.


Effects of undernutrition during suckling on footshock escape behavior and of post-training beta-endorphin administration on inhibitory avoidance task test behavior of young rats. The hypothalamic beta-endorphin system of young Wistar rats of both sexes (21-day-old) responds in a distinct way to behavioral situations when compared to adult rats (90 to 120-day-old). In the present study we investigated whether the post-training amnestic effect of beta-endorphin previously demonstrated in Wistar adult rats is also observed in young (21-day-old) well-nourished and undernourished rats. Rats were undernourished since birth by feeding their dams an 8% casein diet, while well-nourished offspring were fed by dams maintained on a 20% casein diet. beta-endorphin was administered after training in a step-down inhibitory avoidance task using a 0.2- or 0.8-mA footshock. Retention was tested 24 h later. We observed that the dose of beta-endorphin (1 microgram/kg, ip) previously reported to have an amnestic effect on adult rats was ineffective in weanling rats of both nutritional groups. At a higher dose (2 micrograms/kg, ip) and using a 0.2-mA shock, beta-endorphin impaired the retention only of well-nourished rats. Test-to-training difference (in s) in step-down latency for well-nourished beta-endorphin-treated rats was 7 vs 25 s for well-nourished rats treated with saline (P < 0.05). Undernourished rats were hyperreactive to this shock intensity. Footshock escape latency (in s) for undernourished rats was 3.56 vs 5.80 for well-nourished rats (P < 0.05, experiment 1) and 5.01 vs 10.89 (P < 0.05, in experiment 2) and showed better retention than did well-nourished rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of sodium valproate on the open-field behavior of rats. 1. It has been reported that sodium valproate induces a morphine-like withdrawal syndrome in rats. The effects of acute or chronic treatment with sodium valproate on rat behavior was studied in the open-field test. 2. Acute sodium valproate (320 mg/kg, intraperitoneally) decreases the frequency of, and the time spent in grooming even when not modifying locomotion, rearing or defecation (N = 15), either 15 or 60 min after an acute treatment. This effect was not modified (N = 10) by concomitant administration of morphine (2 mg/kg) or naloxone (1 mg/kg). 3. Interruption of prolonged (30 days) valproate treatment with increasing doses of 40 to 320 mg/kg, by gavage, twice daily (N = 10) did not modify rat behavior in the open-field, from the first to the fourteenth day of the test. 4. We conclude that the decreased novelty-induced grooming does not depend on the opioid system and may be related to an anti-anxiety effect of valproate.


Cardiovascular effects on conscious rats of pretreatment with isoproterenol for 3 days. The cardiovascular effect of isoproterenol pretreatment (ISO) mimicking short duration sympathetic hyperactivity was studied in conscious adult male albino rats. Cardiac hypertrophy of right and left ventricles was observed after pretreatment with isoproterenol for 3 days (0.1 mg/kg, 3 times a day), when compared to saline-treated rats. The pressor response to noradrenaline (NOR, 1 to 1000 micrograms/kg, iv) was slightly but consistently larger in the ISO group than in the control group (in mmHg, Control = 109 +/- 3.5; NOR 1 = 114 +/- 3.3; NOR 10 = 116 +/- 4; NOR 100 = 127 +/- 5.6 and NOR 1000 = 152 +/- 7.3 for saline-treated rats and, Control = 106 +/- 5.1; NOR 1 = 112 +/- 5.1; NOR 10 = 118 +/- 5.3; NOR 100 = 134 +/- 4.9 and NOR 1000 = 162 +/- 7.5 for ISO groups). An increased reflex bradycardia was also observed in response to increasing doses of noradrenaline (in bpm, Control = 323 +/- 5.6; NOR 1 = 313 +/- 6.8; NOR 10 = 309 +/- 8.6; NOR 100 = 293 +/- 7.5 and NOR 1000 = 284 +/- 5.3 for saline-treated rats and Control = 316 +/- 4.3; NOR 1 = 294 +/- 4.3; NOR 10 = 289 +/- 3.4; NOR 100 = 267 +/- 4 and NOR 1000 = 251 +/- 2.6 for ISO groups; ANOVa, P < 0.01). There were no significant differences in autonomic tone to the heart between control and isoproterenol-pretreated animals. The present results indicate that isoproterenol treatment induces significant in vivo cardiovascular modifications.


Inhibition of insulin release by Jatrophone. In many countries, including Brazil, extracts of Jatrophona elliptica species are currently used for the treatment of several diseases. Recently it was shown that a purified compound from these plants inhibits contraction of smooth and cardiac muscle in the microM range, probably involving alterations in membrane Ca2+ permeability and/or internal Ca2+ distribution. In collagenase-isolated rat islets and in the absence of glucose, basal insulin secretion measured by radioimmunoassay averaged 122 +/- 13 microU/islet per 90 min (N = 25). At 16.7 mM glucose, the insulin output reached 445 +/- 32 microU/islet per 90 min (N = 27). Jatrophone (1-100 microM/l) caused a dose-related inhibition of glucose-induced insulin release, over basal secretion, with an ID50 close to 8 microM/l. Complete inhibition of insulin release was obtained with 100 microM/l Jatrophone. However, at 100 microM/l (but not at 10 microM/l) concentration, Jatrophone also provoked a reduction in glucose metabolism by the islets which could explain, at least in part, the reduction in insulin secretion. After 120-min incubation, the glucose metabolism, measured by the 14CO2 production, was reduced from 26.58 +/- 3.63 (N = 42) to 7.48 +/- 1.36 (N = 16) pmol/l per islet. In conclusion, at lower concentrations (10 microM/l) Jatrophone could be a valuable tool for the study of the mechanism of insulin release induced either by glucose or other secretagogues.


In vivo effect of glucocorticoids on the rates of pyruvate utilization and lactate formation by incubated lymphocytes. Although the immunosuppressive effect of glucocorticoids has been widely reported, the mechanism of action of these hormones on the immune system has not been fully established. In the present study, the effect of glucocorticoids on the rates of pyruvate utilization and lactate production by lymphocytes obtained from rat mesenteric lymph nodes was investigated. Adult rats were injected with dexamethasone (1 mg/kg, ip) daily for 3 days. A stressed group which received a corresponding volume of 0.9% saline and control rats not injected were also included. All treatments were carried out at 17:00 h. Stress or administration of dexamethasone reduced the rate of pyruvate utilization by 50% and 62%, respectively. The hormonal injection increased the rate of lactate production by 57%, whereas the stressed group showed a 2.4-fold increase of the conversion of pyruvate to lactate, as compared to control rats. These findings suggest that glucocorticoids regulate pyruvate metabolism in lymphocytes and that an additional neurohormonal factor released during stress may also participate in the control of lactate production.


Lessons in long-term care: the benefits of a northern exposure. Canada stands as an important lesson for the United States. At the very minimum it represents the art of the possible. For those interested in long-term care, it suggests that it is possible to include long-term care in a programme of universally accessible care without national bankruptcy. Although Canada has brought medical and social services closer together and has brought acute and chronic care under the same aegis, not all the problems of coordinating such efforts have been solved. For others, the Canadian example shows that care can be provided to all persons who need it without creating uncontrollable expenses or removing families' motivations to care for their own. In so doing, universal coverage creates the opportunity for better coordination of services and ultimately for more efficient care.


Quality assurance in Canada: what are the health professions doing? Although quality assurance (QA) programming in Canada is still at an early stage of development, health professionals are increasingly being called upon to substantiate the value of the services they provide. A body of research showing wide variations in the rates of service provision and significant amounts of inappropriate care have convinced many policy makers about the need to improve quality in the Canadian health system. Recent and severe economic pressures on provincial government funders could foster a more rational approach to resource allocation, including the consideration that better quality care is more efficient care. The bulk of health care in Canada is delivered in the private offices of practitioners, where quality assurance programming is relatively undeveloped. Although some licensing organizations do conduct proactive routine audits of their members' practices, a national survey, based on self-report, indicated that most of these programmes do not conform to recommendations found in the quality assurance literature. Although there have been some new initiatives in Canadian QA, it remains to be seen how these will influence the evolution of quality assurance programming conducted by Canada's health professions.


Management of human resources in health care: the Canadian experience. Each of Canada's ten provinces has a publicly administered system of health insurance, funded by provincial and federal taxes, that is accessible to all citizens and covers all medically necessary services provided by physicians and hospitals. Canadians spend an estimated 9.2 percent of their gross national product on health care (about 2.8 percentage points below US spending), of which three quarters is public-sector spending. According to the Organization for Economic Cooperation and Development, Canada's health status is equal to or better than that of the United States, despite lower per capita health spending. About seven percent of the Canadian labour force works in health care, and attempts to introduce coordinated planning of human resources in health care have not as yet proceeded far. The predominant policy issue here is the supply and the role of physicians. It has been argued that entrenching within the system the fee-for-service method of paying physicians has created a disincentive to the delegation of responsibility to health personnel other than doctors. It is also argued that introduction of government-run health insurance provided the opportunity for human resource planning, but that the decision by governments to act only as the payer resulted in ad-hoc planning approaches. However, governments' concern over health care costs has led to a more direct role by them in the planning of the human resources in health. They are re-examining the autonomy and jurisdictional rights of the professions that deliver health care to Canadians.


