Dye-enhanced laser welding for skin closure.

The use of a laser to weld tissue in combination with a topical photosensitizing dye permits selective delivery of energy to the target tissue. A combination of indocyanine green (IG), absorption peak 780 nm, and the near-infrared (IR) alexandrite laser was studied with albino guinea pig skin. IG was shown to bind to the outer 25 microns of guinea pig dermis and appeared to be bound to collagen. The optical transmittance of full-thickness guinea pig skin in the near IR was 40% indicating that the alexandrite laser should provide adequate tissue penetration. Laser "welding" of skin in vivo was achieved at various concentrations of IG from 0.03 to 3 mg/cc using the alexandrite at 780 nm, 250-microseconds pulse duration, 8 Hz, and a 4-mm spot size. A spectrum of welds was obtained from 1- to 20-W/cm2 average irradiance. Weak welds occurred with no thermal damage obtained at lower irradiances: stronger welds with thermal damage confined to the weld site occurred at higher irradiances. At still higher irradiances, local vaporization occurred with failure to "weld." Thus, there was an optimal range of irradiances for "welding," which varied inversely with dye concentration. Histology confirmed the thermal damage results that were evident clinically. IG dye-enhanced laser welding is possible in skin and with further optimization may have practical application.


Comparative study of laser and scalpel nerve transections.

This investigation was designed to compare standard scalpel transections of the tibial branch of the rat sciatic nerve with those performed using either a milliwatt carbon dioxide (CO2) or a potassium titanyl phosphate (KTP/532) laser. Four transection groups consisted of nerves sectioned with (1) scalpel (control), (2) milliwatt CO2 laser, (3) KTP/532 with microscope attachment, and (4) KTP/532 laser with 400-microns bare fiber. Each laser was used with the same parameters: 10 watts, 0.4-mm spot size, and continuous-wave mode. Horseradish peroxidase (HRP) was applied to the proximal stump for 30 min, and the animals were sacrificed 24 h later. Horseradish peroxidase (HRP)-labeled motoneuron cell bodies in the lumbar spinal cord were then counted. The average numbers of labeled neurons in each group were as follows: group I (n = 14) 518, group II (n = 8) 424, group III (n = 8) 351, and group IV (n = 8) 283. The standard deviations were quite large, however. When all laser transections were pooled and compared with paired scalpel transections, we found a significant difference, both by the paired t-test (P = 0.016) and by the Wilcoxon matched-paired test (P = 0.02). We conclude that laser transection significantly diminishes the number of neurons labeled by the retrograde transport of HRP.


Relationship between serotonergic measures in periphery and the brain of mouse.

Circadian rhythm and the relationship between the concentration of serotonin (5HT) and related substances (5-hydroxyindoleacetic acid; 5HIAA and tryptophan; Trp) in mouse brain, stomach and blood have been studied. All factors underwent circadian changes in the brain and blood. 5HT and 5HIAA levels in the stomach showed no circadian fluctuation. The concentrations of 5HT in the brain and blood did not correlate. Significant correlations were found between other serotonergic parameters analyzed in brain, stomach and blood. A significant negative correlation was observed between brain 5HIAA and blood 5HIAA. The concentration of tryptophan in the brain was correlated with the plasma total tryptophan level. There was fairly significant correlation (p less than 0.06) between brain serotonin and plasma tryptophan levels. The brain serotonin and tryptophan levels were strongly correlated (R = 0.410, p less than 0.03). Significant negative correlation was found between serotonin in the blood and serotonin in the stomach as well as between its level in the brain and in the stomach. The significance of these findings and their relationship to the use of peripheral serotonergic system as a model of neurons are discussed.


Differential effects of galanin and neuropeptide Y on extracellular norepinephrine levels in the paraventricular hypothalamic nucleus of the rat: a microdialysis study.

Evidence suggests that the peptides galanin (GAL) and neuropeptide Y (NPY) interact with the amine norepinephrine (NE) in the hypothalamic paraventricular nucleus (PVN) to stimulate feeding behavior. To directly investigate the nature of these interactions, extracellular levels of PVN NE were monitored in freely-moving rats using the microdialysis/HPLC technique. Following PVN administration of GAL (0.3 nmol), NPY (78 pmol) or Ringer's solution, local NE levels were measured at 20-min intervals for 2 hrs postinjection, under two feeding conditions, namely, in the presence or absence of food. The results demonstrate different effects of these peptides on endogenous NE levels. Following GAL administration, PVN NE levels were enhanced by 80 to 90%, up to 40 min postinjection, independent of food availability. In contrast, following NPY injection, NE levels were significantly reduced 20 min postinjection with food absent, and when food was available, NE levels tended to be enhanced. These results, consistent with pharmacological and biochemical studies, reveal different patterns of peptide-amine interactions in the PVN.


Axonal injury in closed head injury by assault: a quantitative study.

Due to the controversy in the literature regarding the time course of axonal balloon formation in human material, we wished to determine if it was possible to diagnose axonal injury before the development of axonal balloonings. The hypothesis was that the presence of axonal swellings or axonal enlargements associated with a glial reaction could be used as a diagnostic aid in human axonal injury before 12 hours. The brains of eight individuals that survived for less than 48 hours following head injury, and also had evidence of axonal injury using the criteria of Vanezis et al. (1987), were systematically studied by looking at axonal swellings, axonal balloonings, reactive astrocytes, maximum diameter of axonal enlargements and density of axonal enlargements. Controls were eight selected cases without neurological disease. The variables studied were assessed in 25 fields from ten different areas of the brain, using silver stains and immunoperoxidase for glial fibrillary acidic protein (GFAP). Logarithms of one plus the count of each variable were taken from the raw data and these were analysed using percentile distribution and the median, the t-test, Mann-Whitney U test and the Wilcoxon signed rank test. We conclude that quantitation of axonal damage allows the detection of mild degrees of axonal injury that could be overlooked on routine examination, and that the criteria of axonal enlargements, rather than axonal balloonings, are indications of axonal damage, cannot be endorsed with the evidence provided.


[Pancreatic exocrine and endocrine functions stimulated with secretin and thyrotropin-releasing hormone in patients with hyperparathyroidism].

This study was designed to investigate pancreatic exocrine and endocrine secretion stimulated with secretin and thyrotropin-releasing hormone (TRH) in hyperparathyroidism. Pancreatic exocrine secretion during 30 min stimulated by constant secretin infusion of 1U/kg/hour was significantly increased in patients with secondary hyperparathyroidism compared with controls and patients with primary hyperparathyroidism. Intravenous administration of TRH at a dose of 20 micrograms/kg/hour, superimposed on secretin, produced a significant decrease of pancreatic exocrine secretion in both primary and secondary hyperparathyroidism but not in control. Serum insulin, glucagon and secretin levels were significantly higher in the subjects of both primary and secondary hyperparathyroidism than those of controls. Serum glucagon and secretin levels were significantly higher in secondary hyperparathyroidism than primary hyperparathyroidism. The pancreatic endocrine secretion was not influenced by TRH administration. Pancreatic exocrine secretion was not changed by parathyroidectomy in patients with primary hyperparathyroidism. As for endocrine secretion, however, only serum secretin level decreased to the level before parathyroidectomy. In this study, it was speculated that the increase of pancreatic secretion in secondary hyperparathyroidism may be due to hypersecretinemia, and the decrease of exocrine secretion by TRH in primary and secondary hyperparathyroidism may be resulted from the direct effect of TRH on the pancreatic acinar cells.


Regulation by phorbol ester and protein kinase C inhibitors, and by a protein phosphatase inhibitor (okadaic acid), of P-glycoprotein phosphorylation and relationship to drug accumulation in multidrug-resistant human KB cells.

Covalent modification by phosphorylation is a characteristic of the P-glycoproteins expressed in multidrug-resistant cells. This report describes analysis of P-glycoprotein phosphorylation in multidrug-resistant human KB-V1 cells and a study of the relationship of phosphorylation and drug accumulation. In isolated membranes, phosphorylation of P-glycoprotein by purified protein kinase C (PKC) was rapid, and time-dependent dephosphorylation was inhibited by okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases. In 32P-labeled intact KB-V1 cells, P-glycoprotein phosphorylation was stimulated by both 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, and okadaic acid. Two-dimensional thin layer tryptic phosphopeptide maps indicated that the sites of phosphorylation were similar in control, TPA-treated, and okadaic acid-treated cells and that they corresponded to those phosphorylated by PKC in vitro. The protein kinase inhibitor staurosporine, and the PKC-selective inhibitors calphostin C and the alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, inhibited P-glycoprotein phosphorylation in vitro and in intact cells. Drug accumulation assays demonstrated that in KB-V1 cells TPA caused a decrease, whereas staurosporine and calphostin C caused an increase, in accumulation of [3H]vinblastine. These compounds did not significantly alter [3H]vinblastine levels in drug-sensitive KB-3 cells. These results suggest that PKC is chiefly responsible for P-glycoprotein phosphorylation in KB-V1 cells, that membrane-associated protein phosphatases 1 and 2A are active in dephosphorylation of P-glycoprotein, and that phosphorylation of P-glycoprotein may be an important mechanism for modulation of drug-pumping activity.


Determination of the amino acid residues in substance P conferring selectivity and specificity for the rat neurokinin receptors.

We have measured the affinity of various analogs and fragments of the tachykinin substance P for the cloned rat NK1, NK2, and NK3 receptors heterologously expressed in Chinese hamster ovary cells. The hydrophobic carboxyl-terminal pentapeptide sequence substance P-(7-11) binds with similar affinity (2-20 microM) to all three receptors. Our data suggest that addition of one to three amino-terminal residues to this sequence results in the optimization of its interaction within the binding pocket of the NK1 receptor. The addition of Pro-Gln-Gln to the carboxyl-terminal pentapeptide sequence increases affinity for the NK1 receptor, either by providing additional binding interactions or by modifying the conformation of the carboxyl-terminal sequence. This latter hypothesis is supported by the observation that physalaemin and phyllomedusin, which also contain a proline residue in the position analogous to the proline residue 4 of substance P, are also selective for NK1 receptors. Tachykinins that lack this proline have no higher affinity for NK1 than [pGlu] substance P-(6-11). Conversely, addition of Pro-Gln-Gln to the carboxyl-terminal pentapeptide sequence is unfavorable for NK2 and NK3 receptor binding. Preliminary data suggest that tachykinins with high affinity (Kd less than 500 nM) for NK2 receptors contain an aspartate residue in the position analogous to residue 5 of substance P, suggesting that an ionic interaction with the receptor may contribute binding energy. Further experiments will be required to determine the structural determinants of the NK1, NK2, and NK3 receptors responsible for these binding properties.


