Evidence for orthologous seed weight genes in cowpea and mung bean based on RFLP mapping. A well saturated genomic map is a necessity for a breeding program based on marker assisted selection. To this end, we are developing genomic maps for cowpea (Vigna unguiculata 2N = 22) and mung bean (Vigna radiata 2N = 22) based on restriction fragment length polymorphism (RFLP) markers. Using these maps, we have located major quantitative trait loci (QTLs) for seed weight in both species. Two unlinked genomic regions in cowpea contained QTLs accounting for 52.7% of the variation for seed weight. In mung bean there were four unlinked genomic regions accounting for 49.7% of the variation for seed weight. In both cowpea and mung bean the genomic region with the greatest effect on seed weight spanned the same RFLP markers in the same linkage order. This suggests that the QTLs in this genomic region have remained conserved through evolution. This inference is supported by the observation that a significant interaction (i.e., epistasis) was detected between the QTL(s) in the conserved region and an unlinked RFLP marker locus in both species.


[Genetic differentiation of the inhabitants of Mongolia. Geographic distribution of mitochondrial DNA RFLPs and mitotypes in the inhabitants of Mongolia and a population assessment of the mutation rate in the mitochondrial genome]. The geographical distribution of the Asian specific deletion--insertion polymorphisms and or the RFLP's in the V noncoding region and the D-loop and of the mitotypes was analysed in Mongolia. The frequencies of the mtDNA markers demonstrated homogeneity of 18 local groups in Mongolia. The geographical distribution of the mitotypes showed the existence of two ancestral maternal lineages in mongols. There was no significant difference in the average FST values between mitochondrial gene flow and the nuclear gene flow of the Mongolian population. The equality of FST values permit to calculate the mutation rate for the human mtDNA--6.10(-9) per nucleotide per year. The data reveals the Mongolian population is in the equilibrium.


HER-2/neu expression: a major prognostic factor in endometrial cancer. The HER-2/neu oncogene encodes for a specific cell-surface glycoprotein similar to the human growth factor receptor. An analysis of 247 patients with endometrial cancer treated between 1979 and 1983 was performed using an immunoperoxidase technique on paraffin-embedded tissue samples to detect HER-2/neu overexpression. Specimens were graded blindly with regard to HER-2/neu staining intensity. Overexpression of HER-2/neu was identified as strong in 37 patients (15%), mild in 144 (58%), and none in 66 (27%). The 5-year progression-free survival was 56% for the strong, 83% for the mild, and 95% for the nonstaining groups. The strong (P < 0.0001) and the mild (P = 0.028) staining groups were distinct from the nonstaining group in predicting progression-free survival. Likewise, strong overexpression was associated with a poor (51%) overall survival (P < 0.0001). Multivariate analysis revealed that intense overexpression had independent significance in predicting progression-free (P = 0.0003) and overall survival (P < 0.0001). In stage I patients (203), the 5-year progression-free survival was 62% for the strong and 97% for the nonstaining groups (P = 0.0007). This retained independent significance when subjected to multivariate analysis (P = 0.0017). Other significant stage I prognostic factors in multivariate analysis included DNA ploidy, histologic subtype, and histologic grade but not depth of invasion.


Proliferating cell nuclear antigen in epithelial ovarian cancer: relation to results at second-look laparotomy and survival. We determined the proliferative index (PI) of 92 previously untreated advanced epithelial ovarian cancers using PCNA/cyclin immunostaining and image analysis quantitation. In this retrospective study, there was a relationship between tumor PI and 5-year survival. For patients with a tumor PI greater than the median, the estimated 5-year survival was 44%; for patients with a tumor PI below the median, the estimated 5-year survival was 15% (P = 0.003). This may partly reflect sensitivity to chemotherapy, as those patients with more rapidly proliferating tumors were more likely to achieve a pathologic complete response to platinum-based therapy.


C-erbB-2-oncogene expression in breast carcinoma: analysis by S1 nuclease protection assay and immunohistochemistry in relation to clinical parameters. The c-erbB-2 mRNA was detected by the S1 nuclease protection assay and Northern blotting in breast cancer tissues. In contrast to the Northern blot analysis which has been used in all recent publications concerning c-erbB-2 expression on the level of RNA, the S1-nuclease protection assay has distinct advantages with respect to sensitivity, reproducibility, and handling of radioactive probes. We compared the expression of c-erbB-2 in 120 breast carcinomas which were operated in the years 1989-1990 on the level of the mRNA (S1 nuclease protection assay) and the protein (immunohistochemistry), respectively. In general, results obtained with both methods were in good agreement. Only minor differences in classification were observed with 18 samples, all of them belonging to either the moderate or the weak c-erbB-2 expression phenotype. In addition, the level of c-erbB-2-protein was investigated by immunohistochemistry in 271 breast carcinomas which were operated in the years 1984-1987. Comparison of the level of c-erbB-2 expression with the patient history indicates that patients whose tumors had already metastasized to the axillary nodes showed a reduced overall and disease-free survival in the group with pronounced expression of the c-erbB-2 protein. Thus the strong expression of c-erbB-2 oncogene in primary breast cancers appears to be an additional and particular prognostic factor in lymph node positive patients.


Effects of dopaminergic agents on cardiac and renal function in normal man and in patients with congestive heart failure. Dopamine is an effective drug in the management of acute congestive heart failure. Its beneficial action is due to both cardiovascular--peripheral vasodilation and positive inotropy--and renal effects. Dopexamine is one of the newer dopamine agonists. Like dopamine, however, it can only be administered intravenously, and its value in the chronic treatment of congestive heart failure is limited. For this reason, dopamine analogs were developed with oral bioavailability, including levodopa, fenoldopam and biopamine. Although both levodopa and fenoldopam have shown beneficial hemodynamic effects, these agents cannot be recommended for general use in patients with congestive heart failure. Levodopa frequently causes side effects, especially nausea, and fenoldopam induces an increase in neurohumoral activity, which limits its long-term efficacy. Ibopamine has vasodilatory, mild inotropic and diuretic properties and it lowers plasma norepinephrine levels. Since ibopamine is usually well tolerated, it appears to have the most interesting profile of these oral dopaminergic agents.


Matlystatins, new inhibitors of typeIV collagenases from Actinomadura atramentaria. II. Biological activities. Matlystatin A, the main component of matlystatins, inhibits 92 kDa and 72 kDa typeIV collagenases with IC50 values of 0.3 microM and 0.56 microM, respectively, while 7- to 11-fold greater concentrations are required to inhibit thermolysin and aminopeptidase M. The inhibition is reversible and competitive with respect to gelatin. It inhibits the invasion of basement membrane Matrigel by human fibrosarcoma HT1080 dose-dependently with an IC50 value of 21.6 microM.


Mutational analysis of centrin: an EF-hand protein associated with three distinct contractile fibers in the basal body apparatus of Chlamydomonas. Centrin, a 20-kD phosphoprotein with four calcium-binding EF-hands, is present in the centrosome/basal body apparatus of the green alga Chlamydomonas reinhardtii in three distinct locations: the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions. In each location, centrin is found in fibrous structures that display calcium-mediated contraction. The mutant vfl2 has structural defects at all of these locations and is defective for basal body localization and/or segregation. We show that the vfl2 mutation is a G-to-A transition in the centrin structural gene which converts a glutamic acid to a lysine at position 101, the first amino acid of the E-helix of the protein's third EF-hand. This proves that centrin is required to construct the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions, and it demonstrates the importance of amino acid 101 to normal centrin function. Based on immunofluorescence analysis using anti-centrin antibodies, it appears that vfl2 centrin is capable of binding to the basal body but is incapable of polymerizing into filamentous structures. 19 phenotypic revertants of vfl2 were isolated, and 10 of them, each of which had undergone further mutation at codon 101, were examined in detail. At the DNA level, 1 of the 10 was wild type, and the other 9 were pseudorevertants encoding centrins with the amino acids asparagine, threonine, methionine, or isoleucine at position 101. No ultrastructure defects were apparent in the revertants with asparagine or threonine at position 101, but in those with methionine or isoleucine at position 101, the distal striated fibers were found to be incomplete, indicating that different amino acid substitutions at position 101 can differentially affect the assembly of the three distinct centrin-containing fibrous structures associated with the Chlamydomonas centrosome.


Activin A-induced differentiation in K562 cells is associated with a transient hypophosphorylation of RB protein and the concomitant block of cell cycle at G1 phase. The human erythroleukemic cell line, K562, can be induced to differentiate by the addition of activin A, a newly purified protein belonging to the TGF-beta 1 family. The present studies used flow cytometric cell cycle analysis, indirect immunofluorescence staining of the proliferating cell nuclear antigen (PCNA), and thymidine incorporation assay of cell proliferation to study the effects of activin A on the cell cycle during differentiation in K562 cells. Activin A-treated K562 cells were found to undergo a transient block in cell cycle, temporarily halting progression from G1 to S phase. The latter can be observed after approximately 24 hr of incubation with activin A and then disappears after this early stage of induction of differentiation. Cell cycle kinetics analysis using synchronized K562 cells also confirms that in the presence of activin A, K562 cells progress normally through various phases of cell cycle, except that there is prolongation of the G1 phase between 10 to 24 hr of culture. Furthermore, this transient arrest in G1 is correlated with dephosphorylation of a nucleoprotein, the RB gene product, which occurs within 9-24 hr of incubation with activin A; and phosphorylation of RB protein then develops afterward. In addition, these cell cycle-related events are observed to occur earlier than the accumulation of hemoglobins in K562 cells. It is concluded that transient dephosphorylation of RB protein and prolongation of G1 phase of cell cycle precede and accompany erythroid differentiation caused by activin A and chemical inducers, thus constituting part of the mechanism for induction of differentiation in the erythroleukemia cells.


Restriction fragment length polymorphism analysis of HLA-DR- and DQ-linked alleles in multiple sclerosis in Spain. HLA-DR allele subtypes in multiple sclerosis (MS) individuals in Spain were determined by restriction fragment length polymorphism (RFLP) analysis. The well-established association of DRw15, DQw6, Dw2 alleles and MS susceptibility was confirmed. The strength of its association, however, was relatively weak in our MS population and no involvement with any other DR allele was observed. DQw6 increase correlated with the elevation of the involved DR2 subtype. The hypothesis that MS-associated susceptibility genes may be more closely associated with the DQ than the DR region was not supported by our findings which, on the other hand, could be in concordance with the concept that multiple genes contribute to MS susceptibility.


