Immunocytochemical and ultrastructural studies of neuronal and oligodendroglial cytoplasmic inclusions in multiple system atrophy. 2. Oligodendroglial cytoplasmic inclusions. Oligodendroglial cytoplasmic inclusions (OCI) in multiple system atrophy were investigated immunocytochemically and ultrastructurally. Among the 17 cases examined, 16 had OCIs. Almost all OCIs were positive for both alpha B-crystallin and ubiquitin. The antibodies against tubulin, paired helical filament and tau stained OCIs to various extents. Ultrastructurally OCIs consisted of meshworks of granule-associated filaments about 25 nm in diameter that sometimes formed flame-shaped tangle-like structures. Immunoelectron microscopy showed that an epitope of alpha B-crystallin was located on the granule-associated filaments composing OCIs. Our studies further support a cooperative role of alpha B-crystallin, ubiquitin and cytoskeletal protein in the formation of some types of intracytoplasmic inclusions.


Latent herpes simplex virus type 1 in human geniculate ganglia. Viral infection, especially by reactivation of herpes simplex virus (HSV) has been considered to be a possible explanation for the pathogenesis of idiopathic peripheral facial nerve palsy (Bell's palsy). We investigated whether the geniculate ganglia of man contain latent HSV type 1 (HSV-1), and compared the frequency of HSV-infected ganglia and that of latently infected neurons in human geniculate ganglia and in trigeminal ganglia. From autopsy specimens of eight adults 15 geniculate ganglia and 16 trigeminal ganglia were examined by means of in situ hybridization and immunohistochemical staining. The HSV-1 genome was detected in 11 of the 15 (71%) geniculate ganglia and in 13 of the 16 (81%) trigeminal ganglia. No HSV antigen was noted in any of the ganglia. The incidence of latently infected neurons was 0.9% in the trigeminal ganglia and 5.3% in the geniculate ganglia. The difference in percentages between the two types of ganglia was significant. Our results suggest that reactivation of latent HSV in the geniculate ganglia is a probable cause of some cases of herpetic stomatitis and of idiopathic peripheral facial nerve palsy.


A continuous cell line (KK-2) from a supratentorial primitive neuroectodermal tumor. Tumor tissue located in the occipital lobe with hemorrhage was obtained from a 19-year-old patient. Histological examination indicated it to consist of undifferentiated small, round cells without neuronal or glial differentiation, and possibly to be a type of primitive neuroectodermal tumor. The tumor cells were cultured for 3 years and a continuous cell line (KK-2) was established. KK-2 was transplantable to nude mice. With immunocytochemistry, neuron-specific enolase, protein gene product 9.5, vimentin, TUJ1 (a monoclonal antibody specific for neuron-associated class III beta-tubulin isotype) and 6H7 (a monoclonal antibody to NCAM produced by us) were detected. None of the following could be found: glial fibrillary acidic protein, S-100 protein, neurofilament and synaptophysin, calcitonin gene-related peptide, gastrin releasing peptide corticotropin-releasing factor, substance P, somatostatin, chromogranin, aromatic L-amino acid decarboxylase and tyrosine hydroxylase. The original tumor and KK-2 cells obtained after 3 years of culture and transplants in nude mice displayed essentially the same ultrastructural and immunohistochemical characteristics. KK-2 cells showed no differentiation to mature neuronal, glial or ependymal cells. This cell line may possibly serve as a useful model for studying cellular differentiation of human neuroectodermal tumors and normal neuronal development.


Pathomorphologic study of pale bodies in hepatocellular carcinoma. Pathomorphological and immunohistochemical studies were conducted on cases of hepatocellular carcinoma (HCC) with pale bodies (PB). HCC containing PBs was seen in 6 (5.7%) of 106 consecutively resected HCC cases. It was of interest that varying degrees of sclerotic change were found in 4 of the 6 cases and a certain correlation between PBs and sclerotic change of HCC tissue was suggested. Histologically, PBs were identified as a pale amorphous substance with a distinct margin and most of PBs occupied the entire cytoplasm of the cancer cells. PBs were practically negative for periodic-acid Schiff, and were also negative for phosphotungstic acid hematoxylin and orcein stains. Ultrastructurally, PBs were found to be a mass of granular or fibrillar materials having a single-layered limiting membrane, and dilated rough endoplasmic reticular (rER) were also found in the vicinity of PBs, suggesting the presence of a close relationship between rough endoplasmic reticula and PBs. Most PBs were found to be strongly positive for anti-fibrinogen antibody and some of them were weakly positive for anti-albumin, but were solely negative for other antibodies such as anti-HBs antigen, anti-alpha-1-antitrypsin, and anti-ferritin. According to those findings, PBs were thought to be fibrinogens accumulating in cystic rER due to a defective intracellular transport or an excretion disturbance.


Neuronal hyaline inclusions observed in an autopsy case of Behçet's disease. An autopsy case of Behçet's disease is reported. The patient, a 59-year-old Japanese woman, died of intestinal bleeding after a 34-year clinical course of Behçet's disease. She also suffered from recurrent oral aphthous ulcers, erythema nodosum-like cutaneous lesions and genital ulcerated lesions. Autopsy revealed marked atherosclerosis of the aorta and multiple deep ulcerations in the terminal ileum with no significant vascular lesions. Lewy bodies and globular hyaline inclusions in the neurons of the central nervous system were noted, although there were no clinical symptoms of Parkinson's disease throughout the clinical course. These findings appear to suggest that the patient was probably in the preclinical or early stage of Parkinson's disease. However, the presence of Lewy bodies in the 6th decade without any accompanying symptoms is very rare. This case seems to draw attention to the presence of these neuronal inclusions in Behçet's disease.


[Mass spectrometry of pipecuronium bromide and some related compounds]. The mass spectral behaviour of the bis-quaternary salt pipecuronium bromide and some related Cn+ A-n type ammonium salts (n = 1, 2, 3) has been studied and compared. Under EI-MS conditions the evaporation of the samples preceded by in situ dealkylation led to formation of alkyl bromide and amine bases. It has been demonstrated that this technique, completed with quantitation of the relative amount of the decomposition products is applicable to get structure information about the basis part as well as the number and positions of the quaternary centres and the substituents at these centres. The FAB mass spectra of these mono-, bis- and this quaternary ammonium salts were found to be very interesting and provide direct structural information about the salt. They exhibit primary Cn+ intact cations and (Cn+ A-n + 1)+ single charged cluster ions and also fragments characteristic of partial structures at the quaternary centres.


[Determination of the protonation constants of pipecuronium bromide on the basis of optical rotation as a function of pH values]. Pipecuronium bromide molecule contains two slightly basic nitrogen atoms in its two piperazine rings. Their pK values are quite similar to each other in consequence of their almost equivalent surroundings. Although the potentiometric titration used mostly for determination of pK values of organic compounds can be performed in this case too, but the polarimetric titration applied by us is more advantageous. Change of pH considerably influences the values of optical rotation; consequently, the curve obtained in this way is more utilizable than that of potentiometric titration. Protonation of two nitrogen atoms one after the other changes the optical rotation of the molecule in opposite direction. Consequently, a minimum can be seen on the curve of optical rotation versus pH value. The exact pK values were determined on the basis of the estimated parameters of a curve established corresponding to an equation and fitted to the measuring points. The sequence of deprotonation of nitrogen atoms could only be ascertained on the basis of the curves of polarimetric titration of compounds containing one piperazine ring; on the basis of the pK values and of the changing direction of optical rotation.


[Pharmaceutic development of injectable Arduan]. As a consequence of the presence of two ester groups in its molecule the main decomposition route of pipecuronium bromide (active ingredient of Arduan injection) is hydrolysis. In addition to this, autoxidation can also take place. In order to obtain a stable dosage form liophylized ampoules containing mannitol were prepared. Mannitol is not only a filling material but its role is also to decrease the dielectric constant of the solvent water thus increasing the stability of the drug substance before and during the liophylization procedure and after the dissolution of the content of the ampoules for administration. In order to protect the drug molecule against oxidation inert gas atmosphere was used during the production of the ampoules. The preparation is compatible with basic infusion solutions.


[Pharmacologic effects of pipecuronium bromide (Arduan)]. The experimental results in animals suggest that pipecuronium bromide offers the possibility of a neuromuscular blocking agent without side effects for surgical procedures of long duration. Its mechanism of action is twofold: 1. antagonism of acetylcholine effect at neuromuscular junction (postsynaptic nicotine receptors), 2. inhibition of acetylcholine release (presynaptic nicotine receptors). Its neuromuscular blocking potency is somewhat greater (2.0-3.0) than that of pancuronium in all species studied, and the duration of action is twice of that. It has no remarkable cumulative effect. Neostigmine rapidly and completely antagonises the neuromuscular blockade caused by pipecuronium. Certain structural properties (e.g. pipecuronium has no acetylcholine-like fragments in contrast with pancuronium and the interonium distance is also considerably larger than in pancuronium) may predict advantages. This has been proved by low vagal blocking--and ganglion--blocking potencies. On the basis of these a wide margin of safety can be expected in humans as well in preventing cardiovascular side effects. Pipecuronium is also characterized by interactions--only slight interactions--with other drugs used mainly in perioperative period.


[Pharmacokinetics and metabolism of pipecuronium bromide (Arduan)]. The pharmacokinetics of pipecuronium bromide (Arduan) studied in animals (rat, dog, cat) and in humans can be described by a two-compartment open model. The elimination half-life in animals was found to be around 40 min, in humans between 44-137 min. The main route of elimination is the excretion of the unchanged molecule in urine. In kidney patients the elimination becomes longer. The metabolism in the elimination is less important, in rats 3-hydroxy-, 17-hydroxy and 3,17-dihydroxy metabolites have been identified.


[Synthesis of a new neuromuscular blocking agent, pipecuronium bromide (Arduan)]. A series of bisquaternary ammonio steroids having androstane skeleton have been prepared some of which possessed high neuromuscular blocking activity. One of the series 3 alpha, 17 beta-diacetoxy-2 beta, 16 beta-bis (4,4-dimethyl-1-piperazinyl)-5 alpha-androstane dibromide (19, pipecuronium bromide, ARDUAN) has proved to be a clinically useful agent of long duration of action without side effects. The preparation of pipecuronium bromide and its metabolites and the impurities are also described. The structure of 19 and related compounds was elucidated by spectrometric methods IR, NMR and MS.


[Analysis of steroids. Part 44: Analytical investigation of the intermediates of the synthesis of pipecuronium bromide (Arduan)]. The following methods are described for the analytical investigation of the intermediates of the synthesis of pipecuronium bromide (Arduan) (for the numbering of the intermediates and their impurities see Figure 1.). 1. Gas chromatographic methods (capillary GC using fused silica capillaries Ultra-2 and Silar 10C WCOT) for the impurity profiling of intermediates I, II, IV and V including the identification and spectroscopic characterization of their impurities; 2. TLC methods for the similar characterisation of the further intermediates (III, VI, VII and VIII); 3. Gas chromatographic assay methods for IV and V using fused silica capillary technique and internal standards; 4. Potentiometric titration methods for the determination of VII and VIII using 0.1 M hydrochloric acid as the titrant.


