Chronic leukaemias.

Chronic leukaemias. In recent years many subtypes of CLL and some CML variants have been recognized throughout the world by means of careful clinical, epidemiological, immunological, molecular biological and viral studies. Most striking has been the establishment of a close association between certain immunophenotypical subtypes of CLL and infection with HTLV-I and possibly HTLV-II. CLL has consistently been shown to have a strong genetic component and a low incidence among Asians, but a growing body of evidence also links this major leukaemia type with environmental factors including solvents, unidentified farming and other occupational exposures. In contrast, CML is characterized by few genetic associations, relatively homogenous world-wide distribution, greater frequency in Blacks than in Whites, little evidence of viral aetiology, and evidence that exposures to ionizing radiation, benzene and possibly other chemical agents are important aetiological factors. Most studies suggest that acquired rather than genetic factors are of greater importance in the aetiology of CML, but this conclusion is somewhat difficult to reconcile with the relatively small variation in incidence rates internationally. Common to both disorders in most populations are an increasing incidence with age, male predominance, and stability of incidence, survival and mortality over the years, exclusive of improved survival of CML following allogeneic bone marrow transplantation.


Insulin, glucagon and somatostatin in fetal rat islets maintained free-floating in culture: immunocytochemistry and radioimmunoassay. Fetal rat islets maintained free-floating in tissue culture represent a source of B-cells. Because we recently noted the occurrence of other cell types during long-term tissue culture, this in vitro model was used to examine the possible development of non B-cells. The changes in the numbers and percentages of B, A and D-cells in vitro were estimated by counting the hormone-positive cells after immunocytochemical staining. Insulin, glucagon, and somatostatin contents were determined in extracts of the cultured tissue. The experiments described here showed that the cultured islets maintained their viability over a two-week culture period, as evidenced by the increase of both the number of B-cells per islet and the DNA content per islet. During the first few days of culture, immunocytochemically stained free-floating islets indicated the presence of rare A- and D-cells at the periphery of B-cells; thereafter, numerous A- and D-cells were seen interdigitating with B-cells. Expressed per islet, the number of A- and D-cells increased during the culture; within the endocrine cell population, the percentage of these cells increased with time, at the expense of the percentage of B-cells. The glucagon and somatostatin contents of the free-floating islets were also increased. These converging observations suggest that additional non B-cells may have been produced by free-floating islets during long-term tissue culture.


Chronic parastomal ulcers: spectrum of dermatoses. Parastomal ulcers that develop after stoma surgery have reportedly been associated with recurrent inflammatory bowel disease and chronic infection. We report 13 patients with refractory parastomal ulcers, which occurred at a mean of 11 years after surgery. Parastomal ulcers in eight patients were the result of dermatologic conditions (e.g., contact dermatitis, bullous pemphigoid, lichen sclerosus et atrophicus, eczema, or psoriasis) or contact ulcers from dermatitis of the skin around the stoma and faceplate pressure. These ulcers healed after treatment with topical medications at a mean of 4 weeks. Five patients with inflammatory bowel disease had pyoderma gangrenosum ulcerations, which healed with systemic treatment at a mean of 25 weeks. Thus nonpyoderma gangrenosum parastomal ulcerations that occur late after stoma surgery require early enterostomal therapy nursing intervention and dermatologic evaluation, since they respond rapidly to appropriate local therapy.


Differences in intrinsic efficacy of benzodiazepines are reflected in their concentration-EEG effect relationship. 1. The relevance of EEG effect parameters as a measure of the central nervous system effects of benzodiazepines was evaluated. The concentration-EEG effect relationships of the benzodiazepine agonist midazolam, partial agonist bretazenil, antagonist flumazenil and inverse agonist Ro 19-4603 were quantified and compared with the intrinsic efficacy and affinity of these compounds at the gamma-aminobutyric acid (GABA)-benzodiazepine receptor complex. 2. The pharmacokinetics and pharmacodynamics of the compounds were determined after a single intravenous bolus administration of 5 mg kg-1 midazolam, 2.5 mg kg-1 bretazenil, 10 mg kg-1 flumazenil or 2.5 mg kg-1 Ro 19-4603 to male Wistar derived rats. In a separate experiment the distribution between blood, cerebrospinal fluid and brain concentrations of these compounds was determined. A sensitive assay was developed to measure bretazenil and Ro 19-4603 concentrations in small samples of biological fluids. 3. The benzodiazepine-induced changes in amplitudes in the 11.5-30 Hz frequency band, as determined by aperiodic analysis, was used as EEG effect measure. Concentration-EEG effect relationships were derived by a pharmacokinetic-pharmacodynamic modelling procedure and in the case of midazolam, bretazenil and Ro 19-4603 successfully quantified by the sigmoidal Emax model. Large differences in maximal effect of midazolam (Emax = 73 +/- 2 microVs-1), bretazenil (Emax = 19 +/- 1 microVs-1) and Ro 19-4603 (Emax = -6.5 +/- 0.4 microVs-1) were observed, reflecting their differences in intrinsic efficacy. A close correlation was found between the EC50 values based on free drug concentration and receptor affinity as determined by displacement of [3H]-flumazenil in a washed brain homogenate at 37 degrees C. In the concentration range of receptor saturation flumazenil did not produce any changes in the EEG effect measure.4. The study demonstrated that the change in amplitudes in the 11.5-30 Hz frequency band of the EEG is a relevant measure of the pharmacological effect intensity of benzodiazepines, because it seems to reflect their affinity and intrinsic efficacy at the central GABA-benzodiazepine receptor complex.


Interference of cetirizine with the late eosinophil accumulation induced by either PAF or compound 48/80. 1. The effect of topical or systemic treatment with the histamine H1-receptor antagonist, cetirizine, on the rat pleural eosinophil accumulation induced by PAF or compound 48/80 was investigated. The number of pleural resident eosinophils increased 6 h after the intrathoracic (i.t.) injection of PAF (1 microgram/cavity), peaked within 24 h and persisted significantly augmented for at least 96 h. Compound 48/80 (25 micrograms/cavity) also produced a long lasting pleural eosinophilia but this was first noted only 24 h after stimulation. 2. Intraperitoneal pretreatment with cetirizine inhibited eosinophilia induced by either PAF (ED50 = 19 mg kg-1) or compound 48/80 (ED50 = 14 mg kg-1) whereas meclizine, another histamine H1-receptor antagonist, was inactive. 3. Administered locally, cetirizine (5 and 15 micrograms/cavity) also dose-dependently inhibited both PAF- and compound 48/80-induced eosinophilia, providing evidence that its inhibitory effect is not due to any action upon circulating eosinophils. Consistent with this result, incubation of isolated peritoneal eosinophils with cetirizine failed to modify in vitro eosinophil migration caused by PAF. 4. Since the late eosinophilia induced by PAF may depend on the synthesis of a transferable protein mediator, cetirizine was administered to donor or recipient rats in order to determine whether it interferes with the generation or with the expression of this protein. We showed that only the treatment of recipient rats abolished the transfer of the eosinophilotactic activity, indicating that cetirizine does not modify the generation but inhibits the expression of this activity. 5. Our findings indicate that cetirizine is able to inhibit eosinophil accumulation by acting locally. The mechanism is neither related to its recognized ability to antagonize histamine H,-receptors nor to a direct action upon circulating eosinophils.


5-Methylurapidil may discriminate between alpha 1-adrenoceptors with a high affinity for WB4101 in rat lung. The alpha 1-adrenoceptors of rat lung with a high affinity for [3H]-prazosin were subdivided into two populations (high and low affinity sites) by WB4101 and 5-methylurapidil but the proportions were different between both drugs. After pretreatment with chlorethylclonidine, WB4101 recognized only high affinity sites, while 5-methylurapidil still detected high and low affinity sites. These results indicate that alpha 1-adrenoceptors with a high affinity for WB4101 are not homogeneous in the rat lung, suggesting the possible existence of a new alpha 1-adrenoceptor subtype in addition to alpha 1A and alpha 1B subtypes.


The antagonism of benzodiazepine withdrawal effects by the selective cholecystokininB receptor antagonist CI-988. The ability of a selective cholecystokininB (CCKB) receptor antagonist, CI-988, to block benzodiazepine withdrawal effects was examined in mice. The discontinuation of twice daily administration of diazepam (1 mg kg-1, i.p.) induced withdrawal anxiogenesis and a proconvulsant effect. In contrast, no such effects were seen following withdrawal from similar administration of CI-988. However, CI-988 dose-dependently (0.001-1.0 mg kg-1, s.c.) antagonized both the anxiogenesis and the proconvulsant effect following diazepam-withdrawal.


Effects of alpha-methyl-para-tyrosine on monoamine levels in the Japanese quail: sex differences and testosterone effects. Experiments were performed to obtain more information on the regulation by steroids of catecholaminergic systems in the brain of Japanese quail. Dose-response and time-response experiments were first performed to determine optimal conditions for measuring turnover in the quail brain. The norepinephrine and dopamine turnover were then estimated in microdissected brain nuclei of birds that were either sexually mature or gonadectomized or gonadectomized and treated with testosterone. Two major facts that bear direct relationship with the control of masculine reproductive behavior were demonstrated. On one hand, the dopamine turnover in the medial preoptic nucleus, a sexually dimorphic brain structure which is critically implicated in the control of copulatory behavior was much higher in male than in female quail irrespective of the hormonal condition of the birds. On the other hand, norepinephrine concentrations appeared to be higher in several nuclei of the female brain by comparison with males. These sex differences might represent part of the causal factors that underlie the sex dimorphism in reproductive behavior in quail.


Golgi impregnated somatostatin immunoreactive neurons in lamina II of the rat spinal cord. The morphology of somatostatin immunoreactive (SOM-I) neurons in lamina (L) II of the rat spinal cord was determined using a combination of Golgi impregnation and immunohistochemistry. Golgi-impregnated SOM-I neurons that resembled islet, stalked and other cells were observed. Islet cells are considered to be inhibitory interneurons while stalked cells are excitatory and are thought to relay information from primary afferent neurons to L I projection cells. The heterogeneous morphology of SOM-I neurons suggest they have diverse functions.


Generation of a small cell lung cancer variant resistant to lymphokine-activated killer (LAK) cells: association with resistance to a LAK cell-derived, cytostatic factor. Cells of OS2-RA, a human small cell lung cancer line sensitive to lymphokine-activated killer (LAK) cells, were repeatedly cocultured with human LAK cells. Fourteen cycles of the coculture produced a variant, termed OS2-RA-R, capable of growing successfully in the presence of LAK cells. OS2-RA-R showed a moderate resistance to lysis by LAK cells in 4-h 51Cr release assays. OS2-RA-R acted positively as a cold target for lysis of OS2-RA by LAK cells, suggesting no loss of the binding site for LAK cells on the cell surface of the variant. On the other hand, LAK cells were shown to produce a factor capable of suppressing the proliferation of OS2-RA and certain other cell lines but not lymphocytes. Interestingly, OS2-RA-R exhibited a substantial resistance to the cytostatic activity of LAK cell supernatants. The cytostatic factor, eluted at the 57-kDa fraction in gel filtration, showed no activity of interleukin 1, gamma-interferon, transforming growth factor beta, or tumor necrosis factor. These results suggest that LAK cells exhibit antitumor activity through not only rapid cytolysis but also slow-acting cytokine production, and the successful growth of OS2-RA-R in a coculture with LAK cells is the result of acquiring resistance to these two different LAK cell phenomena.