Detection of a new mutation in the beta-myosin heavy chain gene in an individual with hypertrophic cardiomyopathy. Familial hypertrophic cardiomyopathy (FHCM) is an autosomal dominant disease affecting primarily the myocardium. The gene responsible for FHCM has been localized to chromosome 14 in some families and several mutations have been described in the beta-myosin heavy chain (beta MHC), a candidate gene for the disease. We recently identified a family with HCM in whom we did not detect any of the known mutations in the beta MHC gene (the alpha/beta MHC hybrid gene and the missense mutation in exons 13 and 9). However, we did observe a novel 9.5-kb BamHI restriction fragment length polymorphism detected by a beta MHC probe on Southern blots of DNA from the proband of this family. Similarly, a novel 3.8-kb TaqI polymorphism and a novel 4.3-kb HindIII polymorphism were detected on Southern blots of DNA from the same proband. Polymerase chain reaction (PCR) was used to amplify the segment of the beta MHC that was detected by pSC14 probe. PCR amplification of the distal 3'-end of the beta MHC gene yielded an additional product in the DNA template from the proband which was subsequently cloned and sequenced. The sequence analysis showed a 2.4-kb nucleotide deletion involving one allele of the beta MHC gene. The deletion includes part of the intron 39, exon 40 including the 3'-untranslated region and the polyadenylation signal, and part of the beta-alpha MHC intergenic region. This deletion was inherited in Mendelian fashion in an additional three members of this small family of which only the proband has developed clinically diagnosed HCM at a very late onset (age 59 yr), the other three family members are younger and have not developed the disease at the ages of 10, 32, and 33 yr.


Beta cell expression of endogenous xenotropic retrovirus distinguishes diabetes-susceptible NOD/Lt from resistant NON/Lt mice. Endogeneous retroviral expression in beta cells is a feature of prediabetes in nonobese diabetic (NOD) mice. The purpose of this study was to characterize the class-specific pattern of retroviral gene expression in NOD/Lt beta cells versus a related, but diabetes-resistant strain, NON/Lt. Electron microscopic comparison of beta cells from both strains indicated low constitutive expression of the intracisternal type A (IAP) retroviral class. However, NOD beta cells, in contrast to NON beta cells, expressed an additional intracisternal retroviral form resembling a type C particle. Antibodies against both IAP and type C were detected in NOD, with the humoral response to type C, but not IAP, preceding decline in beta cell function. RNA was extracted from freshly isolated islets from NOD and NON males. Comparative Northern blot analysis of total type C retroviral gene expression using a gag-pol DNA probe corroborated expression of endogenous type C proviruses in both NOD and NON islet cells and thymus. Use of class-specific retroviral probes identified the class of expressed endogenous retrovirus distinguishing the two inbred strains. The single ecotropic provirus present in both the NOD and NON genome (Emv-30) was not expressed in islets or thymus of either strain. Comparison of endogenous xenotropic provirus content by Southern blot analysis revealed two unique xenotropic loci (Xmv-65, -66) in NOD; 8.4 and 3.0 kb xenotropic envelope (env) RNA transcripts were detected in NOD, but not NON islets and thymus. NON contained three xenotropic loci common to other inbred strains (Xmv-21, -25, and -28). Both strains were partially characterized for content of recombinant (polytropic and modified polytropic) proviruses. IAP RNA expression was common to both NOD and NON islets and hence could not be specifically associated with the unique intracisternal type C particle found in NOD, but not NON beta cells. In conclusion, this study shows that expression of xenotropic type C but not IAP distinguishes retroviral activity in NOD/Lt versus NON/Lt beta cells. The potential pathogenic role of retroviral gene expression in NOD beta cells is discussed.


Mesangial cell immune injury. Hemodynamic role of leukocyte- and platelet-derived eicosanoids. The role of leukocytes and platelets and of leukocyte- and platelet-derived eicosanoids in mediating acute changes in renal and glomerular hemodynamics was assessed in a model of antibody-induced mesangial cell injury in the rat. After a single intravenous injection (6 mg/kg) of the monoclonal antibody (ER4) against the mesangial cell membrane antigen Thy 1, significant decrements in glomerular filtration rate (GFR) and renal blood flow (RBF) were observed at 1 h, and were associated with increments in glomerular LC (+) leukocyte counts and in the synthesis of thromboxane (Tx)B2, leukotriene (LT)B4, and 12-hydroxyeicosatetraenoic acid (HETE). In rats with immune leukopenia, the rise in glomerular LC (+) leukocytes and in eicosanoid synthesis were abolished and the fall in GFR and RBF after administration of ER4 were completely ameliorated. Likewise, pretreatment of rats with both a thromboxane synthase and a 5-lipoxygenase inhibitor also blocked the fall in GFR and RBF and the rise in glomerular synthesis of TxB2 and LTB4 produced by ER4 without changing glomerular LC (+) leukocyte counts. Selective inhibition of thromboxane or 5-lipoxygenase alone only partially ameliorated the decrements in GFR and RBF produced by ER4. In animals with immune thrombocytopenia, the elevated glomerular synthesis of 12-HETE and fall in RBF but not GFR was ameliorated after administration of ER4. The ER4 antibody-induced fall in GFR was mainly caused by a marked decrement in the ultrafiltration coefficient, Kf, which was dependent on TxA2 and 5-lipoxygenase products, since pretreatment of animals with a thromboxane receptor antagonist or with a 5-lipoxygenase inhibitor partially ameliorated this decrement. Structural changes such as infiltration of glomerular capillaries by leukocytes and endothelial cell damage may also have accounted for the fall in Kf. These observations indicate that in antibody-mediated mesangial cell injury, infiltrating leukocytes and platelets mediate the changes in renal hemodynamics via synthesis of thromboxane and arachidonate 5-lipoxygenation products.


Epidermal growth factor receptor distribution in burn wounds. Implications for growth factor-mediated repair. Epidermal growth factor (EGF) along with several related peptide growth factors has been shown both in vivo and in vitro to accelerate events associated with epidermal wound repair. EGF and transforming growth factor alpha act by binding to a common EGF receptor tyrosine kinase thereby initiating a series of events which ultimately regulate cell proliferation. This study examined the immunohistochemical localization of EGF receptor (EGF-R) in burn wound margins, adjacent proliferating epithelium, and closely associated sweat ducts, sebaceous glands, and hair follicles. Tissue specimens removed during surgical debridement were obtained from full and partial thickness burn wounds in 32 patients with total body surface area burns ranging from 2 to 88%. In the early postburn period (days 2-4), prominent staining for EGF-R was found in undifferentiated, marginal keratinocytes, adjacent proliferating, hypertrophic epithelium, and both marginal and nonmarginal hair follicles, sweat ducts, and sebaceous glands. During the late postburn period (days 5-16), EGF-R was depleted along leading epithelial margins; however, immunoreactive EGF-R remained intensely positive in the hypertrophic epithelium and all skin appendages. Increased detection of immunoreactive EGF-R and the presence of [125I]EGF binding in the hypertrophic epithelium correlated positively with proliferating cell nuclear antigen distributions. Thus, the presence of EGF-R in the appropriate keratinocyte populations suggests a functional role for this receptor during wound repair. Dynamic modulation in EGF receptor distribution during the temporal sequence of repair provides further evidence that an EGF/transforming growth factor alpha/EGF-R-mediated pathway is activated during human wound repair.


Rat glomerular mesangial cells synthesize basic fibroblast growth factor. Release, upregulated synthesis, and mitogenicity in mesangial proliferative glomerulonephritis. Mesangial injury and cell proliferation are frequent findings in various glomerular diseases in man. Previous studies have demonstrated that basic fibroblast growth factor (bFGF) is a potent mesangial cell mitogen in vitro. To further elucidate the role of bFGF in rat mesangial cell (RMC) proliferation, we examined whether RMC synthesize bFGF in vitro and whether bFGF is involved in mesangial proliferation in vivo. Cultured RMC expressed bFGF protein (23, 21.5, and 18 kD forms) and bFGF mRNA, and released biologically active bFGF into the culture medium after antibody- and complement-mediated injury. Normal rat glomeruli in vivo contained no detectable bFGF mRNA, but bFGF protein (23 and 21.5 kD) could be demonstrated, which immunolocalized to the mesangium. Glomerular bFGF decreased markedly during the acute phase of glomerulonephritis induced by anti-Thy 1.1 antibody, compatible with mesangial bFGF release after complement-mediated mesangiolysis. During the subsequent mesangial proliferative phase, glomerular bFGF protein and mRNA increased above normal. Intrarenal infusion of heparin did not affect the bFGF immunostaining of glomeruli at this stage, indicating a predominantly intracellular localization of the bFGF. The capability of bFGF to mediate proliferation in the anti-Thy 1.1 model was further supported by experiments in which intravenous bFGF given 24 h after a subnephritogenic dose of anti-Thy 1.1 antibody led to a 4.9- to 5.1-fold increase in glomerular cell proliferation (with > 60% of the cells identified as mesangial cells by double immunolabeling). No such increase was observed in normal rats injected with bFGF. These data show that mesangial cells produce and release bFGF after injury and that bFGF is mitogenic for injured mesangial cells in vivo. Release of mesangial cell bFGF thus may be an important mechanism involved in the initiation of mesangial cell proliferation in vivo.


Monoaminergic systems in the brainstem and spinal cord of the turtle Pseudemys scripta elegans as revealed by antibodies against serotonin and tyrosine hydroxylase. With the aim of gaining more insight into the monoaminergic regulation of spinal motor systems in the turtle, we have studied the distribution of 5-HT (5-HTir) and tyrosine hydroxylase immunoreactivity (THir) in the brainstem and spinal cord of Pseudemys scripta elegans. 5-HTir cell bodies were located in the midline in nucleus raphe inferior, nucleus raphe superior, and laterally in nuclei reticularis superior and inferior and nucleus reticularis isthmi. THir cell bodies were located in the commissural nucleus, nucleus tractus solitarii, the locus coeruleus-subcoeruleus complex, nuclei reticularis superior and inferior, the pretectal area, and substantia nigra. 5-HTir and THir tracts were found in lateral and ventral bundles superficially in the brainstem. 5-HTir fibers in the spinal cord were located in a large dorsolateral and a smaller ventrolateral tract. In the gray matter, a high concentration of 5-HTir fibers were observed in areas I-IV and in the lateral motor column of cervical and lumbar enlargements. Areas V-VIII and area X were less intensively innervated, with the lowest fibre concentration in areas VII-VIII and area X. Throughout the spinal cord, THir nerve fibres were located in the same areas but with a lower density. Small bipolar 5-HTir and THir cell bodies were found ventromedially to the central canal especially in cervical and lumbosacral segments. Large THir cells were found in area IX in the caudal sacral and coccygeal spinal cord. THir cerebrospinal fluid-contacting cells were also found in the most caudal part of the brainstem and the upper cervical spinal cord. The well developed spinal 5-HT system and the less developed THir system provides an anatomical explanation for the monoaminergic modulation of turtle motoneuron membrane properties, which has been observed in electrophysiological experiments.