[Analysis of steroids. Part 45: Analytical investigation of pipecuronium bromide (Arduan)]. The following methods are described for the analytical investigation of pipecuronium bromide. 1. HPLC method. Of the several systems tried for the separation and quantification of impurities and degradation products the best results were obtained using silica as the stationary phase and 43:43:14 mixture of methanol, acetonitrile and concentrated aqueous ammonia containing 0.1 mole/l each of ammonium chloride and ammonium carbonate as the eluent. The validation of this method is presented. The above described aggressive eluent can be successfully replaced by an ion-pairing system using silica as the stationary phase and 96:4 mixture of acetonitrile and water containing 0.1 mole/l sodium perchlorate as the eluent. 2. Thin-layer chromatography. TLC systems are described for the separation and densitometric quantification of the impurities and degradation products of pipecuronium bromide. 3. Spectrophotometry. Two methods are described. The ester groups of the molecule can be determined by the iron(III)-hydroxamate method while for the ion-pair extraction of the quaternary ammonium steroid picric acid or bromthymol blue are used as the reagents. 4. Titrimetry. In addition to the titration with acetous perchloric acid for the assay of the bulk material a microtitration method is described for the determination of pipecuronium bromide in individual lyophylized ampoules (potentiometric titration with 0.1 M silver nitrate).


[Excerpts from the investigations of pipecuronium bromide with NMR spectroscopy]. The synthesis of Pipecuronium bromide together with the investigation of its impurity profile required the solution of a number of analytical and structural problems. NMR spectroscopical methods received a major role in the structure elucidation of the synthesized compounds. The quantitative assessment of contaminations was also realized by 1H NMR spectroscopy. Out of the large amount of undertaken investigations, here we provide the structural characterization of the contaminating regiosimers of an important intermediate (5 alpha-androst-2-en-17-on) formed in the course of the synthesis, together with the NMR signal assignments of the end-product (Pipecuronium bromide) as measured in D2O and a mixture of CDCl3/DMSO-d6. The structure of a minor contamination of the end-product is also given.


Prostaglandin D2 increases Cl secretion across canine tracheal epithelium through cyclo-oxygenase stimulation and cAMP production. Prostaglandin (PG) D2 is one cyclo-oxygenase product of arachidonic acid metabolites that may play a role in the pathogenesis of asthma. To determine the effect of PGD2 on ion transport by airway epithelium and its mechanism of action, we measured bioelectric properties of canine cultured tracheal epithelium under short-circuit conditions in vitro. PGD2 (10(-7) M) increased short-circuit current (Isc) from 5.5 +/- 1.2 to 14.1 +/- 2.9 microA cm-2 (means +/- SE, P less than 0.01) when added to the mucosal solution, and to 22.2 +/- 3.8 microA cm-2 (P less than 0.001) when added to the submucosal solution, an effect that was accompanied by the corresponding increases in transepithelial potential difference and conductance. These effects were dose-dependent. The PGD2-induced increase in Isc was not altered by preincubation of cells with autonomic antagonists (phentolamine, propranolol, atropine), the lipoxygenase inhibitor AA-861, the protein kinase C inhibitor H-7, or the Na channel blocker amiloride, but it was inhibited by each of indomethacin, piroxicam, the Cl channel blocker diphenylamine-2-carboxylate, the Cl transport inhibitor furosemide, and Cl-free medium. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels were dose-dependently increased by PGD2. These results suggest that PGD2 may selectively stimulate airway epithelial Cl secretion via cyclo-oxygenase- and cAMP-dependent pathway.


The incidence of non-classical 21-hydroxylase deficiency in hirsute adolescent girls. Non-classical adrenal hyperplasia due to 21-hydroxylase enzyme deficiency (NC21OHD) causes hirsutism, acne and menstrual irregularities in women. Clinically, patients with NC21OHD may be indistinguishable from other hyperandrogenic women, as they all present with similar symptoms. An elevated response of cortisol precursors like 17 alpha-hydroxyprogesterone (17-OHP) to ACTH stimulation is a valuable diagnostic criteria. In this study, 32 hirsute adolescent girls, aged 13-19 years, underwent i.v. adrenocorticotrophic hormone (ACTH) (Synacthen 0.25 mg) stimulation test. The results were compared with those of the controls. The plasma levels of 17 alpha-hydroxyprogesterone, cortisol, dehydroepiandrosterone sulphate (DHEA-S), androstenedione, testosterone, follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin were established before, and 60 min after the infusion of ACTH to both patients and controls. Six patients demonstrated an increase in both the 17 alpha-hydroxyprogesterone levels and the 17 alpha-hydroxyprogesterone/cortisol ratio on ACTH stimulation, almost twice that of the mean +/- 2SD in the control group and ten times that in one patient. Six patients with abnormal elevation of 17 alpha-hydroxyprogesterone were considered heterozygotes for 21-hydroxylase enzyme deficiency, and one patient was presumed to have NC21OHD. Human leukocyte antigen (HLA) analysis supported these diagnoses. In this study, the incidence of NC21OHD in hirsute adolescent girls in our population was investigated, and NC21OHD was found in only one of 32 patients.


Ductal carcinoma in situ of the breast: correlation between mammographic calcification and tumor subtype. Histologic subtypes of ductal carcinoma in situ of the breast have been correlated with disease progression after local excision only. This study was undertaken to determine how the predominant type of calcification seen on mammography correlates with the predominant histologic tumor subtype, knowledge that could aid in the development of clinical criteria for selecting patients for appropriate local treatment. A prospective double-blind study was performed to correlate the mammographic and histologic findings in 66 consecutive cases of ductal carcinoma in situ, or ductal carcinoma in situ associated with small invasive foci (n = 11), in which microcalcifications seen on mammograms were found in the ductal carcinoma during histologic evaluation of excisional biopsy specimens. Microcalcifications were categorized as predominantly linear or granular and were correlated with the predominant histologic subtype of ductal carcinoma in situ in the tissue containing the calcifications seen on mammograms. Predominantly linear calcifications were present in 47% (18/38) of ductal comedocarcinomas in situ compared with 18% (5/28) of cribriform, solid, or papillary subtypes (p = .01). Predominantly granular calcifications were present in 53% (20/38) of comedocarcinomas compared with 82% (23/28) of the noncomedo types (p = .01). In 94% (16/17) of cribriform ductal carcinomas in situ, granular microcalcifications were seen on mammograms. Seventy-eight percent (18/23) of linear calcifications in ductal carcinoma in situ were associated with the comedo subtype, whereas 53% (23/43) of the granular calcifications were associated with noncomedo subtypes. We conclude that the comedo subtype of ductal carcinoma in situ is more likely to be accompanied by linear calcifications than are the noncomedo subtypes, and noncomedo ductal carcinoma in situ is more likely to be associated with granular calcifications than is the comedo subtype when microcalcifications are seen on mammograms. However, there is considerable overlap, and the predominant histologic subtype cannot be predicted on the basis of the microcalcification type with a high degree of accuracy.


Malignant biliary obstruction: efficacy of thin-section dynamic CT in determining resectability. Several authorities advocate the use of preoperative angiography to determine the resectability of pancreatic and periampullary tumors, claiming that CT alone is not sufficiently accurate for this purpose. Our objective was to assess the value of CT in predicting surgical resectability in patients with malignant biliary obstruction. We performed a retrospective analysis of 380 consecutive cases of malignant biliary obstruction spanning a 4-year period. Most patients (230) were treated nonoperatively. Sixty-seven patients had surgery, pathologic confirmation of malignancy, and preoperative CT scans available for review. The CT scans were assessed for surgical resectability of tumor by an interpreter who did not know the patient's history. Forty-two patients had pancreatic adenocarcinoma, six had ampullary carcinoma, seven had cholangiocarcinoma, and 12 had other malignant neoplasms. Of 47 patients with tumors thought to be unresectable on the basis of CT findings, 42 had tumors that were found to be unresectable at surgery (positive predictive value, 89%). Of 20 patients with tumors thought to be resectable, 16 had tumors that were surgically resectable (positive predictive value, 80%). CT did not show metastases to duodenal lymph nodes (n = 2), portal vein infiltration (n = 1), and small hepatic metastases (n = 1). Visualization of most of these at angiography would not be expected. The CT finding of infiltration of the periarterial fat around the celiac or superior mesenteric arteries was reliable for predicting surgical unresectability. Lymphadenopathy and infiltration of nonperivascular fat planes were less reliable predictors of unresectability. Although some findings on CT that suggest unresectability are less reliable than others, the accuracy of CT compares favorably with reports on the accuracy of angiography for assessing tumor resectability in cases of malignant biliary obstruction. The addition of angiography to the examination of patients with potentially resectable lesions is not justified when high-quality, thin-section dynamic CT has been performed.


Effects of oral soy phosphatidylcholine on phagocytosis, arachidonate concentrations, and killing by human polymorphonuclear leukocytes. A dietary supplement of linoleic acid (LA) as soy phosphatidylcholine (PC) or as triglyceride on polymorphonuclear leukocyte (PMNL) functions, arachidonate (AA) concentrations, AA release, and leukotriene B4 (LTB4) generation was studied in normal adults. Study 1: Eight subjects were fed PC (27 g) or placebo for 3 d in a blinded crossover experiment with PMNL assays at baseline and 4, 7, and 14 d. Study 2: Subjects were fed equal quantities of LA as PC (18 g, n = 8), safflower (SF, n = 4), or soybean oil (SY, n = 4) with PMNL assays at baseline and 48 h. Study 1: PC increased PMNL phagocytosis and killing of Candida albicans twofold (P less than 0.001) and PMNL phospholipid AA content threefold (P less than 0.001); AA release after Candida albicans stimulation increased 5.3-fold, correlating with PMNL killing (r = 0.932) and phagocytosis (r = 0.872). Study 2: PC, but not SF or SY, produced changes similar to those of study 1. With PMNL exposure to calcium ionophore A23187 or N-formyl-methionyl-leucyl-phenylalanine, PC increased LTB4 generation. Phospholipid LA, in contrast to triglyceride LA, enhanced PMNL phospholipid AA, phagocytosis, and killing.


Frequent rearrangements of retinoic acid receptor alpha gene and myl gene, and rare mutations of RAS and FMS genes in acute promyelocytic leukemia. To investigate leukemogenesis of acute promyelocytic leukemia (APL), we studied the involvements of retinoic acid receptor alpha (RAR alpha) and myl genes, and also the frequency of N-RAS, K-RAS, H-RAS, and FMS point mutations in sixteen patients with APL. By Southern blot analysis, the rearrangements of RAR alpha gene were detected in 13 patients (81.2%), and myl gene in 14 (87.5%). Either RAR alpha or myl gene rearrangements were found in all patients including one with normal karyotype. Breakpoints of both genes were clustered. By direct sequencing, no point mutations were found at codons 12, 13, and 61 of N-, K-, and H-RAS genes, and at codons 301 and 969 of FMS gene. These data indicate that myl-RAR alpha translocation occurs frequently in APL, whereas RAS and FMS mutations are rare in APL. It may be suggested that leukemogenesis of APL is different from other subtypes of acute myelogenous leukemia, and multistep leukemogenesis may not be a prevalent feature in APL.


