Ziskind-Somerfeld Research Award 1992. Endogenous biochemical abnormalities in affective illness: therapeutic versus pathogenic. Examination of the neurobiology of psychiatric illness in general, and of affective disorders in particular, reveals a variety of associated biochemical abnormalities. These have generally been assumed to be part of the pathological process or secondary to it, and thus deserving of therapeutic efforts aimed at reversal. However, recent clinical and preclinical data suggest that some alterations occurring in the affective disorders may be compensatory and adaptive; that is, part of an endogenous therapeutic mechanism rather than part of the evolving disease process. For example, the symptom of sleep loss in depression seems to fall under this rubric inasmuch as sleep deprivation induces mood improvement in depressed patients. Preclinical data are presented that another primary pathological process--the occurrence of kindled seizures--can evoke endogenous compensatory processes that are either anticonvulsant in their own right, or enable the anticonvulsant effects of a drug such as carbamazepine. It may be that some biochemical abnormalities occurring in affective illness are similarly adaptive. As one example, increased thyrotropin-releasing hormone (TRH) has been reported in the cerebrospinal fluid (CSF) of depressed patients. This elevation of TRH and the resulting neuroendocrine profile may be part of an endogenous counter-regulatory process aimed at mood improvement. Again, preclinical seizure models are supportive in that TRH not only is induced following repeated seizures, but also exerts anticonvulsant effects on these same seizures. In an analogous fashion, TRH elevations in depressed patients may also exert ameliorating effects on depressive symptomatology. This formulation presents directly testable hypotheses that could importantly impact on our understanding of the pathophysiology of affective disorders, and suggests novel therapeutic strategies through the enhancement of endogenous compensatory mechanisms.


Cardiovascular, neuroendocrine, and sedative responses to four graded doses of clonidine in a placebo-controlled study. Effects of four doses of the alpha 2-receptor agonist clonidine (CLO) (0.25, 0.5, 1, and 2 micrograms/kg IV) and placebo were studied in seven healthy men who volunteered in a double-blind randomized design in order to delineate possible presynaptic and postsynaptic components in the mechanism of action of CLO. Blood pressure, heart rate, plasma noradrenaline (NOR), plasma 3-methoxy-4-hydroxyphenylglycol (MHPG), plasma growth hormone (GH), and subjective sedation were monitored for a period of 1 hr following infusion of CLO. NOR and MHPG were also analyzed in urine, collected at 1 and 4 hr after the infusions. Dose-dependent decrements were observed in systolic and diastolic blood pressure and plasma NOR levels, and dose-dependent increases in subjective sedation and plasma GH. CLO did not influence plasma MHPG levels, whereas only urinary MHPG excretion was reduced 4 hr after infusion of 2 micrograms/kg CLO. Because no obvious differences between dose-response relations of plasma NOR (believed to be a presynaptic and peripheral effect), blood pressure (believed to be mainly a central presynaptic and postsynaptic effect), and subjective sedation (believed to be a central and probably postsynaptic effect) were observed, our results do not provide simple parameters to discern the multiple mechanisms of action of CLO. However, at a dose of 0.5 micrograms/kg CLO (a dose lower than that generally used) clear effects on plasma NOR, blood pressure, and sedation, but not on plasma GH (a central postsynaptic effect) or urinary MHPG (a presynaptic effect), were observed. When using CLO as a challenge test in psychiatric disorders, a design with 0.5 micrograms/kg CLO, in addition to the traditional 2 micrograms/kg CLO, may provide more information to characterize discrete abnormalities in the noradrenergic system at the level of the brainstem, the pituitary, or the peripheral sympathetic nervous system.


Pain perception in self-injurious patients with borderline personality disorder. Pain ratings during the cold pressor test were significantly lower in female inpatients with borderline personality disorder who report that they do not experience pain during self-injury (BPD-NP group, n = 11), compared with similar patients who report that they do experience pain during self-injury (BPD-P group, n = 11), and normal female subjects (n = 6). Pain ratings were not significantly different in the BPD-P and normal control groups. Self-report ratings of depression, anger, anxiety, and confusion were significantly lower, and ratings of vigor significantly higher following the cold pressor test in the BPD-NP group, but not in the BPD-P group. Only anxiety was significantly lower in the normal control group following the cold pressor test. The implications and limitations of these preliminary findings are discussed.


Beta endorphin levels during heroin, methadone, buprenorphine, and naloxone challenges: preliminary findings. Beta endorphin (BE) is a polypeptide agonist for the brain's endogenous opioid system. Levels of BE are elevated by opioid antagonists such as naloxone and depressed by short-acting agonists such as heroin and morphine; they become normalized during steady-state methadone. Buprenorphine (BUP) is a partial opioid agonist whose effects on BE levels were examined in six former heroin addicts and 14 methadone-maintained patients before and after being switched to sublingual BUP 2 mg daily for 1 month. In six former methadone-treated subjects BE levels also were measured after stopping BUP and after naloxone challenge. Levels of BE were not significantly lower in subjects started on BUP after stopping heroin (n = 6) (8.0 versus 8.1 ng/ml) or in subjects started on BUP after stopping methadone (n = 14) (11.6 vs 15.6 ng/ml). However, BE levels were lower on BUP than after naloxone challenge (n = 6) (7.0 versus 34.9 ng/ml). Levels of BE did not significantly change between the first 2 weeks ("early") and "later," although BE levels on methadone significantly correlated with BE levels on BUP in the "early" but not the "later" phase. The BE levels on BUP also did not differ from BE levels of unmedicated normals.


Neuropsychological function and REM sleep in schizophrenic patients. To test the hypothesis that rapid eye movement (REM) sleep in schizophrenic patients is associated with cognitive function, we studied 18 schizophrenic inpatients by means of electroencephalograms taken during sleep in their own hospital beds after a minimum 2-wk medication withdrawal period. Patients underwent neuropsychological tests to measure memory function and other aspects of cognitive performance. REM sleep measures demonstrated positive and negative correlations with cognition and memory measures, depending on when REM occurred after sleep onset. Minutes of REM sleep and REM density in the first period correlated negatively with performance, while REM minutes occurring after the first REM period correlated positively with neuropsychological performance. Further work should test whether phasic REM sleep regulation at the beginning of the night plays a compensatory role for neuropsychological dysfunction in schizophrenics.


Impaired activity of natural killer cells in patients with acute brucellosis. Researchers have claimed that natural killer (NK) cells are involved in the mechanisms of defense of the host against infections. We have investigated the activity of NK cells in peripheral blood mononuclear cells (PBMNC) from 12 patients for whom acute brucellar infection has been diagnosed and from 14 healthy controls. The sera of eight of the patients were also analyzed 3 months after initiation of a 45-day course of antibiotic treatment, at which time they had no evidence of relapse. PBMNC from patients with acute brucellar infection showed a significantly depressed NK cell activity (P < .01) when compared with those from healthy controls; this depressed activity was not related to a deficient number of NK cells since the numbers of CD56+ and CD16+ cells present in PBMNC were similar in patients and controls. Incubation of PBMNC from patients with acute brucellar infection with recombinant interleukin-2, but not with interferon-gamma, can correct this impaired cytotoxic activity. In treated patients, there was a significant enhancement (P < .05) and normalization of the previously defective NK cell activity. It is concluded that acute brucellar infection is associated with a deficient cytotoxic activity of NK cells that can be overcome by in vitro incubation with interleukin-2 and that reverts to normal after antibiotic treatment.


Anaerobic osteomyelitis in patients with Gaucher's disease. Bone involvement in patients with Gaucher's disease is common, and some of its clinical manifestations may resemble acute osteomyelitis. In addition, many studies have emphasized the high risk of secondary infection when surgical procedures are performed at the site of the involved bone. Nevertheless, these infections have never been well documented in the literature. Presentation of a patient with Gaucher's disease and osteomyelitis due to Prevotella (Bacteroides) melaninogenica provided us the opportunity to review 10 other similar case reports documented in the literature since 1966. This review suggests that acute hematogenic osteomyelitis is an uncommon complication of Gaucher's disease, but it is of interest that in most cases it is due to unusual organisms, particularly anaerobes.


Imported malaria in the Bronx: review of 51 cases recorded from 1986 to 1991. The cases of 51 patients with malaria seen at the Albert Einstein College of Medicine hospitals from January 1986 to June 1991 are reviewed. Thirty-five patients acquired infection on journeys to their country of origin. Of these 35 patients, 83% of whom had lived in the United States for > or = 2 years, only 17% received antimalarial prophylaxis. Ten of the 51 patients were born and raised in the United States, and 70% received prophylaxis (P < .01). Six of the 51 patients were visitors to the United States from areas endemic for malaria. Overall, 64% of patients acquired malaria in West Africa, south of the Sahara; 20% in Asia; 8% in Ecuador; 6% in Haiti; and 4% in the Middle East. The majority of infections were due to Plasmodium falciparum. Six patients traveled to a zone endemic for malaria while pregnant, and none received prophylaxis. In nine of 13 patients who received prophylaxis, there was inadequate dosing or poor compliance. Individuals born in regions endemic for malaria are at high risk of acquiring malaria on return to their countries of origin and are less aware of the need for malaria prophylaxis than are other travelers.


Azole therapy for trichosporonosis: clinical evaluation of eight patients, experimental therapy for murine infection, and review. We studied the in vivo antifungal activity of azoles in humans and in a murine model of disseminated trichosporonosis. Eight patients infected with Trichosporon species were treated with fluconazole, SCH 39304, or miconazole for 2-26 weeks. Four patients had fungemia, two patients had disseminated trichosporonosis, and one patient each had soft-tissue infection and cystitis. Response of trichosporonosis to azoles was seen in all eight patients, although one patient died with disseminated aspergillosis while still receiving SCH 39304. A literature review indicated that responses to ketoconazole or miconazole were noted in four patients with trichosporonosis. In the experimental infection, amphotericin B, SCH 39304, and fluconazole were effective in prolonging survival and reducing fungal counts in the kidneys of mice infected with a clinical strain of Trichosporon beigelii. Fluconazole but not amphotericin B prolonged survival of mice infected with a clinical strain of Trichosporon capitatum. We conclude that azoles represent effective therapy for infection with Trichosporon species.


Persistence of serum antibodies to Borrelia burgdorferi in patients treated for Lyme disease. To determine if antibodies to Borrelia burgdorferi persist after antibiotic treatment, we recalled 32 patients with Lyme disease from a primary care practice a mean of 16 months after treatment and analyzed initial and follow-up serum samples by ELISA and immunoblot assays. Of the eight patients whose initial serum specimens were positive for IgM antibody by ELISA, three had positive titers of IgM antibody at follow-up; of the 23 patients whose initial serum specimens were positive for IgG antibody by ELISA, 19 had positive titers of IgG at follow-up. Of the five patients whose initial serum specimens were positive for IgM antibody by immunoblot, two had positive titers of IgM antibody at follow-up; of the 30 patients whose initial serum specimens were positive for IgG antibody by immunoblot, 29 had positive titers of IgG antibody at follow-up. The bands on the IgG immunoblot remained remarkably constant during the period from analysis of the initial specimen to that of the follow-up specimen. Nine of the 32 patients had persistent or recurrent symptoms, and ELISA and immunoblot were not helpful for identifying these nine patients.