Neuronal associations in the rat suprachiasmatic nucleus demonstrated by immunoelectron microscopy. The synaptic associations of neurons in the suprachiasmatic nucleus (SCN) of rats were examined by single immunolabeling for somatostatin (SRIH) and arginine vasopressin (AVP), and double immunolabeling for SRIH plus AVP and vasoactive intestinal polypeptide (VIP) plus AVP. Single immunolabeling showed that SRIH neurons, which displayed some somatic and dendritic spines, formed synaptic contacts with immunonegative and positive axon terminals. AVP neurons also formed synaptic contacts with both immunonegative and positive axon terminals. The immunonegative terminals contained small, spherical clear vesicles or flattened clear vesicles. A few immunopositive AVP fibers made synapses with immunonegative somatic or dendritic spines. Double immunolabeling showed synaptic associations between SRIH axons and AVP cell bodies or dendritic processes, and between AVP axons and the somata or dendrites of SRIH neurons. These findings suggest a reciprocal relation between the two types of neurons. Synaptic contacts between AVP neurons and VIP axon terminals were also demonstrated. Previously, we found synapses between SRIH axons and VIP neurons. Thus SRIH neurons appeared to regulate AVP and VIP neurons. On the basis of these findings, two possible oscillation systems of the SCN are proposed.


Distribution of modulatory inputs to the stomatogastric ganglion of the crab, Cancer borealis. The pyloric and gastric mill neural networks in the crustacean stomatogastric ganglion receive modulatory inputs from more anteriorly located ganglia via the stomatogastric nerve. In this study we employed biocytin backfilling and immunostaining, as well as electron microscopy, to determine the origin of these inputs in the crab, Cancer borealis. Fiber counts from electron micrographs of sections through the stomatogastric nerve showed that this nerve contains 55-60 medium to large diameter fibers (1-13 microns). These fibers were individually wrapped by several layers of membrane, presumably glial in origin. There was also a single cluster of jointly wrapped, small diameter (< 1 micron) fibers that may originate from peripheral sensory somata. Biocytin backfills revealed that approximately two thirds of the individually wrapped fibers in this nerve originate from somata in the other three ganglia of the stomatogastric nervous system, including the paired commissural ganglia and the single oesophageal ganglion. There were approximately 20 biocytin-labeled somata in each commissural ganglion and 3 somata in the oesophageal ganglion. An additional ten somata were localized to the stomatogastric ganglion itself. This accounts for nearly all of the medium to large diameter fibers in the stomatogastric nerve. We used double-labeling with backfills and immunocytochemistry to determine that there are two proctolin-immunoreactive neurons and four FMRFamide-like immunoreactive neurons among the biocytin-labeled neurons in each commissural ganglion. Both peptides modulate neural network activity in the stomatogastric ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)


Childhood-onset familial porphyria cutanea tarda: effects of therapeutic phlebotomy. Cutaneous fragility at age 2 years with blistering, scarring, milia, and hypertrichosis at age 4 years were noted in an otherwise healthy girl who had no family history of porphyria. Results of porphyrin analyses of urine, serum, and red blood cells revealed a pattern consistent with porphyria cutanea tarda. Red blood cell uroporphyrinogen decarboxylase activity was diminished to approximately 50% of normal in the child and in her mother and maternal grandmother, who were without symptoms; activity was normal in her sister, father, and maternal grandfather. Therapeutic phlebotomies were followed by a biochemical and clinical remission.


[Predictive value of non-specific signs of fetal infection in congenital toxoplasmosis]. To study the predictive value of 9 non-specific signs for congenital toxoplasmosis. The work was carried out as a prospective study comparing non-specific signs in 22 fetuses infected with toxoplasmosis and 59 fetuses free of the disease. Four of the nine parameters were found to be statistically higher in the group of infected fetuses. These are the level of leucocytes (p < 0.05) gamma glutamyl transferase (p < 0.001) total M immunoglobulin (p < 0.0001) and C4 fraction of complement (p < 0.0001). This last sign which is non-specific for infection has never been looked at until now in congenital toxoplasmosis but is seems to be of great importance because its sensitivity is much greater than of all the other parameters that have been used up till now (81%) and the positive predictive value is excellent (72%). On the other hand the value of a raised leucocyte count is a matter for discussion. The presence of even one abnormally raised feature in the fetal blood of these non-specific signs is enough to screen for a population which is of high risk for congenital toxoplasmosis and sufficient to change our attitude to treating the condition.


The identity of Anisakis type II larvae with Anisakis physeteris confirmed by restriction fragment length polymorphism analysis of genomic DNA. The identity of Anisakis type II larvae with adult A. physeteris was confirmed by comparison of restriction fragment length polymorphisms (RFLPs) of 25S ribosomal DNA (rDNA). Patterns of RFLPs in larvae were almost identical with those in adult worms. Directly labelled 25S rDNA might serve as an appropriate probe with highly specific activity for examining RFLPs of larvae and adult worms.


Close range shotgun wounds to the foot.

Close range shotgun wounds to the foot. Presented in a case report of a close range shotgun wound. The nature, description, management, and long-term treatment of these wounds are discussed. Also presented are the reasons for the differences in short- versus long-range shotgun wounds.


Gamma-aminobutyric acid, glutamic acid decarboxylase and tyrosine hydroxylase in rat striatum demonstrated by single and dual immunocytochemistry. By means of dual ultrastructural immunostaining the followings patterns are visualized: gamma-aminobutyric acid (GABA) immunoreactive neurons, dendrites, axons and axon terminals and tyrosine hydroxylase (TH) immunopositive fibers, varicosities and boutons in rat striatum. Additionally single glutamic acid decarboxylase (GAD) immunolabeling is carried out. Four subgroups of GABA and GAD immunoreactive striatal neurons are revealed. These neuronal types are identified on the basis of sectional diameters, nuclear form and nuclear envelope invaginations, quantity of cytoplasm and cell organelles. Plasmalemmal appositions between GABAergic and between GABAergic and immunonegative neurons are observed. All subgroups of striatal GABAergic neurons contact with GABA and GAD immunoreactive, TH immunoreactive and immunonegative boutons. In the striatal neuropil numerous GABAergic, dopaminergic and immunonegative axonal endings synapsed with dendrites and spines are found out. Massive dopaminergic striatal structures using dual immunostaining is evident. Some GABA and GAD immunoreactive dendrites are revealed in direct contact with capillary walls.


Isolated transverse rupture of the peroneus brevis tendon treated with a free split-thickness tendon graft. While tendon ruptures are common, isolated transverse rupture of the peroneus brevis is an unusual occurrence. The etiology and diagnosis will be discussed in this manuscript. Also, the surgical treatment by use of a free split thickness tendon graft will be reviewed.


Identification of a candidate gene responsible for the high blood pressure of spontaneously hypertensive rats. We have recently isolated a gene, designated as the SA gene, which is more than 10 times more abundantly expressed in the kidneys of spontaneously hypertensive rats (SHR) than in those of Wistar-Kyoto (WKY) rats. To address the issue whether the SA gene is one of the genes responsible for the hypertension of SHR, a genetic cosegregation analysis of the blood pressure values with the genotypes in an F2 rat population was undertaken in this study. Male F2 rats were bred from SHR and WKY rats. The genotypes of the SA gene of the F2 rats were determined by utilizing the StuI restriction fragment length polymorphism of the SA gene between SHR and WKY rats. The blood pressure values were determined by the tail-cuff method. The effect of the genotype of the SA gene on the blood pressure of the F2 rats was analysed by one-way analysis of variance. The blood pressure of the F2 rats inheriting two SHR alleles of the SA gene was significantly higher than that of the F2 rats inheriting two WKY alleles. This indicates that the SA gene, or a gene closely linked to it, has a capacity to influence the blood pressure values of the F2 rat population. Further studies to identify functions of the SA gene products will be necessary.


HMEC-1: establishment of an immortalized human microvascular endothelial cell line. The study of human microvascular endothelial cells has been limited, because these cells are difficult to isolate in pure culture, are fastidious in their in vitro growth requirements, and have a very limited lifespan. In order to overcome these difficulties, we have transfected human dermal microvascular endothelial cells (HMEC) with a PBR-322-based plasmid containing the coding region for the simian virus 40 A gene product, large T antigen, and succeeded in immortalizing them. These cells, termed CDC/EU.HMEC-1 (HMEC-1), have been passaged 95 times to date and show no signs of senescence, whereas normal microvascular endothelial cells undergo senescence at passages 8-10. HMEC-1 exhibit typical cobblestone morphology when grown in monolayer culture, express and secrete von Willebrand's Factor, take up acteylated low-density lipoprotein, and rapidly form tubes when cultured on matrigel. HMEC-1 grow to densities three to seven times higher than microvascular endothelial cells and require much less stringent growth medium. HMEC-1 will grow in the absence of human serum, whereas microvascular endothelial cells require culture medium supplemented with 30% human serum. These cells express other cell-surface molecules typically associated with endothelial cells, including CD31 and CD36 and epitopes identified by monoclonal antibodies EN4 and PAL-E. They also express the cell adhesion molecules ICAM-1 and CD44 and following stimulation with interferon-gamma express major histocompatibility complex class II antigens. HMEC-1 specifically bind lymphocytes in cell adhesion assays. Thus HMEC-1 is the first immortalized human microvascular endothelial cell line that retains the morphologic, phenotypic, and functional characteristics of normal human microvascular endothelial cells.


Pulse radiolysis studies of intramolecular electron transfer in model peptides and proteins. IV. Met/S:.Br-->Tyr/O. radical transformation in aqueous solution of H-Tyr-(Pro)n-Met-OH peptides. The intramolecular radical transformation Met/S:.Br-->Tyr/O. in aqueous peptides H-Tyr-(Pro)n-Met-OH, n = 0-3, was investigated in the temperature range of 283-328 K by pulse radiolysis. Corresponding first-order rate constants and thermodynamic parameters of activation of electron transfer, Ea and delta S++, were determined from kinetic data. The rate constants of the reaction were found to decrease exponentially with the number of Pro units and the distance between CR atoms of the terminal amino acids, with a correlation coefficient alpha = 3.2 +/- 0.5 nm-1 at 298 K. Its value appeared to be temperature dependent suggesting the occurrence of thermally induced conformational changes in the peptides. Analysis of experimental data in terms of known conformational properties of the peptides indicates that apparent values of alpha, Ea and delta S++ are probably complicated functions of conformation and thermodynamic stability of the oligoproline bridge, varying with the number of Pro residues, and of intramolecular hydrophobic interactions between side chains of tyrosine and methionine. Estimation of the relative efficiency of electron transfer pathways through the peptide backbone and through direct and/or water mediated contact between groups bearing radical sites led to the conclusion that partitioning of electron transfer along these pathways is likely to occur.


Ethanol radical-induced protein-DNA crosslinking. A radiolysis study. Aqueous solutions of double-stranded DNA from calf thymus and bovine serum albumin (BSA) were irradiated at pH 7 under N2O and N2 in the presence of 10(-1) mol dm-3 ethanol, which was partly 14C-labelled. Ethanol protects DNA from strand breakage by scavenging OH radicals, but ethanol radicals induce protein-DNA crosslinks. Ethanol radicals react readily with BSA mainly by addition. They react also with double-stranded DNA, but produce crosslinking only very slowly. Based on these results the following mechanism is proposed: ethanol radicals bind to BSA producing protein radicals which become crosslinked to DNA.