Circulating thrombomodulin as a novel endothelial cell marker: comparison of its behavior with von Willebrand factor and tissue-type plasminogen activator. Circulating thrombomodulin is a novel endothelial cell marker, which may reflect the endothelial injury. Plasma levels of thrombomodulin were quantitated by an enzyme-linked immunosorbent assay (ELISA) in patients with hematological malignancies, liver disease, diabetes mellitus, collagen disease, thrombotic disease, and disseminated intravascular coagulation (DIC), and the thrombomodulin values were compared with those of von Willebrand factor antigen (vWf:Ag) and tissue-type plasminogen activator (t-PA) which are released from stimulated or damaged endothelial cells. The mean plasma concentrations of thrombomodulin in these disease states were elevated as compared with healthy subjects. A relatively high mean thrombomodulin level was observed in DIC, liver disease, and collagen disease. Abnormally high thrombomodulin values (greater than normal mean value + 3 SD) were found in 32.3% of patients with hematological malignancies, 57.7% of patients with liver disease, 39.3% of patients with diabetes mellitus, 30.0% of patients with collagen disease, 23.1% of patients with thrombotic disease, and 69.0% of patients with DIC. Plasma concentrations of both vWf:Ag and t-PA were also elevated in these patients. On the whole, the plasma thrombomodulin concentration was positively correlated with vWf:Ag (r = 0.441, P less than 0.001) and t-PA (r = 0.398, P less than 0.001). These findings indicate that the elevation of plasma thrombomodulin is frequently seen in a variety of diseases and circulating thrombomodulin is possibly useful for evaluating the endothelial damage in selected disease states.


Elevated neutrophil function in chronic neutrophilic leukemia. A 65-year-old man with marked leukocytosis was admitted for diagnosis and treatment. His peripheral blood leukocyte count was 37,500/microliters and the leukocytes consisted of mature neutrophil-like cells. A high neutrophil alkaline phosphatase score and a normal bone marrow cell karyotype suggested that the patient had chronic neutrophilic leukemia rather than chronic myeloid leukemia. Several neutrophil functions, such as superoxide production, nitroblue tetrazolium reduction activity, and phagocytosis, were elevated. These data and the morphological features (toxic granules and Döhle bodies) indicated that the patient's neutrophils were in an activated stage.


Case report: breast cancer in males--a genetic consideration. For many years, it has been recognized that a portion of female breast cancer is inherited. More recently, the probable contribution of heredity to at least a subset of male breast cancer also has surfaced. This report, which describes affected brothers, a half sister, and the common paternal grandmother, provides further support for the role of genetic factors in male breast cancer. Also noteworthy was the presence of prostate carcinoma in the sibling with bilateral disease and in the unaffected father.


Neuroendocrine effects of intravenous clomipramine in depressed patients and healthy subjects. Neuroendocrine challenge paradigms have been used to asses serotonergic systems in depression, but limitations in the specificity of many of these tests have been noted. In this study, the neuroendocrine responses to acute intravenous administration of the serotonin (5-HT) reuptake inhibitor clomipramine were assessed in depressed patients and matched control subjects. Thirty hospitalized patients who met DSM-III-R criteria for major depression, and 30 healthy control subjects who were matched for age, sex, and season of year for the time of study, received 12.5 mg of intravenously administered clomipramine. The depressed patients demonstrated significant blunting of prolactin responses to clomipramine, as well as trends toward blunted ACTH and cortisol responses. There was no difference between the patient and control groups in growth hormone responses, plasma clomipramine levels, or self-reports of side effects. These data support the hypothesis that depressed patients have abnormal neuroendocrine responses to the intravenous administration of the 5-HT reuptake inhibitor clomipramine. Further study is required to delineate the mechanisms responsible for the abnormal response to intravenously administered clomipramine in depression.


PHLIS: an electronic system for reporting public health data from remote sites. Disease surveillance conducted by the Centers for Disease Control (CDC) in conjunction with state health departments provides databases of information to public health workers. These databases' utility is limited by the lag time from occurrence of disease events until records are available for analysis. We developed the Public Health Laboratory Information System (PHLIS), a PC-based electronic reporting system for entering, editing, and analyzing data locally and for transmitting data electronically to other state or federal offices. Advantages of PHLIS include reduction in paper handling, decrease in lag time between disease incident and availability of information for analysis, ability to rapidly examine data for clusters of disease, downloadable summary tables, data editing at site of input, data analysis capability, increased interaction among participants, and current data for responses to inquiries. PHLIS is available without cost and is transportable to other agencies, states, or countries.


Relationship of tumor cell motility and morphologic patterns. Part 2. Analysis of tumor cell sublines with different motility in vitro. We have previously demonstrated by computer simulation that the relationship of proliferation and motility is likely to influence the morphological pattern of a tumor. Our study of a set of melanocytic skin tumors provided evidence that the assumptions of the model were correct with respect to proliferation. In order to test the validity of the assumptions concerning motility, we injected a tumor cell line with low motility in vitro and a subline of the same tumor cell line with high motility in vitro into the thigh of syngeneic rats. When we evaluated the morphological patterns of the resulting tumors by image analysis and compared them with computer simulations, we found that the differences of the morphological patterns were the same as predicted by the computer model. This finding further supports the concept that estimates of tumor cell proliferation and motility can be derived from the analysis of static histological patterns.


Acrosyringeal adenomatosis (eccrine syringofibroadenoma of Mascaro). A case report and review of the literature. The case of a 63-year-old man with acrosyringeal adenomatosis (eccrine syringofibroadenoma of Mascaro) is reported. Asymptomatic papular lesions had appeared on the dorsum of both of his feet 30 years earlier, then extended gradually and symmetrically to his hands, arms, legs, trunk, and auriculae. Although his hair, teeth, and nails were normal, he had glaucoma, chronic sinusitis, chronic otitis media, chronic pancreatitis, and gastrointestinal polyposis. No similar symptoms were found in the family history. Histopathologically, proliferations of pale epithelial cells extended from acrosyringia into the dermis, forming interconnected epithelial cords, within some of which were ductal structures. A syringoma and syringoma-like structures were closely associated with these lesions. Enzyme-histochemical, immunohistochemical, and ultrastructural studies supported the concept that this case represents a benign tumor derived from the acrosyringium, which is different from eccrine poroma. Acrosyringeal adenomatosis seems to be a more appropriate name for such a lesion than does eccrine syringofibroadenoma (Mascaro).


Linkage of hyperkalaemic periodic paralysis in quarter horses to the horse adult skeletal muscle sodium channel gene. A genetic disease observed in certain Quarter horses is hyperkalaemic periodic paralysis (HYPP). This disease causes attacks of paralysis which can be induced by ingestion of potassium. Recent studies have shown that HYPP in humans is due to single base changes within the adult skeletal muscle sodium channel gene. A large Quarter horse pedigree segregating dominant HYPP was studied to determine if mutations of the sodium channel gene are similarly responsible for HYPP in horses. We used cross-species, PCR-mediated, cDNA cloning and sequencing of the horse adult skeletal muscle sodium channel alpha-subunit gene to identify a polymorphism, and then used this polymorphism to see if the horse sodium channel gene was genetically linked to HYPP in horses. The sodium channel gene was indeed found to be tightly linked to HYPP (LOD = 2.7, theta = 0). Our results suggest that HYPP in horses involves the same gene as the clinically similar human disease, and indicates that these are homologous disorders. The future identification of the specific sodium channel mutation causing HYPP in Quarter horses will permit the development of accurate molecular diagnostics of this condition, as has been recently shown for humans.


Movement characteristics of human spermatozoa collected from different layers of a discontinuous Percoll gradient. In order to evaluate the functional state of human spermatozoa separated on discontinuous Percoll gradient, sperm provided by 15 healthy volunteers were recovered from different layers (60, 70, 80, 90, 100%) of the gradient, then submitted to a complete analysis of sperm movement characteristics. This one was performed using a computer assisted system (Cellsoft, Cryo Resources, Ltd., NY, USA). In all fractions sperm motility was highly maintained during 24 h and all movement characteristics, with the exception of ALH, were different from that observed in seminal plasma. Curvilinear velocity (VCL), linearity (LIN), and percentage of hyper-activated sperm (HA) increased in relation to the Percoll concentration. Nevertheless there was no dramatic difference, concerning motion parameters, between 100%, 90%, and 80% Percoll fractions, which can thereby be pooled in case of insufficient sperm recovering in 100% fraction. On the contrary there is some doubt about the fertilizing ability of spermatozoa recovered in 70% and 60% Percoll layers, since VCL, LIN, and especially HA are severely decreased.


Treatment of adult varicella with oral acyclovir. A randomized, placebo-controlled trial. To assess the efficacy of oral acyclovir in treating adults with varicella and to describe the natural history of adult varicella. Double-blind, placebo-controlled randomized trial. A naval hospital. One hundred forty-eight of 206 consecutive adult active duty Navy and Marine Corps personnel who were hospitalized for isolation and inpatient therapy of varicella and who could be treated within 72 hours of rash onset completed the study. The diagnosis of varicella was confirmed by acute and convalescent serology in 143 of 144 patients with available paired sera. Patients were randomly assigned to receive either acyclovir, 800 mg orally five times per day for 7 days, or an identical placebo. Separate randomization codes were used for patients presenting within 24 hours of rash onset and for those presenting 25 to 72 hours after rash onset. Daily lesion counts, symptom scores, temperature measurements, and laboratory tests were used to monitor the course of the illness. Early treatment (initiated within 24 hours of rash onset) reduced the total time to (100%) crusting from 7.4 to 5.6 days (P = 0.001) and reduced the maximum number of lesions by 46% (P = 0.04). Duration of fever and severity of symptoms were also reduced by early therapy. Late therapy (25 to 72 hours after rash onset) had no effect on the course of illness. Only four patients had pneumonia, and no encephalitis or mortality was noted. Early therapy with oral acyclovir decreases the time to cutaneous healing of adult varicella, decreases the duration of fever, and lessens symptoms. Initiation of therapy after the first day of illness is of no value in uncomplicated cases of adult varicella. The low frequency of serious complications of varicella (pneumonia, encephalitis, or death) precluded any evaluation of the possible effect of acyclovir on these outcomes.