Effects of hexachlorocyclohexanes on gamma-aminobutyric acid receptors expressed in Xenopus oocytes by RNA from mammalian brain and retina.

Poly(A)+ RNA from rat cerebral cortex expresses gamma-aminobutyric acid (GABA)-activated membrane current responses in Xenopus oocytes, mediated by GABAA receptors (IG-Actx). In contrast, RNA from bovine retina expresses GABA responses composed of two pharmacologically distinct Cl- currents, one mediated by GABAA receptors (IG-Aret) and the other by atypical GABA receptors that are resistant to bicuculline and are not activated by baclofen (IG-BR). The pharmacology of the bicuculline/baclofen-insensitive GABA receptors was further investigated by comparing actions of hexachlorocyclohexane (HCH) enantiomers on GABA-activated membrane currents expressed in oocytes by brain and retina RNA. gamma-HCH (lindane) was a potent inhibitor of IG-Actx, with suppression of currents detectable at concentrations as low as 50 nM. The IC50 for gamma-HCH, calculated from inhibitory effects on maximum IG-Actx (current elicited by 3 mM GABA), was 7.3 +/- 3 microM. Inhibitory effects of gamma-HCH on IG-Aret were qualitatively similar to those described for IG-Actx. In contrast, alpha-HCH and delta-HCH induced clear positive modulation of IG-Actx elicited by low (e.g., 10 microM) concentrations of GABA. Thresholds for the modulatory effects of alpha-HCH and delta-HCH were between 100 and 300 nM, with maximum levels of potentiation (5-7-fold) between 20-50 microM. Potentiation of IG-Actx by alpha- and delta-HCH was reversible and largely insensitive to the benzodiazepine antagonist flumazenil (1 microM). Assays on maximum IG-Actx indicated that alpha-HCH (10-100 microM) caused only marginal reductions in response (less than or equal to 15%), whereas delta-HCH had stronger inhibitory effects (IC50, 20-30 microM). At concentrations between 0.1 and 50 microM, beta-HCH induced only 10-25% facilitation of IG-Actx elicited by 10 microM GABA and had no clear effects on maximum responses. IG-BR was also potently inhibited by gamma-HCH. Thresholds for detecting reductions in current were approximately 20 nM, and the IC50 calculated from effects on maximum responses was 5.8 +/- 2 microM. However, neither alpha-HCH nor delta-HCH (1-100 microM) induced any potentiation of IG-BR. alpha-HCH had some weak inhibitory effects that were largely surmountable, whereas delta-HCH and beta-HCH were essentially inactive. These experiments raise the possibility that alpha- and delta-HCH constitute a novel class of GABAA receptor modulators, which might prove to be useful for investigating the mechanisms underlying regulation of GABAA receptors.(ABSTRACT TRUNCATED AT 400 WORDS)


Induction of NADPH-dependent diaphorase and nitric oxide synthase activity in aortic smooth muscle and cultured macrophages.

Lipopolysaccharide (LPS), either alone or in combination with cytokines, induces nitric oxide (NO) synthase activity in cells that normally release little or no NO. In arterial smooth muscle cells and various macrophage cell lines, NO synthase activity is induced after several hours of incubation with LPS. In brain, NADPH-dependent diaphorase activity has been associated with constitutive NO synthase. Here we show that incubation of rat aorta or cultured macrophages with LPS causes a time-dependent induction of NO synthase. The NO synthase activity in both rat aorta and macrophages was calcium independent and inhibited by NG-monomethyl-L-arginine and NG-nitro-L-arginine. We also found that LPS caused a time-dependent induction in NADPH-dependent diaphorase activity in both rat aorta and cultured macrophages. The diaphorase activity was mainly NADPH dependent and NADH independent. NO synthase activity and NADPH-diaphorase activity in crude cytosol from LPS-treated macrophages were found to co-purify, using 2',5'-ADP-Sepharose followed by Superose-6 gel permeation chromatography.


Mutation induction and DNA adduct formation in Chinese hamster ovary cells treated with 6-nitrochrysene, 6-aminochrysene and their metabolites.

6-Nitrochrysene, 6-aminochrysene and several of their metabolites were assayed for mutagenic activity at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in DNA-repair-proficient Chinese hamster ovary (CHO-K1) cells and excision-repair-deficient CHO-UV5 cells. Mutagen-DNA adducts were analyzed by 32P-postlabeling in cells treated under the conditions of the mutagenicity assay and compared with the adduct patterns produced from the in vitro reaction of metabolites of 6-nitrochrysene and 6-aminochrysene with calf-thymus DNA. The mutagenic activities of the test compounds in the presence of a liver homogenate (S9) fraction from Aroclor 1254-pretreated rats, expressed as the number of mutants per 10(6) cells per nmole test compound per ml, in CHO-K1 and CHO-UV5 cells, respectively, were as follows: 6-nitrochrysene, 0.3 and 4; 6-aminochrysene, 35 and 117; 6-nitrochrysene-1,2-dihydrodiol, 1 and 6; 6-aminochrysene-1,2-dihydrodiol, 488 and 644; chrysene (run as a positive control), 12 and 28. 6-Nitrosochrysene was a direct-acting mutagen, yielding 127 and 618 mutants per 10(6) cells per nmole per ml in CHO-K1 and CHO-UV5 cells, respectively. Mutagen-DNA adduct analysis indicated that cells treated with 6-aminochrysene in the presence of S9 or 6-nitrosochrysene in the absence of S9 contained an adduct pattern identical to that derived from the in vitro reaction of N-hydroxy-6-aminochrysene with calf-thymus DNA. Cells treated with 6-aminochrysene-1,2-dihydrodiol plus S9 contained a single mutagen-DNA adduct that was distinct from those derived from N-hydroxy-6-aminochrysene. Based on comparison with previous studies, this adduct is presumed to be derived from 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-aminochrysene. Cells treated with 6-nitrochrysene plus S9 and 6-nitrochrysene-1,2-dihydrodiol plus S9 contained a single major chromatographically identical adduct that was apparently derived from N-hydroxy-6-aminochrysene-1,2-dihydrodiol. The results indicate that 6-nitrochrysene, 6-aminochrysene and their metabolites are mutagenic in CHO cells, but that the major activation pathway for 6-nitrochrysene and 6-nitrochrysene-1,2-dihydrodiol in this system differs from previously described pathways.


Cytogenetic effects of pesticides. IV. Cytogenetic effects of the insecticides Gardona and Dursban.

The cytogenetic effects of the insecticides Gardona and Dursban were investigated. The toxicity and ability of both insecticides to induce chromosome aberrations and sister-chromatid exchange in vitro was tested in a primary culture of mouse spleen cells, in order to assess the potential mutagenicity of both insecticides. The concentrations 10(-7)-10(-3) M were used for testing the toxic effects of the insecticides. Both Gardona and Dursban were toxic to spleen cell cultures and the percentage of viable cells decreased as the concentration of the insecticide was increased. It reached 76.8% and 77.8% of control after treatment with the highest concentration tested (10(-3) M) of Gardona and Dursban respectively. Gardona at 0.25, 0.50, 1.0 and 2.0 micrograms/ml, and Dursban at 0.50, 1.0, 2.0 and 4.0 micrograms/ml were tested for the induction of chromosome aberrations and sister-chromatid exchanges. All of the tested concentrations of both insecticides induced a high percentage of metaphases with chromosomal aberrations in cultured mouse spleen cells after 4-h treatment. The frequency of SCEs/cell increased with increasing concentration of the insecticides. It reached 11.92 +/- 0.14/cell and 13.40 +/- 0.20/cell after treatment with Gardona (2 micrograms/ml) and Dursban (4 micrograms/ml), respectively, compared with 8.2 +/- 0.19/cell and 7.6 +/- 0.15/cell in the solvent control. The presented results indicate that both Gardona and Dursban in the tested concentrations are mutagenic in mouse spleen cell cultures.


Cytogenetic analysis of chromosomal aberrations of peripheral lymphocytes in workers occupationally exposed to nickel.

The authors carried out a cytogenetic examination of chromosomal aberrations of peripheral lymphocytes (100 cells evaluated in each sample) with simultaneous monitoring of the level of exposure by means of determination of nickel in the urine, serum and hair. The series included 21 workers occupationally exposed to nickel at two workshops producing NiO (6 persons) and NiSO4 (15 persons) in a chemical plant. At the same time a comparable control group, i.e., 19 workers of the same chemical plant but without any direct occupational nickel exposure (clerks, service men, etc.), were examined in the same way. In the exposed group chromosomal aberrations of peripheral lymphocytes were detected with an average value of 6.41 +/- 1.9% (range 2-14%); in the group producing NiO it was, on the average, 9.5 +/- 3.2% (range 7-14%) whereas in the NiSO4 production workers it was only 5.2 +/- 1.9% (range 2-10%). There was a dependence of chromosomal aberrations of peripheral lymphocytes on the exposure time and on the nickel content of the biological material. Significantly increased values (in contrast to the normal value of chromosomal aberrations of peripheral lymphocytes, up to 2%) were detected in the control group as well (average value of 4.05 +/- 2.27%, range 1-10%). The authors explain this fact by the nickel-polluted environment of the whole observed chemical plant.


Normal chromosomal aberration frequencies in peripheral lymphocytes of healthy human volunteers exposed to a maximum daily dose of paracetamol in a double blind trial.

Paracetamol (acetaminophen) has been examined for mutagenic potential in numerous studies: gene mutation tests consistently gave negative results while in vitro chromosomal aberration tests showed equally consistently positive effects. In vivo studies for chromosome breaking activity gave clearly negative, equivocal or weakly positive results. In particular two reports have indicated that human volunteers taking a maximum daily dose of paracetamol (3 x 1000 mg over 8 h) exhibited significantly elevated frequencies of chromatid breaks in their peripheral lymphocytes 24 h later. In the one study evaluating the time course, levels returned to normal between 3 and 7 days later. We performed a carefully controlled double-blind study in which volunteers were pre-screened for normal liver function, they all were non-smoking and their diet and environmental exposures were controlled during the study. Cell-cycle kinetics were monitored and paralleled and a placebo group was included. Although a larger number of cells than in the other studies was analysed we were unable to reproduce their findings. No significant increases in structural chromosome aberrations (CA) were found either when the paracetamol group (male, female or both) post-dosing values were compared with pre-dosing values, or when treated groups at any sampling time were compared with the placebo groups. There was not even any evidence that individuals responded to the clastogenic potential of paracetamol or that a group response may have been masked by non-responders. In conjunction with the recently published results of the NTP bioassay, showing no carcinogenic activity in mice and no carcinogenic activity in rats except an increase of mononuclear cell leukaemia in female rats which is of doubtful relevance, the study presented here argues that paracetamol does not pose an unacceptable (if any) genotoxic/carcinogenic risk to man.