Electron transfer from nucleobase electron adducts to 5-bromouracil. Is guanine an ultimate sink for the electron in irradiated DNA? Electron transfer to 5-bromouracil (5-BrU) from nucleobase (N) electron adducts (and their protonated forms) has been studied by product analysis and pulse radiolysis. When an electron is transferred to 5-BrU, the ensuing 5-BrU radical anion rapidly loses a bromide ion; the uracilyl radical thus formed reacts with added t-butanol, yielding uracil. From the uracil yields measured as the function of [N]/[5-BrU] after gamma-radiolysis of Ar-saturated solutions it is concluded that thymine and adenine electron adducts and their heteroatomprotonated forms transfer electrons quantitatively to 5-BrU. Like the electron adduct of adenine, those of cytosine and guanine are rapidly protonated by water. The (protonated) electron adduct of guanine does not transfer an electron to 5-BrU, and in the case of the (protonated) cytosine electron adduct only partial electron transfer is observed. The results can be modelled if the protonated electron adduct (protonated at N(3) or at the amino group) of cytosine, CH., which can transfer its electron to 5-BrU (k approximately 2 x 10(7) dm3 mol-1 s-1) is transformed in a slow tautomerization reaction (k approximately 2.5 x +/- 10(3) s-1) into another form C'H. (possibly protonated at C(6) or C(5)) which does not transfer an electron to 5-BrU. There is also electron transfer from the electron adduct of thymine to cytosine and guanine which serve as electron sinks. The rate constant of electron transfer from the thymine electron adduct to cytosine is about 250 times greater than that of the reverse reaction. The heteroatom-protonated electron-adduct of thymidine transfers an electron to 5-BrU more slowly (k = 2.3 x 10(7) dm3 mol-1 s-1) than the electron-adduct itself (k = 7.2 x 10(8) dm3 mol-1 s-1). Phosphate buffer-induced protonation of the electron-adduct of thymine at carbon (C(6)) prevents electron transfer to 5-BrU. Such phosphate catalysis is also observed as an intramolecular process (k approximately 2 x 10(4) s-1) with thymidine-5'-phosphate but not with the 3'-phosphate. Phosphate-induced protonation at carbon also reduces transfer efficiency for the electron adducts of dinucleoside phosphates such as dTpdT and dTpdA. The data raise the question whether in DNA the guanine moiety may act as the ultimate sink of the electron in competition with other processes such as protonation at C(6) of the thymine electron adduct.


Spectral studies of intermediate species formed in one-electron reactions of bovine liver catalase at room and low temperatures. A comparison with peroxidase reactions. The reactions of native bovine catalase with superoxide and solvated electrons have been investigated using three different methods for generation of these reducing substrates: gamma-radiolysis of oxygenated or deaerated buffer solutions in the presence of an OH radical scavenger; either xanthine or acetaldehyde with xanthine oxidase; and low-temperature (77 K) gamma-radiolysis of buffered ethylene glycol/water solutions with subsequent annealing of samples at 183 K. The first spectral evidence for catalase compound II formation from native catalase via reaction with superoxide was obtained. The results are compared with results for peroxidase compound II or III formation observed under the same experimental conditions. A scheme is proposed to explain these observations involving intermediate formation of catalase compounds I and III and the ferrous enzyme. The one-electron reduction of catalase and peroxidase by radiolytically-generated solvated electrons was compared. In the present study the first absorption spectrum of a high-spin ferrous catalase which has peaks at 561 and 594 nm is reported, in comparison with a hemochromogen low-spin ferrous peroxidase observed under the same experimental conditions (peaks at 527 and 556 nm). Both spectra were recorded at 77 K. Data presented in this work also provide the first spectral evidence indicating the low temperature (183 K) conversion of high-spin ferrous catalase into compound III (oxycatalase) in the presence of dioxygen. Under the same experimental conditions low-spin ferrous peroxidase was converted into the high-spin ferrous form without oxyperoxidase formation.


Mutations caused by gamma-radiation-induced double-strand breaks in a shuttle plasmid replicated in human lymphoblasts. The mutagenicity of open-circular DNA (containing base damage and single-strand breaks) and linear DNA (containing base damage, single-strand breaks, and one double-strand break) produced in vitro by gamma-irradiation of shuttle vector pZ189, was analysed after the plasmid's repair and replication in the human lymphoblast line, GM606. By comparing the survival, mutation frequency, and types of mutations in descendants from the two DNA forms, the effects of the double-strand break were determined. The percentage of viable plasmids from linear DNA was two-fold lower than that from open-circular DNA, 7.8 versus 14.0 (compared with unirradiated, control DNA). The mutation frequency in progenies of the open-circular plasmid was 4.2 +/- 1.7 x 10(-3), compared with 7.8 +/- 0.1 x 10(-3) in progenies of the linear DNA, again, nearly a two-fold difference. Approximately 59% of the mutations from the linear DNA were deletions and 34% were base substitutions. In contrast, only 13% of mutations from open-circular DNA were deletions, but 87% were base substitutions. All recoverable deletions were small, ranging from 1 to 205 base pairs, and the majority contained direct repeats at the deletion junctions, indicating non-homologous recombinations. Thus, mutations found among descendants from the linear and open-circular DNAs were qualitatively similar but quantitatively different. The data suggests that producing one double-strand break in DNA by ionizing radiation causes a two-fold increase in both lethality and mutation frequency.


The yield of fission neutron-induced chromatid aberrations in G2-stage human lymphocytes: effect of caffeine, hydroxyurea and cytosine arabinoside post-irradiation. To evaluate the influence of inhibitors of DNA synthesis/repair on the yield of chromosomal aberrations in the G2 phase of the cell cycle, whole-blood cultures of human lymphocytes were exposed to various doses of fission neutrons or X-rays and treated post-irradiation during the last 2.45 h before harvesting, with 5 mM caffeine, 5 mM hydroxyurea (HU) and 0.05 mM cytosine arabinoside (ara-C). The presence of caffeine and HU strongly potentiated the yield of chromatid-type aberrations induced by both neutrons and X-rays. No potentiating effect, except at the highest dose of neutrons, was observed when irradiated cells were subsequently treated with ara-C. Since ara-C strongly potentiated the frequency of chromatid aberrations induced in G2 lymphocytes by X-rays, the results presented here indicate that fission neutrons produce a smaller proportion of lesions, the repair of which can be inhibited by ara-C, compared with the number produced by X-rays. In addition, neutron-induced mitotic delay was shortened by treatment with caffeine, mainly within the first 2 h after irradiation.


Chromosome damage induced by high-LET alpha-particles in plateau-phase C3H 10T1/2 cells. Chromosome aberrations induced by X-rays and alpha-particles (LET = 177 keV/microns) were observed at the first mitosis in C3H 10T1/2 cells released from density-inhibited cultures. X-radiation induced more chromosome exchanges than breaks (71% vs 27% of total aberrations), while a predominance of breaks (63%) was observed after alpha-irradiation. A linear-quadratic dose-response relationship was obtained for X-rays, while that for alpha-particles was linear. The RBE values for total aberration induction (ranging from 5.1 at low doses to 4.4 at high doses) were very similar to the RBE for cell killing (from 5.2 to 4.3). The RBE for dicentric induction (approximately 2) was much lower than the RBE for the induction of both breaks (from 7 to 6) and interstitial deletions (from 9 to 7). This behaviour supports the hypothesis that chromosome deletions play a major role in the malignant transformation of 10T1/2 cells. A high correlation between cell killing and number of acentric fragments per cell was found. The number of acentrics/cell at the mean lethal dose was about 1.4. This number was reduced to 1.0 when asymmetrical interchanges, which generally result in very small deletions, were subtracted from acentrics. It could be hypothesized that very small deletions could not impair cell survival. However, an alternative hypothesis related to the aneuploid state of C3H 10T1/2 cells can be formulated. Robertsonian translocations were also observed at the first mitosis. The dose-response curve of these translocations appears to be very similar to the dose-response curve for induction of sister chromatid exchanges (observed at the second mitosis) reported by other authors studying the same cell line. This similarity could indicate a general mechanism of action of radiation on the process of recombination of genetic material.


Heavy ion-induced chromosome breakage studied by premature chromosome condensation (PCC) in Syrian hamster embryo cells. We have studied induction and repair of chromosome damage induced by high linear energy transfer (LET) heavy ions in G1/G0 interphase Syrian golden hamster embryo (SHE) cells as revealed by the premature chromosome condensation (PCC) technique. The number of chromosome breaks in condensed chromosomes induced by high LET heavy ions was higher than those induced by 137Cs gamma-rays. Compared with 137Cs gamma rays, the relative biological effectiveness (RBE) for PCC breaks was 1.5 for 35 keV/microns 4He ions, 1.9 for 77keV/microns 4He ions, and 2.5 for 530keV/microns 14N ions. Although 95% of the PCC breaks induced by gamma-rays rejoined during 8 h post-irradiation incubation, only 35-45% of fragments induced by high LET radiations rejoined in the same time. These results suggest that there is a difference, spatial or qualitative, in the initial chromosome damage produced by high LET radiations and low LET radiations.


Factors influencing the DNA content of radiation-induced micronuclei. The distribution of the DNA content of radiation-induced micronuclei was analysed in several cell lines (Chinese hamster, Syrian hamster and mouse NIH-3T3 cells) by flow cytometry. Frequency and DNA content of micronuclei were measured simultaneously using fluorescence and forward scatter signals of micronuclei and nuclei in suspension stained with ethidium bromide. Computerized random breakage of chromosomes and random combination of fragments was performed to compare the measured micronucleus distributions in synchronized cells irradiated during G1-phase with calculated distributions. The measured DNA distribution of radiation-induced micronuclei was found to be influenced by several factors: (1) the DNA distribution and the centromeric index of the chromosomes in the various cell lines; (2) the cell cycle phase at time of micronucleus measurement due to DNA synthesis in micronuclei; (3) the presence of chromosome fragments in micronuclei; and (4) the presence of whole chromosomes in micronuclei. These factors were shown to be responsible for the previously found large radiation-induced micronuclei which could not be explained by the classic assumption only that radiation-induced micronuclei are mainly produced by single acentric fragments.


Accumulation of cAMP in gamma-irradiated thymocytes and internucleosomal DNA fragmentation. Ionizing radiation, glucocorticosteroids and chemical inducers of differentiation (CID) are cytotoxic to thymocytes, and induce internucleosomal DNA fragmentation. Tissue cAMP levels in thymi of irradiated mice were significantly elevated as early as 30 min post-irradiation. In contrast, cAMP content in the liver was not changed significantly up to 1 h post-irradiation, and then some decrease occurred. Irradiation of isolated thymocytes gave essentially the same results as after irradiation of animals, and the elevation in cAMP 30 min after the irradiation, DNA fragmentation and cell death were linearly related to the dose up to 2.5 Gy. The maximal induction of cAMP level occurs in the fractions of radiosensitive cortical thymocytes. In thymocytes all CID tested also induced the increase in cAMP level with concomitant DNA fragmentation. Unlike ionizing radiation, UVC light did not induce cAMP accumulation and DNA fragmentation in thymocytes. Treatment of UV-irradiated cells with But2 cAMP did not result in an increase in DNA fragmentation. Ionizing radiation induced DNA fragmentation and cell death can be prevented by adding the protein kinases inhibitor H-7. Theophylline was shown to reduce the cAMP response, DNA fragmentation and cell death in gamma-irradiated thymocytes, suggesting that the accumulation of cAMP may be partly related to adenosine receptor sites.