Detection of hepatitis C virus infection among cadaver organ donors: evidence for low transmission of disease. To determine the prevalence of antibodies to hepatitis C virus (anti-HCV) and HCV RNA among cadaver organ donors and to correlate these results with donor liver histologic abnormalities and evidence for transmission of disease through organ transplantation. Retrospective testing of stored serum samples from cadaver organ donors for anti-HCV and HCV RNA. Transplantation service of the University of Miami/Jackson Memorial Medical Center and other cooperative medical centers furnishing follow-up data. Of 1096 cadaver organ donors harvested between 1 January 1979 and 28 February 1991, 484 had stored serum samples available for analysis. Recipients of organs from recombinant immunoblot assay (RIBA)-positive donors for whom adequate follow-up was available were also included in the analysis. Samples were tested for anti-HCV by enzyme-linked immunosorbent assay (ELISA). Confirmatory testing was done using a second-generation RIBA. Hepatitis C viral RNA was detected in serum using the polymerase chain reaction. Liver biopsies were obtained from the organ donor and interpreted blindly by a pathologist unaware of the clinical data. Liver chemistry profiles and serum sample analysis for HCV RNA were done for transplant recipients. From the 484 cadaver organ donors, 89 samples (18%; 95% Cl, 15% to 21%) were reactive by ELISA. Of these, 33 (6.8%; Cl, 4.6% to 9%) were RIBA seropositive. Hepatitis C viral RNA sequences were detected in 50% of the RIBA-positive serum samples tested. Liver tissue was available from 24 of the 33 RIBA-positive donors and showed chronic active hepatitis in 16, chronic persistent hepatitis in 2, and no abnormality in 6. Among the 46 recipients of a kidney from a RIBA-positive donor, 13 (28%; Cl, 15% to 41%) developed post-transplant liver disease, of which only 4 cases were highly suggestive of viral transmission from the donor. Little morbidity and no mortality could be attributed to liver disease in this cohort of recipients. These data suggest that HCV transmission by organ transplantation is low and that the consequences of infection are small. If the medical condition of the potential recipient is so serious that other options no longer exist, the use of an organ from an anti-HCV-seropositive donor should be considered.


In vitro histoculture of human tumors with fluorescent dye end-points measured by confocal microscopy: high correlation of in vitro and in vivo chemosensitivity. Tumors from 40 patients and 7 established human xenograft tumor lines were grown in three-dimensional histoculture. A Viable-Cell-Index (VCI) based on fluorescent dyes and Growth Fraction Index (GFI) based on [3H]thymidine incorporation were measured by confocal microscopy and histological autoradiography, respectively, after treatment with cytotoxic agents. Chemosensitivity in vitro with the two methods was correlated with chemosensitivity of the same set of human xenografted tumor lines grown in nude mice. The percent accuracy of in vitro to in vivo correlation with VCI (73%) was higher than GFI (63%). The number of false positives with VCI was 12.1% (4/33), and with GFI was 31.3% (10/32). The results thus indicated that in vitro histoculture with fluorescent vital-dye end-points to measure cell viability is of potential use to determine tumor chemosensitivity.


Expression of transforming growth factor alpha mRNA in benign and malignant tissues derived from gynecologic patients with various proliferative conditions. Growth factors are polypeptides involved in the regulation of normal and malignant cell growth. Transforming growth factor alpha (TGF alpha) is one of such protein growth factors that plays an important role in the regulation of mammalian cell growth. In this study, the expression of TGF alpha mRNA was studied in tissue specimens obtained at the time of surgery from patients with benign and malignant gynecologic proliferative conditions. To analyze TGF alpha mRNA expression we utilized the highly sensitive technique denoted Message Amplification Phenotyping which can detect mRNA in single cells. This technique consists of isolating RNA, reverse transcription of total cellular RNA to produce copy DNA, followed by enzymatic amplification of TGF alpha cDNA fragments using specific TGF alpha primers and polymerase chain reaction. The results showed significant levels to TGF alpha mRNA expression in vulvar (100% of the cases positive), cervical (66% positive), and endometrial (66% positive) carcinomas. Moreover, vulvar condylomas produced by human papilloma virus (HPV) showed the highest levels of TGF alpha mRNA expression of all the pathological tissues examined. In contrast, vulvar melanoma, fibrocystic disease of the breast, and certain ovarian tumors showed undetectable TGF alpha mRNA expression. Normal mesodermal tissues such as myometrium, abdominal rectus muscle, and fallopian tubes were negative for TGF alpha mRNA expression. However, TGF alpha mRNA was present in normal cervix and in normal endometrium. The results showed that TGF alpha mRNA expression is frequently associated with various malignant tumors and HPV-induced lesions of epithelial origin, suggesting that TGF alpha mRNA protein product may be a contributory factor in the progression of these pathological tissue alterations. Finally, TGF alpha mRNA expression was not restricted to malignant cells, suggesting that the TGF alpha mRNA protein product may function as a mitogen in the normal human epithelial tissues examined.


Identification of a streptomycin resistance gene and a partial Tn3 transposon coding for a beta-lactamase in a periodontal strain of Eikenella corrodens. The beta-lactamase gene from a periodontal strain of Eikenella corrodens, resistant to penicillins and streptomycin, was inserted into pBGS9 and transformed into Escherichia coli DH5 alpha. A 4.7-kb insert of pJML1, one of the transformants, was partially sequenced and found to contain the right section of transposon Tn3 from the middle of the TnpR resolvase gene to the right inverted repeat RI(R), including the TEM-1 gene. Sequences identical to RSF1010 were found on either side of the Tn3 sequence. pJML1 also contained a streptomycin resistance gene, probably identical to that of RSF1010. A portion of the pJML1 insert was not homologous to either Tn3 or RSF1010 but was homologous to the chromosomal DNA of E. corrodens ATCC 23834. It is assumed that the insert of pJML1 was derived from the chromosomal DNA of E. corrodens EC-38.


Fluorescent location of brain tumour cells in wax embedded sections. Tumour cells and certain normal cells in wax embedded brain sections possess active isoenzymic forms of a cell surface protease. The normal isoenzymic forms are recognised by protease inhibitors present in normal serum; the tumour isoenzyme remains active after exposure to serum. Fluorescent location of cells possessing active isoenzyme after serum treatment reveals the tumour cells in these sections.


Cytogenetical observations in two cases of polymorphous low-grade adenocarcinoma of the salivary glands. The cytogenetical findings in two cases of cultured salivary polymorphous low-grade adenocarcinoma (PLGA) are reported. The first PLGA was a carcinoma ex pleomorphic adenoma of the parotid gland. This had a pseudodiploid stemline characterized by extensive and complicated structural rearrangements. The chromosomal findings showed many similarities to those in the only previously studied case of PLGA (also a carcinoma ex pleomorphic adenoma of the parotid). The results in both could fit with abnormalities superimposed on those typical of mixed tumors. The second PLGA reported derived from a minor salivary gland. It showed a completely different picture, distinguished by a hypodiploid, almost exclusively numerical, variation and the occurrence of a stemline characterized by only monosomy 22. Etiological differences were proposed as one possible explanation for the cytogenetical differences between the two types of PLGA.


Effects of novel fluoroquinolones on the catalytic activities of eukaryotic topoisomerase II: Influence of the C-8 fluorine group. A previous study (M.J. Robinson, B.A. Martin, T.D. Gootz, P.R. McGuirk, M. Moynihan, J.A. Sutcliffe, and N. Osheroff, J. Biol. Chem. 266:14585-14592, 1991) demonstrated that novel 6,8-difluoroquinolones were potent effectors of eukaryotic topoisomerase II. To determine the contribution of the C-8 fluorine to drug potency, we compared the effects of CP-115,955 [6-fluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid] on the enzymatic activities of Drosophila melanogaster topoisomerase II with those of CP-115,953 (the 6,8-difluoro parent compound of CP-115,955). Removal of the C-8 fluoro group decreased the ability of the quinolone to enhance enzyme-mediated DNA cleavage approximately 2.5-fold. Like its difluorinated counterpart, CP-115,955 increased the levels of cleavage intermediates without impairing the DNA religation reaction of the enzyme. Removal of the C-8 fluorine reduced the ability of the quinolone to inhibit topoisomerase II-catalyzed DNA relaxation. In addition, the cytotoxicity of CP-115,955 towards Chinese hamster ovary cells was decreased compared with that of CP-115,953. These results demonstrate that the C-8 fluorine increases the potency of quinolone derivatives against eukaryotic topoisomerase II and mammalian cells. Further comparisons of CP-115,955 with CP-115,953 and CP-67,804 (the N-1 ethyl-substituted derivative of the difluoro parent compound) indicate that the two intrinsic activities of quinolone-based drugs towards topoisomerase II (i.e., enhancement of DNA cleavage and inhibition of catalytic strand passage) can be differentially influenced by alteration of ring substituents. Finally, correlations between the biochemical and cytological activities of these drugs suggest that the ability to inhibit catalytic strand passage enhances the cytotoxic potential of quinolones towards eukaryotic cells.


Protein kinase C inhibitor (H-7) potentiates antiproliferative effects of a polyamine biosynthesis inhibitor. In this study, a protein kinase C inhibitor, H-7, was found to potentiate the antiproliferative effects of difluoromethyl ornithine (DFMO), inhibitor of the polyamine biosynthesis, on NIH 3T3 and 3T3/SV40 cells in culture. Incubation of the cells with DFMO inhibited the cell growth, whereas the addition of polyamine spermidine to these cells restored the normal rate of cell proliferation with the fact that these cells took up the polyamine from the extracellular medium to compensate the intracellular needs. The addition of H-7 to both the 3T3 and 3T3/SV40 cells, inhibited the cell proliferation, though the level of inhibition was always lower than in those treated with the DFMO alone. The addition of H-7 to the DFMO containing cells potentiated the antiproliferative effects of the latter with the fact that the former inhibited the uptake of the spermidine, though there might be additional targets, like the protein kinase C, involved in the inhibition process.


Characterization of the tet(M) determinant of Tn916: evidence for regulation by transcription attenuation. The nucleotide sequence of the tetracycline resistance determinant tet(M), located on conjugative transposon Tn916 of Enterococcus faecalis, was determined and found to encode a 72,486-dalton protein exhibiting a high degree of homology with other tet(M) determinants. A short open reading frame corresponding to a 28-amino-acid peptide and containing a number of inverted repeat sequences was noted immediately upstream of tet(M), suggesting that regulation might occur by a mechanism involving transcriptional attenuation. Transcription analyses found this to indeed be the case, showing that the expression of tet(M) resulted from an extension of a small transcript representing the upstream leader region into the resistance determinant. Exposure of cells to tetracycline resulted in a significant increase in the amount of tet(M) transcription; this increase could be explained on the basis of increased transcriptional read-through from the upstream transcript. A model suggesting how transcriptional attenuation might operate in this system is presented.