Fairness and feasibility in national health care systems. The issue of National Health Care reform has been discussed from many different perspectives. One of the most fundamental justifications for such reform is based on the notion of horizontal equity. The motivation for equity in medical services use contrasts with the seeming lack of concern for equity in financing. Proposed policy remedies often involve transfers through taxation, though the effects of government taxation often reduce the efficiency of publicly financed health insurance. Further, the effects of heterogeneous preferences complicate the assessment of optimal medical service consumption levels. Ethical justification aside, this paper addresses the notion of fairness in the provision of medical care from an economic perspective. It suggests policies which might be most suitable in achieving such a goal. A paradoxical result of these considerations of fairness is that unequal insurance coverage is requisite to ensure more equal utilization of medical services by the population. The concept of fairness is then developed into a bottom up system of equity in the medical market.


Forces for reforming the U.S. health care system: a review of the cost and access issues. In recent years, a spate of health care reform proposals have emerged on the American agenda. Although the elements of the reform proposals often vary substantially, most of the initiatives are fuelled by two common concerns: rising health care expenditures and a growing uninsured population. National health spending, for example, commands an increasing share of U.S. gross national product despite numerous cost-containment efforts initiated by public and private payers throughout the 1980s. And the uninsured population continues to grow--by an estimated 30 percent between 1978 and 1989. To facilitate understanding of the public policy options being considered to address these concerns, the article examines U.S. health care expenditure data and some of the causes of rising health care costs. The article also discusses the demographic characteristics of the uninsured population, the reasons why they lack health coverage, and the health consequences associated with being uninsured.


Proposals to reform the U.S. health care system: a critical review. The objective is to provide an overview and analysis of the current discussion of ways to reform the U.S. health care system. A common analytic framework is needed to evaluate the alternative approaches that are being advocated. Such a framework, organized around seven general questions, is developed and discussed. The analytic framework is then applied to five specific reform proposals, selected to be representative of the range of options being considered. The results are used to identify the basic choices that are inherent in the current discussion of health system reform. A discussion of the political realities of the health system reform movement in the U.S. concludes that an incremental reform measure will be implemented at the federal level in the near future and that the reform measure will give the federal government increased control over the health care sector. However, the pressure for more fundamental reform will continue to grow.


Pooling international health care expenditure data. The purpose of this paper is twofold. The first is to analyse the statistical relationship between real health care expenditure per capita and aggregate income, public share in finance, age-dependency ratio and inflation. The second purpose deals with methodological problems involved in pooling health care expenditure data. The empirical work is based on pooled cross-sectional, time-series data for 22 OECD countries from 1972 to 1987. Public finance share and inflation were found to be associated with lower per capita health care expenditure. No consistent correlation was found between the age-dependency ratio and health care expenditure. Contrary to results of earlier studies, we found that health care expenditure does not appear to be income (GDP) elastic. However, the results do not appear to be robust to changes in the time periods and countries included.


Appraising the use of contingent valuation. The valuation of treatments and health states has been pursued in a number of ways. Most predominant are contingent valuation (CV), QALYs, and HYEs. CV--that is, willingness to pay and willingness to accept--is the only one of these methods that can be consistent with welfare economic theory, but, as discussed by Gafni (1990), in order to do so three criteria must be met. This article argues that the fulfilment of these criteria is not sufficient to obtain useful results, and some additional criteria are suggested. Several CV studies carried out in the area of health are reviewed, and their compliance or non-compliance, with both sets of criteria, is discussed. Finally, it is argued that, although CV is the more theoretically correct method, it is not a superior tool to QALYs and HYEs, and that the decision as to which is the appropriate valuation method depends on the policy issue at hand.


Are increases in cigarette taxation regressive? Using the latest published data from Tobacco Advisory Council surveys, this paper re-evaluates the question of whether or not increases in cigarette taxation are regressive in the United Kingdom. The extended data set shows no evidence of increasing price-elasticity by social class as found in a major previous study. To the contrary, there appears to be no clear pattern in the price responsiveness of smoking behaviour across different social classes. Increases in cigarette taxation, while reducing smoking levels in all groups, fall most heavily on men and women in the lowest social class. Men and women in social class five can expect to pay eight and eleven times more of a tax increase respectively, than their social class one counterparts. Taken as a proportion of relative incomes, the regressive nature of increases in cigarette taxation is even more pronounced.


Traumatically induced axonal injury: pathogenesis and pathobiological implications. This work reviews the pathobiology of traumatically induced axonal injury. Drawing upon literature gleaned from the experimental and clinical setting, this review attempts to emphasize that, other than the most destructive insults, traumatic brain injury does not typically cause direct mechanical disruption of the axon. Rather, this review documents that with traumatic injury focal, subtle axonal change occurs, and that over time, such change leads to impaired axoplasmic transport, continued axonal swelling, and ultimate disconnection. The initial intra-axonal events that trigger the above described sequence of reactive axonal change are considered with focus on the possibility of either traumatically altered axolemmal permeability, direct cytoskeletal damage/perturbation, or more overt metabolic/functional disturbances. Not only does this review focus on the sequence of traumatically induced axonal change, but also, it considers its attendant consequences in terms of Wallerian degeneration and subsequent deafferentation. The concept that traumatically induced diffuse axonal injury leads to diffuse deafferentation is emphasized together with its pathobiological implications for morbidity and recovery. The potential for either adaptive or maladaptive neuroplasticity subsequent to such diffuse deafferentation is considered in the context of mild, moderate and severe traumatic brain injury.


The pathological diagnosis of specific inflammatory myopathies. Pathological diagnosis of dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM) should be possible in almost all cases when an appropriately involved muscle is biopsied. DM shows characteristic patterns of muscle fiber damage and capillary damage. Lymphocytes and macrophages are seen in PM and IBM partially invading non-necrotic fibers. IBM is also characterized by rimmed vacuoles with membranous whorls, characteristic masses of filaments in cytoplasm and sometimes in nuclei, and grouped atrophic fibers. Muscle fiber damage in PM is more variable. Inflammatory myopathy can be associated with HTLV-1 and HIV infection. In the latter a strong resemblance to PM is reported. Separate, still less well characterized forms of inflammatory myopathy occur in young children.


The generation of cellular diversity in the cerebral cortex. We have studied cell lineage in the rat cerebral cortex using retroviral vectors. With this technique, the virus is used to introduce a marker gene into dividing precursor cells such that their fate can be followed. We have studied cell lineage by using this method in the following ways. First, we have labelled germinal cells of the cerebral cortex in vivo during the period of neurogenesis. Second, we have grown cortical precursor cells in dissociated cell culture, and used the viral labelling technique to follow their development in vitro. Both types of study have shown that by the time neurogenesis is under way, the majority of precursor cells are restricted to the production of a single cell type: neurones, astrocytes, or oligodendrocytes. The only exception is a cell we call the N-O cell because it has the ability to generate both neurones and oligodendrocytes. These data suggest that the ventricular zone, the germinal layer of the embryonic cortex, is a mosaic of different precursor cells each with a different restricted potential. However, this restriction of potential of cortical precursor cells does not extend to their ability to contribute to more than one cortical lamina or cytoarchitectonic area. The precursors of both neurones and grey matter astrocytes contribute cells to multiple layers of the cortex. Moreover, in the hippocampal formation, neuronal precursors can contribute cells to more than one hippocampal field.


Integration site-dependent transgene expression used to mark subpopulations of cells in vivo: an example from the neuromuscular junction. To produce transgenic mice, an exogenous gene is inserted into the germ line, usually by injection of the DNA construct into a pronucleus of a fertilized egg. In most cases the transgene is integrated into the genome at a single random site. Frequently, the transgenes are combinations of regulatory elements from one gene and protein coding sequences from another gene, the reporter. As expected, the promoter in the construct usually controls the expression pattern of the reporter. In some cases, however, transgenes have been constructed with regulatory elements that are not able to direct transcription on their own. In animals containing such transgenes, the expression of the reporter is dependent on endogenous regulatory elements near the chromosomal site of transgene integration. In the present study, an Escherichia coli beta-galactosidase (lacZ) reporter gene linked to a weak promoter was selectively expressed in discrete subpopulations of cells in each of eight independently derived lines of mice. In one line (line 42), which we analyzed in detail, a subset of cells in skeletal muscle were lacZ-positive. Specifically, fibroblasts close to neuromuscular junctions expressed the lacZ-protein, whereas skeletal muscle fibroblasts far from synaptic sites and fibroblasts in other organs were lacZ-negative. Moreover, Schwann cells at nerve terminals were lacZ-positive, whereas Schwann cells in extramuscular nerves were lacZ-negative. These results indicate the existence of differences between perisynaptic and extrasynaptic fibroblasts in normal skeletal muscle. They also demonstrate how such transgenic mice can be used to identify and mark discrete cell populations.


