Extended use of Fluosol emulsion in acute myocardial ischemia treatment.

An intravenous infusion of Fluosol enhanced significantly the t-PA thrombolysis of the arterio-venous shunt made by insertion of 125I-fibrin clot in rabbits. The plasma radioactivity released through thrombolysis increased in both time and dose-dependent manner after the administration of t-PA. Fluosol in combination with t-PA increased the plasma radioactivity, compared with the t-PA treatment alone at the corresponding dosage. The coronary blood flow was markedly reduced to almost zero after the thrombin injection into narrowed LCX with a clamp in open-chest dogs. An intravenous infusion of Fluosol or Pluronic F-68 solution at a dose of 15 ml/kg significantly shortened the thrombolysis time by intracoronary infusion of urokinase alone. While, little change in the QTc interval of ECG and the plasma CPK-MB activity was observed in the Fluosol group in combination with urokinase, suggesting a myocardial protective action of Fluosol possibly due to its oxygen carrying effect.


Combination of treatment with perfluorochemicals and free radical scavengers.

P-cyclohexylmorpholine, given as oxygen carrier of second generation, was tested in two experimental models: the ischemic intestine and the lung under hyperbaric oxygen condition. In both instances in separated groups either alpha-tocopherol or allopurinol was additionally given as oxygen free radical scavenger. The thiobarbituric acid reaction (TBAR) was used as screening test for the occurrence of lipid peroxidation in organ probes; further the content of glutathione in reduced (GSH) and oxidized form (GSSG) was determined. Whereas only slight increases of TBAR substances and GSSG could be found compared to controls, the effect of the administrated scavenging drugs was very impressive: they could suppress nearly totally the effect of an enhanced oxidative stress.


Comparison of the effects of total blood substitution during two different levels of hypothermic cardiac arrest.

The effect of total blood replacement with a solution containing neither hemoglobin nor fluorocarbon was studied under two different levels of hypothermia. Ten dogs were anesthetized and esophageal temperature lowered to about 24 degrees at which time exsanguination began. Upon cardiac arrest and the completion of exsanguination, continuous whole body closed chest extracorporeal circulation of an oxygenated blood substitute was begun. Hematocrit was 1% while core temperature remained less than 10 degrees C for 3 hours of perfusion during which nadirs of 1.3 degrees C (Group I, N = 5) and 7.3 degrees (Group II, N = 5) were achieved. Replacement of the perfusate with whole blood began once the dog was rewarmed to approximately 10 degrees C. All dogs survived the procedure. Two dogs from each group died by the fourth post-operative day but the others survived long term. Group II showed a faster return to normal based on motor behavioral, biochemical and hematological changes. Thus the combination of profound hypothermia and complete blood substitution with a solution lacking any special oxygen carrying molecule, can be tolerated for 3 hours using both levels of hypothermia, however, the warmer one appears to be associated with faster recovery.


Preservation of myocardial function during ischemia with intracoronary perfluoroocytlbromide (Oxygent).

The effects of intracoronary infusion (24 ml/min) of oxygenated perfluoroocytlbromide (PFOB) or autologous blood on regional myocardial function and hemodynamics were studied during a 2 min perfused occlusion in nine open chest dogs to determine if a PFOB infusion could prevent the myocardial dysfunction observed during the balloon occlusion of percutaneous transluminal coronary angioplasty (PTCA). Regional myocardial function was measured with sonomicrometers in the ischemic and non-ischemic zone to determine segment length and percent of systolic shortening (% delta L). Without infusion, the ischemic zone in each animal developed akinesis during a 20 sec coronary occlusion. Each animal underwent a 2 min infusion of blood and PFOB at 24 ml/min. During the 2 min perfused occlusion, the blood ischemic zone % delta L of 14.4 +/- 3.1 versus a PFOB % delta L of 23.4 +/- 2.9. % delta L of blood and PFOB were not as significantly different from control (P less than .05 PFOB vs. blood). The bromine ion in PFOB results in a radiodense compound. Adequate images of the canine left ventricle and coronaries were obtained using DSA. (1) PFOB infusion maintains normal myocardial function during perfused coronary inclusion. (2) PFOB may be used as an oxygen carrying contrast agent to obtain DSA images.


Perfluorochemical reperfusion limits myocardial reperfusion injury after prolonged hypothermic global ischemia.

The ability of an oxygenated perfluorochemical (Fluosol) to limit myocardial reperfusion injury following global hypothermic ischemic insult was investigated. Neonatal piglet hearts were arrested with cold crystalloid cardioplegia and stored for 12 hours in 2 degrees C saline. Reperfusion was carried out using an isolated, blood-perfused, working heart preparation. Hearts were initially reperfused (10 minutes) with either whole blood (WB, n = 6), unmodified perfluorochemical (PFC, n = 8), or aspartate/glutamate-enriched perfluorochemical cardioplegia (PFC+, n = 6), prior to institution of whole blood perfusion, functional evaluation and left ventricular biopsy. A control group (C, n = 7) was evaluated without an intervening period of ischemia. At a left ventricular diastolic pressure of 9 mm Hg WB hearts developed a left-ventricular stroke work index (SWI) of 3.8 +/- 2.3 x 10(3) erg/g (mean +/- standard error of the mean). Under similar conditions, PFC-reperfused hearts achieved a SWI of 14.6 +/- 1.3 x 10(3), significantly greater than that of WB (p less than 0.001). SWI for PFC+ hearts was 19.8 +/- 1.6 x 10(3), significantly increased over that of PFC (p less than 0.01), and not different from values obtained for C (19.2 +/- 0.8 x 10(3)). In addition, PFC-reperfused hearts demonstrated superior maintenance (p less than 0.05) of ATP (2.08 +/- 0.16 umole/g), compared to WB (1.50 +/- 0.19), while preservation of ATP in PFC+ hearts (2.99 +/- 0.12), was significantly increased over that of PFC (p less than 0.001), and not significantly different from that for C (2.68 +/- 0.17).(ABSTRACT TRUNCATED AT 250 WORDS)


Biocompatibility studies with perfluorochemical oxygen carriers.

The effects of injecting either a novel perfluorodecalin (FDC)-based emulsion or various perfluorochemical (PFC) oils on liver cytochromes P-450 (P-450) and aryl esterase (LAE) enzymes in male rats have been studied. Mean P-450 concentration increased (P less than 0.05) following injection of the novel emulsion or FDC. In contrast, LAE increased in response to injection of FDC (P less than 0.05) and the C-16 oil, perfluoroperhydrofluoranthrene (P less than 0.01), added to stabilize the emulsion. No corresponding changes in either P-450 or LAE occurred in rats injected with either perfluorotripropylamine, perfluorotributylamine or perfluorooctylbromide. The "sleeping time" of animals injected with the pentobarbital-based anaesthetic, Equithesin, following pretreatment with the novel emulsion was lower (P less than 0.05) than in saline-injected controls. The results are discussed in relation to the effects of PFCs on liver metabolic functions.


[Febrile syndrome in hospitalized patients].

This study was designed to ascertain if certain characteristics of febrile patients could help to identify infectious or bacteremic conditions. Patients with axillary temperature higher than 37,4 degrees C visiting the emergency room and requiring hospitalization were included in the study. The sample included 345 patients. Infections made up 89% of the causes of fever. The most frequent site of infection was the respiratory system (39%). 13% of hemocultures were positive. Gram negative germs were the most frequent agents. Infectious FS was related with the presence of predisposing factors, duration of fever, erythrocyte sedimentation rate and hemoglobin. Bacteremia was associated to treatment prior hospitalization, average temperature, hemoglobin, AST and urinary sediment. We may conclude that infections are the most frequent cause of FS. We could not found any clinical or analytical parameters that, used together, could help us to identify infectious or bacteremic FS.


[Cases of tetanus in Guipuzcoa].

In order to evaluate the situation of tetanus in Guipúzcoa and to assess the impact of preventive measures, we studied the incidence, mortality and other aspects of the disease during the past three decades. Ninety eight cases were detected (annual average 3,27 SD 2,6), this figure being reduced over time (7,2 cases per year during the period 1962-1966; 1,8 cases per year between 1987 and 1991). 67,3% were men and 32,7% women. We registered 6 cases of neonatal tetanus, all of them prior to 1976. Since 1969, there has been no cases within the 1-14 age group. Half of the cases detected during the last five years (4/9) were intravenously drug-addicts. Among those living in less populated areas (less than 10.000 population), the risk of developing the disease was four times greater. 33,7% of patients died, being the mortality rate higher among newborns and patients died, and patients over 55. Although the situation was not worse than in other similar environments, the incidence of tetanus should be reduced by reinforcing preventive measures.


[Antibiotic treatment for acute episodes of chronic obstructive pulmonary disease].

Antibiotic therapy for acute episodes of chronic obstructive pulmonary disease (COPD) is a controversial issue still not clarified. In order to evaluate the effectivity of the antibiotic therapy, we designed a double-blind controlled and randomized clinical trial, in which 90 patients hospitalized due to an acute episode of COPD were divided into three groups: group I, cotrimoxazole (29 patients); group II, amoxicillin-clavulanic acid (32 patients) and group III, placebo (29 patients). Gasometric and spirometric measures were taken in addition to clinical evaluation at hospital admission and discharge using a numerical valoration system. All patients were treated with common bronchodilators. The three groups were homogeneous at their admission and there were no statistical differences at their discharge. We conclude that antibiotics do not play a relevant role in the improvement of acute episodes of COPD.


Structural determinants of vascular resistance properties in hypertension. Haemodynamic and model analysis.

The average internal radius (ri) of the resistance vessels of the hindquarter (HQ) bed was narrower in renovascular and genetic hypertension than in normotensive controls. The narrowing was approximately uniform over the full range of vasomotor tone, which accounted for the bed's property as an 'amplifier' of vascular resistance (R) (increased slope (S) of the dose-R response curve) and for the elevated R at maximum dilatation (Rmin). In the model we examined the effects on the dose-R curve parameters of altering wall/internal radius (w/ri) ratio, ri and wall 'stiffness' one at a time, whilst the others were held constant: only narrowing of ri led to increases in both S and Rmin; with hypertrophy alone, S increased but Rmin was reduced, whilst increased wall stiffness increased Rmin but lowered S. Thus, for hypertrophy to be associated with rises of both S and Rmin, it must be linked to lumen narrowing, to increased wall stiffness, or to both. Preferential deposition of new material towards the lumen will link hypertrophy to narrowing. It has been suggested that narrowing can occur without hypertrophy ('remodelling'). In the model an increase of only 1-2% WV was required to produce rises in w/ri of congruent to 30-50% when associated with congruent to 10-15% reduction in ri, which is close to the limit of detection. From the literature, the sites of greater narrowing in hypertension extend down to small arteries and large arterioles. The rise in BP upstream from those sites, due to the vascular amplifier, offsets the down-stream effects of vascular narrowing on blood flow and this negative feedback system helps to maintain elevation of BP at a stable level. We also examined developmental rise in R between 4 and 50 weeks, which affected SHR and WKY in the same proportion: structural factors (vascular length of larger arteries, 'rarefaction' of arterioles and capillaries) accounted for only about half the rise in R, and the remainder was probably due to developmental changes in muscle function.