Resistance to N-benzyladriamycin-14-valerate in mouse J774.2 cells: P-glycoprotein expression without reduced N-benzyladriamycin-14-valerate accumulation. N-Benzyladriamycin-14-valerate (AD 198) is a highly lipophilic analogue of Adriamycin with novel cytotoxic mechanisms, greater in vivo antitumor activity, and the ability to circumvent multidrug resistance due to P-glycoprotein-mediated drug efflux or decreased topoisomerase II activity. To identify the mechanism(s) which may confer AD 198 resistance, J774.2 mouse macrophage-like cells were selected for growth in cytotoxic levels of AD 198 (AD 198R). AD 198R cells exhibited over-expression of the mdr1b (P-glycoprotein) gene, cross-resistance to Adriamycin and vinblastine, and potentiation of drug cytotoxicity by verapamil. However, net intracellular accumulation of AD 198 in AD 198R cells was unchanged compared to parental cells, while Adriamycin and vinblastine accumulations were reduced 40% and 95%, respectively. AD 198 was localized in the perinuclear region of the cytoplasm in both parental and AD 198R cells, with additional vesicular compartmentalization in AD 198R cells. Verapamil-induced reversal of AD 198 resistance coincided with some drug redistribution from cytoplasmic vesicles, but without redistribution of AD 198 into the nucleus. These results suggest that AD 198 resistance was not conferred through a P-glycoprotein-mediated reduction in intracellular drug accumulation but through other cytoplasmic mechanisms, including, but not limited to, drug compartmentalization.


Deletion of chromosome 17p loci in breast cancer cells detected by fluorescence in situ hybridization. Allelic loss of tumor suppressor genes on chromosome 17p has been implicated in the progression of breast cancer. This is in principle detectable by fluorescence in situ hybridization if the loss occurs by deletion. In order to determine if detectable deletions occur in primary breast cancer, we used dual-color hybridization with chromosome 17 pericentromeric and region-specific DNA probes to study 19 primary breast cancers. The copy numbers of 17 centromere and 17p13.1 sequences were compared with the loss of heterozygosity (LOH) for probe YNZ22 at 17p13.3 detected by restriction fragment length polymorphism. Nine of 11 cases showing LOH also showed the major population of nuclei with a deletion. The remaining two tumors with LOH were trisomic for both the centromere and 17p13.1 cosmid. In contrast, seven of eight tumors without LOH had no deletions by fluorescence in situ hybridization. These data suggest that the dominant mechanism of allelic loss at 17p in breast cancer is a physical deletion and that analysis of deletions by fluorescence in situ hybridization is a rapid and sensitive approach to studying chromosomal aberrations.


Taxol sensitizes human astrocytoma cells to radiation. Taxol is a chemotherapeutic drug which acts by stabilizing microtubules, preventing normal mitosis and resulting in a block of the cell cycle at G2 and M. The drug is isolated from the yew, Taxus sp. L., and is currently being evaluated in a series of Phase II and Phase III clinical trials. Taxol blocks cells in the most radiosensitive phases of the cell cycle and thus could act as a cell cycle-specific radiosensitizer. We report the results of combined taxol-radiation exposures in the human Grade III astrocytoma cell line, G18. Taxol is a potent inhibitor of G18 cell division; a concentration of 10 nM is cytostatic for a cell population observed for at least two doubling times. Cell survival curves for G18 cells showed a significant concentration-dependent interaction between taxol and radiation. Treatment of G18 cells with a fixed taxol concentration and radiation dose showed the interaction to be dependent on the duration of taxol exposure and consequently the fraction of cells in the G2 or M phase of the cell cycle. The sensitizer enhancement ratio for 10 nM taxol at 10% survival is 1.8 and, for 1 nM taxol, it is 1.2. These results suggest that appropriate combinations of taxol have a more than additive interaction in human tissue culture and may have a role in clinical protocols.


Characterization of multidrug-resistant pituitary tumor cells. These studies were designed to investigate the role of P-glycoprotein in an endocrine cell line. Drug-resistant pituitary cells were obtained by growing GH4C1 cells in the presence of increasing concentrations of colchicine. Cells resistant to colchicine at 0.4 micrograms/ml, termed GH4C1/RC.4, exhibited the multidrug-resistance phenotype, as the LD50 values for colchicine, puromycin, actinomycin D, and doxorubicin were between 8 and 30 times greater than the corresponding values for the parental GH4C1 cells. Verapamil at 10 microM increased the sensitivity of GH4C1/RC.4 cells to colchicine, puromycin, and actinomycin D, almost completely reversing the drug resistance. Flow cytometry and fluorescence microscopy were used to demonstrate that GH4C1/RC.4 cells retained less rhodamine 123 than GH4C1 cells, and that the rate of efflux of rhodamine 123 was much faster for GH4C1/RC.4 cells. Immunocytochemical staining with a monoclonal antibody, C219, to the 170-kilodalton P-glycoprotein showed directly that GH4C1/RC.4 cells overexpress P-glycoprotein. We used drug-resistant pituitary cells to assess the possible role of P-glycoprotein in uptake and efflux of several hormones. At equilibrium, GH4C1 and GH4C1/RC.4 cells bound similar amounts of [125I]L-triiodothyronine and [125I]L-thyroxine, and verapamil did not alter either equilibrium binding or thyroid hormone efflux kinetics. Multidrug-resistant GH4C1/RC.4 cells retained less [3H]hydrocortisone than parental GH4C1 cells at equilibrium, and verapamil increased the equilibrium concentration of [3H]hydrocortisone 3.6-fold. The effect of verapamil was due to its ability to reverse multidrug resistance, since two other chemosensitizers, quinidine and vinblastine, increased [3H]hydrocortisone retention as effectively as verapamil but another calcium channel blocker, nifedipine, had no effect. The drug-resistant GH4C1/RC.4 line synthesized more GH (290%) and much less PRL (5%) than the parent. Hydrocortisone stimulated GH synthesis and inhibited PRL similarly in GH4C1 and GH4C1/RC.4 cells. The results show that the GH4C1/RC.4 line is multidrug-resistant and overexpresses the 170-kilodalton P-glycoprotein and suggest that the P-glycoprotein pump contributes to hydrocortisone kinetics.


Tamoxifen attenuates pulsatile growth hormone secretion: mediation in part by somatostatin. Tamoxifen, a partial competitive antagonist to the estrogen receptor, is a potent inhibitor of the proliferation of experimental mammary carcinoma in the rat and is widely used clinically in the treatment of breast cancer. Blockade of estrogen receptors present on neoplastic cells represents the classic mechanism of action of tamoxifen, but the drug has a variety of other actions that may contribute to its antiproliferative properties. While it is recognized that estrogens play an important role in modulating pulsatile GH release, the effect of antagonists to sex steroid receptors on GH secretory dynamics has not previously been described. In the present study we examined the effect of tamoxifen on pulsatile GH secretion in free-moving adult male and female rats. The drug, when administered in a manner previously shown to be associated with antineoplastic activity, caused a marked suppression of the amplitude of spontaneous GH secretory bursts and significantly reduced mean 6-h plasma GH levels in both sexes compared to those in their respective peanut oil-injected controls. Inhibition of spontaneous GH pulses persisted for up to 7 weeks after tamoxifen administration in both sexes. Immunoneutralization of endogenous somatostatin in tamoxifen-treated male rats completely restored both GH pulse amplitude (121.6 +/- 9.5 vs. 62.5 +/- 13.5 ng/ml in tamoxifen-treated rats given normal sheep serum; P less than 0.02) and mean 6-h plasma GH levels (53.3 +/- 6.6 vs. 17.9 +/- 3.6 ng/ml in normal sheep serum-treated rats; P less than 0.01) to levels observed in our peanut oil-injected controls. These results demonstrate that 1) tamoxifen has potent inhibitory effects on pulsatile GH secretion; and 2) the blunting of GH pulse amplitude by tamoxifen is mediated at least in part by increased release of endogenous somatostatin. These findings motivate further investigation of the clinical significance of tamoxifen-induced suppression of GH secretion in relation to the antineoplastic activity of this commonly used drug.


Purification and sequencing of molluscan insulin-related peptide II from the neuroendocrine light green cells in Lymnaea stagnalis. The growth-controlling neuroendocrine light green cells of the freshwater snail, Lymnaea stagnalis, express a family of genes encoding structurally related, yet distinct, molluscan insulin-related peptides (MIPs). In the present study one of these peptides, MIP II, has been isolated and structurally identified. MIP II is a heterodimer of A and B chains connected by disulfide bonds. Both chains are N-terminally blocked with pyroglutamate. After cleaving of the A and B chains and deblocking with pyroglutamate amino-peptidase their sequences have been determined as: A chain: pQRTTNLVCECCFNYCTPDVVRKYCY and B chain: pQSSCSLSSRPHPRGICGSNLAGFRAFICSNQNSPS. In comparison with the MIP II sequence based on complementary DNA studies, it is clear that the two C-terminal amino acid residues of the B chain are posttranslationally removed. In addition, the glutamic acid residue in A chain was recovered in very low yields during Edman degradation, suggesting that the residue may be posttranslationally modified.


Modulation of glucocorticoid induction of stably transfected tyrosine aminotransferase gene constructs involves elements up-stream of the glucocorticoid-responsive element. Previous studies have documented that the amount of agonist activity expressed by the antiglucocorticoid dexamethasone 21-mesylate (Dex-Mes) for tyrosine aminotransferase (TAT) induction in two rat hepatoma cell lines (Fu5-5 and HTC) is greater in Fu5-5 cells and could be varied in each cell line with changes in cell density. We have proposed that both phenomena are mediated by the binding of a trans-acting factor, the concentration or activity of which is lower in HTC cells. We have now used DNase-I hypersensitivity studies to identify a possible binding site for this factor at around -3.6 kilobases (kb) of the TAT gene. Fu5-5 and HTC cells were then stably transfected with hybrid constructs either with (3.9TATCAT) or without (2.9TATCAT) this region of the TAT gene fused up-stream of a chloramphenicol acetyltransferase (CAT) reporter gene. High levels of Dex-Mes agonist activity for the induction of CAT activity in Fu5-5 cells were seen only with the 3.9TATCAT construct, indicating that the 0.97-kb region unique to this construct controlled the high levels of Dex-Mes agonist activity. Furthermore, variations in Fu5-5 cell density caused major quantitative changes in the amount of Dex-Mes agonist activity only in cells containing the 3.9TATCAT construct, consistent with the same 0.97-kb sequences also controlling the variations in Dex-Mes agonist activity. Additional studies at high and low cell densities revealed that the modulation of Dex-Mes agonist activity for both the endogenous TAT gene and the transfected TAT/CAT gene was not due to changes in the start site of gene transcription. These studies both support our previous hypothesis that modulation of Dex-Mes agonist activity results from changes in a trans-acting factor and localize a necessary cis-acting element to sequences between -3.9 and -2.9 kb of the TAT gene. These studies, thus, define a potentially new element for glucocorticoid regulation of TAT gene transcription.