Capsular types of Streptococcus pneumoniae isolated from blood and CSF during 1982-1987. Knowledge about the type distribution of Streptococcus pneumoniae is fundamental to ensure an effective formulation of pneumococcal vaccine, especially with the possibility of producing a polysaccharide-protein-conjugated vaccine for the prevention of invasive disease in children. During the 6-year period 1982-1987, we received and typed 10,298 isolates from patients with invasive pneumococcal disease: 7,812 (76%) from blood and 2,486 (24%) from CSF. Of all isolates, 81% were recovered from individuals in Europe and 23% were from children. In order of frequency, S. pneumoniae types 6A + 6B, 14, 18C, 19F, 1, 7F, 23F, 19A, 4, and 5 were most commonly isolated from children, and types 3, 1, 14, 7F, 4, 6A + 6B, 8, 23F, 9V, and 19F, from adults. The pneumococcal types in the currently available 23-valent vaccine represented 87% of all isolates in this study, but the proportion of vaccine types varied somewhat with age and source. In all pneumococcal groups included in the vaccine, the vaccine types represented > 80% of the isolates, except in groups 6, 15, and 18.


Extraparenchymal neurocysticercosis: report of five cases and review of management. Neurocysticercosis due to parenchymal cysts carries a good prognosis regardless of therapy. Extraparenchymal neurocysticercosis (including ventricular, spinal, and subarachnoid types) carries a poorer prognosis. Most extraparenchymal cases present with hydrocephalus. Medical treatment alone in doses and schedules developed for parenchymal disease is frequently unsuccessful. For ventricular disease, most cases can be managed with shunting procedures either alone or together with the administration of antiparasitic agents (e.g., praziquantel or albendazole), without extirpation of the cysts. Subarachnoid disease was formerly associated with a case fatality rate of about 50%. However, with the combination of shunting procedures for hydrocephalus, antiparasitic agents, and, in some cases, surgical extirpation of the cysts, the prognosis is much improved. Spinal cysticercosis can be either leptomeningeal (which responds like subarachnoid disease) or intramedullary. For all forms of neurocysticercosis, the role of antiparasitic agents needs to be better defined.


Chronic fatigue syndrome.

Chronic fatigue syndrome. Chronic fatigue syndrome (CFS) is defined by symptoms and diagnosed without any objective diagnostic tests. Risk factors for developing CFS may include infection, psychiatric disorders, and allergies. Modest dysfunction of multiple organ systems, including the immune, central nervous, endocrine, and muscular systems, have been identified in cases of CFS. Symptoms of various organic, psychiatric, and poorly understood disorders overlap those of CFS. There is no known cure for CFS; however, exercise, counseling, and medications may provide symptomatic relief.


beta-Lactam resistance in gram-negative bacteria: global trends and clinical impact. Microbial drug resistance is an inescapable consequence of the utilization of antimicrobial agents in a given environment. Nowhere is the importance of resistance more evident than among agents of the beta-lactam family. Trends toward increased resistance can be seen among fastidious gram-negative bacteria like Haemophilus influenzae, where ampicillin resistance varies from 1% to 64% globally. For Escherichia coli, ampicillin resistance has risen to > or = 50% in high-risk populations, and resistance to third-generation cephalosporins is now being seen in certain areas. Inducible beta-lactamases have been responsible for increasing multiple beta-lactam resistance among certain Enterobacteriaceae and Pseudomonas aeruginosa, and this has been associated with increased use of newer cephalosporins. Xanthomonas maltophilia with its two inducible beta-lactamases is becoming an increasingly important nosocomial pathogen, especially in areas of heavy imipenem utilization. Only through the recognition of factors associated with increasing resistance and the mechanisms responsible can strategies be designed for minimizing beta-lactam resistance.


Antibiotic-induced release of endotoxin: a reappraisal. A three- to 20-fold increase in the total concentration of endotoxin occurs as a consequence of antibiotic action on gram-negative bacteria both in vitro and in vivo. There is considerable overlap between the effect of beta-lactam antibiotics and non-beta-lactam antibiotics. Moreover, there is an unexplained delay between the lethal activity of antibiotics and the release of endotoxin. Hence, the mechanism whereby antibiotic action leads to the release of endotoxin is unclear, and mechanisms other than bacterial lysis warrant consideration. The evidence that the release of endotoxin has clinical importance is conflicting, and the issue is unresolved. However, nonlytic release may have implications for the therapeutic efficacy of antiendotoxin immunotherapy. Although frequently cited in the context of the antibiotic-induced release of endotoxin, a number of important differences pertain to conditions, such as the Jarisch-Herxheimer reaction and the tumor lysis syndrome, for which there is clear evidence of an initial deterioration with effective therapy.


Significant infections due to Bacillus species following abrasions associated with motor vehicle-related trauma. Non-anthracis Bacillus species are ubiquitous gram-positive spore-forming organisms that were once believed to be nonpathogenic but are now recognized as causing a variety of infections. We report a new aspect of trauma associated with bacillus infection: clinically significant infection by Bacillus species in patients who are involved in motor vehicle accidents and sustain injury related to road contact. Cases were evaluated retrospectively from May 1990 through December 1991. Four patients who had documented infections with Bacillus species and who were involved in motor vehicle accidents associated with road trauma were identified during this period. The antibiotic susceptibility profile of the Bacillus species consistently demonstrated resistance to beta-lactam antibiotics. This series of cases illustrates an additional aspect of disease associated with bacteria of the Bacillus species that should be considered for patients who have sustained injuries from motor vehicle accidents associated with road trauma.


Pseudallescheria boydii endocarditis of the pulmonic valve in a liver transplant recipient. We describe a case of Pseudallescheria boydii endocarditis involving the pulmonic valve in an orthotopic liver transplant recipient. The patient required transplantation because of hepatic failure secondary to chronic active hepatitis B. His postoperative course was complicated by surgery for gastric and duodenal ulcers, persistent fever, and, ultimately, sepsis leading to oliguric renal failure. Two days before death, the patient experienced complete heart block, and an echocardiogram revealed pulmonic valve thickening and an endocardial mass along the left side of the septum. At autopsy the patient was found to have a vegetation on the pulmonic valve and a septal abscess. There were multiple fungal emboli found throughout other organs, and P. boydii was obtained on culture. This unique association between pulmonic valve endocarditis and myocardial septal abscess is discussed. In addition, review of the five previous cases of P. boydii endocarditis reveals that this rare infection is associated with immunosuppression and prosthetic devices.


Concomitant esophageal and penile ulcerations healed with steroid therapy in a patient with AIDS: case report. A 50-year-old man with AIDS developed concomitant esophageal and penile ulcerations. An etiology could not be found despite intensive investigation. Both ulcers healed after treatment with corticosteroids. To our knowledge, such a situation has been described only for aphthous ulcerations involving the mouth, oropharynx, or esophagus.


Cefotetan-induced immune hemolytic anemia. Immune hemolytic anemia due to a drug-adsorption mechanism has been described primarily in patients receiving penicillins and first-generation cephalosporins. We describe a patient who developed anemia while receiving intravenous cefotetan. Cefotetan-dependent antibodies were detected in the patient's serum and in an eluate prepared from his red blood cells. The eluate also reacted weakly with red blood cells in the absence of cefotetan, suggesting the concomitant formation of warm-reactive autoantibodies. These observations, in conjunction with clinical and laboratory evidence of extravascular hemolysis, are consistent with drug-induced hemolytic anemia, possibly involving both drug-adsorption and autoantibody formation mechanisms. This case emphasizes the need for increased awareness of hemolytic reactions to all cephalosporins.


Factors influencing prognosis in bacteremia due to gram-negative organisms: evaluation of 448 episodes in a Turkish university hospital. A total of 448 episodes of bacteremia due to gram-negative organisms observed during the 7-year period between 1983 and 1989 at Hacettepe University Hospitals were studied for evaluating the factors influencing the prognosis. The overall mortality rate was 45.0%. The mortality rates were not significantly different in "rapidly fatal" and "ultimately fatal" disease groups (48.3% and 45.5%, respectively), whereas it was significantly less (34.8%) in the "nonfatal" disease group compared with the "rapidly fatal" category. There were great differences in the mortality rates among different diseases within the same disease category. Shock, multi-organ failure, source of infection, hospital service, appropriateness of antibiotic therapy, and place of acquisition of infection were found to affect prognosis significantly in multivariate analysis. In conclusion, the identification of prognostic factors is a further step for making necessary interventions in reducing the mortality rate associated with bacteremia due to gram-negative organisms. Underlying disease is still an important prognostic factor; however, a new approach is needed for classification of underlying diseases.


Acute renal failure in patients with severe falciparum malaria. Since 1988 in this referral center for severe cases of malaria for South Vietnam, a specialist team has managed malaria-associated renal failure (MARF) with peritoneal dialysis, and the mortality rate of MARF has fallen from 75% (78 of 104) to 26% (27 of 104) (P < .0002). Sixty-four patients with MARF (of whom 12 died) were compared to 66 patients with severe malaria whose serum creatinine levels remained < 250 mumol/L (six died). MARF had the clinical and biochemical features of acute tubular necrosis and was significantly associated with liver dysfunction (P < .05). A fatal outcome was associated significantly with anuria, a short history of illness, multisystem involvement, and high parasitemia. Most patients died from complications related to renal failure. Recovery of renal function was unrelated to parasitemia or hemoglobinuria; the median (range) time until urine output exceeded 20 mL/(kg.d) was 4 (0-19) days, and the time (mean +/- SD) for serum creatinine level to return to normal was 17 +/- 6 days. MARF can be managed effectively by prompt and careful peritoneal dialysis, but more effective dialysis or diafiltration might reduce the mortality rate further.


QSAR, diagnostic statistics and molecular modelling of 1,4-dihydropyridine calcium antagonists: a difficult road ahead. Quantitative structure-activity relationships of a series of substituted 1,4-dihydropyridine calcium channel antagonists were studied. The analysis is difficult because of the problem of multicollinearity of substituent parameters, a high-leverage point, and position-dependent grouped observations. Canonical regression appears to be the method of choice. With respect to a maximum activity, it was shown that the following rank order of substituent parameters exists: Lipophilicity approximately ortho-position > inductivity > minimum width > meta-position. The molecular conformation of antagonists does not differ markedly (with exception of nifedipine derivatives and nimodipine), but differences seem to exist between the antagonists and the activator BAY K 8644.


Evaluation of some N-acyl analogues of 3,5-bis(arylidene)-4-piperidones for cytotoxic activity. The synthesis of fourteen N-acyl derivatives of two 3,5-bis(arylidene)-4-piperidones was accomplished and these compounds were evaluated against L1210 leukemia cells in vitro. With one exception, the compounds had IC50 values of less than 10 microM and six of them had IC50 figures in the 0.2-0.6 microM range which were comparable to a reference drug melphalan. Twelve of the sixteen compounds showed specificity for human leukemia cell lines in the NCI in vitro screen. Studies using 1H NMR spectroscopy revealed that solutions of three N-acetylated compounds underwent deamination and possibly other reactions, the deaminated product itself being unstable in the solvent used.