[Epidemiological study of acute poisoning within the population of Zaragoza].

Were chosen for analysis 1443 acute poisonings, that were treated in the Emergency Services of the Miguel Servet Hospital and the University Hospital of Zaragoza between the years 1988 and 89. The great part of the patients didn't required hospitable ingress, it was observed a predominance of the voluntary poisonings with predominance in people under 40 years. Among the drug poisonings were the benzodiazepines the drug more used and among nonpharmacologic agent, alcohol and the narcotics. The treatment more used was the symptomatic (53%) and was contrast the decrease of gastric lavage (15%) and forced diuresis (10%).


[Mediastinal tuberculosis as a cause of fever of unknown origin].

Tuberculosis is the most frequent cause of fever from unknown etiology. On the other hand, mediastinal tuberculous adenopathy (TBM), without associated pulmonary affection is a rare form of presentation among adults, generally evolving with sustained fever until a thoracic radiology is performed showing mediastinal enlargement. We present a case which started as fever from unknown etiology (FUE), given that, according to the thoracic radiology performed one month after the onset of fever, no mediastinal affection was observed.


[Comments with respect to 2 patients with anti-Jo 1 antibodies].

Anti-Jo 1 antibodies define a subgroup of patients with polymyositis, which are characterized by the presence of extra-muscular involvement as pulmonary fibrosis, although Raynaud's phenomena, arthritis, tenosynovitis, pleuritis and pericarditis may also be present. Given the fact that extra-muscular signs may precede the clinical diagnosis of polymyositis, as in the two cases that we present here, these antibodies may help us to diagnose the disease before the onset of myositis.


[Hypothyroidism state with psychomotor agitation and fast atrial fibrillation: a rare form of presentation].

Schizoid psychosis and atrial fibrillation are two rare signs of hypothyroidism which may suggest the opposite condition, hyperthyroidism, with the associated risk of adopting a wrong therapeutical approach. Recently, we have treated a patient in which those two mentioned circumstances were present. In this paper, we review psychiatric signs and electrocardiographic disorders associated to hypothyroidism.


Access of blood-borne vasoconstrictors to the arteriolar smooth muscle.

In vitro experiments have shown that luminally applied water-soluble vasoactive materials have limited access to arteriolar smooth muscle cells, and as a result, the responses to such agents applied luminally are less than the responses to those applied adventitially. To determine the extent to which this 'compartmentation' influences arteriolar responsiveness to blood-borne water-soluble vasoconstrictors in vivo, we applied phenylephrine, vasopressin and angiotension II to arterioles in the hamster cheek pouch both by luminal perfusion, and by topical application to the arteriolar smooth muscle via micropipettes. The arterioles were about 2 orders of magnitude more sensitive to these water-soluble vasoconstrictors when they were applied topically than when they were applied luminally. In contrast, the arterioles were almost equally sensitive to the lipid-soluble alpha 1-adrenoceptor agonist SKF 89748-A applied by either route. The venular wall appears to be much less effective as a barrier than the arteriolar endothelium. Phenylephrine and vasopressin both elicited large arteriolar constrictions when perfused through venules in close proximity to the arteriole, and these constrictions were larger than those observed when the drug was applied to the arteriole's own lumen. Our observations confirm that the arteriolar endothelium can inhibit the direct access of water-soluble blood-borne agents to the arteriolar smooth muscle in vivo, and they suggest that the capillaries and venules could be the primary routes of access for water-soluble agents from the blood to the arteriolar smooth muscle.


[Lymphoproliferative syndromes associated with rheumatoid arthritis].

Rheumatoid arthritis predispose to several lymphoproliferative syndromes, some of them are of doubtful neoplastic type, as the lymphoproliferative disease of granular lymphocytes, whereas others are clearly malignant, as non-Hodgkin's lymphomas and multiple myeloma. In this paper, we review the potential etiological agents, mainly the reduction of the "natural killer" activity and immunity disorders against virical oncogenes. The onset of monoclonal gammopathy in rheumatoid patients is also stressed, due to its potential prognosis value in the development of lymphoid neoplasias.


Alteration of arteriolar responses to serotonin by two intravenous anesthetics.

The effects of serotonin (5-HT) on the microvasculature of cremaster muscles were compared in decerebrate, ketamine- and pentobarbital-anesthetized rats. 5-HT-induced constriction of large distributing arterioles was enhanced in the ketamine- and pentobarbital-anesthetized rats. Precapillary arterioles of pentobarbital-anesthetized animals were more sensitive to 5-HT-induced dilation than either decerebrate or ketamine-anesthetized animals. The response of vessels to two 5-HT receptor antagonists (methysergide and LY53857) were unchanged by the anesthetics. Our findings suggest that both ketamine and pentobarbital enhance the microvascular response to 5-HT, but that these changes are not due to an alteration of receptor sensitivity.


[Infections in the diabetic. Comparative study of infections in the foot and other locations].

Given the high morbi-mortality of foot infections among the diabetics and the poor knowledge of their predictive, clinical and evolutive factors, we have retrospectively studied a group of patients with these characteristics, comparing them with infections among diabetics affecting other locations. We studied 66 infections among diabetics: 34 patients with diabetic's foot and 32 with infections at other locations: 20 pyelonephritis and 12 pneumonias. Medical records were obtained in all cases and all patients underwent a complete physical exploration in order to assess their risk factors. We observed as a significant predictive factor of diabetic's foot, diabetes type I, with an evolution longer than 10 years, neuropathy, vasculopathy or retinopathy. From the clinical point of view and compared with the other infections, these patients showed longer hospitalization, greater initial clinical severity, glucemias higher than 200 mgr/l., anemia and high GSR. Ethiologically, the infection of diabetic's foot was polymicrobian in 42.3% of all cases, being S. aureus the microorganism more frequently isolated. On the contrary, in infections at other locations, monomicrobian flora was more frequent, being E. coli the most frequent in pyelonephritis and S. pneumoniae in pneumonias. The evolution was satisfactory in all cases, with a close medical and surgical combined treatment and the appropriate use of antibiotic combinations, mainly clindamicine + tobramicine in the diabetic's foot and cefuroxime in the other locations.


[Meningitis from Listeria monocytogenes in the adult].

We describe the characteristics of 6 adult patients with meningitis by Listeria monocytogenes. Five of them had previous disease and only one of these was being treated with immunosuppressors. All of them presented meningeal syndrome with cephalorhachidean fluid's pleocytosis and five of them, polymorphonuclear predominance. The empirical treatment was correct only in two cases. The mortality rate reached 50%.


[Seroprevalence of Q fever among the adult population of Lanzarote (Canary Islands)].

Q fever is an endemic zoonosis in the Canary Islands. In 1986, we detected, in a pilot study, residual antibodies of the infection in 3% of the population from Lanzarote. In 1989, we performed a new study in order to assess seroprevalence of Q fever among the adult native population from the island. We studied 390 human serums obtained from an statistically representative sample. Age ranged from 30 to 64 years. Out of 390 serums, 196 (50.25%) were obtained from men and 194 (49.74%) from women. The serological technique used was the fixation of complement using Coxiella burnetii antigens in phase II. Titres equal or higher than 1/8 were considered positive. No statistically significant differences were observed with regard to seroprevalence rates considering sex, age, nor living in or outside the island's capital city. However, when dividing the island's territory in three areas (north, centre and south), and assessing independently their respective seroprevalences, we observed relatively higher seroprevalences in the furthest areas (13.3% in the north and 13.5% in the south) than in the central area (4.7%), although only the higher seroprevalence in the south reached statistical significance when compared with the mean prevalence. Probably, these observations indicate that, although Q fever is extended all over the island, it is a more frequent infection in rural areas of Lanzarote, at the north and the south, than in the central area, where the main urban areas are located.


[Study of smoking habit among the adult population of Aragón].

We determined the prevalence of tobacco consumption among the general population and established its distribution considering socioeconomical variables. We performed a cross-sectional study with 2781 subjects from 93 rural and urban areas, selected by randomized, census-based, multi-staged and stratified sampling. 40.9% of the population from Aragon are regular smokers (58.6 of men and 23% of women). Heavy smokers comprise 17.2% of the total (30.1% of men and 3.8% of women). The higher proportion of smokers in observed among single men over 40 and single women under 40. The percentage of smokers is lower among 20 to 29-years-old young people from high socio-economical than from low levels. In general, the mean percentage of smokers among men and, specially, among women from rural areas is higher than in the rural areas. In Aragon, tobacco is a risk factor affecting 41% of the adult population and, specially, young people, men, single persons from both sexes and living in urban areas. It seems that young people with a high socio-economical level are becoming smokers at a lower frequency.


[Prolonged febrile syndrome as a manifestation of Coxiella burnetii infection, presentation of infrequent clinical cases].

We present two cases of infection by Coxiella burnetii which developed with sustained fever symptoms. During its evolution, the first case presented granulomatous hepatitis, whereas the second case presented left Cosofemoral Arthritis. We describe the clinical-evolutive characteristics of these clinical forms and within the evolution of the chronic forms of Q fever.


[Cerebellar syndrome as first manifestation of late onset systemic lupus erythematosus].

Clinical characteristics of a 70-year-old women affected by an erythematous systemic lupus (ESL) of late onset, whose first main manifestation was the compromise of the central nervous system as cerebellar syndrome and which exhibited a good response to treatment, is described. Pathogenic and diagnostic aspects of the neurological manifestations of ESL are described.


[Pre-sternal invasion as first manifestation of lung epidermoid carcinoma].

Lung epidermoid carcinoma is a tumor of central origin and slow growth which tends to invade locally; however, it is rare to find a presternal tumoration produced by local invasion as one of its manifestations. The aim of this communication is to present a case and analyze its natural history.


[Pheochromocytoma: a motley tumor].

Four patients admitted to our hospital with different symptomatology are studied: a 9-years-old boy with hyperhidrosis; a 47-years-old woman with arterial hypertension and two young males, 25 and 36-years-old, respectively, with thoracic pain. In all cases, the presence of pheochromocytoma was suspected. One of them died due to left ventricular failure with acute lung edema. The other three patients were diagnosed by hormonal determinations, detecting a supra-renal body with abdominal echography and computerized axial tomography and undergoing surgery. Currently, they are asymptomatic.


[Omeprazole and maintenance treatment: new concepts for traditional therapies?].

Duodenal ulcer and reflux oesophagitis are a chronic diseases characterized by remission and relapses. If no active prophylactic treatment is given after healing around 70-100% of the patients have a relapse during the first year. Various therapeutic strategies have been suggested to maintain the disease in remission. Continuous maintenance treatment with H2 blockers has proved to be effective in reducing the spontaneous recurrence rate of duodenal ulcers but the results are disappointing in reflux oesophagitis. Initials studies on different treatment regimen of omeprazole for maintenance treatment have indicated promising results. The efficacy and safety of this drug have been evaluated in this article.