Cytogenetic studies of workers from the rubber industry.

Lymphocytes from 21 vulcanizers from a tire factory were analyzed for the presence of chromosome aberrations (CAs), sister-chromatid exchanges (SCEs) and for proliferation indices (PIs). An increase in the frequencies of CAs and SCEs and a decrease in PIs were observed. Cytogenetic parameters were correlated with age, smoking habits, and duration of occupational exposure. From the present study it can be concluded that cigarette smoking enhances the genotoxic effects of mixtures of chemicals to which workers in the rubber industry are exposed.


Baseline and phosphoramide mustard-induced sister-chromatid exchanges in pharmacists handling anti-cancer drugs.

Determinations of baseline and mutagen-induced sister-chromatid exchanges (SCE) have been used as indicators of previous mutagen exposure in several human populations. Mutagen-induced SCE is based on the premise that a genetic outcome may depend not only on a present exposure, but also on a cell's "memory" of previous exposure. The genotoxicity of some anti-cancer drugs including cyclophosphamide (CP) has been studied by determining baseline and mutagen-induced SCE in peripheral blood lymphocytes in treated cancer patients. This study examined the in vivo genotoxic effects of occupational exposure to anti-cancer drug handling by relating baseline and phosphoramide mustard (PM) -induced SCE levels with duration of anti-cancer drug handling as a surrogate for anti-cancer drug exposure dose. The mean baseline SCE for the population was 5.19 +/- 0.17 and was not correlated with duration of drug handling. However, a strong correlation was demonstrated between inducible SCE values and life-time duration of drug handling with r = 0.63 (p less than 0.0001 for low-dose PM challenge (0.1 mg/ml PM) and r = 0.67 (p less than 0.0001) for high-dose PM challenge (0.25 mg/ml PM). A similar relationship was seen for PM-induced SCE and duration of anti-cancer drug handling for the workers' present job with correlations obtained being r = 0.63 (p less than 0.0001) for low-dose PM and r = 0.59 (p less than 0.0001) for high dose PM. The short-lived nature of the baseline SCE lesion is discussed as a limitation in population surveillance studies, as it reflects primarily recent mutagen exposure and persists only for days to weeks after exposure. The induced SCE measure is postulated to provide an integrating dosimeter of remote previous exposure, improving upon the current limitation of the baseline SCE measure and allowing the "unmasking" of previous exposure in a provocative framework.


Induction of micronuclei in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol.

Tetrachlorohydroquinone, a metabolite of the fungicide pentachlorophenol, induced significant dose-related increases in micronuclei in V79 Chinese hamster cells without exogenous metabolic activation. The lowest observed effective dose was 10 microM, where the relative survival was about 62%. At the highest dose tested, 20 microM, the relative survival was about 8% and the frequency of cells with micronuclei was about 6 times the solvent control frequency. The induction of micronuclei by tetrachlorohydroquinone was significantly inhibited by the hydroxyl radical scavenger dimethyl sulfoxide at 5% (v/v).


Chinese medicinal herbs modulate mutagenesis, DNA binding and metabolism of aflatoxin B1.

Oldenlandia diffusa (OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors while Astragalus membranaceus (AM) and Ligustrum lucidum (LL) are often used as an adjunct in cancer therapy. In this study, we determined the effects of aqueous extracts of these four herbs on aflatoxin B1 (AFB1)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver 9000 x g supernatant as the activation system. The effects of these herbs on [3H]AFB1 binding to calf-thymus DNA were assessed. Organosoluble and water-soluble metabolites of AFB1 were extracted and analyzed by high-performance liquid chromatography (HPLC). Mutagenesis assays revealed that all of these herbs produced a concentration-dependent inhibition of histidine-independent revertant (His+) colonies induced by AFB1. At a concentration of 1.5 mg/plate, SB and OD in combination exhibited an additive effect. The trend of inhibition of these four herbs on AFB1-induced mutagenesis was: SB greater than LL greater than AM. LL, OD and SB significantly inhibited AFB1 binding to DNA, reduced AFB1-DNA adduct formation, and also significantly decreased the formation of organosoluble metabolites of AFB1. Our data suggest that these Chinese medicinal herbs possess cancer chemopreventive properties.


Induction of micronuclei in BALB/c-3T3 cells by selected chemicals and complex mixtures.

The genotoxicity of benzo[a]pyrene, cyclophosphamide, 2-aminoanthracene, 2-nitrofluorene, nitrosated coal-dust extracts, and cigarette-smoke condensate were tested with the micronucleus assay using an established mammalian cell line. The results showed that all chemicals and complex mixtures studied induced micronuclei in BALB/c-3T3 cells. These results indicate that BALB/c-3T3 cells are capable of activating certain promutagens and procarcinogens. It seems, therefore, that in addition to cell transformation, the micronucleus assay in BALB/c-3T3 cells without an exogenous activation system may be useful for in vitro studies to detect genotoxic chemicals and complex mixtures.


Micronucleated cells in nasal mucosa of formaldehyde-exposed workers.

The frequency of micronuclei (MN) and cytology of respiratory nasal mucosa cells were evaluated in 15 non-smokers exposed to formaldehyde in a plywood factory. Each subject was paired with a control matched for age and sex. Mean levels of exposure to formaldehyde ranged from about 0.1 mg/m3 in the sawmill and shearing-press departments to 0.39 mg/m3 in the warehouse area. There was a contemporary exposure to low levels of wood dust (inspirable mass ranged from 0.23 mg/m3 in the warehouse to 0.73 mg/m3 during sawing operations). Nasal respiratory cell samples were collected by an otorhinolaryngologist near the inner turbinate using a brush for endocervical cytology. After staining (Feulgen plus Fast Green and Papanicolaou's method for MN analysis and cytology, respectively), about 6000 cells were screened for micronuclei and scored in parallel for cytology according to a histopathological scale. A higher frequency of micronucleated cells was observed in the exposed group than in the controls (0.90 +/- 0.47 vs. 0.25 +/- 0.22, Mann-Whitney U test: p less than 0.01). Cytological examination indicated chronic phlogosis in the nasal respiratory mucosa of plywood factory workers, with a high frequency of squamous metaplasia cells (mean score 2.3 +/- 0.5 vs. 1.6 +/- 0.5 in the control group, Mann-Whitney U test: p less than 0.01).


Evaluation of genotoxicity of tert.-butylhydroquinone in an hepatocyte-mediated assay with V79 Chinese hamster lung cells and in strain D7 of Saccharomyces cerevisiae.

tert.-Butylhydroquinone (TBHQ) has been reported to be genotoxic in some short-term assays but non-genotoxic in others. We have examined cytotoxicity and genotoxicity of TBHQ, a principal metabolite of the phenolic antioxidant 2(3)-tert.-butyl-4-hydroxyanisole (BHA), in an hepatocyte-mediated assay with V79 Chinese hamster lung cells including both sister-chromatid exchange (SCE) and thioguanine-resistance (TGR) endpoints. The ability of BHA and of TBHQ to elicit a genotoxic response in Saccharomyces cerevisiae strain D7 was also investigated. In V79 cytotoxicity tests, TBHQ without hepatocytes produced a 50% reduction in colony formation at 4.2 micrograms/ml and was lethal to 100% of the cells at concentrations above 5 micrograms/ml. At partially cytotoxic dose levels, (0.17-3.4 micrograms/ml of medium), TBHQ sometimes increased significantly the frequency of SCE. TBHQ also produced sporadic statistically significant increases in the mutation frequency at the HGPRTase (TGR) gene locus when tested alone or with activation by rat or hamster hepatocytes. Mitotic gene conversion and reverse mutation were not induced in strain D7 of Saccharomyces cerevisiae by exposure to BHA or to TBHQ for 4 h at concentrations as high as 200 micrograms/ml for BHA or 500 micrograms/ml for TBHQ, either alone or with activation by rat-liver S9. Incubation of the yeast cells with BHA or TBHQ for 24 h in growth medium without activation also did not induce genotoxic activity. The slight and sporadic response to TBHQ in the V79 test system may indicate weak genotoxicity which is sensitive to slight differences in test conditions. The classification and test strategies adopted for compounds such as TBHQ could have important implications for regulatory decisions and for the validation of short-term tests.


Evaluation of the genotoxic effects of a folk medicine, Petiveria alliacea (Anamu).

Crude extract from a plant known as Petiveria alliacea (Anamu) is used extensively as folk medicine in developing countries like Colombia, South America. Although the plant is known to contain toxic ingredients potential adverse health effects from its use have not been adequately evaluated. We investigated its genotoxic activities by conducting a sister chromatid exchange (SCE) assay using cells in vitro and in vivo. Lymphocytes from humans were treated at 24 h after initiation of culture for 6 h with alcohol extract from the folk medicine. Concentrations of 0, 10, 100, 250, 275, 500, 750, and 1000 micrograms/ml of the extract were used. Significant dose-dependent increase of SCE (3.7-7.4 SCE per cell) were observed (analysis of variances, p less than 0.01). Delay in cell proliferation but not inhibition of mitosis was also observed. In another experiment, mice were exposed once orally to 1x, 200x, 300x and 400x the human daily consumption dose of Anamu. The induction of sister chromatid exchanges in bone marrow cells were investigated. We observed a significant dose dependent increase of SCE compared with the saline control (2.15-4.53; p less than 0.01) and compared with the solvent control (3.04-4.53; p less than 0.01). Our data suggest, therefore, that the folk medicine contains mutagenic and potentially carcinogenic agents although the medicine is not a potent mutagen. Individuals who consume large amounts of this drug may be at risk for development of health problems. Further studies with cells from exposed individuals and from experimental animals should be conducted to provide a better evaluation of health risk from the use of this drug.


Cytogenetic studies of mice exposed to styrene by inhalation.

