Effect of foil material on the apparent yield of the 124Xe(p, x)123I reaction. The effect of the foil material on the recovery of Xe-123 from the irradiation cell used for the measurement of the production of I-123 from the proton reaction on xenon-124 has been studied. The metal used for the foil can have a significant effect on the amount of Xe-123 which can be cryogenically pumped out of the irradiation cell at 30 min after the end of irradiation. Foils made from several metals and alloys were tested. Of these, the effect is most pronounced with "Havar" alloy foil where only 58% of the theoretically available activity was recovered. This effect should be considered and tested in the measurement of reaction cross-sections in irradiation cells and targets which have metal foils.


Large scale preparation strategy for labelling of [123I]-SCH 23982, a dopamine D1 receptor binding agent. The labelling of the D1 antagonist SCH 23982 with 123I was studied in detail by following the nucleophilic and electrophilic approaches and the reaction conditions were optimized. The product was purified by reversed phase HPLC with a phosphoric acid/EtOH mixture which simply has to be neutralized and diluted before injection. Its binding was tested in vitro with rat striatal membranes proving the high affinity to D1 and very low affinity to D2 receptors.


Evaluation of the Lawrence Livermore National Laboratory (LLNL) torso phantom by bone densitometry and x-ray. The recent Workshop on Standard Phantoms recommended that the LLNL torso phantom be adopted as a calibration standard for the quantitation of in vivo radioactivity. This phantom was designed for the calibration of systems for the detection of x-rays of less than 20 keV. The anthropomorphic characteristics and tissue substitute composition of the phantom were assessed with techniques using photons of higher energy. Dual photon absorptometry at 42 and 100 keV showed that the phantom was representative of in vivo tissue composition. Chest radiography showed that the phantom was representative of a human even though the stomach, GI tract and the scapulae were not present and air gaps were observed at organ boundaries.


Calibration of bone mineral and heavy metal measurements using doped wall-less gel phantoms of arbitrary form. Cylindrical gel phantoms of 2% agarose concentration were used as wall-less phantoms of arbitrary shape for calibration purposes in non-destructive measurements of bone mineral and heavy metal concentration using XRF. The counts of the coherently and incoherently scattered photons with no sample present should be subtracted as "background" to get a linear relationship between the ratio of the coherently and incoherently scattered photons and the bone mineral concentration for phantoms of arbitrary diameters.


A new, semi-automated system for the micro-scale synthesis of [195mPt]cisplatin suitable for clinical studies. A new, semi-automated system for the microscale synthesis of [195mPt]cisplatin has been developed. Radiochemical yields of up to 70% of pure [195mPt]cisplatin can be obtained routinely e.g. 555-629 MBq (15-17 mCi) from 925 MBq (25 mCi) of [195mPt]Pt metal. Chemical losses during synthesis, and radiation exposure of personnel, have been minimized. These increased yields are the consequence of the elimination of most transfers, and enhanced yields at most steps: ammination losses decreased from 27 to 7%, and at the diiodo- to di-acquo- conversion, from 34 to 11%. The versatility of such a system is discussed.


Simultaneous spectrophotometric estimation of thorium and calcium in urine. A simple and rapid method for the simultaneous estimation of thorium and calcium in urine was developed. Thorium and calcium were coprecipitated as oxalates at pH 3-4 and were determined using Thoronol as reagent. A multi-wave-length linear regression analysis procedure was applied. Thorium recovery was found to be 78.3% +/- 5.1% (1 sigma) and that of calcium was 93.7 +/- 3.0% (1 sigma). The minimum detection limit for thorium was found to be 2.54 mBq/dm3. The analytical results of calcium agree very well with those determined by EDTA titrimetry.


Is there a place for radiation therapy in the management of hepatoblastomas and hepatocellular carcinomas in children? From May 1978 to August 1988, 15 children with a primary malignant liver tumor received radiation therapy as part of their management at the Institut Gustave-Roussy. Age ranged from 4 months to 13 years. The male to female ratio was 1.5. Eleven patients had a histologically proven hepatoblastoma, two a hepatocellular carcinoma, and histology was not documented in two. Resection of the primary liver tumor was performed in nine cases, and all patients also received sequential chemotherapy, generally preoperative and alternating vincristine, doxorubicin, cyclophosphamide with vincristine, cyclophosphamide, and cis-platinum. Radiotherapy was performed postoperatively in eight incompletely resected patients. Six of eight are alive and free of disease 4-83 months following treatment (median 39 months) and 11-98 months since diagnosis (median 45 months). All but one were treated to limited fields to a total dose of 25-45 Gy (median 40 Gy). One patient became resectable by a combination of 24 Gy to the whole liver and concomitant 5FU and Cis-Platinum and remains with no evidence of disease 68 months following radiation therapy. Of four unresectable primaries, only one was controlled by radiotherapy. Neither of two children with pulmonary metastases were controlled by whole lung irradiation to a dose of 18 and 20 Gy, respectively, and one still remains stable 41 months after resection of a residual metastatic nodule. Neither of two hepatocellular carcinomas were controlled by doses up to 40 Gy. This small series suggests that in hepatoblastoma, radiotherapy to a total of 25-45 Gy fractionated doses, combined with chemotherapy, can play a role in selected inoperable children and also in those with minimal postoperative residues below 2 cm. It also indicates that in hepatocellular carcinoma, radiotherapy is ineffective in this dose-range.


Induction chemotherapy for non-small cell lung cancer with clinically evident mediastinal node metastases: the role of postoperative radiotherapy. Survival for clinical Stage IIIa (T1-3, N2) non-small cell lung cancer is very poor because of poor local disease control and systemic spread. To address these shortcomings, we initiated a treatment program with induction chemotherapy, surgery, and postoperative radiation reserved for patients with residual disease at thoracotomy. Between 1984 and 1986, 41 patients with clinically evident N2 disease were treated with induction chemotherapy followed by resection and the selective use of intraoperative brachytherapy. All patients with tumor in the resection specimen received two cycles of chemotherapy and 15 patients received radiation therapy. With a median follow-up of 5.4 years, overall survival is 27% at 3 years, and 12% at 5 years. Despite the adverse selection process median survival is 19 months for patients receiving postoperative radiation therapy, and 22 months for the more favorable patients not requiring radiation therapy, supporting the selective use of postoperative radiation in this setting. In summary, this treatment has yielded good median survival and long-term survival for some of the patients. However, the ultimate value of this approach can only be determined by prospective trials which compare it to standard therapy.


Postoperative radiotherapy for malignant fibrous histiocytoma. Between 1974 and 1989, 49 patients with histologically confirmed malignant fibrous histiocytoma received postoperative radiotherapy at the Mallinckrodt Institute of Radiology for primary (41) or recurrent (8) disease. Median age of the patients was 63 years, and the median follow-up period was 41 months. Patients were grouped according to the 1988 AJC staging classification: stage IA (one patient), stage IIA (4 patients), stage IIB (9 patients), stage IIIA (15 patients), stage IIIB (18 patients), and stage IVA (2 patients). Eight tumors (16%) were in the pelvis, 8 (16%) in the trunk, 4 (8%) in the head and neck, and 29 (60%) in the extremities. Primary surgical procedures included incisional biopsy (4 patients), excisional biopsy (19), narrow margin excision (14), wide local excision (9), and removal of the entire compartment (3). Based on pathology reports, the margins of resection were classified as positive in 23 (5 gross, 18 microscopic), 5 close, 11 negative, and 10 unknown. Patients were irradiated with shrinking field technique; the median radiation dose was 6000 cGy, with more than 95% of patients receiving at least 4500 cGy. In addition, seven patients received postoperative chemotherapy. The 5-year overall survival rate was 62%, disease-free survival 64%, local control 68%, and freedom-from-distant metastasis 85%. Thirteen patients had local recurrences, with greater than 75% recurring within 3 years. Sites of local recurrence were as follows: trunk (3), pelvis (3), lower extremities (4), and head and neck (3). There appears to be a correlation of local failure with positive surgical margin: of 23 patients with positive margins, 9 (39%) had local recurrences, whereas 1 of 11 patients (9%) with negative margins had local recurrence. Three of 13 patients with persistent or recurrent disease were salvaged by additional treatment, rendering ultimate local control in 80% (39/49). Thirty-four of 36 patients with local control obtained good to excellent function. Two patients were found to have grade 3 complications: 1 patient had edema of the extremity, and the other developed necrotic skin ulcer that was successfully treated with hyperbaric oxygen. Five patients developed distant metastases, with 80% occurring within 2 years. In summary, adequate but conservative surgery with postoperative radiotherapy for malignant fibrous histiocytoma can achieve local tumor control as well as preservation of functional limbs with acceptable morbidity in a large proportion of patients.


Pharmacology of benzodiazepine hypnotics. The "revolution" in pharmacologic treatment of insomnia began in 1970 with the availability of flurazepam, the first of the benzodiazepine hypnotics. Flurazepam largely replaced all other hypnotics during the decade of the 1970s. The second revolution began in the early 1980s as shorter half-life hypnotics, triazolam and temazepam, became available and began to replace flurazepam. The decade of the 1990s will probably see a more balanced pattern of benzodiazepine hypnotic use, as well as use of newer nonbenzodiazepine hypnotics. Among available benzodiazepines, all have the capacity to produce dose- and concentration-dependent sedation, drowsiness, performance impairment, and amnesia. Benzodiazepine-induced amnestic effects are characterized by either impairment of information acquisition, impairment of consolidation and storage, or both. In general, apparent clinical differences among benzodiazepine hypnotics are explained by differences in pharmacokinetic properties of absorption, distribution, elimination, and clearance.


Atypical peripheral neuropathies.

Atypical peripheral neuropathies. Systemic disease and metabolic imbalance may be associated with peripheral nerve focal compressive neuropathies. The nerve may be primarily involved, or symptoms may result from chronic tenosynovitis or synovitis. We review the more commonly occurring compressive neuropathies associated with underlying systemic disorders.