From chance to choice in the generation of neural cell lines. Despite the central importance of cell lines in contemporary studies in cellular and molecular biology, many areas of potential investigation remain impeded by the limited number of lines available and by the difficulty in generating new lines of interest. Thus, there has been a constant pressure to develop improved methods for obtaining cell lines of particular interest. This review examines some of the problems associated with in vitro approaches to cell line generation. In addition, two different ways in which transgenic animals can be used to overcome the limitations of in vitro production of cell lines are discussed. In the first approach, specific promoters are utilized to target expression of immortalizing genes to cells of interest. The second approach is concerned with development of a strain of transgenic animals (the H-2KbtsA58 transgenic mouse) designed to obviate the need for identification of cell-type specific promoters, and in which it is theoretically possible to directly generate conditionally immortal cell lines from any tissue of the body by simple dissection and growth of cells in appropriate tissue culture conditions. Finally, approaches are also discussed in which investigations on the control of precursor differentiation have been applied so as to bypass the need for expression of activated immortalizing oncogenes in the generation of large quantities of conditionally immortalized cells with the capacity to undergo normal differentiation in vitro and in vivo.


Retrovirus-mediated oncogene transfer into neural transplants. A gene transfer model was developed which allows for the identification of transformation pathways in the developing nervous system. Transforming genes were introduced into fetal brain transplants using embryonic CNS as donor tissue and replication-defective retroviral vectors as genetic vehicles. This technique relies on the extraordinary organotypic differentiation capacity of neural grafts and the expression of retrovirally transmitted genes in various cell types of CNS transplants. In contrast to transgenic animals but analogous to sporadic tumor formation, target cells for the retroviral vector develop in an environment of unmodified neural tissue. We have introduced a number of neurotropic oncogenes into fetal brain transplants including genes with an associated tyrosine kinase activity (polyoma medium T, v-src), a novel member of the fibroblast growth factor (fgf) gene family and the SV40 large T antigen. These experiments have demonstrated a significant transformation potential of oncogenes in specific target cells of the brain, provided evidence for a dominant complementary transforming effect of simultaneously expressed ras and myc genes in neural precursor cells and have yielded intriguing model systems for human CNS neoplasms such as the cerebellar medulloblastoma. This review describes the transplantation model, demonstrates several striking phenotypes induced by oncogene expression in neural grafts and elaborates on future prospects of this experimental approach.


Human foamy virus: an underestimated neuropathogen? Human foamy virus (HFV) is a recently characterized retrovirus which was originally isolated from patients with various neoplastic and degenerative diseases. However, until today it has not been possible to identify HFV as the causative agent of any disease and little is known about its prevalence in human populations. Like HTLV and HIV, HFV encodes the three structural retroviral genes, gag, pol and env, and an additional region containing three open reading frames, bel-1 to bel-3. Bel-1 activates transcription of the long terminal repeat of HFV and HIV. In order to study the consequences of expressing HFV regulatory genes and to investigate a possible pathogenic potential of HFV, we have introduced parts of the HFV genome into the germ line of mice. Our studies with transgenic mice demonstrate that HFV transgenes encompassing the bel region are transiently transcribed between midgestation and birth at moderate levels in various tissues. Expression is then suppressed, but resumes after a latency of several weeks in a restricted range of tissues and leads to extensive accumulation of HFV transcripts in single cells. This is associated with a progressive degenerative disease of the central nervous system and of striated muscle. These findings provide the first evidence of a disease induced by HFV and suggest that HFV might also act as a human pathogen in neurological diseases. Moreover, the transgenic mouse model will be useful for studying the molecular basis of HFV-induced neurotoxicity, the role of individual disease-associated HFV genes and the regulation of retroviral latency.


The N-myc proto-oncogene: developmental expression and in vivo site-directed mutagenesis. The N-myc proto-oncogene is a member of the superfamily of transcription factors. In mammals, expression of this gene is predominantly restricted to the developing embryo. Specifically, the level of expression is highest in differentiating epithelial components of the embryo including those of the developing brain, kidney and lung. The observation that N-myc is expressed in differentiating but not terminally differentiated structures suggests that these genes may function in the maintenance of cells in a determined or proliferative state. Available evidence suggests that when N-myc expression is down-regulated, cells progress through differentiation and acquire their terminal phenotype. N-myc expression is also correlated with poor prognosis in a number of tumor systems. Since malignant tumors are usually poorly differentiated, this may reflect the role that N-myc plays in preventing differentiation of otherwise determined cells. In vivo site-directed mutagenesis by homologous recombination has made it possible to introduce a variety of mutations into mice. This review summarizes this technology and describes our initial results in the characterization of mice that lack a functional N-myc gene. Specifically, we have observed that in the absence of a functional N-myc gene, embryos arrest in midgestation. This body of work demonstrates that this gene is not required for normal development until the onset of organogenesis.


New morphological approaches to the study of mitochondrial encephalomyopathies. Molecular genetics, biochemistry, immunology and morphology, are being applied in a coordinated fashion to unveil the molecular basis of the mitochondrial encephalomyopathies. Mutations of mitochondrial DNA (mtDNA) have been found in well characterized clinical groups of these disorders. New and old morphologic methods have been applied to investigate muscle biopsies from patients with mtDNA mutations. Important observations have been made on the cellular localization of normal and mutated mtDNA and on the expression of mtDNA-encoded polypeptides. These observations have provided insight into the pathogenesis of respiratory chain enzyme deficiency at the level of individual muscle fibers. Application of immunocytochemical and in situ hybridization techniques at the electron microscopic level will extend these studies to the level of individual mitochondria.


Defects of mitochondrial DNA.

Defects of mitochondrial DNA. In the past few years several syndromes have been associated with lesions of the human mitochondrial DNA. MtDNA is a small, circular extra-nuclear chromosome encoding essential components of the respiratory chain. MtDNA-related syndromes can be divided into two groups: mitochondrial encephalomyopathies, characterized by the presence of ragged-red fibres (RRF) as the morphological hallmark, or "pure" encephalopathies with no gross morphological abnormalities in muscle. The first group includes myoclonic epilepsy with ragged-red fibres (MERRF), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Kearns-Sayre syndrome (KSS), chronic progressive external ophthalmoplegia (CPEO) and a new entity, maternally inherited myopathy and cardiomyopathy. The second group includes Leber's Hereditary Optic Neuroretinopathy (LHON) and the newly described ataxia-retinitis pigmentosa-dementia complex. Three kinds of molecular lesions have been identified: point mutations of protein encoding mtDNA-genes (similar to yeast mit- mutations); point mutations of mtDNA-tRNA genes (similar to yeast syn- mutations); and large-scale rearrangements of mtDNA (similar to yeast rho- mutations). In general, "mit-" mutations are responsible for non-RRF encephalopathies, while "syn-" and "rho-" mutations are associated with mitochondrial encephalomyopathies with RRF. Furthermore, point mutations (mit- and syn-) are usually maternally- inherited, while large-scale mtDNA rearrangements are either sporadic or inherited as mendelian traits. In most cases, the molecular detection of the known defects of mtDNA can be carried out by non-invasive techniques, thus making it an easy and relatively inexpensive procedure in the differential diagnosis of the mitochondrial disorders, a rapidly expanding area of clinical neurology.


Disorders associated with multiple deletions of mitochondrial DNA. Multiple deletions of mitochondrial DNA (mtDNA) have recently been described in a number of patients with neurological disorders. Most cases have been clinically characterized by autosomal dominant inheritance, adult onset, and a slowly progressive course with external ophthalmoplegia and muscle weakness. Some patients have had evidence of central or peripheral nervous system involvement or episodes of myoglobinuria. Muscle biopsy findings include ragged-red fibres (RRF), muscle fibres with absent COX-activity and abundant abnormal mitochondria with paracrystalline inclusions. Biochemically, a generalized reduction in the activities of mtDNA-encoded enzymes is observed in skeletal muscle. Southern blotting or PCR analysis reveal multiple populations of deleted mtDNA. The deletions occur at multiple sites between the replication initiation sites, involving a large portion of mtDNA, and most deletions seem to be flanked by direct sequence repeats, shown to be "hot spots" in the case of single large deletions. Apparently, a defect in a nuclear gene results in multiple deletions of mtDNA. Both clinical, genetic and molecular genetic observations indicate heterogeneity of this new disease category, apparently based on a disturbance in the "cross-talk" between the nuclear and the mitochondrial genomes.