[Clinical syndromes in HIV infection (1st of 2 parts)].

The spectrum of clinical manifestations associated to HIV infection is very broad. In addition to processes that may be attributed to the virus itself, multiple opportunistic infections can develop while the immunodeficiency develops. Any organ or system of the organism may be affected by these processes. The main clinical syndromes in AIDS and the differential diagnosis of their potential etiologies are reviewed.


Spectrotypes of anti-DNA antibodies show that anti-DNA-secreting B-cell clones of SLE patients are restricted in number, stable and long lived.

We have investigated the number of B-lymphocyte clones secreting anti-ssDNA antibodies in SLE patients and a chronic active hepatitis patient by isoelectric focusing and reverse immunoblotting of serum antibodies. Individual clones can be identified by the unique pattern of bands produced by their antibodies (the clonotype). Using this technique, we have shown that the anti-DNA response of the majority of SLE patients is clonally restricted, in many cases only a single B-cell clone responding. We have also measured qualitative and quantitative changes in expression of B-cell clones and shown that these clones are remarkably stable with lifespans of up to six years or more. These results are in agreement with previous observations of clonal restriction of the anti-DNA response in three mouse models of SLE and in addition show that, unlike the mouse models, human anti-DNA-secreting B-cell clones are extremely stable and long-lived. The implications of these results for models of initiation and regulation of the autoimmune response are discussed.


Analysis of carbohydrate residues on human thyroid peroxidase (TPO) and thyroglobulin (Tg) and effects of deglycosylation, reduction and unfolding on autoantibody binding.

The contribution of carbohydrate residues and peptide chain conformation to autoantibody binding sites on human thyroid peroxidase (TPO) and thyroglobulin (Tg) has been investigated. In addition the nature of carbohydrate residues associated with human TPO has been studied. 125I-labelled human TPO and Tg were treated with the following glycosidases: EndoD, EndoH, neuraminidase, O-glycanase, neuraminidase followed by O-glycanase and PNGaseF. Thereafter binding to different sera containing TPO autoantibodies and Tg autoantibodies was assessed using solid phase protein A to separate antibody-bound and free labelled antigens. In addition, labelled Tg and TPO were treated with reducing agent (dithiothreitol) or sodium acetate buffer pH 7.5, 5.5 and 3.2 (followed by neutralisation with 2 M Tris pH 8.3) prior to antibody binding studies. Furthermore, the effect of deglycosylation and treatment with acid buffers on TPO enzyme activity was studied. The nature of carbohydrate residues associated with hTPO was analysed by assessment of the effects of different glycosidases on 125I-TPO mobility on SDS-PAGE followed by autoradiography and by the use of lectins. Deglycosylation of labelled Tg and TPO had no clear effect on Tg and TPO autoantibody binding. Reduction of labelled Tg and TPO resulted in almost complete loss of autoantibody binding with all sera studied. Furthermore, adjusting the pH of labelled TPO or Tg transiently to pH 5.5 lowered autoantibody binding in the case of all the sera and the effect was more marked at pH 3.2. TPO enzyme activity (guaiacol assay) of unlabelled TPO was decreased after treatment with EndoH but not with other glycosidases. The low pH buffers affected unlabelled TPO enzyme activity measured by iodide assay. Treatment of 125I-labelled TPO with EndoH, neuraminidase and PNGaseF caused marked changes in the double band pattern characteristic of TPO on analysis by SDS gel electrophoresis (TPO doublet). Analysis of changes in the mobility of the 2 bands of the doublet after treatment with different glycosidases and binding studies with lectins indicated that both high mannose and complex type sugar residues were associated with hTPO. The high mannose type residues were associated mostly with the lower band of the hTPO doublet whereas complex type residues were associated mostly with the upper band. Overall, our studies indicate that (1) the major autoantibody binding sites on hTPO and hTg are conformational, (2) sugar residues do not appear to be important in forming the autoantibody binding sites on hTPO and hTg, and (3) both high mannose type and complex type sugar residues are associated with hTPO.


Non-receptor muscle antibodies in myasthenia gravis are of IgG1 and IgG4 subclasses.

The IgG subclass distribution of non-receptor muscle antibodies was examined in 15 myasthenia gravis (MG) sera, employing an indirect haemagglutination-immunofluorescence technique. Four sera contained only IgG1, 4 contained only IgG4 and 7 contained both IgG1 and IgG4 muscle antibodies. IgG2 and IgG3 antibodies were not found. Among 11 patients with a defined thymus pathology 8 had thymoma and 3 had thymic atrophy, but there was no correlation between antibody subclass pattern and thymic pathology. Patients with both IgG1 and IgG4 antibodies tended to have the longest disease duration. We conclude that IgG non-receptor muscle antibodies in MG are of the IgG1 and/or IgG4 subclasses, irrespective of thymic pathology.


Target organ susceptibility and autoantibody production in an animal model of spontaneous autoimmune thyroiditis.

F1-hybrids of Obese strain (OS) chickens, afflicted with spontaneous autoimmune thyroiditis (SAT), and normal, inbred CB chickens, do not develop severe thyroiditis. About 50% of these crosses show circulating autoantibodies to thyroglobulin (TgAAb), but the thyroid glands are only slightly infiltrated, suggesting that the target organ is not susceptible to autoimmune attack. In the present study we show that despite this mild infiltration TgAAb are only synthesized by lymphoid cells within the thyroid gland. Furthermore, we demonstrate that immunization with chicken thyroglobulin (Tg) in complete Freund's adjuvant causes severe experimental autoimmune thyroiditis (EAT) in F1(OSxCB) hybrids.


Genetic association between natural autoantibody responses to histones and DNA in murine lupus.

Genetic regulation of the spontaneous anti-histone antibody production in systemic lupus erythematosus (SLE) was studied using the H-2-congenic and T cell receptor beta chain gene complex (TCR beta)-congenic NZB and NZW strains and their crosses. We found that the original, parental H-2d/d NZB mice produced significantly higher titers of serum IgM class anti-histone antibodies than did the congenic H-2d/z or H-2z/z NZB mice. However, none of these three NZB strains produced IgG antibodies. The NZW strain of any H-2 haplotype did not produce IgM and IgG anti-histone antibodies. The IgG anti-histone antibodies were produced only by H-2d/z heterozygous NZB x NZW F1, but not by homozygous H-2z/z or H-2d/d NZB x NZW F1 mice. In studies using (NZB x NZW) F1 x NZB backcross mice, only the progeny having both H-2d/z and NZW-type TCR beta genotypes produced high amounts of IgG antibodies. There was a tight linkage between the NZW-type TCR beta and the production of IgG anti-histone antibodies in TCR beta-congenic NZB x NZW F1 mice. All these findings were in keeping with our preceding observations on the genetic regulation of anti-DNA antibodies in these mice and suggest that certain common mechanisms such as super-antigen-mediated or common idiotope-mediated regulations may underlie the production of these two distinct autoantibodies in NZB x NZW F1 mice.


[Diagnostic possibilities of transcranial magnetic stimulation in diseases of the cervical spine].

We investigated the question as to whether transcranial magnetic stimulation of nerve structures can be used to obtain an objective description of motor impairment in humans with cervical nerve root compression or myelopathies. We were able to show that paresis is correlated with an increase in the latency of the evoked muscle potentials. Application of the method in the fields of orthopaedics and neurosurgery permits a description of motor deficits in cervical compression radiculopathy and myelopathy. Although the value of the method for orthopaedic and neurosurgical purposes is not yet completely clear, our experience does indicate interesting possibilities in the diagnostic evaluation of diseases of the cervical spine.


[Measuring intracardiac impedance for the determination of sympathetic nerve activity in frequency-adapted electrostimulation--Part 2: Clinical results].

The results of a multicenter clinical study involving patients receiving the first ANS controlled rate adaptive pacemaker are presented. In the patients with primary or secondary chronotropic insufficiency, it is possible to reestablish the closed loop control system that includes the baroreceptors, the medulla oblongata, the cardiac output and the mean arterial blood pressure. This system serves to keep the blood pressure constant in the face of changing demands on the circulation. Utilizing intracardiac impedance measurements, the myocardial contractility can be determined, which contains information about the current sympathetic tone, and thus represents an excellent physiological input for a rate adaptive mechanism. The results presented are taken from a study population of over 200 patients. The objective evaluation of this new approach was performed echocardiographically, by ergometry and 24-hour Holter monitoring.


[Reduction of the number of recorded EEG channels for routine monitoring in the intensive care unit].

Monitoring patients in the intensive care unit with the aid of the conventional electroencephalogram employing a large number of recording channels is rather difficult, and can be laborious. This imposes limits on the routine application of this method. To investigate the possibility of developing a new monitoring device for easier application in the ICU, we aimed to establish whether the relevant information provided by a multi-channel EEG could be found in a subgroup of channels, thus reducing the number of channels required. Preferably those channels should be identified for use which are least contaminated by artefacts under routine conditions in the ICU. A total of 150 EEG recordings from the intensive care unit were inspected visually for the presence of artefacts. The derivations C3-P3 and C4-P4 proved to be least contaminated, at 35% and 39%, respectively. In these derivations visual assessment of the EEG was found to be impossible due to artefacts in only 4 and 5%, of all cases, respectively. A data set comprising 52 EEG segments with the fewest possible artefacts, was analysed using time series methods. On the basis of multivariate autoregressive processes, a measure was derived which describes the loss of information associated with a reduction in the number of EEG channels. The computation of the information loss for several channel combinations revealed that the derivations F3-C3, C3-P3 and A1-Cz represent a good compromise between information content, number of channels and frequency of artefacts. Practical experience shows that, at least for the control of sedation, a further reduction to a single channel should be possible.(ABSTRACT TRUNCATED AT 250 WORDS)


[Measuring intracardiac impedance for determining sympathetic nerve activity in frequency-adjustment electrostimulation--1: Biomedical technical principles].

Modern Pacemaker technology makes it possible to adapt the pacing rate to hemodynamic requirements. The most ambitious approach aims at restoring the physiological closed-loop system by utilizing the information supplied by the Autonomic Nervous System and extracted from myocardial contractile performance. Measurement is accomplished by the impedance method using the stimulating electrode as the measuring electrode. The Ventricular Inotropic Parameter (VIP) has been identified as an ANS-dependent parameter. A special detection algorithm, the Regional Effective Slope Quantity (RQ), with a high ANS sensitivity has been developed specially for the purpose. Rate adaptation is achieved by using an individually-adjustable Inotropic Index (II). The concept has been evaluated in a multicenter study employing a standardized exercise protocol. The clinical results will be presented in Part 2 of this paper.


[A comparative study of the characteristics of femoral hip prosthesis heads--study and results ceramic-coated hip joint heads of titanium].