Organization of the zona incerta in the macaque: a Nissl and Golgi study. The zona incerta has been implicated in the control of the initiation of saccadic eye movements in the primate. Complex interactions within the zona incerta must take place to integrate its varied inputs and to produce a coherent efferent signal in order for this function to occur. However, whether the anatomical substrates exist within the zona incerta to allow this integration to take place has not been established. The zona incerta in monkeys (Macaca mulatta) was examined in frontally, horizontally, and sagittally sectioned preparations stained for Nissl, myelinated fibers, or cytochrome oxidase, or impregnated by the Golgi technique. This nucleus can be separated into dorsal and ventral laminae on the basis of staining and morphological differences between these two subdivisions. Neurons are more densely packed, more darkly stained, and larger in the ventral lamina. In addition, the neuropil of the ventral lamina is much more intensely stained after cytochrome oxidase histochemistry. Two neuronal types, principal cells and interneurons, were identified on the basis of neuronal cell body, dendritic, and axonal features in Golgi-impregnated preparations. Principal cells have fusiform or polygonal somata (long axis from 18 to 40 microns) and dendrites that extend for up to 750 microns within the lamina in which the cell bodies are located. Putative local interneurons have small (12-16 microns), round or oval cell bodies with wavy dendrites (up to 400 microns). Numerous multilobed appendages and axon-like processes originate from these dendrites and make apparent contacts with other interneurons or with dendrites of principal cells. Dendrites of most neurons in both laminae are oriented preferentially along the principal axis, dorsolateral-to-ventromedial, of the nucleus. Therefore, within the limits of light microscopy, the zona incerta appears to possess the morphological heterogeneity to form complex intrinsic interactions. These interactions are hypothesized to form the integrative substrate for the large array of incertal inputs that are utilized to produce an efferent signal involved in the initiation of saccadic eye movements.


Human papillomavirus type 11 in multiple squamous cell carcinomas in a patient with subacute cutaneous lupus erythematosus. Squamous cell carcinoma (SCC) is a rare complication of cutaneous lupus erythematosus (LE). Aside from the documented role of ultraviolet light, SCC may arise in areas of chronic scarring, such as in lesions of discoid LE. Iatrogenic immunosuppression associated with organ transplantation also results in a predisposition to SCC, often with multiple tumors. A role for certain human papillomaviruses (HPV) in the development of squamous cell carcinoma has been documented; specifically, HPV types 5 and 8 are detected in SCCs in patients with epidermodysplasia verruciformis and in recipients of organ transplants. HPV-11 is generally found in benign genital condylomata or laryngeal papillomas, but has not yet been associated with malignancy. We describe a patient with non-scarring cutaneous LE who was treated with azathioprine and prednisone and developed multiple SCCs. HPV-11 DNA and the target oncogenes neu and Ki-ras were detected in tumor tissue with the polymerase chain reaction. The HPV may have been involved in tumor induction and the azathioprine may have been involved in tumor promotion.


Effects of deuterium oxide and temperature on heart rate in Drosophila melanogaster. A non-intrusive optical technique has been developed to monitor heartbeat in late third-instar Drosophila larvae. Heartbeat in this insect is an oscillation that is not temperature compensated. Deuterium oxide lengthens the period of a number of high and low frequency oscillators and clocks in a variety of organisms. To determine whether deuterium affects heart rate, flies were raised on proteated and deuterated media and their heartbeat was monitored at four temperatures ranging from 18 to 33 degrees C. The rate of heartbeat increased linearly with increasing temperature, and decreased with increasing concentrations of deuterium. There was a significant interaction between temperature and deuterium: the higher the concentration of deuterium oxide the less temperature-sensitive was the heart rate. Raising temperatures also increased the amount of "noise" in the rhythm: signal-to-noise ratio, which characterizes the amount of power in a rhythmic signal, decreased with increasing temperatures. Deuterium oxide had no effect on signal-to-noise ratio.


Antibacterial and antiviral activity of camel milk protective proteins. Lysozyme (LZ), lactoferrin (LF), lactoperoxidase (LP), immunoglobulin G and secretory immunoglobulin A were extracted from camel milk. The activity of these protective proteins was assayed against Lactococcus lactis subsp. cremoris, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and rotavirus. Comparative activities of egg white LZ, bovine LZ and bovine LF are also presented. The antibacterial activity spectrum of camel milk LZ was similar to that of egg white LZ, and differed from bovine milk LZ. Bovine and camel milk LF antibacterial activity spectra were similar. The camel milk LP was bacteriostatic against the Gram-positive strains and was bactericidal against Gram-negative cultures. The immunoglobulins had little effect against the bacteria but high titres of antibodies against rotavirus were found in camel milk. The LP system was ineffective against rotavirus.


The role of albumin in developing rodent dental enamel: a possible explanation for white spot hypoplasia. The uptake of serum albumin by maturation-stage rodent enamel and the resulting effects on the growth of enamel crystallites were investigated in vitro. Albumin uptake was demonstrated by means of gel electrophoresis and confirmed by Western blotting with use of monoclonal antibodies. Measurement of crystal size was carried out by direct TEM measurement of enamel crystallite outlines after incubations in metastable solutions of calcium phosphate. The ability of endogenous enamel enzymes to degrade albumin was investigated by substrate-specific zymography. The results showed that albumin could be taken up by maturation-stage enamel and produce inhibition of crystallite growth. There was no detectable proteolytic activity in the enamel against albumin substrate, which suggests that albumin entering enamel by extravasation in vivo may produce incomplete tissue maturation, resulting in a white, opaque appearance on eruption.


Glomus tumor imaging: use of MRI for localization of occult lesions. Magnetic resonance imaging has been performed in six patients with glomus tumors of the hand and correlated with clinical surgical histopathologic findings and with angiography in one case. Two of the patients had obscure pain without the classic clinical and radiologic findings of glomus tumors. The MRI examinations depicted the tumors in excellent detail and facilitated diagnosis in the two patients with atypical presentation. In all six cases the diagnoses were confirmed at surgery. The MRI proved valuable as a noninvasive and accurate means for the early diagnosis of occult glomus tumors.


Keloid formation in a simple syndactyly release: a case report. Keloid formation on the palms of the hands and soles of the feet is rare. A keloid developed 2 months after release of simple syndactyly in a young black child. The process of keloid formation is not completely understood, and surgical correction is not effective.


Evaluation and management of upper extremity neuropathies in Charcot-Marie-Tooth disease. The evaluation and treatment of five patients with upper extremity neuropathies secondary to Charcot-Marie-Tooth disease were reviewed with emphasis on age at onset of Charcot-Marie-Tooth disease and upper extremity deformities, clinical findings, signs of associated nerve compression, and outcome of surgical treatment. The onset of the disease generally occurred in the first or second decade of life. The onset of upper extremity symptoms lagged behind by an average of 8 years. All patients had intrinsic minus hands with decreased sensibility. Three of five patients had clinical or electrophysiologic evidence of associated nerve compression syndromes. Treatment with standard tendon transfers, nerve compression releases, soft tissue releases, and joint fusions resulted in subjectively improved function in three of four patients undergoing reconstruction. Release of six compression neuropathies in one patient provided excellent pain relief, but the underlying neuropathy progressed. Pessimism regarding reconstructive surgery in the patient with upper extremity neuropathies secondary to Charcot-Marie-Tooth disease is unwarranted.


Dinitrophenyl (DNP) hapten sandwich staining (DHSS) procedure. A 10 year review of its principle reagents and applications. Over the past 10 years an immunoperoxidase method using dinitrophenyl (DNP) hapten-labelled primary or secondary probes has been devised. Its widely successful application in research and diagnostic work has depended upon the development of certain key reagents. These include a novel non-deleterious DNP labelling compound, a unique multivalent monoclonal bridge antibody, and an efficient DNP hapten substituted or anti-DNP linked marker enzyme. In this article the development of these reagents and various modifications of the basic technique are reviewed in conjunction with the special applications accruing from their use.


A multiple luminescent procedure for the detection of different papillomaviruses on dot blots. We developed a method based on the use of various luminescent systems for identification of several nucleic acid sequences on the same dot blots. In a simultaneous assay, the target DNA molecules were hybridized with different DNA probes. These probes were labelled with biotin or digoxigenin or directly coupled to enzymes such as glucose-6-phosphate dehydrogenase, peroxidase or alkaline phosphatase. After hybridization, these labels were detected by luminescent reactions using an amplified camera. Rapid detection and specific identification of pathogenic agents such as human papillomaviruses (HPV) could be performed in a single step by this procedure. Polymerase chain reaction was carried out using general primers and virus types were identified on dot blots using short HPV 11, 16 and 18 specific oligonucleotides.


The role of pathology in pelvic ileal reservoir surgery. The increasing use and success of pelvic ileal reservoir surgery places an ever more demanding role onto the surgical pathologist. Close surgical and pathological cooperation is critical for preoperative patient selection, for intraoperative diagnostic confirmation and for postoperative follow up. The long term consequences of pouch construction are, as yet, unknown, but there is much evidence to substantiate the need for life-long surveillance by clinical, endoscopic and pathological means to maximise the benefits of the pelvic ileal reservoir to the patient. This paper reviews the pathological input into these aspects of pelvic ileal reservoir surgery together with a critical appraisal of the likely long term consequences of pelvic ileal reservoir construction.


Collagenolytic activity in experimental intestinal anastomoses. Differences between small and large bowel and evidence for the presence of collagenase. Collagen degradation is thought to be an integral part of the healing sequence of intestinal anastomoses, but almost nothing is known about the enzyme activities involved. We have studied collagenolytic activities, extracted from 1 day-old intestinal anastomoses in the rat. Using either soluble type I collagen or fibrillar type I or type III collagen as a substrate, activities measured in extracts from anastomotic segments were compared to those in extracts from uninjured intestine, removed at operation: in all cases, the collagenolytic activity in anastomotic extracts was significantly higher. This increase was significantly more pronounced in large bowel than in small bowel. The activities were strongly inhibited by serum and metallo-chelating compounds. Analysis, by means of SDS-polyacrylamide gel electrophoresis, of the reaction products of the degradation of fibrillar type I collagen by the extracts revealed the presence of a multitude of fragments, amongst them TcA fragments characteristic for the activity of mammalian collagenase. Thus, the degradative capacity towards various collagen substrates is enhanced in the anastomotic area during the first postoperative period and a true mammalian collagenase is one of the enzymes present.


Detection of occult nasopharyngeal primary tumours by means of in situ hybridization. Detection of nasopharyngeal carcinoma primaries in patients presenting with neck node metastases may sometimes demand considerable efforts. By using the 'in situ hybridization' technique, we manage to identify the Epstein-Barr virus in neck metastases secondary to nasopharyngeal carcinomas. We propose that such identification in neck node metastases where the primary lesion is unknown indicates a nasopharyngeal primary.


Superoxide generation by the human polymorphonuclear leukocyte in response to latex beads. In this study, superoxide formation was not immediately detected when polymorphonuclear leukocytes were treated with linoleyl alcohol-coated, carboxy-modified latex beads. However, all other measures of neutrophil activation were present. Superoxide was not detected until 30 min after the initial exposure to beads. However, an O2(-)-producing, NADPH-dependent oxidase is active 15 min after exposure. When polymorphonuclear leukocytes are pretreated with cytochalasin B, superoxide production is detected immediately after exposure to the beads. Superoxide secretion after treatment with linoleyl alcohol-coated latex beads is compared with the response to other latex beads. The results imply that neutrophils form a phagolysosome around linoleyl alcohol-coated latex beads that is tightly sealed and does not allow superoxide to escape into the medium where it could be detected by the reduction of ferricytochrome c.