Synthesis and antinociceptive activity of 1-[2-(pyridyl)ethyl] and 1-[2-(dihydropyridyl)ethyl] analogues of fentanyl. The syntheses and antinociceptive activities of all three isomeric 1-[2-(pyridyl)ethyl]-4-(propionanilido)-piperidine isosteres (11a-c) of fentanyl (1) are described. The 2- (11a), 3- (11b) and 4-pyridyl (11c) isomers exhibited 10, 2 and 0.2 times the antinociceptive activity of fentanyl, respectively. The ED50 values for 11a, 11b, 11c and fentanyl in the rat 4% NaCl-induced writhing test were 0.00023, 0.00085, 0.0087 and 0.0021 mg/kg sc, respectively. The 3-pyridyl (11b) and 4-pyridyl (11c) compounds were further elaborated to the 6-phenyl-1,6-dihydropyridine (12), C-2 H, Me, n-Bu and Ph 1,2-dihydropyridine (13a-d) analogues having a phenoxycarbonyl substituent on the dihydropyridine ring nitrogen. The most active compound in this series was 1-(2-[3-(1-phenoxycarbonyl-6-phenyl-1,6-dihydropyridyl)ethyl ])-4- (propionanilido)piperidine (12), which provided a 58% inhibition of writhing at a dose of 0.4 mg/kg sc. 1-(2-[4-(1-phenoxycarbonyl-1,2-dihydropyridyl)ethyl])-4- (propionanilido)piperidine (13a) exhibited an ED50 of 1.3 mg/kg sc, indicating a decrease in antinociceptive activity of about a 100 fold relative to the parent 4-pyridyl compound (11c). The dihydropyridine analogues 12 and 13 exhibit substantial antinociceptive activity relative to meperidine (ED50 = 0.6 mg/kg sc). The muscular rigidity effect induced by the pyridine compounds (11a-c) at a dose of 4 mg/kg sc, was not illicited by the dihydropyridine analogues at the same dose, or at a high dose of 40 mg/kg sc (13a). Compounds 12 and 13 may therefore be useful lead compounds for the development of more useful 4-anilidopiperidines if the antinociceptive activity can be dissociated from the muscular rigidity effect.


Synthesis and calcium channel antagonist activity of dialkyl 1,4-dihydro-2,6-dimethyl-4-[3-(1-methoxy-carbonyl-4-substituted-1,4- dihydro-pyridyl)]-3,5-pyridinedicarboxylates. A novel class of dialkyl 1,4-dihydro-2,6-dimethyl-4-(3-[1- methoxycarbonyl-4-(H, Me, n-Bu or Ph)-1,4-dihydropyridyl])-3,5-pyridinedicarboxylates (3-29) were synthesized and evaluated as calcium channel antagonists using the muscarinic receptor mediated Ca2+ dependent contraction of guinea pig ileal longitudinal smooth muscle (GPILSM). The differences in activity among members of this new class of compounds was less than one log unit (IC50 range of 8.25 x 10(-6) to 4.36 x 10(-7) M), relative to the reference drug nifedipine (IC50 = 1.43 x 10(-8) M). Compounds possessing symmetrical C-3(5) diethoxycarbonyl ester substituents generally exhibited optimum activity. The R3 substituent (H, Me, n-Bu, Ph) at the 4-position of the 4-[3-(1-methoxycarbonyl-1,4-dihydropyridyl)] moiety was a determinant of activity. In symmetrical diester compounds, a R3 H substituent provided optimum activity for Me, i-Bu and t-Bu dialkyl ester analogues, whereas a R3 Ph substituent provided optimum activity for Et and i-Pr symmetrical diesters. The test results indicate the 4-[3-(1-methoxycarbonyl-4-substituted-1,4-dihydropyridyl)] substituent in this new class of compounds is bioisosteric with a 4-(3-nitrophenyl), or a 4-(3-pyridyl) substituent in classical 1,4-dihydropyridine antagonists. Compounds possessing large symmetrical dialkyl ester substituents (i-Bu, t-Bu), in conjunction with a 4-[3-(1-methoxycarbonyl-4-methyl-1,4-dihydropyridyl)] substituent, permanently inhibited recovery of the KCl-induced response in GPILSM.


Formation, characterization, and toxicity of the glutathione and cysteine conjugates of toxic heptapeptide microcystins. Microcystins LR, YR, and RR, cyclic heptapeptide hepatotoxins produced by cyanobacteria, were synthetically converted into glutathione (GSH) and cysteine (Cys) conjugates. Fast atom bombardment mass spectra showed [M + H]+ ions corresponding to GSH and Cys conjugates of microcystins LR, YR, and RR for the obtained compounds. 1H NMR spectral analyses revealed that two singlet signals of olefinic protons of N-methyldehydroalanine (Mdha) in microcystins disappeared in the conjugates, confirming that thiols of GSH and Cys added nucleophilically to the alpha, beta-unsaturated carbonyl of the Mdha moiety. On examination of the 50% lethal dose (LD50) with intravenous injection using mice, both GSH and Cys conjugates showed reduction in toxicity compared with microcystins, but their toxicity still remained. Microcystin LR and its GSH conjugate were separated and identified in a standard mixture by using a frit-fast atom bombardment liquid chromatography/mass spectrometry (Frit-FAB LC/MS) method. Obtained conjugates in the present study would be important compounds as the standard samples for study of metabolism of microcystins, and the Frit-FAB LC/MS method would be applicable to mass spectrometric identification of metabolites of microcystins.


A study of inactivation reactions of N-acetylcysteine with mucochloric acid, a mutagenic product of the chlorination of humic substances in water. The Salmonella typhimurium (TA100) mutagenic compound, mucochloric acid [3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA)], was inactivated by in vitro N-acetylcysteine (NAC). The reaction of MCA with NAC at pH7 was second order and gave products 4, 5, and 6a that resulted from the displacement of chlorine from C-3 or C-4 of MCA. The sodium borohydride treatment of product 4 gave the same product (7) as was obtained by treating 3,4-dichloro-2(5H)-furanone with NAC. The treatment of MCA with (R)-(+)-cysteine gave the bicyclic product 9a, in which the two chlorine atoms of MCA were still present. This product was slightly more mutagenic than MCA, whereas product 5 was less mutagenic than MCA and product 4 was nonmutagenic in the Salmonella typhimurium (TA100) assay.


Synthesis of 2'-deoxy-7,8-dihydro-8-oxoguanosine and 2'-deoxy-7,8-dihydro-8-oxoadenosine and their incorporation into oligomeric DNA. Reliable methods have been developed for the synthesis of the 3'-O-[(diisopropylamino) (2-cyanoethoxy)phosphino]-5'-O-(4,4'- dimethoxytrityl) derivatives of 2'-deoxy-7,8-dihydro-8-oxoguanosine (8-oxo-dGuo, 1) and 2'-deoxy-7,8-dihydro-8-oxoadenosine (8-oxo-dAdo, 2), and for the efficient incorporation of the latter into oligomeric DNA. Both methods rely on the conversion of the 2'-deoxy-8-bromopurine nucleosides 3 and 10 to their corresponding 2'-deoxy-8-(benzyloxy) nucleosides 4 and 12 followed by catalytic hydrogenation to generate the 8-oxo function at the C-8 position. The preparation of the phosphoramidites 8 and 19 required for the synthesis of a series of DNA oligomers was carried out under strictly anhydrous conditions. Failure to keep the systems dry resulted in great difficulties during the purification procedures, and erratic results when DNA synthesis was attempted. In the preparation of the DNA itself, it was found to be extremely important during the ammonia deprotection step to add an antioxidant. Otherwise aerial oxidation resulted in almost complete loss of the oligomer. However, when these special conditions were followed, oligomeric DNA containing 8-oxo-dGuo and 8-oxo-dAdo residues could be prepared in excellent yield. Analysis of selected DNA oligomers by enzymatic degradation and mass spectroscopic analysis confirmed the designated sequences and compositions.


Studies on 1,2,3,6-tetrahydropyridine derivatives as potential monoamine oxidase inactivators. The Parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and close structural analogs are the only known cyclic tertiary amines with good monoamine oxidase substrate properties. In addition to its excellent substrate properties, MPTP is a mechanism-based inactivator of monoamine oxidase B (MAO-B). In an attempt to exploit the special interactions between this cyclic tertiary allylamine and MAO-B, we have initiated studies to evaluate the enzymatic and biological properties of MPTP analogs bearing functional groups which are known to mediate the metabolism-dependent inactivation of this enzyme. This paper describes the synthesis, enzyme substrate/inhibitor properties, and neurotoxic/neuroprotective properties of 1-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine, the corresponding acyclic secondary amine (E)-4-cyclopropyl-2-phenyl-2-butene, and 4-cyclopropyl-1-methyl-1,2,3,6-tetrahydropyridine.


Metabolism of nicotine by human liver microsomes: stereoselective formation of trans-nicotine N'-oxide. Liver microsomes from humans catalyze the NADPH-dependent oxidation of (S)-nicotine. The principal product is the 5'-carbon atom oxidation product, nicotine delta 1',5'-iminium ion, which is efficiently converted to the gamma-lactam derivative cotinine in the presence of aldehyde oxidase. Another major product is nicotine N'-oxide. In contrast to previous reports describing in vitro or in vivo studies, formation of only trans-nicotine N'-oxide was observed. Demethylation of nicotine was not observed. Studies on the biochemical mechanism of nicotine 5-carbon atom oxidation strongly implicate one major cytochrome P-450 isoenzyme (i.e., P-450 2A6) as largely responsible for delta 1',5'-iminium ion formation. Stereoselective formation of trans-nicotine N'-oxide may be catalyzed in large part by the flavin-containing monooxygenase (form II). These conclusions are based on the effects of alternate substrates for the flavin-containing monooxygenase, heat inactivation studies, immunoblot studies, and selective substrates for cytochromes P-450. The results suggest that (S)-nicotine trans N'-oxygenation and delta 1',5'-iminium ion formation may be selective probes of human liver flavin-containing monooxygenase form II and cytochrome P-450 2A6 activities, respectively, useful for in vivo phenotyping of humans.


1,N2-ethenodeoxyguanosine: properties and formation in chloroacetaldehyde-treated polynucleotides and DNA. 1,N2-Etheno-2'-deoxyguanosine (1,N2-epsilon dGuo), not previously reported as a product of chloroacetaldehyde (CAA) reaction, has been synthesized and characterized. Reaction of deoxyguanosine with CAA in dimethylformamide in the presence of K2CO3 led to preparation of pure 1,N2-epsilon dGuo with 55% yield. pKa values are 2.2 and 9.2. The anionic form of the compound exhibits weak but defined fluorescence; the intensity is similar to that of N2,3-etheno-2'-deoxyguanosine (N2,3-epsilon dGuo) at neutrality. The stability of the glycosyl bond of 1,N2-epsilon dGuo (t1/2 = 2.3 h at 37 degrees C, pH 1) is 10-fold greater than of unmodified deoxyguanosine and at least one thousand-fold greater than of isomeric N2,3-epsilon dGuo. Reaction of CAA with model polynucleotides indicates that hydrogen bonding of guanine residues in the double-stranded structures is, as expected, an important factor in the formation of 1,N2-ethenoguanine. In contrast, the formation of isomeric N2,3-ethenoguanine is relatively independent of whether the DNA is single- or double-stranded. In salmon sperm DNA, reacted with CAA at neutrality, the formation of 1,N2-ethenoguanine could be demonstrated. However, we find the efficiency of formation of this adduct in double-stranded DNA to be lower than that of all other etheno derivatives.