Transfer of cadmium from a sandy acidic soil to man: a population study. This population study included 230 subjects (age range 20-83 years) who consumed vegetables grown in kitchen gardens on a sandy acidic soil (mean pH approximately 6.3). The study investigated the association between the Cd (cadmium) levels in blood and urine and the Cd concentration in the soil (range 0.2-44 ppm). Seventy-six subjects were current smokers and 122 participants lived in a district with known Cd pollution. Urinary Cd in the 230 subjects averaged 8.7 nmole/24 hr, (range 1.3 to 47 nmole/24 hr) after age adjustment positively correlated with the Cd level in the soil; a twofold increase of the Cd concentration in the soil was accompanied by a 7% rise in urinary Cd in men (R2 = 0.05; P = 0.04) and by a 4% rise in women (R2 = 0.02; P = 0.05). Blood Cd averaged 11.5 nmole/liter (range 1.8-41 nmole/liter) and was negatively associated with the Cd level in the soil. After adjustment for significant covariates (smoking and serum gamma-glutamyl transpeptidase in both sexes, and age and serum ferritin in women), a twofold increase in the Cd concentration in the soil was accompanied by a 6% decrease in blood Cd in men (R2 = 0.03; P = 0.09) and by a 10% decrease in women (R2 = 0.06; P less than 0.01). In conclusion, in a rural population, consuming vegetables grown on a sandy acidic soil, 2 to 4% of the variance of urinary Cd was directly related to the Cd level in the soil. The negative correlation with blood Cd, a measure of more recent exposure, was biased by the implementation of preventive measures in the polluted district.


Gadolinium ion inhibits loss of metabolites induced by osmotic shock and large stretch-activated channels in bacteria. Bacteria subjected to a hypotonic osmotic shock lose internal ions and also metabolites, without lysis of the cells. We show that the presence in the shock medium, at submillimolar concentrations, of the ion gadolinium, recently shown to block stretch-activated channels in Xenopus oocytes [Yang, X.-C. & Sachs, F. (1989) Science 243, 1068-1071], was sufficient to inhibit shock-induced release of metabolites such as lactose and ATP in Escherichia coli and ATP in Streptococcus faecalis. Moreover, gadolinium was observed, in patch-clamp experiments, to inhibit the giant stretch-activated channels of E. coli, S. faecalis. and Bacillus subtilis. Taken together, these data suggest that stretch-activated channels are localized in the cytoplasmic membrane of Gram-negative and Gram-positive bacteria, where they control the efflux of osmotic solutes, thus probably playing a major role in the response to hypotonic osmotic shock.


GHox-7: a chicken homeobox-containing gene expressed in a fashion consistent with a role in patterning events during embryonic chick limb development. Homeobox-containing (HOX) genes are thought to be involved in the regulation of pattern formation and specification of positional information during vertebrate limb development. We report the isolation from a chick limb bud cDNA library of several overlapping chicken HOX cDNAs, which on the basis of their nucleotide and deduced amino acid sequences have been identified as corresponding to the chicken cognate of mouse Hox-7.1. The gene encoding these chicken (Gallus) HOX cDNAs has been designated GHox-7, and is a member of a family of vertebrate HOX genes that are highly similar in sequence to the Drosophila msh gene. GHox-7 encodes an mRNA transcripts of about 1.8-2.0 kb that is expressed at early stages of chick limb development. In situ hybridization analysis has revealed that GHox-7 is expressed in limb bud mesoderm in a temporal and spatial fashion. This is consistent with its involvement in specifying anterior positional identity and/or in the response of limb mesenchymal cells to the apical ectodermal ridge (AER), which directs polarized proximodistal limb outgrowth. At early stages (stages 20-21) of chick limb development when positional values along the anterior-posterior (A-P) axis are being specified, GHox-7 exhibits an asymmetric arc of expression extending from the anterior border of the limb bud to the mesenchymal cells directly subjacent to the AER.(ABSTRACT TRUNCATED AT 250 WORDS)


Expression of the murine Dlx-1 homeobox gene during facial, ocular and limb development. We have analysed the expression pattern of the mouse homeobox containing gene Dlx-1. This gene harbors a homeodomain related to that found in the Drosophila distal-less (dll) gene. In addition to its expression in the developing forebrain, Dlx-1 is transcribed in several structures containing cells of neural crest origin such as the facial mesenchyme and various elements of the peripheral nervous system. Dlx-1 transcripts are also detected in the differentiating retina and during limb morphogenesis, in the apical ectodermal ridge. The possible involvement of Dlx-1 during facial, ocular and limb development is discussed.


Chronic toxicity and carcinogenicity studies with the beta-adrenoceptor antagonist levobunolol. The chronic toxicity and carcinogenicity of levobunolol, a nonselective beta-adrenoceptor antagonist, was evaluated in Swiss mice and Wistar rats. The drug was administered in the diet to mice at 0, 12, 50, and 200 mg/kg/day for 80 weeks and to rats at 0, 0.5, 2, 5, 30, and 180 mg/kg/day for 2 years. In mice, uterine leiomyomas were present in 4 of 50 females at 200 mg/kg but not in any other group. The incidences of other tumor types, as well as pathologic findings, were comparable among groups. In rats, significant body weight gain suppression occurred at 5, 30, and 180 mg/kg. Brown discoloration of perianal fur and steel-gray discoloration of hairless skin were evident in high-dose rats. A generalized steel-gray discoloration of internal organs and tissues occurred in the 30 and 180 mg/kg groups. No other differences between treated and control groups were evident. The clinical relevance of the increased incidence of uterine leiomyoma in mice is questionable because it occurred only in one species at more than 200 times the projected therapeutic dose.


The ocular pathology of Norrie disease in a fetus of 11 weeks' gestational age. The ocular pathology of Norrie disease was studied for the first time in a fetus of 11 weeks' gestation, following prenatal diagnosis using genetic markers for Norrie disease and elective abortion. The eyes were histologically normal, with no evidence of primary neuroectodermal maldevelopment of the retina, previously postulated to be the cause of the ocular changes. We believe that the retinal and other manifestations of Norrie disease are the result of a primary abnormality of vascular proliferation, probably in relation to persistent hyperplastic primary vitreous after approximately 14 weeks' gestation. We postulate that the ocular and otological effects of Norrie disease may be due to a genetically mediated abnormality of secretion of, or sensitivity to, angiogenic growth factors at endodermal-neuroectodermal interfaces during fetal and postnatal development.


[Study of the signal transduction system of platelet alpha 2-adrenergic receptors in endogenous depression]. Researches in the mechanisms of action of antidepressant drugs have recently suggested that endogenous depression is related to an altered sensitivity of alpha-2 adrenergic receptors. Since this hypothesis is difficult to study in the central nervous system of human subjects directly, the alpha-2 adrenergic receptors on peripheral-blood platelets have been used as an accessible and convenient marker of the receptor function. Although the inhibition of adenylate cyclase, via the GTP-binding protein termed Gi, was believed to be the exclusive mechanism of alpha-2 adrenergic receptor action in the platelet, this concept has become less tenable. We have recently indicated that epinephrine stimulates phosphoinositide (PI) hydrolysis by activating alpha-2 adrenergic receptors in human platelets [Life Sci., 741-747 44 (1989)]. This method involves the measurement of the accumulation of [3H] -inositol-1-phosphate (IP-1) as an index of PI hydrolysis; lithium is added to inhibit the metabolism of IP-1, thus giving an enhanced signal. In addition, this PI response elicited by epinephrine was found to be inhibited in a concentration-dependent manner by treatment of platelets with dibutyryl cyclic AMP and 8-bromo-cyclic GMP which are known as potent inhibitors for platelet activation, and may therefore be a useful biochemical index for the study of the signal transduction system of alpha -2 adrenergic receptors. In the present study, the platelet alpha-2 adrenergic receptor-mediated PI responses were assessed in 15 unmedicated patients with endogenous depression and 15 age -and sex-matched control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)


[Clinical evaluation of N-acetyl-beta-D-glucosaminidase on prediction of diabetic nephropathy]. To assess whether urinary N-acetyl-beta-D-glucosaminidase (NAG) could be used as a predictor of diabetic nephropathy, renal tubular enzymes such as NAG and gamma-glutamyl transpeptidase (gamma GTP), albumin, total protein and beta 2-microglobulin (BMG) in urine and/or serum were measured in various stages of diabetic nephropathy. As a predictor of diabetic nephropathy, urinary NAG was the most useful indicator among of them. Urinary gamma GTP had no clinical benefit on early detection of diabetic nephropathy although in cis-platin induced nephrotoxicity both urinary gamma GTP and NAG increased in parallel. Increase of urinary NAG appeared in diabetic patients prior to clinical proteinuria. With appearance of proteinuria, urinary NAG more increased. Urinary NAG correlated significantly with HbAlc and BMG in serum (sBMG). It is therefore needed for clinical application of urinary NAG as a predictor of diabetic nephropathy that control states of blood glucose in the patients should be considered. However, the results of sequential measurements of urinary NAG, sBMG and HbAlc in 78 diabetic patients for 18-month period showed that only urinary NAG was a responsible factor for elevation of sBMG known as an indicator of deterioration of renal function. These results indicate that renal tubular damage may already exist in early-stage of diabetic nephropathy, and that increase of urinary NAG activity is a useful predictor of diabetic nephropathy.


Effect of adenosine and inosine on carbon tetrachloride-induced liver damage in rats. Liver damage induced in rats by carbon tetrachloride (CCL4) was obvious macroscopically as well as microscopically in stained sections. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma glutamyl transpeptidase (gamma-GT) were also significantly raised. Adenosine and inosine effectively countered the damage when these were given before and during the period during which CCl4 produces the typical damage. The beneficial effect was seen in biochemical as well as pathological studies.


Some aspects of vascular pharmacology of frog (Rana tigrina). Vascular autonomic receptors in amphibians exhibit difference from more evolved mammalian species. Vascular perfusion studies in frog indicate constrictions by prominent muscarinic but rudimentary nicotinic constrictive regulation by cholinergic systems. Difference from classical effect-patterns of pharmacological interventions, observed in the study, make room to visualise complexity of additional regulatory mechanisms.


[Psoriasis and beta-blockade].

[Psoriasis and beta-blockade]. Psoriasiform skin eruptions during treatment with beta-adrenergic blocking drugs have recently attracted increasing attention and led the Bundesgesundheitsamt [German Office of Public Health] to publish general information on this issue. Today's knowledge on the clinical picture, pharmacology, and the pathogenesis is discussed, with reference to the latest experimental data. The blockade of keratinocyte beta-adrenergic receptors may play a key role in the aetiopathology of this unwanted side-effect.


