Doxorubicin-loaded nanospheres bypass tumor cell multidrug resistance.

We have demonstrated that in vitro resistance of tumor cells to doxorubicin (Dox) can be fully circumvented by using doxorubicin-loaded nanospheres (Dox-NS), consisting of biodegradable polyisohexylcyanoacrylate polymers of 300 nm diameter and containing 2.83 mg of Dox per 31.5 mg of polymer. Five different multidrug-resistant cell lines, characterized by mdr1 amplification, were used in this study: Dox-R-MCF7, a human breast adenocarcinoma; SKVBL1, a human ovarian adenocarcinoma; K562-R, a human erythroleukemia; and two murine lines: P388-Adr-R, a monocytic leukemia of DBA2 mouse, and LR73MDR, a Chinese hamster ovarian cell line. These lines were 38.7, 210, 232, 143 and 20 times more resistant than their corresponding sensitive counterparts, respectively. Using Dox-NS, we obtained complete reversion of drug resistance in vitro, i.e. cell growth inhibition comparable with that obtained with sensitive cells exposed to free Dox. In vivo, we significantly prolonged the survival of DBA2 mice which had previously received P388-Adr-R cells by i.p. injections of Dox-NS, while free Dox injection was ineffective toward this rapidly growing tumor. (Prolongation of survival time: 115% vs 167% after Dox vs Dox-NS treatment, respectively.) Using the MCF7 cell line and its resistant variant, we studied the intracellular concentration and the cytoplasmic and nuclear distribution of Dox by laser microspectrofluorometry (LMSF). In sensitive cells, we observed a similar accumulation and distribution of Dox whatever the form of Dox delivery, i.e. whether free or carried by nanospheres. Analysis by LMSF showed that 99% of intranuclear Dox was bound to DNA after treatment with both forms of Dox. Of Dox, 81 and 83% were found in the intranuclear compartment of sensitive cells incubated with free Dox and Dox-NS, respectively. Resistant cells incubated with Dox-NS accumulated the same amount of Dox as sensitive cells incubated with free Dox or with Dox-NS. Dox, when loaded in nanospheres, bypasses the efflux mechanism responsible for multidrug resistance. LMSF analysis showed that Dox, transported and released by nanospheres, interacts with DNA identically in sensitive and resistant cells.


Phenylethanolaminotetralines compete with [3H]dihydroalprenolol binding to rat colon membranes without evidencing atypical beta-adrenergic sites.

[3H]Dihydroalprenolol ([3H]DHA) specific binding (determined by the difference in the presence and absence of 20 microM (-)isoprenaline) to rat colon membranes was saturable (Bmax = 39.6 fmol/mg protein), of high affinity (Kd = 0.87 nM) and stereospecific (IC50 330 and 3510 nM for (-)- and (+)isoprenaline, respectively); the Hill coefficient was close to one, indicating binding homogeneity. [3H]DHA (0.6 nM) specific binding was potently inhibited (Ki range 1.9-3.3 nM) by the non-selective beta-adrenoceptor antagonists pindolol, alprenolol, but not by the non-adrenergic compounds 5-hydroxytryptamine, 8-hydroxydipropylaminotetraline, methysergide, dopamine and verapamil (Ki greater than 10,000 nM). The selective beta 1- and beta 2-adrenoceptor antagonists CGP 20,712A and ICI 118,551 resulted in biphasic competition binding curves, whose low and high affinity components were compatible with two populations of binding sites accounting for about 75 (beta 2) and 25% (beta 1) of total sites. The relative competing potencies of reference adrenergic agonists also suggested a prevalence of beta 2-adrenergic sites. The new agonists phenylethanolaminotetralines (PEATs), highly selective for the atypical beta-adrenoceptors whose abundance in rat colon has been confirmed by comprehensive functional studies, had variable affinity for the [3H]DHA-labelled sites depending on chirality, but with no substantial correlation with their pharmacological potency. Only 40% of [3H]DHA binding, at a concentration about 10 times its Kd for high affinity sites (beta 1 and beta 2), was prevented by saturating concentrations of isoprenaline. Under this condition, the representative PEAT, SR 58611A, highly potent and selective for atypical beta-adrenoceptors in functional tests, and its pharmacologically inactive enantiomer, both inhibited the residual binding equipotently. In conclusion, [3H]DHA binding did not detect atypical beta-adrenoceptor sites in rat colon membranes, most probably because of its weaker affinity for them than for the coexisting beta 1 and beta 2 sites. PEAT stereoisomers proved essential for assessing both the stereospecificity and the functional significance of this atypical binding and to compare their affinity for [3H]DHA-labelled sites and pharmacological potency.


Taking the thymus to pieces.

Complex in vitro and in vivo techniques are being combined to unlock the remaining secrets of the thymus. In this report from a recent thymus workshop*, Bruno Kyewski and Thomas Hünig describe the genetic manipulations aimed at clarifying the mechanisms of T-cell selection and lineage commitment, and the use of organ culture and immunohistology to identify the thymic microenvironments in which these events take place.


Alterations to the cytoskeleton of erythrocytes infected with frog erythrocytic virus: a fluorescence and electron microscopic study.

Erythrocytes of bullfrogs (Rana catesbeiana) infected with frog erythrocytic virus are spheroid and their nucleus is displaced. In contrast, uninfected cells are ellipsoid and have a centralized nucleus. Fluorescent staining revealed that these changes are correlated with alterations to components of the erythrocyte cytoskeleton. Uninfected erythrocytes contained a broad, continuous marginal band of microtubules, which appeared thinner and interrupted in infected cells. The described disruption of microtubules was associated with an inability to polymerize the tubulin pool with the addition of 12 microM taxol. The arrangement of submembranous microfilaments in uninfected erythrocytes was not significantly altered in infected cells. Vimentin filaments were distributed throughout the cytoplasm and around the nucleus of uninfected cells, and concentrated at the cell and nuclear peripheries. Cytoplasmic pockets that did not contain vimentin filaments were associated with the viral assembly site(s) in infected cells. These data suggest that the distortion of viral-infected erythrocytes could be due, in part, to an irreversible depolymerization of microtubules of the marginal band and a reorganization of the vimentin filament network.


Immunological abnormalities in the natural history of HIV infection: mechanisms and clinical relevance.

Infection with the human immunodeficiency virus (HIV) ultimately results in profound immunodeficiency characterized by severe depletion of CD4+ T helper cells. In symptomatic infection a general perturbance of immune function is observed. Here recent insights in the sequence of events in progression to AIDS is reviewed. Following seroconversion a rapid persistent loss of inducible B cell function is observed. In addition, in long term infection, antigen-presenting cell functions of monocytes and dendritic cells are increasingly affected. T-cell non-responsiveness, preceding CD4 cell loss, appears to be induced through several different, sequential mechanisms. In early infection, the in-vivo deletion of memory cells can account for the in-vitro decreased responsiveness. Later on in infection, when the balance between memory and naive T cells is normalized, both CD4 and CD8 cells are non-responsive to nominal antigen and low dose anti-CD3 monoclonal antibodies. This anergy is at the level of IL-2 gene expression since early signal transduction events following CD2 and CD2 receptor occupancy are normal. This state of anergy, probably due to inappropriate activation in vivo, may be related to programmed cell death (PCD) observed in vitro for both CD8 and CD4 cells reflecting a systemic interference with maturation and differentiation of T cells. In progression to symptomatic infection, the proportion of non-responsive CD8 cells with immature or activated phenotypes increases and in about fifty percent of the cases, CD4 cell decline may accelerate in association with emergence of syncytium-inducing HIV variants. During this progressive stage, anti-CD3 reactivity is severely decreased, and alloantigen reactivity and finally the capacity to respond to phytohemagglutinin (PHA) are affected. These functional parameters appear useful for staging of HIV-infected individuals and for evaluation of anti-viral therapy.


The programmed cell death theory of AIDS pathogenesis: implications, testable predictions, and confrontation with experimental findings.

The programmed cell death theory of acquired immunodeficiency syndrome (AIDS) pathogenesis postulates that most immunological and non-immunological defects in human immunodeficiency virus (HIV)-infected people are related to a single mechanism, the inappropriate expression in mature CD4+ T cells and other cell-populations such as neurons of an activation-induced physiological cell suicide program that plays an essential role during embryogenesis in the maturation of both the immune and the nervous systems. The theory is discussed in the context of a series of recent experimental findings indicating that mature T cells can, as immature thymocytes, undergo programmed cell death in response to T-cell receptor mobilization in various physiological and pathological circumstances including murine and human acquired immunodeficiency of retroviral origin. These findings provide new insights into the pathogenesis of AIDS and may have implications for the design of therapeutic strategies.


Effects of concentration reduction and partial replacement of paraquat by diquat on human toxicity: a clinical survey.

Paraquat poisoning was studied in 174 patients over a 12-month period when a new, low concentration paraquat product (4.5% w/v paraquat ion mixed with 4.5% w/v diquat ion; 63 cases) replaced the original high concentration paraquat product (20% w/v paraquat ion only; 111 cases). In both groups approximately 60% of the patients died from circulatory failure accompanied by multiple organ failure within a week of ingesting the products. However, a remarkable reduction in late deaths from respiratory failure was noted in the new product group (17.1% vs 6.3%). This was reflected in this group's improved survival (23.4% vs 34.9%). The improvement in survival seems to be attributable to the dilution of paraquat with diquat which seems to have a different toxicological profile to paraquat.


Pharmacokinetics of fluazifop-butyl in human volunteers. II: Dermal dosing.

1. The absorption of the herbicide fluazifop-butyl (f-b), has been determined from plasma and urine measurements in groups of six male volunteers following dermal administration of 2.5, 25 and 250 micrograms cm-2 from standardized formulations containing 0.05, 0.5 and 5.0% (w/v) fluazifop-butyl to a skin area of 800 cm2. 2. Urinary excretion rate of the principal metabolite fluazifop, following dosing with the 5% formulation, was described by a two-compartment pharmacokinetic model; the average elimination half-lives of initial and terminal phases were 18 h and approximately 70 h, respectively. For the other dose levels the elimination half-life was estimated to be 17 h; urine concentrations at later time points were too low to characterize a second compartment. 3. The estimated total fluazifop-butyl absorbed was 8.0, 3.4 and 1.6% of the applied dose for the 0.05, 0.5 and 5.0% formulations, respectively. 4. Up to 50% of the applied fluazifop-butyl was readily removed by skin washing and the majority of the remainder was transferred to clothing during the 24 h following application. 5. When six volunteers were given a daily dermal dose of the 0.5% formulation for five consecutive days, the plasma and urinary excretion kinetics of fluazifop could be accurately predicted by simple mathematical extrapolation of the kinetic data from the single exposure study at the equivalent daily dose. 6. It is concluded that fluazifop-butyl is only slowly and poorly absorbed through human skin and has a low potential to accumulate in man.


Sex hormones and epididymal sperm parameters in rats following sub-chronic treatment with hexavalent chromium.