Today, only hard/soft cup and femoral head combinations are employed for hip joint prostheses. Highly polished ceramic is a material with very good tribological properties for femoral heads, being highly resistant to mechanical wear and tear, and highly resistant to chemical reactions in the biological environment. The advantage of metal heads, in contrast, undoubtedly lies in their resistance to breakage and the ease with which their geometry can be modified with respect, for example, to antirotation angle and neck length. The ideal material for femoral heads is a combination of the two materials. The new multi-layer combination of titanium-niobium oxide/nitride ceramic coating applied to a prehardened titanium head combines the positive material properties in an ideal manner. Femoral heads made of CoCrMo, oxide-hardened titanium, aluminium oxide or multilayer titanium-niobium ceramic were compared by means of friction an wear and tear tests. The TiNb-ceramic-metal heads showed similar abrasion at the surface as the ceramic heads. At the high loads of more than 400 kp, which may also be reached under physiological conditions, the specially coated titanium-ceramic heads proved to be superior in terms of resistance to fracture and tribological properties.


[Model for immunologic testing of biomaterials].

Corrosion products and electric fields are capable of changing proteins to antigens, thus permitting the immunological system to identify the biomaterial as foreign. The reaction between corrosion products and a macro-molecule also leads to an antigen (carrier antigen), such as conformational changes of a macro-molecule, e.g. a protein, caused by the electric field at the implant surface (modified macro-molecule antigen). While the sensitivity to corrosion and the effectiveness of galvanic elements is measurable by electrochemical methods, suitable methods of determining the field strength in the vicinity of biomaterial surfaces are still unavailable. The influence of the double layer of uncoated and coated titanium surfaces on the conformation of proteins and their conversion to antigens are investigated with polyclonal antibodies capable of identifying the unchanged protein despite adsorption to the surface. 14C-marked Bovine Serum Albumin serves as a model protein. Determination of the total number of protein molecules adsorbed is effected via the detection of the emitted electrons. The quotient of the concentration of natural proteins to the concentration of adsorbed molecules gives the biocompatibility index, which is independent of the surface area, and gives an indication of the expected biocompatibility of the material. The results of the biological tests of titanium and two coating materials on titanium were confirmed in an animal experiment. It is possible that in the future immunological tests may replace experiments in animals.


Motions studies of the human alpha 1-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and polarization of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tryptophan residues.

Dynamics studies on tryptophan residues of human alpha 1-acid glycoprotein (orosomucoid) and of 2-p-toluidinylnaphthalene-6-sulfonate bound to the protein are performed. Excitation at the red edge of the absorption spectrum of the tryptophan does not lead to a shift of the fluorescence emission maximum of the fluorophore. This reveals that Trp residues present motions with respect to their microenvironment. This is confirmed by polarization studies as a function of temperature. Excitation at the red edge of the absorption spectrum of TNS leads to an important shift (15 nm) of the fluorescence emission maximum of the probe. This reveals that emission of TNS occurs before relaxation of the amino-acids dipole occurs. Emission from a non-relaxed state means that TNS molecules are bound tightly to the protein, a result confirmed by polarization studies.


[Possibilities of myoelectric control of artificial limb prostheses].

This article summarizes the main possibilities for myoelectrical control of prostheses and other rehabilitation devices. In this context fundamental cybernetic aspects, the question of achieving optimal differentiation of antagonistic signals, and 16 possibilities of myoelectrical control, ranging from simple n-state threshold detectors to rate sensitive controllers, to intelligent controllers and pattern recognition techniques, are considered.


Fluorescence lifetime distribution of 1,8-anilinonaphthalenesulfonate (ANS) in reversed micelles detected by frequency domain fluorometry.

The fluorescence emission decay of ANS (1,8-anilinonaphthalenesulfonate) in reversed AOT (sodium bis-(2-ethyl-1-hexy)sulfosuccinate) micelles at different water contents was investigated by frequency domain fluorometry. The whole ANS emission decay in reversed AOT micelles could not be fitted in terms of discrete lifetime values, i.e., mono-exponential and bi-exponential models. Better fits were obtained when using continuous unimodal Lorentzian lifetime distributions. This was interpreted as arising from the reorientation processes of water molecules around the excited state of ANS or probe exchange among different probe locations, occurring on a time scale longer than fluorophore lifetime. The dependence of ANS fluorescence anisotropy on the emission wavelength was consistent with the existence of a great emission heterogeneity especially for inverted micelles having reduced H2O/AOT molar ratio. Finally, the observation that the distribution width decreases with increasing temperature and/or micelle size suggested that fast processes of water dipolar reorganization around the fluorophore are facilitated under these conditions.


Different superstructural features of the complexes between spermine and the light responsive elements of the two pea genes rbcS-3A and rbcS-3.6. Gel electrophoresis and circular dichroism studies.

Two light responsive elements (LREs), DNA sequences, 62 base pairs long, relevant to the light control during transcription of the pea genes rbcS-3A and rbcS-3.6, were synthesized and ligated to obtain multimers with defined superstructural features. Their gel electrophoretic mobilities were studied in the presence of the tetracation, spermine, since it was previously suggested, on the basis of theoretical analysis, that spermine can increase DNA bending and thus could be useful in revealing DNA superstructural features. In fact, the difference between the curvatures of the two LREs, derived from gel electrophoresis retardation ratios, increases in the presence of spermine. Circular dichroism spectra of the complexes between spermine and the two LREs, at different neutralization ratios, show that the polyamine is able to induce the formation of asymmetric arrangements of complexes of molecules. The chirality of these complexes appears dramatically different for the two LREs, suggesting that their different superstructural features give rise to different interactions with the polyamine.


Three new microcystins, cyclic heptapeptide hepatotoxins, from Nostoc sp. strain 152.

Three new cyclic heptapeptide hepatotoxins, [D-Ser1,ADMAdda5]microcystin-LR (1), [D-Asp3,-ADMAdda5]microcystin-LHar (2), and [ADMAdda5,Mser7]microcystin-LR (3), were isolated from the cyanobacterium (blue-green alga) Nostoc sp. strain 152, together with four known microcystins, [ADMAdda5]microcystin-LR (4), [ADMAdda5]microcystin-LHar (5), [D-Asp3,-ADMAdda5]microcystin-LR (6), and [DMAdda5]microcystin-LR (7). The structures of new microcystins were assigned on the basis of high-resolution fast atom bombardment mass spectrometry (HR FABMS), collisionally induced tandem FABMS (FABMS/MS), amino acid analysis, and gas chromatography (GC) on a chiral capillary column. All three new toxins contained 9-acetoxy-3-amino-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADMAdda) instead of the corresponding 9-methoxyl derivative (Adda), while 7 contains the corresponding 9-hydroxy analog (DMAdda). Compound 1 is the first microcystin reported that contains D-serine (D-Ser) in lieu of the D-alanine (D-Ala) unit which was thought to be an invariable amino acid component of the microcystins. Compound 2 has L-homoarginine (Har) instead of L-arginine (L-Arg) in 6 and D-aspartic acid (D-Asp) instead of D-erythro-beta-methylaspartic acid (D-MeAsp) in 5. Compound 3, the N-methylserine (Mser) variant of the N-methyldehydroalanine unit in 4, would be a biosynthetic precursor of 4.


Glutathione conjugation of aflatoxin B1 exo- and endo-epoxides by rat and human glutathione S-transferases.

Much evidence supports the view that the rate of conjugation of glutathione (GSH) with aflatoxin B1 (AFB1) exo-epoxide is an important factor in determining the species variation in risk to aflatoxins and that induction of GSH S-transferases can yield a significant protective effect. An assay has been developed in which the enzymatic formation of the conjugates of GSH and AFB1 exo-epoxide and the recently described AFB1 endo-epoxide is measured directly. 1H NMR spectra are reported for both the AFB1 exo- and endo-epoxide-GSH conjugates. Structural assignments were made by comparison with AFB1 exo- and endo-epoxide-ethanethiol conjugates, for which nuclear Overhauser effects were measured to establish relative configurations. The endo-epoxide was found to be a good substrate for GSH conjugate formation in rat liver cytosol while mouse liver cytosol conjugated the exo-epoxide almost exclusively. Human liver cytosol conjugated both epoxide isomers to much lower extents than did cytosols prepared from rats or mice. Purified rat GSH S-transferases catalyzed the formation of the AFB1 exo-epoxide-GSH conjugate in the order 1-1 approximately 4-4 approximately 3-3 greater than 2-2 greater than 4-6 (7-7 and 8-8 did not form the exo-epoxide-GSH conjugate at levels above the nonenzymatic rate). The only rat GSH S-transferases that conjugated the endo-epoxide were 4-4 and 4-6, with 4-4 being the more active.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of DNA adducts formed in vitro by reaction of N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline at the C-8 and N2 atoms of guanine.

The covalent binding of the carcinogenic N-hydroxy metabolites of 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) to deoxynucleosides and DNA was investigated in vitro. Two major adducts were formed by the reaction of the N-acetoxy derivatives of IQ and MeIQx with deoxyguanosine (dG); however, no adducts were formed with deoxycytidine, deoxyadenosine, or thymidine. From proton NMR and mass spectroscopic characterization the adducts were identified as 5-(deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-N2-IQ),N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo-[4,5-f]q uinoline (dG-C8-IQ), 5-(deoxyguanosin-N2-yl)-2-amino-3,8-dimethylimidazo[4,5-f]qu inoxaline (dG-N2-MeIQx), and N-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]qui noxaline (dG-C8-MeIQx). The level of dG-C8 adducts was approximately 8-10 times greater than the amount of dG-N2 adducts formed from the reaction of dG with the N-acetoxy derivatives of IQ and MeIQx. The C-8-substituted dG adduct was also the major adduct formed from reactions of DNA with N-acetoxy-IQ and N-acetoxy-MeIQx. Approximately 60-80% of the bound carcinogens were recovered from DNA as dG-C8 adducts upon enzymatic digestion. The dG-N2 adducts also were detected and accounted for approximately 4% of the bound IQ and 10% of the bound MeIQx. These results suggest that the relative contributions of the nitrenium and carbenium ion resonance forms as well as DNA macromolecular structure are major determinants for DNA adduct substitution sites. Investigations on adduct conformation of 1H NMR spectroscopy revealed that the anti form is preferred for the dG-N2 adducts of IQ and MeIQx, while the syn form is preferred for the dG-C8 adducts. The possible role of these adducts in the initiation of carcinogenesis is discussed.


Characterization of isothiocyanates, thioureas, and other lysine adduction products in carbon disulfide-treated peptides and protein.