Disorders associated with depletion of mitochondrial DNA. Quantitative defects of mtDNA have been recently described in patients with fatal mitochondrial disease of early infancy or mitochondrial myopathy of childhood. There was variable tissue expression and depletion of up to 98% of mtDNA in affected tissues. Pedigree analysis was compatible with mendelian inheritance, suggesting faulty communication between nuclear and mitochondrial genomes, but the primary molecular lesion is unknown. In muscle, morphological studies allowed to correlate mtDNA depletion, absence of mtDNA-encoded peptides, mitochondrial proliferation, and loss of cytochrome c oxidase (COX) activity in individual fibers.


Mitochondria and ageing.

Mitochondria and ageing. This work reviews the role of mitochondria in the ageing process and summarizes pathomorphological biochemical and molecular genetic data. The pathophysiological mechanisms underlying the phenomenon of ageing are only partly understood. There is, however, increasing evidence that mitochondria are essentially involved. In various tissues of various species a decline in the respiratory chain capacity is seen with ageing. Enzyme histochemistry of cytochrome c oxidase (complex IV of the respiratory chain) has revealed an age-related increase of randomly distributed defective fibres/cells in the skeletal and heart muscle the random pattern probably indicating cellular heterogeneity of the ageing process. Observed deletions of mitochondrial DNA during ageing may represent one causative factor. Similar to primary mitochondrial myopathies point mutations and depletion of the mtDNA are probably also involved. There is some evidence that damage of the mitochondrial genome and of other mitochondrial structures might be due to increased oxygen radical production during ageing. The role of nuclear influences on the degeneration of mitochondrial function remains, however, also to be determined. Nevertheless, the decline of respiratory chain function with ageing represents an important factor for the decline of functional organ reserve capacity in senescence.


Mitochondrial encephalomyopathies: defects of nuclear DNA. The term "mitochondrial diseases" encompasses a heterogeneous group of disorders in which a primary mitochondrial dysfunction is suspected or proven by morphologic, genetic, or biochemical criteria. Clinically, these progressive disorders usually affect muscle, either alone (mitochondrial myopathies) or in combination with other systems, most often brain (encephalomyopathies). Mitochondria are unique among intracellular organelles in that mitochondrial proteins are encoded by two genomes, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). The vast majority of mitochondrial proteins are encoded by the nuclear genome, whereas mtDNA (a circular, double stranded 16.5 kb molecule) encodes only 13 polypeptides, all of them subunits of respiratory chain complexes. In addition to structural genes, mtDNA also codes for 22 transfer RNAs and two ribosomal RNAs. Our understanding of mitochondrial diseases has grown at an impressive rate in the past few years, and most of the progress has been in the area of mtDNA genetics, where several mtDNA mutations have been associated with specific diseases (reviewed in this issue by Zeviani et al.). In comparison, our understanding of mitochondrial disorders due to nDNA lesions has lagged behind and, to date, molecular defects of nuclear genes have been documented in only a few patients. We will review which alterations in the nuclear genome can cause mitochondrial disorders and which criteria are useful in identifying such mutations. While several examples will be provided, this is not intended as a complete review of the subject.


Primary central nervous system lymphoma. Primary central nervous system lymphomas (PCNSL) are uncommon neoplasms accounting for less than 2% of brain tumours. Their incidence appears to be increasing across a wide age range, in both immunocompetent and immunosuppressed populations. Particular risk groups include those with congenital and acquired immunodeficiencies and transplant recipients. The spread of the AIDS epidemic has seen large numbers of complicating PCNSL develop. Epstein-Barr virus infection appears to play a role in the development of these lymphomas in the immunosuppressed population. The aetiology of these tumours in the immunocompetent is uncertain. Their tendency to remain within the nervous system is not well understood but may be a function of CNS binding molecules carried by lymphocytes. Clinically PCNSL may present with a wide variety of signs and symptoms and has a capacity to mimic many other neurological conditions. Radiologically they appear as hyperdense homogenous deposits in subcortical white matter. Although most lesions are intermediate or high grade B cell lymphomas, T cell lymphomas are being recognised with increasing frequency. Immunohistochemistry and genotypic analysis have an important role in accurately characterising PCNSL, particularly in stereotactic biopsies. Involvement of multiple areas of the neuraxis, the eye and multiple intracranial sites can occur in the absence of obvious systemic lymphoma. The role of surgery in their treatment is uncertain. A combination of radiotherapy and chemotherapy can increase the length of survival. The prognosis, however, remains poor in comparison with nodal lymphomas, and particularly so in those with AIDS.


Drainage of brain extracellular fluid into blood and deep cervical lymph and its immunological significance. Cerebral extracellular fluids drain from brain to blood across the arachnoid villi and to lymph along certain cranial nerves (primarily olfactory) and spinal nerve root ganglia. Quantification of the connection to lymph in rabbit, cat and sheep, using radiolabelled albumin as a marker of flow, indicates that a minimum of 14 to 47% of protein injected into different regions of brain or cerebrospinal fluid passes through lymph. The magnitude of the outflow to lymph is at variance with the general assumption that the absence of conventional lymphatics from the brain interrupts the afferent arm of the immune response to brain antigens. The immune response to antigens (albumin or myelin basic protein) introduced into the central nervous system (CNS) has been analysed using a rat model with normal brain barrier permeability. The micro-injection of antigen into brain or cerebrospinal fluid elicits a humoral immune response, with antibody production in cervical lymph nodes and spleen, and also affects cell-mediated immunity. Furthermore, antigen may be more immunogenic when administered into the CNS than into conventional extracerebral sites. Clearly, the afferent arm of the immune response to antigens, within the CNS, is intact. Modern studies suggest that the efferent arm is also intact with passage of activated lymphocytes into the brain. Results support a new view of CNS immunology which incorporates continuous and highly regulated communication between the brain and the immune system in both health and disease.


Pathways of fluid drainage from the brain--morphological aspects and immunological significance in rat and man. There is firm physiological evidence for the lymphatic drainage of interstitial fluid and cerebrospinal fluid from the brains of rats, rabbits and cats. The object of this review, is to describe firstly the morphological aspects of lymphatic drainage pathways from the rat brain and secondly, to explore through scanning and transmission electron microscope techniques, the possibility of similar lymphatic drainage pathways in man. Interstitial and oedema fluid spreads diffusely through the white matter in the rat and appears to drain into the ventricular cerebrospinal fluid. In grey matter, however, tracers pass along perivascular spaces to the surface of the brain and into the cerebrospinal fluid. Paravascular compartments in the subarachnoid space follow the course of major arterial branches to the circle of Willis and thence along the ethmoidal arteries to the cribriform plate of the ethmoid bone. Particulate tracers, such as Indian ink, enter channels in the arachnoid beneath the olfactory bulbs and connect directly with nasal lymphatics through channels which pass through holes in the cribriform plate. Proteins and other solutes may also drain along other cranial nerves. Thus, there is a bulk flow pathway for interstitial and cerebrospinal fluid from the rat brain into cervical lymphatics. In man, it is probable that diffuse interstitial drainage of fluid from the white matter occurs in a similar way to that in the rat. Furthermore, the anatomical pathways exist by which bulk drainage of fluid could occur along perivascular spaces from the grey matter into perivascular spaces of the leptomeningeal arteries and thence into the cerebrospinal fluid (CSF).(ABSTRACT TRUNCATED AT 250 WORDS)


The autosomal dominant dystonias.

The autosomal dominant dystonias. Dystonia is a term used to describe a specific set of abnormal movements that can occur as a symptom of a variety of neurologic disorders, but also as a disease entity in its own right. This review focuses on the primary dystonias and delineates the genetic contribution to these disorders. Included is a description of the well recognized forms of primary dystonias which manifest autosomal dominant inheritance, especially the "classic" type of early onset, generalized torsion dystonia, but also other clinically distinct forms such as myoclonic dystonia, paroxysmal dystonia, and DOPA-responsive dystonia. Also, a summary of the molecular genetic studies pertinent to these disorders and a discussion of the implications of recent genetic research for delineating the wide spectrum of this phenotypically and genetically heterogeneous group of diseases are forthcoming.