The data for the in vivo genotoxicity of styrene (STY) are equivocal. To evaluate the clastogenicity and sister-chromatid exchange (SCE)-inducing potential of STY in vivo under carefully controlled conditions, B6C3F1 female mice were exposed by inhalation for 6 h/day for 14 consecutive days to either 0, 125, 250 or 500 ppm STY. One day after the final exposure, peripheral blood, spleen, and lungs were removed and cells were cultured for the analysis of micronucleus (MN) induction using the cytochalasin B-block method, chromosome breakage, and SCE induction. Peripheral blood smears were also made for scoring MN in erythrocytes. There was a significant concentration-related elevation of SCE frequency in lymphocytes from the spleen and the peripheral blood as well as in cells from the lung. However, no statistically significant concentration-related increases were found in the frequency of chromosome aberrations in the cultured splenocytes or lung cells, and no significant increases in MN frequencies were observed in binucleated splenocytes or normochromatic erythrocytes in peripheral blood smears.


Genotoxicity of 'gudakhu', a tobacco preparation. I. In mice in vivo.

'Gudakhu' is a paste-like tobacco preparation used widely in Orissa and neighbouring states of India. During use it is rubbed over the teeth and gum with a finger tip. Besides tobacco, it contains molasses, lime, red soil and water. The genotoxic potential of acetone extract of gudakhu was evaluated in mice in vivo using the chromosome aberration assay, micronucleus test and SCE analysis following single as well as long-term repeated treatment. The animals received an aqueous suspension of the extract via the oral route. Gudakhu extract induced significantly high frequencies, compared to controls, of chromosome aberrations, micronuclei (MN) and SCEs. Single treatment with different doses clearly revealed a distinct dose-dependent increase of the effects in all the assays. Analysis of MN in regenerated hepatocytes also indicated a significant positive correlation between time-course of chronic treatment and frequencies of micronucleated cells. But incidences of chromosome aberrations, MN and SCEs in bone marrow cells following repeated treatment for different periods did not differ greatly from each other; and these repeated treatment data, particularly in the MNT in bone marrow cells and the SCE assay, also did not differ markedly from the respective single treatment data for the same dose. This was probably due to the proliferative nature of the bone marrow cells.


Mutagenicity of quinolines in Salmonella typhimurium TA100. A QSAR study based on hydrophobicity and molecular orbital determinants.

The mutagenicity of 33 quinolines in the Salmonella test using TA98 and TA100 cells has been reported. Significant activity was found only with TA100 cells. Quantitative structure-activity relationships (QSAR) could be formulated using molecular orbital parameters or Hammett constants and hydrophobic parameters for those compounds with substituents in the 6, 7 and 8 positions. The QSAR points to the 2-position on the quinoline ring as being the site for activation by S9 oxidation.


On the induction of umu gene expression in Salmonella typhimurium strain TA1535/pSK1002 by some nitrofurans.

Several nitrofurans were found to induce umu gene expression in Salmonella typhimurium TA1535/pSK1002 as defined on the basis of at least a 2-fold increase of beta-galactosidase activity over the background level. beta-Galactosidase activity increased with increasing concentrations of the chemical, attained a maximum at a concentration which was different for different nitrofurans used, and then gradually decreased with a further increase of the nitrofuran concentration. The umu gene expression test revealed that the genotoxic activity was highest for furazolidone and lowest for 5-nitro-2-furaldehyde.


Mutagenic activity of dichloroethylamino derivatives of nitronaphthofuran and some nitrobenzofurans in the Salmonella/microsome assay.

The mutagenic activity of five dichloroethylamino 2-nitrobenzofuran derivatives and one dichloroethylamino 2-nitronaphthofuran derivative was analysed in the Salmonella/microsome assay. We investigated the influence of the position of the dichloroethylamino and/or the methoxy groups on the mutagenic activity of these nitro arenofurans in S. typhimurium strain TA100 and its variant TA100NR, deficient in nitroreductase. Without metabolic activation 7-[bis(2-chloroethyl)amino]-2-nitronaphtho[2,1-b]furan (1), 4-[bis(2-chloroethyl)amino]-7-methoxy-2-nitrobenzofuran (2), 7-[bis(2-chloroethyl)amino]-4-methoxy-2-nitrobenzofuran (5) and 6-[bis(2-chloroethyl)amino]-2-nitrobenzofuran (6) are mutagenic in TA100, while 4-[bis(2-chloroethyl)amino]-5-methoxy-2-nitrobenzofuran (4) is weakly mutagenic and 5-[bis(2-chloroethyl)-amino]-2-nitrobenzofuran (3) toxic. In the NR deficient strain compounds 1, 3 and 6 are strong mutagens and 4 is weakly positive. The two isomers 2 and 5 are negative in that strain. The naphthofuran derivative 1 is highly mutagenic in the absence of S9 mix in both strains considered, but less than R7000 (7). A decrease in the electronic polarity of compound 1 versus compound 7 according to the hypothesis developed by Royer et al. is a possible explanation. After exogenous metabolic activation by S9 mix all the compounds tested are highly mutagenic in both Salmonella strains. The position of the dichloroethylamino group and/or the presence of a methoxyl on the alpha-nitroarenofuran derivatives seem to modify the activity of bacterial as well as exogenous nitroreductases or other activating enzymes.


Chromosome aberrations reduced in whole-body irradiated mice by pretreatment with cyanide.

Male mice exposed to single, whole-body 60Co irradiation, were injected intraperitoneally with a non-toxic dose of KCN, 2 min or 20 min prior to irradiation. Bone-marrow cells were examined for chromatid breaks and chromosome aberrations (CA) at different times post-irradiation. The 2 min but not the 20 min treated mice had a marked reduction in chromatid breaks and chromosome aberrations. A study was made of mice exposed to 3.0 Gy (1.8 Gy/min), treated with KCN 2 min prior to irradiation and examined 5 min to 30 d post-irradiation. After 5 min there were no significant changes in frequency of CA. Subsequently, the incidence of CA in the KCN-treated group was reduced compared to the irradiated controls. By the 30th day, however, CA frequencies had returned to control levels in all groups. No effect of KCN treatment was observed in the white or red blood cells. The cytogenetic results were posited to be a function of the relative inhibition and recovery times of cyanide affected cytochrome oxidase, DNA synthesis, and ATP.


Insect sex chromosomes, XI. 3H-TdR induces random aberrations in the X chromosome(s) of Gryllotalpa fossor (Orthoptera).

The pattern of titrated thymidine (3H-TdR), a direct precursor of DNA, induced aberrations on the X chromosome of Gryllotalpa fossor was examined. 3H-TdR produced aberrations randomly distributed over the entire length of the X chromosome; breaks were observed in both the eu- and the heterochromatic arms of the X chromosome in both the sexes. Since the eu- and the heterochromatic arms cannot be distinguished cytologically in this insect, the presence of aberrations on both arms of the same X chromosome in the male and damage to both X chromosomes in the female indicate that both euchromatic and heterochromatic regions (facultative or constitutive) are equally liable to aberrations induced by H-TdR. This is in contrast to the non-random induction of aberrations by 3H-UdR, which causes chromosome damage due to the proximity of the labeled RNA to the DNA template during transcription.


Inhibition of mutagenesis by p-aminobenzoic acid as a nitrite scavenger.

Nitrite treatment enhances the direct-acting mutagenicity of various foodstuffs (e.g., chicken, bloater, the soybean flour 'kinako', and Ban-Ban-Chi sauce) on Salmonella typhimurium TA100. p-Aminobenzoic acid (PABA) and glutathione (GSH) reduced this mutagenicity; on the other hand, thioproline (TPRO) increased it. PABA seemed more effective than TPRO in scavenging nitrite ion. In analysis of the reactions of PABA and sodium nitrite under acidic conditions (pH 3.0), p-hydroxybenzoic acid (PHBA) was identified as a major reaction product. The reaction seems to involve two steps, diazotization and diazonium substitution. PHBA was not mutagenic to four strains (TA97, TA98, TA100 and TA102) of S. typhimurium with or without S9 mix.


Ethylene dibromide: negative results with the mouse dominant lethal assay and the electrophoretic specific-locus test.

Ethylene dibromide (1,2-dibromoethane; EDB) was tested for the induction of dominant lethal and electrophoretically-detectable specific-locus mutations in the germ cells of DBA/2J male mice. Males were treated with a single intraperitoneal injection of 100 mg/kg EDB and mated to two C57BL/6J females. In the dominant lethal assay, matings were carried out to measure the effect of EDB on meiotic and postmeiotic stages; germ cells representing spermatogonial stem cells were analyzed in the electrophoretic specific-locus test. Neither of these germ cell tests produced any evidence that EDB is a germ cell mutagen. It appears from these data and those reported in the literature that EDB, a genotoxic carcinogen that affects male fertility in some mammalian species, is not mutagenic in the germ cells of the male mouse.


Cytotoxicity of tin on human peripheral lymphocytes in vitro.

The comparative effects of inorganic and organic tin compounds on chromosomes were assessed in human peripheral blood lymphocytes of healthy donors 20-40 years of age. The endpoints observed were chromosomal abnormalities, sister-chromatid exchanges (SCEs) and cell cycle kinetics. The maximum concentrations which reduced the replicative index by about 50%, of stannic chloride and trimethyltin chloride were 40 micrograms and 2 micrograms per culture respectively. The tested doses were 20 micrograms and 10 micrograms of stannic chloride and 1 microgram and 0.5 microgram of trimethyltin chloride. Both doses of stannic chloride induced a much higher frequency of chromosomal abnormalities (P less than 0.05-P less than 0.001) and a greater reduction of cell cycle kinetics than the corresponding relative doses of trimethyltin chloride. The frequencies of SCEs/cell induced by the latter were, however, slightly higher than those induced by the former.


Low temperature between conditioning and challenge treatment prevents the 'adaptive response' of Vicia faba root tip meristem cells.

When the temperature during intertreatment time (2 h) between conditioning and challenge treatment of Vicia faba root tip meristems with either triethylenemelamine or maleic hydrazide was reduced from 24 degrees C to 12 degrees C no adaptive response occurred any more. The yield of metaphases with chromatid aberrations under these circumstances was similar to that observed after challenge treatment alone, i.e., no reduction occurred. This indicates that the metabolic state of the cells is of critical importance for the presence or absence of adaptive responses.


Increased mutagenicity of some nitroimidazoles by non-mutagenic nitrotoluene on Klebsiella pneumoniae (fluctuation test).