The reactions of cis-[Pt(NH3)2(H2O)2]2+ with L-(+)-cystathionine and seleno-L-methionine: potential relevance to the molecular basis of cisplatin toxicity. NMR spectroscopic studies indicate that the hydrolysis product of cisplatin, cis-[Pt(NH3)2-(H2O)2]2+, reacts readily with the important intracellular thiol L-(+)-cystathionine and the amino acid derivative seleno-L-methionine. In both cases, the formation of six-membered mononuclear S,N- or Se, N-chelate rings was established on the basis of [1H], [13C], [77Se], [195Pt], and [13C]-(1H) DEPT (distortionless enhancement by polarization transfer), COSY (correlation spectroscopy), heterocorrelation, and NOE (nuclear Overhauser effect) difference NMR experiments. The formation of these products suggests that related in vivo processes may play a significant role in the toxicity of cisplatin. The potential loss of NH3 in such platinum complexes may lead to additional products over time.


Structures of three new homotyrosine-containing microcystins and a new homophenylalanine variant from Anabaena sp. strain 66. A hepatotoxic strain of cyanobacterium Anabaena sp. 66 was isolated from a hepatotoxic water bloom sample in Lake Kiikkara, Finland. Four cyclic heptapeptide hepatotoxins were isolated and purified by HPLC from cultured cells of this organism. The structures of three new homotyrosine (Hty) containing toxins, [Dha7]microcystin-HtyR (Dha = dehydroalanine) (1), [D-Asp3,Dha7]microcystin-HtyR (2), and [L-Ser7]microcystin-HtyR (3), were assigned, based upon amino acid analyses using both a Waters Pico Tag HPLC system and chiral capillary GC, 1H NMR, fast atom bombardment mass spectrometry (FABMS), and collisionally induced tandem FABMS. A new homophenylalanine (Hph) variant of 1, [Dha7]microcystin-HphR (4), was also obtained as a minor component. Compound 3 is most likely a biosynthetic precursor of 1. The four new toxins did not have an N-methyl group at the dehydroamino acid or its precursor unit.


Damage to human alpha-1-proteinase inhibitor by aqueous cigarette tar extracts and the formation of methionine sulfoxide. The effects of aqueous extracts of cigarette tar (ACT) on human alpha-1-proteinase inhibitor (alpha 1PI) are examined by determining alterations in the elastase inhibitory capacity (EIC), amino acid residue content, and electrophoretic behavior of the protein. Hydrogen peroxide generated in ACT by autoxidative processes accounts for the major portion of the loss of EIC. This is indicated by several lines of evidence, including the fact that anaerobic incubations of ACT with alpha 1PI cause negligible loss of EIC. The hydrogen peroxide content of the ACT was estimated by measuring the ability of the extracts to oxidize methionine to methionine sulfoxide; hydrogen peroxide concentrations that model those in ACT cause a similar loss in EIC. Exposure of alpha 1PI to ACT leads to methionine sulfoxide as the only detectable amino acid residue modification and explains the loss of EIC. This is the first report to directly demonstrate methionine sulfoxide formation in alpha 1PI exposed to cigarette smoke components in vitro. Similar amounts of methionine sulfoxide are found in alpha 1PI exposed to hydrogen peroxide at the concentrations predicted to be formed in ACT. Nondenaturing PAGE reveals that alpha 1PI exposed to ACT, but not hydrogen peroxide, shows changes in electrophoretic behavior; the changes are nonoxidative in nature and are not related to the loss of EIC. Studies on the effect of chelators on ACT-mediated damage to alpha 1PI indicate some role for metal ions; however, none of the chelators completely protect alpha 1PI.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparison of metabolism and toxicity to the structure of the anticancer agent sulofenur and related sulfonylureas. The metabolic formation of p-chloroaniline from the oncolytic agent sulofenur [N-(5-indanesulfonyl)-N'-(4-chlorophenyl)urea, LY186641,] and from similar diaryl-substituted sulfonylureas, and its possible relevance to the compound's toxicity, was studied. In previous studies it was found that significant amounts of metabolites such as 2-amino-5-chlorophenyl sulfate (II), which is also a metabolite of p-chloroaniline, are formed from sulofenur in mice, rats, monkeys, and humans. The metabolism of N-(4-tolyl)-N'-(2-hydroxy-4-chlorophenyl)-urea (V) was studied, and V was not found to be an intermediate in the metabolic formation of II from the sulfonylurea N-(4-tolyl)-N'-(4-chlorophenyl)urea (LY181984, III). The amounts of this p-chloroaniline metabolite (II) formed in C3H mice from a series of diarylsulfonylureas were found to correlate with the compound's propensities to form methemoglobin, one notable toxicity of p-chloroaniline. This metabolism was also found to correlate with the structure of the arylsulfonyl moiety of the sulfonylurea. Other evidence supports the hypothesis that p-chloroaniline is directly formed by metabolism of sulfofenur and similar diarylsulfonylureas as well. Metabolic formation of p-chloroaniline thus appears to be a plausible explanation for the methemoglobinemia and anemia found to be dose-limiting toxicities of sulofenur in Phase I trials.


Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone pulmonary metabolism and tumorigenicity in mice by analogues of the investigational chemotherapeutic drug 4-ipomeanol. 4-Ipomeanol (IPO) is an investigational chemotherapeutic drug with specific toxicity toward the lung. It is metabolically activated to reactive intermediates by cytochrome P450 enzymes present in Clara cells. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a highly carcinogenic tobacco-specific nitrosamine with organo-specificity for the lung. Like IPO, which it resembles structurally, it is metabolically activated by cytochrome P450 enzymes of rat Clara cells. We synthesized nontoxic analogues of IPO and tested their activities as inhibitors of the metabolism and tumorigenicity of NNK. The IPO analogues synthesized were 4-hydroxy-1-phenyl-1-pentanone (HPP), 7-hydroxy-1-phenyl-1-octanone (HPO), 4-hydroxy-1-(2-thienyl)-1-pentanone (HTP), and 4-hydroxy-1-(3-pyridyl)-1-pentanone (HPYP). When added to A/J mouse lung microsomal incubations, all compounds significantly inhibited the oxidative pathways of NNK metabolism--alpha-hydroxylation and pyridine N-oxidation--to varying extents. Inhibition of carbonyl reduction of NNK was generally less effective. Inhibition of alpha-hydroxylation by IPO, HPP, and HTP was more pronounced in incubations with lung microsomes than with liver microsomes. None of the IPO analogues showed significant toxicity when given to A/J mice at a dose of 25 mumol; IPO itself was lethal at this dose. HPP and HPO, at doses of 25 mumol, significantly inhibited lung tumor multiplicity in mice treated with NNK; the other analogues and IPO itself were ineffective. The results of this study provide new leads for development of inhibitors of NNK metabolism and chemical probes for the active site of P450 enzymes in Clara cells.


Neuropathy target esterase inhibitors: 2-alkyl-, 2-alkoxy-, and 2-(aryloxy)-4H-1,3,2-benzodioxaphosphorin 2-oxides. The standard probes used earlier to study neuropathy target esterase (NTE) are N,N'-diisopropyl phosphorofluorodiamidate (mipafox), diisopropyl phosphorofluoridate (DFP), 2-(2-methylphenoxy)-4H-1,3,2-benzodioxaphosphorin 2-oxide (2-CH3C6H4O-BDPO) (the neurotoxic metabolite of tri-o-cresyl phosphate), and dipentyl 2,2-dichlorovinyl phosphate (DDP) with I50s for hen brain enzyme of 7000, 700, 29, and 3 nM, respectively. NTE phosphorylated by DFP and DDP is proposed to undergo alkylation on aging, and this probably also occurs with 2-CH3C6H4O-BDPO. Optimized probes for NTE should meet the following specifications: highest potency achievable; rapid aging perhaps associated with alkylation; preferably a phosphonate so there are only two leaving groups. An attempt was made to achieve these goals in the 4H-1,3,2-benzodioxaphosphorin 2-oxide series by synthesis of 49 analogs systematically varied in the 2-alkyl, 2-alkoxy, or 2-(aryloxy) substituent. Special precautions are required in synthesis of BDPO derivatives because of their potential hazard on human exposure. Thirty of these compounds had NTE I50s lower than 3 nM. Representative high-potency NTE inhibitors in each series are [2-substituent,I50 (nM) for hen and human brain NTE, respectively]: octyl, 0.25 and 0.18; nonyloxy, 0.89 and 0.98; 4-propylphenoxy, 0.82 and 0.77. In comparing these compounds, although the octyl analog is the most potent in vitro NTE inhibitor, the propylphenoxy compound is the most effective in vivo NTE inhibitor and delayed neurotoxicant in hens. These benzodioxaphosphorins are improved probes for investigations on NTE phosphorylation and alkylation in relation to delayed neurotoxicity.


Identification of individual benzo[c]phenanthrene dihydrodiol epoxide-DNA adducts by the 32P-postlabeling assay. Purine deoxyribonucleoside 3'-phosphates were reacted separately with the four configurational isomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide. Products resulting from the cis and trans opening of the epoxide ring by the exocyclic amino groups of deoxyadenosine and deoxyguanosine 3'-phosphates were separated by high-pressure liquid chromatography and identified by comparison of the observed circular dichroism spectra with the known spectra for the corresponding nucleoside adducts. The 16 structurally identified benzo[c]phenanthrene-purine deoxyribonucleoside 3'-phosphate adducts were then separately postlabeled according to the Randerath method, and the positions of the individual bisphosphates were mapped by thin-layer chromatography. Chromatographic conditions were developed that allowed separation of the four adducts for 3 of the 4 dihydrodiol epoxide isomers.