Testicular atrophy and reduced epididymal sperm count are known to occur after i.p. administration of high doses of hexavalent chromium to rats. The effect of 0.5 mg kg-1 hexavalent chromium injected i.p. 5 d a week for 8 weeks was investigated in male Wistar rats. A significant reduction in epididymal sperm motility was found at the end of the exposure period. The reduction was reversed after an unexposed period of a further 8 weeks. In addition, a decrease in serum testosterone and an increase in FSH were found at the end of the exposure period. The results indicate that a number of mechanisms may be involved in the deleterious effects of chromate on male fecundity.


Sex hormones and semen quality in welders exposed to hexavalent chromium.

Recent experimental studies in rodents document the spermatotoxic effects of water-soluble hexavalent chromium. Welders comprise, worldwide, a major occupational group with acknowledged exposure to chromium. This study examines the relationship between semen quality and chromium in the urine and blood of a population of 30 tungsten inert gas (TIG) stainless steel welders, 30 mild steel welders and 47 non-welding workers. Each subject provided two to three semen samples. The chromium concentration ranged from 0.17 to 4.74 nmol mmol1 creatinine (median 1.08) in post-shift spot urine and from 6.0 to 46.4 nmol l-1 in blood. None of several semen parameters deteriorated with increasing level of internal exposure to chromium. Low-level exposure to hexavalent chromium associated with TIG stainless steel and mild steel welding do not appear to be a major hazard for human spermatogenesis.


Long-term anticonvulsant therapy worsens outcome in paracetamol-induced fulminant hepatic failure.

1. Paracetamol hepatotoxicity has been found to be potentiated by anticonvulsant drugs in animal experiments; isolated case reports in humans suggest that long-term anticonvulsant therapy may also adversely influence outcome following overdose. 2. We compared the clinical course, after paracetamol overdose, of 18 patients on long-term anticonvulsant therapy with corresponding features in two published series of paracetamol-induced fulminant hepatic failure from this unit: 297 patients seen between 1973 and 1985 and a further 99 between October 1986 and April 1988. 3. Mortality in those patients who were taking anticonvulsants, but who did not receive N-acetylcysteine, was higher than in either of these series (93.3% vs 64.6% and vs 57.9%, P less than 0.025). Although not statistically significant, there were also trends towards more severe coma (grade 3 or 4: 93.3% vs 75.4%, 1986-88), acidosis (pH less than 7.30: 40% vs 22.6%, 1973-85) and coagulopathy (prothrombin time greater than 100 s: 53.3% vs 33.7%, 1973-85). In the small number of patients given N-acetylcysteine, mortality was similar to that in the 1986-88 series (1/3 vs 15/42). 4. We conclude that chronic use of anticonvulsants enhances clinical features of paracetamol toxicity and discuss possible mechanisms by which this could be mediated.


Sulphur derivative of hexachlorobenzene in human urine.

Pentachlorothiophenol, a sulphur derivative of the widespread environmental pollutant hexachlorobenzene (HCB) has been detected and quantified in the urine of a human general population with high body burden of HCB. The sulphur derivative was analysed by GLC-MS as pentachlorothioanisole (PCTA) after hydrolysis and methylation of the respective conjugate and was found in 100% of the samples (n = 40) with a mean concentration of 1.85 +/- 0.98 ng ml-1 (mean +/- s.d., range 0.58-4.50 ng ml-1). No correlation with urinary pentachlorophenol (PCP) and no sex-related differences were found. The derivative may originate from the biotransformation of HCB stored in tissues and may be a useful maker of HCB metabolism in humans.


Chromosomal aberrations and sister-chromatid exchange frequencies in workers occupationally exposed to textile dyes.

The peripheral lymphocytes of 11 male and seven female workers occupationally exposed to textile dyes were studied for cytogenetic change. A significant increase in the frequency of chromosomal aberrations and sister chromatid exchanges were recorded regardless of the duration of the workers' exposure to the dyes.


Chronic low-dose exposure of sodium nitrite in VM-strain mice: central nervous system changes.

1. There is suggestive evidence that nitrite may be a causative factor in cerebral glioma. 2. To test this hypothesis we selected the VM mouse strain, known for its susceptibility to spontaneous glioma formation, and exposed 300 animals to 0.2% sodium nitrite in their drinking water. One hundred of this group were exposed both in utero and throughout their adult lives. The remaining 200 animals received nitrite from the time of weaning. A further 200 mice were used as controls and received distilled water. 3. All animals were maintained until their natural death and were then subjected to autopsy and routine histological examination. 4. There was no excess of nervous system tumours in the experimental groups.


Subacute toxicity of pentavalent antimony compounds in rats.

A subacute toxicity study of pentavalent antimony (Sb) compounds, sodium stibogluconate (SSG) and meglumine antimoniate (MA) was carried out in rats. Three groups of 10 rats each were treated with saline (control group), 300 mg Sb kg-1 d-1 or 900 mg Sb kg-1 d-1 of SSG for 30 d. A parallel study of similar type was conducted for MA. Compared with controls, drug-treated rats showed an impairment of feeding habits and retardation of weight gain (P less than 0.01) during the treatment period. In both SSG- and MA-treated rats there was a dose-related reduction in haemoglobin concentration (P less than 0.001), and hematocrit (P less than 0.001). Red cell count was reduced in SSG-treated rats only. Both drugs, however, significantly raised the white cell count (P less than 0.05). These changes were more pronounced with SSG them with MA. There was no change in MCV, MCH and MCHC. SSG, 900 mg Sb kg-1 d-1, significantly raised AST (P less than 0.005), ALT (P less than 0.01) and alkaline phosphatase activity (P less than 0.01). SSG-treated rats also had raised BUN (P less than 0.01) and creatinine (P less than 0.001), but no significant change in bilirubin levels. MA significantly raised AST (P less than 0.01), ALT (P less than 0.01), BUN (P less than 0.001) and serum creatinine levels (P less than 0.001), but had no appreciable effect on bilirubin and alkaline phosphatase levels. Both SSG and MA decreased blood glucose levels (P less than 0.01) and induced proteinuria.(ABSTRACT TRUNCATED AT 250 WORDS)


Anterograde amnesia in triazolam overdose despite flumazenil treatment: a case report.

Anterograde amnesia, possibly accompanied by acute brain syndrome, is a potential side-effect of certain benzodiazepines, particularly triazolam. Flumazenil is a benzodiazepine antagonist that is highly effective in reversing the central nervous system effects of benzodiazepine overdose. We report a case of triazolam overdose resulting in anterograde amnesia after flumazenil administration had restored clear consciousness. The defect in memory may have been due to too little flumazenil being given or failure of memory consolidation affected by the character of triazolam during the induced lucent period. We feel that physicians should be aware of the potential occurrence of acute brain syndrome in patients with benzodiazepine overdose despite treatment with flumazenil.


Indomethacin and protein binding of methotrexate.

Indomethacin, a non-steroidal anti-inflammatory drug is known to increase the efficacy and toxicity of methotrexate, the widely used anti-cancer drug in man. The mechanism for this interaction has not been clearly established. However, since these drugs bind with albumin, a possible displacement of methotrexate by indomethacin from albumin might explain this interaction. To investigate the possible interaction an in-vitro protein-binding displacement study was carried out in 17 normal volunteers and in two groups of eight cancer patients. One group of patients had active disease and the other was in complete clinical remission. Serum samples were obtained and protein levels estimated. The protein binding of methotrexate was measured alone and with indomethacin using equilibrium dialysis. Statistical analysis of results suggested that the binding of methotrexate is not influenced by indomethacin, confirming that methotrexate is not displaced by indomethacin.


Pertussis toxin reverses prostaglandin E2- and somatostatin-induced inhibition of rat parietal cell H(+)-production.

In enzymatically dispersed enriched rat parietal cells we studied the effect of pertussis toxin on prostaglandin E2 (PGE2)- or somatostatin-induced inhibition of H(+)-production. Parietal cells were incubated in parallel in the absence (control cells) and presence of pertussis toxin (250 ng/ml; 4 h). [14C]Aminopyrine accumulation by both pertussis toxin-treated and control cells was used as an indirect measure of H(+)-production after stimulation with either histamine, forskolin or dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) alone and in the presence of PGE2 (10(-9)-10(-7) M) or somatostatin (10(-9)-10(-6) M). PGE2 inhibited histamine- and forskolin-stimulated [14C]aminopyrine accumulation but failed to alter the response to dbcAMP. Somatostatin was less effective and less potent than PGE2 in inhibiting stimulation by histamine or forskolin and reduced the response to dbcAMP. Pertussis toxin completely reversed inhibition by both PGE2 and somatostatin on histamine- and forskolin-stimulated H(+)-production but failed to affect inhibition by somatostatin of the response to dbcAMP. After incubation of crude control cell membranes with [32P]NAD+, pertussis toxin catalysed the incorporation of [32P]adenosine diphosphate (ADP)-ribose into a membrane protein of molecular weight of 41,000, the known molecular weight of the inhibitory subunit of adenylate cyclase (Gi alpha). Pertussis toxin treatment of parietal cells prior to the preparation of crude membranes almost completely prevented subsequent pertussis toxin-catalysed [32P]ADP ribosylation of the 41,000 molecular weight protein.(ABSTRACT TRUNCATED AT 250 WORDS)


Contribution of the gamma-carboxyl group of Glu-43(beta) to the alkaline Bohr effect of hemoglobin A.

Glu-43(beta) of hemoglobin A exhibits a high degree of chemical reactivity around neutral pH for amidation with nucleophiles in the presence of carbodiimide. Such a reactivity is unusual for the side-chain carboxyl groups of proteins. In addition, the reactivity of Glu-43(beta) is also sensitive to the ligation state of the protein [Rao, M. J., & Acharya, A. S. (1991) J. Protein Chem. 10, 129-138]. The influence of deoxygenation of hemoglobin A on the chemical reactivity of the gamma-carboxyl group of Glu-43(beta) has now been investigated as a function of pH (from 5.5 to 7.5). The chemical reactivity of Glu-43(beta) for amidation increases upon deoxygenation only when the modification reaction is carried out above pH 6.0. The pH-chemical reactivity profile of the amidation of hemoglobin A in the deoxy conformation reflects an apparent pKa of 7.0 for the gamma-carboxyl group of Glu-43(beta). This pKa is considerably higher than the pKa of 6.35 for the oxy conformation. The deoxy conformational transition mediated increase in the pKa of the gamma-carboxyl group of Glu-43(beta) implicates this carboxyl group as an alkaline Bohr group. The amidated derivative of hemoglobin A with 2 mol of glycine ethyl ester covalently bound to the protein was isolated by CM-cellulose chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)


Prothrombin Salakta: substitution of glutamic acid-466 by alanine reduces the fibrinogen clotting activity and the esterase activity.

Structural studies on a hereditary abnormal prothrombin, prothrombin Salakta, have been performed to identify the difference responsible for its reduced fibrinogen clotting activity and its reduced esterase activity. Amino acid composition and sequence analyses of a peptide isolated from a lysylendopeptidase digest of the abnormal thrombin indicated that Glu-466 had been replaced by Ala. This amino acid substitution can result from a single nucleotide change in the codon for Glu-466 (GAG----GCG). The model building and the molecular dynamics simulation of thrombin Salakta suggest that the Glu-466----Ala substitution would change the proper conformation around the substrate binding site containing Trp-468, which is a unique surface loop on the thrombin molecule. This is the experimental and theoretical evidence supporting the role of the surface loop containing Trp-468 for the proper conformation of the substrate binding site.