Heterogeneity in the circulating neutrophil pool: studies on subpopulations separated by continuous flow electrophoresis. Isolated blood neutrophils from healthy individuals have been separated by continuous flow electrophoresis (CFE) as a Gaussian-shaped profile extending over 12-15 fractions, on the basis of differences in cell surface electrical charge. The fractions were pooled into three or four subpopulations; the mean electrophoretic mobilities of the least and most electronegative cells were 0.96 and 1.22 microns/sec/V/cm, respectively. Each pool of neutrophils was analyzed for functional and biochemical differences. Expression of respiratory burst in terms of the rate of superoxide production by the least and most electronegative cells to fixed concentrations of N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10(-7) M) or phorbol myristate acetate (PMA, 1 microgram/ml) revealed that the least electronegative cells generated superoxide anion (O2-) at approximately twice the rate of the most electronegative cells. However, when lower concentrations of fMLP were used (1-5 x 10(-9) M), the most electronegative cells were most active. The least electronegative cells were also the most active in terms of phagocytosis and chemotaxis. In accordance with these differences in motile function, the basal F-actin content of the least electronegative cell pool was greater than the F-actin levels found in the most electronegative cells and remained so upon stimulation with fMLP. Such neutrophil heterogeneity as detected by CFE may be important in selective margination and recruitment of cells to inflammatory foci and sites of infection and may in part represent subsets of cells within the circulation that are primed in vivo to respond to inflammatory stimuli.


[The new heparins].

[The new heparins]. Low molecular weight heparins are increasingly prescribed in France. Prepared from standard heparin by depolymerisation, they show a markedly decreased anti IIa activity and a anti Xa/anti IIa ratio ranging from 2 to 4. Their mode of action in the coagulation system is still not well known and it is difficult to explain the mechanism of their antithrombotic effect, demonstrated in vivo. They seem to inhibit the first traces of thrombin and then counteract the priming and amplification of coagulation. Their fibrinolytic activity is also a disputed question, but seems to be lower than that of standard heparin. The pharmacological studies show a venous as well as arterial antithrombotic activity of a low molecular weight heparin on several animal models, a lower but not negligible bleeding risk as compared to unfractionated heparin. Furthermore heparin fragments have a weak interaction with platelets, which allow to foresee a greater efficacy of LMWH than standard heparin in arterial thrombosis. Some very rare cases of thrombocytopenia in patients treated with LMW heparins have been recently reported. The compared pharmacokinetics of heparins gave proof of a renal elimination of low molecular weight heparin and a bio availability of about 90% after subcutaneous injection. Many clinical studies allowed to define indications of heparin fragments in prophylactic treatment after surgery as well as in medical patients and in curative treatment in case of deep vein thrombosis. However, others studies must be carried out to define the real efficacy of such a treatment during pulmonary embolism, disseminated intravascular coagulation and myocardial infraction, or during thrombotic complications after vascular surgery.


Glucocorticoid inhibition of stimulus-evoked adrenocorticotrophin release caused by suppression of intracellular calcium signals. Stress provokes a cohort of homeostatic reflexes by the central nervous, the immune as well as the metabolic control systems of the body. These powerful adaptive responses, which can cause a collapse of body homeostasis in the absence of feedback inhibition, are suppressed by adrenal glucocorticoid hormones. A prominent and physiologically significant early action of glucocorticoids that requires the induction of newly synthesized messenger RNA and protein is the suppression of ACTH release by anterior pituitary corticotroph cells. It is demonstrated here that glucocorticoids inhibit stimulated ACTH secretion in pituitary corticotroph tumour (AtT-20) cells by reducing stimulus-evoked intracellular free calcium transients. Thus, the data show for the first time that intracellular calcium signals may be modified by rapidly induced proteins. It is proposed that this is a general mechanism that underlies the early inhibitory effects of glucocorticoids during stress in various types of cell.


Lipopolysaccharide is able to bypass corticotrophin-releasing factor in affecting plasma ACTH and corticosterone levels: evidence from rats with lesions of the paraventricular nucleus. Stimulation of the immune system or experimental conditions (bacterial lipopolysaccharide (LPS) treatment) provoke a broad spectrum of physiological responses. It was recently shown that one of them is the activation of the hypothalamic-pituitary-adrenal (HPA) axis. The mechanism and the site or sites through which LPS stimulates the HPA axis are not well understood. To establish whether the effect of bacterial LPS is related in vivo to the presence of hypothalamic hypophysiotrophic peptides (corticotrophin-releasing factor-41, arginine vasopressin, etc.), plasma ACTH and corticosterone levels were monitored in intact and sham-operated rats, and in rats with paraventricular nucleus lesions in order to remove the main source of these neuropeptides. Evidence was obtained that 4 h after treatment, LPS was able to activate the hypophysial-adrenal system in the absence of hypophysiotrophic neuropeptides of paraventricular origin. It is suggested that, in vivo, LPS could have a direct effect on the pituitary gland or that it acts through an extrapituitary, non-paraventricular pathway to activate the HPA axis.


The role of calcitonin gene-related peptide in the regulation of anion secretion by the rat and human epididymis. A study was carried out to investigate the role of the calcitonin gene-related peptide (CGRP) in the regulation of electrolyte transport in the rat and human epididymis. In monolayer cultures derived from the rat cauda epididymal cells, CGRP stimulated the short-circuit current (SCC) in a dose-dependent manner with the EC50 (concentration required to produce 50% of the response) at 15 nmol/l. This effect of CGRP was seen when the peptide was added to the basolateral aspect of the cells; apical addition having negligible effect. The CGRP-induced rise in the SCC was dependent on the presence of chloride in the bathing solution. Calcitonin had no effect on the SCC and did not affect the CGRP-induced rise in the SCC. The effect of CGRP on secretion was inhibited in a competitive fashion by the CGRP receptor antagonist CGRP(8-37). In contrast to bradykinin, angiotensin II and endothelin I, the effect of CGRP was independent of prostaglandin synthesis. Measurement of intracellular adenosine 3':5'-cyclic monophosphate showed a time- and dose-dependent increase upon stimulation with CGRP. CGRP also stimulated the SCC in monolayers grown from the human epididymis. The current could be inhibited by apical application of the chloride channel blocker, diphenylamine-2-carboxylate. Immunoreactive CGRP was found in the epithelia of rat and human cauda epididymidis. It is suggested that CGRP may regulate the electrolyte and fluid secretion in the epididymis, thereby providing an optimal microenvironment for the maturation and storage of spermatozoa.


Phosphate transport by fibroblasts from patients with hypophosphataemic vitamin D-resistant rickets. It is accepted that renal phosphate wasting is the basis of hypophosphataemia in vitamin D-resistant hypophosphataemic rickets (VDRR). Abnormal renal adaptation to phosphate deprivation has also been reported in these patients. We studied sodium-dependent phosphate transport and its modulation by phosphate deprivation in skin fibroblasts cultured from healthy subjects and patients with VDRR. Control fibroblasts exhibited high-affinity sodium-dependent phosphate transport (77 +/- 12 mumol/l) which resembled the ubiquitous transport of renal and non-renal cells. Phosphate deprivation (incubation in low phosphate medium) increased the maximal velocity (Vmax) of the transport by 2.7-fold after 24 h, with no change in the affinity. The increase in Vmax was dependent on gene transcription and protein synthesis. The sodium-dependent phosphate transport exhibited in fibroblasts from VDRR patients did not significantly differ from that of control subjects, except that the Vmax of the phosphate transport was higher in cells from patients with VDRR under normal and phosphate-deprivation conditions, although the difference was significant only after 24 h of phosphate deprivation (Vmax: 22.6 +/- 2.4 pmol/mg protein per s in VDRR vs 16 +/- 3.6 pmol/mg protein per s in controls, P less than 0.05). These data demonstrate that sodium-coupled phosphate transport in human skin fibroblasts has the properties of ubiquitous sodium-phosphate co-transport and show that this transport is not deficient in patients with VDRR. Indeed paradoxically the Vmax was 40% higher in VDRR than in control subjects after 24 h of phosphate deprivation. The transport must be either different from that of kidney cells responsible for the phosphate leak, or differently modulated.(ABSTRACT TRUNCATED AT 250 WORDS)


Alpha-melanocyte-stimulating hormone stimulates protein kinase C activity in murine B16 melanoma. The effect of alpha-melanocyte-stimulating hormone (alpha-MSH) on protein kinase C activity and distribution was investigated in murine B16 F1 melanoma cells. alpha-MSH was found to induce an increased association of protein kinase C (PKC) activity with the particulate fraction of the cells, with an associated loss of enzyme activity from the soluble fraction. The peak response to alpha-MSH occurred between 20 and 60 min of incubation time, and enzyme activities redistributed to those seen in the control cells over the following 12 to 24 h. The average response to alpha-MSH (1 nmol/l) was an approximate 2.5-fold increase in the percentage of enzyme activity associated with the membrane within 1 h of exposure to alpha-MSH; the particulate enzyme activity represented 19.2 +/- 4.4% of total activity in the absence of alpha-MSH and 50.7 +/- 4.7% (means +/- S.E.M., n = 9, P less than 0.005) in the presence of alpha-MSH (1 nmol/l). Cells which had a relatively small percentage of their PKC activity on the membrane initially were significantly (P less than 0.01) more responsive to alpha-MSH stimulation than cells which initially had a relatively large percentage of PKC activity on the membrane. The association of PKC activity with the membrane showed some evidence of being dose-related to alpha-MSH. This is the first report, to the best of our knowledge, of alpha-MSH activating PKC.


The effects of recombinant human interleukin (IL)-1 alpha, IL-1 beta or IL-6 on hypothalamo-pituitary-adrenal axis activation. We have investigated the effects of recombinant human interleukin (IL)-1 alpha, IL-1 beta and IL-6 on the activation of the hypothalamo-pituitary-adrenal axis. We have determined the effects of a single i.p. injection of cytokine on circulating ACTH and corticosterone levels, corticotrophin-releasing factor (CRF) mRNA in the parvocellular cells of the paraventricular nucleus and pro-opiomelanocortin (POMC) mRNA in the anterior pituitary at both 4 h and 24 h after injection. IL-1 alpha had no effect on any of the parameters measured at either time-point. In contrast, IL-1 beta increased CRF mRNA in the parvocellular paraventricular nucleus and POMC mRNA in the anterior pituitary 4 h after injection. Plasma ACTH and corticosterone were increased at 4 h and circulating ACTH was still increased at 24 h after treatment with IL-1 beta. IL-6 had no effect on message levels but did increase circulating ACTH and corticosterone levels both 4 h and 24 h after injection. The mechanism responsible for the increase in circulating ACTH after IL-6 injection is unclear but would appear to be different from that which is activated by IL-1 beta which also results in increased CRF and POMC gene expression.