Cloning and characterization of the groESL operon from Bacillus subtilis. The sequence of the 10 N-terminal amino acids of a Bacillus subtilis protein that cross-reacts with antibody to Escherichia coli GroEL was used to design a set of degenerate oligonucleotide probes. These probes identified a clone which carries almost the entire groESL operon from a B. subtilis subgenomic library. By chromosomal walking, an additional fragment carrying the 3' end of groESL and its flanking sequence was isolated. Sequence analysis revealed two open reading frames (ORFs) in the cloned DNA. The upstream ORF encodes a 10-kDa protein which has 47% amino acid identity with E. coli GroES. The downstream ORF encodes a 58-kDa protein which is 62% identical to E. coli GroEL. A 2.1-kb groESL mRNA from B. subtilis was detected independently by Northern (RNA) blot analyses with a groES- and a groEL-specific probe. This demonstrated that groES and groEL are in an operon. The groESL promoter was located by using a promoter-probing plasmid, and the apparent transcription start site was mapped by primer extension analysis. The same promoter is utilized under normal and heat shock conditions. This promoter has the same features as a typical sigma A promoter. A strain in which the groESL operon was under the control of the sucrose-inducible sacB promoter was created. With this strain, it was possible to show that both groES and groEL are essential genes under both normal and heat shock conditions.


Cloning, sequencing, mapping, and transcriptional analysis of the groESL operon from Bacillus subtilis. Using a gene probe of the Escherichia coli groEL gene, a 1.8-kb HindIII fragment of chromosomal DNA of Bacillus subtilis was cloned. Upstream sequences were isolated as a 3-kb PstI fragment. Sequencing of 2,525 bp revealed two open reading frames in the order groES groEL. Alignment of the GroES and GroEL proteins with those of eight other eubacteria revealed 50 to 65% and 72 to 84% sequence similarity, respectively. Primer extension studies revealed one potential transcription start site preceding the groESL operon (S) which was activated upon temperature upshift. Northern (RNA) analysis led to the detection of two mRNA species of 2.2 and 1.5 kb. RNA dot blot experiments revealed an at least 10-fold increase in the amount of specific mRNA from 0 to 5 min postinduction, remaining at this high level for 10 min and then decreasing. A 9-bp inverted repeat within the 5' leader region of the mRNA might be involved in regulation of the heat shock response. By using PBS1 transduction, the groESL operon was mapped at about 342 degrees.


Biosynthetic precursors of deazaflavins. The incorporation of 13C- and 14C-labeled precursors into 5-deaza-7,8-didemethyl-8-hydroxyriboflavin (factor F0) was studied with growing cells of Methanobacterium thermoautotrophicum. 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione was incorporated into the deazaflavin and into riboflavin without dilution. Tyrosine and 4-hydroxyphenylpyruvate were incorporated into the deazaflavin and into cellular protein. 4-Hydroxybenzaldehyde was not incorporated. A reaction mechanism is proposed for the formation of the deazaflavin chromophore from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and tyrosine or 4-hydroxyphenylpyruvate.


Development of multipurpose peroxisomes in Candida boidinii grown in oleic acid-methanol limited continuous cultures. We have studied the development and metabolic significance of peroxisomes in the yeast Candida boidinii following adaptation of the organism to cultivation conditions which require the simultaneous presence and activity of two independent peroxisome-mediated pathways for growth. After the addition of methanol to oleic acid-grown cells at late exponentional growth, a number of new small peroxisomes developed which, apart from the presence of beta-oxidation enzymes, were characterized by the presence of enzymes involved in methanol metabolism (alcohol oxidase and dihydroxyacetone synthase). The latter proteins, however, were absent in the larger organelles which were originally present in the oleic acid-grown cells prior to the addition of methanol and which contained only enzymes of the beta-oxidation pathway. Subsequent experiments on cells from continuous cultures grown on a mixture of oleic acid and methanol at steady-state conditions revealed that both the enzymes of the beta-oxidation pathway and those involved in methanol metabolism were found in one and the same compartment. Thus, under these conditions the cells contained peroxisomes which were concurrently involved in the metabolism of two different carbon sources simultaneously used for growth. Our results indicated that the heterogeneity in the peroxisomal population of a single cell, observed in the transient state following the addition of methanol, is only temporary and due to heterogeneity among these organelles with respect to their capacity to incorporate newly synthesized matrix proteins.


Proline biosynthesis in Saccharomyces cerevisiae: molecular analysis of the PRO1 gene, which encodes gamma-glutamyl kinase. The PRO1 gene of Saccharomyces cerevisiae encodes the 428-amino-acid protein gamma-glutamyl kinase (ATP:L-glutamate 5-phosphotransferase, EC 2.7.2.11), which catalyzes the first step in proline biosynthesis. Amino acid sequence comparison revealed significant homology between the yeast and Escherichia coli gamma-glutamyl kinases throughout their lengths. Four close matches to the consensus sequence for GCN4 protein binding and one close match to the RAP1 protein-binding site were found in the PRO1 upstream region. The response of the PRO1 gene to changes in the growth medium was analyzed by measurement of steady-state mRNA levels and of beta-galactosidase activity encoded by a PRO1-lacZ gene fusion. PRO1 expression was not repressed by exogenous proline and was not induced by the presence of glutamate in the growth medium. Although expression of the PRO1 gene did not change in response to histidine starvation, both steady-state PRO1 mRNA levels and beta-galactosidase activities were elevated in a gcd1 strain and reduced in a gcn4 strain. In addition, a pro1 bradytrophic strain became completely auxotrophic for proline in a gcn4 strain background. These results indicate that PRO1 is regulated by the general amino acid control system.


Characterization of a major polymorphic tandem repeat in Mycobacterium tuberculosis and its potential use in the epidemiology of Mycobacterium kansasii and Mycobacterium gordonae. In this study, the occurrence of repeated DNA sequences in the chromosome of Mycobacterium tuberculosis was investigated systematically. By screening a M. tuberculosis lambda gt-11 gene library with labeled total chromosomal DNA, five strongly hybridizing recombinants were selected, and these contained DNA sequences that were present in multiple copies in the chromosome of M. tuberculosis. These recombinants all contained repeated sequences belonging to a single family of repetitive DNA, which shares homology with a previously described repeated sequence present in recombinant pPH7301. Sequences analysis of pPH7301 showed the presence of a 10-bp sequence that was tandemly repeated and invariably separated by 5-bp unique spacer sequences. Southern blot analysis revealed that the majority of the repeated DNA in M. tuberculosis is composed of this family of repetitive DNA. Because the 10-bp repeats are slightly heterogeneous in sequence, we designated this DNA as a major polymorphic tandem repeat, MPTR. The presence of this repeated sequence in various other mycobacterial species was investigated. Among the MPTR-containing mycobacterial species the chromosomal location of the repetitive DNA is highly variable. The potential use of this polymorphism in the epidemiology of mycobacterioses is discussed.


Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) beta-galactosidase from Escherichia coli. The covalent intermediate formed during catalysis by the lac Z beta-galactosidase from Escherichia coli can be trapped by reaction of the enzyme with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-galactopyranoside, thereby inactivating the enzyme. Kinetic parameters for this inactivation process with the holo- and apo-enzymes have been determined. The intermediate so formed turns over only very slowly (t1/2 = 11.5 h) resulting in reactivation of the enzyme. The nucleophilic amino acid involved has been identified as Glu-537 by using a tritium-labeled inactivator to label the enzyme, then cleaving the labeled protein into peptides and purifying and sequencing the labeled peptide. This residue is conserved in five homologous beta-galactosidases and is different from that (Glu-461) proposed to be the nucleophile (Herrchen, M., and Legler, G. (1984) Eur. J. Biochem. 138, 527-531) on the basis of affinity labeling studies with conduritol C cis-epoxide. A role for glutamic acid residue 461 as the acid/base catalyst is proposed and justified.


Identification of Gln726 in nidogen as the amine acceptor in transglutaminase-catalyzed cross-linking of laminin-nidogen complexes. The laminin-nidogen complex, the most abundant noncollagenous component of basement membranes, was recently shown to be a specific substrate for tissue transglutaminase (Aeschlimann, D., and Paulsson, M. (1991) J. Biol. Chem. 266, 15308-15317). Saturation experiments to determine the number of amine acceptor site(s) indicated a single reactive Gln residue in nidogen and none in laminin. Murine nidogen was labeled with [3H]putrescine in the tissue transglutaminase-catalyzed reaction, and two major radioactively labeled fragments, T70 and T40, were isolated after limited trypsin digestion. NH2-terminal sequencing showed that T40 is contained in T70 and corresponds to the rodlike structure of nidogen, made up of epidermal growth factor-like repeats. Three radioactively labeled peptides, obtained by extensive trypsin digestion of reduced and alkylated T40, were sequenced. In all a single residue, Gln726, was found to contain label. Sequencing of additional peptides, obtained after further treatment of the largest radioactively labeled peptide with endoproteinase Asp-N, gave the same result. Gln726 is located in an exposed loop between the second and the third EGF-like repeat in nidogen. This site is also conserved in the human sequence.


Regulation of heme-controlled eukaryotic polypeptide chain initiation factor 2 alpha-subunit kinase of reticulocyte lysates. We have obtained highly purified preparations of the heme-controlled eukaryotic initiation factor 2 alpha-subunit (eIF-2 alpha) kinase (HCI) from rabbit reticulocyte lysates containing five different polypeptides. One of these is a 87-kDa (p87) phosphopeptide which appears to show an autokinase activity. The controlled digestion with trypsin of HCI preparations leads to the suggestion that phosphorylation of p87 is not needed for kinase activity and, furthermore, that another 89-kDa polypeptide could be the kinase catalytic subunit. In agreement with this, monoclonal antibodies directed against p87 do not interfere with eIF-2 alpha kinase activity. Moreover, the anti-p87 antibodies and those directed against the mammalian 90-kDa heat shock protein recognize the same p87 polypeptide from rabbit reticulocyte lysates. Upon incubation of the HCI preparation with hemin (5-10 microM), the eIF-2 alpha kinase is converted into an inactive form and appears to become associated with related peptides forming high molecular weight complexes which can be reversibly activated by 2-mercaptoethanol. The maintenance of the integrity of the porphyrin ring is absolutely required for kinase inactivation and although the presence of metal ion is not essential, the iron and cobalt metalloporphyrins are more effective than protoporphyrin IX. The formation of the inactive form of HCI by hemin is prevented by either N-ethylmaleimide, monoclonal antibodies directed against p87, or phosphorylation of p87. The data strongly suggest that hemin regulates eIF-2 alpha kinase activity by promoting formation of the inactive dimer HCI.p87 via disulfide bonds and direct binding of hemin. A model of HCI regulation is discussed.