Carbon disulfide (CS2) is an industrial solvent used in rayon production and as an organic synthetic precursor. It is also a member of the class of neuropathy-inducing xenobiotics known as the "neurofilament (NF) neurotoxicants". Current hypotheses propose direct reaction of CS2 with NF lysine epsilon-amine moieties as a step in the mechanism of this neuropathy. In this study, covalent CS2 binding in a lysine-containing dipeptide and in bovine serum albumin (BSA) in vitro was characterized. Dipeptide and BSA, incubated with 14CS2, exhibited stable incorporation of radioactivity after removal of unbound CS2 and reincubation in physiological buffer for up to 10 days. In contrast, free thiol levels decreased from a maximum immediately following CS2 exposure to near-base-line levels after 10 days, consistent with time-dependent conversion of initially formed N-substituted dithiocarbamate adducts into secondary products. HPLC/thermospray-MS and HPLC/UV photodiode-array analysis of CS2-dipeptide adducts confirmed dithiocarbamate formation and demonstrated their conversion into N-alkylisothiocyanates and, ultimately, N,N'-disubstituted thioureas and ureas. The results of UV spectrophotometry of CS2-treated BSA were also consistent with loss of dithiocarbamate and appearance of thioureas. Similar time-dependent formation of these products, in addition to N,N'-disubstituted thiuram disulfides, was demonstrated in CS2-treated BSA by means of 13C-NMR spectroscopy. SDS-PAGE analysis of adducted protein revealed a discrete, higher mobility band, likely representing a specific intramolecular cross-link. In contrast, no evidence for intermolecular protein cross-linking was obtained. Identical results were obtained with cysteinyl-blocked BSA, indicating the lack of formation of N,S-dialkyldithiocarbamate (dithiourethane) cross-links in these preparations.(ABSTRACT TRUNCATED AT 250 WORDS)


A comparative toxicological investigation of perfluorocarboxylic acids in rats by fluorine-19 NMR spectroscopy.

Male Fischer-344 rats administered a single intraperitoneal dose of perfluoro-n-octanoic acid (PFOA) or perfluoro-n-decanoic acid (PFDA) display a similar "wasting toxicity" characteristic of perfluorocarboxylic acids, with marked differences in temporal expression. Food/water consumption and urine output were monitored daily in PFOA-treated, PFDA-treated, and control rats. Fluorine-19 nuclear magnetic resonance (NMR) spectroscopy was used to monitor these fluorocarbons and possible fluoro metabolites in vivo, and to correlate differences in elimination with differences in effective toxicity. The data reveal a prolonged hypophagic response to PFDA and a more acute but transient response associated with PFOA treatment. PFOA causes a greater decline in food consumption than PFDA within the first 24 h postdose. PFOA-treated rats also show a ca. 2.5-fold increase in urine output on day 1, with only a slight increase in water consumption. In contrast to PFDA, PFOA-treated rats recover from hypophagia within 8 days. Fluorine-19 NMR spectra of various bodily fluids and liver in vivo display resonances of the parent PFOA or PFDA compounds and do not reveal any evidence of metabolism. Inorganic fluoride from dietary sources is detected in urine from both exposed and control rats. Differences in the route of excretion of PFOA vs PFDA are apparent from the spectral signal-to-noise ratio. The data suggest that PFOA is more readily excreted in the urine while PFDA is preferentially carried in bile. These apparent differences in elimination may account for their observed differences in effective toxicity. The acute transient toxicity and higher LD50 associated with PFOA may result from its rapid renal clearance.(ABSTRACT TRUNCATED AT 250 WORDS)


Ozonation of methyl oleate in hexane, in a thin film, in SDS micelles, and in distearoylphosphatidylcholine liposomes: yields and properties of the Criegee ozonide.

We here explore the use of Criegee ozonides as markers of ozone damage to lipids in systems that model pulmonary surfactant, lung lining fluids, and/or pulmonary membranes. Ozonation of methyl oleate in hexane gives an 89% yield of the Criegee ozonide. The presence of water should reduce the yield of this ozonide, and as expected, small but significant yields of Criegee ozonides are formed when the ozonation of methyl oleate is carried out as a film over phosphate buffer, in aqueous micelles of sodium dodecyl sulfate (SDS), or in distearoyl-L-alpha-phosphatidylcholine (DSPC) liposomes spiked with methyl oleate. Analysis utilizes reversed-phase HPLC and 1H NMR. The total yield of ozonides in 0.02 M SDS micelles exposed to 26 ppm ozone for 3 h at pH 7.4 and 22 degrees C is 11%; 7.5% is the normal ozonide, methyl 5-octyl-1,2,4-trioxolane-3-octanoate (MOO2) (ca. 4.2% trans and ca. 3.3% cis), and 3.5% is accounted for by the two cross ozonides, 3,5-dioctyl-1,2,4-trioxolane (MOO1) and dimethyl 1,2,4-trioxolane-3,5-dioctanoate (MOO3). No significant difference of ozonide yields is observed for ozonations with 26 ppm and 1.2% (12,000 ppm) ozone. The conversion of methyl oleate increaes with increasing SDS concentration. Approximately comparable yields of ozonides also are found in the ozonation of methyl oleate in DSPC liposomes although yields are not quantified. The ozonides slowly hydrolyze at pH 7.4 and 37 degrees C with half-lives for trans-MOO2 and cis-MOO2 in 0.10 M SDS micelles of 23 and 6 days, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Modeling cyanide release from nitriles: prediction of cytochrome P450 mediated acute nitrile toxicity.

A mechanism-based model for prediction of acute nitrile toxicity was developed using octanol-water partition coefficients (log P) and estimated rates of alpha-hydrogen atom abstraction as variables. Relative rates of hydrogen atom abstraction were derived from differences in heats of formation for ground-state and radical geometries and radical ionization potentials. Calculated energies of activation for all potential sites of oxidation for a given nitrile were used to estimate partitioning of metabolites among multiple oxidative pathways. logP and the resulting corrected rate constants for alpha-carbon oxidation were effective variables in an acute toxicity model of structurally diverse nitriles. The pharmacokinetics of substrate disposition is discussed in the context of multiple metabolic pathways. Structure-toxicity relationships are also discussed.


Comparative metabolism of dibenz[a,j]anthracene and 7-methyldibenz[a,j]anthracene in primary cultures of mouse keratinocytes.

The identification of several metabolites formed from dibenz[aj]anthracene (DB[aj]A) and 7-methyldibenz[aj]anthracene (7MeDB[aj]A) in primary cultures of mouse keratinocytes is presented. The metabolites were analyzed by coelution with known synthetic standards using high-pressure liquid chromatography. The metabolite identifications were further facilitated by comparisons of fluorescence excitation and emission spectra obtained for isolated metabolites with those for the synthetic standards. Both DB[aj]A and its 7-methyl analog were converted by mouse keratinocytes to the corresponding 3,4- as well as 5,6-dihydrodiols. The 5,6-dihydrodiol of DB[aj]A was the major intracellular metabolite found at all time points up to 24 h. By 48 h, the relative proportion of both intracellular dihydrodiols had decreased, and they were found in approximately equal proportions. In contrast, 7MeDB[aj]A-3,4- dihydrodiol was the major intracellular metabolite of 7MeDB[aj]A at all time points examined up to 48 h. The decreased intracellular retention of DB[aj]A-3,4-dihydrodiol was due, in part, to glucuronide conjugation and subsequent excretion of this metabolite. In this regard, both the 3,4- as well as the 5,6-dihydrodiols of DB[aj]A were found as glucuronides in the extracellular medium, whereas no such conjugates were detected extracellularly in cultures exposed to the 7-methyl derivative. The chromatographic profiles of cell- and medium-associated metabolites from both hydrocarbons also exhibited several other metabolite peaks that remain to be identified. The observed differences in dihydrodiol metabolism by mouse keratinocytes could explain, in part, the greater biological activity of 7MeDB[aj]A relative to DB[aj]A.


Differential effects of thiols on DNA modifications via alkylation and Michael addition by alpha-acetoxy-N-nitrosopyrrolidine.

The hepatocarcinogen NPYR is metabolically activated by alpha-hydroxylation mediated by cytochrome P-450 enzymes to yield a 4-oxobutylating agent and 2-butenal (crotonaldehyde). Both are reactive intermediates capable of modifying DNA with guanine either by simple alkylation or by Michael type addition, respectively. In order to assess the roles of these pathways in NPYR tumorigenesis, we are interested in identifying agents which can selectively modify one of these two pathways. In this study, we examined the effects of three thiols--(mesna), glutathione (Glu), and N-acetylcysteine (Nac)--on DNA adduct formation by alpha-acetoxyNPYR, a stable precursor of alpha-hydroxyNPYR. Calf thymus DNA isolated from incubation of alpha-acetoxyNPYR with or without thiol was hydrolyzed and analyzed for the adducts formed by alkylation (adducts 1 and 2) and Michael addition (adducts 3-5). The results showed that the addition of mesna completely blocked the formation of the crotonaldehyde-derived adducts 3-5, whereas it exerted little effect on the formation of the alkylated adducts 1 and 2. These results indicate the preferential conjugation of mesna with crotonaldehyde. In contrast, Nac had little selectivity on adduct formation; levels of adducts 1 to 5 were were reduced by 36-75%. These results suggest that Nac conjugated with both alkylating agent and crotonaldehyde. Similar to mesna, Glu blocked the formation of the crotonaldehyde-derived adducts (adducts 3-5) efficiently. However, unlike mesna, Glu inhibited the formation of adduct 1, while it did not inhibit the formation of adduct 2, although both adducts are presumably derived from the 4-oxobutylating agent.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of urinary metabolites from [1,2,methoxy-13C]-2-methoxyethanol in mice using 13C nuclear magnetic resonance spectroscopy.

2-Methoxyethanol (2-ME) is an industrial solvent that induces developmental and testicular toxicity in laboratory animals. Oxidation of 2-ME to 2-methoxyacetic acid (2-MAA) is required for the generation of these adverse effects. The urinary metabolites of 2-ME were investigated to characterize the fate of 2-ME and 2-MAA. 13C NMR spectroscopy was used to detect and assign metabolites in the urine of pregnant CD-1 mice following administration of 250 mg/kg of [1,2,methoxy-13C]-2-ME. Two-dimensional NMR methods were used to correlate signals from the labeled carbons in each 2-ME metabolite and to determine the number of hydrogens attached to each carbon. Structures were assigned from the NMR data together with calculated values of shift for biochemically feasible metabolites and by comparison to standards. Pathways involved in forming metabolites assigned in this study include transformation of 2-ME via ethylene glycol, conjugation with glucuronide or sulfate, and oxidation to 2-MAA. Additional metabolites were assigned that can be formed from further conversion of 2-MAA to glycine and glucuronide conjugates, as well as metabolites derived from the incorporation of 2-methoxyacetyl CoA derivatives into intermediary metabolism. Elucidation of the further metabolism of 2-MAA may be important for understanding the mechanisms by which 2-ME induces adverse effects.


Crystal and molecular structure of the doubly unsaturated dehydropeptide Ac-delta Phe-Ala-delta Phe-NH-Me.