Simultaneous determination of common benzodiazepines in blood using capillary gas chromatography.

Blood samples were extracted with n-butyl acetate, and the extracts analysed by capillary gas chromatography using DB-1 and DB-1701 capillary columns with electron-capture detection. The DB-1701 column was found to give better separation of different benzodiazepines (BZDs). Recoveries ranged from 79 to 98%. Detection limits ranged from 0.005 to 0.015 microM for triazolam and flunitrazepam, and from 0.02 to 0.1 microM for other BZDs. Data on accuracy and precision are given for diazepam, desmethyldiazepam, flunitrazepam and nitrazepam.


Lithium chloride stimulates catecholamine synthesis and secretion in cultured bovine adrenal medullary cells.

We examined the effects of lithium treatment on the synthesis and secretion of catecholamines in cultured bovine adrenal medullary cells. The treatment of cells with lithium (0.5-4 mmol/L) for 7 days caused an increase in basal and carbachol-stimulated synthesis of 14C-catecholamines from [14C]-tyrosine but not from [14C]-DOPA. Lithium treatment (4 mmol/L, 7 days) increased the activity of tyrosine hydroxylase in the cells. Lithium treatment (2-4 mmol/L, 7 days) also enhanced the secretion of catecholamines caused by carbachol, although the carbachol-induced influx of 45Ca2+ was reduced. Lithium (4 mmol/L, 7 days) potentiated the secretion of catecholamines evoked by the Ca2+ (1 mumol/L) from cells that were permeabilized by digitonin. The activity of protein kinase C in a soluble fraction was increased in lithium-treated cells (4 mmol/L, 7 days). These results demonstrate that lithium treatment increases the synthesis and secretion of catecholamines and the activity of protein kinase C in cultured adrenal medullary cells.


Expression patterns of engrailed-like proteins in the chick embryo.

The protein products of both of the identified chick engrailed-like (En) genes, chick En-1 and chick En-2, are localized in cells of the developing brain, mandibular arch, spinal cord, dermatome, and ventral limb bud ectoderm, as demonstrated by labeling with the polyclonal antiserum alpha Enhb-1 developed by Davis et al. (Development 111:281-298, 1991). A subpopulation of cephalic neural crest cells is also En-protein-positive. The monoclonal antibody 4D9 recognizes the chick En-2 gene product exclusively (Patel et al.: Cell 58:955-968, 1989; Davis et al., 1991) and colocalizes with chick En-2 mRNA in the developing head region of the chick embryo as shown by in situ hybridization (Gardner et al.: J. Neurosci. Res. 21:426-437, 1988). In the present study we examine the pattern of alpha Enhb-1 and 4D9 localization throughout the chick embryo from the first appearance of antibody (Ab)-positive cells at stage 8 (Hamburger and Hamilton: J. Morphol. 88:49-92, 1951) through stage 28 (1-5.5 days). We compare the localization patterns of the two Abs to each other, as well as to the localization of the monoclonal Ab, HNK-1, which recognizes many neural crest cells, using double- and triple-label fluorescence immunohistochemistry. Most En protein-positive cells in the path of neural crest cell migration are not HNK-1 positive. In detailed examination of alpha Enhb-1 and 4D9 localization, we find previously undetected patterns of En protein localization in the prechordal plate, hindbrain, myotome, ventral body-wall mesoderm, and extraembryonic membranes. Based upon these observations we propose: 1) that En expression in the mesoderm may be induced through interaction with En expressing cells in the neuroectoderm; 2) that En expression in the head mesenchyme is associated with somitomere 4; and 3) that En expression may be involved in epithelial-mesenchymal cell transformations.


[Results of computerized tomography after Whipple operation of chronic pancreatitis].

53 patients were controlled with CT after Whipple resection. The median age of the patients was 49 years, the median time after surgery nine years. Diagnostic criteria were the identification of the residual pancreas, signs of pancreatic atrophy and radiological signs of chronic pancreatitis. CT was performed with slice thickness of 4 mm and steps of 4 mm. The residual pancreas was visible in all 53 cases. Pancreatic atrophy was found in 26 cases, in four of these the residual pancreas was only 2 mm in size. Signs of chronic pancreatitis were found in 16 cases. By comparing preoperative and postoperative CT-scans, seven progresses of pancreatitis were detected. Signs of acute pancreatitis were not found in the 53 patients. Fatty liver degeneration was found in 7.5%. We found that CT is the most reliable technique to visualize the pancreas after Whipple resection. CT should be performed in all patients with clinical symptoms of an acute pancreatitis. CT should also be performed in patients with residual pancreas pseudocysts. There is no indication for CT in the routine examination of asymptomatic patients after Whipple resection.


Recreational MDMA use in Sydney: a profile of 'Ecstacy' users and their experiences with the drug.

'Ecstasy' (3,4-methylenedioxymethamphetamine or MDMA) is a recreational drug that is gaining popularity world wide. There is a paucity of research regarding the ways in which Ecstasy is used and the nature of its effects. A 'snowball' peer network technique was used to recruit 100 users who completed anonymous questionnaires. The research revealed that Ecstasy is primarily used by infrequent recreational drug users for 'fun' at dance parties and social gatherings. The primary reported effects of Ecstasy were a 'positive mood state' and feelings of intimacy and closeness to others. The secondary effects of Ecstasy were the stimulant effects of energy and activation, and the psychedelic effects of insight and perceptual and sensual enhancement. Ecstasy was reported to share the properties of both amphetamines and hallucinogens in the nature of its side effects and residual effects which were no more severe than those of the latter two classes of drug. It appeared Ecstasy was not conductive to regular and frequent use, because tolerance was reported to develop to the positive effects of Ecstasy, while negative effects increased with use. Although few problems associated with the recreational use of Ecstasy have surfaced to date, animal research has shown it to be neurotoxic to serotonergic nerve terminals. Caution must be observed until further research can determine the level of hazard in humans.


Blood stem cell transplants come of age.

In this report we review meeting highlights. Clearly there has been substantial progress in the laboratory and clinic with regard to blood stem cells. Having proven that these transplants work, it is now time to investigate biologic features in greater detail. Also needed are randomized trials evaluating several of the issues we raise such as whether the better results of blood cell autotransplants are due to more effective treatment or subject selection and whether in vitro removal of tumor cells is necessary or effective.


HLA-DR and DQ matching by DNA restriction fragment length polymorphism methods and the outcome of mixed lymphocyte reaction tests in unrelated bone marrow donor searches. The IMUST Study.

The use of DNA restriction fragment length polymorphism (DNA-RFLP) typing for HLA-DR and DQ genes was assessed in 96 patients who were HLA-A,B and DR matched by serology with one or more potential unrelated marrow donors (UD). Two hundred recipient-donor pairs from 10 transplant centres in the UK were studied. DNA-RFLP revealed serological errors in HLA-DR typing and identified additional recipient-donor mismatches. Of the 200 allegedly serologically matched pairs, 55 (28%) were mismatched for HLA-DR and/or DQ by DNA-RFLP typing. Of the 200 pairs, 68 (34%) mixed lymphocyte reactions (MLR) were negative and 132 (66%) positive. There was a significant correlation between DNA-RFLP defined mismatch and MLR positivity. However quantitative studies revealed no trend towards increasing MLR relative response index (RRI) with cumulative RFLP defined mismatch (i.e. for DR plus DQ compared with DR or DQ alone). RFLP matching for HLA-DR and DQ failed to predict a negative MLR. The RRI in the graft-versus-host direction was greater in RFLP matched pairs who carried HLA-DR4 than in matched pairs lacking DR4, suggesting that failure of RFLP to characterize DR4 subtypes was one reason why routine prediction of a negative MLR was not possible. Despite these limitations we have shown that DNA-RFLP is a reliable method for HLA-DR, DQ typing in routine UD searches in diverse clinical centres. By reducing the number of UD tested in MLR, RFLP typing can substantially reduce the work involved in donor selection.


Growth retardation and depigmentation of hair after high-dose busulfan and congenic hematopoietic cell transplantation in mice.

Busulfan, a myeloablative but non-immunosuppressive alkylating agent, is used extensively in clinical bone marrow transplantation (BMT), but the effects of high-dose administration have not been previously evaluated in preclinical BMT settings with young murine recipients. We compared the survival and growth of C57BL/6 mice given graded single doses of busulfan (10-100 mg/kg) or total body irradiation (TBI; 900 cGy) at age 9 days and hematopoietic cell transplantation (HCT; transplantation of congenic bone marrow and spleen cells) 24 h later. The 30-day survival was 87-100% in mice transplanted after 10-40 mg/kg busulfan and 79% after TBI, but fell to 54% and 33%, respectively, after 80 mg/kg and 100 mg/kg busulfan, suggesting that this latter dosage range represents the LD50 for single-dose busulfan in young C57BL/6 mice given stem cell rescue. The weights of 10-week-old mice given HCT after lower doses of busulfan ranged from 87% of control at 10 mg/kg to 64-69% of control in mice conditioned with 35-65 mg/kg busulfan or TBI. Impairment of weight gain was most striking (approximately 50% of control) in mice transplanted after 80-100 mg/kg busulfan. Despite retardation of somatic growth, the brain weights of busulfan-conditioned mice remained at least 90% of control, and there were no obvious neuropathological alterations in the brains of these animals. All mice treated with at least 20 mg/kg busulfan or TBI lost hair by 3-4 weeks after transplant.(ABSTRACT TRUNCATED AT 250 WORDS)


In situ hybridization histochemistry: a new method for processing material stored for several years.

We describe here a protocol developed to detect specific mRNAs by in situ hybridization using tissue sections that were not treated to inactivate RNase and were stored in cryoprotectant solution for several years. Brains from rats, monkeys and humans were sectioned at 50 microns and stored free floating in an ethylene glycol based cryoprotective solution at -20 degrees C. Rat brain sections were kept in cryoprotective solution for 3 days, 1 month and 2 months. Control sections were cut and mounted immediately on gelatin-coated slides and stored at -80 degrees C. Monkey brain sections were stored in cryoprotective solution for up to 5 years. Human sections were tested after storage for one year. Oligonucleotide probes that were complementary to human preproenkephalin mRNA (amino acid sequences 130-145), rat preproenkephalin mRNA (sequences 388-435) and rat tyrosine hydroxylase mRNA (sequences 1441-1488) were labeled with 35S-dATP and terminal deoxynucleotidyl transferase. To prevent possible RNase contamination from mounting the tissue sections onto gelatin coated slides, the in situ hybridization was performed in sterile culture dishes. Following each step, solutions were aspirated out of the dish. The amount of probe necessary for each section was 45 microliters (rat), 450 microliters (monkey), and 350 microliters (human). Using this protocol, the detection of specific mRNAs in rat brain sections was more specific with less non-specific background as compared to control sections that were processed after they were mounted onto gelatin-coated sides. Excellent resolution was also obtained from monkey brain sections that were stored in cryoprotectant for up to 31 months and in human brain sections stored for 12 months.(ABSTRACT TRUNCATED AT 250 WORDS)


Evidence for presynaptic dopamine mechanisms underlying amphetamine-conditioned locomotion.