Release of interleukin-6 by human thyroid epithelial cells immortalized by simian virus 40 DNA transfection. Interleukin-6 (IL-6) has actions on a variety of endocrine tissues. The cytokine is secreted by cells of the anterior pituitary and endocrine pancreas and has recently been shown to be produced by cultures of thyroid epithelial cells. In this study we have examined some of the factors which regulate IL-6 release from an immortalized human thyroid line (HTori3). IL-6 release over 24 h was stimulated by TSH (5000 microU/ml), by forskolin (0.01 mmol/l), by fetal calf serum (1-20%) and by epidermal growth factor (20 ng/ml). Stimulation was also apparent with gamma-interferon and with tumour necrosis factor at concentrations known to enhance class II major histocompatibility antigen expression by thyroid epithelium. The most potent factor tested was interleukin-1 (IL-1), which controls IL-6 release from other cell types. Threefold stimulation was found with 1 U/ml rising to 350-fold with 1000 U/ml. The effect of IL-1 took 2 h to develop and was blocked by cycloheximide (100 mumol/l). Stimulation was not markedly inhibited by pertussis toxin. Many of the actions of IL-1 are mediated by prostaglandin E2 (PGE2). At concentrations as low as 30 nmol/l, PGE2 stimulated IL-6 release but the maximum stimulation obtained with PGE2 was only threefold. The effect of IL-1 was not inhibited by indomethacin. These data provide further evidence that IL-6 is produced by human thyrocytes. The effect of IL-1 has not been demonstrated previously. Stimulation of IL-6 release by IL-1 did not appear to be mediated by prostaglandin.(ABSTRACT TRUNCATED AT 250 WORDS)


A comparison of the clearance of ovine and human corticotrophin-releasing hormone (CRH) in man and sheep: a possible role for CRH-binding protein. A specific binding protein for human corticotrophin-releasing hormone (hCRH), which does not bind to the ovine hormone (oCRH), has recently been demonstrated in human plasma. No such binding protein has been found in sheep plasma. We have investigated the half-life of human and ovine CRH in man and in sheep. Peptides were measured directly in plasma with two-site immunoradiometric assays, as these assays are unaffected by the presence of inactivated peptide fragments. In man, the half-life of hCRH (30.5 +/- 3.3 min; mean +/- S.E.M.) was significantly (P less than 0.001) less than that of oCRH (42.8 +/- 6.4 min). In sheep, there was no significant difference between the half-life of hCRH (46.5 +/- 7.2 min) and that of oCRH (39.8 +/- 10.1 min); these half-lives were also significantly (P less than 0.001) longer than that of hCRH in man. One possible explanation for the shorter half-life of hCRH in man is that the clearance of hCRH is enhanced by CRH-binding protein, although other binding proteins often have the opposite effect. Peak ACTH and cortisol responses occurred earlier in sheep than in man, although no differences were found in the response times to oCRH or hCRH within either species. The responses were more sustained in sheep than in man, and the previously reported biphasic response was only seen in some of the sheep and not in man. Absolute responses to either peptide were greater in sheep than in man; however, in man an 8.1-fold rise in ACTH was measured in response to oCRH, while hCRH gave a significantly (P = 0.043) smaller 4.4-fold response.(ABSTRACT TRUNCATED AT 250 WORDS)


Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies. Epstein-Barr virus (EBV), a human herpes virus with oncogenic potential, persists in B lymphoid tissues and is controlled by virus-specific cytotoxic T lymphocyte (CTL) surveillance. On reactivation in vitro, these CTLs recognize EBV-transformed lymphoblastoid cell lines (LCLs) in an HLA class I antigen-restricted fashion, but the viral antigens providing target epitopes for such recognition remain largely undefined. Here we have tested EBV-induced polyclonal CTL preparations from 16 virus-immune donors on appropriate fibroblast targets in which the eight EBV latent proteins normally found in LCLs (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, leader protein [LP], and latent membrane protein [LMP] 1 and 2) have been expressed individually from recombinant vaccinia virus vectors. Most donors gave multicomponent responses with two or more separate reactivities against different viral antigens. Although precise target antigen choice was clearly influenced by the donor's HLA class I type, a subset of latent proteins, namely EBNA 3A, 3B, and 3C, provided the dominant targets on a range of HLA backgrounds; thus, 15 of 16 donors gave CTL responses that contained reactivities to one or more proteins of this subset. Examples of responses to other latent proteins, namely LMP 2 and EBNA 2, were detected through specific HLA determinants, but we did not observe reactivities to EBNA 1, EBNA LP, or LMP 1. The bulk polyclonal CTL response in one donor, and components of that response in others, did not map to any of the known latent proteins, suggesting that other viral target antigens remain to be identified. This work has important implications for CTL control over EBV-positive malignancies where virus gene expression is often limited to specific subsets of latent proteins.


Developmental regulation of thymocyte susceptibility to deletion by "self"-peptide. The specificity of the T cell receptor (TCR) repertoire for foreign peptide bound to self-major histocompatibility complex (MHC) molecules is determined in large part by positive and negative selection processes in the thymus, yet the mechanisms of these selection events remain unknown. Using in vitro organ culture of thymi isolated from mice transgenic for a TCR-alpha/beta specific for cytochrome c peptide bound to I-Ek, we analyzed the developmental timing of negative selection (deletion). On the basis of the experiments described below, we conclude that all CD4+8+ thymocytes, and only CD4+8+ thymocytes, are susceptible to negative selection mediated by the cytochrome c peptide antigen. First, we found that deletion of thymocytes resulting from addition of the cytochrome c peptide to the thymic organ cultures can occur at the earliest stage of TCR, CD4, and CD8 coexpression. Second, we found that CD4+8+ thymocytes isolated from positively selecting or nonselecting MHC haplotypes were equally efficiently deleted in vitro, suggesting that positive selection is not a prerequisite for deletion. Third, we examined the effects of TCR/ligand avidity on the developmental timing of deletion by varying the concentration of cytochrome c peptide added to the organ cultures. We detected deletion only at the CD4+8+ stage: intermediate concentrations of peptide that resulted in partial deletion of CD4+8+ cells did not eliminate the appearance of mature CD4+8- cells. Finally, we found that CD4+8- thymocytes were resistant to deletion as well as activation by peptide antigen added to the intact organ cultures. Nevertheless, the CD4+8- thymocytes isolated from the peptide-treated organ cultures responded vigorously to peptide presented by spleen cells in vitro. Thus, the T cells were tolerant of (but not anergized by) self-antigen encountered in thymic organ culture. Together, these results indicate that thymocytes susceptible to negative selection are not developmentally distinct from those susceptible to positive selection, and further, that the thymic microenvironment plays a role in regulating the outcome of TCR/ligand interactions.


A monkey model for Epstein Barr virus-associated lymphomagenesis in human acquired immunodeficiency syndrome. High-grade malignant nonHodgkin's lymphomas--five lymphoblastic, three pleomorphic, and two immunoblastic--developed in 10/25 cynomolgus monkeys (Macaca fascicularis) followed for up to 746 d after infection with simian immunodeficiency virus, strain SIVsm. These lymphomas were shown to be associated with an Epstein-Barr (EB)-like cynomolgus B-lymphotropic herpesvirus (CBLV) by electron microscopy, by Southern blot hybridization with probes against human EBV, and by the expression of antigens corresponding to EBV-associated nuclear antigens (EBNAs) involved in human B cells transformation. Southern blot demonstration of immunoglobulin gene rearrangements and homogeneous EBV episomes indicated that all the lymphomas were CBLV-associated monoclonal B cell proliferations. Our findings suggest that these tumors correspond to the EBV-associated malignant lymphomas in acquired immunodeficiency syndrome with respect to clinical, morphological, phenotypic, and genotypic characteristics. The particular susceptibility of SIVsm immunodeficient cynomolgus monkeys for CBLV-associated lymphomagenesis appears therefore a useful model for EBV-associated lymphomas in humans.


Elevated secretion of reactive nitrogen and oxygen intermediates by inflammatory leukocytes in hyperacute experimental autoimmune encephalomyelitis: enhancement by the soluble products of encephalitogenic T cells. Perivascular lesions within the central nervous system (CNS) of rats with hyperacute experimental autoimmune encephalomyelitis (HEAE) contained large numbers of peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMN), cells enzymatically capable of producing reactive nitrogen and oxygen intermediates (RNI and ROI), which, in excess, are mediators of tissue damage. PBMC and PMN isolated from the CNS and periphery of HEAE-affected rats secreted significantly (p less than 0.01-0.0001) elevated levels of ROI and RNI compared with that of similar cell populations from pertussis- and saline-treated control animals. Coincubation of systemically derived PBMC and PMN with antigen-stimulated myelin basic protein-specific T cell lines led to further increases in ROI and RNI output of between 15.3 and 83.1%, an effect that could be largely attributed to heat-labile, soluble products released by these T cell lines. Our studies suggest a putative neuropathological role for ROI and RNI in HEAE, which may be mediated via cytokines emanating from autoreactive T lymphocytes.


Alpha-1-adrenergic modulation of K and Cl transport in bovine retinal pigment epithelium. Intracellular microelectrode techniques were used to characterize the electrical responses of the bovine retinal pigment epithelium (RPE)-choroid to epinephrine (EP) and several other catecholamines that are putative paracrine signals between the neural retina and the RPE. Nanomolar amounts of EP or norepinephrine (NEP), added to the apical bath, caused a series of conductance and voltage changes, first at the basolateral or choroid-facing membrane and then at the apical or retina-facing membrane. The relative potency of several adrenergic agonists and antagonists indicates that EP modulation of RPE transport begins with the activation of apical alpha-1-adrenergic receptors. The membrane-permeable calcium (Ca2+) buffer, amyl-BAPTA (1,2-bis(o-aminophenoxy)-ethane-N,N,N',N' tetraacetic acid) inhibited the EP-induced voltage and conductance changes by approximately 50-80%, implicating [Ca2+]i as a second messenger. This conclusion is supported by experiments using the Ca2+ ionophore A23187, which mimics the effects of EP. The basolateral membrane voltage response to EP was blocked by lowering cell Cl, by the presence of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the basal bath, and by current clamping VB to the Cl equilibrium potential. In the latter experiments the EP-induced conductance changes were unaltered, indicating that EP increases basolateral membrane Cl conductance independent of voltage. The EP-induced change in basolateral Cl conductance was followed by a secondary decrease in apical membrane K conductance (approximately 50%) as measured by delta [K]o-induced diffusion potentials. Decreasing apical K from 5 to 2 mM in the presence of EP mimicked the effect of light on RPE apical and basolateral membrane voltage. These results indicate that EP may be an important paracrine signal that provides exquisite control of RPE physiology.