In vitro antistaphylococcal activities of two investigative fluoroquinolones, CI-960 and WIN 57273, compared with those of ciprofloxacin, mupirocin (pseudomonic acid), and peptide-class antimicrobial agents. By using broth microdilution, 373 clinical isolates of staphylococci were studied to determine their susceptibilities to CI-960, WIN 57273, ciprofloxacin, mupirocin, vancomycin, teicoplanin, and ramoplanin. Test strains comprised 179 strains of Staphylococcus aureus and 194 strains of coagulase-negative species. Strains of S. aureus were susceptible to CI-960, which had a mode MIC of 0.032 micrograms/ml and an MIC for 90% of the strains of 2 micrograms/ml. CI-960 was equally active against methicillin-susceptible and -resistant S. aureus strains as well as ciprofloxacin-resistant strains. Similarly, WIN 57273 was highly active, with a mode MIC of 0.008 micrograms/ml and an MIC for 90% of the strains of 1 micrograms/ml. No cross-resistance to CI-960 and WIN 57273 among ciprofloxacin-resistant strains was detected. Mupirocin was four- to eightfold more active than ramoplanin, vancomycin, and teicoplanin. With regard to coagulase-negative staphylococci, CI-960 and WIN 57273 were the most active of the test compounds, inhibiting all strains at 0.5 and 1 micrograms/ml, respectively. Against the same strains, mupirocin was fourfold more active than ramoplanin and eightfold more active than vancomycin. Five strains of S. haemolyticus were found to be resistant to ciprofloxacin, while resistance to teicoplanin was found among strains of S. epidermidis, S. haemolyticus, S. hominis, S. saprophyticus, S. simulans, S. warneri, and S. xylosus.


Efficacy and safety of temafloxacin versus those of amoxicillin in hospitalized adults with community-acquired pneumonia. Temafloxacin, a new fluoroquinolone, was compared with amoxicillin in the treatment of adult hospitalized patients with community-acquired pneumonia. In this double-blind, multicenter study, patients were randomly assigned to treatment with temafloxacin at 600 mg twice daily (n = 125) or amoxicillin at 500 mg three times daily (n = 121); the average duration of treatment was 10 days. Clinical recovery rates were similar for patients treated with temafloxacin and amoxicillin (89 and 85%), as were bacterial eradication rates (99 and 97%). This was also true for subgroups of patients with pneumococcal pneumonia (n = 100), nonpneumococcal pneumonia (n = 122), or atypical pneumonia (n = 12). Outcomes for temafloxacin- and amoxicillin-treated patients were also similar in terms of defervescence, improvement in leukocytosis, and radiographic evidence of infection. The frequency and severity of adverse events were similar in both groups, consisting primarily of digestive disorders and skin manifestations. We conclude that temafloxacin may be recommended as an alternative antibacterial drug for patients with suspected pneumococcal pneumonia who fail to respond to benzylpenicillin or amoxicillin when the incidence of multiresistant pneumococcal strains is low. In countries where the incidence of these strains is high, temafloxacin may also be recommended.


Susceptibilities of penicillin-susceptible and -resistant strains of Streptococcus pneumoniae to RP 59500, vancomycin, erythromycin, PD 131628, sparfloxacin, temafloxacin, win 57273, ofloxacin, and ciprofloxacin. The MICs of four new quinolones, sparfloxacin (AT-4140, CI-978), PD 131628 (the active form of the prodrug CI-990), temafloxacin, and Win 57273, compared with those of ciprofloxacin and ofloxacin were tested against 53 penicillin-susceptible, 35 penicillin intermediate-resistant, and 51 penicillin-resistant pneumococci. Susceptibility to RP 59500, a new streptogramin, was also tested and compared with those to the quinolones, erythromycin, and vancomycin. All MICs were determined by a standardized agar dilution method by using Mueller-Hinton agar supplemented with sheep blood. Quinolone, vancomycin, and RP 59500 susceptibilities were not affected by susceptibility or resistance to penicillin. For Win 57273, the MICs for 50% (MIC50) and 90% (MIC90) of strains tested were 0.015 and 0.03 micrograms/ml, respectively. MIC50S of both sparfloxacin and PD 131628 were 0.25 micrograms/ml, and MIC90S were 0.5 micrograms/ml. The MIC50 of temafloxacin was 0.5 micrograms/ml, and the MIC90 was 1.0 micrograms/ml. By comparison, ofloxacin and ciprofloxacin both yielded MIC50S of 1.0 micrograms/ml and MIC90s of 2.0 micrograms/ml. RP 59500 yielded an MIC50 of 0.5 microgram/ml and an MIC90 of 1.0 microgram/ml and was only 1 doubling dilution less active against 17 erythromycin-resistant strains. Vancomycin was active against all strains (MIC50, 0.25 microgram/ml; MIC90, 0.5 microgram/ml). All four experimental quinolones as well as RP 59500 show promise for therapy of infections with penicillin-resistant and -susceptible pneumococci.


Lack of effect of carbonyl cyanide m-chlorophenylhydrazone on KB-5246 accumulation by Staphylococcus aureus. The accumulation of KB-5246 in a quinolone-susceptible strain of Staphylococcus aureus was about 70 times that of norfloxacin. Carbonyl cyanide m-chlorophenylhydrazone increased the accumulation of norfloxacin about eightfold, but it did not influence that of KB-5246. The low efflux of KB-5246 from S. aureus may contribute to its potent antibacterial activity.


Relative beta-lactamase- and transpeptidase-inhibitory activities of the new quinolone WIN-57273 in Staphylococcus aureus. The new quinolone WIN-57273 was shown to inhibit Staphylococcus aureus beta-lactamase activity noncompetitively in vitro with an apparent Ki value of 0.5 mM. MICs of penicillin G for a highly quinolone-resistant, beta-lactamase-negative strain in the presence of exogenous beta-lactamase decreased considerably when subinhibitory concentrations of WIN-57273 were added. Furthermore, the attachment transpeptidase reaction, investigated on whole cells of S. aureus, was impeded by WIN-57273 concentrations of greater than or equal to 30 microM. While these interactions suggest a novel mechanism of action for this compound, they are probably not relevant to the overall antibacterial potency of WIN-57273.


Changes in cAMP-dependent phosphorylation of specific cytosol protein in the peri- and postmenopausal ovary. Changes in cAMP-dependent phosphorylation of 53 kDa protein in a human ovary with respect to the aging process were studied. The phosphorylation of endogenous proteins in ovarian cytosol decreased with aging. However, ovarian aging was accompanied by an increase in the cAMP-dependent phosphorylation of 53 kDa specific protein while the amount of this protein decreased with aging. The plasma gonadotropin levels of women with regular menstrual periods were significantly different from those of women whose menstrual periods had in the recent past become irregular (FSH: p less than 0.01; LH: p less than 0.05); however, there was no significant difference in the plasma estradiol levels between these two groups. Our data show the possible correlations between the non-steroidal ovarian factor (53 kDa protein) and/or its phosphorylation by cAMP-dependent protein kinase and the ovarian aging process.


Role of macrophages in peripheral nerve degeneration and repair. A cut or crush injury to a peripheral nerve results in the degeneration of that portion of the axon isolated from the cell body. The rapid degeneration of this distal segment was for many years believed to be a process intrinsic to the nerve. It was believed that Schwann cells both phagocytosed degenerating axons and myelin sheaths and also provided growth factors to promote regeneration of the damaged axons. In recent years, it has become apparent that the degenerating distal segment is invaded by monocytes from the blood. We will review the evidence that these recruited macrophages play a role in both degeneration and regeneration of peripheral nerve axons after injury and consider whether the slow degeneration and poor monocyte recruitment in the central nervous system may contribute to the poor regeneration there.


Phorbol esters stimulate phosphoinositide phosphorylation and phosphatidylcholine metabolism in brain microvessels. The effects of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on phosphoinositide metabolism in cerebral microvessels were examined. Treatment of microvessels with TPA evoked a dose-dependent increase in the 32P-orthophosphate incorporation into polyphosphoinositides, phosphatidic acid (PA) and phosphatidylcholine (PC). The effect on PC was found only after a lag period. Experiments with membranes isolated from microvessels indicated a TPA-induced activation of phosphoinositide kinases. Evidence that this effect was mediated by protein kinase C (PKC) activation was provided by the reversal of the effect in the presence of staurosporine. Concomitantly with these observations, an increase of diacylglycerol (DAG) production was evoked without formation of inositol phosphates. Therefore, we suggest that TPA also stimulates PC metabolism. These results support a regulatory role for PKC in phospholipid signaling pathways in the blood-brain barrier (BBB).


The effects of arachidonic acid and its CoA ester on the catabolism of prostaglandin E2 in rabbit kidney cortex. The effects of arachidonic acid and arachidonoyl CoA on the catabolism of prostaglandin E2 in a 105000 x g supernatant fraction from rabbit kidney cortex were examined. Arachidonic acid reduced the 15-hydroxy prostaglandin dehydrogenase activity by 50% at 20 microM, while arachidonoyl CoA showed weak inhibition for the enzyme activity (15% at 20 microM). However, arachidonoyl CoA strongly inhibited the prostaglandin delta 13 reductase activity, the concentration required for 50% inhibition being about 3 microM. The dehydrogenase inhibition by arachidonic acid was non-competitive with regard to NAD+ and prostaglandin E2, respectively. Arachidonoyl CoA was also a non-competitive inhibitor for the reductase with regard to NADH and 15-keto prostaglandin E2, respectively. These results suggest that arachidonic acid and arachidonoyl CoA can be important modulating factors in prostaglandin catabolism by the kidney cortex.


Effects of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate on porcine heart protein phosphatase 2A. Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate are apparent noncompetitive inhibitors of porcine protein phosphatase 2A2 having Ki values of 0.38 and 0.56 mM, respectively. The inhibitory effects were on the catalytic subunit and were not substrate directed. In addition, fructose 2,6-bisphosphate caused a time-dependent inactivation of phosphatase activity toward phosphorylase a. This inactivation was antagonized by MnCl2. The fructose 2,6-bisphosphate-inactivated enzyme had increased p-nitrophenyl phosphate phosphatase activity. These effects are similar to the known effects of ATP on type 2A phosphatases.


[Ligands possessing affinity to specific DNA base pair sequences. IX. Synthesis of netropsin and distamycin A analogs having sarcolysin residues or a platinum(II) atom]. In search for compounds capable of forming covalent bonds with DNA AT-pair clusters, distamycin A and netropsin analogues containing DL-sarcolysin or platinum (II) atom at the N-terminus of the molecule were synthesized, as well as bis-netropsin and bis-distamycin in which two netropsin- or distamycin-like fragments are bound via a cis-diammineplatinum (II) residue. It is shown that these substances can be used for the DNA selective cleavage.


Cytomegalovirus infection.

Cytomegalovirus infection. Post-transfusion CMV infection most frequently results in asymptomatic seroconversion. Among immunocompetent patients only seronegative pregnant women require such products because of the risk of fetal CMV infection. In selected groups of immunocompromised patients, significant disease can occur. It is desirable to provide blood and blood components with reduced CMV risk to the following patients: seronegative infants weighting less than 1200 g at birth, seronegative bone marrow transplant patients who receive marrow from seronegative donors and seronegative renal transplant patients receiving kidneys from seronegative donors. Heart and liver transplantation seronegative patients may receive seronegative blood if the donor is seronegative. CMV--seronegative HIV infected cases may also be transfused with CMV--seronegative blood.