Rats with a history of receiving (+)-amphetamine in a specific environment exhibit a conditioned psychomotor response when subsequently placed in that environment without drug treatment. Previous work has shown that while the unconditioned effects of amphetamine can be blocked by dopamine D1 or D2 receptor antagonists or with alpha-methyl-p-tyrosine, conditioned locomotion is not influenced by these treatments. In the present experiment, alpha-methyl-p-tyrosine (50 mg/kg, s.c.) was given in conjunction with amphetamine (1.5 mg/kg, s.c.) for 8 days before testing for conditioned locomotion. alpha-Methyl-p-tyrosine completely blocked amphetamine-induced locomotion but only attenuated amphetamine-conditioned locomotion. Reserpine (reduced over the 8 days from 2.5 to 1.25 mg/kg, i.p.) did not block amphetamine-induced locomotion; indeed, potentiation of amphetamine-induced locomotor activity was observed on the last 3 days of treatment. Reserpine treatment in conjunction with alpha-methyl-p-tyrosine treatment blocked amphetamine-induced locomotion for the first 4 days only, with full recovery of amphetamine-induced unconditioned locomotion by the last treatment day. Reserpine alone had no effect on amphetamine-conditioned locomotion, but completely blocked amphetamine-conditioned locomotion when given with alpha-methyl-p-tyrosine. It is concluded that the alpha-methyl-p-tyrosine-sensitive pool of dopamine mediates the immediate psychomotor effects of amphetamine, but that both the alpha-methyl-p-tyrosine- and reserpine-sensitive pools of dopamine are involved in the establishment of amphetamine-conditioned locomotion. In addition, the occurrence of amphetamine-conditioned locomotion is independent of the direct effects of amphetamine on locomotion.


Interaction between thyrotropin-releasing hormone (TRH) and NMDA-receptor-mediated responses in hypoglossal motoneurones.

The effect of thyrotropin-releasing hormone (TRH) on the responses to excitatory amino acids was investigated in hypoglossal motoneurones in an in vitro preparation of the brainstem from guinea pigs using current clamp and discontinuous single electrode voltage clamp (dSEVC). Bath application of 20-50 microM TRH markedly potentiated the response to iontophoretically applied NMDA, whereas no potentiation of the response to glutamate, aspartate or quisqualic acid was seen. Voltage clamp experiments showed that TRH did not increase the current flowing through NMDA channels, thus a direct modulatory role of TRH on NMDA channels was not a likely explanation of the potentiation. Voltage clamp studies of the current-voltage relationship showed that the potentiation of the response to NMDA and lack of potentiation of the response to quisqualic acid was a result of an interaction between the actions of TRH and the amino acids on the electroresponsive profile of the membrane. Endogenous NMDA receptor activation was produced by tetanic stimulation of the reticular formation dorsolaterally to the hypoglossal nucleus, evoking large APV sensitive EPSPs in the presence of CNQX, a non-NMDA blocker. The amplitude and duration of these potentials were increased at more positive membrane potentials in response to TRH. It is concluded that TRH can act as a neuromodulator-potentiating the response to NMDA receptor activation-simply by changing the electroresponsive properties of the membrane.


Amphetamine regulation of mesolimbic dopamine/cholecystokinin neurotransmission.

The effects of acute and repeated amphetamine administration on mesolimbic dopamine (DA) neurons was assessed by studying DA and cholecystokinin (CCK) release in the nucleus accumbens (Acc), as well as effects on mRNA genes regulating DA and CCK synthesis in ventral tegmental area (VTA) cells in rats. Amphetamine (1.5 mg/kg) markedly increased extracellular levels of DA in the medial Acc (assessed by in vivo microdialysis) in drug-naive animals, about twice the amount released in animals repeatedly administered the drug for the previous 7 days (twice daily). CCK overflow was found to mirror the DA responses in that the very transient elevation of CCK monitored in drug-naive animals was attenuated in those with prior amphetamine use. The attenuation of both DA and CCK overflow in the medial Acc was found to be associated with a decrease in the number of CCK mRNA-positive VTA neurons (assessed by in situ hybridization histochemistry). Although the number of cells expressing CCK mRNA were decreased, the gene expression in those positive CCK and tyrosine hydroxylase mRNA cells in the VTA was significantly increased. The CCK mRNA neurons in the VTA were positively identified as those projecting to the medial Acc by the local perfusion of Fluoro-gold retrograde tracer via microdialysis probes located in the Acc.


Regulation of excitatory amino acid release by N-methyl-D-aspartate receptors in rat striatum: in vivo microdialysis studies.

The microdialysis technique was utilized to study the effects of N-methyl-D-aspartate (NMDA) receptor ligands on the in vivo release of endogenous glutamate (Glu) and aspartate (Asp) from the rat striatum. Addition of NMDA (250 and 500 microM) to the dialysis perfusion solution resulted in a striking dose-dependent increase in extracellular concentrations of Glu and Asp in the striatum. The NMDA-induced effects were reduced in a dose-related way by prior perfusion with 75 microM dizocilpine (MK-801), a non-competitive NMDA receptor antagonist. MK-801, at 75 microM, produced no changes on basal levels of Glu and Asp. However, 100 microM MK-801 did increase Glu and Asp extracellular concentrations. Local infusion with 500 microM D-serine, an agonist at the glycine site associated to the NMDA receptor, significantly increased basal level of Glu, but not Asp. Such D-serine-induced effects were reduced by 7-Cl-kynurenic acid (200 microM), a selective blocker of the glycine site present in the NMDA receptor. It is proposed that activation of NMDA receptors by endogenous Glu and Asp enhances the subsequent release of these excitatory amino acids in the striatum. Part of these NMDA receptors might be located presynaptically on cortico-striatal nerve endings. In addition, postsynaptic NMDA receptors present in the striatum may also indirectly modulate the release of Glu and Asp, through trans-synaptic mechanism.


Adrenal hormone effects on hippocampal excitatory amino acid binding.

The influence of short-term adrenalectomy or corticosterone treatment on the binding of glutamate receptor subtypes in the rat hippocampus was explored using the technique of in vitro autoradiography. Analysis of NMDA, kainate and AMPA binding in the hippocampus was conducted on the brains of control, adrenalectomized, and adrenalectomized animals given corticosterone treatment. In addition, serum corticosterone levels were determined by RIA. No striking effects of acute adrenalectomy on the distribution or density of any glutamate receptor subtype were observed in the hippocampus. Adrenalectomy had a small but significant effect on kainate binding in the stratum lucidum and stratum radiatum of CA3 in the first experiment, but no effect in follow-up experiments. Short-term treatment with stress levels of corticosterone had no effect on the binding of NMDA or kainate in any hippocampal subfield. However, a small effect of high doses of corticosterone (CORT) was observed on AMPA binding in one subregion. Although the hippocampus is a target for glucocorticoids and uses excitatory amino acids as a primary neurotransmitter, transient manipulation of adrenal hormone levels did not directly modulate excitatory amino acid receptor binding.


Dopamine receptor antagonists increase markedly the quantity of retrograde transport of HRP in the rat masseteric motoneuron.

Horseradish peroxidase (HRP) was injected, bilaterally, into the rat masseter muscle, subsequent to an intramuscular or intraperitoneal injection of one of five dopamine antagonists (chlorpromazine and haloperidol as the D1 and D2 receptor antagonist, SCH 23390 as the specific D1 receptor antagonist, sulpiride and domperidone as the specific D2 receptor antagonist). Control rats received an injection of a corresponding vehicle solution. After a survival period of 16 h, the brainstem was cut into 60 microns cryosections and processed with the TMB technique. The amount of retrogradely transported HRP was quantitatively measured in terms of the amount of HRP reaction product present in the motoneuron by the method which we have developed using an image processing system combined with a light microscope and a TV camera. Chlorpromazine, haloperidol, SCH 23390 and sulpiride significantly raised the quantity of retrograde transport of HRP. On the contrary, domperidone which can not penetrate the blood-brain barrier showed no significant change in the amount of the retrograde transport. In addition, an intravenous injection of chlorpromazine (8 mg/kg) was found to increase the amplitude of monosynaptic masseteric reflex EMG activity evoked by stimulations of the mesencephalic trigeminal nucleus. These results suggest that a possible regulatory system involving the dopamine receptor in the uptake and retrograde transport of HRP from axon terminals to cell bodies of the masseteric motoneuron exists in higher order neurons which make synaptic contact with the motoneuron.


Calcium-dependent release of glutamate from human cerebral cortex.

The release of the excitatory amino acids glutamate and aspartate from human neocortex was investigated in vitro by utilizing brain tissue removed during anterior temporal lobectomies for tumor or epilepsy. Depolarization (50 mM K+) increased the glutamate release to 291% of control (809 pmol/mg/min) during blocked synaptic transmission and to 669% (1859 pmol/mg/min) when synaptic transmission was not blocked. Aspartate release increased to 141% (326 pmol/mg/min) and 178% (412 pmol/mg/min) respectively. The difference between release with and without blocked synaptic transmission was statistically significant only for glutamate (P less than 0.01). These data provides evidence for a Ca(2+)-dependent release of glutamate, supporting a possible role of this amino acid as a neurotransmitter in human neocortex.


Inhibition of energy metabolism by 3-nitropropionic acid activates ATP-sensitive potassium channels.

3-Nitropropionic acid (1 mM), which inhibits succinate dehydrogenase activity and reduces cellular energy, produces in the pyramidal cell layer of the hippocampal region CA1 a hyperpolarization for variable lengths of time before evoking an irreversible depolarization. Hyperpolarization is caused by an increased potassium conductance that is attenuated by glibenclamide (1-10 microM), a selective antagonist of ATP-sensitive potassium channels; in contrast, diazoxide (0.5 mM), an agonist at this channel, induces a hyperpolarization in CA1 neurons of rat hippocampal slices. The transient hyperpolarization after prolonged (ca. 1 h) application of 3-NPA is followed by a depolarization that is incompletely reversed by brief application of the glutamate antagonists (D-2-amino-5-phosphonopentanoic acid (APV), 6,7-dichloroquinoxaline-2,3-dione (CNQX), 3-(+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), 7-chloro-kynurenic acid (7Cl-KYN)). Early application of glibenclamide (within the initial 5 min) blocked or reduced hyperpolarization and accelerated the depolarization. These data suggest that metabolic inhibition by 3-NPA initially activates ATP-sensitive potassium channels. Events other than activation of glutamate receptors participate in the final depolarization resulting from uncoupling of oxidative phosphorylation.


Somatostatin receptors increase in the olfactory bulb of developing pups after perinatal exposure to cocaine.