LAN-1: a human neuroblastoma cell line with M1 and M3 muscarinic receptor subtypes coupled to intracellular Ca2+ elevation and lacking Ca2+ channels activated by membrane depolarization. The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.


Identification of an activator of the microtubule-associated protein 2 kinases ERK1 and ERK2 in PC12 cells stimulated with nerve growth factor or bradykinin. Treatment of PC12 pheochromocytoma cells with nerve growth factor (NGF) or bradykinin leads to the activation of extracellular signal-regulated kinases ERK1 and ERK2, two isozymes of microtubule-associated protein 2 (MAP) kinase that are present in numerous cell lines and regulated by diverse extracellular signals. The activation of MAP kinase is associated with its phosphorylation on tyrosine and threonine residues, both of which are required for activity. In the present studies, we have identified a factor in extracts of PC12 cells treated with NGF or bradykinin, named MAP kinase activator, that, when reconstituted with inactive MAP kinase from untreated cells, dramatically increased MAP kinase activity. Activation of MAP kinase in vitro by this factor required MgATP and was associated with the phosphorylation of a 42- (ERK1) and 44-kDa (ERK2) polypeptide. Incorporation of 32P into ERK1 and ERK2 occurred primarily on tyrosine and threonine residues and was associated with a single tryptic peptide, which is identical to one whose phosphorylation is increased by treatment of intact PC12 cells with NGF. Thus, the MAP kinase activator identified in PC12 cells is likely to be a physiologically important intermediate in the signaling pathways activated by NGF and bradykinin. Moreover, stimulation of the activator by NGF and bradykinin suggests that tyrosine kinase receptors and guanine nucleotide-binding protein-coupled receptors are both capable of regulating these pathways.


In vivo evidence that lithium inactivates Gi modulation of adenylate cyclase in brain. In vivo microdialysis of cyclic AMP from prefrontal cortex complemented by ex vivo measures was used to investigate the possibility that lithium produces functional changes in G proteins that could account for its effects on adenylate cyclase activity. Four weeks of lithium administration (serum lithium concentration of 0.85 +/- 0.05 mM; n = 11) significantly increased the basal cyclic AMP content in dialysate from prefrontal cortex of anesthetized rats. Forskolin infused through the probe increased dialysate cyclic AMP, but the magnitude of this increase was unaffected by chronic lithium administration. Inactivation of the inhibitory guanine nucleotide binding protein Gi with pertussis toxin increased dialysate cyclic AMP in control rats, as did stimulation with cholera toxin (which activates the stimulatory guanine nucleotide binding protein Gs). The effect of pertussis toxin was abolished following chronic lithium, whereas the increase in cyclic AMP after cholera toxin was enhanced. In vitro pertussis toxin-catalyzed ADP ribosylation of alpha i (and alpha o) was increased by 20% in prefrontal cortex from lithium-treated rats, but the alpha i and alpha s contents (as determined by immunoblot) as well as the cholera toxin-catalyzed ADP ribosylation of alpha s were unchanged. Taken together, these results suggest that chronic lithium administration may interfere with the dissociation of Gi into its active components and thereby remove a tonic inhibitory influence on adenylate cyclase, with resultant enhanced basal and cholera toxin-stimulated adenylate cyclase activity.


A cyclic AMP mechanism mediates the serotonin-induced increase in glutamic acid decarboxylase activity in the preoptic area and hypothalamus. Previous studies have shown that the injection of 5-hydroxytryptamine (5-HT) into the third ventricle of rats on the afternoon of proestrus increases glutamic acid decarboxylase (GAD) activity in the preoptic area and the hypothalamus. In the present report we examine the adenylate cyclase-cyclic AMP (cAMP) system as mediator of that effect. The increase in GAD activity induced by intraventricular injection of 5-HT was completely blocked by injecting an antiserum against cAMP into the third ventricle 30 min earlier, whereas an injection of serum from normal rabbits was ineffective. On the contrary, activation of adenylate cyclase activity by intraventricular injection of forskolin increased GAD activity, an effect that was also blocked by anti-cAMP serum. Anti-cAMP serum also lowered GAD activity in the preoptic area and hypothalamus when injected on the morning of proestrus but not when injected in the afternoon, when the values of GAD activity were already low. The results suggest that a cAMP mechanism may be involved in the changes in preoptic-area and hypothalamic GAD activity such as the rise in enzyme activity induced by intraventricular injection of 5-HT.


Cerebral acid buffering capacity at different ages measured in vivo by 31P and 1H nuclear magnetic resonance spectroscopy. Cerebral acidosis occurring during ischemia has been proposed as one determinant of tissue damage. Newborn animals appear to be less susceptible to ischemic tissue damage than adults. One possible component of ischemic tolerance could derive from maturational differences in the extent of acid production and buffering in newborns compared to adults. The purpose of this study was to measure the dependency of acid production on the blood plasma glucose concentrations and acid buffering capacity of piglets at different stages of development. Complete ischemia was induced in 29 piglets ranging in postconceptual age from 111 to 156 days (normal term conception, 115 days). Brain buffering capacity during the first 30 min of ischemia was quantified in vivo, via 31P and 1H nuclear magnetic resonance (NMR) spectroscopy, by measuring the change in intracellular brain pH for a given change in the concentration of compounds that contribute to the production of hydrogen ions. Animals from all four age groups showed a similar linear correlation between preischemia blood glucose concentration and intracellular pH after 30 min of ischemia. For each animal the slope of the plot of intracellular pH versus cerebral buffer base deficit was used to calculate the buffer capacity. Using data obtained over the entire 30 min of ischemia, there was no difference in the mean buffer capacity of the different age groups, nor was there a significant correlation between buffer capacity and age. However, there was a significant increase in buffer capacity for the intracellular pH range 6.6-6.0, compared to 7.0-6.6, for all age groups. No significant differences in buffer capacity for these two pH ranges were observed between any of the age groups. Acid buffering capacity was also measured by performing pH titrations on brain tissue homogenized in the presence of inhibitors of glycolysis and creatine kinase. Plots of homogenate pH versus buffer base deficit showed a nonlinear trend similar to that seen in vivo, indicating an increase in buffer capacity as intracellular pH decreases. A comparison of newborn and 1-month-old brain tissue frozen under control conditions or after 45 min of ischemia revealed no differences that could be attributed to age and a slight decrease in buffer capacity of ischemic brain compared to control brain tissue homogenates. There was no difference between the brain buffering capacity measured in vivo using 31P and 1H NMR and that measured in vitro using brain homogenates.


Metabotropic glutamate receptor activation produces extrapyramidal motor system activation that is mediated by striatal dopamine. Little is known about the in vivo function of the GTP-binding protein-coupled "metabotropic" excitatory amino acid (EAA) receptor. In vitro studies on agonist-induced brain phosphoinositide hydrolysis have shown that (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid is a highly selective and efficacious metabotropic EAA agonist. We have recently reported that in vivo unilateral intrastriatal injection of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid induces transient extrapyramidal motor activation that manifests itself as contralateral turning. In this study, we fully characterized the onset of turning behavior following intrastriatal (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid injection and the possible involvement of striatal dopamine neurons in the mediation of this effect. Rats were anesthetized with the short-acting agent halothane to allow for rapid surgical recovery and thus early behavioral measurements. Intrastriatal (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1 mumol/2 microliters) produced an incremental increase in contralateral turning starting at 1 h and plateauing 3-6 h after injection (peak effect, 39.1 +/- 6.7 rotations per 5 min). Dopamine depletion with alpha-methyl-DL-p-tyrosine (250 mg/kg i.p., 80% depletion) resulted in greater than 85% inhibition of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid-induced contralateral turning. The dopamine antagonist haloperidol (0.3 mg/kg i.p.) produced 48% inhibition of the (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid response. In time course studies, turning behavior correlated with increases in levels of the dopamine metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid. These results suggest a functional interaction between the metabotropic EAA receptor and the dopaminergic system in the striatum.


Evidence for metabotropic excitatory amino acid receptor heterogeneity: developmental and brain regional studies. To examine whether multiple subtypes of the excitatory amino acid (EAA) receptor coupled to phosphoinositide (PPI) hydrolysis exist, we have pharmacologically characterized the PPI response in neonatal and adult rat brain. Activation of PPI hydrolysis was determined by the accumulation of [3H]inositol monophosphate in brain slices prelabeled with [3H]inositol. In neonatal hippocampus, D,L-2-amino-3-phosphonopropionic acid (AP3; 1 mM) inhibited the cis-1-aminocyclopentane-1,3-dicarboxylic acid (IUPAC nomenclature; ACPD; 100 microM)- and quisqualate (Quis; 100 microM)-stimulated PPI hydrolysis by 73 and 66%, respectively, but had no effect in neonatal cerebellum. In adult hippocampus, AP3 stimulated PPI hydrolysis with potency and efficacy comparable to those of Quis and ACPD and completely masked the Quis concentration-response curve. In adult cerebellum, only Quis behaved as a full agonist on the PPI response. The Quis concentration-response curve was shifted rightward with a fourfold decrease in potency in the presence of ACPD (5 mM), whereas it was nearly additive with the PPI response induced by AP3 (5 mM). Thus, our data reveal significant developmental and brain regional differences in metabotropic EAA receptor responses and support the notion that this receptor is heterogeneous, in both a regionally specific and a developmentally dependent manner.


Protein phosphatases 1 and 2A dephosphorylate B-50 in presynaptic plasma membranes from rat brain. The protein B-50 is dephosphorylated in rat cortical synaptic plasma membranes (SPM) by protein phosphatase type 1 and 2A (PP-1 and PP-2A)-like activities. The present studies further demonstrate that B-50 is dephosphorylated not only by a spontaneously active PP-1-like enzyme, but also by a latent form after pretreatment of SPM with 0.2 mM cobalt/20 micrograms of trypsin/ml. The activity revealed by cobalt/trypsin was inhibited by inhibitor-2 and by high concentrations (microM) of okadaic acid, identifying it as a latent form of PP-1. In the presence of inhibitor-2 to block PP-1, histone H1 (16-64 micrograms/ml) and spermine (2 mM) increased B-50 dephosphorylation. This sensitivity to polycations and the reversal of their effects on B-50 dephosphorylation by 2 nM okadaic acid are indicative of PP-2A-like activity. PP-1- and PP-2A-like activities from SPM were further displayed by using exogenous phosphorylase alpha and histone H1 as substrates. Both PP-1 and PP-2A in rat SPM were immunologically identified with monospecific antibodies against the C-termini of catalytic subunits of rabbit skeletal muscle PP-1 and PP-2A. Okadaic acid-induced alteration of B-50 phosphorylation, consistent with inhibition of protein phosphatase activity, was demonstrated in rat cortical synaptosomes after immunoprecipitation with affinity-purified anti-B-50 immunoglobulin G. These results provide further evidence that SPM-bound PP-1 and PP-2A-like enzymes that share considerable similarities with their cytosolic counterparts may act as physiologically important phosphatases for B-50.