Purification and characterization of the neu/erb B2 ligand-growth factor from bovine kidney. A neu/erb B2 ligand growth factor (NEL-GF) was purified to homogeneity from bovine kidney by a procedure involving ammonium sulfate fractionation (35-70% saturation) followed by sequential column chromatography on DEAE-cellulose (DE52), Sulfadex (sulfated Sephadex G-50), heparin-Sepharose 4B, and Superdex 75 (fast protein liquid chromatography). NEL-GF was found to be a 25-kDa polypeptide according to the analysis by gel filtration on Superdex 75 and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NEL-GF stimulated the tyrosine-specific autophosphorylation of the neu/erb B2 gene product purified by immunoabsorbent and tyrosine-specific phosphorylation of the neu/erb B2 gene product in intact dihydrofolate reductase (DHFR/G-8 cells (NIH 3T3 cells transfected with rat c-neu). NEL-GF also down-regulated the cell surface neu/erb B2 gene product in DHFR/G-8 cells. NEL-GF was mitogenic toward NIH 3T3 cells, DHFR/G-8 cells, A431 cells (human epidermoid carcinoma cells), and SK-BR-3 cells (human breast carcinoma cells) but inactive toward bovine aorta endothelial cells. NEL-GF was sensitive to 0.1% trifluoroacetic acid but resistant to 5% beta-mercaptoethanol and appeared to be distinct from a neu protein-specific activating factor (Davis, J. G., Hamuro, J., Shim, C. Y., Samanta, A., Greene, M. I., and Dobashi, K. (1991) Biochem. Biophys. Res. Commun. 179, 1536-1542) and a 30-kDa glycoprotein which competed with a monoclonal antibody for binding to the neu/erb B2 gene product (Lupu, R., Colomer, R., Zugmaier, G., Sarup, J., Shepard, M., Slamon, D., and Lippman, M. E. (1990) Science 249, 1552-1555).


A mutation in GroEL interferes with protein folding by reducing the rate of discharge of sequestered polypeptides. GroEL140, a mutant Escherichia coli chaperonin unable to support bacteriophage lambda head assembly, was purified to near homogeneity and compared to wild type GroEL (cpn60). GroEL140 exhibited a 1.5-fold lower ATPase activity relative to the wild type protein. The hydrolysis of ATP by both polypeptides was fully inhibited by an excess of ATP gamma S and partially inhibited by ADP and 5'-adenylylimidodiphosphate, suggesting that adenine nucleotides display different affinities for the ATP binding site of chaperonins. GroEL140 was more sensitive to trypsin digestion compared to wild type GroEL indicating that the mutation destabilized the conformation of the mutant. The proteolytic susceptibility of both chaperonins was similarly enhanced upon addition of ATP, ADP or non-hydrolyzable ATP analogs, providing evidence (i) of a conformational change in the chaperonin structure which is likely to drive the protein discharge process, and (ii) that hydrolysis of ATP is not required to achieve topological modifications. GroEL140 retained its ability to bind non-native ribulose bisphosphate carboxylase/oxygenase (Rbu-P2-carboxylase), but released bound proteins upon addition of ATP and GroES (cpn 10) 6-7-fold less efficiently compared to GroEL. This functional defect was shown to be related to a suboptimal, but not an absence of, interaction with GroES since (i) GroEL140 and GroES were unable to form a complex isolatable by size exclusion chromatography, and (ii) increasing the incubation time or the concentration of GroES enhanced the amount of refolded Rbu-P2-carboxylase discharged from GroEL140-Rbu-P2-carboxylase binary complexes. Pulse-chase experiments involving a double immunoabsorption technique confirmed that Rbu-P2-carboxylase remained associated two times longer with GroEL140 than with GroEL in vivo.


Liver giant mitochondria revisited.

Liver giant mitochondria revisited. To examine the correlation between the severity of alcohol induced liver damage and the presence of intracytoplasmic red bodies (defined as periodic acid-Schiff diastase negative, globular, hyaline cytoplasmic inclusions larger in size than the hepatocyte nucleolus). To investigate the incidence of intracytoplasmic red bodies (ICRBs) in non-alcoholic liver disease. Liver biopsy specimens from 53 patients with alcoholic liver disease and 50 patients with a variety of nonalcohol related liver diseases were examined by light microscopy for the presence of ICRBs. For the 53 patients with alcoholic liver disease an assessment of recent alcohol consumption was made indirectly from measurements of red cell volume (MCV) and plasma gamma-glutamyl transferase (GGT). In addition, 10 liver biopsies with alcohol induced changes and ICRBs were examined by electron microscopy for the presence of mitochondrial aberrations including enlargement. ICRBs were detected in 18 of the 53 liver biopsy specimens showing alcohol induced changes, and were more abundant in those showing more advanced changes. Those patients whose liver specimens contained ICRBs were found to have a significantly higher mean plasma GGT activity and mean MCV than those individuals whose liver biopsy specimens did not contain ICRBs. Two of the 50 liver biopsy specimens showing non-alcohol induced changes contained ICRBs. Giant mitochondria were not detected by electron microscopy, but this may reflect sampling. The results of this study indicate that ICRBs are definitely associated with alcoholic liver disease and are more likely to be found in liver biopsy specimens showing more advanced alcohol induced damage, and when recent alcohol consumption has been high.


PCNA and Ki67 expression in breast carcinoma: correlations with clinical and biological variables. To investigate the expression of two cell cycle related antigens (proliferating cell nuclear antigen (PCNA) and Ki67 related antigen) in a series of breast cancers; and the possible correlations between the PCNA and Ki67 labelling indexes (PCNA-LI and Ki67-LI) and their associations with other biological and clinicopathological variables. Ninety six ductal and 10 lobular carcinoma specimens were investigated. Samples were fixed in formalin and in Methacarnoy for localisation of PCNA. Ki67 was immunostained on frozen sections. The PCNA-LI and Ki67-LI were evaluated in relation to tumour size, mitotic count, histological grade, nodal state as well as receptor content and altered expression of the p53 gene. PCNA-LI did not correlate with Ki67-LI, nor was it associated with any other variable examined. A high KI67-LI (above the median value of 13.5) was associated with high grade and mitotic count, negative receptor content, and altered expression of the p53 gene, but not with other variables. The PCNA-LI does not seem to be a substitute for the Ki67-LI in evaluating the growth fraction in breast cancer.


c-erbB-2 oncogene product expression and prognosis in gastric carcinoma. The prognostic value of c-erbB-2 protein expression was assessed retrospectively in 87 "curative" gastrectomy specimens from patients with gastric carcinoma. Tumours were stained immunohistochemically with the specific antibody 21N. Eight (9%) cases had strong membrane staining, all of which were of the intestinal type, and lymph node metastases, which showed concordance of staining in seven cases. In contrast to studies in breast cancer, positive cases showed a trend towards better five year survival, but this did not reach significance.


Antiallergic effect of epinastine (WAL 801 CL) on immediate hypersensitivity reactions: (II). Antagonistic effect of epinastine on chemical mediators, mainly antihistaminic and anti-PAF effects. Anti-histamine and anti-PAF effects of epinastine were tested in rats, guinea pigs and rabbits. Epinastine showed a potent histamine H1-blocking effect, but the potency was slightly less than that of ketotifen in histamine-induced contraction of guinea pig ileum and histamine-induced cutaneous reactions in rats. In histamine-induced dye leakage into the nasal cavity tested in rats, the drug was slightly more potent than ketotifen and azelastine. Epinastine as well as ketotifen suppressed rabbit platelet aggregation induced by PAF at higher concentrations compared with WEB 2086, a specific PAF-antagonist. In the bronchospasm induced by PAF in guinea pigs, epinastine was more effective than ketotifen in inhibiting the bronchoconstriction, while it showed no remarkable effect on the hypotension induced by PAF. Epinastine caused a potent antagonistic effect on LTC4-induced contraction of isolated guinea pig trachea. In conclusion, the potent anti-histamine, anti-PAF and anti-LT effects of epinastine may significantly contribute to its antiallergic activity.


Effects of taxol on the macrophage function. Interactions with some immunological parameters. The effects of Taxol on some immunological parameters were investigated, in vitro, in the murine macrophage cell line J774.1. Mitochondrial dehydrogenase activity and (3H) TdR incorporation were shown to be increased in Taxol treated cells. Likewise, Taxol induced TNF alpha secretion. But Taxol seemed to be a weak inducer of the two interleukins IL-1 and IL-2, since their detection occurred late in the cell culture supernatants.


Abnormalities of glucocorticoid metabolism and the renin-angiotensin system: a four-corners approach to the identification of genetic determinants of blood pressure. To assess the feasibility and utility of a new method to identify factors associated with increased predisposition to high blood pressure in young people. Eight hundred and sixty-four people aged 16-24 years and their parents. Ladywell Medical Centre, Edinburgh, Scotland, UK. Blood pressure was measured in 864 young adults and in both of their parents. Four groups of approximately 50 offspring were selected from the corners of a scatter diagram, with offspring blood pressure scores on one axis and combined parental blood pressure scores on the other. Blood and urine samples were taken for biochemical and genetic analyses. Two groups of offspring had parents with high blood pressure and two groups had parents with low blood pressure. When parental blood pressure was low, comparison of offspring with high and low blood pressure revealed significantly higher mean body mass index in offspring with high blood pressure, but no significant elevation of biochemical or hormonal variables. When parental blood pressure was high, comparison of offspring with high and low blood pressure also revealed a significant difference in body mass index, but in addition, offspring with high blood pressure and high parental blood pressure had higher levels of angiotensinogen, cortisol and 18-OH corticosterone. Restriction fragment length polymorphism analysis revealed that 27% of offspring at the greatest genetic risk (high personal and parental blood pressure) were homozygous for the larger allele of the glucocorticoid receptor gene compared with only 9% of those at lowest genetic risk (low personal and parental blood pressure). The combined biochemical and genetic findings suggest that abnormalities of glucocorticoid metabolism and the renin-angiotensin system may help to explain genetic predisposition to high blood pressure. The new sampling method is practicable and could be applied to the investigation of other continuously distributed variables which show familial aggregation.