The effects of chronic prenatal and/or postnatal exposure to cocaine on somatostatin concentration and receptors were studied in the olfactory bulbs of rat pups at birth and at 15 days old. Wistar rats were injected subcutaneously with single daily doses of 40 mg cocaine hydrochloride/kg from days 7 to 19 of gestation, from day 7 of gestation to day 15 postpartum or from parturation to day 15 postpartum. Fetal exposure to cocaine decreased SS concentrations in the olfactory bulb of the newborn pups while prenatal-plus-postnatal exposure increased this parameter. Administration of cocaine only during lactation did not induce any change. Exposure during gestation or during nursing induced an increase in the total number of somatostatin receptors and a decrease in the affinity constant in the olfactory bulb of newborn and 15-day-old pups. These results suggest that the development of somatostatin receptors in the olfactory bulb can be altered by prenatal and/or nursing period exposure to cocaine.


Cellular events accompanying regression of skin recurrences of breast carcinomas treated with intralesional injections of natural interferons alpha and gamma.

Cutaneous recurrences of breast carcinomas were treated with 10 i.l. injections of nIFNs alpha and gamma delivered in combination (7 lesions) or singly (11 with nIFN-alpha, one with nIFN-gamma). Histologically confirmed complete regressions occurred in 5 of 7 lesions treated with nIFN-alpha/nIFN-gamma and in 5 of 11 recurrences injected with nIFN-alpha alone. In all cases specimens were obtained before and after therapy. In addition, in some cases (4 treated with nIFN-alpha/nIFN-gamma, 2 with nIFN-alpha, one with nIFN-gamma) multiple recurrences were injected simultaneously and were excised 24 h after 1, 3, and 10 injections and 21 days after completion of therapy. The main findings observed in the treated lesions undergoing complete and partial regressions included: (a) inhibition of mitotic activity and up-regulation of antigenic expression (mammary epithelial membrane antigen, intercellular adhesion molecule 1, HLA-DR) by the carcinoma cells; (b) activation of macrophages and dendrocytes with marked expression of HLA-DR and HLA-A,B,C; (c) infiltration of the dermis and tumors by activated T-lymphocytes (CD3+, CD4+, CD8+); (d) questionable participation by B-lymphocytes and natural killer cells; (e) activation of endothelium with enhancement of antigenic expression (intercellular adhesion molecule 1, HLA-DR), procoagulant activity, and vascular permeability. The responses elicited by nIFN-alpha/nIFN-gamma were greater than those caused by either IFN used alone. It appears that in these patients the IFNs exerted an antiproliferative action and potentiated a cell-mediated immunological response liminally present in the neoplastic tissues prior to therapy.


Reversal of multidrug resistance by two novel indole derivatives.

Two new fused indoles were found to overcome multidrug resistance in P388/Adr cells in vitro. These agents potentiated the cytotoxicity of the antitumor drugs Adriamycin, vinblastine, and vincristine in multidrug-resistant cells with no effect on drug-sensitive parent P388 cells. They significantly increased the ATP-dependent accumulation of [3H]-vinblastine and inhibited efflux of the labeled drug from resistant cells. These compounds also inhibited photoaffinity labeling of P-glycoprotein by [3H]azidopine in P388/Adr cells and membranes isolated from these cells. In addition, the calcium antagonist activity of these compounds was very weak compared with that of verapamil. These data suggest that the compounds reported here may specifically overcome multidrug resistance without the serious hypotensive effects associated with calcium antagonists and that this activity may be independent of their ability to block calcium transport.


Differential susceptibility of cultured human melanoma cell lines to enhancement by retinoic acid of intercellular adhesion molecule 1 expression.

The potential role of intercellular adhesion molecule 1 (ICAM-1) in the biology of human melanoma cells has stimulated interest in the characterization of its modulation. The present study has shown that the differentiating agent retinoic acid (RA) up-regulates ICAM-1 expression by melanoma cells in a dose- and time-dependent fashion. The enhancement of ICAM-1 cell surface expression is paralleled by an increase in ICAM-1 mRNA. Therefore, ICAM-1 represents an additional gene which may be transcriptionally regulated by RA. The five melanoma cell lines tested displayed a differential susceptibility to the modulation of ICAM-1 expression by RA, since the cell line MeWo did not change in its ICAM-1 expression following incubation with RA. Nevertheless, RA-insensitive as well as RA-sensitive melanoma cell lines displayed a higher increase in ICAM-1 expression following incubation with RA and cytokines than following incubation with each of them. Analysis of the distribution in the melanoma cell lines of retinoic acid receptors (RARs) showed a relationship between susceptibility to a RA-mediated increase of ICAM-1 expression and RAR beta expression, suggesting that the latter receptor may play a role in the phenomenon. RAR alpha and RAR gamma were present in RA-sensitive and -insensitive melanoma cell lines, suggesting that they play a role in the enhancement by RA of cytokine-mediated up-regulation of ICAM-1 expression. The melanoma cell lines we have described may represent a useful system for investigating the role of RAR in the regulation of gene expression and the mechanism(s) which underlie this effect.


Tissue localization of methotrexate-monoclonal-IgM immunoconjugates: anti-SSEA-1 and MOPC 104E in mouse teratocarcinomas and normal tissues.

Methotrexate (MTX) was coupled to the tumor-targeting monoclonal IgM, anti-SSEA-1 and the non-targeting myeloma IgM, MOPC 104E. At 24-h intervals following injection, drug deposition in MH-15 teratocarcinomas and in several normal tissues was followed by immunoperoxidase microscopy using the M16 monoclonal antibody to MTX. MTX-anti-SSEA-1 was deposited on the surface and in the interior of living tumor cells 24 h after injection; at 48 h and after, only low-level binding to necrotic tissue was found. There was no significant gradation in staining from the outside to the interior of the tumors. In tumors, the control MOPC 104E immunoconjugate was detectable only in necrotic tissue. Binding to SSEA-1-expressing normal tissues was undetectable, except for pericryptal fibroblasts in the small intestine. No significant pathology was found in normal tissues that are SSEA-1 positive. High levels of the immunoconjugate were detected in the liver, where MTX was found predominantly in Kupffer cells and possibly in hepatocytes; again, no significant morphological changes were associated with this retention. Thus tumor-associated antigens can be suitable targets for antibody-drug conjugates even when present in normal tissues and in large quantities, provided that the antigens in normal tissues are inaccessible. Moreover, deposition in viable tumor tissue can be assessed using monoclonal antibodies to methotrexate.


Immunoscintigraphic localization of renal tumours in an extracorporeal perfusion model with a monoclonal antibody against gamma-glutamyltransferase.

Monoclonal antibody 138H11 against human gamma-glutamyltransferase has been shown to react immunohistochemically with 98% of all tested clear-cell type and chromophilic renal cell carcinomas, but not with renal chromophobic carcinomas, Duct-Bellini carcinomas or oncocytomas. In normal kidney the target epitopes of mAb 138H11 are located in the luminal brush-border membrane of proximal tubule cells, whereas in renal carcinomas the epitopes are found surrounding the whole tumour cells. These results form the basis of the present immunoscintigraphic study designed to evaluate mAb 138H11 in an extracorporeal perfusion model. Immediately after nephrectomy, human tumour-bearing kidneys were perfused with 99mTc-labelled mAb 138H11 in Euro-Collins solution. High specific uptake in 4/4 renal clear cell carcinomas could be demonstrated by planar immunoscintigraphy and single-photon-emission computed tomography, "regions of interest" investigation and immunohistochemistry. In contrast, a perfused oncocytoma showed up as an unlabelled lesion. The results indicate a possible use for mAb 138H11 in immunoscintigraphy or even therapy, provided high tumour uptake can be confirmed in patients.


Left ventricular geometry in Takayasu arteritis complicated by severe aortic regurgitation.

Although it has been reported that the aortic regurgitation (AR) of patients with Takayasu arteritis is due to dilatation of the aortic ring, the geometry of the left ventricle (LV) has not been described. We compared the cardiac findings in patients with Takayasu arteritis (TA) and severe AR with those of patients having severe AR of other origins. Echocardiographically, wall thickness (WT) and the concentric hypertrophic ratio (WT/WT + left ventricular end-diastolic dimension) were greater in patients with TA. It is concluded that the LV of the TA patients revealed concentric hypertrophy even when AR was severe.


Involvement of the Th1 subset of CD4+ T cells in acquired immunity to mouse infection with Trypanosoma equiperdum.

Heat- or merthiolate-inactivated Trypanosoma equiperdum was administered to recipient mice that were subsequently challenged with viable inocula of the same stabilate. Only mice inoculated with merthiolate-killed parasites were completely protected from a challenge inoculum of 10(3) trypanosomes, an effect that was abolished by prior immunosuppression of mice. Immune sera from protected animals contained high levels of interferon (IFN)-gamma and specific IgG2a antibodies. Spleen cells from these mice produced high amounts of interleukin (IL)-2 and IFN-gamma in vitro in response to specific antigen or concanavalin A, whereas splenocytes from mice receiving heat-killed parasites produced high amounts of IL-6. In contrast, the production of tumor necrosis factor (TNF)-alpha and colony-stimulating activity (CSA) was not significantly different in mice receiving either killed parasite preparation. The protection in immunized mice was associated with the detection of strong delayed-type hypersensitivity (DTH) to T. equiperdum antigens, an effect that could be adoptively transferred onto naive recipients by specifically immune CD4+ lymphocytes. These results suggest that the development of protective immunity in mice to T. equiperdum by our immunization protocol may involve the activity of helper/DTH T cells, particularly those of the Th1 subset.


Regulation of the expression of leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) on a human eosinophilic leukemia cell line EoL-3.

The effects of several cytokines and phorbol myristate acetate (PMA) on LFA-1 and ICAM-1 expression on a human eosinophilic leukemia cell line, EoL-3, were investigated and compared with those of a human monocytic leukemia cell line, U937. EoL-3 cells expressed large amounts of LFA-1 and small amounts of ICAM-1, and their expression was regulated similarly in EoL-3 cells and U937 cells. Interferon-gamma (IFN-gamma) enhanced ICAM-1 expression but not LFA-1 expression, and PMA augmented both LFA-1 and ICAM-1 expression. IFN-gamma and PMA showed an additive effect on ICAM-1 expression. These results collectively suggest that expression of LFA-1 and ICAM-1 is regulated differently and that IFN-gamma and PMA regulate the expression through different mechanisms. PMA but not IFN-gamma induced homotypic adhesion of EoL-3 and U937 cells, suggesting that PMA but not IFN-gamma activated the adhesive function of these cells. Staurosporin, an inhibitor of protein kinases (PKs), partly suppressed IFN-gamma- and PMA-augmented expression of ICAM-1 on EoL-3 and U937 cells, but did not affect PMA-augmented LFA-1 expression, suggesting that staurosporin-sensitive PKs are involved in IFN-gamma- and PMA-augmented ICAM-1 expression but not in PMA-augmented LFA-1 expression. The role of protein kinase C (PK-C) in these mechanisms was not revealed because a PK-C inhibitor, H-7, did not show any definitive effect on IFN-gamma- and PMA-induced expression of LFA-1 and ICAM-1. Moreover, cyclic AMP (cAMP)- and cGMP-dependent pathways were not shown to be involved in the augmentation of the expression of these molecules.