Identification of N-(Deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine as the major adduct formed by the food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, with DNA. The covalent binding of the N-acetoxy-, N-hydroxy-, and nitro derivatives of the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to 2'-deoxyribonucleosides or DNA was investigated in vitro and in vivo. N-Acetoxy-PhIP reacted with deoxyguanosine (dG), but not with the other deoxyribonucleosides, to form N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP), whose structure was determined by NMR and mass spectral analyses and by ultraviolet absorption and pH-solvent partitioning characteristics. While reaction of N-acetoxy-PhIP with calf thymus DNA at pH 5.0 yielded 5.38 +/- 1.16 nmol of bound PhIP residues/mg of DNA, N-hydroxy-PhIP gave only 0.13-0.23 nmol binding/mg of DNA under identical reaction conditions. Nitro-PhIP produced no detectable binding under these conditions. HPLC analysis of 1-butanol extracts of enzymatically hydrolyzed DNA that had been modified by N-acetoxy-PhIP in vitro showed a major adduct which coeluted with and had an ultraviolet absorption and a mass spectrum that were identical to that of authentic dG-C8-PhIP. 32P-Postlabeling analysis of DNA isolated from colon, pancreas, lung, heart, and liver of rats treated orally with PhIP revealed the presence of a major PhIP-DNA adduct. This adduct had chromatographic properties identical to that of the 32P-labeled bis(phosphate) derivative of dG-C8-PhIP and represented 35-45% of the total adducts.(ABSTRACT TRUNCATED AT 250 WORDS)


[Effects of N-phthalamoyl-L-glutaminic acid, a selective agonist of NMDA receptor, on binding of 3H-L-glutamate with synaptic membranes of the hippocampus]. In the present investigation the interaction of a novel selective NMDA receptors agonist, N-phthalamoyl-L-glutamic acid (PhGA), with the synaptic membranes preparation of human hippocampus was examined against NMDA. It was established that there are two binding sites of 3H-L-Glu, Kd1 = 0.35 +/- 0.11 nM, Bmax1 = 6.5 +/- 2.3 pmol/mg and Kd2 = 51 +/- 12 nM, Bmax2 = 98 +/- 17 pmol/mg. The inhibition constants (Ki) were calculated for NMDA and PhGA and were equal: Ki(NMDA) = 19 microM, Ki (PhGA) = 13 microM, respectively. It was concluded that PhGA is the partial agonist of the NMDA receptors.


[Biogenic amines in brain structures of rats of different zoo-social rank during immobilization stress]. The content of monoamines and their metabolites in different parts of the brain: mesencephalic reticular formation, locus coeruleus, sensomotor cortex was studied by high-performance liquid chromatography in rats with different zoo-social position. Content of dopamine and serotonin in the brain structures studied was found to be different in dominants and subdominants. Maximal changes of monoamines under immobilization stress were observed in dominants.


Reaction of alpha-acetoxy-N-nitrosopiperidine with deoxyguanosine: oxygen-dependent formation of 4-oxo-2-pentenal and a 1,N2-ethenodeoxyguanosine adduct. The six-membered heterocyclic nitrosamine N-nitrosopiperidine (NPIP) is an esophageal carcinogen in the rat whereas its five-membered homologue N-nitrosopyrrolidine (NPYR) is a liver carcinogen. These contrasting organo-specificities may be due to differences between NPIP and NPYR in their metabolic activation to intermediates which bind to DNA. Previous studies have shown that the metabolic activation of NPYR to DNA binding products occurs through alpha-hydroxylation. DNA adducts of NPIP have not been characterized. Therefore, we began our studies by investigating the reaction of alpha-acetoxyNPIP with deoxyguanosine. A major adduct, detected by high-performance liquid chromatography with UV detection, was characterized by its UV, 1H-NMR, and MS as 7-(2-oxopropyl)-5,9-dihydro-9-oxo-3-beta-D-deoxyribofuranosylimidazo+ ++[1,2-a] purine. This 7-(2-oxopropyl)-substituted 1,N2-ethenodeoxyguanosine adduct was formed by reaction of 4-oxo-2-pentenal (3-acetylacrolein) with the 1 and N2 positions of deoxyguanosine. Since the formation of 4-oxo-2-pentenal from alpha-acetoxyNPIP was unexpected, we investigated the solvolysis of alpha-acetoxyNPIP in more detail. Major products formed in incubations of alpha-acetoxyNPIP for 7-24 h in phosphate buffer (pH 7.0) at 37 degrees C included 4-oxo-2-pentenal (11-21% yield), 4-hydroxypentanal (18-22%), and 5-hydroxypentanal (27-29%). The formation of 4-oxo-2-pentenal required O2. The results of this study demonstrate some unique features of the chemistry of alpha-acetoxyNPIP and the resulting deoxyguanosine adducts which may be related to the carcinogenic activity of NPIP.


[Hypoxemia, acid-base balance shifts and blood system reactions in adaptation mechanisms]. Experiments were carried out on nonanesthetized and anesthetized dogs with open chest. Intravenous norepinephrine infusion (0.1 microgram/kg body weight/min during 25 minutes) simulated physiological stimulation of sympathetic nervous system. In all the cases it was associated with a significant arterial oxygen tension reduction that could be considered as a reaction with an antioxidant trend. At the same time the acid--base balance parameters shift directed to metabolic acidosis was seen that correlated with the hypoxemia significance. Recorded blood picture changes--hemoglobin and hematocrit increase as well as blood sedimentation rate lowering--improved both oxygen transport and utilization.


Identification of a cysteinyl adduct of oxidized 3-methylindole from goat lung and human liver microsomal proteins. 3-Methylindole is a selective pneumotoxin that is oxidized by cytochrome P-450 enzymes to a reactive intermediate. 3-Methyleneidolenine, a methylene imine electrophile, is the postulated reactive intermediate, and it binds to proteins, a reaction that probably initiates the pneumotoxicity of 3-methylindole. Thioether adducts of this electrophile are formed with glutathione in vitro, but the identity of the adducted electrophile with amino acid residues of microsomal proteins had not previously been determined. 3-Methylindole was incubated with NADPH and goat lung or human liver microsomal proteins, and the proteins were hydrolyzed. 3-(Cystein-S-ylmethyl)indole was isolated and identified as the major amino acid adduct of 3-methyleneindolenine, demonstrating that cysteine thiols preferentially attack the exocyclic methylene position and result in a covalently (thioether) attached 3-methylindole residue to these pulmonary and hepatic proteins. These results demonstrate that the putative methylene imine intermediate is indeed the active electrophile that binds to proteins and presumably initiates the toxic events.


[Sex differences in response to adrenaline in white rats]. Dynamics of changes of and plasma corticosterone was studied in both male and female rats after intraperitoneal injections of adrenaline at a dose of 20 micrograms per 100 g body weight. The control rats were injected with saline. Animals were decapitated 10, 30, 60, 120, 180 and 240 min after the injections. The specific effect of adrenaline was revealed in the first 10 min of adrenaline injection. This effect was significantly increased to 30-60 min after termination of saline--induced activation of the pituitary-adrenal axis. Both saline and adrenaline caused more significant increases in corticosterone levels in female rats than in male ones. There was a significant delay in the return of corticosterone to resting levels in males compared to that in females. It is supposed that the almost two-fold difference in peak plasma corticosterone concentrations observed after stressors may be associated with increased responsiveness of the female hypothalamus with respect to adrenaline secretion.


Pentahaloethane-based chlorofluorocarbon substitutes and halothane: correlation of in vivo hepatic protein trifluoroacetylation and urinary trifluoroacetic acid excretion with calculated enthalpies of activation. The hydrochlorofluorocarbons (HCFCs) 2,2-dichloro-1,1,1-trifluoroethane (HCFC-123) and 2-chloro-1,1,1,2-tetrafluoroethane (HCFC-124) and the hydrofluorocarbon (HFC) pentafluoroethane (HFC-125) are being developed as substitutes for chlorofluorocarbons that deplete stratospheric ozone. The structural similarity of these HCFCs and HFCs to halothane, which is hepatotoxic under certain circumstances, indicates that the metabolism and cellular interactions of HCFCs and HFCs must be explored. In a previous study [Harris et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1407], similar patterns of trifluoroacetylated proteins (TFA-proteins) were detected by immunoblotting with anti-TFA-protein antibodies in livers of rats exposed to halothane or HCFC-123. The present study extends these results and demonstrates that in vivo TFA-protein formation resulting from a 6-h exposure to a 1% atmosphere of these compounds follows the trend: halothane approximately HCFC-123 much greater than HFC-124, greater than HFC-125. The calculated enthalpies of activation of halothane, HCFC-123, HCFC-124, and HFC-125 paralleled the observed rate of trifluoroacetic acid excretion in HCFC- or HFC-exposed rats. Exposure of rats to a range of HCFC-123 concentrations indicated that TFA-protein formation was saturated at an exposure concentration between 0.01% and 0.1% HCFC-123. Deuteration of HCFC-123 decreased TFA-protein formation in vivo. Urinary trifluoroacetic acid excretion by treated rats correlated with the levels of TFA-proteins found after each of these treatments. No TFA-proteins were detected in hepatic fractions from rats given 1,1,1,2-tetrafluoroethane (HFC-134a), which is not metabolized to a trifluoroacetyl halide.(ABSTRACT TRUNCATED AT 250 WORDS)


[Use of thermo- and acid-stable proteinase inhibitor to suppress proteolytic activation in pulmonary inflammation]. A comparative study of thermo-acid stable proteinase inhibitor from rabbit serum and commercial drug contrykal was made on rats and in vitro experiments. Both inhibitors in equal doses effectively suppress proteolytic activity during initiation of lung inflammation. It was shown that the thermo-acid stable inhibitor possesses higher than contrykal, antielastase activity. A high effect of the acid stable inhibitors in the treatment of lung disease is suggested.


Metabolism of benzimidazoline-2-thiones by rat hepatic microsomes and hog liver flavin-containing monooxygenase. The metabolism of benzimidazoline-2-thione (I) and the 1-methyl (II) and 1,3-dimethyl (III) derivatives was studied to elucidate the mechanisms of hepatic oxidation for this class of thionosulfur-containing xenobiotics. NADPH-dependent metabolism of I, II, and III to the corresponding benzimidazoles Ia, IIa, and IIIa, respectively, was observed in dexamethasone-pretreated rat hepatic microsomes. III was the only thiocarbamide converted to an amide metabolite (IIIb). The effects of heat and 1-aminobenzotriazole pretreatment suggested that rat hepatic microsomal metabolism of I was catalyzed by the flavin-containing monoxygenase (FMO) only and that of II and III by both FMO and cytochrome P450 isozymes (P450). Addition of 5.0 mM glutathione (GSH) blocked formation of all metabolites from I, II, and III. Highly purified hog liver FMO catalyzed formation of all metabolites observed in rat hepatic microsomal systems. Incubation of III with either rat liver microsomes or with highly purified hog liver FMO in the presence of [18O]water led to ca. 50% incorporation of [18O] into IIIb. When [18O] molecular oxygen was used, ca. 8% incorporation of [18O] into IIIb was observed. Highly purified hog liver FMO also converted I-III to chemically reactive species that covalently bound to protein thiols. In the presence of hog liver FMO, the covalent binding pattern of radiolabeled I-III to bovine serum albumin was essentially identical to that observed for rat hepatic microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)


[Effects of blood platelets on generation of free radicals by leukocytes]. The paper is concerned with studies on generation of free radicals by donor leukocytes in plasma rich or poor in platelets. NBT test and chemiluminescence were employed. Platelets are shown to inhibit generation of free radicals. Changes in free radicals generation were studied under stimulation by zymosan and addition of washed platelet fraction. The conclusion is made that platelets inhibit leukocyte generation of free radicals by adsorption factors necessary for this process from plasma.