Properties of amino acid neurotransmitter receptors of embryonic cortical neurons when activated by exogenous and endogenous agonists. 1. The properties of receptors for amino acid neurotransmitters expressed by developing cortical neurons were studied with the use of whole-cell recording in the intact cerebral cortex of embryonic turtles in vitro. The inhibitory agonist gamma-aminobutyric acid (GABA) and the excitatory agonist glutamate were focally applied to single cells under voltage clamp, and the ionic dependence, voltage dependence, and pharmacological sensitivity of the responses were characterized. The responses mediated by a glutamate receptor subtype, the N-methyl-D-aspartate (NMDA) receptor, produced by glutamate and by evoked release of an endogenous excitatory agonist, were compared further. Fluctuation analysis was used to characterize the properties of the NMDA channels and the mechanism of action of receptor antagonists. 2. When postmitotic neurons first appeared at stage 15, all neurons tested responded to GABA with a current that reversed at the equilibrium potential for chloride ions and that was sensitive to the GABAA receptor antagonist bicuculline methiodide (BMI). As development proceeded, an increasing proportion of neurons also responded with a BMI-insensitive current that reversed near the equilibrium potential for potassium ions. This current was blocked by the GABAB receptor antagonist 3-amino-2-propyl phosponic acid (phaclofen). The GABAB agonist baclofen, however, failed to produce a detectable postsynaptic current. 3. Neurons at stage 15 showed a biphasic response to glutamate that reversed at the equilibrium potential for cations. All neurons tested showed a slow, sustained response associated with an increase in current variance compared with background, and, as development proceeded, an increasing proportion also exhibited a fast, transient response. Both fast and slow responses varied linearly with voltage in the absence of Mg2+ ions, but the addition of Mg2+ ions to the bathing medium attenuated the slow response at hyperpolarized potentials. As a result, the current-voltage relation of the slow response in the presence of Mg2+ ions exhibited a region of negative slope conductance, like that of currents mediated by NMDA receptors. 4. The fast and slow responses to glutamate differed in their pharmacological sensitivity. The fast responses were sensitive to the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whereas the slow responses were sensitive to the NMDA receptor antagonist D(-)-2-amino-5-phosphonovalerate (D-APV). 5. When cells were held at -70 mV, glutamate evoked a fluctuating current consisting of channel currents with a mean open time, tau, of 4.42 +/- 0.47 (SE) ms in early postmitotic neurons at stage 15 and 4.99 +/- 0.38 ms at stages 17-20.(ABSTRACT TRUNCATED AT 400 WORDS)


Which anti-hypertensive to add to a beta-blocker: ACE inhibitor or diuretic? Thirty-eight patients already treated with atenolol 50 mg once daily were randomly assigned to treatment with either hydrochlorothiazide (12.5-25 mg once daily) or lisinopril (10-20 mg once daily) for 8 weeks in a double-blind crossover study. Eight weeks' treatment with the combination of ACE inhibitor and beta-blocker or the diuretic and beta-blocker produced falls in blood pressure (lying: -8.4 +/- 15.4/ -7.3 +/- 80 mmHg and -6.1 +/- 15.3/ -5.2 +/- 8.8 mmHg [mean +/- SD] for lisinopril and hydrochlorothiazide respectively; standing: -10.2 +/- 14.2/8.2 +/- 9.2 mmHg and -6.8 +/- 14/ -6.3 +/- 10.3 mmHg for lisinopril and hydrochlorothiazide respectively) which were not statistically significantly different. Heart rate was significantly increased on the combination of beta-blocker and diuretic (lying: +4.3 +/- 10.7; standing: +3.2 +/- 10.0 beats/min) compared with a fall on beta-blocker+ACE inhibitor (lying; -0.5 +/- 7.6; standing: -1.5 +/- 7.4). Both therapeutic regimens were equally well tolerated. These results suggest that where patients fail to respond to monotherapy with a beta-blocker the addition of an ACE inhibitor may be as effective as the more traditional option of diuretic therapy.


Synthesis and antiallergic activity of 11-(aminoalkylidene)-6,11-dihydrodibenz[b,e]oxepin derivatives. A new series of 11-substituted 6,11-dihydrodibenz[b,e]oxepin-2-carboxylic acid derivatives was synthesized and demonstrated to be orally active antiallergic agents. These compounds are structurally related to 1 (KW-4994), which we had reported previously to be a new antiallergic agent. Most compounds synthesized exhibited potent inhibitory effects on 48-h homologous passive cutaneous anaphylaxis (PCA) in rats and on IgG1-mediated bronchoconstriction in guinea pigs. Additionally, compounds possessing a terminal carboxyl group at the 2-position of the dibenz[b,e]oxepin ring system exhibited inhibitory effects on specific [3H]pyrilamine binding to guinea pig cerebellum histamine H1 receptors, whereas these demonstrated negligible effects on specific [3H]QNB binding to rat striatum muscarinic acetylcholine M1 receptors. Structure-activity relationship studies revealed that the following key elements were required for enhanced antiallergic activities: (1) a 3-(dimethylamino)propylidene group as the side chain at the 11-position, (2) a terminal carboxyl moiety at the 2-position, and (3) a dibenzoxepin ring system. Among the compounds synthesized, (Z)-11-[3-(dimethylamino)propylidene]-6,11-dihydrodibenz [b,e]oxepin-2-acetic acid hydrochloride (16) was selected for further evaluation. It had an ED50 value of 0.049 mg/kg po in the PCA test in rats and an ID50 value of 0.030 mg/kg po in inhibiting anaphylactic bronchoconstriction in guinea pigs. Furthermore, it had a Ki value of 16 +/- 0.35 nM for the histamine H1 receptor, while it exhibited negligible CNS side effects up to a dose of 600 mg/kg po. Compound 16 is now under clinical evaluation as KW-4679.


H2 receptor antagonist or H. pylori eradication? To assess the healing and relapse rate of duodenal ulcer (DU) treated with H2 receptor antagonists in helicobacter pylori (HP) positive vs negative cases, we analysed 95 cases of endoscopically proven duodenal ulcer. H. pylori colonization was found in 73 (77%) patients before treatment. No difference was observed in the pre-treatment characteristics between patients with HP positive and HP negative duodenal ulcers. Healing rates with H2 receptor antagonist at 8 weeks were 90% and 91% respectively (NS). No difference in HP colonization was found between patients with and without healed ulcerie, 77% and 78% respectively. Relapse rate within 1 year was 50% in patients with HP positive vs 73% with HP negative cases. We conclude that duodenal ulcer healing and relapse rate is related to acid inhibition rather than HP colonization.


Morphine responsiveness of chronic pain: double-blind randomised crossover study with patient-controlled analgesia. There is controversy about whether the lack of response of some chronic pain to opioid treatment is absolute or relative. It is widely believed that nociceptive pain is responsive to opioids whereas neuropathic pain tends not to be. We have used a method of patient-controlled analgesia (PCA) with simultaneous nurse-observer measurement of analgesia, mood, and adverse effects to address these issues. Ten patients with chronic pain were given morphine at two concentrations (10 and 30 mg/ml) by PCA in two separate sessions in a double-blind randomised crossover study. Before the study a clinical judgment was made as to whether each pain was nociceptive or neuropathic. Seven patients showed good analgesic responses (more than 70 mm pain relief on a visual-analogue scale) of pain at rest, two patients poor responses (less than 30 mm pain relief), and one a moderate response with both concentrations (30-70 mm pain relief). The response to morphine was consistent (greater and faster relief with the higher concentration) in nine patients. Two patients had pain on movement that responded moderately to low-concentration morphine and well to the higher concentration. All patients with pains judged to be nociceptive showed good analgesic responses compared with half of those with neuropathic pain. There was no evidence that analgesic responses in patients with neuropathic pain were due to changes in mood. This PCA method is a quick and efficient tool to determine the consistency of the analgesic response. Such consistency can guide the clinician as to whether continued or higher-dose opioid treatment will produce good analgesia. An inconsistent response points to the use of other pain-relieving strategies.


Use of nonoxynol-9 and reduction in rate of gonococcal and chlamydial cervical infections. The spermicide nonoxynol-9 (N-9) has been used as a contraceptive for over 30 years, but the use of a vaginal spermicide and condoms for the prevention of sexually transmitted infections has not been examined in randomised studies. We report a single-blind randomised field trial to assess the effect of N-9 film on the rate of gonococcal and chlamydial cervical infection in women at high risk of these diseases. 343 women were randomly assigned to use either condoms and N-9 (186 women) or condoms and a placebo (157). Compliance with condom use was much the same in the two groups. Overall, N-9 reduced the rate of cervical infection by 25% (rate ratio [RR] 0.75, 95% confidence interval [Cl] 0.5-1.1); in women who used N-9 for more than 75% of their coital acts the infection rate was reduced by 40% (RR 95% Cl 0.3-1.0). The rate of yeast vulvovaginitis or genital ulcers was not higher in N-9 users than in placebo users, but the rate of symptomatic irritation was increased by 70% (RR 95% Cl 1.1-2.6) among N-9 users. Condom use was more protective against cervical infection than N-9 use. The rate of infection was 50% (RR 95% Cl 0.3-0.7) lower with 75% than with 0-50% condom compliance. The use of a vaginal N-9 spermicide with condoms whenever possible seems to be a better strategy than the use of condoms only for prevention of gonococcal and chlamydial cervical infection.


Debrisoquine hydroxylase gene polymorphism and susceptibility to Parkinson's disease. The pathogenesis of Parkinson's disease may be influenced by genetic and environmental factors. Cytochrome P450 mono-oxygenases help to protect against toxic environmental compounds and individual variations in cytochrome P450 expression might, therefore, influence susceptibility to environmentally linked diseases. The frequency of mutant CYP2D6 alleles was studied in 229 patients with Parkinson's disease and 720 controls. Individuals with a metabolic defect in the cytochrome P450 CYP2D6-debrisoquine hydroxylase gene with the poor metaboliser phenotype had a 2.54-fold (95% Cl 1.51-4.28) increased risk of Parkinson's disease. Determination of CYP2D6 phenotype and genotype may help to identify those at greatest risk of Parkinson's disease and may also help to identify the environmental or metabolic agents involved in the pathogenesis of this disease.


Autoantibodies in pet dogs owned by patients with systemic lupus erythematosus. Systemic lupus erythematosus (SLE) occurs in human beings and in dogs. There is evidence to suggest that the disease is caused by a transmissible factor that may cross the species barrier. We investigated this possibility by looking for autoantibodies and serum protein abnormalities in serum samples from 15 pet dogs owned by patients with SLE and comparing results with those obtained with serum from 10 healthy dogs kept in a controlled environment and 9 dogs with autoimmune diseases. We used standard immunological assays modified for use with canine samples. Compared with the normal dogs, increased amounts of antibody to double-stranded DNA were found in serum from the SLE patients' dogs (median optical density [405 nm] = 0.117 vs 0.299; p = 0.0001), this latter amount being similar to that found in serum from the autoimmune group (0.201; p = 0.6). Abnormal serum protein electrophoresis patterns were found in samples from the autoimmune and SLE patients' dogs but not the normal dogs. These findings support the idea that exposure to common environmental factors or transmissible agents may have a role in causing SLE.


Side-branch occlusion during percutaneous transluminal coronary angioplasty. Concentrations of creatine kinase (CK) MB mass and cardiac troponin T were measured in serial peripheral venous blood samples from 21 patients who underwent percutaneous transluminal coronary angioplasty (PTCA). Angiography showed side-branch occlusion during PTCA without clinical signs of myocardial injury in 5 patients. After PTCA, CKMB mass concentrations were substantially higher than normal in all 5 patients with side-branch occlusion, and troponin T concentrations were high in 3. By contrast, only 2 patients and 1 patient, respectively, without side-branch occlusion had slight rises in CKMB and troponin T. Release of the contractile protein troponin T reflects more severe damage to myocytes than simple leakage of CKMB. Therefore, myocardial damage induced by side-branch occlusion can be graded by measurement of troponin T in plasma.