Developmental expression of the proenkephalin and prosomatostatin genes in cultured cortical and cerebellar astrocytes.

Astrocytes were prepared from rats of 4 ages, embryonic day 20, postnatal days 3 and 8, and adult, in order to study the developmental time course of expression of enkephalin and somatostatin (SS). Glial fibrillary acidic protein (GFAP) content was constant in both cortical and cerebellar astrocytes prepared from all ages. SS mRNA and peptide decreased over this developmental time course in cerebellar astrocytes; the time course of changes in SS mRNA paralleled that for rat cerebellum. Proenkephalin (PE) mRNA increased about 3-fold in cerebellar astrocytes from embryonic day 20 to adult but remained constant in cortical astrocytes; in contrast, PE mRNA showed a 10- to 12-fold increase in rat cerebellum and cortex developmentally. For both cerebellar and cortical astrocytes, free met-enkephalin decreased from embryonic day 20 to adult, whereas total met-enkephalin (measured following trypsin-carboxypeptidase B digestion of the extracts) increased. These results suggest (1) that there is a developmental regulation of the expression of both enkephalin and SS peptides in astrocytes, and (2) that the regulation occurs at the level of transcription for SS but at the level of precursor processing for PE. Possible trophic functions for astrocyte-derived peptides early in CNS development are discussed.


N-methyl-D-aspartate effects on the growth, morphology and cytoskeleton of individual neurons in vitro.

Short-term (up to 5 h post-plating) cerebellar granule cell cultures were prepared from the week-old rat and maintained in a micro-incubator during time lapse video microscopy to examine normal and N-methyl-D-aspartate (NMDA)-evoked neurite extension. In untreated cultures growth of neurites was stochastic but proceeded at an average rate of 12.0 +/- 1.4 microns/h. Growth cone morphology was variable. The classical filopodia and lamellipodia possessing tips were motile or non-motile, while those processes ending in a club shape were rarely seen to extend. Individual growth cones passed through several different morphologies during growth. New processes extended more rapidly (13.0 +/- 1.7 microns/h) than those already present (9.0 +/- 0.5 microns/h). Addition of the NMDA receptor antagonist, aminophosphonovalerate (APV), caused a marked retraction of pre-existing processes. Stimulation of the receptor with 50 microM NMDA caused a marked increase in growth rate compared to controls (15.0 microns/h and 1.7 microns/h, respectively). When the presence of actin-rich structures was examined using rhodamine-phalloidin labelling it was found that NMDA increased the proportion of neuronal processes that possessed a growth cone by 28%. Conversely, inhibition of NMDA receptor activity with APV reduced the formation of lamellipodia from neuronal cell bodies.


Developmental expression of somatostatin receptors in the rat retina.

The ontogeny of somatostatin receptor binding was studied in developing rat retina using the iodinated derivative of the somatostatin analog, SMS 204-090. Specific binding of the ligand was seen as early embryonic day (E) 15 in the region of the inner neuroblastic layer. At E19 binding was localized to the ganglion cell and developing inner plexiform layers. At postnatal day (P) 2, there was diminished binding on autoradiography in this region. At P11, binding was more intense in the inner plexiform layer, and there was discernible binding in the outer plexiform layer. In the adult retina, the binding was seen clearly in two distinct bands corresponding to the inner plexiform layer and the outer plexiform layer. There was a single saturable binding site with the dissociation constant (Kd) of 0.25 +/- 0.04 nM. Binding sites were fairly constant throughout development except for a significant decline during the first postnatal week (Bmax = 1.8). These results demonstrate the early appearance of somatostatin receptors in the rat retina with high levels present embryologically followed by a brief decline in the early postnatal period with a return to high levels by synapse formation (P11). These receptor data parallel previous reports of the appearance of the somatostatin mRNA and peptide in rat retina.


Pharmacokinetic optimisation of antiemetic therapy.

Antiemetic drugs are used to treat nausea and vomiting due to a variety of causes and have a wide range of pharmacological properties. The choice of drug will, therefore, depend in part on the condition being treated. The drugs can be classified as dopamine antagonists (including phenothiazines and nonphenothiazines), corticosteroids, cannabinoids, benzodiazepines, serotonin antagonists, antihistamines and anticholinergics. There is very little evidence of a relationship between plasma drug concentrations and either their efficacy or the incidence of adverse effects with most antiemetic drugs. With drugs for which concentration-effect studies have been performed, e.g. the benzodiazepines and antihistamines, the effects monitored have not been directly relevant to their use as antiemetics. Antiemetics are used widely, for example, in cancer chemotherapy. Nonetheless, apart from metoclopramide, little work has been done on the influence of indicators of systemic disease on the pharmacokinetics of antiemetic drugs.


Effects of acute febrile infectious diseases on the oral pharmacokinetics and effects of nitrendipine enantiomers and of bisoprolol.

In 2 longitudinal studies with 10 patients each, the stereoselective pharmacokinetics of nitrendipine and the pharmacokinetics of racemic (rac) bisoprolol (both 20mg orally) were investigated during acute febrile infectious diseases and at least 6 weeks later in the healthy state. The area under the plasma concentration-time curve (AUC) and peak plasma concentration (Cmax) of rac-nitrendipine were increased in the infectious state by 89% [95% confidence interval (CI): 24 to 187%] and 95% (95% CI: 22 to 209%), respectively. Similar increases were observed for both S- and R-nitrendipine. Nitrendipine exhibited stereoselective pharmacokinetics in both the healthy state and the infectious state, but the mean ratios of S:R AUC values [healthy: 1.79 (95% CI: 1.36 to 2.11); infectious: 1.87 (95% CI: 1.62 to 2.11)] were not different. The elimination half-life, protein binding and haemodynamic effects of nitrendipine also did not differ between the infectious and the healthy state. The mechanism for the disease effects may be related to suppression of hepatic cytochrome P450 activity by mediators of inflammatory reactions. On the other hand, none of the pharmacokinetic parameters, including nonrenal clearance, of rac-bisoprolol was changed during febrile infectious disease, indicating specificity in the effects of acute febrile disease on oxidative drug metabolism.


Regulation of Na+/H+ exchange and pH in erythrocytes of fish.

1. The function of trout RBC Na+/H+ antiport is unrelated to cell volume or cell pH regulation. Its role is to improve oxygen transport capacity when the supply of oxygen becomes limited. 2. Antiport activation, mediated by cAMP, promotes complex changes in blood pH which have been analyzed in vivo and in vitro. 3. The regulation of antiport (activation, desensitization, control by molecular oxygen and by a newly discovered cytosolic protein, arrestin) is presented. 4. Molecular cloning of the antiport shows that two typical site motifs of phosphorylation by cAMP-dependent protein kinase are localized on the cytoplasmic region.


Cell volume and pH regulation by the Amphiuma red blood cell: a model for hypoxia-induced cell injury.

The Amphiuma red blood cell is one of the model systems employed early in the study of vertebrate cell volume regulation. Following both cell swelling and shrinkage the Amphiuma red blood cell demonstrates volume regulation to virtual completion in 90-120 min. When swollen the Amphiuma red blood cell loses K, Cl and osmotically obliged water, while following shrinkage volume regulation is the result of Na, Cl and therefore water uptake. The main contribution of the Amphiuma red cell as a model is that it was the first cell in which volume regulation was demonstrated to be electroneutral and more specifically that K/H and Na/H exchangers were responsible for regulation following cell swelling and shrinkage, respectively. Additionally, the Amphiuma red blood cell K/H and Na/H exchangers have been demonstrated to function in a pH regulatory capacity. The latter observation in turn led to the demonstration of the mutually exclusive and contradictory nature of volume and pH regulation predicted upon Na/H exchanger activity. These observations prompted our recent investigations of the Na/H exchanger as a contributor to hypoxia-induced cell damage, using the rabbit heart as a model. These studies illustrated that Na, and Ca imbalances characteristic of hypoxia-induced cell damage are ultimately referable to the Na/H exchanger's function in a pH regulatory capacity, which contributes fundamentally to cell volume and Ca derangement and ultimately cell injury.


Diversities of transport of sodium in rodent red cells.

1. Na-K pumps of rodent red cells reveal variations among species in terms of kinetic properties such as ouabain sensitivity, Na/K coupling and temperature sensitivity and variations within an individual organism related to such physiological challenges as K deficiency, calorie deficiency and seasonal changes in temperature. 2. Passive Na entry among rodents collectively occurs through the same routes as in red cells of other mammals, but red cells of hamsters, rats and thirteen-lined ground squirrels lack or are deficient in an amiloride-sensitive, shrinkage-activated Na-H exchange. 3. In guinea-pig this pathway appears to be both activated and uncoupled by cooling from 37 to 20 degrees C. 4. Red cells of rodents in general and hamsters in particular are rich in a Na-Mg exchange pathway. In hamsters, this appears to be the only amiloride-sensitive pathway in simple media. 5. In hamster cells, Na entry through the amiloride-sensitive Mg-activated pathway exhibits the same kinetics as previously shown for Na activation of Mg extrusion.


Volume-activated cation transport in dog red cells: detection and transduction of the volume stimulus.

1. Carnivore red cells lose their Na/K pump capability as they develop from erythroblasts to reticulocytes to mature cells. They defend their fluid volume by utilizing the Ca/Na exchanger as a Na extrusion pump, the energy for which is ultimately derived from active Ca transport. 2. Swelling-induced [K-Cl] cotransport and shrinkage-induced Na/H exchange are regulated in a coordinated fashion in dog red cells. Circumstantial evidence points to a regulatory protein kinase-phosphatase system. 3. Dog red cells detect changes in their fluid volume, not by virtue of membrane distortion, but by alterations in the concentrations of cytoplasmic macromolecules induced by swelling or shrinkage.


Ferret red cells: Na/Ca exchange and Na-K-Cl cotransport.

The primary pathway for K influx in ferret red cells is the Na-K-Cl cotransporter and the primary pathway for Ca influx is the Na/Ca exchanger. This makes ferret red cells favorable models for the study of these two transport systems. The evidence that Na/Ca exchange is of primary importance for steady state cell volume regulation and the Na-K-Cl cotransport has a minor role is presented. The approaches to, and results of, the determination of the stoichiometry, of the mechanism, and of the regulation by ATP and Mg, for Na/Ca exchange is contrasted with that taken for Na-K-Cl cotransport.


Ion transport in sheep red blood cells.

There is a polymorphism (HK/LK or high potassium/low potassium) of the cation concentrations in sheep red cells which also affects the cation transport pathways in these cells. The current status of understanding of two of these pathways in LK red cells, the Na/K pump and the K-Cl cotransporter, is summarized here. Recent results are presented on stimulation of the Na/K pump by insulin-like growth factor, and on the transduction mechanism by which changes in cell volume modulate the K-Cl cotransporter.


Gill (Na+ +K+)-ATPase involvement and regulation during salmonid adaptation to salt water.