Hereditary Lewy-body parkinsonism and evidence for a genetic etiology of Parkinson's disease. The first systematic genetic study of Parkinson's disease (PD) was carried out by Mjönes in 1949. His results indicated autosomal dominant transmission with 60% penetrance. These conclusions, however, were long discounted because oligosymptomatic and atypical relatives were counted as secondary cases without clear justification. Subsequent surveys of patient and twin studies failed to confirm evidence of familial concentration and the hypothesis of a genetic etiology was over-shadowed by interest in possible environmental neurotoxins. A growing accumulation of pedigrees of histologically confirmed Lewy-body PD over the past several years has refocused attention on genetic factors. Fluorodopa positron emission tomography (PET) studies in oligosymptomatic co-twins of probands and recent clinicopathologic studies of atypical cases have provided retrospective support for Mjönes' methodology. A survey of a personal series of PD patients showed that the majority of those for whom pedigree information was available were familial. This along with another recently reported series confirm Mjönes' data showing similar segregation ratios for siblings and parents, a low ratio of maternal:paternal transmission and a marked asymmetry in the distribution of ancestral secondary cases. These findings favor monogenic autosomal dominant inheritance and show reason to argue against a multifactorial etiology or heteroplasmy. The clinical evidence justifies searching for gene loci linked to PD. Available methods are briefly outlined. Preliminary investigations have examined possible allelic associations, e.g., with alleles of MAO-A and debrisoquine hydroxylase and linkage to the tyrosine hydroxylase gene on chromosome 11p15.5 has been excluded in one study of juvenile familial parkinsonism. Linkage mapping studies are presently underway.


New insights into the clinical features, pathogenesis and molecular genetics of Huntington disease. Traditionally, a clinical diagnosis of Huntington disease (HD) presents no problems in patients with a positive family history, consistent with autosomal dominant inheritance, chorea or other extrapyramidal motor signs, and progressive mental decline. However, due to the slowly progressive nature of the disease and the slow evolution of signs and symptoms, it is often difficult to determine when at risk individuals are showing early signs. Moreover, the clinical recognition of both early and late-onset cases, and of choreic patients in whom a family history is lacking, presents special diagnostic challenges. In recent years, much progress has been made in the recognition of early clinical signs of the disease. Factors which have contributed to this understanding include the longitudinal study of large cohorts of at-risk individuals, particularly in Venezuela, the data from predictive testing programs, and the application of positron emission tomography (PET)-scanning to individuals without overt chorea. We are now able to identify persons at risk as being affected before they display overt and obvious involuntary movements.


Molecular genetics and neuropathology of Charcot-Marie-Tooth disease type 1A. Charcot-Marie-Tooth (CMT) syndrome describes a genetically and clinically heterogeneous group of polyneuropathies. Electrophysiologically, at least two types of CMT can be distinguished; CMT1 which has decreased nerve conduction velocities (NCV) and CMT2 which has normal or near normal NCV with decreased amplitudes. For CMT1, three gene loci (on chromosomes 1, 17 and the X chromosome) have been mapped. The locus on chromosome 17, CMT type 1A (CMT1A), is responsible for the most common form of CMT which has recently been shown to be associated with a large DNA duplication. Recent data demonstrates that the CMT1A phenotype results from an inherited DNA rearrangement and a gene dosage effect. The trembler (Tr) and allelic tremblerJ (TrJ) mice have been proposed as animal models for CMT. Tr has similar electrophysiological and neuropathological features to CMT1 patients and maps to mouse chromosome 11 in a region of conserved synteny with human chromosome 17p. Tr and TrJ have recently been shown to have different point mutations in regions encoding putative transmembrane domains of the myelin specific protein PMP-22. The human peripheral nerve-specific PMP-22 gene maps within the CMT1A duplication. PMP-22 is thus a candidate gene for CMT1A. This paper describes the molecular genetics of CMT1A and sural nerve pathology in CMT1A patients with the CMT1A duplication.


Menkes disease: an X-linked neurological disorder of the copper metabolism. Menkes disease is an X-linked, recessive disturbance of copper metabolism associated with a progressive clinical course and abnormal hair. The disease is dominated by neurological symptoms combined with connective tissue manifestations, most of which can be explained by the lack of important copper enzymes. Despite excessive accumulation of the metal in various tissues, a functional copper deficiency is evident, probably caused by a defective intracellular copper transport protein of unknown nature. The molecular basis of the copper disturbance has proven difficult to define and will most likely have to await cloning of the gene. The chromosomal region of interest has now been narrowed down to a sub-band on the long arm of the chromosome (Xq13.3), and positional cloning is in progress in a number of laboratories including our own. Identification of the Menkes gene will be of importance for our understanding of the cellular handling of copper and other trace elements.


Oskar and Cécile Vogt, Lenin's brain and the bumble-bees of the Black Forest. Oskar Vogt (1870-1955) was a prominent German neurologist and neuroanatomist with a strong interest in the pathogenesis of brain diseases. Together with his wife Cécile (1875-1962), he published landmark papers on the cyto- and myelo-architecture of the brain and the functional anatomy of the basal ganglia. He developed the concept of pathoclisis, i.e., the selective vulnerability of specific neuronal populations in the CNS. In the 1920's, Vogt created a multi-disciplinary brain research institute, the Kaiser-Wilhelm-Institut für Hirnforschung in Berlin-Buch. After Lenin's death in 1924, Oskar Vogt was called to Moscow where he formed a new brain research institute, with the main purpose to investigate the revolutionary's brain. After being dismissed from office by the Nazi government in 1937, the Vogts continued their work in a privately funded institute in Neustadt, the Black Forest.


Outpatient percutaneous transluminal angioplasty for peripheral arterial disease. Percutaneous transluminal angioplasty (PTA) of peripheral arterial stenoses or occlusions has become a widely recognized therapy in patients with disabling arterial insufficiency of the lower limbs. In most institutions, the procedure requires a two-day hospital stay, and official guidelines do not recommend ambulatory practice. Therefore, we reviewed our experience with outpatient PTA from 1986 through 1991. PTA was performed in an outpatient setting in 141 subjects suffering from peripheral arterial insufficiency (mostly intermittent claudication), which represents 51% of the peripheral PTAs performed in our institution during this period (n = 276). Immediate technical success was achieved in 127 (90%), resulting in 112 (79%) with clinical improvement at 10 days. Complications were recorded in 21 (15%) procedures, most of them being minor (n = 14, 10%). Fourteen patients (10%) required prolonged observation. None of the major complications (n = 7, 5%) and only one (0.7%) (groin haematoma with one-day hospital stay) of the minor side effects were attributed to the ambulatory aspect of the procedure. Outpatient PTA may be performed in selected patients suffering from symptoms of peripheral arterial disease without additional risk.


"Catch-up" growth in steroid dependent dermatomyositis treated with cyclosporin-A. To improve the short-term growth, and hopefully final height attainment, of children with dermatomyositis treated with chronic daily corticosteroid regimens. We have examined the growth of nine pre-pubertal children (7F, 2M), mean age 8.9 (range, 5.7-12.3) years, who had severe juvenile dermatomyositis and required persistent corticosteroid therapy. All were treated with cyclosporin-A. During a mean period of 1.3 years (range 1.0-1.7) cyclosporin-A treatment was associated with a marked decrease in the dosage of prednisolone, from a mean of 12.4 to 6.6 mg/day (p < 0.02), and an increase in height velocity standard deviation score from a mean of -1.37 to +1.39 at 6 months (p < 0.02) and -0.33 at one year. During the first year of treatment with cyclosporin-A, seven patients were able to either withdraw or reduce their prednisolone dose. No side effects of treatment were observed. Cyclosporin-A was a useful treatment modality in juvenile dermatomyositis and allowed a decreased dose of corticosteroids and associated "catch-up" growth.


Asymptomatic renal-vein thrombosis in adult nephrotic syndrome ultrasonography and urinary fibrin-fibrinogen products: a prospective study. The diagnosis of renal vein thrombosis (RVT), a frequent complication of adult nephrotic syndrome (NS), is generally made by means of invasive methods, i.e. renal venography, venous time of renal arteriography and, more recently, computed tomography (CT). We undertook a prospective study to evaluate the use of Doppler ultrasonography (DUS) and urinary fibrin-fibrinogen degradation products (FDPU) for the diagnosis of asymptomatic RVT. Thirty-one adult NS with non proliferative glomerulonephritis were studied. Reference procedures [(selective renal arteriography (n = 18) and renal vein CT (n = 13)] were performed blindly within a few days (48 hours in 17 patients) of renal vein DUS (search for a lack of venous flow) and measurement of FDPU (5 micrograms/min) (in 24 patients). DUS was not interpretable in one patient and positive in nine. Of these 9 patients, RVT was detected by reference methods in only two (sensitivity: 1, specificity: 0.75; positive predictive value: 0.22; negative predictive value: 1). Increased FDPU was observed in 4 patients, 2 of whom had an RVT (sensitivity: 1, specificity: 0.9; positive predictive value: 0.5; negative predictive value: 1). We conclude that DUS and FDPU are helpful for screening of RVT in asymptomatic NS patients; their negativity allow further radiological investigations to be avoided while positive results must be confirmed by reference methods.