Gingival sequestration of nifedipine in nifedipine-induced gingival overgrowth. The mechanism of gingival overgrowth associated with long-term use of nifedipine and of other drugs that affect calcium homoeostasis, such as cyclosporin and phenytoin, is unknown. With an ultrasensitive assay, we measured the pharmacokinetics of nifedipine in plasma and gingival crevicular fluid (GCF) of nine patients receiving this drug for angina and hypertension. In seven patients, the maximum nifedipine concentration was in the range 15-316 (mean 84 [SD 105]) times greater in GCF than in plasma. The two patients with low (undetectable) GCF nifedipine did not have overgrowth. We propose that gingival tissues sequester nifedipine and that the very high nifedipine concentrations predispose the tissues to overgrowth.


Central-nervous-system dysfunction after warm or hypothermic cardiopulmonary bypass. The increasing popularity of warm heart surgery led us to assess the effect of temperature during cardiopulmonary bypass (CPB) on neuropsychological function after coronary surgery. 34 patients enrolled in a randomised trial of normothermic versus hypothermic CPB were subjected to a battery of psychomotor and memory tests before and after their operations. The mean nasopharyngeal temperature for warm CPB was 34.7 (SD 0.5) degrees C and that for hypothermic CPB was 27.8 (2.0) degrees C. In all seven neuropsychological tests the postoperative scores were better in the warm CPB than in the hypothermic group, although only one difference achieved significance (trial-making test A; p less than 0.023). Thus, neurological function after normothermic CPB seems to be no worse than that after hypothermic procedures.


Cumulative conception and livebirth rates after in-vitro fertilisation. Cumulative conception and livebirth rates related to age and cause of infertility provide the most useful estimate of success after in-vitro fertilisation (IVF), but limited data are available. It is also uncertain whether the probability of pregnancy, livebirth, and pregnancy failure changes with repeated treatment cycles. To assess the effects of patients' age and cause of infertility on these outcomes, we studied the results of 5055 consecutive IVF cycles (773 clinical pregnancies, 518 livebirths) undertaken on 2735 patients in a single IVF unit. Cumulative conception and livebirth rates were analysed by the life-table approach and differences in rates between age-groups and between causes of infertility were measured by the log-rank test and logistic regression modelling. Both conception and livebirth rates per cycle declined with age (p less than 0.001), and cumulative conception and livebirth rates after five treatment cycles were about 54% and 45%, respectively, at 20-34 years, compared with 38.7% and 28.9% at 35-39 years and 20.2% and 14.4% at greater than or equal to 40 years. The two rates were significantly different between causal groups (p less than 0.001 and p = 0.02, respectively) and were lowest in patients with male infertility or multiple infertility factors. The pregnancy failure rate was higher (p = 0.006) in women over the age of 34 years and there was a significant decline in the chances of pregnancy and livebirth per cycle with successive treatment cycles.(ABSTRACT TRUNCATED AT 250 WORDS)


Critical study of consensus analysis.

Critical study of consensus analysis. Consensus analysis has been proposed as a statistical method by which the efficacy of clinical and laboratory tests of inflammatory activity can be assessed. This technique is claimed to overcome the need for an external "gold standard" as a reference method; instead, the consensus of all tests is used as the gold standard. We have evaluated the reliability of consensus analysis using data collected from patients with Crohn's disease. Our results demonstrate that the technique depends strongly on the correlation structure underlying the set of measures of disease used for analysis. This observation was supported by a series of conventional cluster analyses of the same set of variables. Furthermore, slight modifications of the algorithm had profound effects on the final result. We conclude that for the evaluation of tests of inflammatory activity, an external reference method, albeit an imperfect one, remains indispensable.


Effects of the alpha 1-adrenergic agonist cirazoline on locomotion and brown adipose tissue thermogenesis in the rat. Anorexia is induced by injection of alpha 1-adrenergic receptor agonists into the hypothalamic paraventricular nucleus (PVN) in rats. Of the agonists tested to date, cirazoline is the most potent when administered either into the PVN or systemically. The present experiments assess the effects of systemically administered cirazoline, at doses that suppress food intake, on dopamine and norepinephrine systems as evident in locomotion and stereotypy and in the induction of brown adipose tissue (BAT) thermogenesis. In Experiment 1, adult male rats were treated with either vehicle (0) or 0.05, 0.1, 0.2 or 0.4 mg/kg cirazoline (IP) prior to 30 minutes assessment of horizontal and vertical locomotion and stereotypy in Omnitech activity chambers. Horizontal activity and stereotypy were significantly suppressed at 0.05 mg/kg cirazoline but these effects waned at higher cirazoline doses. In Experiment 2, interscapular BAT temperature in adult male rats was monitored for 30 minutes after injection (IP) of either vehicle or 0.4 mg/kg cirazoline. Cirazoline, at 0.4 mg/kg did not influence BAT temperature whereas a positive control treatment (phenylpropanolamine: 40 mg/kg) rapidly increased BAT temperature during a 15 minute period after injection. These results suggest that cirazoline-induced anorexia is not the result of competing motor responses and that this drug, at a dose that produces maximal suppression of feeding, does not alter BAT thermogenesis.


Subchronic treatment with metformin produces anorectic effect and reduces hyperinsulinemia in genetically obese Zucker rats. The effect of subchronic metformin treatment on food intake, weight gain and plasma and tissue hormone levels was investigated in genetically obese male Zucker rats and in their lean controls. Metformin hydrochloride (320 mg/kg/day for 14 days in the drinking water) significantly reduced 24 hour food intake both after one and two weeks treatment in obese rats. In contrast, metformin had only a transient effect on food intake in lean animals. The reduced food intake was associated with body weight decrease, particularly in obese rats. Metformin markedly reduced also the hyperinsulinemia of the obese animals without altering their plasma glucose or pancreatic insulin content which may reflect an improved insulin sensitivity after metformin treatment. Metformin did not change plasma corticosterone levels or insulin and somatostatin concentrations in the pancreas. Metformin reduced pyloric region somatostatin content in lean rats. It is concluded that metformin has an anorectic effect and reduces body weight and hyperinsulinemia in genetically obese Zucker rat.


[Effect of trans-ACPD, a specific agonist of glutamate interacting with metabotropic receptors, on synaptic transmission in the rat hippocampus]. Trans-ACPD, a cyclic analogue of glutamate, has been studied for its influence on field potentials and excitatory postsynaptic currents (EPSCs) in the CA1 layer. Being applied in concentration 50 microM and above, trans-ACPD completely and reversibly inhibited excitatory postsynaptic field potentials but has no effect on EPSCs. Trans-ACPD in the same concentration reversibly reduced the amplitude of antidromic population spike in the CA1 layer, but has an insignificant effect on antidromic population spike in the CA3 layer.


[DNA markers linked to Huntington's disease (D4S10 and D4S95) in Spanish families: preliminary results]. We have studied six Spanish families with at least one affected member with Huntington's disease, using RFLPs from probes linked to the gene. Our results suggest a genetic homogeneity in our population and show a 100% of informativity with the probes and enzymes used.


Early stages of motor neuron differentiation revealed by expression of homeobox gene Islet-1. Motor neurons in the embryonic chick spinal cord express a homeobox gene, Islet-1, soon after their final mitotic division and before the appearance of other differentiated motor neuron properties. The expression of Islet-1 by neural cells is regulated by inductive signals from the floor plate and notochord. These results establish Islet-1 as the earliest marker of developing motor neurons. The molecular nature of the Islet-1 protein suggests that it may be involved in the establishment of motor neuron fate.


[Effects of thyroxine and methimazolum on monamine transmitters in preoptic/anterior hypothalamic area and peripheral serum in rats]. The contents of monamine transmitters in preoptic/anterior hypothalamic (PO/AH) area and in the serum of rats injected with thyroxine (T4, 1 mg/100 g body wt/d, for 10 d, hypodermic i.) and fed with methimazolum (10 mg/100 g body wt/d, for 15 d) were assayed by high pressure liquid chromatography with electrochemical detector (HPLC-ECD). It was found that the content of dopamine and homovanillic acid in PO/AH area showed significant increase (P less than 0.01) associated with a slight rise (P greater than 0.05) of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), and with no change of norepinephrine in the thyroxine group. After fed with methimazolum, the content of norepinephrine decreased significantly (P less than 0.05), but no obvious changes occurred in dopamine, homovanillic acid, 5-HT and 5-HIAA. By synchronous analysing of monamine transmitters in peripheral serum, it was showed that there was no significant linear relationship between the changes of monamine transmitters in the brain and in the serum. The correlation between thyroxine and methimazolum on the content of monamine transmitters and on the change of body temperature was also discussed.


Calcitonin immunoreactivity and hypercalcitoninemia in two patients with sporadic, nonfamilial, gastroenteropancreatic neuroendocrine tumors. Hypercalcitoninemia in gastroenteropancreatic tumors associated with calcitonin immunoreactivity is rare. We report here two patients in whom pancreatic neuroendocrine tumors both contained and secreted immunoreactive calcitonin. Both patients experienced elevated basal calcitonin immunoreactivity. The peak responses of immunoreactive calcitonin occurred 5 minutes after pentagastrin administration in these two patients and were 30% and 180% above basal concentrations corresponding to peak increments of 0.39 and 8.78 ng/ml, respectively. The immunoreactive calcitonin response to pentagastrin in these two patients was not significantly different from that seen among five patients with medullary carcinoma of the thyroid gland. It does not appear that immunoreactive calcitonin responses to pentagastrin stimulation will discriminate between patients with medullary carcinoma of the thyroid gland and those with nonfamilial, gastroenteropancreatic neuroendocrine tumors that express calcitonin immunoreactivity. In patients with secretory diarrhea and/or flushing, an elevated level of immunoreactive calcitonin, in the absence of a thyroid mass in the neck, may herald the presence of a gastroenteropancreatic neuroendocrine tumor.


Hormonal and neural blockade prevents the postoperative increase in amino acid clearance and urea synthesis. The combined effect of continuous blockade of glucagon and cortisol by somatostatin and etomidate and thoracic epidural analgesia on hepatic conversion of amino nitrogen was studied in eight patients who underwent elective cholecystectomy on day 1 after operation and was compared with 16 patients who underwent operation without blockade. Surgery increased the plasma clearance of total alpha-amino nitrogen from 5.2 +/- 0.3 to 6.6 +/- 0.3 ml/sec (mean +/- sem; p less than 0.05). This increase was due to increased elimination by the liver, because the hepatic effectiveness for amino nitrogen conversion measured by the functional hepatic nitrogen clearance increased from 9 +/- 2 to 16 +/- 4 ml/sec (p less than 0.05). In contrast, during the combined neural and hormonal blockade, surgery decreased the plasma clearance of amino nitrogen from 5.3 +/- 0.3 to 3.9 +/- 0.3 ml/sec (p less than 0.05), and the blockade prevented the postoperative increase in functional hepatic nitrogen clearance. The results suggest that glucagon, cortisol, and afferent neural reflexes are mediators of the hepatic contribution to catabolism after operation.