Inositol 1,4,5-trisphosphate receptor immunoreactivity in SH-SY5Y human neuroblastoma cells is reduced by chronic muscarinic receptor activation. Inositol 1,4,5-trisphosphate (InsP3) receptor immunoreactivity in SH-SY5Y human neuroblastoma cells was monitored with a monoclonal antibody raised against the mouse cerebellar InsP3 receptor. Recognition of a protein corresponding to the InsP3 receptor (molecular mass, approximately 275 kDa) was inhibited markedly following exposure of cells to 0.1 mM carbachol. This effect was half-maximal and maximal at approximately 2 and approximately 6 h, respectively; was blocked by atropine; but was not mimicked by thapsigargin, K+, or phorbol 12-myristate 13-acetate. However, the decrease in immunoreactivity following exposure of cells to carbachol for 5 h was blocked if the extracellular Ca2+ concentration was reduced from 1.3 mM to 200 nM. This manipulation also reduced markedly carbachol-induced increases in InsP3 concentration at 5 h. These data indicate that chronic muscarinic stimulation of phosphoinositide hydrolysis reduces InsP3 receptor concentration in SH-SY5Y cells, perhaps via a mechanism that involves prolonged elevation of InsP3 levels.


Involvement of growth-associated protein-43 with irreversible neurite outgrowth by dibutyryl cyclic AMP and phorbol ester in NG108-15 cells. Simultaneous treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) and dibutyryl cyclic AMP (diBu-cAMP) for 72 h induced neurites in NG108-15 cells significantly longer than treatment with each alone. Treatment for 72 h with both drugs induced irreversible neurite extension and a decline in protein kinase C activity, although neurites extended by diBu-cAMP alone disappeared after the withdrawal of the drug. The expression of growth-associated protein-43 (GAP-43) mRNA was also observed by a combined application of TPA and diBu-cAMP. The increased level of GAP-43 mRNA induced by treatment with both drugs for 72 h was maintained at least 24 h after withdrawal of the drugs. In cells transfected with GAP-43 cDNA, neurites induced by treatment with diBu-cAMP alone for 72 h were maintained at least 48 h after removal of the drugs. These results suggest that GAP-43 could be involved in the maintenance of elongated neurites and that a decline in protein kinase C activity may be involved in the accumulation of GAP-43.


Distinct developmental patterns of expression of rat alpha 1, alpha 5, gamma 2S, and gamma 2L gamma-aminobutyric acidA receptor subunit mRNAs in vivo and in vitro. We have quantitated the alpha 1, alpha 5, gamma 2S, and gamma 2L gamma-aminobutyric acidA (GABAA) receptor subunit mRNAs in the maturing cerebellum in vivo and in cerebellar granule neurons differentiating in vitro. Absolute amounts of mRNA were measured by reverse transcription and competitive polymerase chain reaction (PCR) analysis with appropriate internal standards. The alpha 1 and gamma 2L mRNA content increased continuously during postnatal cerebellar maturation and their changes with time matched very closely those of the cerebellar granule cells differentiating in vitro. The gamma 2S subunit mRNA showed a relatively constant pattern of expression both in vivo and in vitro, with comparable absolute concentrations in both developmental paradigms. The alpha 5 mRNA was initially high in vivo and decreased (eight-fold) to adult levels as postnatal cerebellar development progressed. In vitro the amount of alpha 5 GABAA receptor subunit mRNA was higher than in vivo at 3 days, increased by more than twofold by 8 days, and declined to approximately the initial values at 23 and 28 days in vitro. Collectively, the results indicate that the alpha 1, alpha 5, gamma 2S, and gamma 2L GABAA receptor subunit mRNAs are regulated differentially in a temporal manner during in vivo and in vitro maturation. Moreover, a comparison of the ontogenetic profiles of the gamma 2S and gamma 2L mRNAs indicates that alternative splicing of the gamma 2 primary RNA transcript is regulated developmentally during postnatal maturation of the rat cerebellum.


Localization of the peripheral-type benzodiazepine binding site to mitochondria of human glioma cells. Subcellular fractionation was performed on human U251 glioblastoma cultures. In all subcellular fractions, the binding of the peripheral benzodiazepine ligand, [3H]PK 11195, correlated with the specific activity of monoamine oxidase (r = 0.95, p less than 0.001) and succinate dehydrogenase (r = 0.93, p less than 0.001), two mitochondrial enzymes. The specific activity of plasma membrane and nuclear markers correlated poorly with the presence of PK 11195 binding sites. These data support the mitochondrion as the primary location of peripheral-type benzodiazepine binding sites (PBBS) in human glioma cells. Mitochondria-rich preparations were then assayed for [3H]Ro5-4964 binding. Six nM [3H]Ro5-4964 failed to specifically bind to human U251 mitochondria, but bound vigorously to mitochondria from rat C6 glioma. These data indicate that the low affinity of Ro5-4864 for PBBS in human glioma cells compared to those in rat is due to interspecies receptor variation rather than impaired drug transport into human cells.


The treatment of adult supratentorial high grade astrocytomas. From 1 January, 1982 until 31 December, 1987 260 adult patients were referred to the Cancer Control Agency of B.C. with high grade supratentorial astrocytomas. Multifocal disease on presentation was present in 17 cases (6.5%). Their survival is poor and whole brain radiotherapy is required. All other cases had unifocal disease, but eight did not receive radiotherapy. The 235 cases who received radiotherapy were subject to univariate and multivariate analyses according to extent of surgery, age, Kernohan and WHO grading, Karnofsky performance status, whole brain treatment, partial brain treatment, total dose and neuroret. Age is an extremely important predictor of survival (P approximately equal to 0). The pathologic appearance of glioblastoma (WHO grade) as well as the Karnofsky performance status were also important independent factors in predicting survival (P = 0.016, 0.027 respectively) on Cox multivariate analysis. Dose and neurorets were significant factors only in cases where the performance status was not recorded, suggesting that dose was selected according to the patient's condition and age. In this analysis it was found that localized radiation fields may be used rather than whole brain without jeopardizing survival.


Chromosomal localization of human glutamate receptor genes. The chromosomal localization of human glutamate receptor genes (GluR1-4) has been established using PCR with DNA isolated from mapping panels of Chinese hamster-human hybrid cell lines and high-resolution fluorescent in situ suppression hybridization. This was accomplished with genomic clones containing putative human homologs of rat GluR 1-4 isolated by high-stringency screening of a cosmid library with the rat cDNAs encoding GluR1-4. The locations of GluR1-4, respectively, are 5q32-33, 4q32-33, Xq25-26, and 11q22-23. Evidence implicating glutamatergic synapses in a diversity of physiologic and pathologic processes together with concordance of the chromosomal locales and results of linkage analyses establishes GluR3 and GluR4 as candidate genes for a number of nervous system disorders including the oculocerebral-renal syndrome of Lowe and a form of manic-depressive illness.


Regulation of somatodendritic dopamine release in the ventral tegmental area by opioids and GABA: an in vivo microdialysis study. Microdialysis of the ventral tegmental area in conscious rats was used to evaluate the influence of opioids and GABA agonists on extracellular levels of GABA and somatodendritically released dopamine. The administration of morphine through the dialysis probe elicited significant, dose-dependent increases in the levels of extracellular dopamine and significantly reduced the extracellular concentration of GABA. In contrast, a dose-dependent decrease in somatodendritic extracellular dopamine was produced following the administration of the GABAB agonist baclofen. The increase in dopamine levels elicited by morphine (100 microM) was completely blocked by either baclofen (100 microM) coadministration or peripheral injection of naloxone (2 mg/kg, i.p.). Application of the GABAA agonist muscimol produced a significant increase in both extracellular levels of dopamine and locomotor activity. The present results, together with other electrophysiological, neurochemical, and behavioral data, support a hypothesis that stimulation of mu-opioid or GABAA receptors inhibits the activity of GABAergic afferents to dopamine neurons, thereby removing tonic inhibitory regulation, whereas stimulation of GABAB receptors directly inhibits dopamine neurons.


Differential effects of serotonin, FMRFamide, and small cardioactive peptide on multiple, distributed processes modulating sensorimotor synaptic transmission in Aplysia. At least two processes contribute to the modulation by 5-HT of the connections between sensory neurons and motor neurons in Aplysia. The first involves broadening of the presynaptic spike through modulation of 5-HT-sensitive K+ channels that leads to elevated levels of intracellular Ca2+ and increased release of transmitter. A second process (or set of processes) apparently accounts for the amount of facilitation not produced by presynaptic spike broadening. This spike duration-independent (SDI) process is particularly prominent in depressed synapses. We used a protocol in which spikes were prebroadened into a range of durations in which further spike broadening by itself has little or no effect on facilitation of the EPSP.5-HT produced pronounced facilitation in depressed synapses under these conditions. Another modulatory agent, small cardioactive peptide (SCPb), also broadened spikes in sensory neurons but did not produce facilitation comparable to that produced by 5-HT. These results indicate that 5-HT activates the SDI process whereas SCPb fails to do the same. A 5 min preexposure to the modulatory peptide FMRFamide inhibited 5-HT-induced activation of the SDI process, whereas a 1 min preexposure did not. Another process(es) that may modulate synaptic efficacy in sensorimotor synapses involves changes in the properties of the motor (follower) neuron, such as input resistance. FMRFamide decreased the input resistance of postsynaptic neurons. This action could contribute to the effects of FMRFamide when administered alone, but it did not appear to be responsible for the inhibitory action of FMRFamide on 5-HT-induced facilitation. Neither 5-HT nor SCPb had a clear effect on input resistance. The actions of these three agents, therefore, seem to be differentially distributed among various pre- and postsynaptic processes involved in the modulation of synaptic transmission.