The dehydropeptide Ac-delta Phe-L-Ala-delta Phe-NH-Me, containing two dehydro-phenylalanine (delta Phe) residues, crystallizes from methanol/water in space group P2(1)2(1)2(1), with a = 12.508 (2), b = 12.746 (1) and c = 15.465 (9). In the crystalline state, the peptide chain assumes a right-handed 3(10)-helical conformation stabilized by two intramolecular hydrogen bonds, between the N-terminal acetyl group and the NH of delta Phe3, and between the CO of delta Phe1 and the NH of the C-terminal methylamide group, respectively. The two consecutive 10-membered rings formed by the hydrogen bonds have torsion angles quite close to the standard values for type III beta-bends. delta Phe1 is located in the (i + 1) position of the first beta-bend, while delta Phe2 is located in the (i + 2) position of the other beta-bend. In the crystal, the molecules are linked head to tail by intermolecular hydrogen bonds to form long helical chains. The axes of the helices are parallel to the c axis, but neighboring helices run in antiparallel directions. This crystal packing is similar to the packing motifs frequently observed in Aib-containing peptides.


Hydration of DNA bases: analysis of crystallographic data.

We present a systematic analysis of water structure around nucleic acid bases. We have examined 28 crystal structures of oligonucleotides, and have studied the patterns of water around the four bases, guanine, cytosine, adenine, and thymine. The geometries of water positions were calculated up to 4.00 A from base atoms. We have found conformation-dependent differences in both the geometry and extent of hydration of the bases.


Alkylation of DNA by 1,3-dialkyl-3-acyltriazenes: correlation of biological activity with chemical behavior.

The reactions of calf thymus DNA with four 1,3-dialkyl-3-acyltriazenes were studied alone or in the presence of pig liver esterase in pH 7.4 phosphate buffer for varying lengths of time. The best alkylating agent in the absence of esterase was determined to be 1,3-dimethyl-3-carbethoxytriazene (DMC), followed in order by 1-(2-hydroxyethyl)-3-methyl-3-carbethoxytriazene (HMC), 1-(2-hydroxyethyl)-3-methyl-3-acetyltriazene (HMA), and 1-(2- chloroethyl)-3-methyl-3-carbethoxytriazene (CMC). This order is the same as that for the rate of decomposition of the various acyltriazenes in pH 7.5 phosphate buffer. The extent of calf thymus DNA alkylation by CMC was found to be dependent on both the reaction buffer and the ionic strength of the medium. Alkylation by CMC alone in low ionic strength glycine buffer produced large quantities of 7-(2-chloroethyl)guanine and 7-(2-hydroxyethyl)guanine. The products of DNA alkylation observed at neutral pH are consistent with N(2)-N(3) heterolysis of the triazene, resulting in the N(1) alkyldiazonium ion as the sole alkylating species. In the presence of esterase, CMC showed an enhanced rate of product formation. Furthermore, the product distribution shifted dramatically from mainly hydroxyethylation to predominantly methylation. CMC is postulated to undergo initial enzymatic deacylation, leading to two different alkyldiazonium ions which competitively alkylate DNA. HMC, on the other hand, was little affected by the esterase. The enzyme-catalyzed reaction showed a small increase in methylation and a smaller decrease in hydroxyethylation.(ABSTRACT TRUNCATED AT 250 WORDS)


Kinetics of folding of alpha alpha-tropomyosin subsequences.

The kinetics of folding random coils of alpha alpha-tropomyson (Tm) subsequences to two-chain coiled coils was studied by stopped-flow CD. Subsequences studied were those comprising residues 11-127 (11Tm127), 142-281 (142Tm281), 1-189 (1Tm189), and 190-284 (190Tm284) of the parent 284-residue alpha-tropomyosin chain. Unlike the parent, subsequences 1Tm189 and 11Tm127 fold within the dead time of the instrument (less than 0.04 s). Like the parent, subsequences 142Tm281 and 190Tm284 fold in two phases. In the fast phase, 45% and 32%, respectively, of the equilibrium helical content form. In the time-resolvable, first-order slow phase (k-1 = 2.7 s at 20 degrees C for 142Tm281 and k-1 = 2.0 s at 15 degrees C for 190Tm284), the remaining structure forms. Neither reduced 142Tm281 nor 190Tm284 show any dependence of the rate on concentration, so chain association occurs in the fast phase. Like the parent 142Tm281 forms more helical content in the fast phase when cross-linked at C-190, and the remaining structure forms slowly with rate parameters similar to those of the reduced species. Comparison of the folding behavior of C- and N-terminal subsequences with that of the parent protein suggests that the slow phase in the parent is caused by a folding bottleneck somewhere nearer the C-terminus. However, rapid association and partial folding near the N-terminus is not necessary for prompt folding, since even 190Tm284 chains associate and partially fold very rapidly (less than 0.04 s), and then complete the folding in seconds.


An in vitro model for the in vivo mobilization of cadmium by chelating agents using 113Cd-NMR spectroscopy.

An in vitro method, based on 113Cd-NMR spectroscopy, that provides an alternative to the use of animals for an initial screening of cadmium antagonists is presented. The relative values of the effective stability constants of potential chelating antagonists for cadmium are estimated by using 113Cd-NMR spectroscopy to determine the concentrations of the cadmium species involved in appropriate competitive equilibria. This is accomplished via an examination of the competition between the proposed antagonist and EDTA (ethylenediaminetetraacetic acid) for cadmium-113; previously, EDTA has been shown to be capable of removing cadmium from such in vivo binding sites as metallothionein. The reactions proceed via the stepwise addition of three dithiocarbamate groups to the cadmium accompanied by the concurrent stepwise release of donor groups from the EDTA. The resulting 113Cd-NMR data allow for the determination of the overall stability constant for the complex formed between cadmium and N-methyl-D-glucamine dithiocarbamate, iminodiacetic acid dithiocarbamate, proline dithiocarbamate, sarcosine dithiocarbamate. The use of 113Cd-NMR spectroscopy has the potential for providing direct evidence on the effectiveness of chelate antagonists to compete with endogenous ligands for other toxic metal ions. This technique could prove very useful for other compounds that are not stable enough toward acid and/or base to be examined by standard titrimetric methods.


Molecular structure of cyclo[-(D-Val-L-Hyi-L-Val-D-Hyi)2-] revealed by x-ray analysis.

The crystal structure of a synthetic analogue of valinomycin, cyclo[-(D-Val-L-Hyi-L-Val-D-Hyi)2-] (octa-meso-valinomycin) (I) (C40H68N4O12.1.5.C4H8O2, M(r) = 937.01 + 88.10), has been determined. Crystals grown from dioxane are monoclinic, space group P2(1)/a, with cell parameters a = 21.487 (8), b = 16.836 (5), c = 16.089 (4) A, beta = 111.70 (4), and Z = 4. The atomic coordinates for nonhydrogen atoms were refined in the anisotropic thermal motion approximation. H atom positions were included in the structure factor calculations at their geometrically expected positions. Values of the standard and weighted R factors after refinement are 0.11 and 0.13, respectively. The conformation of the depsipeptide crystallized from dioxane is different from that crystallized from chloroform (II). The molecule adopts a rectangular shape with two type IV beta-turns containing a hydrogen bond and possesses pseudorotational symmetry. The side chains are located on the molecular periphery. The orientation of the carbonyl groups of the molecule is not conducive for efficient metal-ion coordination and in the observed conformation cannot behave as an ionophore. In the crystal the molecules form infinite chains parallel to the c axis, and are stabilized by two intermolecular hydrogen bonds that are shorter and have better geometry than the intramolecular hydrogen bonds. A phi/psi plot for dodecadepsipeptides with a (DLLD)3 sequence has well-defined areas for Val and Hyi residues only in cases when the crystals have been grown from nonpolar or medium-polar solvents. The phi/psi plot for octadepsipeptides crystallized from chloroform (II) shows this behavior also.(ABSTRACT TRUNCATED AT 250 WORDS)


Dihydroxylated mercapturic acid metabolites of bromobenzene.

Bromobenzene is metabolized to electrophilic epoxides and quinones which covalently bind to protein sulfur nucleophiles, yet no quinone-derived mercapturic acid metabolites of bromobenzene have been reported. To search for them, phenobarbital-induced Sprague-Dawley rats were dosed (0.5-1.5 mmol/kg, ip) with either bromobenzene, 2-, 3-, or 4-bromophenol (BP), 3- or 4-bromocatechol (BC), or 2-bromohydroquinone (BHQ). Urine (0-24 h) was alkaline permethylated (2 N NaOH/CH3I/80 degrees C), and the resulting thioanisole derivatives were analyzed by GC/MS. Three dimethoxythioanisoles and eight bromodimethoxythioanisoles were formed by alkaline permethylation of urine of rats treated with bromobenzene or 3-BP; alkaline permethylation of urine from rats in other treatment groups afforded characteristic subsets of these derivatives. The major thioanisole from all groups except 3-BC or 4-BC was 2,5-dimethoxythioanisole, which arises from (2,5-dihydroxyphenyl)mercapturic acid. The latter was isolated from rat urine and is the first debrominated metabolite of bromobenzene reported to date. Its formation from both 4-bromophenol and BHQ requires two parallel mechanisms for bromine loss: (1) nucleophilic addition to 1,4-benzoquinone formed by hydroxylative debromination of 4-bromophenol and (2) nucleophilic displacement of bromine from 2-bromo-1,4-benzoquinone by sulfur. The yields of quinone-derived mercapturic acids formed from bromobenzene are quite low (less than 1% of dose) compared to the high yields of epoxide-derived mercapturic acids (40% of dose). Potential reasons for this are discussed.


Vibrational CD studies of interchain hydrogen-bonded tripodal peptides.

The solution conformations of three trispeptides--L,L,L-1,3,5-C6H3[CH2NHCOCH(X)-NHBoc++ +]3, X = CH3 (Ala) or CH2CH(CH3)2 (Leu), and L,L,L-N(CH2CH2NHCOCH[CH2-CH(CH3)2]NHBoc)3--have been determined from their ir and vibrational CD (VCD) spectra in the NH stretching and carbonyl stretching regions in apolar solution. The compounds containing L-Leu are shown to occur primarily in a propeller conformation with C3 symmetry that is stabilized by interchain hydrogen bonds. Through application of the coupled oscillator model of VCD, a right-handed sense for the hydrogen-bonded chains in the propeller is deduced, in agreement with previous empirical force field calculations. The spectra also provide evidence for interchain association between two chains, resulting in a C10-ring. For chains not involved in interchain association, the spectra reveal the presence of C7-rings within a chain. The trispeptide containing L-Ala is found to occur primarily in a random form.


Examination of diols and diol epoxides of polycyclic aromatic hydrocarbons as substrates for rat liver dihydrodiol dehydrogenase.