In the fluctuation test the mutation frequency of Klebsiella pneumoniae by the 5-nitroimidazoles panidazole and dimetridazole was increased by adding the non-mutagenic substances 4-nitrotoluene or toluene-4-sulfonamide. This effect was not found with 1-methyl-5-nitroimidazole, metronidazole, ronidazole, nimorazole and 1-methyl-2-hydroxymethyl-5-nitroimidazole. It is suggested that the molecules of panidazole or dimetridazole form some association, which is destroyed by 4-nitrotoluene or toluene-4-sulfonamide, thus increasing the concentration of mutagenic particles.


Simultaneous analysis of chromosome damage and aneuploidy in cytokinesis-blocked V79 Chinese hamster lung cells using an antikinetochore antibody.

A modified antikinetochore antibody technique was established in the V79 Chinese hamster lung cells to simultaneously analyze chromosome damage and aneuploidy induced by various agents. The method involved sequential treatment of slides with crest serum, fluoresceinated goat-antihuman and swine-antigoat antibodies, and propidium iodide. In this method, cytoplasm (green), nuclei or micronuclei (red), and kinetochores (yellow), are identified using the same filter setting under blue excitation (440-490 nm) with a barrier filter at 520 nm. Using this method, three agents, vinblastine (VB), X-rays, and methyl methanesulfonate (MMS) were tested for micronucleus/aneuploidy induction. An aneugen, VB and a clastogen, X-rays, induced predominantly kinetochore positive (K+) and negative (K-) micronucleated binucleate (MNBN) cells, respectively, in a dose-dependent fashion. An alkylating agent, MMS, produced both K+ and K- MNBN cells. These results are comparable with the results reported in the literature on these compounds using various methods and thus demonstrate the usefulness of this assay in distinguishing clastogenicity from aneugenicity.


Solvent extraction efficiencies of mutagenic components from diesel particles.

In order to determine the efficiency of organic solvent extractions of fresh diesel-engine exhaust particles a series of Soxhlet extractions were set up using single and sequential extractions of fresh diesel particles with solvents of increasing polarities. Single extractions were carried out with methylene chloride, methanol, acetone and acetonitrile. A single filter was sequentially extracted with methylene chloride followed by methanol, acetone and acetonitrile. Methylene chloride extracted the most mutagenicity relative to the other three solvents. In addition, methylene chloride extracted 97% of the total extracted mutagenicity from a sequential series of extractions. Therefore, we conclude that of the solvents tested methylene chloride extracts the highest proportion of mutagens from fresh diesel exhaust and little mutagenicity is lost using methylene chloride as the only extraction solvent.


Glycyrrhiza glabra extract as an effector of interception in Escherichia coli K12+.

Glycyrrhiza glabra polar lipid extract contains a number of flavonoids and related chemical compounds. Studies on the effectiveness of Glycyrrhiza glabra polar lipid extract in intercepting reactive molecules generated from the illumination of the photosensitizers rose bengal and phenosafranin indicate that it is effective in preventing cytotoxicity against E. coli K12+ in a dose-related fashion using illuminated rose bengal. Since only a modest scavenging of singlet oxygen generated from phenosafranin is observed, the effects of the extracts are less related to singlet oxygen-mediated oxidation of substrate (type II reactions) than non-singlet oxygen-mediated oxidation of substrate (type I reactions). Elevated levels of glutathione observed in exponentially growing cells of E. coli K12 were also observed.


Genotoxicity of N-acryloyl-N'-phenylpiperazine, a redox activator for acrylic resin polymerization.

N-Acryloyl-N'-phenylpiperazine is a promoter of redox reactions synthesized recently, and proposed as an activator for the polymerization of acrylic resins for biomedical use. The chemical was analyzed for different genotoxicity endpoints, to obtain both information on its possible mutagenic/carcinogenic potential and a model analysis of a tertiary arylamine, which belongs to a class of chemicals commonly used as polymerization accelerators in the biomaterial field. The genotoxicity endpoints considered were: gene mutation in the Salmonella test; structural and numerical chromosome alterations in Chinese hamster V79 cells, evaluated by the micronucleus test together with an immunofluorescent staining specific for kinetochore proteins; in vitro and in vivo DNA damage, evaluated in V79 cells and in mouse liver by the alkaline DNA elution technique. On the whole, the results indicate that N-acryloyl-N'-phenylpiperazine is to be regarded not so much as a DNA-damaging agent, but as a genomic mutagen. Indeed, it was not mutagenic in Salmonella (though its toxicity did not allow testing concentrations over 70 micrograms/plate), and it was weakly positive in inducing chromosomal fragmentation in vitro (one positive, not dose-related, result out of five different doses tested) and in vivo DNA damage (increases in DNA elution rate never doubling control values). The chemical was, however, clearly positive (with dose-dependent effects up to about 25 times the control value) in causing numerical chromosome alterations, at the maximal non-toxic doses.


Effect of the antiarrhythmic agent moricizine on survival after myocardial infarction.

The Cardiac Arrhythmia Suppression Trial (CAST) tested the hypothesis that the suppression of asymptomatic or mildly symptomatic ventricular premature depolarizations in survivors of myocardial infarction would decrease the number of deaths from ventricular arrhythmias and improve overall survival. The second CAST study (CAST-II) tested this hypothesis with a comparison of moricizine and placebo. CAST-II was divided into two blinded, randomized phases: an early, 14-day exposure phase that evaluated the risk of starting treatment with moricizine after myocardial infarction (1325 patients), and a long-term phase that evaluated the effect of moricizine on survival after myocardial infarction in patients whose ventricular premature depolarizations were either adequately suppressed by moricizine (1155 patients) or only partially suppressed (219 patients). CAST-II was stopped early because the first 14-day period of treatment with moricizine after a myocardial infarction was associated with excess mortality (17 of 665 patients died or had cardiac arrests), as compared with no treatment or placebo (3 of 660 patients died or had cardiac arrests); and estimates of conditional power indicated that it was highly unlikely (less than 8 percent chance) that a survival benefit from moricizine could be observed if the trial were completed. At the completion of the long-term phase, there were 49 deaths or cardiac arrests due to arrhythmias in patients assigned to moricizine, and 42 in patients assigned to placebo (adjusted P = 0.40). As with the antiarrhythmic agents used in CAST-I (flecainide and encainide), the use of moricizine in CAST-II to suppress asymptomatic or mildly symptomatic ventricular premature depolarizations to try to reduce mortality after myocardial infarction is not only ineffective but also harmful.


Developmental regulation of NMDA receptor-mediated synaptic currents at a central synapse.

The central nervous system has extraordinary plasticity in early life. This is thought to involve N-methyl-D-aspartate (NMDA) receptors which, along with the non-NMDA receptors, mediate fast excitatory synaptic transmission. Although NMDA receptors may be transiently enhanced early in life, it has not been possible to demonstrate directly a functional change in the NMDA receptor-mediated synaptic response because of the voltage-dependence of the NMDA conductance and the overlapping inhibitory synaptic conductances. Here I report that the duration of evoked NMDA-receptor-mediated excitatory postsynaptic currents (e.p.s.cs) in the superior colliculus is several times longer at early developmental stages compared to that measured in older animals. In contrast, the amplitude of NMDA-receptor-mediated miniature e.p.s.cs does not change during development. The kinetic response of excised membrane patches to a brief activation of NMDA receptors is similar to that of the NMDA e.p.s.c, which suggests that the time course of the NMDA e.p.s.c. in the superior colliculus reflects slow NMDA channel properties as in the hippocampus. Therefore, these data indicate that the molecular properties of NMDA receptors are developmentally regulated and thus may be controlling the ability of synapses to change in early life.


FK-506- and CsA-sensitive activation of the interleukin-2 promoter by calcineurin.

Antigen recognition by the T-cell receptor (TCR) initiates events including lymphokine gene transcription, particularly interleukin-2, that lead to T-cell activation. The immunosuppressive drugs, cyclosporin A (CsA) and FK-506, prevent T-cell proliferation by inhibiting a Ca(2+)-dependent event required for induction of interleukin-2 transcription. Complexes of FK-506 or CsA and their respective intracellular binding proteins inhibit the calmodulin-dependent protein phosphatase, calcineurin, in vitro. The pharmacological relevance of this observation to immunosuppression or drug toxicity is undetermined. Calcineurin, although present in lymphocytes, has not been implicated in TCR-mediated activation of lymphokine genes or in transcriptional regulation in general. Here we report that transfection of a calcineurin catalytic subunit increases the 50% inhibitory concentration (IC50) of the immunosuppressants FK-506 and CsA, and that a mutant subunit acts in synergy with phorbol ester alone to activate the interleukin-2 promoter in a drug-sensitive manner. These results implicate calcineurin as a component of the TCR signal transduction pathway by demonstrating its role in the drug-sensitive activation of the interleukin-2 promoter.


Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation.

The immunosuppressive drugs cyclosporin A (CsA) and FK506 both interfere with a Ca(2+)-sensitive T-cell signal transduction pathway, thereby preventing the activation of specific transcription factors (such as NF-AT and NF-IL2A) involved in lymphokine gene expression. CsA and FK506 seem to act by interaction with their cognate intracellular receptors, cyclophilin and FKBP, respectively (see ref. 11 for review). The Ca2+/calmodulin-regulated phosphatase calcineurin is a major target of drug-isomerase complexes in vitro. We have therefore tested the hypothesis that this interaction is responsible for the in vivo effects of CsA/FK506. We report here that overexpression of calcineurin in Jurkat cells renders them more resistant to the effects of CsA and FK506 and augments both NFAT- and NFIL2A-dependent transcription. These results identify calcineurin as a key enzyme in the T-cell signal transduction cascade and provide biological evidence to support the notion that the interaction of drug-isomerase complexes with calcineurin underlies the molecular basis of CsA/FK506-mediated immunosuppression.


New human gene encoding a positive modulator of HIV Tat-mediated transactivation.

The human immunodeficiency virus-1 (HIV-1) protein Tat is a potent activator of virus gene expression. Tat functions through a sequence known as TAR, located immediately downstream of the transcription start site in the long terminal repeat. Several observations suggest that cellular factors cooperate with Tat in the overall transactivating process. We have isolated a human complementary DNA from the new gene MSS1, which may encode such a cellular factor, by transcomplementation of a yeast sgv1- mutant. The MSS1 protein shares 42% sequence identity with the human TBP-1 protein, which binds Tat in vitro and suppresses Tat-mediated transactivation in vivo (ref. 6). We report here that the levels of HIV activation by Tat correlate with endogenous levels of MSS1 messenger RNA. Furthermore, we provide evidence that expression of MSS1 enhances the Tat-mediated transactivation. Our results suggest that MSS1 has a key role in activation of HIV genes regulated by Tat.