HLA-DP antigen and Takayasu arteritis.

HLA-DP antigen and Takayasu arteritis. Sixty-four patients with Takayasu arteritis and 317 healthy individuals in the Japanese population were examined for HLA-A, -B and -C alleles by serological typing and for HLA-DR, DQ and DP alleles by DNA typing using PCR/SSOP analysis. The frequencies of HLA-Bw52, DRB1*1502, DRB5*0102, DQA1*0103, DQB1*0601 and DPB1*0901 alleles were significantly increased and the frequencies of HLA-Bw54, DRB1*0405, DRB4*0101, DQA1*0301, DQB1*0401 alleles were significantly decreased. Strong linkage disequilibria among the increased alleles and among the decreased alleles were evident in the Japanese population. Therefore, the combination or haplotype of HLA-Bw52-DRB1*1502-DRB5*0102-DQA1*0103-DQB1*0601 -DPA1*02-DPB1*0901 may confer susceptibility to Takayasu arteritis while another combination or haplotype of HLA-Bw54-DRB1*0405-DRB4*0101-DQA1*0301-DQB1++ +*0401 may confer resistance to the disease. Because this is the first evidence for the association between an HLA-DP allele and Takayasu arteritis, we examined the nucleotide sequences of the DPB1*0901 allele from a patient and her healthy relatives and found no difference. The disease is therefore not caused by a mutated DPB1 gene.


Sequence differences between HLA-B and TNF distinguish different MHC ancestral haplotypes. The HLA-B locus is extremely polymorphic. We have sequenced a region, CL, telomeric of HLA-B that also shows a high degree of allelic variation which we have shown previously by RFLP analysis. The polymorphism can be accounted for by sequence variation in duplicated, reiterated sequence elements called geometric elements. Comparison of the CL1 and CL2 sequences from the 57.1, 8.1, 18.2 and 7.1 ancestral haplotypes revealed that the lengths of the elements vary, both between the duplicated loci within a haplotype and between haplotypes, apparently because certain sequences are inserted or deleted. It is possible, using the polymerase chain reaction, to amplify these elements in genomic DNA from ancestral haplotypes for which sequence data of the CL region are not available and to obtain gel patterns which are characteristic of different ancestral haplotypes. The most striking feature of the data is the fact that the majority of the CL patterns are haplospecific; i.e. have a particular pattern that is unique for a particular ancestral haplotype and can be used to type these ancestral haplotypes. At least 12 different allelic patterns have been identified within a panel of 29 cell lines representing 16 ancestral haplotypes. For these 16 ancestral haplotypes, all examples of each haplotype have the same CL pattern. The haplotypic nature of the patterns confirms that ancestral haplotypes are conserved chromosomal segments and that coding and non-coding sequences are identical by descent from a remote ancestor.


Studies of the HLA class II alleles involved in human responses to ragweed allergens Ambrosia artemisiifolia V (Ra5S) and Ambrosia trifida V (Ra5G). Previous studies have associated skin test sensitivity and specific IgE response to Ambrosia artemisiifolia V (Amb a V) with HLA-DR2, and to Ambrosia trifida V (Amb t V) with HLA-DRw52 haplotypes in atopic individuals. Using HLA class II typing by restriction fragment length polymorphism (RFLP) analysis with DRB, DQB and DQA DNA probes to define the HLA-D alleles, we have demonstrated the association of the DQw6 in 16 out of 16 (100%) Amb a V-responsive individuals, compared to 3 out of 18 (17%) ragweed-sensitive but Amb a V-nonresponsive individuals (p = 5.7 x 10(-6), RR greater than 75). We suggest that the DQw6 association with Amb a V sensitivity may be a reflection of an association with the DQA*0102 allele. This suggests an association of a particular HLA class II allele with an immune response to a well-characterized antigen (Amb a V). The HLA-DRw52 haplotypes in the Amb t V-sensitive individuals are not of one particular subtype. The HLA-DRw52 association with Amb t V sensitivity may reside in homologous DRB1 alleles linked on HLA-DRw52-bearing haplotypes.


Pharmacokinetics and patient-controlled analgesia. With patient control over dosage regimens as in PCA, the pharmacodynamic properties of the opioid analgesic agents emerge as being of greater importance than the pharmacokinetic properties in obtaining a salutary result. However, the prescription of opioid analgesic agents for PCA is more complex than for the same drugs under conventional control. There are major differences in the physicochemical, receptor selectivity and pharmacokinetic properties among opioid analgesic agents. Improved understanding of these differences, it is contended, can lead to more optimized strategies for the clinical management of pain whether under conventional or patient control. The impact of these properties on the components of the PCA prescription is discussed.


Patient-controlled analgesia in children. The use of patient-controlled analgesia in children and adolescents undergoing major surgery is safe and effective provided that patients are carefully selected, adequate information is provided to patients, adequate training is given to hospital staff and efficacy as well as side-effects are appropriately monitored. Practical guidelines and examples of complications are given.


[Effects of alpha adrenoceptor agonists and antagonists on palpebral fissure and lacrimation in mice]. Ip methoxamine (Met), norepinephrine (NE), xylazine (Xyl), and clonidine (Clo) produced dose-related reversal of ptosis induced by reserpine. These agonists had equivalent Emax values, however, the order of potency was: Clo greater than Met approximately NE greater than Xyl. Met-induced antiptotic action was competitively inhibited by prazosin (Pra) (apparent pA2 = 6.86), but not by idazoxan (Ida); Xyl-induced antiptotic action was competitively inhibited by Ida (apparent pA2 = 6.39), but inhibited by Pra in a noncompetitive manner. These results suggested that the eyelids of mice had both alpha-1 and alpha-2 adrenoceptors. In mice lacrimal gland, Met, and NE elicited dose-related lacrimal secretions, however, Xyl and Clo had no such reaction. Lacrimal secretions elicited by Met or NE were inhibited by Pra, but not by Ida. These results suggested that the lacrimal gland of mice had only alpha-1 adrenoceptor.


[Central analgesic action of calcitonin and its relationship with central monoamine transmitters]. The analgesic action of calcitonin (0.25 MRC units.kg-1) injected into lateral cerebral ventricle was investigated in rats. The pain threshold was evaluated by the tail-flick test. The influences of icv naloxone 5 micrograms/rat, a blocker of opiate receptor, on the analgesic action of calcitonin were observed. The results showed that icv calcitonin produced a significant analgesic action, which was reversed by naloxone. While the pain threshold was raised by calcitonin, the contents of central monoamines (5-HT, NE, DA) in brain (diencephalon, brain stem) were examined by fluorophotometry, which were increased remarkably. It is suggested that calcitonin-induced analgesia is related to the opiate receptors and the contents of 5-HT, NE, and DA in CNS.


Acute and long-term effects on myocardial ischemia of intermittent and continuous transdermal nitrate therapy in stable angina. The aim of this study was to compare the efficacy and safety of continuous and intermittent transdermal nitrate therapy using ambulatory electrocardiographic (Holter) monitoring. Eighty-five patients with stable angina pectoris and positive exercise test results participated during their concomitant antiischemic medication in a randomized open trial lasting 12 weeks. After a 3-week run-in period with continuous therapy (10 mg/24 hours), patients were randomized to either continuous- or intermittent-therapy groups. In the intermittent-therapy group the patients removed their patch at night (the mean patch-off period was 10 hours). Forty-eight-hour Holter monitoring was performed in each patient after randomization, and again after 2 and 12 weeks. Eighteen patients withdrew, 9 in each group. A total of 11,194 hours of electrocardiography were recorded and 607 ischemic episodes were detected, of which 79% were asymptomatic and 95% appeared during daytime. The number of ischemic episodes per 48 hours with intermittent therapy was 3.1 +/- 0.7 (mean +/- SEM) after randomization, 1.8 +/- 0.4 at 2 weeks and 2.0 +/- 0.6 at 12 weeks. With continuous therapy the respective numbers were 3.8 +/- 1.1, 3.5 +/- 0.9 and 4.2 +/- 1.2. The differences were not statistically significant because a large number of patients (30%) had no ischemic episodes on Holter recording. However, when examining 47 patients with episodes during the study, the number of episodes was significantly reduced in the intermittent-therapy group (p less than 0.05 at 12 weeks). The changes in asymptomatic and symptomatic episodes were concordant. No changes and differences between the treatment groups were seen in nighttime episodes.(ABSTRACT TRUNCATED AT 250 WORDS)


Composition and in vitro corrosion of orthodontic appliances. The high incidence of nickel allergy and the increasing use of nickel-containing dental biomaterials has been of growing concern. The purpose of this investigation was to analyze different types of alloys used in orthodontics, and to study whether nickel and chromium will be released from these alloys when stored in physiologic saline. Face-bows, brackets, molar bands, and arch wires were analyzed. Most of the different parts in the face-bows, brackets, and molar bands were similar to conventional 18/8 stainless steel. Except the wires, most appliances included a variable amount of silver solder, the greatest in face-bows. After 14 days in 0.9% sodium chloride (NaCl), the largest amount of nickel and chromium were leached out from the face-bows and the least amount from the arch wires. Soldered stainless steel face-bows seemed to be very susceptible to corrosion. The release of nickel seemed to be related to both the composition and the method of manufacture of the appliances, but the release was not proportional to the nickel content.


Recombination of 4p16 DNA markers in an unusual family with Huntington disease. The Huntington disease (HD) mutation has been localized to human chromosome 4p16, in a 6-Mb region between the D4S10 locus and the 4p telomere. In a report by Robbins et al., a family was identified in which an affected individual failed to inherit three alleles within the 6-Mb region originating from the parental HD chromosome. To explain these results, it was suggested that the HD locus (HD) lies close to the telomere and that a recombination event took place between HD and the most telomeric marker examined, D4S90. As a test of this telomere hypothesis, we examined six members of this family, five of whom are affected with HD, for the segregation of 12 polymorphic markers from 4p16, including D4S169, which lies within 80 kb of the 4p telomere. We separated, in somatic cell hybrids, the chromosomes 4 from each family member, to determine the phase of marker alleles on each chromosome. We excluded nonpaternity by performing DNA fingerprint analyses on all six family members, and we found no evidence for chromosomal rearrangements when we used high-resolution karyotype analysis. We found that two affected siblings, including one of the patients originally described by Robbins et al., inherited alleles from the non-HD chromosome 4 of their affected parents, throughout the 6-Mb region. We found that a third affected sibling, also studied by Robbins et al., inherited alleles from the HD chromosome 4 of the affected parent, throughout the 6-Mb region. Finally, we found that a fourth sibling, who is likely affected with HD, has both a recombination event within the 6-Mb region and an additional recombination event in a more centromeric region of the short arm of chromosome 4. Our results argue against a telomeric location for HD and suggest that the HD mutation in this family is either associated with DNA predisposed to double recombination and/or gene conversion within the 6-Mb region or is in a gene that is outside this region and that is different from that mutated in most other families with HD.