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) can suppress the formation of anti-diol epoxides that arise from the metabolic activation of PAH by oxidizing their precursor trans-dihydrodiols to o-quinones [Smithgall, T.E., et al. (1988) J. Biol. Chem. 263, 1814-1820]. DD has also been found to reduce the mutagenicity of benz[a]anthracene (+/-)-anti-8,9-dihydrodiol 10,11-epoxide [(+/-)-anti-BADE] in the Ames test (Glatt, H.R., et al. (1982) Science 215, 1507-1509), suggesting that anti-diol epoxides are substrates for this enzyme. In this study, attempts have been made to demonstrate directly that diol epoxides of PAH are substrates for homogeneous DD. Spectrophotometric assays indicate that high concentrations of the stable anti-diol epoxides, naphthalene (+/-)-anti-1,2-dihydrodiol 3,4-epoxide (10 mM) and (+/-)-anti-BADE (20 microM; limit of solubility) were not oxidized by micromolar concentrations of enzyme. By contrast, 20 microM (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene [(+/-)-trans-BP-diol] was oxidized by 50-fold less enzyme. Using a reverse-phase high-performance liquid chromatography (RP-HPLC)-based assay [1,3-3H]-(+/-)-trans-BP-diol could be almost completely oxidized by DD in the presence of NADP+. Using a similar assay, [1,3-3H]benzo[a]pyrene (+/-)-anti-7,8-dihydrodiol 9,10-epoxide [(+/-)-anti-BPDE], and unlabeled (+/-)-syn-BPDE and (+/-)-anti-BADE were tested as substrates for DD.(ABSTRACT TRUNCATED AT 250 WORDS)


Conformational states of a TT mismatch from molecular dynamics simulation of duplex d (CGCGATTCGCG).

The TT mismatch region in duplex d (CGCGATTCGCG) was studied using a 500-ps molecular dynamics (MD) simulation in water, and a series of 1-ps MD simulations and energy minimizations in vacuum. The DNA maintained its duplex structure, although the mismatch region showed significantly higher flexibility than the GC regions. The predominant conformation in the 500-ps MD simulation involved an average -42 degrees propeller twist between T6 and T'6, and a -22 degree buckle between A5 and T'7. One hydrogen bond was formed between T6 and T'6, and another between T6 and the O2 of T'7, with both Watson-Crick hydrogen bonds between A5 and T'7 remaining intact. The minimizations resulted in conformations with the equivalent hydrogen-bonding pattern, as well as ones with "wobble pair" hydrogen bonds between T6 and T'6. However, the wobble pair conformation was found to be unstable in the water simulation.


Flow linear dichroism spectra of four filamentous bacteriophages: DNA and coat protein contributions.

In this study, we have separated the contributions of DNA and protein to the absorption and linear dichroism (LD) of each of four phages: fd, IKe, Pf1, and Pf3. We have found that the DNA packaged in each of the phages is hypochromic relative to the purified single-stranded DNA, suggesting that bases are stacked in all of the phages. We have oriented the phages by flow and for the first time report the intrinsic LD from 320 to 190 nm for each of these phages. From the intrinsic LD of the phages and the isotropic absorption of the individual components, we have determined the reduced dichroism of the DNA within the phages and, subsequently, the maximum angle of inclination of the DNA bases (from the helix axis) for the packaged DNA. The maximum angles were 63 degrees and 64 degrees for the DNAs of class I phages fd and IKe, respectively. The angles were significantly less, 51 degrees and 49 degrees, for the DNAs of the class II phages Pf1 and Pf3, respectively. Thus, the two classes of phage differ in the structures of their packaged DNA, the DNA bases of the class II phages being more parallel to the long axis of the phage than are the DNA bases of the class I phages.


Crystal and molecular structure of the depsipeptide ionophore hexadecaisoleucinomycin, cyclo-[(D-Ile-L-Lac-L-Ile-D-Hyi)4-] (C80H136N8O24).

The crystal structure of a synthetic depsipeptide ionophore hexadecaisoleucinomycin, cyclo [-(D-Ile-L-Lac-L-Ile-D-Hyi)4-] (C80H136N8O24), has been determined by single crystal x-ray diffraction techniques. The crystals are orthorhombic, space group P2(1)2(1)2(1), number of molecules per unit cell z = 4, and cell parameters a = 11,195, b = 17.853, c = 54.835 A. The values of the standard (R) and weighted (Rw) discrepancy factors after refinement are 0.122 and 0.135, respectively. The structure is characterized by an elongated bracelet form with a twofold axis of pseudosymmetry. It is stabilized by eight intramolecular 4----1 hydrogen bonds between the amide C = O and N - H groups. The ester carbonyls are directed toward the inside of the molecule, their oxygen atoms forming an ellipsoidal internal cavity. The side chains are located on the molecular periphery. The conformational states of hexadecaisoleucinomycin in solution are discussed in the light of the data obtained.


Studies of DNA dumbbells. I. Melting curves of 17 DNA dumbbells with different duplex stem sequences linked by T4 endloops: evaluation of the nearest-neighbor stacking interactions in DNA.

Seventeen DNA dumbbells were constructed that have duplex sequences ranging in length from 14 to 18 base pairs linked on the ends by T4 single-strand loops. Fifteen of the molecules have the core duplexes with the sequences 5'G-T-A-T-C-C-(W-X-Y-Z)-G-G-A-T-A-C3', where (W-X-Y-Z) represents a unique combination of A.T, T.A, G.C, and C.G base pairs. The remaining two molecules have the central sequence (W-X-Y-Z) = A-C and A-C-A-C-A-C. These duplex sequences were designed such that the central sequences include different combinations of the 10 possible nearest-neighbor (n-n) stacks in DNA. In this sense the set of molecules is complete and serves as a model system for evaluating sequence-dependent local stability of DNA. Optical melting curves of the samples were collected in 25, 55, 85, and 115 mM [Na+], and showed, regardless of solvent ionic strength, that the transition temperatures of the dumbbells vary by as much as 14 degrees for different molecules of the set. Results of melting experiments analyzed in terms of a n-n sequence-dependent model allowed evaluation of nine independent linear combinations of the n-n stacking interactions in DNA as a function of solvent ionic strength. Although there are in principle 10 possible different n-n interactions in DNA, these 10 are not linearly independent and therefore can not be uniquely determined. For molecules with ends, there are 9 linearly independent combinations, as opposed to circular or semiinfinite repeating copolymers where only 8 linear combinations of the 10 possible n-n interactions are linearly independent. The n-n interactions are presented as combinations of the deviations from average stacking for the 5'-3' base-pair doublets, delta Gi, and reveal several interesting features: (1) Titratable changes in the values of delta Gi with changing salt environment are observed. In all salts the most stable unique combination is delta G4 = (delta GGpC+delta GCpG)/2, and the least stable is the GpG/CpC stack, delta G2 = delta GGpG/CpC. (2) The chi 2 values of the fits of the evaluated delta Gi's to experimental data increased with decreasing [Na+], suggesting that significant interactions beyond nearest neighbors become more pronounced, particularly at 25 nM Na+.(ABSTRACT TRUNCATED AT 400 WORDS)


Studies of DNA dumbbells. II. Construction and characterization of DNA dumbbells with a 16 base-pair duplex stem and Tn end loops (n = 2, 3, 4, 6, 8, 10, 14).

The preparation and characterization of DNA dumbbells that contain the 16 base-pair duplex sequences 5'G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C3' (set 1) and 5'G-C-A-T-C-A-T-C-G-A-T-G-A-T-G-C3' (set 2) are reported. The dumbbells of set 1 have the duplex stem nucleated on both ends by Tn (n = 2, 3, 4, 6, 8, 10, and 14) loops. The dumbbells of set 2 have Tn (n = 2, 4, 8, 10) end loops. For the molecules of set 1, effects of end loop size on the electrophoretic mobility, CD and UV absorbance spectra, and cleavage by restriction enzymes, were investigated. Effects of loop size on the CD spectra and restriction enzyme cleavage of the molecules of set 2 were also examined. Optical melting curves of the molecules of set 1 were collected as a function of sodium ion concentration from 30 to 120 mM. These investigations revealed that as loop size decreases, the electrophoretic mobilities, rates of enzyme cleavage, and optical melting temperatures increase. For end loops with at least three T's the observed increases are inversely proportional to loop size. The behavior of the dumbbell with T2 end loops departs from this linear dependence and is anomalous in every experimental context. For molecules with end loops comprised of at least four T's CD spectra were virtually indistinguishable. However, these spectra differed considerably from the CD spectrum of the T2-looped molecule. The CD spectrum of the dumbbell with T3 end loops displayed features common to the dumbbells with larger loops and T2 end loops. Thermodynamic evidence that the terminal G.C base pairs (bps) nucleating the T2 end loops were intact was obtained from a comparison of the melting temperature of this molecule with that of a DNA dumbbell containing the 14 central bps of the set 1 duplex sequence linked instead by end loops comprised of the four base sequence, C-T-T-C. The tm of this latter molecule was determined to be 9 degrees C less than that of the former dumbbell assumed to contain a 16-bp stem and T2 end loops.(ABSTRACT TRUNCATED AT 400 WORDS)


Studies of DNA dumbbells. III. Theoretical analysis of optical melting curves of dumbbells with a 16 base-pair duplex stem and Tn end loops (n = 2, 3, 4, 6, 8, 10, 14).

Optical melting curves of seven DNA dumbbells with the 16 base-pair duplex sequence 5'G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C3' linked on both ends by Tn (n = 2, 3, 4, 6, 8, 10, and 14) loops measured in 30, 70, and 120 mM Na+ are analyzed in terms of the numerically exact statistical thermodynamic model of DNA melting. The construction and characterization of these molecules were described in the previous paper (Amaratunga et al., 1992). As was recently reported for hairpins (T. M. Paner, M. Amaratunga, M. J. Doktycz, and A. S. Benight, 1990, Biopolymers, Vol. 29, pp. 1715-1734) theoretically calculated melting curves were fitted to experimental curves by simultaneously adjusting the parameters representing loop and circle formation to optimize the fits. The systematically determined empirical parameters provide evaluations of the free energies of hairpin loop formation delta Gloop (n) and single-strand circles delta Gcircle (N), as a function of end loop size, n = 2-14, and circle size, N = 32 + 2n. The dependence of these quantities on solvent ionic strength over the range from 30 to 120 mM Na+ was evaluated. An approximately analytical expression for the partition function Q(T) of the dumbbells was formulated that allowed a means for determining the transition enthalpy delta H degrees and entropy delta S degrees for every dumbbell, revealing the dependence of these quantities on loop size. In this multistate approach a manifold of partially melted intermediate microstates are considered and therefore no assumptions regarding the nature of the melting transitions (that they are two-state) are required. The transition thermodynamic parameters were also determined from a van't Hoff analysis of the melting curves. Comparisons between the results of the multistate analysis and the two-state van't Hoff analysis revealed significant differences for the dumbbells with larger end loops, indicating that the melting transitions of the larger looped dumbbells deviate considerably from two-state behavior. Results are then compared with published melting studies of much larger DNA dumbbells (D. B. Naritsin and Y. L. Lyubchenko, 1990, Journal of Biomolecular Structure and Dynamics, Vol. 8, pp. 1-13), of small hairpins (Paner et al., 1990; M. J. Doktycz, T. M. Paner, M. Amaratunga and A. S. Benight, 1990, Biopolymers, Vol. 30, pp. 829-845) and another dumbbell (A. S. Benight, J. M. Schurr, P. F. Flynn, B. R. Reid, and D. E. Wemmer, 1988) Journal of Molecular Biology, Vol. 200, pp. 377-399).(ABSTRACT TRUNCATED AT 400 WORDS)


The ever-evolving epidemiologic concepts of human immunodeficiency virus infections.