Are endogenous retroviruses involved in human autoimmune disease? A role for viruses in the etiopathogenesis of human autoimmune diseases has long been suspected but has not yet been proven. In Sjögren's syndrome (SS), there is continuing experimental support for the possible involvement of Epstein-Barr virus. Since the advent of AIDS, there is also great interest in retroviruses and autoimmune disease. We previously reported that 30% of SS patients and 36% of systemic lupus erythematosus (SLE) patients have serum antibodies to the p24 gag protein of HIV-1. We now report that two mechanisms classic for retroviruses (molecular mimicry and immunosuppression) may be operative in SS and SLE. The p24 gag protein shares a proline-rich epitope with the Sm nucleoprotein to which many SLE patients have antibodies. The impaired lymphocyte activation seen in peripheral blood T cells in SS patients is also seen in a human T cell line infected with an A-type retroviral particle linked to SS. Many studies suggest that endogenous retroviral sequences are important in immunoregulation. We now suggest that endogenous retroviral sequences may also be important in the etiology and pathogenesis of SS and SLE.


Differential effect of tumor necrosis factor on proliferation of primary human keratinocytes and cell lines containing human papillomavirus types 16 and 18. Keratinocytes immortalized by human papillomaviruses (HPV) 16 and 18 are partially resistant to the inhibition of proliferation exerted by transforming growth factor-beta (TGF-beta). To determine if this finding reflects a generalized resistance to inhibitory cytokines, we studied the effect of tumor necrosis factor-alpha (TNF-alpha) on subconfluent cultures of both normal and HPV-immortalized human foreskin keratinocytes. Whereas primary and HPV-16-immortalized keratinocytes were sensitive to TNF-alpha, HPV-18-immortalized keratinocytes (and those immortalized by simian virus 40) were resistant to the inhibitory effects of this cytokine. The ability of HPV-18 to induce a more resistant phenotype correlated with its more potent in vitro transforming activity and its apparent association with more aggressive tumors. Interestingly, the state of TNF-induced growth inhibition in normal or HPV-16-immortalized keratinocytes was not accompanied by a reduction in the expression of c-myc RNA or protein. This contrasts sharply with the ability of TGF-beta to inhibit c-myc RNA expression in normal cells. Evidently, the resistance of HPV-immortalized keratinocytes to TNF-alpha and TGF-beta proceeds along different regulatory pathways.


Role of mutations at codon 61 of the c-Ha-ras gene during diethylnitrosamine-induced hepatocarcinogenesis in C3H/He mice. Liver tumors of certain strains of mice frequently contain mutations at codon 61 of the c-Ha-ras gene. In our study, we investigated the significance of these mutations in the carcinogenic process. Male C3H/He mice received a single injection of diethylnitrosamine (DEN) on day 15 after birth, and groups of animals were killed at various time intervals between 11 and 52 wk after treatment. At the earlier time points (11-29 wk), we analyzed microdissected tissue from precancerous glucose-6-phosphatase-deficient liver lesions larger than approximately 200 microns in diameter, for the presence and pattern of c-Ha-ras codon 61 mutations. In parallel, the growth characteristics of these liver lesions were studied by pulse labeling with [3H]thymidine and by determining the size distribution of the lesions. At the later time points (42-52 wk after DEN treatment), liver tumors were dissected and also analyzed for the presence of c-Ha-ras mutations. We found mutations to be already present in some of the enzyme-altered liver lesions at weeks 11-29, suggesting that the mutations occurred early in the carcinogenic process. Whereas about 10% of the precancerous focal liver lesions showed mutations in the c-Ha-ras gene, the mutation frequency was increased to about 50% in the later-appearing hepatocellular adenomas and carcinomas, suggesting that c-Ha-ras codon 61 mutations may provide a selective advantage to the mutated cell clones.


Controllability and duration of stress alter central nervous system depressant-induced sleep time in rats. Rats were exposed to either 80 escapable shocks or yoked inescapable shocks and then injected with several hypnotic doses of sodium pentobarbital, midazolam, or ethanol; their sleep-time duration was compared with that of naive controls. Inescapable shock exposure resulted in a significant increase in ethanol-induced sleep time compared with the escapable shock and naive control groups. Both escape and yoked groups showed an increase in barbiturate-induced sleep time compared with controls, although no difference was observed for midazolam. Acute stress (twenty 5-s inescapable shocks) did not alter the depressant-induced sleep time for any of the drugs tested. These results illustrate the importance of psychological aspects of stress and its influence on the potency of certain depressants.


Amiloride sensitivity of the chorda tympani response to sodium chloride in sodium-depleted Wistar rats. Peripheral gustatory mechanisms that may contribute to the expression of sodium (Na) appetite have been a focus of interest for many years. Because amiloride-sensitive Na transport is involved in the generation of neural signals in response to NaCl stimulation, the present study assessed whether changes in amiloride sensitivity of the neural response to NaCl accompany the induction of a Na appetite in the rat. Na deprivation was achieved by acute depletion with the diuretic furosemide. The magnitude of the whole-nerve chorda tympani response to 0.5 M NaCl was reduced in Na-depleted, compared with Na-replete, rats, which provides qualified support for previous reports that the induction of a Na appetite is associated with reduced neural responses to NaCl. However, changes in sensitivity to the specific Na channel blocker amiloride hydrochloride as a result of Na depletion were not evident. These findings suggest that the behavioral and neural changes that occur after Na depletion are not based on changes in amiloride sensitivity in the taste bud.


Otologic effects of interferon beta serine in experimental rhinovirus colds. To evaluate the efficacy and otologic effects of recombinant interferon beta serine for experimental rhinovirus colds, 38 healthy adults received nasal drops of recombinant interferon beta serine, 12 x 10(6) U, or placebo three times daily for 4.3 days beginning 36 hours after infection. Illness rates and severity did not differ between the groups, but the frequency of virus shedding was reduced on the fourth (37% vs 74%) and sixth (11% vs 42%) postchallenge days in recipients of recombinant interferon beta serine. Abnormal eustachian tube function in at least one ear was identified by the inflation-deflation test during 44% of observations in 13 infected recipients of recombinant interferon beta serine compared with 62% of observations in five placebo recipients. Tympanometry revealed middle-ear pressure abnormalities (less than -50 or greater than 20 mm H2O) in at least one ear during 18% of observations in recipients of recombinant interferon beta serine compared with 38% of observations in placebo recipients. These results suggest that antiviral therapy may alter the course of middle-ear dysfunction associated with experimental colds.


Peptide mapping and microsequencing of proteins separated by SDS-PAGE after limited in situ acid hydrolysis. A method is described for the isolation of peptide fragments from proteins separated by polyacrylamide gel electrophoresis. After completion of the electrophoresis step, gels are stained with Ponceau S or Coomassie Blue. Gel portions containing protein stained with Ponceau S are excised and transferred to borosilicate glass digestion tubes containing 0.9 ml of 1 mM NaOH or 5 mM Na2HPO4. After complete dissociation of the dye from the protein, 0.1 ml of 20% formic acid is added and the protein is hydrolyzed in situ at 112 degrees C for four hours. Subsequently the acid solution is made 10% in acetonitrile and chromatographed as such on a C18 (C4) reversed-phase column using an appropriate large-volume sample loading syringe and injection loop. Proteins stained with Coomassie Blue can be hydrolyzed in situ after complete removal of the dye with an aqueous solution containing 40% acetone, 10% triethylamine and 5% acetic acid. The gel slices are next washed with HPLC-grade water and protein is hydrolyzed in 2% formic acid under standard conditions. Gel-related contaminants do not interfere with the peptide separation under the proper conditions of HPLC analysis.


Strategies for automated sequencing of human mitochondrial DNA directly from PCR products. A rapid, robust and sensitive method has been developed for the amplification and direct sequencing of human mitochondrial DNA. A 403-bp hypervariable segment was amplified by two successive rounds of nested PCR. This was then sequenced by the dideoxy chain termination method using dye-labeled universal sequencing primers in conjunction with an automated DNA sequencer. This paper describes the assessment of four different strategies for this amplification and sequencing process. Optimal results were obtained by immobilizing the biotinylated PCR product on Dynabeads followed by solid-phase sequencing with Sequenase. Degraded samples and those containing trace amounts of DNA such as extracts from hair shafts can be analyzed by this method.


Rat mammary myoepithelial-like cells in culture possess kinetically distinct low-affinity receptors for fibroblast growth factor that modulate growth stimulatory responses. The rat mammary myoepithelial-like cell line Rama 401 possesses 46,000 high-affinity receptors (Kd 52 pM) and 2.8 x 10(6) low-affinity receptors (Kd 24 nM) for basic fibroblast growth factor (bFGF) per cell. Heparin or heparinase pretreatment of the cells inhibits the specific binding of [125I]-bFGF by over 70%, and abolishes binding to the low-affinity sites. Dissociation experiments suggest that there are three kinetically distinct low-affinity receptors, with dissociation rate constants of 3.8 s-1, 0.067 s-1 and 0.0018 s-1. Consistent with the presence of low-affinity receptors possessing a slow dissociation rate constant, exogenously added bFGF bound to the low-affinity receptor can stimulate DNA synthesis in Rama 401 cells without being released into the bulk of the culture medium. These results suggest that the low-affinity receptors on Rama 401 cells are heparan sulfate glycosaminoglycans (HSGAGs) and that their ability to modulate the action of bFGF may result from their diverse range of dissociation rate constants. A cell line, Rama 401ts, derived from Rama 401 by transformation with a temperature sensitive src gene, deposits less extracellular matrix at the permissive temperature of 34 degrees C than at the non-permissive temperature of 41 degrees C. Whilst the binding of [125I]-bFGF to Rama 401ts cells at 41 degrees C is identical to that observed with the parental Rama 401 cells, at 34 degrees C there are fewer low-affinity receptors. These results suggest the (HSGAGs) low-affinity receptors on Rama 401 cells are associated at least in part with the extracellular matrix.


Kupffer cells express type I TGF-beta receptors, migrate to TGF-beta and participate in streptococcal cell wall induced hepatic granuloma formation. Intraperitoneal injection of Group A streptococcal cell wall (SCW) fragments into female Lewis rats results in the induction of an acute hepatic inflammation that progresses to granulomatous lesions. Kupffer cells have been shown to rapidly clear circulating SCW which triggers production of TGF-beta. In this study, we examined Kupffer cells for the expression of TGF-beta receptors to determine if these cells might be modulated in an autocrine/paracrine fashion by TGF-beta during SCW-hepatic inflammation. By receptor crosslinking and subsequent SDS-PAGE analysis we demonstrate that Kupffer cells express Type I TGF-beta receptors, but not Types II and III. Scatchard analysis indicated a receptor density of approximately 1100 receptors per cell. Functionally, TGF-beta was found to be chemotactic for Kupffer cells in vitro and this chemotactic response was higher in cells isolated from rats 1-21 days post SCW-injection. Although TGF-beta 1 mRNA is constitutively expressed by Kupffer cells, in vitro stimulation of the cultures with purified TGF-beta augments the expression of TGF-beta 1 mRNA and protein synthesis suggesting autocrine/paracrine regulation. These results indicate that TGF beta secreted by Kupffer cells during SCW-induced hepatic inflammation may amplify its own expression and regulate Kupffer cell functions relevant to the formation of granulomatous lesions within the liver.