Molecular diversity of the NMDA receptor channel.

Two novel subunits of the mouse NMDA receptor channel, the epsilon 2 and epsilon 3 subunits, have been identified by cloning and expression of complementary DNAs. The heteromeric epsilon 1/zeta 1, epsilon 2/zeta 1 and epsilon 3/zeta 1 NMDA receptor channels exhibit distinct functional properties in affinities for agonists and sensitivities to competitive antagonists and Mg2+ block. In contrast to the wide distribution of the epsilon 1 and zeta 1 subunit messenger RNAs in the brain, the epsilon 2 subunit mRNA is expressed only in the forebrain and the epsilon 3 subunit mRNA is found predominantly in the cerebellum. The epsilon 1/zeta 1 and epsilon 2/zeta 1 channels expressed in Xenopus oocytes, but not the epsilon 3/zeta 1 channel, are activated by treatment with 12-O-tetradecanoylphorbol 13-acetate. These findings suggest that the molecular diversity of the epsilon subunit family underlies the functional heterogeneity of the NMDA receptor channel.


RNA substrate binding site in the catalytic core of the Tetrahymena ribozyme.

In catalysis by group I introns, the helix (P1) containing the RNA cleavage site must be positioned next to the guanosine binding site. We have identified a conserved adenine in the catalytic core that contributes to the stability of this arrangement and propose that it accepts a hydrogen bond from a specific 2'-OH in P1. Such base-backbone tertiary interactions may be generally important to the organization of RNA tertiary structure.


Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen.

Immunization with myelin basic protein (MBP) induces experimental allergic encephalomyelitis (EAE), a prototype of CD4+ T-cell mediated autoimmune disease. In rodents, MBP-reactive T-cell clones are specific for a single, dominant determinant on MBP and use a highly restricted number of T-cell receptor genes. Accordingly, EAE has been prevented by various receptor-specific treatments, suggesting similar strategies may be useful for therapy of human autoimmune disease. Here we report that in (SJL x B10.PL)F1 mice, immune dominance of a single determinant, MBP:Ac1-11, is confined to the inductive phase of EAE. In mice with chronic EAE, several additional determinants of MBP in peptides 35-47, 81-100 and 121-140 recall proliferative responses. Most importantly, reactivity to the latter determinants was also detected after induction of EAE with MBP peptide Ac1-11 alone; this demonstrates priming by endogenous MBP determinants. Thus, determinants of MBP that are cryptic after primary immunization can become immunogenic in the course of EAE. Diversification of the autoreactive T-cell repertoire due to 'determinant spreading' has major implications for the pathogenesis of, and the therapeutic approach to, T-cell driven autoimmune disease.


Protein images obtained by STM, AFM and TEM.

Scanning tunnelling microscopy and atomic force microscopy, one scanning the tunnelling current and the other the repulsive atomic force between same and probe, can give high-quality surface topographies of proteins, which have been difficult to obtain by more conventional methods such as transmission electron microscopy.


The treatment of intracranial malignant gliomas using orally administered tamoxifen therapy: preliminary results in a series of "failed" patients.

In vitro studies have shown that the nonsteroidal antiestrogen tamoxifen can suppress deoxyribonucleic acid synthesis and cell proliferation in cultured human gliomas. This growth suppression is independent of its antiestrogenic properties. Tamoxifen may act through the inhibition of the enzyme protein kinase C, which transduces mitogenic signals from the cell surface to the nucleus. Based on these preclinical studies, we initiated a clinical trial of orally administered tamoxifen, 20 mg twice daily, to patients with recurrent, progressive malignant gliomas who were not candidates for other "failed" protocols, such as brachytherapy. No limits were placed on age, Karnofsky Performance Score (KPS), or expected survival. Thirty-two patients were entered in the study, 29 with a glioblastoma multiforme and 3 with an anaplastic astrocytoma. The mean age of the group was 48 years, and the mean KPS was 65. Median survival of the entire cohort from the onset of tamoxifen therapy was 17 weeks; the median survival of those patients with an initial KPS of 70 or more was 21 weeks. Seven patients survived for more than 6 months with no change in their baseline computed tomographic scans or KPS on tamoxifen, including 2 patients with computed tomographic evidence of regression during the course of therapy. There were no significant patient-reported side effects of the treatment. Three patients had thromboembolic complications during tamoxifen administration. We conclude that tamoxifen can be administered safely to these patients and may show some efficacy against glial neoplasms.


Keloids of the external ear.

Keloid formation is a rare complication of normal scar tissue development, also to the surgeon who performs surgery on the external ear. We are presenting 4 cases of keloid formation which occurred after ear piercing in 2 cases and after otoplasty in another 2 cases. The treatment of keloid formation caused difficulties and is not unequivocal. Therefore we wish to present our experiences as we have seen no cases of keloid formation after otoplasty described in the literature.


Fractionation and antigenic characterization of organelles of Eimeria tenella sporozoites.

Sporozoites of Eimeria tenella were disrupted by sonication and subcellular fractions were separated by sucrose gradient ultracentrifugation. Fractions from gradients were characterized by electron microscopical appearance and their polypeptide and antigenic profiles determined by PAGE and Western blotting with antisera to sporozoites and 1st- and 2nd-generation merozoites. Fractions containing micronemes, rhoptries or membranes showed markedly different polypeptide content and antigenic reactivity. Microneme epitopes were strongly conserved between sporozoites and 2nd-generation merozoites whereas the majority of rhoptry epitopes and many membrane epitopes were sporozoite specific. The only polypeptide of sporozoites which was strongly recognized by antisera raised to 1st generation merozoites was a microneme antigen of molecular weight approximately 100 kDa.


Effects of three anthelmintics on the tegument of Hymenolepis fraterna (Cestoda).

The in vivo effects of the anthelmintics taenifugin, VUFB 14170 and VUFB 15269 on the tegument of Hymenolepis fraterna have been examined by SEM, TEM and cytochemistry. The drugs were given to H. fraterna-infected mice on the 14th day post-infection in a single oral dose of 150, 200 and 200 mg/kg, respectively. By 72 h post-treatment, the drug-induced pathomorphological changes in the tegument indicated that all three drugs had a significant effect. Changes were most pronounced on the brush border and in the intercellular material. On the apical surface, there was blebbing as well as accumulation of membrane fragments over the microthrix tips and erosion of the brush border. The intercellular material was changed in structure, showing increased electron density in some areas and oedema of the intercellular spaces in other areas. There were also fractures of the tegument of variable depth, sometimes reaching to the parenchyma. These results suggest altered tegumental integrity and, occasionally, complete disruption of the selective permeability barrier created by the normal tegument. This suggestion is further supported by the penetration of ruthenium red into some tegumental areas and its distribution into the intercellular spaces, down to the parenchyma. The intrategumental lysosomes also appeared to be significantly activated. There was evidence of autophagy in both distal cytoplasm and tegumental cells. Mature and gravid proglottides were more susceptible to drug damage than those in the anterior strobila and neck.


A small-conductance charybdotoxin-sensitive, apamin-resistant Ca(2+)-activated K+ channel in aortic smooth muscle cells (A7r5 line and primary culture).

A small conductance K+ channel was identified in smooth muscle cells of the rat aortic cell line A7r5 and also in rat aortic smooth muscle cells in primary culture, using conventional single-channel recording techniques. The single-channel conductance shows no rectification, either in the range -70 to +40 mV under asymmetrical conditions (9.1 pS), or in the range -100 to +50 mV in symmetrical 150 mM K+ (37 pS). Channel activity is reversibly inhibited by extracellular application of charybdotoxin, with a concentration of 8 nM producing half-maximal inhibition. It is unaffected by apamin or scyllatoxin. Channel activity depends on the presence of free Ca2+ on the cytosolic face of the membrane, with an activation zone between 0.1 and 1 microM. This small-conductance, charybdotoxin-sensitive, Ca(2+)-regulated K+ channel is activated by vasoconstrictors such as vasopressin and endothelin.


Paradoxical decrease in cytosolic calcium with increasing depolarization by potassium in guinea-pig mesotubarium smooth muscle.

The free intracellular Ca2+ concentration ([Ca2+]i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. [Ca2+]i and force were maximal at a relatively low (30 mM) concentration of extracellular K+ ([K+]o), and declined at 90 and 140 mM K+. Plateau values of both [Ca2+]i and force were higher in the presence of 5.10(-6) M ryanodine, indicating that the sarcoplasmic reticulum (SR) contributes to the decline with depolarization. Force and [Ca2+]i at 90 mM K+ were both lower then the high-K+ solution was applied after a period in 30 mM K+ than after a period in normal solution (5.9 mM K+), consistent with inactivation of Ca2+ channels during prolonged depolarization. Addition of carbachol to the depolarized muscle caused a maintained increase in force without maintained increase in [Ca2+]i. We conclude that the decrease in force at increased [K+]o (the "calcium-potassium paradox") is due to a membrane-potential-mediated decrease in [Ca2+]i and, to a lesser extent, to desensitization of the contractile-regulatory apparatus to Ca2+.


Cross-bridge stiffness in Ca(2+)-activated skinned single muscle fibres.

Tension transients, in response to small and rapid length changes (completed within 40 microseconds), were obtained from skinned single frog muscle fibres incubated in activating solutions with varying concentrations of Ca2+. The first 2 ms of these transients were described by a linear model in which the fibre is regarded as a rod composed of infinitesimally small, identical segments containing a mass, one undamped elastic element and in the case of relaxed fibres two damped elastic elements in series, or in the case of activated fibres three such elastic elements in series. The stiffness of activated fibres, expressed in elastic constants or apparent elastic constants, increased with increasing concentrations of Ca2+. All the damped elastic constants that were necessary to describe the tension responses of activated fibres were proportional to isometric tension. However, the undamped elastic constant did not increase linearly with increasing isometric tension. Equatorial X-ray diffraction patterns were obtained from single frog muscle fibres under similar conditions as under which the tension transients were obtained. The filament spacing (d10) of Ca(2+)-activated single frog muscle fibres decreased with increasing isometric force, whereas the intensity ratio (I11/I10) increased linearly with increasing isometric force. From experiments in which dextran (MW 200,000 Da) was added, it followed that such a change in filament spacing would modify passive stiffness. The d10 value of relaxed fibres decreased and stiffness increased with increasing concentrations of the polymer dextran, whereas I11/I10 remained constant. The relation of stiffness and filament spacing with concentration of dextran was used to eliminate the effect of decreased filament spacing on stiffness of activated fibres. After correction for changes in filament spacing the undamped complicance C1, normalized to tension, was not constant, but increased with increasing isometric tension. If we assume that isometric tension is proportional to the number of force generating cross-bridges, this means that only part of the undamped compliance of activated fibres is located in the cross-bridges.