1. The involvement of gill (Na+ +K+)-ATPase in salmonid adaptation to salt water (SW) is discussed. 2. Gill (Na+ +K+)-ATPase increase during SW adaptation is mainly related to the increased number and complexity of chloride cells deputed to salt extrusion. 3. The temporal relationships between serum peaks of thyroid hormones, cortisol, growth hormone, prolactin and gill (Na+ +K+)-ATPase rise during salmonid smoltification, suggest a hormonal involvement in the enzyme stimulation and thus in the acquirement of SW tolerance. 4. Literature on gill (Na+ +K+)-ATPase response to hormonal treatment is reviewed. The effects produced on gill (Na+ +K+)-ATPase and chloride cells by exogenous hormones point out a complex inter-relationship between the hormones considered. The mechanisms involved in hormonal regulation of the enzyme remain a matter of debate.


Hemosomegenesis and hemoglobin biosynthesis in vertebrates.

1. Ultrastructural observations on maturing rabbit embryo erythroid cells led to the finding of hemoglobinized organelles distinguishable from mitochondria due to their highly dense matrix, two or three longitudinally arranged double lamellae, and smaller diameters. Intraorganellar 50-60 A particles identical to those contained in the hemoglobinized cytoplasm were found. 2. Their hemoglobin (Hb) content was demonstrated by electrophoresis of the concentrated supernatant from the isolated, washed, and osmotically lysed organellar fraction. We have proposed that these organelles are the sites for heme integration into the globin (G) polypeptide chains and subunits assembly. The term hemosome has been suggested for such entities. 3. This hypothesis has been sustained by several analytical and experimental works based on the postulation that hemosomes should be found at higher frequencies where the Hb biosynthesis rate is more intensive, or where the induction of this biosynthesis is always dependent on the formation of hemosomes. 4. Maturing erythroid cells of the circulating embryo blood contain hemosomes in higher frequency than in liver erythroid cells, coinciding with the higher Hb biosynthesis rate in peripheral blood than in the liver. In bleeding anemia, the decay of Hb concentration parallels the reduction of the mean number of hemosomes per reticulocyte, in comparison with normal reticulocytes. 5. In HeLa cells and epithelial cultured cells induced to synthesize Hb, it was shown that this biosynthesis is ever concomitant with the formation of hemosomes and depends on the presence of erythropoietin, as occurs in erythroid cells. 6. Studies on hemosomegenesis and Hb biosynthesis experimentally effected in epithelial cultured cells, allowed the interpretation of the sequence of events leading to hemosome formation in maturing erythroid cells. Simultaneously with iron uptake, mitochondria differentiate to lamellated bodies and, successively, expansions rise for ferruginous compounds and G polypeptides gathering, followed by prehemosome vesicles formation, which condense and change to prohemosomes that afterwards evolve to hemosomes. 7. These dynamics, and organellar Hb have been detected in immature erythrocytes of mammalians, including humans, avians, reptilians, amphibians and representative fish specimens. It appears that these events occur in the erythrocytary maturation of all vertebrate classes.


Prolactin and the onset of mammary extraction of plasma triacylglycerols during lactogenesis in the goat.

1. In untreated goats, the onset of mammary extraction of circulating triacylglycerols (TG) at parturition occurred shortly after the peak of prolactin concentration in circulating plasma. Suppressing this peak of systemic prolactin, by acutely treating with bromocriptine, delayed the onset of mammary extraction of TG and secretion of long-chain TG fatty acids. 2. Regular unilateral removal of secretion pre-partum, which brought about an early local onset of TG extraction and secretion of long-chain TG fatty acids in this treated half of the udder, was not associated with an increased local concentration of prolactin in secretion in this gland.


Comparison of the subcellular distribution of alveolar surfactant in two mammalian species of similar body weight: cat and rabbit.

1. We studied the total amount and subcellular distribution of alveolar surfactant, extracted through bronchoalveolar lavage of anesthetized cats and rabbits. This was correlated to several morphometric and ventilatory variables of these animals. 2. Lung weight was significantly larger in the cat while respiratory frequency and minute ventilation were significantly larger in the rabbit. No significant differences were observed in tidal volume, total lung capacity, P(a)O2, P(a)CO2 and pH(a). 3. While both species had similar protein contents in the bronchoalveolar lavage, rabbits had larger phospholipid contents, mostly distributed in the lighter, more active subfractions. 4. With regard to the estimated values obtained from allometric equations derived for mammals, the rabbit presented a lung weight of nearly one-third of the estimated one, an exceedingly larger minute ventilation (by nearly 60%) and a respiratory frequency twice the calculated one. 5. We suggest that the different distribution of alveolar surfactant in these species may be explained by disparities in their ventilatory demands, the rabbit having a higher respiratory frequency and a larger minute ventilation, performed by a mass of lung tissue lower than that corresponding to its body mass.


Determination of transepithelial (mucosal and serosal) electrical potentials in toad skin. Action of chemical agents.

1. Potential differences across the mucosal or outer, and the serosal or inner, membranes of the toad skin (M and S) were recorded separately. Total potential difference across the skin (T) and the short-circuit current (SCC) were recorded by means of the classical Ussing method. 2. The independent determination of the M and the S is of importance in the elucidation of the mechanism of action of agents which alter ion fluxes across the skin. 3. The percentage values of the M and the S obtained in toad skins during the summer were similar to the percentage values obtained by microelectrode impalement of cells. 4. Angiotensin II (AII) and antidiuretic hormone (ADH) increased T with a notable rise in M and a slight increase in S. These agents act mainly by increasing mucosal membrane permeability to Na+ since M is principally affected. 5. Amiloride and ouabain reversed M, decreased T and increased S above T. The reversal of M might be explained by the flow of a cation to the mucosal aspect or of an anion to the cell interior. 6. These results show that the effects of several agents on the toad skin potential may be analysed independently across the mucosal and serosal membranes and reflect the behaviour of the entire tissue rather than of a single cell.


Age-related changes in GSH content of eyes in mice--a comparison of senescence-accelerated mouse (SAM) and C57BL/J mice.

1. Age-related changes in glutathione (GSH) content of eye lenses were investigated in senescence-accelerated mouse (SAM) and C57BL/J mice. 2. The decrease of GSH content with aging is markedly observed in SAM strains. 3. The oxidized glutathione (GSSG) content of eyes increased significantly with aging in SAM. 4. Ophthalmic changes, including cataract, increased with age in SAM alone. 5. The decrease of GSH content and the increase of GSH oxidation may be involved in the pathogenesis of cataract in SAM.


Adrenergic receptor-mediated increase of intracellular Ca2+ concentration in isolated bovine corneal epithelial cells.

1. We determined if Ca2+ is a second messenger for adrenergic receptor-effector coupling in bovine corneal epithelial cells. 2. Methoxamine (10(-5) M) selectively increased intracellular Ca2+ concentration (Cai) by 65%. This increase was only partially suppressed through the removal of extracellular Ca2+ or pretreatment with 10(-6) M verapamil. 3. The beta-adrenergic-mediated increases in Cai were entirely dependent on extracellular Ca2+. These increases were directly elicited through stimulation of adenylate cyclase because 10(-6) M isoproterenol and the active analogues of forskolin (10(-5) M) all elevated Cai. 4. Therefore, increases in Cai serve a second messenger function for alpha-1 and beta-adrenergic receptor-effector coupling.


Blood parameters in draught oxen during work: relationship to physical fitness.

1. Four Zebu and four Simmental oxen were submitted to moderate and exhaustive work. Venous blood samples were taken before, immediately after and 30 min after work and assayed for several blood parameters. 2. Draught work led to a decrease in carbon dioxide (pvCO2) and increases in pH, oxygen (pvO2), triglycerides, free fatty acids (FFA) and lactate. 3. Zebu oxen had higher pvCO2 and FFA and lower pH, pvO2 and lactate in response to exercise. 4. Ratios of individual draught power output and values of pvO2 and lactate after work enable the identification of fit and/or weak individuals.


Hematological and plasma chemical characteristics in beech marten (Martes foina).

1. The normal values of hematological and clinical-chemical variables in plasma of 21 males and 27 females of beech marten (Martes foina) have been illustrated and compared to corresponding normal values in mink (Mustela vison) and ferret (Mustela putorius). 2. The number of erythrocytes, the hematocrit value, and the hemoglobin concentration were higher in male than in female beech marten. 3. Divergences according to age in the investigated variables have been found. 4. The erythrocytes in beech marten are clearly smaller in size and volume and have a lower mean corpuscular hemoglobin than the erythrocytes in mink and ferret.


Serum-cell interactions in transmission of sarcoma in the soft shell clam, Mya arenaria L.

1. Serum proteins from sarcomatous soft shell clams, Mya arenaria L., enhanced transmission of sarcoma. 2. Sarcoma cells were isolated and administered to the recipients at the same cell density in different sarcoma-protein-free diluents: seawater, serum from normal clams, heat-treated sarcoma serum or protease-digested sarcoma serum. 3. Transmission in these groups was significantly slower than in the group where cells were administered in intact sarcoma serum, demonstrating that the tumor promoting factors in the serum were heat-sensitive proteins. 4. Normal hemocytes administered in sarcoma serum caused mortality but not sarcoma transmission, suggesting the presence of cytotoxic factors in sarcoma serum.


Autonomic basis for hypoxia-induced hyperglycaemia in toads (Bufo paracnemis).

1. Toads were exposed to steady hypoxic conditions (inspired PO2 = 40 mmHg) for 60 min. 2. Within the exposure time, glucose concentrations rose from about 30 mg% to a steady level of 45 mg%. The development of hyperglycaemia reached a stable level within 40 min. 3. This effect was eliminated by treatment with either propranolol or atropine, suggesting combined cholinergic and adrenergic activation.


Urate excretion by the cat kidney.

1. The renal handling of urate by the cat kidney was investigated during continuous infusion of urate. 2. Fractional urate excretion (FE(UA)) in cats was 0.57 +/- 0.04, indicating net reabsorption of urate. In contrast, FE(UA) in rabbits was 1.76 +/- 0.08, reflecting net secretion of urate. 3. Fractional PAH excretion (FE(PAH)) was 3.94 +/- 0.26 in cats and 4.12 +/- 0.76 in rabbits, showing net secretion in both species. 4. FE(UA) in cats was dependent on urine flow, but was independent of plasma urate concentration. 5. The urate excretion in cats was enhanced by probenecid, but was insensitive to PAH and PZA. 6. The PAH excretion in cats was reduced by probenecid, but was unaltered by urate and PZA. 7. These results indicate that urate is handled in the cat kidney by a unique transport system which is distinct from that for organic anions.


Is sodium necessary for stimulus-secretion coupling in toad (Caudiverbera caudiverbera) adrenal chromaffin cells?

1. Chromaffin cells of the toad were used to investigate the effects of total replacement of extracellular Na+ by monovalent cations or sucrose on secretion of catecholamine (CA). 2. K+, Rb+ or Cs+ in place of Na+ produced an immediate secretory response, which are dependent on extracellular Ca2+ and it was blocked by Co2+. Li+ or choline+ did not affect basal secretion. 3. Isosmotic replacement of Na+ by sucrose caused CA secretion even in the absence of external Ca2+ or in the presence of Ca-channel blockers. 4. Tetraethylammonium decreased the extent of CA release produced by either K+ or Rb+. 5. The secretagogue effect of Na+/K+, Na+/Rb+ or Na+/Cs+ replacement could be explained by a depolarization of the cell membrane, which ultimately will cause Ca2+ influx through voltage-dependent Ca-channels. However, the present results indicate that Na+ may be sufficient but not necessary for CA secretion.