Estrogen suppresses mu-opioid- and GABAB-mediated hyperpolarization of hypothalamic arcuate neurons. The effects of estrogen on the response of hypothalamic arcuate neurons to mu-opioid and GABAB agonists were investigated. Intracellular recordings were made from arcuate neurons in slices prepared from ovariectomized guinea pigs that were pretreated with estrogen or vehicle. Estrogen shifted the dose-response curve to the mu-opioid agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) by 3.4-fold; the EC50 for DAMGO was 240 +/- 25 nM in estrogen-treated females versus 70 +/- 12 nM in the controls. The maximal hyperpolarization induced by DAMGO was equivalent in neurons from both groups. The Ke for the naloxone antagonism of the DAMGO response was similar in both groups, which would indicate that the affinity of the mu-receptor was unchanged. To explore where in the receptor/G-protein/K+ channel cascade estrogen may be acting to attenuate the mu-opioid-mediated hyperpolarization, the response to the GABAB agonist baclofen was also tested. Estrogen treatment also shifted the dose-response curve for the baclofen-induced hyperpolarization by 3.3-fold without altering the maximum hyperpolarization; the EC50 shifted from 11.0 +/- 4.0 microM to 36.0 +/- 5.0 microM. All of the neurons were identified after linking the intracellular biocytin with streptavidin-FITC, and a subpopulation of cells in both groups were immunoreactive for beta-endorphin. We conclude that estrogen decreases the functional coupling of the mu-opioid and GABAB receptors to the inwardly rectifying K+ channel possibly through an action on the G-protein.


Somatic gene transfer of nerve growth factor promotes the survival of axotomized septal neurons and the regeneration of their axons in adult rats. Intracerebral grafts consisting of primary fibroblasts genetically engineered to express NGF were used to assess the regenerative capacity of cholinergic neurons of the adult rat septum. Our data reveal that NGF-producing grafts sustain a significantly higher proportion of NGF receptor-immunoreactive septal neurons following axotomy (approximately 65-75%) than do grafts of noninfected fibroblasts. In addition, NGF promotes the regeneration of septal axons. Following the ablation of cholinergic septal projections to the hippocampus, NGF-producing grafts placed within the lesion cavity contain large numbers of AChE-positive axons; control grafts, on the other hand, lack such cholinergic axons. Ultrastructural examination reveals that unmyelinated axons within NGF-producing grafts use many different substrates for growth, including astrocytes and components of the extracellular matrix. Grafts of control fibroblasts possess the same cellular and matrix substrates but contain only a small population of axons, probably of peripheral origin. AChE-positive axons growing through NGF-producing grafts provide a new topographically organized input to the deafferented hippocampal dentate gyrus. Furthermore, regenerating septal axons terminate predominantly on the dendritic processes of granular neurons. The dentate gyrus ipsilateral to grafts of noninfected fibroblasts, on the other hand, remains devoid of AChE-positive fibers. From these results, we conclude that the availability of NGF is a necessary requirement to sustain axotomized cholinergic septal neurons and to promote axon regeneration and cholinergic reinnervation of dentate granular neurons by these lesioned neurons. The presence of many permissive substrates (e.g., astrocytes, basal lamina, and collagen) alone, however, is not sufficient to induce axon regrowth from adult septal neurons.


Primary osteosarcoma of the thyroid gland. Primary extraosseous osteosarcoma of the thyroid gland is a rare tumor which is associated with a poor prognosis. In this report, we discuss such a tumor and its recurrence in a 78-yr-old female with multinodular goiter, focusing on the imaging evaluation of this unusual tumor.


An application of item response theory to alexithymia assessment among abstinent alcoholics. An item response theory (IRT) model identified three dimensions assessed by the Toronto Alexithymia Scale (TAS) in a sample of 130 male applicants for inpatient care at a Veterans Administration (VA) medical center alcoholism treatment program. A unidimensional solution did not capture all of alexithymia's theoretical features. Subjects with lower alexithymia scores gave positive responses to items tapping emotional awareness deficits; only those with higher alexithymia scores gave positive responses to items tapping external, operative cognitive style. Thus, a total TAS score may not represent alexithymia accurately in substance-abusing patient populations.


Blunted ACTH response to hypoglycemic stress in depressed patients but not in patients with schizophrenia. In this study, 7 hospitalized patients with major depression (MD), 5 hospitalized patients with schizophrenia (S), and 13 control subjects (C) were administered 0.15 units/kg of regular insulin at 1600 h by intravenous bolus infusion. ACTH, cortisol, and glucose levels were measured intermittently for 2h following infusion. Baseline ACTH, cortisol and glucose levels were similar in Cs, MDs, and Ss. The mean glucose nadir was equivalent for Cs, patients with MD, and patients with S. Patients with MD had a blunted ACTH response (F = 3.28; df = 12,126; p = .0004) and cortisol response (F = 4.20; df = 12,132; p = .0001) to hypoglycemia when compared to Cs and patients with S. Carroll Depression Rating Scale scores in patients with S (23 +/- 10) were similar to patients with MD (30 +/- 8) and significantly higher than in controls (1 +/- 2) (F = 55.2; df = 2.22; p = .0001). These findings suggest that patients with MD show different ACTH and cortisol responses to hypoglycemic stress which are not explained by negative feedback of baseline ACTH or cortisol, glucose nadir, or the number of depressive symptoms per se.


Demonstration of cytomegalovirus (CMV) DNA and anti-CMV response in the synovial membrane and serum of patients with rheumatoid arthritis. One hundred forty-seven synovial membranes were investigated for the presence of human cytomegalovirus (CMV). In 11 of 83 synovial membranes of patients with rheumatoid arthritis (RA), but only in 2 of 64 synovial membranes from patients with other joint disorders, CMV-DNA could be detected by the polymerase chain reaction (PCR) technique (p less than 0.05). In contrast, the percentage of seropositive patients was not significantly different between the 2 groups of patients and no blood sample from any patient tested revealed the presence of CMV by PCR. Only in patients with RA and CMV positive synovial membranes was the titer of CMV specific antibodies in the synovia higher than in the serum. Thus, the local presence of CMV in the synovial membrane and associated local antiviral responses might participate in the pathogenesis of synovitis in a minority of patients with RA.


Potential role of Epstein-Barr virus in Sjögren's syndrome and rheumatoid arthritis. In the pathogenesis of Sjögren's syndrome (SS), a role for Epstein-Barr virus (EBV) has been suggested because; (a) EBV is present in salivary gland epithelial cells of healthy individuals, and exaggerated immune responses against EBV could play a role in the destruction of salivary glands in SS; (b) SS salivary gland biopsies contain increased levels of EBV DNA compared to normal salivary glands, indicating viral reactivation and inability of lymphoid infiltrates to control EBV replication in patients with SS; and (c) salivary gland epithelial cells in patients with SS express high levels of HLA-DR antigens and may present EBV associated antigens to immune T cells in patients with SS. Therefore, SS may represent a situation where genetically predisposed individuals (i.e., HLA-DR3-DQA4-DQB2) have a persistent but ineffectual T cell immune response against EBV at its site of latency. In the case of rheumatoid arthritis (RA), evidence for a potential role of EBV includes the following: (a) EBV encoded proteins share antigenic and sequence similarity to proteins found in the synovial tissues. These crossreactive proteins include EBV protein gp110 (BALF-4) and the beta chain of HLA-DR4. Also, the human and mycobacterial 65 kDa heat shock proteins have a sequence similar to that of EBV encoded proteins; (b) patients with RA have increased frequency and levels of antibodies against specific epitopes on EBV encoded EBNA-1 (BKRF-1) and EBNA-3 (BERF-1) antigens; and (c) lymphocytes from patients with RA have decreased ability to limit outgrowth of autologous EBV infected lymphocytes, probably due to defects in release of interferon gamma.(ABSTRACT TRUNCATED AT 250 WORDS)


Lentivirus induced arthritis in animals. Retroviral arthritis in sheep and goats depends on persistent infection in the animals. Virus is latent in macrophage precursor cells and viral replication is initiated when these cells are induced to differentiate. Antiviral antibodies and cytokines modulate the efficiency of viral gene product expression. Specific cytokines induced during replication of the lentivirus in mononuclear cells are also responsible for directing infected cells from peripheral blood through the vascular endothelium to particular tissues. Cytokines induced by other infectious agents such as bacteria, mycoplasma or protozoa, may also contribute to this chemotactic process. Once in the tissue, macrophages interact with lymphocytes to induce an inflammatory cascade with further production of cytokines which enhances expression of class II major histocompatibility complex antigens and proliferation of B and CD8 lymphocytes. In addition, immune complexes between viral glycoproteins and immunoglobulins are produced locally and probably lead to further enhancement of pathological changes in the tissues.


Immunomodulating functions of tumor necrosis factor and interleukin 1 inhibitors. Cytokines play a crucial role in the inflammatory and immune responses. The activity of cytokines is counterbalanced by specific inhibitors with some functioning as receptor antagonists. Inhibitors to interleukin 1 and tumor necrosis factor may have therapeutic potential in conditions such as inflammatory arthritis, diabetes mellitus, disseminated intravascular coagulopathy and septic shock. The ability to modulate host defenses with cytokines and cytokine antagonists may also have applications in the fields of transplantation, oncohematology and immunodeficiency.


Interleukin 1 and tumor necrosis factor signal transduction mechanisms: potential targets for pharmacological control of inflammation. Interleukin 1 and tumor necrosis factor activate serine/threonine kinase activity in cells within minutes of receptor interaction. Kinases increasing phosphorylation of the small heat shock protein (HSP 27) and epidermal growth factor receptor have been identified and shown to be activated independent of protein kinase A or C. Such kinases are also implicated in activation of the transcription factor nuclear factor kappa B. The cytokines have novel signal transduction pathways that could offer potential therapeutic targets.


Structure-activity study of hCGRP8-37, a calcitonin gene-related peptide receptor antagonist. A structure-activity study was carried out to determine the importance of the N-terminal amino acids of hCGRP8-37 in binding and antagonistic activity to CGRP receptors. Therefore, fragments of hCGRP8-37 as well as analogs obtained by the replacement of residues 9-12 by L-alanine were synthesized by solid-phase peptide synthesis, using BOP as a coupling reagent. The affinities of the peptides to CGRP receptors were evaluated in the rat brain, guinea pig atrium, and guinea pig vas deferens membrane preparations. Their antagonistic activities were measured in the guinea pig atria and rat vas deferens bioassays. The pharmacological characterization showed that arginine-11 and leucine-12 play a crucial role for the affinity of hCGRP8-37. Interestingly, it was observed that [Ala11]hCGRP8-37 was able to potentiate the twitch response of the electrically stimulated rat vas deferens. On the other hand, the substantial antagonistic potencies of analogs [Ala9]-, [Ala10]-, and [Ala12]hCGRP8-37, as compared to those of the fragments hCGRP10-37, hCGRP11-37, and hCGRP12-37, suggest that the side chains of Thr-9, His-10, and Leu-12 assume mainly a structural role. Accordingly, the conformational characterization of these peptides by circular dichroism spectroscopy revealed that the residues 9-12 are important for the integrity of the amphiphilic alpha-helix of hCGRP8-37.