Determinants for binding of a 40 kDa protein to the leaders of yeast mitochondrial mRNAs.

An abundant yeast mitochondrial 40 kDa protein (p40) binds with high specificity to the 5'-untranslated region of cytochrome c oxidase subunit II (COX2) mRNA. Using mobility shift and competition assays, we show here that purified p40 complexes with the leaders of all eight mitochondrial mRNAs of Saccharomyces cerevisiae. The location of the protein binding site on the different leaders is not conserved with respect to the AUG start codon. In vitro RNA footprint and deletion experiments have been used to define the p40-binding site on the leaders of COX1 and ATP9 mRNAs. Nucleotides at, and near, a single stranded region are protected or exposed for DEPC modification by binding of p40 to these leaders. Removal of this region from the COX1 messenger shows that it is essential for the protein-RNA interaction. While no obvious sequence similarity can be detected between the single stranded regions in different leaders, a nearby helical segment is conserved. A consensus model for p40-RNA interactions is presented and the possible biological function of p40 is discussed.


Extensive phosphorothioate substitution yields highly active and nuclease-resistant hairpin ribozymes.

The catalytic function of the hairpin ribozyme has been investigated by modification-interference analysis of both ribozyme and substrate, using ribonucleoside phosphorothioates. Thiophosphate substitutions in two ribozyme domains were examined by using a novel and highly efficient two-piece ribozyme assembled from two independently synthesized oligoribonucleotides. The catalytic proficiency of the two-piece construct (KM = 48 nM, kcat = 2.3 min-1) is nearly identical to that of the one-piece ribozyme. The two-piece ribozyme is essentially unaffected by substitution with thiophosphates 5' to all guanosines, cytidines, and uridines. In contrast, incorporation of multiple adenosine phosphorothioates in the 5' domain of the ribozyme decreases ribozyme activity by a factor of 25. Modification-interference experiments using ribozymes partially substituted with adenosine phosphorothioate suggest that thiophosphates 5' to A7, A9 and A10 interfere with cleavage to a greater extent than substitutions at other sites within the molecule, but the effect is modest. Within the substrate, phosphorothioate substitution does not directly interfere with cleavage, rather, increasing thiophosphate content decreases the stability of the ribozyme-substrate complex. We describe the construction of a hairpin ribozyme containing dinucleotide extensions at its 5' and 3' ends. Full substitution of this molecule with G and C phosphorothioates results in a ribozyme with greatly enhanced stability against cellular ribonucleases without significant loss of catalytic efficiency.


Competition of aminoacyl-tRNA synthetases for tRNA ensures the accuracy of aminoacylation.

The accuracy of protein biosynthesis rests on the high fidelity with which aminoacyl-tRNA synthetases discriminate between tRNAs. Correct aminoacylation depends not only on identity elements (nucleotides in certain positions) in tRNA (1), but also on competition between different synthetases for a given tRNA (2). Here we describe in vivo and in vitro experiments which demonstrate how variations in the levels of synthetases and tRNA affect the accuracy of aminoacylation. We show in vivo that concurrent overexpression of Escherichia coli tyrosyl-tRNA synthetase abolishes misacylation of supF tRNA(Tyr) with glutamine in vivo by overproduced glutaminyl-tRNA synthetase. In an in vitro competition assay, we have confirmed that the overproduction mischarging phenomenon observed in vivo is due to competition between the synthetases at the level of aminoacylation. Likewise, we have been able to examine the role competition plays in the identity of a non-suppressor tRNA of ambiguous identity, tRNA(Glu). Finally, with this assay, we show that the identity of a tRNA and the accuracy with which it is recognized depend on the relative affinities of the synthetases for the tRNA. The in vitro competition assay represents a general method of obtaining qualitative information on tRNA identity in a competitive environment (usually only found in vivo) during a defined step in protein biosynthesis, aminoacylation. In addition, we show that the discriminator base (position 73) and the first base of the anticodon are important for recognition by E. coli tyrosyl-tRNA synthetase.


Can early bacterial complications of aspiration with respiratory failure be predicted?

We studied the early infectious complications of all children admitted for intensive care over a six-year period who were at high risk of having severe aspiration of gastric or pharyngeal secretions. Patients were only analyzed if they required mechanical ventilation for acute respiratory failure, had a blood culture obtained in the first 48 hours, and survived at least 24 hours. Infections were identified by positive blood cultures. Possible infections were defined as fever (over 38.5 degrees C), abnormal leukocyte count (greater than 10,000 or less than 5000), and a potential pathogen in tracheal secretions. Patients' diagnoses included near-drowning (13), aspirated foreign body (5), observed aspiration of gastric contents in a hospitalized patient (2), and hydrocarbon aspiration (1). Of 21 high-risk patients, five (23.8%) had infections and two (9.5%) had possible infections in the first 48 hours. In contrast, no late infections were seen. Infected patients tended to be older (P less than 0.05). No diagnostic features in the first two days of hospitalization reliably identified those who would develop early infections (P greater than 0.05). Since early life-threatening infection is common and cannot be reliably predicted by clinical signs, we recommend aggressive bacteriologic surveillance and the administration of IV antibiotics on admission to all patients in respiratory failure requiring mechanical ventilation after presumed aspiration of gastric or pharyngeal secretions.


Inducing tricuspid regurgitation as palliation.

We describe a technique for palliative decompression of the hypertensive right ventricle seen in pulmonary atresia with intact ventricular septum (PAIVS), utilizing a bioptome catheter for partial tricuspid avulsion.


Prostate cancer. Finding and managing it.

Despite development of new technology for the diagnosis and treatment of prostate cancer, the age-adjusted mortality rate for the disease has not declined for nearly half a century. Physicians now have the ability to diagnose very small tumors in asymptomatic patients, but it remains to be determined which subset of patients would benefit most from early identification and treatment. Studies that are currently under way hopefully will clarify what screening protocols, if any, actually reduce the mortality and morbidity that are associated with prostate cancer and its treatment. Until such evidence becomes available, screening measures for the general population are unwarranted, with the possible exception of digital rectal examination performed as part of other healthcare services. Prostate-specific antigen (PSA) determination may have a place in screening patients whose family history puts them at increased risk. In patients with symptoms or findings suggestive of prostate cancer, diagnosis and staging can be accomplished more accurately and easily than ever before with a combination of PSA determination, transrectal ultrasound, ultrasound-guided tissue sampling using a biopsy gun, and the judicious use of imaging techniques such as bone scans, magnetic resonance imaging, and computed tomography. New treatment methods are being studied, and the options available, especially for management of disease progression, are expected to increase. Monitoring of treated patients has been greatly facilitated by determination of PSA levels, which are predictive of both long- and short-term prognosis. Although much work remains to be done, we may finally be on the verge of making real progress in control of prostate cancer.


Testicular cancer. Role of primary care physicians in screening and education.

Cure rates for testicular cancer have improved dramatically over the years, but early diagnosis is still essential. Primary care physicians have a responsibility to include testicular examination as part of cancer screening and to educate young male patients about the frequency of testicular cancer in their age-group and on how to perform self-examination. A high index of suspicion and careful bimanual palpation are crucial in evaluation of complaints referable to the scrotum. Prompt urologic consultation must be obtained if a tumor is suspected.


Improved performance in a prenatal screening programme for Down's syndrome incorporating serum-free hCG subunit analyses.

A prenatal screening programme for Down's syndrome potentially detecting 76 per cent of affected pregnancies in the South Australian general population at an amniocentesis rate of 3.9 per cent was designed following analysis of mid-trimester serum samples from 57 women who carried an affected fetus. This equates to one affected pregnancy being detected for 41 chromosomal analyses performed. For the experimental series, 75.4 per cent of affected pregnancies were detected, while 4.1 per cent of control specimens produced estimated risk odds consistent with further action. A maternal risk odds of birth of a Down's syndrome fetus of 1:420 was taken as the decision value, which is the prevalence of Down's syndrome births to 35-year-old mothers in South Australia. This screening performance was achieved by investigating combinations of serum analytes not previously reported and by refining the calculation of maternal risk odds to include selective weighting of indicator analytes. Combination of the measurements of free alpha-subunits and beta-subunits of chorionic gonadotrophin, alpha-fetoprotein, unconjugated oestriol, and placental lactogen was found to be most effective in indicating Down's syndrome fetuses. In all combinations of analytes tested, replacing the measurements of free alpha-subunits and free beta-subunits of chorionic gonadotrophin with the measurement of intact chorionic gonadotropin produced a less effective screen.


Maternal serum alpha-fetoprotein levels and fetal outcome in early second-trimester oligohydramnios.

Early second-trimester oligohydramnios was associated with normal maternal serum alpha-fetoprotein (MSAFP) levels in nine out of 26 cases (35 per cent). Congenital malformations of the fetal urinary tract resulting in fetal anuria were present in nine cases; in seven of them, normal MSAFP levels were measured. In contrast, normal MSAFP levels were established in only 2 out of the 17 cases without fetal malformations. These data suggest that fetal urine is the major source of elevated AFP in the maternal compartment in early second-trimester oligohydramnios. This is further supported by the lack of any relationship between concentrations of MSAFP non-reactive with Concanavalin A, originating mainly from the yolk sac-derived amniotic fluid AFP pool, and the presence of fetal diuresis. Three out of 26 women had experienced early second-trimester oligohydramnios in a previous pregnancy, suggesting the existence of a recurrence risk for this condition.


High levels of maternal serum alpha-fetoprotein and human chorionic gonadotrophins leading to the diagnosis of combined neural tube defect and partial mole.