Thermoregulation and evaporative cooling in the cicada Okanagodes gracilis (Homoptera: Cicadidae).

1. Okanagodes gracilis uses a combination of physiological and behavioral mechanisms to regulate body temperature (Tb) to a prescribed range. 2. High thermal tolerances (48.6 degrees C Tb) and evaporative cooling permit the species to remain active during the hottest parts of the day. 3. The regression of Tb on ambient temperature (Ta) (Y = 0.142X + 34.63) intersects the isothermal line at 40.4 degrees C, below the shade-seeking value of 41.2 degrees C. 4. In the laboratory, weight (water) loss is faster at higher (46 degrees C) than at lower (43 degrees C) temperatures; the cicadas were able to survive mass losses of 25% in the laboratory. 5. Pores in the dorsal thorax and abdomen are the probable sites of water loss. 6. O. gracilis is the first cicada reported that is able to continue activity while simultaneously feeding and evaporatively cooling. 7. Behavioral mechanisms of thermoregulation and the possible thermoregulatory value of the species' coloration are discussed.


Digestive enzyme levels and histopathology of pancreas disease in farmed Atlantic salmon (Salmo salar).

1. The pancreatic digestive enzyme activities of trypsin and chymotrypsin were assessed in vitro and were found to correlate well with the histological changes characteristic of pancreas disease (PD) in farmed Atlantic salmon (Salmo salar). 2. Pancreatic enzyme activity was assessed in vivo using the chymotrypsin specific substrate N-benzoyl-L-tyrosyl-p-aminobenzoic acid. In all cases, significantly less p-aminobenzoic acid was excreted by fish later found to be suffering from PD. 3. It is concluded that the in vitro digestive enzyme assay was effective in diagnosing PD with the in vivo method, indicating further promise for assessing exocrine pancreatic function.


L-tryptophan alleviates fatty liver and modifies hepatic microsomal mixed function oxidase in laying hens.

1. Three experiments were conducted to study the effect of dietary L-tryptophan supplementation (250-1000 ppm) on lipid accumulation, an occurrence of hemorrhages and microsomal mixed function oxidase in the liver of laying hens. 2. Dietary L-tryptophan supplementation resulted in significant decreases in hepatic lipids, in particular triglyceride, and occurrence of hemorrhage in laying hens. 3. Hepatic lipid accumulation by estrogen injection in starved-refed growing chicks decreased as dietary tryptophan content increased. 4. Supplementation of L-tryptophan at 1000 mg/kg diet enhanced alanine aminotransferase activity in the hepatic tissue and at 500 mg/kg diet, increased cytochrome b5, a component of the mixed function oxidase, in the hepatic microsomes. 5. These results demonstrate that L-tryptophan alleviates fatty liver in laying hens and modifies microsomal mixed function oxidase in the liver.


Pleiotropic, multidrug-resistant phenotype and P-glycoprotein: a review.

This article is a brief review of recent knowledge about the pleiotropic resistant phenotype--multidrug-resistant (MDR) phenotype--which is responsible for cross-resistance in cancer chemotherapy. Thus it is possible to explain chemoresistance to vinca alkaloids, podophyllotoxins and anthracyclines. The MDR phenotype is associated with reduced intracellular drug accumulation by drug efflux, utilizing transmembrane P-glycoprotein (Gp170). The overexpression of Gp170 in normal and cancer tissues and the genetics are reported.


7-substituted pterins in humans with suspected pterin-4a-carbinolamine dehydratase deficiency. Mechanism of formation via non-enzymatic transformation from 6-substituted pterins.

A recently described new form of hyperphenylalaninemia is characterized by the excretion of 7-substituted isomers of biopterin and neopterin and 7-oxo-biopterin in the urine of patients. It has been shown that the 7-substituted isomers of biopterin and neopterin derive from L-tetrahydrobiopterin and D-tetrahydroneopterin and are formed during hydroxylation of phenylalanine to tyrosine with rat liver dehydratase-free phenylalanine hydroxylase. We have now obtained identical results using human phenylalanine hydroxylase. The identity of the pterin formed in vitro and derived from L-tetrahydrobiopterin as 7-(1',2'-dihydroxypropyl)pterin was proven by gas-chromatography mass spectrometry. Tetrahydroneopterin and 6-hydroxymethyltetrahydropterin also are converted to their corresponding 7-substituted isomers and serve as cofactors in the phenylalanine hydroxylase reaction. Dihydroneopterin is converted by dihydrofolate reductase to the tetrahydro form which is biologically active as a cofactor for the aromatic amino acid monooxygenases. The 6-substituted pterin to 7-substituted pterin conversion occurs in the absence of pterin-4a-carbinolamine dehydratase and is shown to be a nonenzymatic process. 7-Tetrahydrobiopterin is both a substrate (cofactor) and a competitive inhibitor with 6-tetrahydrobiopterin (Ki approximately 8 microM) in the phenylalanine hydroxylase reaction. For the first time, the formation of 7-substituted pterins from their 6-substituted isomers has been demonstrated with tyrosine hydroxylase, another important mammalian enzyme which functions in the hydroxylation of phenylalanine and tyrosine.


Purification of the main somatostatin-degrading proteases from rat and pig brains, their action on other neuropeptides, and their identification as endopeptidases 24.15 and 24.16.

The main somatostatin-degrading proteases were purified from rat and pig brain homogenates and characterized as thiol- and metal-dependent endoproteases. Two types of proteases with apparent native and subunit molecular masses of 70 kDa and 68 kDa could be differentiated in both species. Beside somatostatin, both hydrolyzed several other neuropeptides with chain lengths between 8 and 30 amino acid residues. Cleavage sites were generally similar or identical, but some clear exceptions were observed for enzymes from both species which could be used to differentiate between the two proteases. The 68-kDa protease cleaved somatostatin at three bonds (Asn5-Phe6, Phe6-Phe7 and Thr10-Phe11) and neurotensin only at the Arg8-Arg9 bond, whereas the 70-kDa protease digested somatostatin at only two bonds (Phe6-Phe7 and Thr10-Phe11) and neurotensin as well as acetylneurotensin-(8-13) additionally (pig protease) or almost exclusively (rat protease) at the Pro10-Tyr11 bond. Relative rates for the digestions of various peptides were, however, more dependent on the species than on the type of protease. Cleavage sites for angiotensin II, bradykinin, dynorphin, gonadoliberin and substance P were, apart from different rates, identical for both proteases. In both species the 68-kDa protease was found to be mainly, but not exclusively, soluble and not membrane-associated, whereas the inverse was detected for the 70-kDa protease. Based on distinct molecular and catalytic properties, the 68-kDa protease is supposed to be congruent with the endopeptidase 24.15 (EC 3.4.24.15), the 70-kDa protease with endopeptidase 24.16 (EC 3.4.24.16, neurotensin-degrading endopeptidase). This investigation demonstrates that both proteases hydrolyze various neuropeptides with similar cleavage sites, but with species-dependent activity. Species-independent distinctions are the exclusive action of endopeptidase 24.16 on acetylneurotensin-(8-13) and liberation of free Phe from somatostatin only by endopeptidase 24.15.


Maturation of Xenopus laevis oocyte by progesterone requires poly(A) tail elongation of mRNA.

Meiotic maturation of Xenopus laevis oocytes by progesterone requires translation of stored maternal mRNAs. We investigated the role of poly(A) tail elongation of mRNAs during this process using cordycepin, which inhibits poly(A) tail elongation of mRNAs. When oocytes were treated with the buffer containing 10 mM cordycepin for 12 h, concentration of 3'-dATP in cytosol of oocytes increased to 0.7 mM, while that of ATP remained constant at around 1.2 mM. Incorporation of [32P]AMP into poly(A) mRNA was inhibited almost completely by this treatment. Progesterone-induced germinal vesicle breakdown (GVBD) was also abolished. Dose dependence of inhibition of progesterone-induced GVBD on cordycepin was similar to that of [32P]AMP incorporation into poly(A) mRNA. However, maturation-promoting factor-induced GVBD was unaffected by treatment of oocytes with cordycepin. Furthermore, the inhibition of GVBD by cordycepin was rescued by removal of cordycepin even in the presence of actinomycin D. Therefore, we concluded that poly(A) tail elongation of mRNA is required for induction of meiotic maturation of X. laevis oocytes. In addition, progesterone induced a 2.7-fold activation of [32P]AMP incorporation into the poly(A) tail of mRNA after a lag period of 3 h whereas GVBD was induced after 6-8 h from the progesterone treatment. Syntheses of most of the proteins were unaffected by treatment of oocytes with progesterone or cordycepin. However, syntheses of several proteins were increased or decreased by progesterone and cordycepin treatment.


Microvillar enzyme activity in amniotic fluid, extraembryonic coelomic fluid and maternal serum in the first trimester of pregnancy.

The activities of two microvillar enzymes, gamma-glutamyl transpeptidase and total alkaline phosphatase, have been measured in samples of amniotic fluid and extraembryonic coelomic fluid obtained by high-resolution transvaginal ultrasound-guided amniocentesis from 40 women between 7 and 12 weeks of gestation. There was a highly significant difference between gamma-glutamyl transpeptidase activity in amniotic fluid (median level 31 U/l; range 2-409 U/l) and extraembryonic coelomic fluid (median level 2 U/l; range less than 2-16 U/l) (P less than 0.001; Mann-Whitney U-test). Alkaline phosphatase activity was not detected in 84% of amniotic fluid samples and 97% of extraembryonic coelomic fluid samples. No difference was found between total alkaline phosphatase activity in these fluids (P = 0.14; Mann-Whitney U-test). Enzyme activities in amniotic fluid increased with gestational age. A significant linear correlation was found between amniotic fluid gamma-glutamyl transpeptidase activity and stage of gestation (r = 0.86; P less than 0.001) and total alkaline phosphatase activity in amniotic fluid and stage of gestation (r = 0.66; P less than 0.001).


Regulated expression of vasopressin gene by cAMP and phorbol ester in primary rat fetal hypothalamic cultures.

Using dispersed cultures of fetal rat hypothalami, we studied the effects of forskolin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), activators of protein kinase A and C, respectively, upon vasopressin (VP) secretion, VP mRNA expression and VP mRNA poly(A) tail length. Forskolin stimulated the VP mRNA content and peptide secretion 2.6-fold and induced an increase in the poly(A) tail length of approximately 90 nucleotides. TPA induced an increase in VP mRNA size and stimulated 1.9-fold the secretion of VP without an increase in VP mRNA content. Depolarization with potassium induced an increase in the VP peptide secreted of 2.2-fold, with no effect on the VP mRNA content or size. Increased osmolality had no effect on either VP peptide or VP mRNA. We conclude that VP expression in cultured fetal rat hypothalamic cells is regulated via both protein kinase A and protein kinase C pathways.