[Isoform pattern of inducible HSP 70 in the rat myocardium after heat shock]. The hsp 70 content was measured in the myocardium of a control rat group, in the group of rats 24 hours following a heat shock and in the group of rats 48 hours after a heat shock. In 24 hours after the heat shock, a major inducible hsp 70 with molecular weight of 71 kDa and pI about 5.8 occurred which was utterly absent in myocardial cytosol from control animals. In addition, there was an increase in polypeptide fraction with molecular weight of 73 kDa and pI about 5.6 (HSX73). In 48 hours after the heat shock, first the inducible hsp 70 with molecular weight of 71 kDa and pI about 5.8 disappeared which was found in 24 hours; secondly, HSX73 decreased to the control level and, thirdly, several isoforms pronouncedly accumulated with molecular weight of about 71 kDa and pI ranging from 5.9 to 6.3. Thus, the isoform composition of stress proteins induced by heat shock strongly depends on the time after the stress exposure. Furthermore, the accumulation of more acidic isoforms precedes the accumulation of alkaline ones.


[Prevention of postischemic lesions of the myocardium using liposomes]. The experiments on dogs showed that 60-min blood flow restriction in the left coronary artery branch resulted in pumping and contractile heart dysfunctions. The removal of the blood flow barrier caused reinforcement of the above dysfunctions. The administration of 50 mg/kg liposome prior to reperfusion improved pumping and contractile heart functions and allowed maintenance of stable hemodynamics during the reperfusion.


[Effects of different doses of perfluorocarbon emulsion on hemodynamics and contractility of ischemic heart]. The effect of different doses of perfluorocarbon emulsion (5, 10, and 15 ml/kg) on the hemodynamics and contractility of heart was studied on anesthetized dogs. The emulsion was introduced intravenously by the 60th minute of acute myocardium ischemia caused by partial coronary occlusion. When pO2 = 120 mm Hg, the emulsion was efficient only at doses of 10 and 15 ml/kg (an increase in cardiac ejection, in the rate of contraction and relaxation of the myocardium, reduction of vascular resistance). However, the efficiency of the emulsion at a dose of 15 ml/kg was lower, possibly, due to hypervolemia and cardiodepressive effect of introduction of excess quantity of the surface-active substance proxanol, a component of the emulsion.


[Activity of antioxidants in serum of animals with experimental crush syndrome]. Ischemia of soft tissues in crush syndrome results in activation of free radical oxidation processes, which negatively effect the functions of many biological systems. The presence of antioxidant system in the body can inhibit the action of free radicals. Antiradical effect of this system is seen in vitamin K, ubiquinone and ascorbic acid. The results of the experiment showed that the quantity of vitamin K, ubiquinone and ascorbic acid increases in the serum of blood after decompression of soft tissues in 14 hours, three and seven days. This fact supports an active participation of antioxidant system in the pathogenesis of crush syndrome.


[Determination of esterase activity of human and animal serine proteinases using fluorogenic esters of amino acids as substrates]. Two fluorogenic derivatives of amino acids are proposed as substrates for the purpose of enzymatic assay: N-benzyloxycarbonyl-phenylalanine-4-methyl umbelliferyl ester (substrate-1) and tert-butyloxycarbonyl-alanine-4-methyl-umbelliferyl ester (substrate-II). Chymotrypsin-like (hydrolysis of substrate-1), elastase-like (hydrolysis of substrate-II) esterase activity of bovine pancreatic chymotrypsin, activities of cathepsin G and elastase from human, porcine and rat neutrophils and esterase activity of human, porcine and rat serum were assayed. Differences in the level of chymotrypsin-like and elastase-like activities of human, porcine and rat serum were established. Activities of purified elastase and cathepsin G from human and animal neutrophils were shown to have no significant distinctions.


[Cytoplasmic casein kinase 2 and its substrate proteins in the brain in Alzheimer's disease]. The absence of casein kinase 2 on blots of temporal cortex extracts from Alzheimer's disease patients (ADP) was shown using antiserum to casein kinase 2. Casein kinase 2 activity towards endogenous substrates and casein is 2-5 times less in ADP brain in comparison to normal controls. The fractions of heparin-binding proteins, containing protein substrates for phosphorylation, were isolated from temporal cortex of ADP and normal controls. The total amount of heparin-binding proteins from ADP brains is less than from control brains, and the polypeptide composition of these fractions is much more poop.


[Modulating effects of adenosine on the process of human platelet activation]. The inhibitory effect of adenosine on aggregation of human platelets activated by platelet activating factor (PAF), ADP and serotonin (5-HT) were examined using native platelets from blood of volunteers. Platelet aggregation was determined by Born's method. Effective adenosine concentrations (IC50) which had inhibited platelet aggregation were found to be 0.63 +/- 0.11, 1.47 +/- 0.31 and 0.64 +/- 0.18 microM, respectively. It was shown that 10 microM adenosine inhibited PAF-induced platelet aggregation completely. The same adenosine concentration blocked ADP- and 5-HT-induced aggregation only partially. Adenosine is physiological inhibitor of human platelet aggregation in administration of PAF, ADP and 5-HT. Specific characteristics of adenosine modulating effect on these ligands was elicited.


[Induction of monooxygenase system and incorporation of radioactivity from 2-14C-lysine into proteins of rat liver microsomes under the action of phenobarbital and the background of lysine, methionine, threonine and vitamin A, C and E deficiencies]. The effect of diet on induction of monooxygenases and distribution of radioactivity from 2-14-C-lysine in fractions of liver homogenate, muscle homogenate and blood of male rats treated with phenobarbital (80 mg/kg, three days) was studied. 2-14-C-lysine was injected intraperitoneally 24 h before the first injection of phenobarbital. It was demonstrated that monooxygenase induction, increase of relative liver weight and incorporation of radioactivity from 2-14-C-lysine into fractions of liver homogenate in phenobarbital-treated rats fed diet deficient in lysine, methionine, threonine and vitamins A, C and E were more pronounced as compared with the similarly treated rats which were fed a balanced diet. The possibility of mobilization of deficient essential components to liver from other organs and tissues for maintenance of monooxygenase induction is discussed.


[A combined effect of cortisol and concanavalin A on calcium ion contents in thymic lymphocytes]. The influence of hydrocortisone (1-10 microM) on mitogen-induced Ca level was studied in cytoplasma thymocytes of rat. The investigation was conducted with the use of fluorescence technique (FURA-2AM). A specific Ca-blocking effect of the hormone was observed in the thymocytes of intact animals. Hydrocortisone did not change Ca-response of mitogen-stimulated hormone-resistant thymic lymphocytes. A conclusion was made about applicability of this method to assess the response of the cells to glucocorticoids in vitro.


[Study of the role of different types of glutamate receptors in spatial memory in rats]. A comparative study was performed to assess effects of different antagonists of glutamate receptors on spatial discrimination of rats in 8-arm radial and water mazes. Noncompetitive NMDA receptor blocker MK-801 in the dose 0.10 mg/kg and higher impaired the accuracy of performance in radial maze, whereas in the doses 0.05--0.30 mg/kg in water maze. Quisqualate IAMPA- and 2-amino-4-phosphonobutyrate (2-APB) I antagonists glutamate diethyl ester (250--500 mg/kg) and 2-APB (50--100 mg/kg), respectively, only increased time to complete radial maze performance and failed to alter choice accuracy. Both antagonists at the same doses did not affect performance of water maze task. The involvement of each type of glutamate receptors in different stages of spatial discrimination process in rats was discussed.


[Spectral analysis of the effects of nomifensine on bioelectric activity of the rat brain]. The influence of nomifensine on bioelectrical activity of sensorimotor cortex, dorsal hippocamp and lateral hypothalamus in conscious rats in free behavior was studied. The pharmacological and EEG analysis of nomifensine action on EEG power spectra by Fourier technique was measured. It was established that nomifensine evoked an increase and stabilization of the dominant peak in EEG spectra in the left and right cortex and in the left hippocamp, while in the other ranges of frequency a decrease was observed. The effects on hypothalamus was opposite--EEG power spectra decreased in all the ranges. The authors conclude that nomifensine evokes higher level of wakefulness, vigilance of the animals. This is likely to underlie neurophysiological mechanisms of optimal behavior due to stimulants of CNS, including nomifensine.


[Effects of chorionic gonadotropin on dynamics of primary immune response]. The effect of chorionic gonadotropin (CG) on primary immune response was estimated according to the level of direct and indirect plaque-forming cells (PFC) on day 5, 8 and 12 after immunization of non-castrated and ovariectomized female mice of CBA strain. It was established, that on the 5th day CG (40-200 IU) did not influence the direct PFC level in ovariectomized animals, but stimulated them in non-ovariectomized mice (40 IU). In ovariectomized animals the selective immunodepressive effect of hormone on the IgG-PFC formation processes has been revealed. The CG effect depended on the time of PFC number examination as well as on the hormone dose. In non-castrated animals, where immunomodulating CG effects are partially mediated by ovarian hormones, the injection of hormone only in the dose of 200 IU significantly lowered the number of IgM and IgG-PFC. It is suggested, that sex steroids on the late stages of PFC formation, when the processes of isotype antibody synthesis switch take place, appear to be synergists of CG immunodepressive effect.


[Lymphocyte subpopulation state of the human lung during prenatal development]. Distribution of surface lymphocyte markers was assessed in foetus lung by cytofluorimetry using monoclonal antibodies produced by "Ortho Diagnostic systems" and "Becton Dickinson". Seven lymphocyte subpopulations were detected in developing lungs even at the pseudoglandular stage (8-15 weeks). There was a significant increase in these lymphocyte subpopulations in canalicular stage. Quantity of T3+ cells increased 8-fold and B-cells 10-fold. There was a tendency to increased number of cortical thymocytes (T6+), and to decreased ratio T6+/T3+ lymphocytes. The ratio of immunoregulatory lymphocyte subpopulations (T4+/T8+) resembled that of these subpopulations in adult human lung rather than in cord blood. In canalicular stage the share of lung lymphoid cells in S-phase was decreased.


[Role of histamine receptors of mouse and guinea pig peritoneal leukocytes in the pathogenetic effect of Yersinia pestis adenylate cyclase]. The effect of purified preparation of adenylate cyclase isolated from Y. pestis on peritoneal leukocytes of white mice and guinea pigs was investigated. Y. pestis adenylate cyclase was shown to accomplish its pathogenic action via histamine-specific receptors on the surface of eukaryotic cells. The involvement of H1 and H2 histamine receptors on target cells in the adenylate cyclase action leading to development of plague infection is discussed.


[Quantitative determination of anthracycline antibiotic binding with DNA]. A new method of quantitative intravital assessment of anthracyclines accumulation and binding with DNA in alive cells has been developed. For this purpose DNA specific fluorescent dye Hoechst 33258 was used. Extent of fluorescence quenching of bound with DNA dye correlated with binding of daunomycin with cell DNA after mixing solutions of the drug and cells. It allowed us to determine quantity of drug molecules bound with DNA in the cells during the time.