Regulation of gene expression by vitamin A: the role of nuclear retinoic acid receptors. Over the past five years, a wealth of information has accumulated concerning the molecular mechanisms mediating RA effects on gene expression. The molecular cloning of the 3 retinoic acid receptors (RARs), of their 16 or so different isoforms, and of the 3 retinoid X receptors (RXRs) as well as the identification of at least 2 different active isomers of RA (all trans- and 9-cis-RA) and of several different CRABPs and CRBPs now provide the essential tools to explain the pleiotropy that has become associated with RA effects. In the years to come, a concentrated effort to delineate the complex interactions between the various components of the retinoid signal transduction system should shed light on the mechanisms underlying pattern formation during vertebrate development and point to new ways in which retinoids can be exploited therapeutically.


Development of antiviral treatment strategies in murine models. Murine models with type C murine leukemia viruses have been used to develop major new prophylactic and therapeutic strategies in vaccination, drug therapy of acute virus exposure and chronic viremia, combination therapy, prevention of maternal transmission, and therapy targeted to the central nervous system. Transgenic mice expressing either the whole human immunodeficiency virus type 1 (HIV-1) provirus or subgenomic sequences allow the in vivo analysis of selected HIV-1 functions. The full replicative cycle of HIV-1 can be studied in human/mouse chimerae which were created by transplanting human hematolymphoid cells into SCID mice. The chimeric SCID mouse models have been used successfully to evaluate anti-HIV-1 drugs. The role of the various murine retrovirus systems in the development of anti-HIV-1 and anti-AIDS therapies is summarized.


Effects of the stereochemical orientation of phenethylamines and imidazolines on alpha-adrenergic receptor-mediated DNA synthesis in primary cultured rat hepatocytes. The hepatic alpha 1-adrenergic receptor mediates a variety of hepatic functions including respiration, glycogenolysis, gluconeogenesis, and growth. We have utilized a rat primary hepatocyte culture system to show that the alpha 1-adrenergic receptor can be activated in a stereoselective manner by a series of phenethylamines and catecholimidazolines resulting in the stimulation of DNA synthesis as determined by [3H]thymidine incorporation. The phenethylamines adhered to the Easson-Stedman hypothesis with a rank order of potency of (-)-(R)-norepinephrine (NE) greater than (+)-(S)-NE greater than the desoxy analog dopamine (DA) for the stimulation of DNA synthesis. However, the 2-substituted catecholimidazolines did not follow this trend and demonstrated an order of potency of the desoxy analog 3,4-dihydroxybenzyl imidazoline (DHT) greater than or equal to (-)-(R)-2-(3,4,alpha-trihydroxybenzyl)imidazoline (TBI) greater than (+)-(S)-TBI. 4-Substituted catecholimidazolines were less potent as inducers of DNA synthesis than the corresponding 2-substituted analogs with an order of potency of (+)-(R)-4-(3,4-dihydroxybenzyl)imidazoline (DBI) greater than (+,-)-(R,S)-DBI greater than (-)-(S)-DBI. When the beta-hydroxyl moiety of NE is replaced with an amino group as in 3,4-dihydroxyphenylethylenediamine, the isomers are less active than the beta-hydroxylated analogs and also demonstrate no stereoselectivity for the stimulation of DNA synthesis. These results demonstrate that the hepatic alpha 1-adrenergic receptor can recognize various isomeric forms of these compounds and that hepatocellular growth can be modulated in a stereoselective manner by phenethylamines and imidazolines.


Retinoic acid and its rearranged receptor in the etiology and treatment of acute promyelocytic leukemia. All-trans retinoic acid can induce complete remissions in patients with acute promyelocytic leukemia. The balanced chromosomal translocation t(15;17)(q22;q12-21) of this malignancy is now known to involve the nuclear retinoic acid receptor-alpha (RAR-alpha) on the long arm of chromosome 17 and a novel gene on the long arm of chromosome 15, designated PML (previously called myl). A unique fusion mRNA, PML/RAR-alpha, is produced. Paradoxically, this rearrangement of RAR-alpha results in clinical sensitivity to retinoic acid. Despite its efficacy, retinoic acid therapy has side effect, including a syndrome of hyperleukocytosis and respiratory distress in some patients. Remissions induced and maintained by continuous all-trans-retinoic treatment are not durable in most of these patients. Retinoic acid therapy for acute promyelocytic leukemia represents a unique example where a molecular defect may be involved both in the pathogenesis and treatment of a malignancy.


The role of radiotherapy in patients with advanced seminomatous germ cell tumors. Controversies in management. Part 2. The role of radiotherapy in the management of advanced seminomatous germ cell tumors remains controversial. The authors review the available literature, and recommend radiotherapy for stage II seminoma with bulky abdominal metastases (greater than 10 cm) under the following circumstances: As primary therapy only if the patient cannot or will not be treated with chemotherapy; as an adjuvant following chemotherapy in patients with residual masses of any size. Similar recommendations are made regarding the role of radiotherapy in the management of extragonadal seminoma outside the central nervous system.


Insulin-like growth factor 1 receptors in human breast tumour: localisation and quantification by histo-autoradiographic analysis. To assess the precise role of IGF1 in benign and malignant breast diseases, we analysed the tissular localisation, characterised, and quantified specific insulin-like growth factor 1 (IGF1) binding sites in these heterogenous tissues, using histo-autoradiographic analysis (HAA). The 125I-IGF1 binding was performed on frozen tissue sections and analysed using 3H Ultrofilm autoradiography coupled to computerised image analysis. Competitive binding experiments using unlabelled IGF1, IGF2 and insulin showed that the tissues exhibited typical type I IGF binding sites. This specificity was confirmed by the use of alpha IR-3 monoclonal antibody, as inhibitor of 125I-IGF1 binding. IGF1 binding sites were detected in 18 human primary breast cancers, 12 benign breast tumours and two normal breast tissues. Using HAA we found that the human breast carcinomas studied exhibit a specific and high binding capacity for 125I-IGF1 exclusively localised on the proliferative epithelial component. The 125I-IGF1 binding activity of benign breast tumours or normal breast tissue was significantly lower than in cancerous tissues. There was a significant correlation between IGF1-R concentrations detected with HAA and those detected with a classical biochemical method. Moreover, HAA could be useful in further detailing whether a tumour is IGF1-R positive or negative HAA appears to be a useful method for the detection of growth factor receptors, specially in small biopsy specimens.


Molecular and biological properties of an abrin A chain immunotoxin designed for therapy of human small cell lung cancer. An immunotoxin (IT) comprising abrin A chain attached to the mouse monoclonal antibody SWA11, recognising a cell surface antigen highly associated with human small cell lung cancer (SCLC), was synthesised using a hindered disulphide crosslinker, N-succinimidyl 3-(2-pyridyldithio) butyrate (SPDB), and purified by Blue Sepharose CL-6B affinity chromatography. The IT preparation contained monomeric conjugate, composed of one abrin A chain molecule linked to one SWA11 molecule, and was free from unconjugated A chain or antibody. The IT fully retained the cell-binding capacity of the antibody component and the ribosome-inactivating activity of the A chain. In cytotoxicity assays using the SW2 SCLC cell line in tissue culture, SWA11-SPDB-abrin A chain inhibited the incorporation of 3H-leucine by 50% at a concentration of 10 pM and by 99% at a concentration of 1 nM. The anti-tumour efficacy of the IT was tested in nude mice bearing established s.c. solid SW2 tumour xenografts. A single i.v. injection of SWA11-SPDB-abrin A chain at a non-toxic dose induced a significant 7 to 10 day growth delay that could not be matched by administration of equivalent doses of either unconjugated SWA11 or abrin A chain alone. The results of this study indicate that the antigen recognised by SWA11 is an effective target for therapy of SCLC with A chain ITs in vivo.


Cisplatin therapy in infants: short and long-term morbidity. The tolerance to and toxicity of cisplatin treatment was retrospectively studied in 30 infants. A total of 191 courses were given with a median of six per child and a median cumulative dose of 400 mg m-2. Electrolyte disturbances were noted in 15/23 infants (38:144 courses): hypomagnesaemia, which was dose related, in 10/23 (25/144 courses), hyponatraemia in 6/23 (7:144 courses), hypercalcaemia in 4/23 (6/144 courses), and hypocalcaemia in 3/23 (4/144 courses). Seizures occurred in two infants. Vomiting followed 31/191 courses and neutropenic febrile episodes 23/191 courses. Median survival is 6 years 1 month. Six children have died of progressive malignancy. Glomerular filtration rate was less than 80 ml min-1 per 1.73 m2 in 15/29 children, at or within a month of the end of treatment; of ten retested at follow-up, eight had increased to more than 80 ml min-1 per 1.73 m2 (P = 0.027). High-frequency hearing loss was observed in 10/28 children, but was only significant in five (four grade 2 and one grade 3). the long-term toxicity of cisplatin in infants, at this dose range and schedule, is no more severe than in older children.


The aetiology of cancer in the very young. Epidemiological studies of cancer in young children have implicated a number of environmental factors, which need to be studied in more detail, but it is probably fair to say that the main benefit of these studies has come from the negative findings, which have served to exclude (or at least place an upper limit on the role of) potential risk factors. Our inability to identify environmental causes could mean either that the environment does not substantially affect cancer incidence in young children, or that we are simply not looking in the right places. Most attention has naturally been focused on the known and suspected environmental carcinogens and mutagens. Based on the data summarised in this paper, one possibility is that the most important mutagen is endogenous: 5-methyl-cytosine. If so, factors increasing cancer risk could be those which increase the rate of spontaneous deamination, or impair the efficiency of the excision repair enzymes, or regulate the processes of CpG methylation and demethylation. These factors could still be classed as mutagens in their own right, since they would lead to an increase in uncorrected point mutations, but they would be distinctive in a number of ways. Firstly, since the role of methylation in bacteria is very different from that in eukaryotic cells the Ames mutagenicity assay could not be relied upon to detect methylation-mediated mutagens. Furthermore the risk may be highly age dependent, reflecting changes in methylation patterns with growth and cellular differentiation. Agents which disrupted the imprinting process in the testis would not be detectable by animal carcinogenicity tests unless specifically looked for.(ABSTRACT TRUNCATED AT 250 WORDS)


Human estrogen receptor regulation in a yeast model system and studies on receptor agonists and antagonists. An expression system that utilized yeast copper metallothionein promoter and ubiquitin fusion technology to express the human estrogen receptor gene in yeast is described. We have studied the biochemical and transcriptional regulatory properties of the human estrogen receptor. The biochemical properties of the yeast expressed receptors are identical to the receptors isolated from human tissue. Estradiol mediated activation of transcription by the receptor was studied by a reporter beta-galactosidase gene where expression was under the control of estrogen response elements. Using this expression system and a hyperpermeable yeast strain we have studied the effects of various antiestrogens on the regulation of estrogen receptor function. We demonstrate that tamoxifen and ICI 164,384 are capable of binding to the receptor but neither antiestrogen was able to block the estradiol mediated increase in transcription. In fact, both antiestrogens exerted weak agonist activity in this system.