Detecting medical problems in dentistry: a survey of 4,087 patients in The Netherlands. This study was conducted to determine whether a medical history could be an effective means of detecting medical problems in dental patients. A risk-related patient-administered medical questionnaire was completed by 4,087 patients in 47 dental practices. The data collected were summarized by means of descriptive statistics; the number of medical problems and the relationship between these problems and the age of the patient were analyzed. In all, 37.2% of these patients reported at least one medical problem. Hypertension, chronic bronchitis, allergies and medication were the items most frequently mentioned. The frequencies of heart disease, hypertension, endocrinologic and neurologic disorders increased with age, while allergies and chronic obstructive pulmonary disease were evenly distributed over the different age groups. The differences between the health of the patients in the various dental practices were not significant. On the basis of these results, it can be concluded that taking a medical history by means of a patient-administered questionnaire is an effective method of detecting medical problems of dental patients.


Unexplained fever and chronic fatigue: abnormal circadian temperature pattern. Standard clinical and biological investigations can be used to determine the origin of persistent and moderate fever in a large number of otherwise asymptomatic patients. However, in a small proportion of cases, isolated fever and fatigue persist despite the absence of detectable organic malfunction. This study was conducted to investigate the circadian thermic pattern in patients with apparently unexplainable fever and chronic fatigue and in those with fever of recognized origin. We recorded central temperature continuously for 24 hours in patients with moderate fever of both unexplained and recognized origin, and in a control group of healthy volunteers. A Fourier series was used for harmonic analysis. Thermic patterns specific to the three groups were identified by statistical and factorial analysis. The patients with fever of unknown origin and chronic fatigue were clearly characterized in terms of the phase, amplitude of the first (fundamental) harmonic and minimum circadian temperature. The abnormal central temperature pattern in these patients may prove to be an important step in the management of febrile patients.


Hip surgery skin cellulitis.

Hip surgery skin cellulitis. Of 583 erysipelas/cellulitis seen in our Department between 1981 and 1991, six (1.04%) were localized to the buttock and hip. Five patients had had a dynamic hip screw implanted for coxarthritis on the side where the skin infection developed. This does not appear to be secondary to superinfection of the surgical wound as the erysipelas/cellulitis occurred weeks (7 to 520) after surgery. The possible surgical compromise of the venous/lymphatic circulation appears to be the causative factor. In all patients, the course was satisfactory upon systemic antibiotic therapy and no recurrence was seen after a mean follow up of 28 months.


Effects of mild-to-moderate hyperglycaemia per se on glucose production and uptake in the elderly. In order to better understand the mechanisms responsible for the diminished glucose tolerance that occurs in the elderly, the present study aimed at investigating the effect of mild hyperglycaemia on glucose production and uptake in a group of aged subjects. For comparison, a group of young subjects was simultaneously investigated. Seven aged (71.8 +/- 2.3 yrs) and seven young (25.5 +/- 1.7 yrs) healthy non-obese subjects underwent two hyperglycaemic glucose-clamps having as targets plasma glucose levels 7.5 and 10.0 mmol/L. Contemporary infusion of D-[3-3H]-glucose allowed determination of glucose turnover parameters in basal conditions and during the clamps. Endogenous pancreatic secretion was inhibited by somatostatin (8.3 micrograms/min) while glucagon (67 ng/min) and insulin (0.15 mU/kg/min) were replaced by exogenous infusions. In basal conditions, glucose uptake (12.9 +/- 0.5 vs 14.4 +/- 0.4 mumol/kg/min; p < 0.05) and glucose metabolic clearance rate (2.58 +/- 0.15 vs 3.35 +/- 0.10 ml/kg/min; p < 0.01) were lower in elderly vs young subjects. In the hyperglycaemic glucose-clamps, we observed, in the elderly subjects, the persistence of a greater glucose production during mild (7.5 mmol/L) (11.6 +/- 0.4 vs 9.7 +/- 0.2 mumol/kg/min; p < 0.005) but not moderate (10 mmol/L) (3.5 +/- 0.1 vs 3.4 +/- 0.1 mumol/kg/min; NS) hyperglycaemia. In contrast, glucose-induced glucose uptake and glucose metabolic clearance rate were similarly affected by glucose infusions in both groups of subjects. Moreover, in elderly but not in young subjects, basal glucose disappearance rate was significantly negatively correlated with fasting plasma glucose levels (r = -0.84; p < 0.01). In the basal state, glucose uptake and glucose metabolic clearance rate are slightly impaired in elderly, compared to young subjects. Furthermore, in the elderly, endogenous glucose production is less suppressed by mild hyperglycaemia i.e. 7.5 mmol/L, than it is in young people. Such impairment in the inhibition of endogenous glucose production is not seen when blood glucose attains 10 mmol/L. We suggest that impairment in glucose tolerance in the elderly results from both reduced glucose uptake (in basal conditions) and excessive glucose production (at mild hyperglycaemic levels).


Mobilization of nonconjugative pBR322-derivative plasmids from laboratory strains of Escherichia coli to bacteria isolated from seawater. Mobilization of derivatives of plasmids pBR322 and pBR325 was shown to occur between Escherichia coli K12 strains in LB-broth at 37 degrees C, provided a mobilizer plasmid (F') was present either together with the nonconjugative plasmid or in a second donor strain. Evidence from restriction endonuclease analysis suggested that the mobilization was facilitated by a transposition phenomenon involving the "gamma-delta" sequence of F'. It was shown that mobilization of a derivative of pBR325 from E. coli K12 to bacteria isolated from seawater occurred in incubations in both LB-broth and filtered seawater and that Pseudomonas sp., Enterobacter aerogenes, Klebsiella oxytoca, E. coli, and Citrobacter amalonaticus isolates were recipient-active.


Behavior in agricultural soils of a recombinant Pseudomonas bacterium that simultaneously degrades alkyl- and haloaromatics. Pseudomonas sp. FR1 (pFRC20P) is a recombinant bacterium able simultaneously to degrade alkyl- and haloaromatics due to the xylXYZL and xylS genes from the TOL plasmid borne on the bacterial chromosome, and to the gene encoding for a gamma-lactone isomerase from Alcaligenes eutrophus on pFRC20P. The survival of this strain in sterile soils was shown to depend on the physicochemical properties of the soil. The recombinant information was stable in bacteria introduced in soils and conferred selective advantage to the host bacterium when the soils were amended with low amounts of p-methylbenzoate. However, relatively high amounts of this chemical significantly reduced survival of the bacterium. Survival was more seriously affected by chlorobenzoates than by methylbenzoates. Evolution of 14CO2 from p-methyl-14C-benzoate in soils confirmed that Pseudomonas sp. FR1 (pFRC20P) functions in a complex system in accordance with its design. Survival of Pseudomonas sp. FR1 (pFRC20P) was better at 4 degrees C and 25 degrees C than at 37 degrees C. On plates, the pFRC20P plasmid was mobilized by helper plasmids to P. putida at a frequency of about 10(-6) transconjugants per recipient. However, no transfer of recombinant DNA integrated on the chromosome was found, nor was lateral transfer of the recombinant DNA borne by Pseudomonas sp. FR1 (pFRC20P) detected in soils.


Identification of closely related Agrobacterium vitis isolates by chromosomal DNA probes. Crown gall formation on grapevine by Agrobacterium vitis is an important plant disease in many regions of the world. On grapevine, octopine/cucumopine (o/c) strains are widely distributed. Here we describe two chromosomal sequences of o/c strains of 15 and 20 kb, which reveal a high degree of polymorphism in different o/c isolates. Part of the polymorphism is due to the presence or absence of a 2.2-kb-long repeated sequence, a homolog of which is also found on the octopine Ti plasmid of Ach5, immediately to the left of the left TL-region border. The occurrence and distribution of this repeat in different o/c isolates make it possible to reconstruct the evolution of these strains. The chromosomal DNA sequences outside the repeats also differ in various isolates. The two probes described here can be used to identify strains from a collection, classify new isolates, or trace a given isolate under experimental release conditions.


Bioluminescence-based detection of genetically engineered microorganisms in nonsterile river water. The luminescence genes of the marine bacterium Vibrio fischeri were cloned into a lac expression vector and introduced into Escherichia coli and Pseudomonas putida. Survival of the cells in river water samples was monitored by light measurements. Whereas E. coli survived in sterilized river water for more than 29 days, it died off in nonsterile river water after 9 to 13 days. The engineered P. putida cells survived in nonsterile river water for more than 137 days. The detection limit for E. coli was 11 cells/ml.