Cytotoxicity of 125I decay in the DNA double strand break repair deficient mutant cell line, xrs-5. The survival of parental Chinese hamster ovary (CHO) K1 cells and the DNA double strand break (DSB) repair deficient mutant, xrs-5 was determined after accumulation of 125I decays. Both CHO and xrs-5 cells were extremely sensitive to accumulated 125I decays. The D0 values for CHO and xrs-5 cells were 40 and approximately 7 decays per cell, respectively. The difference in cell survival between CHO and xrs-5 cells was not due to differences in overall 125IUdR incorporation, differences in labelling index (LI) or differences in plating efficiency (PE). Relative biological effectiveness (RBE) values calculated relative to 137Cs gamma radiation survival values (D0 and D10) were higher in xrs-5 cells compared with CHO cells. Although both CHO and xrs-5 cells have high RBE values that correspond to a high sensitivity of CHO and xrs-5 cells to 125I decay. The higher RBE observed for xrs-5 cells in combination with the known repair defect in xrs-5 cells support the idea that unrepaired DNA double strand breaks are lethal to the cell.


Brain atrophy after foetal exposure to very low doses of ionizing radiation. Acute, high dose-rate, exposure of the rat embryo on day 15 post-conception (PC) causes a reduction of brain weight in adult life that is proportional to the dose received. Doses as low as 10 mGy of 600 keV neutrons, from a Van de Graaff accelerator, or 100 mGy of 250 kV X-rays are capable of eliciting a significant effect. The relative biological effectiveness for acute neutron exposure compared with 250 kV X-rays was 3.5. A brain weight reduction was also observed after gamma-ray exposures protracted over 4 or 6 days, during cerebral corticogenesis. The dose-rate reduction factor was only 1.5 for exposure from days 12 to 16 PC and 3.3 for exposure from days 14 to 20 PC. In relation with the decrease in brain weight, the cingulum bundle, a myelinated structure associated with the corpus callosum, displayed a significant reduction in size. The implications of these observations for human exposures are discussed.


Time course for the hazard of radiation-induced pneumonitis death in mice. The form of the hazard function for radiation-induced pneumonitis death in mice was investigated. 'Hazard' refers to the instantaneous failure rate at a specified time, conditional upon non-failure to that time. Thus, the hazard function describes the time profile for the risk of pneumonitis death among still-surviving subjects. Single-dose lethality data from nine previously published studies involving irradiation of the lung were combined. Sufficient data were then available to estimate the hazard for eight different dose groups (dose range 12-15 Gy). The results of this study suggest that there are multiple distinct peaks in the hazard function for radiation pneumonitis, corresponding to distinct waves of death separated by an average interval of 33 days. The times of the peak hazards are dose dependent, with the peak hazards occurring earlier after larger doses, and the values of the hazards at the peaks are also dose dependent, with larger doses corresponding to a greater risk of death. The implications of a multiply-peaked hazard function for the possible mechanisms of response to whole-lung irradiation are discussed.


Rearrangements of the upstream regulatory region of human papillomavirus type 6 can be found in both Buschke-Löwenstein tumours and in condylomata acuminata. Clinically malignant Buschke-Löwenstein tumours and benign condylomata acuminata are caused by human papillomaviruses (HPVs), predominantly HPV-6 and -11. In some cases, the HPV-6 genomes found in Buschke-Löwenstein tumours and in verrucous carcinomas differ from HPV-6b isolated from a benign genital wart, by rearrangements of the upstream regulatory region (URR). To evaluate the frequency and role of mutations of the URR of HPV-6 we analysed 42 condylomata acuminata and four Buschke-Löwenstein tumours by the polymerase chain reaction and restriction enzyme cleavage. Using only four different restriction enzymes we could demonstrate four distinct restriction patterns, indicating that naturally occurring HPV-6 isolates display a high degree of DNA polymorphism within the URR. One Buschke-Löwenstein tumour and two condylomata acuminata yielded rearranged URRs with DNA duplications. All three lesions harboured multiple HPV-6 variants, suggesting that cellular or environmental factors facilitate the development of rearrangements. Therefore, rearrangements of the URR may represent only secondary events in condylomata acuminata and Buschke-Löwenstein tumours which do not necessarily confer a higher malignant potential to the infected cell.


Detection of proteinase K-resistant prion protein and infectivity in mouse spleen by 2 weeks after scrapie agent inoculation. The sequential accumulation of the protease-resistant form of the endogenous prion protein (PrP-res) was compared to levels of scrapie infectivity in the spleen and brain of scrapie-infected mice at various times after inoculation. In mouse spleen PrP-res was detected 1 week after inoculation, and increased 65-fold between 1 and 3 weeks post-inoculation and an additional 15-fold during the next 17 weeks. Infectivity in spleen reached a maximum plateau level by 3 weeks. In contrast, in mouse brain PrP-res was not detected until 8 weeks after inoculation and then increased 200-fold during the next 12 weeks. During this same time, infectivity increased approximately 10,000-fold. Therefore, in both spleen and brain of scrapie-infected mice accumulation of PrP-res and infectivity appear to be associated. However, it was not possible to show quantitative correlations between PrP-res detection and infectivity, perhaps owing to the inaccuracy of the infectivity assay.


Excitotoxic cell death.

Excitotoxic cell death. Excitotoxicity refers to the ability of glutamate or related excitatory amino acids to mediate the death of central neurons under certain conditions, for example, after intense exposure. Such excitotoxic neuronal death may contribute to the pathogenesis of brain or spinal cord injury associated with several human disease states. Excitotoxicity has substantial cellular specificity and, in most cases, is mediated by glutamate receptors. On average, NMDA receptors activation may be able to trigger lethal injury more rapidly than AMPA or kainate receptor activation, perhaps reflecting a greater ability to induce calcium influx and subsequent cellular calcium overload. It is possible that excitotoxic death may share some mechanisms with other forms of neuronal death.


P-glycoprotein expression in brain tumors. Overexpression of P-glycoprotein (P-gp) in cancer cells can result in resistance to several chemotherapy agents (multidrug resistance) including doxorubicin and vincristine. The drugs to which resistance develops also penetrate the blood brain barrier poorly. P-gp expression in brain capillary endothelial cells suggests that P-gp may restrict drug entry into brain tumors and thus be another mechanism of drug resistance. To seek evidence for either of these roles in the drug resistance of brain tumors, we examined the location of expression of P-gp in 49 brain tumors, using an anti-P-gp mouse monoclonal antibody and immunohistochemistry. P-gp expression was observed in tumor cells of two glioblastomas and a meningeal sarcoma but not in low-grade primary or metastatic tumors. In low-grade primary tumors, P-gp was present in all vascular endothelial cells. In the vascular endothelial cells of anaplastic primary brain tumors and brain metastases, P-gp expression was heterogeneous or absent. These findings are consistent with a role for P-gp in the resistance of some brain tumors to chemotherapy agents.


The erbB-3 gene in human pancreatic cancer. Abnormalities of the type 1 growth factor receptor family have been implicated in the pathogenesis of pancreatic cancer. There is evidence for a potential autocrine loop involving overexpression of the epidermal growth factor (EGF) receptor and its ligands, as well as overexpression of the erbB-2 receptor. A third member of this receptor family, erbB-3, has recently been recognized and found to be abnormally expressed in some types of human cancer. In this study we show that overexpression of the erbB-3 protein occurs very frequently in carcinoma of the exocrine pancreas and also in chronic pancreatitis. We found no evidence of amplification or rearrangement of the erbB-3 gene by Southern blot analysis of DNA from pancreatic cancer cells lines.


Production and characterization of a polyclonal antibody to the c-erbB-3 protein: examination of c-erbB-3 protein expression in adenocarcinomas. A polyclonal rabbit antibody was raised to the c-erbB-3 protein using a synthetic peptide corresponding to amino acids 1229-1241 of the predicted protein sequence of c-erbB-3. In Western blot analysis this antibody detects a single band at approximately 165 kD in a c-erbB-3 transfected (293/HER-3) human cell line. c-erbB-3 protein expression was then examined in a variety of adenocarcinomas. Expression of c-erbB-3 protein was indicated by membrane and/or cytoplasmic tumour cell immunoreactivity in formalin-fixed, paraffin-embedded tissue sections. c-erbB-3 protein was detected in a series of 13 out of 14 primary breast carcinomas, 3 of 5 gastric adenocarcinomas, 8 of 9 colonic adenocarcinomas, 2 of 9 prostatic adenocarcinomas, 0 of 6 renal cell carcinomas, 1 of 4 primary lung adenocarcinomas, and 5 of 7 endometrial adenocarcinomas. Immunohistochemical expression of the c-erbB-3 protein appears to be a relatively common event in adenocarcinomas, and further studies are now warranted to establish the role of the c-erbB-3 protein in neoplasia.


Epidermotropism of T cells correlates with intercellular adhesion molecule (ICAM1) expression in human papillomavirus (HPV)-induced lesions. Adhesion molecules play an important role in inflammatory reactions. Among them, ICAM1, a ligand for the lymphocyte function-associated antigen (FLA1) of leucocytes, may be expressed by antigen-presenting cells and keratinocytes in various inflammatory disorders. As cell-mediated immune responses play a great role in HPV infections, we investigated the expression of ICAM1 and correlated it with the presence of LFA1-positive cells by immunohistochemistry on serial frozen sections of a series of non-regressing cutaneous and mucosal HPV-induced lesions. ICAM1 expression by keratinocytes was observed only in intensely infiltrated lesions of condylomas and laryngeal papillomas. Its induction was usually correlated with the presence of LFA1-positive cells (mainly CD8-positive cells) which were in close apposition to ICAM1-positive proliferative epithelial cells expressing also, in some cases, HLA-DR antigen. ICAM1 was not correlated with the presence of HPV DNA or viral antigen. In moderately infiltrated lesions, keratinocytes did not express ICAM1, and LFA1-positive cells were not observed in the epidermis. In all lesions, ICAM1 was more intense on endothelial cells than in normal skin; infiltrating cells (lymphocytes and dendritic cells) may also express this antigen but intraepithelial Langerhans cells were devoid of any labelling. These studies provide further evidence that T-lymphocyte mechanisms are important in the host response to HPV-induced lesions. ICAM1 expression correlates with a lesional infiltrate but not with HPV infection and probably results in a more efficient initiation of the immune reaction.