Human immunodeficiency virus infection has now reached epidemic proportions in both industrialized and nonindustrialized countries. Two preventive measures remain of questionable benefit: mandatory testing and partner notification. Sexually transmitted diseases and cervical ectopy may be risk factors for heterosexual transmission, now the most frequent mode of transmission of human immunodeficiency virus worldwide. Smokable freebase cocaine, the use of which is increasing in many cities in industrialized countries, is associated with acquisition of sexually transmitted diseases and human immunodeficiency virus infection. In addition to perinatal transmission of human immunodeficiency virus, postnatal transmission via breastfeeding has been demonstrated in association with a recent acquisition of human immunodeficiency virus by the mother. Intriguingly, in multiple pregnancies, first-born twins of human immunodeficiency virus-infected mothers could be at higher risk of infection than second-born twins. Nosocomial transmission of human immunodeficiency virus, both from infected patients to health professionals and from infected health professionals to patients, is a matter of concern and justifies preventive measures.


Pelvic inflammatory disease.

Pelvic inflammatory disease is a common serious complication of the sexually transmitted pathogens Neisseria gonorrhoeae and Chlamydia trachomatis. There are more than 800,000 cases of pelvic inflammatory disease annually accounting for approximately 200,000 hospital admissions for acute and chronic infections. Early accurate diagnosis and treatment are essential to prevent the serious sequelae including ectopic pregnancy, tubal disease infertility, chronic pain, and disability requiring multiple hospitalizations and surgery. Although clinical models to aid in the diagnosis and management of pelvic inflammatory disease have been developed by numerous investigators, all have lacked the sensitivity and specificity to be helpful to the clinician. Laparoscopy, considered by many to be the "gold standard" for diagnosis, is underutilized, and the definition of pelvic infection differs between investigators. Improved patient compliance and safety may be seen if single-agent therapy for acute pelvic inflammatory disease becomes a reality. In a small prospective randomized study, oral ofloxacin was as effective as cefoxitin plus doxycycline for outpatient treatment of chlamydial and gonococcal pelvic inflammatory disease. Treatment of tuboovarian abscess appears to be successful with single agent and combination therapy. Risk factors for developing postabortion endometritis continue to be identified, and the most efficacious prophylactic antibiotic regimen has not been determined to date.


New trends in combined use of gonadotropin-releasing hormone antagonists with gonadotropins or pulsatile gonadotropin-releasing hormone in ovulation induction and assisted reproductive technologies.

The use of gonadotropin-releasing hormone agonists as adjunctive therapy with gonadotropins for ovulation induction in in vitro fertilization and other assisted reproductive technologies has become common clinical practice. With the recent advent of potent gonadotropin-releasing hormone antagonists free from the marked histamine-release effects that stymied earlier compounds, an attractive alternative method may be available. We have established the feasibility of combining gonadotropin-releasing hormone antagonist-induced inhibition of endogenous gonadotropins with exogenous gonadotropin therapy for ovulation induction in a nonhuman primate model. Here, the principal benefits to be gained from using the gonadotropin-releasing hormone antagonist rather than the gonadotropin-releasing hormone agonist are the immediate inhibition of pituitary gonadotropin secretion without the "flare effect," which brings greater safety and convenience for patients and the medical team and saves time and money. We have also recently demonstrated the feasibility of combining gonadotropin-releasing hormone antagonist with pulsatile gonadotropin-releasing hormone therapy for the controlled restoration of gonadotropin secretion and gonadal steroidogenesis culminating in apparently normal (singleton) ovulatory cycles. This is feasible only with gonadotropin-releasing hormone antagonists because, unlike gonadotropin-releasing hormone agonists, they achieve control of the pituitary-ovarian axis without down regulation of the gonadotropin-releasing hormone receptor system. This capacity to override gonadotropin-releasing hormone antagonist-induced suppression of pituitary-ovarian function may allow new treatment modalities to be employed for women who suffer from chronic hyperandrogenemia with polycystic ovarian disease.


Current technology of oocyte retrieval.

Transvaginal follicle puncture under sonographic control has become the method of choice for ovum retrieval worldwide. Because it is the current technology of the 1990s, there are very few reports in the literature on other methods of oocyte retrieval. Furthermore, perhaps because of the simplicity of transvaginal follicle puncture under sonographic control, there are only a few reports on the technique itself in the current literature. This review classifies the papers on hand according to general studies of the subject, appropriate anesthesia, necessity of follicle flushing for oocyte retrieval, vaginal disinfection and the evaluation of the risk following transvaginal oocyte retrieval, and new techniques in connection with the harvesting of oocytes.


Fertilization and early embryonic development.

Whether one carefully and meticulously records detailed observations in the laboratory, or jumps feet first into new and exciting areas of investigation, each scientist contributes to the advances being made in the control of human reproduction. The study of spermatozoa, oocytes, sperm-oocyte interaction, preembryo growth and development, and early pregnancy rewards us with breakthroughs in innovative technology and promotes paths of better understanding. One cannot search through the massive amounts of currently available literature without desiring to try "this" technique, expound on "that" one, take someone else's work a little further, or look at one's own results from a different angle. Presented here are some informative data, most published within the past year. Oocyte maturational competence and integrity, sperm function and quality, preembryo culture conditions, preembryo morphology, and ovarian age all emerge as factors important for interpreting a patient's chance of achieving pregnancy.


Male factor infertility and assisted reproductive technologies.

Male infertility has many facets that need to be evaluated in an initial screening. Once these are known, recommendations become clearer for aggressively pursuing assisted reproductive techniques. Progress has been made in defining more clearly the severe male factor: concentration less than 5 x 10(6) sperm/mL, motility less than 10%, morphology less than 4% normal forms (by strict criteria), and recovered swim-up sperm less than 1.5 x 10(6). Sperm preparation techniques may be improved by use of Percoll separation medium, and morphology may be used in prediction of cleavage rate in in vitro fertilization. Hopefully, these techniques will lead to more specific guidelines for successful use of assisted reproductive technology in male factor patients.


Micromanipulation of gametes and embryos in preimplantation genetic diagnosis and assisted fertilization.

Recent advances in micromanipulation and biopsy of gametes and embryos have made it possible to develop new approaches for early genetic diagnosis and prevention of genetic disease and for treatment of severe male-factor infertility. Preimplantation diagnosis of a number of X-linked and autosomal recessive disorders has been performed, using polar body sampling and blastomere biopsy, coupled with polymerase chain reaction. Blastocyst biopsy has also been performed in human embryos; however, there has been no clinical application so far. Existing data have not shown any detrimental effect of micromanipulation and biopsy involved in the preimplantation development of the human embryo. The existing experience on micromanipulation of gametes (zona-opening procedures, subzonal sperm insertion, and sperm microinjection into the ooplasm) has also demonstrated the clinical usefulness in assisted fertilization, suggesting a possible selective application of various micromanipulation techniques and their combinations in male infertility.


Cryopreservation of semen, oocytes, and embryos.

Human embryo cryopreservation is widely applied by programs of assisted reproductive technology. However, recent surveys have shown that relatively few in vitro fertilization programs in the United States substantially increase their per retrieval delivery rates through cryopreservation. While embryos may be successfully frozen and thawed at a range of developmental stages using several different cryoprotectants, the majority of in vitro fertilization programs use 1,2 propanediol to freeze early (pronuclear four-cell) embryos. Ultrarapid freezing techniques are under continued study; they have not yet been shown to offer any clinical advantage over slow freezing protocols. Although interest in oocyte cryopreservation remains high, experimental evidence shows that cryoinjury to human oocytes results in chromosome sorting defects, reduced fertilization, and compromised embryonic development. Micromanipulation of mammalian oocytes appears to have no deleterious effect on subsequent cryopreservation.


Oocyte donation.

Use of donor oocytes is becoming an increasingly important facet of assisted reproductive technology programs. Candidates for donor oocytes include women with premature ovarian failure or severe genetic disorders, women who respond poorly to human menopausal gonadotropin, and women older than 40 years who do not conceive with used of other therapies. The coordination of donor and recipient of oocytes is facilitated by pituitary desensitization of both parties using gonadotropin-releasing-hormone agonists. Pregnancy rates are much higher than that achieved with traditional in vitro fertilization and embryo transfer, possibly because of the young age of the donors and better endometrial receptivity.


Endometrial receptivity and the luteal phase.

Endometrial receptivity to the implanting blastocyst determines whether pregnancy occurs. Whether fertilization is initiated in vivo or in vitro, the receptivity of the luteal-phase endometrium results from the input of interacting hormonal, growth, and immunologic factors. Endometrial adequacy for implantation has traditionally been assessed by the endometrial biopsy. Newer methods used to study the endometrium, such as ultrasound imaging and measurement of endometrial proteins, have increased our understanding yet have demonstrated the limits of our knowledge. This review is designed to analyze the available literature for concepts and scientific facts to aid in our understanding of the complex events required in preparation of a receptive endometrium. Special attention is focused on the effect of supraphysiologic levels of steroid hormones on the endometrium in ovulation induction. The effect of both embryo quality and luteal-phase support are also reviewed.


Gamete and zygote intrafallopian transfers and related techniques.

Until recently, most authors reported superior results (ie, higher implantation and pregnancy rates) with gamete intrafallopian transfer (GIFT) and zygote intrafallopian transfer (ZIFT) compared with results using in vitro fertilization-embryo transfer (IVF-ET). According to these investigators, the advantages of tubal over uterine transfer are related mainly to a stable tubal environment and a more appropriate arrival time of the embryo into the uterine cavity. However, more recently, the use of IVF-ET has been increasingly extended to etiologies other than tubal infertility. Indeed, the recent simplification of this technique and the achievement of pregnancy rates comparable to those obtained with tubal transfers have seriously questioned the value of ZIFT and any other type of tubal embryo transfer. As discussed in this review, the results obtained with various transfer procedures in nontubal infertility must still prove ZIFT to be a more effective procedure than IVF-ET. Efforts to develop transcervical methods of transfer to the tube have not translated into higher pregnancy rates than those with IVF-ET. On the other hand, laboratory conditions appear to affect embryos in ways not corrected by the tubal milieu. Negative effects of laboratory conditions on embryos are confirmed by differences in results between GIFT and ZIFT that are accentuated with age. The results obtained with GIFT in patients 40 years of age and older seem to emphasize not only the high compliance of the endometrial receptivity but also the relevance of the biologic potential of the embryos in the process of implantation at this age.