(+-)-carbocyclic 5'-nor-2'-deoxyguanosine and related purine derivatives: synthesis and antiviral properties. Beginning with 3-cyclopenten-1-ylamine hydrochloride, the 5'-nor derivatives of carbocyclic 2'-deoxyguanosine (2), 2'-deoxyadenosine (3), and 2,6-diaminopurine 2'-deoxyribofuranoside (4) have been prepared. These compounds were evaluated for antiviral potential versus herpes simplex virus, varicella-zoster virus, cytomegalovirus, vaccinia virus, vesicular stomatitis virus, and human immunodeficiency virus and found to lack activity. Also, compounds 2-4 were virtually nontoxic toward the host (human diploid fibroblast ESM and HEL) cells. These biological properties may be due to the inability of 2-4 to be phosphorylated to the requisite nucleotide level that is likely to be necessary for biological activity by correlation to carbocyclic 2'-deoxyguanosine (1), which possesses significant antiviral properties as a result of conversion to its 5'-triphosphate derivative.


Benzodiazepine receptor affinity and interaction of some N-(indol-3-ylglyoxylyl)amine derivatives. Several derivatives, in which tryptamine, tyramine, and dopamine moieties are linked to the indole nucleus by an oxalyl bridge, were tested for their ability to displace the specific binding of [3H]flunitrazepam from bovine brain membranes. GABA ratio and in vivo tests for the most potent compounds showed they behave as inverse agonists at the benzodiazepine receptor (BzR). To better define the structure-activity relationship (SAR) of this kind of ligand, several phenylethylamine derivatives were synthesized to evaluate their affinity to BzR. Some of these derivatives (17, 21, 24, 26, and 30) were found to exhibit high affinity (Ki = 0.51-0.085 microM) for BzR and possessed a partial agonist activity, although their chemical structure is closely related to tryptamine 2-6, tyramine 7-11, and dopamine 12-16 derivatives. A different interaction of these ligands to the receptor site is hypothesized. Moreover, all the prepared 1-methyl derivatives exhibited very low binding affinity to BzR.


Synthesis and receptor binding properties of fluoro- and iodo-substituted high affinity sigma receptor ligands: identification of potential PET and SPECT sigma receptor imaging agents. Unlabeled fluoro- and iodo-substituted ligands exhibiting very high affinity and selectivity for sigma receptors were synthesized based on three different structural classes of sigma receptor ligands. These compounds were evaluated for sigma receptor affinity and specificity in order to assess their potential as PET/SPECT imaging agents. Thus, (+)- and (-)-N-(5-fluoro-1-pentyl)normetazocines [(+)- and (-)-4] based on the (+)-benzomorphan class of sigma ligands were synthesized via N-alkylation of optically pure (+)- and (-)-normetazocine with 5-[(methylsulfonyl)oxy]-1-pentyl fluoride (11). (+)- and (-)-4 displaced [3H](+)-3-PPP with Ki values of 0.29 and 73.6 nM and [3H](+)-pentazocine with Ki values of 10.5 and 38.9 nM, respectively. The second class of PET/SPECT ligands was based upon the N-(arylethyl)-N-alkyl-2-(1-pyrrolidinyl)ethylamine class of sigma ligands; N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-(3-fluoro-1-propyl)-2-(1- pyrrolidinyl)ethylamine (5) was obtained via N-alkylation of N-[2-(3,4-dichlorophenyl)-1-ethyl]-2-(1-pyrrolidinyl)ethylamine (14) with 3-fluoropropyl p-toluenesulfonate. 5 exhibited Ki values of 4.22 and 5.07 nM for displacement of [3H](+)-3-PPP and [3H](+)-pentazocine, respectively, comparable with the parent N-propyl compound. Attempts to synthesize N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-[3- [(methylsulfonyl)oxy]-1-propyl]-2-(1-pyrrolidinyl)ethylamine (26), a precursor to 5 that could conceivably be converted to [18F]-5 by treatment with 18F-, proved unsuccessful. The sequence of regioselective nitration, catalytic hydrogenation, and diazotization followed by NaI quench of N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-methyl-2-(1- pyrrolidinyl)ethylamine (2) afforded the iodinated ethylenediamine N-[2-(2-iodo-4,5-dichlorophenyl)-1-ethyl]-N-methyl-2-(1- pyrrolidinyl)ethylamine (8), a potential SPECT ligand for sigma receptors. This compound showed an affinity of 0.54 nM ([3H](+)-3-PPP) comparable with the parent compound 2 (Ki = 0.34 nM, [3H](+)-3-PPP). Ligand 8 exhibited a similar potency against [3H](+)-pentazocine. The third class of high-affinity sigma receptor ligands was rationalized based on rearrangement of the bonds in ethylenediamine 2 to give 1-[2-(3,4-dichlorophenyl)-1-ethyl]-4-(1-propyl)piperazine (3). This compound exhibited very high affinity (Ki = 0.31 nM, [3H](+)-3-PPP) and selectivity for sigma receptors.(ABSTRACT TRUNCATED AT 400 WORDS)


Synthesis, configuration, and calcium modulatory properties of enantiomerically pure 5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylates. Enantiomerically pure hexahydroquinolinones of the structural type 9 were prepared by a variation of the Hantzsch synthesis in which an optically active acetoacetate served as a chiral auxiliary reagent. Determinations of the de and ee values are described. The absolute configurations of the optically pure products were characterized by single-crystal X-ray analysis. The antipodes 9a and 9b exhibited calcium antagonistic activities on smooth musculature; the (S)-(-)-enantiomer 9b was the more potent compound with regard to the EC50 values which differed by a factor of 100; the intrinsic activity of 9b was 1.2, compared with a value of 0.54 for 9a. On the other hand, R-(+)-9a exerted positive inotropic effects on electrically stimulated atria. The cause of these effects is discussed.


Selective reversible and irreversible ligands for the kappa opioid receptor. (+-)-(5 beta,7 alpha,8 beta)-3,4-Dichloro-N-methyl-N-[3-methylene-2- oxo-8-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-7-yl]benzeneacetamide (14) and its (5 alpha,7 alpha,8 beta) diastereomer 15 have been synthesized from 1,4-cyclohexanedione monoethylene ketal (1) in 10 steps. Compound 14, which we have designated SMBU-1, was found to bind with moderate affinity (Ki = 109 nM) and good selectivity (mu/kappa = 29) to the kappa opioid receptor, while 15 was only 1/10 as potent as a kappa ligand. Preincubation of brain membranes with 14 resulted in wash-resistant inhibition of kappa-receptor binding (69 +/- 6% of control at 10(-6) M). The ketone precursor trans-N-methyl-N-[5-oxo-2-(1- pyrrolidinyl)cyclohexyl]benzeneacetamide (12) showed a higher kappa-affinity (Ki = 78 nM) and a much higher kappa-selectivity (mu/kappa = 166) than 14. Compound 10, the ethylene ketal precursor of 12, exhibited a similar receptor binding profile to 14, with increased kappa-selectivity (mu/kappa = 55), while ketal 11, being a regioisomer of 10 and an oxygen isostere of the kappa-selective analgesic spiradoline (U-62,066), demonstrated the highest kappa-affinity (Ki = 1.5 nM) and kappa-selectivity (mu/kappa = 468) observed in this series.


Chemical and enzyme-mediated oxidation of the serotonergic neurotoxin 5,7-dihydroxytryptamine: mechanistic insights. The oxidation chemistry and biochemistry of the serotonergic neurotoxin 5,7-dihydroxytryptamine (1) has been studied under anaerobic and aerobic conditions in aqueous solution at physiological pH. Under anaerobic conditions, one-electron oxidants (ferricytochrome c, peroxidase/H2O2, ceruloplasmin, Cu2+) generate a radical intermediate. Dimerization of the C(6)-centered resonance form of this radical followed by secondary oxidations yields 3-(2-aminoethyl)-6-[3-(2-aminoethyl)-1,7-dihydro- 5-hydroxy-7-oxo-6H-indol-6-ylidene]-1-H-indole-5,7(4H,6H)-dione. Under aerobic conditions, molecular O2 attacks the C(4)-centered 1 radical to yield a hydroperoxy radical which decomposes to 5-hydroxytryptamine-4,7-dione (2). Autoxidation of 1 proceeds by primary attack by molecular O2 on a C(4)-centered carbanion to form a superoxide-radical complex. This rearranges to a C(4)-centered hydroperoxide which decomposes to 2. A C(6)-centered carbanion of 1 combines with 2 to give, ultimately, 6,6'-bi-5-hydroxytryptamine-4,7-dione (3). Trace concentrations of transition metal ions (Fe3+, Fe2+, Cu2+, Mn2+) catalyze the autoxidation of 1 by catalytic cycles in which a hydroperoxide intermediate plays key roles. A byproduct of the transition metal-catalyzed oxidation of 1 is superoxide, O2-. Because of its enormous basicity O2- facilitates deprotonation of 1. The C(4)-centered carbanion so produced is oxidized by molecular O2 or by the hydroperoxy radical (HO2) to give radical intermediates and thence 2 and 3. Mechanistic pathways leading to the various products of oxidation of 1 are proposed and the potential roles of oxidation reactions of the indolamine are related to its neurodegenerative properties.


2-Acetylpyridine thiocarbonohydrazones. Potent inactivators of herpes simplex virus ribonucleotide reductase. A series of 2-acetylpyridine thiocarbonohydrazones was synthesized for evaluation as potential antiherpetic agents. The compounds were prepared by the condensation of 2-acetylpyridine with thiocarbonohydrazide followed by treatment with isocyanates or isothiocyanates. Many were found that were potent inactivators of ribonucleotide reductase encoded by HSV-1 and weaker inactivators of human enzyme. Several thiocarbonohydrazones (e.g. 38 and 39) inactivated HSV-1 ribonucleotide reductase at rate constants as much as seven times that of lead compound 2. In general, those substituted with weak electron-attracting groups offered the best combination of potency and apparent selective activity against the HSV-1 enzyme. Seven new thiocarbonohydrazones (21, 25, 31, 36, 38, 39, and 40) were apparently greater than 50-fold more selective than 2 against HSV-1 ribonucleotide reductase versus human enzyme. The results indicated new compounds worthy of further study as potentiators of acyclovir in combination topical treatment of herpes virus infections.