Effect of cromakalim on the smooth muscle of the cat gastric antrum. The effect of the K(+)-channel opener cromakalim (BRL 34915) on the electrical and contractile activity of the smooth muscle of the cat gastric antrum has been studied. Cromakalim induced a concentration-dependent inhibition of the contractions and shortening of the sustained partial repolarization phase of the plateau action potential. High concentrations of cromakalim produced hyperpolarization and shortening of the repolarization and depolarization phases of the plateau action potential. The K(+)-channel blockers 4-aminopyridine (10(-2) M) and tetraethylammonium (10(-2) M) decreased the effect of cromakalim on the phasic contractions, while glibenclamide (5 x 10(-5) M) completely abolished it. We suggested that the inhibitory effect of cromakalim on the electrical and contractile activity of the gastric antrum smooth muscle is due to the cromakalim-induced increase of the outward K(+)-current through glibenclamide-dependent K(+)-channels.


Calcium antagonism by cobalt ions on contraction of guinea-pig taenia coli. In guinea-pig taenia coli, cobalt ions (Co2+) inhibited the tonic response to a highly concentrated K+ solution (high-K+, 40 mM) more strongly than the phasic response. Co2+ displaced Ca2+ concentration-response curves to the right, inhibited the increase in tissue calcium content caused by high-K+, and inhibited Ca2+ binding at low affinity sites more than at high affinity sites. After treatment with Co2+, the tonic tension caused by high-K+ was not restored by a wash with normal medium, but it was restored by a wash with EDTA. The cobalt remaining in muscles was almost completely eliminated after a 20-30 min wash with EDTA. The results suggest that Co2+ binds chiefly to the surface membrane of taenia coli. Co2+ probably reduced the tension in response to high-K+ mainly by inhibiting Ca2+ influx rather than by affecting Ca2+ release.


Ultrastructural alterations and DNA synthesis of renal cell nuclei following cisplatin or carboplatin injection in rats. To clarify the difference in nephrotoxicity between cisplatin and carboplatin, ultrastructural alterations and DNA synthesis of renal cell nuclei were studied in Sprague-Dawley rats which had received intravenously either cisplatin or carboplatin at an equitoxic dose. Twelve hours after cisplatin injection, nucleolar segregation accompanied by aggregated nuclear heterochromatin was observed in the third segment of the proximal tubules. Seventy-two hours after cisplatin injection, nuclear damage was more widespread while regenerative cells were also observed. Nuclear damage was not observed in the carboplatin-treated rats. Nuclear DNA synthesis of renal cells was suppressed at 8, 12 and 24 h and was accelerated at 72 h after cisplatin injection. Carboplatin did not suppress nuclear DNA synthesis at any time. The results indicate that cisplatin, but not carboplatin, can affect the renal cell nuclei. Cisplatin-induced nephrotoxicity is related to its effects on renal cell nuclei.


Selective bronchodilators from 1-(5'-oxohexyl)xanthines. A series of twenty one 1-(5'-oxohexyl)xanthines substituted with alkyl chains at the N3 and N7 positions of the xanthine nucleus were prepared and their relaxant activity in guinea-pig isolated tracheal muscle and positive chronotropic activity in isolated right atrium of guinea-pig were compared. The tracheal relaxant activities were markedly increased with alkyl chain length at the N3 position, but decreased by the N7 alkylation. The positive chronotropic activities in the right atrium were increased by introduction of an n-propyl group at the N3 position but decreased by substitution of longer alkyl chains, and the action on the heart was diminished by N7 substitution. The activities of compounds on cAMP-phosphodiesterase (PDE) and binding of [3H]8-cyclopentyl-1,3-dipropylxanthine were measured in the homogenate of tracheal muscle and the membrane preparation of cerebral cortex, respectively. No relationship among tracheal muscle relaxant activity, cAMP-PDE inhibitory activity and adenosine antagonism of these xanthines was observed, and other action mechanisms should be considered for their relaxant activities. This study indicated that N3 alkylation is important for the selectivity for tracheal muscle, while the introduction of long alkyl chains such as n-butyl and n-pentyl groups at the N3 and N7 positions diminished the potency for the right atrium in guinea-pigs. 3-n-Pentyl- and 7-methyl-3-n-pentyl-1-(5'-oxohexyl)xanthines showed much higher bronchoselectivity than oxpentifylline and theophylline.


Pharmacokinetic interactions between isoniazid and theophylline in rats. Pharmacokinetic interactions between isoniazid and theophylline were studied in male Wistar rats, 206 +/- 17 g. Concomitant oral administration of 2 x 5 mg kg-1 isoniazid accelerated slightly the disposition of theophylline (10 mg kg-1, i.v.) whereas 2 x 25 mg kg-1 isoniazid slowed it marginally. The differences in distribution volume, systemic clearance and area under the concentration-time curve (AUC) between the high and the low dose, however, were statistically significant. One week pretreatment with 10 mg kg-1 isoniazid tended towards inhibition (significant decrease of systemic clearance, increase of AUC) and 50 mg kg-1 to acceleration (decrease of half-life, mean residence time and AUC, increase of systemic clearance) of theophylline disposition. After oral pretreatment with 20 mg kg-1 theophylline, neither the kinetics of free isoniazid (50 mg kg-1, i.v.) and the amount acetylated nor the acetylation indices differed from the controls. There was no evidence that concomitant or subacute administration of different doses of isoniazid affects major metabolic pathways of theophylline or that prolonged theophylline treatment interacts with the N-acetylation capacity.


Effect of uraemia and anephric state on the pharmacokinetics of tenoxicam in the rat. Renal alterations, uraemia and nephrotic syndrome induced in experimental animals caused a reduction in the plasma albumin concentration of 25 and 30%, respectively. As a result of this decrease, the unbound fraction of tenoxicam in uraemic rats (0.06 +/- 0.02) and in anephric rats (0.11 +/- 0.08) increased with respect to the control group (0.03 +/- 0.004). The induced hypoalbuminaemia did not modify the blood to plasma concentration ratio. Both plasma clearance (CL) and apparent volume of distribution at steady-state (Vdss) rose significantly with the increase in the unbound fraction: (Vdss 55 +/- 6 mL (control rats); 69 +/- 12 mL (uraemic rats); 96 +/- 30 mL (anephric rats); CL = 7 +/- 1 mL h-1 (control rats); 12 +/- 4 mL h-1 (uraemic rats); 15 +/- 7 mL h-1 (anephric rats)). Tenoxicam elimination was found to be restrictive, with an extraction ratio less than 0.1 in the three groups. The induction of nephrotic syndrome was observed to have a significant effect on intrinsic metabolic activity, intrinsic clearance of tenoxicam being reduced by 30% in the anephric rats (161 +/- 38 mL h-1) with respect to the values obtained in the control group (228 +/- 22 mL h-1).


Pharmacokinetic study of gadolinium-DOTA in control and streptozocin diabetic rats. The pharmacokinetics of gadolinium tetraazacyclododecanetetraacetic acid (Gd-DOTA), a contrast agent used in magnetic resonance imaging, have been evaluated in control and streptozocin-diabetic rats of different ages. In control rats, an age-related decrease in the Gd-DOTA elimination rate was noted, supported by a significantly lower apparent total body clearance and a significantly higher mean residence time. In diabetic rats, a similar but less important age-related change in the mean residence time and the apparent total body clearance was observed. Regardless of age-related differences in the pharmacokinetic parameters, a diabetic state induced several alterations in the Gd-DOTA pharmacokinetic parameters. The apparent total body clearance was significantly higher and the mean residence time significantly lower in diabetic rats indicating a higher elimination rate of Gd-DOTA. An important age-related increase in the volume of distribution at steady-state was noted in diabetic rats.


Disposition of heptabarbitone in the rat: identification of a new metabolite by tandem mass spectrometry. The purpose of this study was to elucidate the structure of a metabolite of heptabarbitone, the occurrence of which was reported previously in a number of pharmacokinetic and pharmacodynamic modelling studies. By application of thermospray liquid chromatography (tandem) mass spectrometry, the identity of the metabolite was proposed to be 5-ethyl-5-(1',[3' or 6']-cycloheptadienyl)-barbituric acid. By measuring the ratios between the areas under the concentration time curves of the metabolite and heptabarbitone after administration of three different intravenous dosages of heptabarbitone, it was shown that the exposure to the metabolite is directly correlated with the exposure of heptabarbitone.


Binding of [3H]haloperidol to dopamine D2 receptors in the rat striatum. The present study was designed to examine the properties of [3H]haloperidol binding to dopamine D2-receptors in rat striatum membranes, displacement potencies of various chemicals and differences between the affinities of various chemicals and two new 5-hydroxytryptamine (5-HT2) receptor antagonists, MCI-9042, (+/-)-2-(dimethylamino)-1-[[o-(m-methoxyphenetyl)phenoxy]methyl]et hyl hydrogen succinate hydrochloride and one of its metabolites. The plots of specific binding for the striatum membranes obtained from the Scatchard analysis using [3H]haloperidol were monophasic when non-specific binding was determined with 10 microM chlorpromazine, and the Kd and Bmax values were 7.42 +/- 1.03 nM and 1.58 +/- 0.20 pmol (mg protein)-1 (n = 10), respectively. The displacement potencies of D2 receptor, 5-HT2 receptor, histamine H1-receptor, and adrenoceptor antagonists were characterized by [3H]haloperidol binding to D2 receptors. The pKi values of a new antiplatelet agent, MCI-9042, and its metabolite were 5.02 and 5.53, respectively, and these values were lower than those of the D2-receptor antagonists, fluphenazine, spiperone, haloperidol, prochlorperazine, chlorpromazine, thioridazine, and sulpiride. They were also lower than the pKi values of the 5-HT2-receptor antagonists, pirenperone, ketanserin, methysergide, and mianserin. We conclude that the binding site of [3H]haloperidol in the rat striatum is the D2 receptor, that MCI-9042 and its metabolite have lower affinities for D2 receptors than for 5-HT2 receptors, and that this radioreceptor assay is useful for assessing the affinities of various agents.


Effect of fenbufen on the entry of new quinolones, norfloxacin and ofloxacin, into the central nervous system in rats. The entry of two new quinolone antibacterial agents, norfloxacin and ofloxacin, into the central nervous system (CNS) of rats, and the effect of fenbufen on this was investigated. At various times after the administration of a bolus intravenous dose of norfloxacin or ofloxacin (10 mg kg-1) with or without fenbufen (20 mg kg-1), serum and cerebrospinal fluid (CSF) samples and whole brain were collected from the rats and the concentration of norfloxacin or ofloxacin in each sample was determined. Serum concentrations of both quinolones declined biexponentially with time and were significantly elevated by coadministration with fenbufen at the terminal phase. The fractions of these quinolones bound to serum protein were not altered by coadministration with fenbufen. Coadministered fenbufen raised the brain concentrations of both quinolones but did not affect their brain to serum unbound concentration ratios. In contrast, CSF to serum unbound concentration ratios as well as CSF concentrations of norfloxacin and ofloxacin were elevated by coadministration with fenbufen. Apparent diffusional clearances between blood and CSF of norfloxacin and ofloxacin estimated by the physiological model analysis increased by 1.9 and 2.6 times, respectively, after coadministration with fenbufen. These findings suggest that coadministered fenbufen may facilitate the entry of norfloxacin and ofloxacin into the CNS.