[Differences in anti-oncogene p53 expression in human monocytes and lymphocytes in vitro]. The p53 gene has been associated with malignant transformation as well as "anti-oncogene" activity. In the present report expression of p53 in resting and activated human blood monocytes and lymphocytes is analyzed. It is found that human monocytes freshly isolated by continuous percoll gradient centrifugation contained detectable level of p53 mRNA. Stimulation of monocytes by potent activation inducer Staphylococcus Aureus Cowan I for 3-5 hr caused disappearance of r53 mRNA. In contrast, induction of high level of TNF-alpha mRNA was detected. Addition of cycloheximide had no effect on p53 mRNA content in stimulated monocytes, and caused disappearance of mRNA in resting cells. In lymphocytes cultures p53 mRNA was absent in freshly isolated cells and in resting lymphocytes cultured for 20 hr. Activation of lymphocytes by lectin caused accumulation of p53 mRNA. We suggest that r53 gene regulation and functions might be different in human monocytes and lymphocytes.


[Physicochemical and functional properties of hemoglobin of mice infected with Ehrlich's carcinoma]. Injection of white mice with Ehrlich's carcinoma triggers an increase in the mice blood erythrocyte fraction suspended in the 14% sucrose concentration zone. As established by acid erythrograms, a quantitative increase of this red blood cell population is due to an increased rate of erythroblast maturation and occurrence of immature cell forms in the blood stream. As shown by alkali denaturation of hemoglobin, tumour development causes an increase in alkali-resistant hemoglobin fraction in the erythrocytes. On the basis of the data on alkali denaturation of hemoglobin, it is suggested that in the infected mice increased rate of erythrocyte maturation go in line with selective binding of alkali resistant hemoglobin fraction to red cell membrane.


[Mouse serum inhibition of cytotoxicity of goat antibodies against mouse thymocytes]. It is shown that the serum of Balb/c, C57Bl/6 and F1(C57Bl/6 x CBA) mice inhibits cytotoxicity of goat antithymocyte antibodies. The addition of the serum into the incubation medium increases the proportion of alive cells from -5% up to 95%. Cytotoxicity was also inhibited by the globulin fraction of the mice serum. It is suggested that normal mice serum contains factors which block cytotoxicity of antibodies against antigen host determinants.


[Development of long-term post-tetanic potentiation and changes in S-100 protein contents in hippocampal slices of rats in different functional states]. Male rats of the strains with low (LE) high excitability (HE) of the nervous system have been used in this study. Half of the animals of each strain were neurotized in accordance with the Hecht's scheme. In the hippocampal slices of the non-neurotized LE rats there was a significant increase of the populational spike amplitude during development of LTP as compared with the opposite group of the animals. The LTP formation in the LE strain of rats caused a decrease in the S-100 protein content in the water-soluble, and an increase in the membrane-bound fraction of the protein. Similar results we have observed with the non-inbred Wistar rats but not with the HE strain of the animals. The levels of the water-soluble S-100 protein fraction were also higher in the hippocampuses and entorenal cortices, but not in the cerebellae of the LE strain, as compared with the HE strain of the rats. No differences have been found in the membrane-bound fraction of S-100 protein.


[Effects of acyclic analogs of atrial natriuretic factor on proliferative processes of the epithelial tissue in white rats]. The effect of atrial natriuretic factor (ANF) synthetic linear truncated analogues AP-H-6-OH and AP-FOR-6-OH on corneal, skin, duodenum and colon epithelium proliferation has been studied on male rats. The epithelium mitotic activity and DNA synthesis were evaluated 4 and 24 h after intraperitoneal injection of 10 or 100 micrograms/kg peptides. In a dose of 10 micrograms/kg both ANF synthetic analogues inhibited proliferation processes in corneal epithelium, but activated the DNA synthesis in duodenum and colon epithelium. AP-FOR-6-OH (10 micrograms/kg) decreased the mitotic activity of skin epithelium and increased the silver grain density over the cell nuclei at the same time. 100 micrograms/kg ANF analogues stimulated cell mitogenesis in all organs studied. According to the data obtained ANF linear truncated analogues influence on epithelium proliferation is similar to effector of previously studied cyclic atriopeptin AP II.


[Fast transport of proteins in the axons of spinal cord motor neurons in rats adapted and unadapted to physical loading]. The prolonged nonintensive swimming without load (12+ +2 h) provoked a decrease of the rate of fast axonal transport in the motor fibres of the sciatic nerve by 18% and of the overall amount of transported proteins 2-fold as compared with control. The relatively short-term but more intensive activity (swimming with the load 1/11 of body weight during 60+ +10 min.) results in an increase of the rate by 10% and the overall amount of fast transported proteins 2-fold. Daily swimming (3 h. without load and 10-15 min. with load) during 20 days returns the above parametres to the controls. The compensative-adaptive changes of fast anterograde transport are the fact that may be involved in trophic communication between neuron and target cell.


[Proliferation and differentiation of nerve cells of the human NN. supraopticus et paraventricularis transplanted to the brain of adult rats]. Human fetal hypothalamic neurosecretory cells (NSC) were studied after transplantation into the third ventricle of the adult rat brain. 3H-thymidine uptake data have shown that the time of origin and proliferation of NSC of grafted tissue and of supraoptic and paraventricular nuclei in normal embryogenesis was similar. Specific staining revealed that the start of neurosecretory function in grafted tissue corresponded to the date when NSC began functioning in normal ontogenesis.


[Effects of male sex hormones on specific uptake and release of 3H-serotonin in the rat hypothalamus in vitro]. With the use of "isotopic method" a study was made of the main parameters of functional activity of serotoninergic elements of hypothalamus--the specific uptake and release of 5-OT. The animals used were sexually mature rats castrated on the first postnatal day. In sexually mature intact males the specific uptake of 3H-5-OT by serotoninergic structures of the anterior hypothalamus was significantly lower than in females. Castration of animals on the first day of life resulted in the increase of specific 5-OT uptake in sexually mature males up to that observed in females. There were no differences between the sexes in the rate of spontaneous release of 5-OT. However, response to K(+)-depolarization in the anterior hypothalamus of intact males was significantly lower than that in females. In the hypothalamus of males castrated neonatally the amplitude of the response to the effect of the depolarizing agent was increase up to the level observed in females. By the results obtained it is indicated that elimination of the effect of male hormones on the first postnatal day results in the increase of 5-OT uptake and release in the hypothalamus of sexually mature rat males.


[In vitro phenomenon of beta cell migration from fetal calf pseudoislets]. Pseudoislets--pancreatic microfragments containing a lot of beta-cells and capable of insulin secretion in vitro-were obtained from 12 fetal calf pancreata by the use of collagenase. Morphological and functional changes of the pseudoislets were studied during culture. We found a rapid migration of beta-cells out of the pseudoislets to the bottom of plastic tissue culture plates. This process was accompanied by a significant decrease of insulin-secreting capacity of the floating microfragments. This should be taken into consideration in cases when pseudoislets are prepared for transplantation in order to avoid beta-cell loss.


Should women with node-negative breast cancer receive adjuvant chemotherapy?--Insights from a decision analysis model. The use of adjuvant chemotherapy in women with node-negative breast cancer has been controversial and actively debated since the 1988 National Cancer Institute Clinical Alert. We developed a decision analysis model that used the results of available randomized controlled trials to assess the potential clinical and financial effects of using adjuvant chemotherapy for groups of 45-year-old and 60-year-old women. Using the baseline assumptions, we found that chemotherapy increases quality adjusted life expectancy and survival by a substantial amount at a cost comparable to most accepted medical interventions. The model highlights the uncertainties in duration of benefit from therapy, the need for refinements in risk stratification, the importance of patient preferences about toxicity and benefit, and the need for accurate cost-accounting for oncologic therapies. Decision analysis complements other methods for information gathering, analysis, and synthesis used in clinical research. With the increasing focus on the effectiveness of medical interventions, decision analysis will be an important tool for oncologists to understand.


Proliferative activity of breast cancers increases in the course of genetic evolution as defined by cytogenetic analysis. The prognostic value of proliferative activity and its relationship with steroid hormone receptors and histopathological grade have been demonstrated for breast cancers. However, nothing is known about the underlying mechanisms. In order to understand the chronology of the appearance of increased proliferative activities, we used a series of 760 consecutive breast cancers for which we had obtained S-phase fractions (SPFs) by DNA flow cytometry. When the absolute difference from a DNA index of 1.00 was compared to SPFs, a significant positive correlation was obtained (r = 0.39, p < 0.0001), indicating that the probability of observing a high SPF increases when tumors progressively deviate from diploidy. A highly significant correlation was observed for the hyperploid group when hypertetraploid tumors were excluded, as the SPFs increased progressively as the DNA indices decreased from 2.00 to 1.30. This observation suggested a relationship with the evolution of chromosomal abnormalities as determined by cytogenetic analysis. Indeed, in a subset of 52 cases for which sufficient metaphases were available, there was a highly significant correlation between the SPF values and the proportion of rearranged chromosomes in the tumor cells (r = 0.60, p < 0.0001). When SPFs were separated into low or high using the median value (4.5%), a correlation also existed with the genetic evolution, since they increased from diploidy to hypodiploidy and then, after endoreduplication, from tetraploidy towards triploidy, as determined by the chromosome counts. Our results substantiate the relationship between proliferative activity and steroid hormone receptors which follow the same model.(ABSTRACT TRUNCATED AT 250 WORDS)


Contraceptive steroids and the mammary gland: is there a hazard?--Insights from animal studies. The safety of synthetic steroid hormones to be used for contraception in the human female is tested in rats, beagle dogs, and (once marketing starts) in monkeys. Because early studies did not show a mammary tumor stimulating effect in the human, in contrast to findings in the dog, many objections have been raised to the use of the dog for these toxicity studies. It has been claimed that the dog is unique in its sensitivity to the mammary tumor promoting effect of progestins and that this tumorigenic effect results from progestin-induced growth hormone (GH) induction. A thorough review of the literature does not support these claims. Tumor stimulatory effects of progesterone or synthetic progestins can be observed under some conditions in rodents as well as in cats and monkeys. In addition, recent evidence suggests a role for progesterone in mammary tumorigenesis in the human, and contraceptive steroids may also not be completely without risk. While the suggested role for GH in dog mammary tumorigenesis is far from proven, such a role cannot be excluded in the other species. Whether tumor stimulatory effects of sex steroids are based upon induction of proliferation in target cells or upon genotoxic effects or both is not yet certain.


Breast tumor-derived factors stimulate reduction of estrone to estradiol in nonmalignant breast tissue. This study examines the paracrine influence by human breast carcinoma cells (UISO-BCA-1) on nonmalignant breast tissue in vitro. The 17 beta-OH-SDH-mediated reductive pathway (estrone-->estradiol) was significantly increased in nonmalignant breast tissue coincubated with human breast carcinoma cells, compared to control tissues incubated in the media alone. No influence on the enzyme activity was noticed in coincubated breast cancer cells. Preincubation of breast cancer cells with estradiol (10(-8) M) significantly decreased the enzyme activity in coincubated nonmalignant breast tissue, which was restored to control levels by addition of R5020 (10(-8) M), tamoxifen (10(-6) M), or a combination of both. In nonmalignant tissues incubated in the presence of growth factor TGF alpha, enzyme activity was reduced to between 46% and 76%. No other growth factors (IGF I, IGF II, PDGF) influenced enzyme activity. In nonmalignant tissues incubated with malignant tumor cytosol, enzyme activity was increased in 16% cases, inhibited in 21%, and not significantly changed in 63%. The data from the present study suggest that factors produced by breast carcinoma cells may influence interconversion of estradiol in nonmalignant tissue. In patients, factors produced by malignant tumor mass may have paracrine influence on surrounding nonmalignant breast tissue and, thereby, may influence the estrogen availability to tumor mass.