OmpC and OmpF porins influence viability and culturability of Escherichia coli cells incubated in seawater. The contribution of the major outer membrane porins OmpF and OmpC to the maintenance of viability and culturability of Escherichia coli cells in seawater was analyzed using isogenic mutant strains lacking one or both porins. Cells that possessed OmpF and OmpC survived better than those lacking one or both of them. However, the results differed, depending on whether the cells were adapted to high osmolarity or not before transfer to seawater. When cells were grown at low osmolarity, survival was largely influenced by porins, the OmpF+ strains surviving better than those lacking this porin. Addition of an OmpF plasmid to OmpF- OmpC- cells also improved their viability. When grown at high osmolarity, the role of porins was less critical since both the viability and culturability of the cells increased. However, cells that expressed only OmpC showed the most dramatic loss of viability. Cells lacking both OmpF and OmpC exhibited a higher loss of viability and culturability in seawater. Regarding the influence of porins on survival, these results show that the conditions that prevail during the growth of cells before their transfer to seawater are highly influential: cells that express the porin corresponding to the growth conditions they are in at the time of transfer survive better.


Recombinant interferon gamma for treatment of chronic granulomatous disease and other disorders. The chemistry, biological activity, and pharmacokinetics of gamma-interferon and recombinant interferon gamma are reviewed, and the agent's clinical efficacy, adverse effects, and dosage and administration for the treatment of chronic granulomatous disease (CGD) and other disorders are described. Endogenous gamma-interferon is a 166-amino-acid protein encoded by a single gene on chromosome 12. Recombinant human interferon gamma is purified from Escherichia coli as a monomer containing 139 amino acids. Gamma-interferon has antiviral, immunomodulatory, and antiproliferative activity. Serum concentrations of recombinant interferon gamma increase in proportion to the dose. Clearance after i.m. or s.c. administration fits a two-compartment model. The half-life is 3.5-7.5 hours, and bioavailability is 89%. Evidence that recombinant interferon gamma can enhance phagocytic oxidative metabolism led to its evaluation for use in the treatment of CGD. Clinical studies showed that the agent decreases the frequency of serious infections in patients with CGD. Recombinant interferon gamma has shown only limited success in the treatment of metastatic renal cell carcinoma (RCC), both as a single agent and in combination with recombinant interferon alfa. Similarly, although interferons appear to be able to change cytogenetic abnormalities in some patients with Philadelphia chromosome-positive chronic myelogenous leukemia, therapy with recombinant interferon gamma has led to minimal success. However, the agent has produced some encouraging results in atopic dermatitis. The adverse effects of recombinant interferon gamma in patients with CGD usually consist only of fever, chills, headache, and erythema. The recommended dosage in CGD-afflicted children whose body surface area is greater than 0.5 sq m is 50 micrograms/sq m given by s.c. injection three times a week for life. Recombinant interferon gamma has given new hope to patients with CGD. Although the drug is very expensive, the cost may be offset by fewer hospitalizations to treat infection.


Management of psychotropic-induced hyperprolactinemia. The effects of individual psychotropic medications on serum prolactin concentrations are described, and recommendations for dealing with adverse effects are provided. Hyperprolactinemia can result in galactorrhea, amenorrhea, irregular menses, and anovulation; in men, impotence and azoospermia, with or without lactation and gynecomastia, can occur. Antipsychotics may block dopamine receptors in the pituitary prolactin-secreting cells and prevent dopamine-induced reduction of prolactin release. The magnitude of the increase in prolactin concentration correlates with the amount of antipsychotic drug given. The treatment of choice is reduction of the antipsychotic dosage or discontinuation of therapy. If adjustments to the antipsychotic dosage fail to resolve symptoms, the dopamine agonists bromocriptine and amantadine may be tried. Antidepressants may produce elevated serum prolactin concentrations, especially with long-term administration. However, the frequency of antidepressant-induced hyperprolactinemia is much lower than that seen with antipsychotics, and serious adverse clinical effects are uncommon. Other psychotropic drugs such as lithium, valproic acid, buspirone, carbamazepine, and benzodiazepines either are only rarely associated with symptomatic hyperprolactinemia or do not produce clinically important changes in prolactin concentrations. Antipsychotic drugs are the psychotropic agents most likely to cause symptomatic hyperprolactinemia. Bromocriptine or amantadine may provide symptomatic relief if withdrawal or adjustment of the antipsychotic dosage does not eliminate the symptoms.


Oral anticoagulant therapy in patients undergoing dental surgery. The literature on dental surgery in patients receiving oral anticoagulants is reviewed, and methods of managing anticoagulant therapy to minimize the risk of complications are discussed. Although blood loss during and after oral surgery in patients receiving oral anticoagulant drugs can be substantial, research indicates that most bleeding incidents are not serious and can be controlled by local measures. Studies of 241 anticoagulant-treated patients undergoing more than 500 dental extractions during the 1950s and 1960s showed that only 9 had postoperative bleeding. More recent studies indicate that continued anticoagulation can increase the frequency of prolonged bleeding and delay wound healing. An antifibrinolytic mouthwash containing tranexamic acid can effectively suppress postoperative bleeding. Gelatin sponges, oxidized cellulose, and microcrystalline collagen are other useful hemostatic agents. A reduction in the intensity of anticoagulation therapy has been recommended; the prothrombin time should be measured shortly before the procedure in such patients. In many patients the duration of subtherapeutic anticoagulation must be minimized to reduce the possibility of thromboembolism. An option for high-risk patients is to switch them to heparin. Each patient must be evaluated individually, and the level of risk of the dental procedure and the risk of thromboembolism should be taken into account. In patients taking oral anticoagulants who must undergo dental surgery, careful control of the intensity of anticoagulation and improved methods of local hemostasis can minimize the risk of hemorrhagic complications and thromboembolism.


The postantibiotic effect.

The postantibiotic effect. The factors that affect the presence and duration of the post-antibiotic effect (PAE) for different antimicrobial agents are described, and the clinical importance of the PAE is discussed. Proposed mechanisms by which the PAE occurs include both nonlethal damage induced by the antimicrobial agent and a limited persistence of the antimicrobial agent at the bacterial binding site. The specific microorganism-antimicrobial combination is the most important factor to influence the presence and duration of the PAE. Additional factors are antimicrobial combinations and experimental conditions, including the antimicrobial concentration and the length of the antimicrobial exposure. Most antimicrobial agents produce a PAE when tested against gram-positive cocci. However, against gram-negative bacilli, beta-lactam antibiotics (except for imipenem) have a minimal, or even a negative, PAE. Aminoglycosides, inhibitors of protein and nucleic acid synthesis, and fluoroquinolones have PAEs against gram-negative bacteria that range from one to four hours. In vivo PAEs are generally longer than in vitro PAEs for the same microorganism-antimicrobial combination. In human studies, aminoglycosides, which have an extended PAE against gram-negative bacilli, have been effective when given in once-daily dosing regimens that allow serum drug concentrations to fall below the minimum inhibitory concentration. Extending the dosing interval of an antimicrobial agent that has a PAE has several potential advantages, among them reduced cost, less toxicity, and better compliance among outpatients receiving antimicrobial therapy. Although data are limited, animal and human studies provide support for the clinical importance of the PAE. Further research into the impact of the PAE on antimicrobial dosing, efficacy, toxicity, and costs is warranted.


Microbial production of citric acid from salad oil. Strain CCU-U2-N4-41 was derived from a wild strain of yeast CCU-087. This strain can produce large amounts of citric acid from salad oil. The optimal medium for citric acid production is one liter of medium containing salad oil 100 g, yeast extract 2.0 g, CH3COONH4 3.0 g, KH2PO4 5.0 g, MgSO4 500 ppm, 1 ppm each of MnSO(4).5 H2O and CuSO(4).2 H2O, pH: 7.0. The optimal culture conditions are: temperature: 28 degrees C; agitation: 110 rpm; 100 mL medium in 500-mL Hinton flask; 1.5% CaCO3 added after 12 hr incubation. With 96 hr cultivation 71.2 g/L of citric acid can be produced. Citric acid produced was compared with authentic citric acid by melting point determination, IR, NMR and Mass. From the spectra, it was found that they were identical.