Enantioselective pharmacokinetics of alpha-fluoromethylhistidine in rats and its comparison with histidine. The enantiomer-specific pharmacokinetics of histidine and its analogue, alpha-fluoromethylhistidine (FMH), were investigated in rats. After bolus intravenous administration of each enantiomer of histidine or FMH at a dose of 40.3 mg kg-1 as free base equivalents, the plasma concentrations of L-histidine, D-histidine, (S)-FMH and (R)-FMH decreased biexponentially with half-lives of 39.2, 20.8, 32.8 and 25.0 min, respectively, in the elimination phase. Although the concentration of L-histidine in the plasma was lower than that of D-histidine, there was no large difference in plasma concentration-time curves of the enantiomers of FMH. The apparent total clearance of L-histidine from rat plasma was about 4 times that of D-histidine or the enantiomers of FMH. L-Histidine was quickly transferred to the peripheral tissues where the concentrations also decrease biphasically. L-Histidine penetrated more rapidly into the brain than either its D-enantiomer or a compound closely related in structure such as FMH. However, the disappearance of L-histidine from the various brain regions was very rapid. In contrast, brain/plasma ratios of D-histidine and (S)-FMH increased continuously after injection of these compounds, indicating that D-histidine or (S)-FMH partitioned into the brain and was very slowly removed from the brain; (R)-FMH was not distributed to the brain. These results suggested stereoselectivity in disposition of histidine and FMH enantiomers in rats.


Gastric H+, K(+)-ATPase inhibition by catechins. Five catechins, (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin and (-)-epigallocatechin gallate, inhibited gastric H+, K(+)-ATPase activity with IC50 values ranging from 1.7 x 10(-4) to 6.9 x 10(-8) M, with (-)-epigallocatechin gallate as the most potent inhibitor. The intensity of inhibitor activity paralleled the number of phenolic hydroxy groups in the molecule. The inhibition of the enzyme by (-)-epicatechin was competitive with respect to ATP and noncompetitive with respect to K+. These findings suggest that the anti-secretory and anti-ulcerogenic effects of catechins previously reported, are due to their inhibitory activity on gastric H+, K(+)-ATPase.


Gastric anti-ulcer activity of silymarin, a lipoxygenase inhibitor, in rats. Oral treatment with silymarin was found to be effective in the prevention of gastric ulceration induced by cold-restraint stress, in rats. Statistically significant ulcer index values with respect to the control group, were observed. In 6 h pyloric-ligated animals silymarin showed a significant reduction in the number and severity of the ulcers; however, it did not alter the gastric secretion volume or acidity although histamine concentration was significantly decreased. In absolute ethanol-induced ulcers, treatment with silymarin 1 or 2 h before the anti-ulcerogenic agent, did not prevent the formation of gastric lesions. Furthermore, the hexosamine content was decreased significantly, but the total protein output was enhanced, showing similar values to those with the standard drug, carbenoxolone. These results suggest that the anti-ulcerogenic effect of silymarin could be related to its inhibitory mechanism of enzymatic peroxidation by the lipoxygenase pathway, avoiding leukotriene synthesis.


High molecular weight protein aggregates formed in the liver of the rat following large doses of paracetamol. Paracetamol (200 and 500 mg kg-1) was given intraperitoneally to rats pretreated with 3-methylcholanthrene for 3 days. Glutamic oxalacetic acid transaminase (GOT) activity in plasma increased in rats receiving 500 mg kg-1 paracetamol. Plasma GOT activity was low at the dose of 200 mg kg-1, but the same dose to diethyl maleate pretreated rats increased the GOT activity. High mol. wt protein aggregates were found to be formed in liver homogenates and microsomes of rats which showed high plasma GOT activity, accompanied by depletion of hepatic glutathione. The formation of protein aggregates in the liver of rats following large doses of paracetamol suggests a contribution of lipid peroxidation to paracetamol-induced hepatotoxicity.


In-vitro protein binding interaction between a metabolite of triflusal, 2-hydroxy-4-trifluoromethylbenzoic acid and other drugs. 2-Hydroxy-4-trifluoromethylbenzoic acid (HTB) is the main active metabolite of triflusal, an antiplatelet drug. The in-vitro binding of HTB to human serum was studied in the presence of different drugs. The results indicate that no statistically significant changes are observed in the HTB binding in the presence of caffeine, theophylline, glisentide, enalapril, cimetidine or warfarin. The free fraction of HTB increases significantly in the presence of the non-steroidal anti-inflammatory drugs studied: diclofenac, ibuprofen, indomethacin, naproxen, piroxicam and salicylic acid. At high concentrations, HTB displaces these anti-inflammatory drugs and also glisentide and warfarin from their protein binding sites.


The effects of calcitonin nasal preparations and their excipients on mucociliary clearance in an ex-vivo frog palate test. The topical tolerability of the commercial preparation of 1-7 Asu-eel and salmon calcitonin with 2% ammonium glycyrrhyzinate and 0.01% benzalkonium chloride, respectively, and of their excipients mixture in solution with increasing concentrations of ammonium glycyrrhyzinate and benzalkonium chloride, respectively, were assessed by investigating their effects on the mucociliary transport velocity in the ex-vivo frog palate preparation. This preparation provides an integrated biological model readily usable in the laboratory which closely resembles human nasal mucociliary clearance mechanism and can be used for rapid testing and toxicity of agents proposed for topical administration in the upper and lower airways. Frog-Ringer control, 1-7 Asu-eel and salmon calcitonin commercial spray preparations and the excipients plus 2% ammonium glycyrrhyzinate and plus 0.01% benzalkonium chloride did not modify significantly the mucociliary transport velocity, confirming their very good tolerability on ciliated epithelium. Higher concentrations of ammonium glycyrrhyzinate (10 and 20%) caused significant slowing, on average -32 and -55%, respectively. Higher concentrations of benzalkonium chloride (0.05 and 0.1%) also caused significant slowing, on average, -43.5 and -87%, respectively.


The binding of the antimalarial arteether to human plasma proteins in-vitro. The binding of the novel antimalarial drug, arteether, to human plasma, pure albumin and alpha 1-acid glycoprotein has been investigated by ultrafiltration, using [14C]arteether. The protein binding in plasma obtained from 11 healthy male subjects ranged from 73.4 to 81.8% bound, with a mean of 78.7 +/- 2.1%. The binding of drug in plasma was mainly accounted for by binding to albumin and alpha 1-acid glycoprotein. Scatchard analysis of the binding data revealed that the binding affinity of arteether to alpha 1-acid glycoprotein is much greater (20-fold) than that to albumin. This suggests that alpha 1-acid glycoprotein is the more important binding protein in plasma. This may have clinical importance due to alterations in plasma protein binding in patients with malaria, as the concentration of alpha 1-acid glycoprotein is markedly increased during malarial infection.


The Ginkgo biloba extract, EGb761, increases synaptosomal uptake of 5-hydroxytryptamine: in-vitro and ex-vivo studies. The Ginkgo biloba extract (EGb 761) added to a synaptosomal fraction prepared from mice cerebral cortex modified [3H]5-hydroxytryptamine ([3H]5-HT) uptake in a biphasic manner. Between 4 and 16 micrograms mL-1 EGb 761 increased significantly the [3H]5-HT uptake (maximum + 23%). A similar increase was also obtained when synaptosomes were prepared from the cortex of mice treated orally with EGb 761, either acutely (100 mg kg-1, 14 h and 2 h before death) or semi-chronically (2 x 100 mg-1 kg daily for 4 consecutive days). The in-vitro increase in [3H]5-HT uptake induced by EGb 761 was not observed in the presence of 10(-6) M clomipramine, a 5-HT-uptake inhibitor. EGb 761 did not increase [3H]dopamine uptake by synaptosomes prepared from striatum of mice. We investigated different fractions of EGb 761 in order to determine the compounds inducing the increase in [3H]5-HT uptake. The BN 52063 extract (corresponding to the EGb 761 devoid of flavonoid substances) did not increase [3H]5-HT uptake. The Cp 202 extract (corresponding to the EGb 761 devoid of terpenic substances and containing mostly flavonoid substances) increased [3H]5-HT uptake. Among the flavonoids, quercetin has been tested and had no effect on the [3H]5-HT uptake. Since at the usual therapeutic doses of EGb 761, the effective concentrations of the components responsible for this increase are likely to be reached in the brain, one may suggest that this effect could contribute to the therapeutic effect of EGb 761.


The effects of labetalol and dilevalol on isolated cardiovascular preparations of the guinea-pig and rat. Differing effects of labetalol and dilevalol on cardiovascular preparations have been reported. I have studied the effects of labetalol and dilevalol on the contractile responses of the rat and guinea-pig left atria and rat portal vein. On the guinea-pig left atria low concentrations of labetalol (> or = 10(-8) M) and of dilevalol (> or = 10(-7) M) inhibited to a small extent the responses to electrical cardiac stimulation, which is indicative of membrane stabilizing activity. Labetalol (> or = 3 x 10(-8) M) and dilevalol (> or = 10(-8) M) caused surmountable antagonism of the isoprenaline responses of the atria and the pA2 values were 8.60 and 8.98 at the beta 1-adrenoceptors of the rat left atria and 7.90 and 8.31, respectively, on the guinea-pig left atria which has functional beta 1- and beta 2-adrenoceptors. Labetalol and dilevalol (both at > or = 10(-7) M) attenuated the spontaneous contractile activity of the rat portal vein and the attenuation to labetalol at 10(-6) M was abolished by ICI 118,551 which illustrates that the labetalol-induced attenuation is beta 2-adrenoceptor mediated. The isoprenaline attenuation responses of the portal vein were inhibited by labetalol and dilevalol (both at > or = 10(-7) M) and the pA2 value for the labetalol at beta 2-adrenoceptors was 7.59. It is concluded that labetalol and dilevalol are beta 1-adrenoceptor selective antagonists.