Influence of aminoglutethimide on plasma oestrogen levels in breast cancer patients on 4-hydroxyandrostenedione treatment. The clinical and biochemical effects of combined treatment with the two aromatase inhibitors aminoglutethimide and 4-hydroxyandrostenedione were evaluated in 10 patients suffering from advanced breast cancer. All patients had become resistant to treatment with one of the drugs before having combined treatment. Seven patients progressing on 4-hydroxyandrostenedione who had aminoglutethimide added to their treatment and achieved a further suppression of plasma oestradiol by a mean of 40.0% (p < 0.05). Plasma oestrone was suppressed by a mean of 40.6% (p < 0.025) and plasma oestrone sulphate was suppressed by a mean of 63.6% (p < 0.025). Two of the patients, neither of whom had responded to 4-hydroxyandrostenedione alone, experienced objective tumour regression when aminoglutethimide was given in concert. Three patients progressing on aminoglutethimide who had 4-hydroxyandrostenedione added showed no further suppression of their plasma oestrogen levels, and no tumour regression was observed. These findings suggest a dose-response relationship between plasma oestrogen suppression at low postmenopausal levels and objective tumour response in breast cancer.


Cytosol cathepsin-D content and proliferative activity of human breast cancer. The Comitato Italiano per il Controllo di Qualita del Laboratorio in Oncologia. Mitogenic properties have been demonstrated in vitro for the lysosomal acidic protease cathepsin-D (cath-D). We investigated possible relationships between cath-D cytosol cell content and tumor proliferative activity in a series of 129 operable breast cancer patients. For total cytosol cath-D evaluation, a solid phase two-site immunoradiometric assay was utilized on tumor cell cytosol obtained for hormone receptor assay (DCC method). The percentage of S-phase cells was analyzed by 3H-thymidine autoradiographic assay. Median 3H-thymidine Labeling Index (3H-Tdr-LI) of the series was 2.7%; median cath-D content resulted 57 pmol/mg of protein cytosol and was significantly higher in node-positive with respect to the node-negative subgroup (p < 0.03). When classified in low, intermediate or high tumor cath-D content and slow or fast proliferative activity (cut-off: median values of the series), no significant agreement was found between the two variables. Statistical analysis, however, showed that a significant inverse correlation existed in node positive tumors between cath-D and 3H-Tdr-LI values which was even more evident in N-positive high estrogen receptor-positive (ER+) cases (coefficient of correlation = 0.6828; p = 0.0001). Cytosol cath-D content cannot be generally proposed as a direct marker of proliferative activity for operable breast cancer.


Immunohistochemical reaction to human milk fat globule antibodies and breast cancer differentiation. Immunohistochemical studies of human breast cancers using human milk fat globule (HMFG) antibodies are divergent regarding the association with histologic differentiation. To clarify this association 392 tumors were assessed using two antibodies, HMFG-1 and HMFG-2. In half the tumors studied the tissue reaction to both antibodies was the same and in 63% the site of reaction was identical. Using both univariate and multivariate statistics, a highly significant association was recorded between membrane staining and well differentiated cancers and cytoplasmic staining and poor differentiation. Well differentiated tumors contained significantly more reactive cells. Complementing these findings was the association between age at diagnosis, tumor stage, and estrogen receptor status and differentiation. It is possible that immunohistochemical analysis with HMFG antibodies will provide better characterization of tumor differentiation than morphologic interpretation.


Secretion of breast gross cystic disease fluid proteins by T47D breast cancer cells in culture--modulation by steroid hormones. The effect of steroid hormones on modulating the secretion rates of three human breast gross cystic disease fluid proteins (GCDFP-15, GCDFP-24, and GCDFP-44) by T47D breast carcinoma cells in tissue culture was evaluated. Androgens (dihydrotestosterone or fluoxymesterone) were capable of stimulating the secretion rates for all three GCDFP's while showing a minimal trend toward slowing the growth rate of T47D cells. This is the first study which shows that androgens can specifically stimulate all three of the major breast GCDFP's concomitantly. Progesterone, and three synthetic progestins, all showed inhibition of the growth rate of T47D cells while causing enhancement of the secretion of GCDFP-15 and GCDFP-44, and only minimal effect on the secretion rate of GCDFP-24. Estradiol was essentially neutral to the growth rate of the T47D cells in our test system. Estradiol did cause a mild enhancement of GCDFP-44 secretion rate, with no appreciable effect on GCDFP-15 or GCDFP-24 secretion rates. These findings suggest that an androgenic stimulus may be involved in the secretion of GCDFP's associated with breast gross cystic disease.


Characterization of two human mammary carcinomas, MT-1 and MT-3, suitable for in vivo testing of ether lipids and their derivatives. The human mammary carcinomas MT-1 and MT-3 originate from surgical material and were transplanted in nude mice. Both tumors have been classified as estradiol- and progesterone receptor-negative. Therapeutic doses of hormones and anti-hormones remained without growth inhibitory effect. MT-1 and MT-3 proved to be sensitive to conventional cytostatic drugs used for treatment of mammary carcinomas; striking is their sensitivity to ether lipids. Therefore, they are considered suitable tumor models for this class of substances.


Control of proliferation of MCF-7 breast cancer cells in a commercial preparation of charcoal-stripped adult bovine serum. A commercial preparation of charcoal-stripped adult bovine serum was used to culture MCF-7 cells in estrogen-free media. Use of this stripped adult bovine serum represents an alternative to calf serum which is in more limited supply, and saves charcoal-stripping of serum in the laboratory, which can be a rate-limiting step in the preparation of materials for estrogen-free tissue culture. MCF-7 cell proliferation was controlled by estrogens, epidermal growth factor (EGF) and lithium chloride in adult bovine serum as well as in standard media prepared with charcoal-stripped calf serum, and approximately the same fold-increase in response to the tested agents was observed in the two sera. Although the growth rates were lower in media prepared with adult bovine serum, MCF-7 cells in both media exhibited the same sensitivities in dose-responses to these three mitogens. Levels of estrogen and progesterone receptors, and the magnitude of estrogen-dependent stimulation of the progesterone receptors, were similar in cells maintained in both sera. Therefore, a commercially stripped adult bovine serum can be used to replace calf serum in the study of estrogenic responses and the control of proliferation in MCF-7 breast cancer cells.


An indirect mechanism by which a protein from the male gonad hastens salivary gland degeneration in the female ixodid tick, Amblyomma hebraeum. In the adult female tick, Amblyomma hebraeum Koch (Acari: Ixodidae), salivary gland degeneration is triggered by an ecdysteroid, provided the female is above a critical weight (approximately 300-400 mg). In mated females, salivary gland degeneration is virtually complete within 4 days of detachment from the host. In virgin females, salivary gland degeneration is delayed by 4 days. This delay can be reversed by the injection of a male reproductive tract homogenate directly into the hemocoel. In this study, we consider a possible mechanism of action for this "male factor." Once mated, male factor likely gains access to its target tissue(s) as a humoral factor. Male factor, however, appears not to act by sensitizing the salivary glands to the action of ecdysteroids. Instead, it appears to act by accelerating the appearance of ecdysteroids in the hemolymph.


Sequence accuracy of large DNA sequencing projects. Very little information has been accumulated regarding the likely accuracy of final or consensus DNA sequence data. With the large-scale efforts anticipated for the Human Genome Project, the subjective determination of final sequence must eventually be replaced with more objective, automatic methods. This will require a much better understanding of the nature of error in raw sequencing data and its impact on the determination of the final sequence. This paper describes a start at defining the error model of large-scale sequencing efforts based on random subcloning strategies.


An estimate of the sequencing error frequency in the DNA sequence databases. We have examined vector sequences fortuitously present in the EMBL sequence database as contaminating parts of submitted sequences, and found a sequencing error frequency of 3.55% in this subset of release 27 of the database. We discuss the possibility that this value may be representative for corresponding errors in the database as a whole.


Closely linked H2B genes in the marine copepod, Tigriopus californicus indicate a recent gene duplication or gene conversion event. Two nonallelic histone gene clusters were characterized in the marine copepod, Tigriopus californicus. The DNA sequence of one of the clusters reveals six genes in the contiguous arrangement of H2B, H1, H3, H4, H2B and H2A. The order of genes within the second cluster is H3, H4, H2B and H2A. There is no evidence for the presence of an H1 gene in this cluster. Comparison of the three copepod H2B genes reveals a high degree of similarity between the 5' upstream regions and between the amino terminal halves of the two H2B genes found within the same cluster. From these data we infer that gene duplication and/or gene conversion events occurred within this cluster in the recent past.


The nucleotide sequence of the human transcription factor HTF4a cDNA. A partial cDNA has previously been isolated that encodes HTF4, a new member of the basic helix-loop-helix family of DNA binding proteins. We have reconstructed a full cDNA, designated HTF4a cDNA, which spans the entire HTF4 coding region. The 5' end of the reconstructed cDNA includes an open reading frame encoding a 117-residue polypeptide. Three closely spaced ATG codons in the major open reading frame may potentially serve as sites of initiation for HTF4a synthesis, to yield proteins that contain 682, 675, and 657 amino acid residues, respectively. Pairwise dot matrix analysis indicates that there are at least three distinct human genes which encode proteins related to the Drosophila daughterless.


The nucleotide sequence between genes 31 and 30 of bacteriophage T4. The nucleotide sequence of the 2994 bp T4 phage DNA fragment between genes 31 and 30 is presented. The fragment contains 7 complete open reading frames in the direction of early transcription and two early promoters, PE128.6 and PE128.2, which we show to cause difficulties in cloning DNA from this genomic region. Our data complete the nucleotide sequence and the organization of genes in the genomic region between T4 genes 31 and 30.


Nucleotide sequence of bovine interleukin-6 cDNA. We report the cloning of bovine interleukin-6 (IL-6) cDNA. The clone was isolated from a bovine-leukemia virus (BLV)-induced B cell-lymphosarcoma cDNA library cloned in the bacteriophage lambda gt11. The cDNA encodes a full length IL-6 protein made of 208 amino acids with 65, 53, 42 and 42% homology to published sequences of porcine, human, mouse and rat IL-6, respectively. The significance of IL-6 expression in a BLV-induced tumor is briefly discussed.


Prognosing melanomas: the argyrophilic nucleolar organizer region approach. Staining of argyrophilic nucleolar organizer regions (AgNORs) is a histological technique that has been assayed on a vast array of cutaneous tumors in order to help distinguish benign from malignant lesions. In a number of papers, malignant melanomas have been found to have a higher number of black nucleolar and extranucleolar dots (i.e. AgNORs) than benign nevi, including Spitz nevus. The technique may be used for prognostic purposes, as a recent paper suggests. Melanomas with more than 3.62 AgNORs/cell have a higher probability to develop metastasis than melanomas with fewer AgNORs. The problems such an apparently simple, fast and reproducible technique may pose are discussed in detail.