Isolation and partial purification of cytochrome oxidases from Bacillus thuringiensis subsp. israelensis HD-567. Analyses of the cell membrane fractions by spectral absorbance revealed the presence of cytochrome c and three cytochrome oxidases in Bacillus thuringiensis subsp. israelensis HD-567-cytochromes a+a3, d, and o. A modified procedure was used to purify the cytochrome c:o complex from this organism. The oxidase complex was first solubilized from a sonic-disrupted cell membrane fraction (R3 fraction) using deoxycholate and KCl. The resulting soluble fraction was further purified by Sephadex G-50 gel filtration and DEAE ionexchange chromatography. TMPD oxidase specific activity and cytochrome concentration were assayed to monitor the purification procedures. The F7 fraction (obtained after G-50 chromatography) contained cytochrome c (0.44 nmole/mg protein), cytochrome a+a3 (0.26 nmole/mg protein), and cytochrome o (0.38 nmole/mg protein), which had 6-fold, 3.4-fold and 18.9-fold increase, respectively, by comparison with the original R3 fraction. The TMPD oxidase specific activity of the F7 fraction also increased 4.8 fold. The F8 fraction (obtained after the final DEAE chromatography) contained a cytochrome c:o complex only (cytochrome c, 0.46 nmole/mg protein; cytochrome o, 0.16 nmole/mg protein), but no cytochrome a+a3 was found. Both the TMPD oxidase specific activity and the cytochrome o were attenuated greatly in comparison with the F7 fraction. SDS-PAGE analysis revealed that the F7 fraction contained numerous protein components, while the F8 fraction contained only a prominent major protein of 113.5 kD thought to be the cytochrome c:o complex, and a minor polypeptide (MW = 65.8 kD). Although the final DEAE procedure removed many undesired polypeptides, TMPD oxidase activity and cytochrome o component were also lost greatly. Kinetic studies of this cytochrome c:o complex is in progress in our laboratory.


In vitro antibacterial activities of ticarcillin alone and ticarcillin plus clavulanic acid against beta-lactamase producing and non-producing microorganisms. A total of 818 clinical bacterial isolates were tested for the production of beta-lactamase by rapid chromogenic cephalosporin method and for the susceptibility to ticarcillin alone and in combination with clavulanic acid (2 micrograms/mL) by agar dilution method. These included 83 strains of methicillin-sensitive Staphylococcus aureus (MSSA), 31 of methicillin-resistant S. aureus (MRSA), 49 of Neisseria gonorrhoeae, 58 of Haemophilus influenzae, 112 of Escherichia coli, 118 of Klebsiella pneumoniae, 58 of Proteus mirabilis, 30 of Proteus vulgaris, 60 of Serratia marcescens, 113 of Enterobacter cloacae, 60 of Pseudomonas aeruginosa and 46 of Bacteroides fragilis. The results revealed that 46.6% of P. mirabilis, 53.4% of H. influenzae, 57.1% of N. gonorrhoeae, 80% of P. vulgaris, 83.9% of MRSA, 85.6% of MSSA, 87.5% of E. coli, 91.7% of S. marcescens, 95.7% of B. fragilis, 98.2% of E. cloacae, and 100% of K. pneumoniae and P. aeruginosa strains produced beta-lactamase. In general, beta-lactamase nonproducers were more susceptible to ticarcillin than beta-lactamase producers. The ranges of minimum inhibitory concentrations (MICs) of ticarcillin for beta-lactamase nonproducers of MSSA, MRSA, H. influenzae, E. coli, P. vulgaris, S. marcescens, E. cloacae, B. fragilis and beta-lactamase producers of MSSA, H. influenzae strains were all within the in vitro susceptible range. The presence of clavulanic acid resulted in a significant enhancement of the antibacterial activity of ticarcillin against beta-lactamase producers of MRSA, N. gonorrhoeae, E. coli, K. pneumoniae, P. mirabilis, P. vulgaris and B. fragilis strains. Clavulanic acid had no synergistic activity for ticarcillin against S. marcescens, P. aeruginosa and E. cloacae.


[Evaluation of two kinds of enzyme linked immunosorbent assay method for clonorchiasis]. In this present study, adult worm antigen of Clonorchis sinensis and the indirect and double sandwich enzyme linked immunosorbent assay (ELISA) were used to detect the serum antibody of clonorchiasis patients from the endemic area of Miao-Li county. The results of indirect ELISA revealed a good correlation between the egg stool count and IgG strength of patients sera. Focusing on the different immunoglobulin classes in serum, the patient group has significantly higher level than that of control group, and the IgG level was distinguishly higher than the other Igs. The result also showed that the sensitivity and specificity on the detection of IgG for clonorchiasis patient were 83.23% and 100%, respectively. After praziquantel treatment, the antibody titer of patients decreased in accord with the time period, and the decrease rate of antibody titer in patients with moderate infection was more closely related to the time after treatment than those in the other degree infection groups. When double sandwich ELISA method was used to detect serum antibody to C. sinensis in patients with the different parasitic infection, it was shown that antibody levels in patients with clonorchiasis were significantly higher than those in the un-identical parasitic infection patients. Both of the sensitivity and specificity by using this method were 100%. Based on the results of the indirect and double sandwich ELISA methods, it is recommended that uses of the double sandwich ELISA incorporated with the indirect ELISA may give a sufficient results on the epidemiological study of human clonorchiasis. It may also be recommended that double sandwich ELISA method can be used for a mass survey and indirect ELISA method for further detection in the patients with clonorchiasis.


Detection and differentiation of dengue-1 from Japanese encephalitis virus infections by ABC MAC-ELISA. An IgM antibody capture enzyme linked immunosorbent assay using avidin biotin complex system (ABC MAC-ELISA) was established for the detection and differentiation of dengue-1 and Japanese encephalitis virus infections. The cut-off value of MAC-ELISA was based on the mean value of optical density at 490 nm of seven negative human sera carried along in each assay multiplied by 2.1. The specificity of MAC-ELISA tested on 200 healthy enrolled male serum was 99.5% and 98.0% for dengue-1 and Japanese encephalitis IgM, respectively. Two hundred and sixty-six acute or followed-up dengue serum specimens which were identified to possess dengue-1 virus by virus isolation technique using C6/36 cell line and monoclonal antibody immunofluorescent assay (IFA) were tested by MAC-ELISA for IgM antibodies to dengue-1 and Japanese encephalitis virus infection. The positivity of IgM antibody for serum collected from day 1 to day 7 after onset of the disease was 0.0%, 0.0%, 7.6% 14.2%, 25.0%, and 77.7% by each consecutive day, correspondingly, for those collected from day 8 to two month was 96.7% overall. By comparison the P/N value of dengue-1 IgM to JE IgM, 98.5% and 96.8% of the dengue-1 and Japanese encephalitis suspected serum specimens could be distinguished from each other, respectively. MAC-ELISA is a convenient, rapid, sensitive and specific method for the detection and differentiation of dengue-1 from Japanese encephalitis virus infections.


Autoantibodies and related immunity of leprosy patients from leprosarium in Taiwan. Autoantibodies and related immunological examinations were measured in 60 leprosy patients from a leprosarium in Taiwan. Thirty-one lepromatous type, 24 tuberculoid type and 5 borderline type patients were identified. The measured autoantibodies included antinuclear antibodies, anti-nDNA, anti-cardiolipin and rheumatoid factor. Serum protein electrophoresis and immunofixation were also performed to detect the monoclonal and polyclonal status of immunoglobulins. Circulating immune complex and complements were also quantitated. Delayed type skin tests were performed during patients' visits. A higher frequency of autoantibodies, especially the antinuclear antibodies and anticardiolipin antibodies, were detected in lepromatous type patients. Higher levels of circulating immune complex and frequency of polyclonal and monoclonal gammopathy were also noted in lepromatous type patients. Anergy skin tests were only noted in lepromatous type patients. It was concluded that the more impared cell-mediated immunity in leprosy patients, with lepromatous type in particular, the greater the production of autoantibodies.


Increased production of interleukin-6 and autoantibodies in patients with Sjögren's syndrome. The occurrence of autoantibodies including antinuclear antibody, anti-nDNA, anti-Sm, anti-RNP, anti-SSA, anti-SSB, anticardiolipin (aCL) antibody, M-Proteins, and interleukin-6 (IL-6) and circulating immune complex (CIC) were studied in 18 patients with primary Sjögren's syndrome (SS) and 20 patients with secondary SS. Another 15 healthy individuals were served as a control group. The differences between primary and secondary SS in these autoantibodies, IL-6 and M-protein were also compared. The result showed a high incidence of autoantibodies and CIC in patients with SS. Higher frequencies of anti-nDNA and anti-Sm antibodies were noted in patients with secondary SS. There were also elevated levels of IL-6, much higher in primary than in secondary SS. In addition, higher frequencies of M-protein were detected in patients with primary SS. In conclusion, through immune dysregulations, higher levels of IL-6 were found in patients with SS. Autoantibodies produced in these patients might be derived from IL-6 stimulated B cells.