Effects of 9-ene-tetrahydrocannabinol on uterine estrogenicity in the mouse. Several early (Phase I) and late (Phase II) estrogenic effects of 9-ene-tetrahydrocannabinol (THC) were examined in the adult mouse uterus. An injection of THC (2.5 or 10 mg/kg body wt) in ovariectomized mice neither stimulated uterine water imbibition or accumulation of [125I]bovine serum albumin (Phase I responses) at 6 h, nor antagonized these Phase I responses elicited by estradiol-17 beta (E2). With respect to Phase II responses, although single injections of THC (2.5, 5.0 and 10 mg/kg body wt) alone were ineffective in influencing uterine weight at 24 h or incorporation of [3H]thymidine at 18 h, this drug interfered with these responses elicited by E2 in a dose-dependent manner. In contrast, an injection of THC in progesterone (P4)-primed ovariectomized mice modestly enhanced (61%) uterine incorporation of [3H]thymidine. However, E2-stimulated uterine thymidine incorporation in P4-primed ovariectomized mice was antagonized by THC treatment. Effects of THC on blastocyst implantation were examined. Single or multiple injections of various doses of THC neither induced implantation in P4-primed delayed implanting mice, nor interfered with E2-induced implantation. Furthermore, daily injections of THC (10 mg/kg body wt) during the peri-implantation period had no apparent adverse effects on implantation, or on experimentally induced decidualization (deciduomata). The data suggest that THC is neither pro- nor antiestrogenic with respect to Phase I responses. However as regards Phase II responses, THC is modestly pro-estrogenic in the P4-treated uterus, but is anti-estrogenic in the presence of E2. These estrogen agonistic/antagonistic effects of THC on uterine Phase II responses do not adversely affect the process of implantation and decidualization.


Type II oestrogen binding site is associated with the major 4S oestrogen receptor isoform in breast tumours. Using high resolution isoelectric focusing we have been able to identify a low affinity/high capacity oestrogen binding protein, which exhibits an apparent pI of 7.0. Using this system it can be separated from the previously described high affinity oestrogen receptor (ER) isoforms which focus at pI 6.1, 6.3, 6.6 and 6.8. The pI 7.0 protein was detected in 30/30 breast tumours analysed and had the binding characteristics of the cytoplasmic Type II ER (Kd = 88 +/- 8 nM). The concentration of this protein was shown to be significantly correlated with the concentration of the pI 6.6 species, which represents the major 4S isoform. It is not related to any other isoform of ER, and is expressed independently of the progesterone receptor. The importance of this observed relationship with respect to ER function remains obscure, but it may provide new insights into the role of the Type II oestrogen binding site in breast cancer.


A potent bombesin receptor antagonist inhibits bombesin-stimulated growth of mouse colon cancer cells in vitro: absence of autocrine effects. Bombesin (BBS) exerts significant effects on the growth of a mouse colon cancer cell line (MC-26) in vitro. The presence of specific binding sites on MC-26 cells for gastrin-releasing peptide (GRP)/BBS-related peptides was recently reported by us. In the present study, we determined that the transcript size of the mRNA species that codes for GRP receptors is 9 kilobase pairs, which is similar to that reported for mouse Swiss 3T3 cells, using the complementary DNA probe for the GRP receptor gene from mouse Swiss 3T3 cells. We next examined the effects of potent GRP receptor antagonists, D-Phe6, bombesin(6-13)-propylamide (D-Phe6,BN(6-13)PA) and Leu13-psi-(CH2NH)Leu14-bombesin (LL-BBS), on BBS-stimulated growth of MC-26 cells in vitro. A possible autocrine role of GRP in the growth of MC-26 cells was also investigated. MC-26 cells were inoculated s.c. into male BALB/c mice, and tumors were harvested 21-28 days postinoculation. Both D-Phe6,BN(6-13)PA and LL-BBS significantly inhibited the binding of 125I-GRP to MC-26 tumor membranes in a dose-dependent manner, with 50% inhibitory concentrations of 4.5 +/- 0.52 nM and 87 +/- 6 nM, respectively. D-Phe6,BN(6-13)PA similarly inhibited the specific binding of 125I-GRP, cross-linked to a approximately 80 kilodalton binding protein on the MC-26 tumor membranes. In order to determine whether the BBS receptor antagonist, D-Phe6,BN(6-13)PA, functioned as an antagonist or an agonist of biological functions, we measured the bioefficacy of D-Phe6,BN(6-13)PA.(ABSTRACT TRUNCATED AT 250 WORDS)


Molecular conformation of ubiquitinated structures and the implications for regulatory function. The molecular conformation of ubiquitinated structures and the validity of the N-end rule were examined by simulating the molecular mechanics to ascertain the global energy-minimized structure. We examined the chemical linkage involved in attaching the ubiquitin carboxyl terminus to the N-terminus of three different x-hexapeptides, where x is the amino group of the acceptor peptide--either valine, arginine or glutamic acid--(x-K linkage) and to the epsilon-amino group of lysine of the acceptor hexapeptide x-glu1-his2-lys3-gly4-lys5-val6 (K-K linkage) through the formation of an isopeptide bond. Changes in conformation and molecular stability of the multi-ubiquitinated structures were determined by energy-minimization procedures using the SYBYL program developed by Tripos Associates. In the x-K linkage, the ubiquitin molecule is stretched in the beta-pleated sheets and beta-turns while the alpha-helices expand, as the molecule continues to unfold linearly. In the K-K linkage, the ubiquitin molecules have turned into a u-shaped, semi-circular alignment, contracting into a compact, folded structure.


Polyadenylic: polyuridylic acid-induced determinants of host resistance to cytomegalovirus and their potentiation by hyperthermia. The ability of spleen cells from poly A:poly U-treated mice to inhibit murine cytomegalovirus (MCMV) replication in confluent monolayer cells of secondary mouse embryo fibroblasts (MEFs) cultured at 37 and 40 degrees C was investigated. When spleen cells from BALB/c mice injected 48 h earlier with poly A:poly U were added to MEFs infected 2 h previously with MCMV, 37% less plaques were observed than in cultures containing control cells. Of interest, the poly A:poly U-induced antiviral activity at the elevated temperature (40 degrees C) resulted in a further drop to 61% in MCMV-induced plaques compared to those of the normothermic (37 degrees C) cultures. The antiviral function of spleen cells induced by poly A:poly U was evident in the supernatant fluid when cultured for 48 h at 37 degrees C. MCMV-induced plaques were reduced to 52 and 5% of controls in the plaque assays performed at 37 and 40 degrees C, respectively. Supernatant fluids generated at 40 degrees C, however, inhibited MCMV replication only when incubated at 40 degrees C. No direct inhibitory effect of the supernatant fluids on MCMV was evident; rather, inhibition was effected directly on the MEFs. The NK cell fraction of spleen cells from poly A:poly U-treated mice alone showed only a slight inhibitory effect at 40 degrees C. However, in the presence of the supernatant fluid from poly A:poly U-exposed spleen cells, the antiviral activity of NK cells was significantly increased both at 37 and 40 degrees C. The cellular source of the culture fluid showing poly A:poly U-induced antiviral activity appeared to be in the T-cell population. It was completely neutralized by monoclonal anti-IFN gamma antibody but not by anti-IFN beta, anti-IL4, anti-transforming growth factor, or anti-prostaglandin E2. In conclusion, these data document the ability of spleen cells from poly A:poly U-treated mice to inhibit MCMV replication and this activity is potentiated by hyperthermic conditions. The antiviral function of poly A:poly U-treated spleen cells appeared to be due mainly to the action of IFN gamma produced by T cells. The enhanced antiviral activity by hyperthermia appeared to be related to the action of IFN gamma rather than its production.


Binding of ethylenediamine to phosphatidylserine is inhibitory to Na+/K(+)-ATPase. Covalent linkage of ethylenediamine with the Na+/K(+)-ATPase complex from rabbit kidney outer medulla by the use of the water-soluble carbodiimide, N-ethyl,N'-(3-dimethylaminopropyl)carbodiimide, resulted in a 73% reaction with phosphatidylserine and only 27% with carboxylic groups in the proteic component of the enzyme. Condensation products from the reaction between phosphatidylserine and ethylenediamine, N-(O-phosphatidylseryl)ethylenediamine, N,N'-bis(O-phosphatidylseryl)ethylenediamine and its intermediary product O-phosphatidyl-[N,N'-bis(seryl)]ethylenediamine, were synthesised. Symmetrically substituted ethylenediamine was the most likely condensation product of ethylenediamine with endogenous phosphatidylserine. The synthesised lipids were incorporated in proteoliposomes containing Na+/K(+)-ATPase and only the addition of the phospholipid phosphatidylcholine. The ratio of phospholipid to protein was 52 (w/w). These proteoliposomes were perforated by the addition of 0.5% cholate and both the Na(+)-dependent phosphorylation level and its dependence on Na+, Mg2+ and ATP were measured. Phosphatidylcholine alone increased the half-maximal activation concentration for Na+ ([Na+]0.5) from 0.2 to 1-2 mM, for Mg2+ from 0.1 to 0.8 microM and for ATP from 0.02 to 0.3 microM. The Ki for K+ (in the absence of Na+) was unaffected: 12.8 microM vs. 12.5 microM in the non-reconstituted system. Replacing 10 mol% of phosphatidylcholine by phosphatidylethanolamine: or phosphatidylserine had no significant effect on [Na+]0.5: 1.1 and 0.7 mM, respectively. Replacing 5 mol% phosphatidylcholine by the bis(phosphatidylseryl) substituent of ethylenediamine further increased [Na+]0.5 to 13.7 mM, while half-maximal activation concentrations for Mg2+ and ATP were unaltered. The mono-phosphatidylseryl derivatives of ethylenediamine, each 5 mol%, also increased [Na+]0.5, but to a lesser extent (3.2-3.8 mM). In addition to their competitive effects, the phosphatidylseryl-substituted ethylenediamine compounds exerted a slowly-increasing non-competitive inhibition, not only in phosphorylation, but also in overall ATPase activity, which was reduced, although not abolished, by exogenous protein (bovine serum albumin). A detergent-like action in the usual sense is unlikely since liposomes containing these lipids remained intact. These studies prove that phospholipids are not only required for optimal activity of this transport enzyme, but in excess or in compositions deviating from the normal, may also be inhibitory.


A new surfactant series, the N-alkylamino-1-deoxylactitols: application for extraction of 'op' opiate receptors from frog brain. A new series of surfactants, the N-alkylamino-1-deoxylactitols, was prepared and employed to extract 'op' opiate receptors from frog brain. These surfactants are both cheap and convenient to prepare. Receptors were reproducibly extracted in a good yield using N-nonylamino-1-deoxylactitol. This derivative, which was not denaturing during the extraction process, could thus be used instead of the more costly digitonin, whose rather variable purity affects yield.