Fab-bound colchicine appears to adopt Fab fragment disposition in rats. The disposition of colchicine-specific Fab fragments and the effect of Fab fragment administration on the disposition of colchicine were studied in anaesthetized bile duct-cannulated rats. One group of rats (n = 6) received a 125I-Fab dose of 38 mg kg-1 i.v. The plasma disposition was characterized by a volume of distribution of 179 +/- 48 mL kg-1, total body clearance of 1.02 +/- 0.07 mL min-1 kg-1, t1/2 alpha of 0.17 +/- 0.03 h and t1/2 beta of 1.3 +/- 0.3 h. Fab fragments were in part excreted by the renal route (15.6 +/- 6% of the Fab dose), while biliary excretion was a minor route (< 2% of the Fab dose). Two other groups of rats received 15 micrograms kg-1 colchicine (n = 6) or 15 micrograms kg-1 colchicine plus 38 mg kg-1 colchicine-specific Fab fragments (n = 6) by intravenous infusion. Pharmacokinetics of colchicine was markedly altered in the Fab-colchicine-treated rats. In this group, distribution volume and total body clearance of colchicine were decreased by factors of 22 and 10, respectively, compared with the values in the colchicine-treated group and were very similar to those of Fab fragments. An 80% reduction of cumulative biliary excretion of colchicine was observed in Fab-colchicine-treated rats (P < 0.01). The fraction of colchicine dose excreted by the urinary route was 38 +/- 6.9 and 9 +/- 0.7% respectively in Fab-colchicine- and colchicine-treated groups (P < 0.01). These data show that during Fab treatment, colchicine followed the elimination kinetics of Fab fragments.(ABSTRACT TRUNCATED AT 250 WORDS)


Spectrophotometric method for the determination of carbidopa using neotetrazolium chloride. A new high-sensitive spectrophotometric method for the determination of microquantities of carbidopa is described. The method is based on the reduction of neotetrazolium chloride by carbidopa in an alkaline ethanolic medium. The formazan formed exhibits an absorption maximum at lambda = 507 nm, with an apparent molar absorptivity of epsilon = 6.63 x 10(4) M-1 cm-1 and a corresponding Sandell's sensitivity of 3.68 ng cm-2. Beer's law is obeyed over the range of 0.10-6.0 micrograms mL-1 while the optimum concentration range is 0.125-5.0 micrograms mL-1. The regression line equation was calculated as: A = 0.271C + 0.0023 with a correlation coefficient of 1.0000 (n = 28). The accuracy and the precision of the method were considered as very satisfactory. The results obtained from the determination of carbidopa using both the described procedure and the corresponding USP. XXII and BP 1988 clinical methods were statistically compared by means of Student's t-test as well as by the variance ratio F-test, and no significant difference was observed.


Simultaneous determination of bufadienolides in the traditional Chinese medicine preparation, liu-shen-wan, by liquid chromatography. The bufadienolide compounds (bufalin, cinobufagin and resibufogenin), major constituents of Chansu in Liu-Shen-Wan (LSW), were determined by reverse phase high performance liquid chromatography. The procedure involves a preliminary extraction of the bufadienolides from LSW with chloroform using ultrasonication and subsequent evaporation to dryness of the chloroform extract. The residue of the chloroform extract was dissolved in methanol and separated on a Merck LiChrosorb RP-18 column. Methanol: water (74:26) was used as mobile phase. The compounds were satisfactorily separated with good chromatographic peaks. Good coefficients of correlation (r > 0.999) were obtained from the calibration of peak areas with concentrations for the 3 bufadienolides. Results of analysis showed that there were differences between the contents of bufadienolides in 11 LSW samples of different origin available to the public in Hong Kong where at present there is no legal control over the sale of traditional Chinese medicines. The variability of quantities of bufadienolides in Chansu may be a hazard to the public.


Uptake of cefadroxil derivatives in rat intestinal brush-border membrane vesicles. Uptake of cefadroxil and its two acetyl-derivatives, N-acetyl- and O-acetyl-cefadroxil, into the brush-border membrane vesicles (BBMV) was measured at [pH]o = 5.5, 7.4 and [pH]i = 7.4. Both acetyl-derivatives showed a significantly slower uptake than cefadroxil at [pH]o = 5.5 and 7.4. Cefadroxil and the two derivatives showed a higher uptake rate in the presence of an inward H+ gradient ([pH]o = 5.5, [pH]i = 7.4). At [pH]o = 5.5, uptake of cefadroxil into BBMV was inhibited by N-acetyl-, O-acetyl-, N-BOC-, and N-BOC-O-acetyl-cefadroxil, but not by cephalothin and cefuroxime. At [pH]o = 7.4, no inhibition of cefadroxil uptake was evident for any inhibitors. There were two different transporters responsible for the uptake of cefadroxil at pH 5.5 and 7.4. One is the H(+)-coupled dipeptide transport system, and the other is the neutral pH-preferring system. The alpha-amino group may be essential for the transport of cefadroxil by both transport systems. Although the phenolic group in the side chain is not an essential functional group of beta-lactam antibiotics, an additional derivation on the phenolic group of cefadroxil also inhibited both the H(+)-coupled dipeptide transport system and the neutral pH-preferring transport system.


Effects of nicarbazin on intestinal digestion and absorption of nutrients in the rabbit. Nicarbazin is an anticoccidial drug, used mainly in birds, which has shown several other effects including inhibition of growth and feed efficiency in poultry, and stimulation of sugar and amino acid intestinal absorption in rabbit. The present work was designed to determine whether nicarbazin added to the feed, affects growth and feed intake in rabbit, and whether the continuous ingestion of nicarbazin can alter the mechanisms of intestinal nutrient absorption or digestion in this species. Nicarbazin, administered at the recommended dose (125 ppm) had no harmful effects either on growth or on feed intake of animals. After treatment for one month with nicarbazin at the dose of 125 ppm added to the feed, rabbits displayed a higher transport ability of both D-glucose and L-leucine through the enterocyte plasma membranes than did untreated rabbits. These animals also showed higher specific activities of two brush-border enzymes, sucrase and aminopeptidase N, than the control animals.


Inhibitory effect of prostaglandin E1 on laurate-induced peripheral vascular occlusive sequelae in rabbits: optimized topical formulation with beta-cyclodextrin derivative and penetration enhancer HPE-101. Prostaglandin E1 (PGE1) and its inclusion complexes with beta-cyclodextrin (beta-CyD) and O-carboxymethyl-O-ethyl-beta-cyclodextrin (CME-beta-CyD) were made as topical preparations. The PGE1 preparations, when applied with a penetration enhancer, 1-[2-(decylthio)ethyl]azacyclopentane-2-one (HPE-101), markedly increased the regional blood flow in the ear of rabbits and were longer acting than when administered by the intravenous route. Topical application of the PGE1 preparations significantly protected rabbits against laurate-induced peripheral vascular occlusive sequelae; the protective potency increased in the order of PGE1 alone = beta-CyD complex < CME-beta-CyD complex preparation. The PGE1 preparations elicited skin reactions such as erythema and oedema depending on their vasodilating actions. These reactions disappeared gradually after removal of the preparations, and hence may not be serious obstacles for their safe use. These results suggest that combinations of CME-beta-CyD and HPE-101 work synergistically to facilitate the entry of PGE1 into the skin, and consequently enhance the therapeutic potential of PGE1 in the topical preparation tested.


Decrease by psychotropic drugs and local anaesthetics of membrane fluidity measured by fluorescence anisotropy in Escherichia coli. The effects of psychotropic drugs and local anaesthetics on the fluidity of Escherichia coli cell membranes were examined. Chlorpromazine was shown to increase 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy, indicating that it decreased the membrane fluidity. This increase was significant at a temperature of more than 24 degrees C. Dibucaine, lignocaine, imipramine, tetracaine and procaine also increased the fluorescence anisotropy.


The 3,3'-bis-pyridinium mono-oximes as antidotes against organophosphorous intoxication. In an attempt to develop effective antidotes against organophosphorous intoxication, three new structurally related bispyridinium mono-oximes with a 2-oxopropane bridge were synthesized. The compounds were evaluated for in-vivo therapeutic protection and cholinesterase reactivation in blood and various brain regions. In neuromuscular function studies against diisopropyl-fluorophosphate and isopropyl methylphosphonofluoridate poisoning, the oximes produced significant protection. The compounds produced marked peripheral reactivation and beneficial effects on neuromuscular transmission. The reactivation of cholinesterase in cerebral cortex by the oximes, in spite of their being quaternary salts is a notable feature.


Some features of Cannabis plants grown in the United Kingdom from seeds of known origin. The cannabinoid content of UK-grown plants (up to the 6th generation) from Moroccan, Sri Lankan and Zambian seedstock was determined by TLC, GLC and HPLC. All plants from the 5th and 6th series resembled their parents, and UK-grown plants were always much greener than those grown overseas. Cannabinoid content remained broadly typical of the source countries. However, tetrahydrocannabinolic acid (THCA) consistently predominated over tetrahydrocannabinol (THC) to a far greater extent than in the original plants; the THCA/THC ratio was 17 in UK-grown plants compared with 2.0 in the plants from the original areas. Two types of plant emerged from the Moroccan seedstock, one tending to increased cannabidiol (CBD), the other tending to zero levels of this component. The first generation Sri Lankan plants revealed one type of plant with an increased CBD/THC ratio (1.7 compared with 0.11) but this returned to the original value in the succeeding generations. Other Sri Lankan plants had low or undetectable levels of CBD. Moroccan and Sri Lankan CBD-rich plants did not contain cannabichromene, although this cannabinoid was found in THC-rich plants. Zambian plants did not appear to show such a pattern. Zambian seedstock plants had total tetrahydrocannabivarin (diol and acid) levels greater than THC but the ratio was progressively reversed in succeeding generations. The study concludes that the ratios of particular cannabinoids is greatly influenced by the environment.


Transfer of heparin across the human perfused placental lobule. A system of dual perfusion of an isolated lobule of term human placenta was used as a model to study the transfer of heparin from maternal to foetal circulation. The metabolic viability of the system was assessed by measuring beta-HCG and alkaline phosphatase levels in both maternal and foetal perfusates. Creatinine and antipyrine were used as markers to determine juxtaposition of the maternal and foetal circulations. Results of this study indicate that following administration of a single bolus dose of heparin into the maternal circulation, its concentration declined slowly from 99.01 +/- 2.98 at 15 min to 97.23 +/- 4.12% and transfer of heparin in the foetal circulation was linear and increased from 0.10% +/- 0.05% at 15 min to 0.46 +/- 0.19% over a period of 120 min. The maternal (MAUC) and foetal (FAUC) concentration-time integrals were found to be 70160 +/- 1332 and 340 +/- 30 int. units min mL-1, respectively. Placental permeability of heparin and creatinine, calculated as the ratio of foetal concentration to the integral maternal-foetal concentration difference, was 8.65 x 10(-5) +/- 0.80 x 10(-5) and 0.033 +/- 0.006 mL min-1 g-1 of perfused placental weight, respectively. These data suggest that heparin was transferred from the maternal to the foetal circulation in small quantities.


The effects of various peptides on human isolated gut muscle. The effects of eleven peptides of gastrointestinal origin have been studied on the contraction, relaxation and spontaneous activity of circular and longitudinal muscle strips from different regions of the human gastrointestinal tract. The effects varied with the peptides and sometimes with the region and muscle layer. There was either contraction, no effect, or relaxation and/or inhibition of an acetylcholine-induced contraction. Responses to some peptides are consistent with the possibility that they may contribute directly to the control of motility: galanin, neurotensin and substance P might be involved in contraction, and vasoactive intestinal peptide, peptide histidine isoleucine and peptide histidine methionine might be inhibitory transmitters.