A case of combined partial mole and neural tube defect is presented. The detection of high levels of both maternal serum (MS) alpha-fetoprotein (AFP) and human chorionic gonadotrophin (hCG) during the second trimester led to the ultrasonic demonstration of anencephaly, omphalocele, and partial mole. This is the first report of combined elevation of MSAFP and MShCG.


Interleukin 4 induces the maturation of idiotype-specific regulatory B cell populations.

CD5+ B cells have been shown to be disproportionately associated with autoimmune diseases and transformation. In many cases, their apparent ability to bypass self-tolerance is manifested by the production of autoantibodies. These observations, plus the hypothesis that CD5+ B cells represent a distinct B cell lineage, encourage studies into the conditions and factors that influence their development. In the present study, we employed a well-established assay for murine CD5+ B cell function, i.e., their ability to augment the responses of IgHb-linked idiotypic determinants on anti-(4-hydroxy-3-nitrophenyl) acetyl (NP) antibody (Nbb) idiotype-bearing CD5- B cells to a T-independent antigen, together with multiple methods of cell surface phenotyping, to evaluate the potential for interleukin (IL) 4 to effect maturation of CD5+ B cells, CD5+, IgM+, Thy-1-, and NPb idiotype-specific cells panned on antibody-coated petri dishes or sorted by flow cytometry from spleen were capable of augmenting NPb idiotypic responses of NP-KLH-primed responder cells to NP-Ficoll. Splenic B cell populations depleted of CD5+ B cells failed to affect idiotype expression even after 2 days in culture, a time when a small percentage of CD5+ B cells appeared to be regenerated. However, idiotype-specific regulatory activity could be restored in CD5- splenic B cell populations by culture for 2 days with recombinant IL-4. Cells responsible for idiotype-specific regulatory activity after culture with IL-4 were in fact CD5+, IgM+, and Thy-1.2- B cells, demonstrating that IL-4 can drive the functional, if not the phenotypic, maturation of splenic B cells associated with the CD5+ B cell lineage. The results illustrate one possible mechanism by which T cells could control the maturation of cells belonging to the CD5+ B cell lineage.


Pericranial healing and the temporalis myo-osseous flap in the rabbit model.

The purpose of this animal study was to determine the rate of revascularization of a temporalis myo-osseous (TMO) flap after pericranial elevation. In 24 rabbits, the right pericranium was raised in entirety through a bicoronal flap at the first operation. The pericranium was then reapproximated in situ. The pericranium was allowed to heal for 1 to 28 days before the second operation. At the second operation, through the same bicoronal flap, right and left temporalis myo-osseous flaps were raised. The left temporalis myo-osseous flap served as a control. Revascularization and viability of the temporalis myo-osseous flaps were studied by using technetium bone scans, india ink injection studies, and histologic study. Results demonstrated that 4 days following pericranial elevation, the temporalis myo-osseous flap is viable and revascularized by the pericranium. Immediate bone scanning and india ink injection showed patent pericranial circulation to the osseous portion of the temporalis myo-osseous flap at 4 days. Histologic study confirmed the viability of the temporalis myo-osseous flap. In conclusion, after pericranial elevation, pericranial healing and revascularization were complete at 4 days. This allowed a viable temporalis myo-osseous flap to be raised successfully at this time.


Stereoselective behavioral effects of Lu 19-005 in monkeys: relation to binding at cocaine recognition sites.

The effects of the monoamine uptake inhibitor Lu 19-005 ((+/-)-trans-3-(3,4-dichlorophenyl)-N-methyl-1-indanamine) and its (+) and (-) enantiomers, Lu 20-042 and Lu 20-043, were compared with those of cocaine and the selective dopamine uptake inhibitor GBR 12909 (1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine) in behavioral and radioligand binding experiments. Behavioral experiments were conducted in groups of squirrel monkeys trained under fixed-interval schedules of reinforcement in which responding was maintained either by presentation of food or by termination of a visual stimulus associated with mild electric shock. Radioligand binding studies were conducted using [3H]CFT and [3H]GBR 12935 to label elements of the dopamine uptake system in caudate-putamen membranes of cynomolgus monkeys. All drugs produced dose-related increases in response rate under the fixed-interval schedules. Lu 19-005, Lu 20-042, and Lu 20-043 had relatively slow onsets (approximately 2 h) and relatively long durations of action, with effects persisting for two or more days following administration. Stereoselectivity was evident in the behavioral effects of the enantiomers of Lu 19-005, with Lu 20-042 being approximately 14 times more potent than Lu 20-043. In radioligand binding experiments, Lu 19-005 and its enantiomers were potent inhibitors of specifically bound [3H]CFT and [3H]GBR 12935. As in behavioral experiments, Lu 20-042 was more potent than Lu 20-043. The degree of stereoselectivity, however, varied with the temperature of the assay medium.(ABSTRACT TRUNCATED AT 250 WORDS)


Impaired acquisition of temporal differentiation performance following lesions of the ascending 5-hydroxytryptaminergic pathways.

Nineteen rats received injections of 5,7-dihydroxytryptamine into the dorsal and median raphe nuclei; 16 rats received sham injections. The rats underwent 50 daily training sessions under an interresponse-time-greater-than-15-seconds (IRT greater than 15 s) schedule of sucrose reinforcement. The lesioned group showed impaired acquisition of temporal differentiation, in that their response rates remained significantly higher and their obtained reinforcement frequencies significantly lower than those of the control (sham-lesioned) group. Comparison of the IRT frequency distributions obtained from the two groups during the last 5 days of training showed that the lesioned group produced a significantly higher proportion of very short IRTs (less than 3 s) than the control group; when these short IRTs were disregarded, the lesioned group displayed a significantly lower mean IRT and a significantly higher coefficient of variation than the control group. The levels of 5-hydroxytryptamine (5HT) and 5-hydroxyindoleacetic acid in the parietal cortex, hippocampus, amygdala, nucleus accumbens and hypothalamus were markedly reduced in the lesioned group, but the levels of noradrenaline and dopamine were not significantly affected by the lesion. The results suggest that destruction of the ascending 5HTergic pathways may reduce animals' capacity to inhibit positively reinforced operant behaviour, and may impair temporal discrimination.


Human umbilical vein endothelial cells synthesize S-protein (vitronectin) in vitro.

S-protein, also named vitronectin, is a multifunctional glycoprotein with molecular weight (MW) of about 75 kDa and a serum concentration of 0.14-0.60 mg/ml. It is synthesized mainly in the liver, but synthesis has also been found in monocytes/macrophages. We used human umbilical vein endothelial cells (EC) which were incubated with agarose beads, an activator of the alternative complement pathway. By radioimmunoassay (RIA) based on monoclonal and polyclonal S-protein antibodies, we detected S-protein on harvested agarose beads. The time-dependent increase in the amount of S-protein was significantly reduced by the presence of cycloheximide (10 micrograms/ml) in the cell cultures. We also found a strong binding of S-protein antibodies to agarose beads preincubated in native serum, which was strongly reduced (70-80%) by inactivation of the alternative complement pathway (50 degrees C, 20 min). Our results show that EC synthesize S-protein in vitro.


Reactivity and self-association in vivo of a human monoclonal IgG rheumatoid factor.

In order to study the pathogenic potential of IgG rheumatoid factor (IgG-RF) we generated a human monoclonal IgG4-RF-producing cell line, OR-1, by Epstein-Barr virus transformation of B cells derived from a healthy donor. Characterization of OR-1 RF specificity demonstrated that this RF binds only to IgG and not to dsDNA or seven different proteins tested. Although both OR-1 RF and C1q bind to the Fc part of IgG, no influence could be observed of OR-1 RF on the complement-fixing potential of heat-aggregated IgG, suggesting that OR-1 RF does not interfere with C1q binding to IgG. This was confirmed by blocking studies which showed that binding of OR-1 RF to IgG could be prevented by Staphylococcal protein A (SpA), but not by C1q. Comparison of OR-1 RF with SpA regarding their ability to bind to IgG derived from different species and human IgG subclasses demonstrated that OR-1 RF and SpA have an identical IgG specificity. The possibility that a structural homology exists between SpA and OR-1 RF was ruled out, however, by using affinity-purified chicken anti-SpA antibodies, which were not able to bind to OR-1 RF. The potential of self-recognition of OR-1 RF in vivo was examined by injecting OR-1 cells in SCID mice. Two months after injection IgG-RF was present in the circulation in monomeric, dimeric and polymeric forms whereas circulating IgG without RF activity, derived from an injected control cell line, was present in the monomeric form only. In vitro studies indicated that IgG-RF is secreted in monomeric form and that polymerization is a concentration-dependent phenomenon. The fact that IgG-RF is able to form immune complexes in vivo indicates that IgG-RF has a pathogenic potential by itself and therefore IgG-RF may play a role in the pathogenesis of RA.


[Assessment of the nephrotoxicity of amikacin in patients with cystic fibrosis].

Cystic fibrosis patients are at risk for nephrotoxic effects of aminoglycosides. Fifteen cystic fibrosis patients were admitted to hospital with 18 acute exacerbations of pulmonary symptoms associated with the isolation of Pseudomonas aeruginosa from sputum. They were treated intravenously with amikacin and ceftazidime for 14 days. Urinary excretion of N-acetyl-beta-D-glucosaminidase and alpha 1-microglobulin, two markers of tubular damage, and of albumin, a marker of glomerular permeability, was studied before and during treatment. Urinary activity of N-acetyl-beta-D-glucosaminidase and excretion of alpha 1-microglobulin was normal before amikacin treatment in approximately two thirds of patients and pathologically increased at the end of the study in 95%. Urinary albumin excretion was always normal before amikacin treatment and failed to increase consistently during treatment. The pattern of urinary protein excretion observed in the study before and during treatment with amikacin indicates a selective tubular toxicity.


Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor.

A 3.5 angstrom resolution electron density map of the HIV-1 reverse transcriptase heterodimer complexed with nevirapine, a drug with potential for treatment of AIDS, reveals an asymmetric dimer. The polymerase (pol) domain of the 66-kilodalton subunit has a large cleft analogous to that of the Klenow fragment of Escherichia coli DNA polymerase I. However, the 51-kilodalton subunit of identical sequence has no such cleft because the four subdomains of the pol domain occupy completely different relative positions. Two of the four pol subdomains appear to be structurally related to subdomains of the Klenow fragment, including one containing the catalytic site. The subdomain that appears likely to bind the template strand at the pol active site has a different structure in the two polymerases. Duplex A-form RNA-DNA hybrid can be model-built into the cleft that runs between the ribonuclease H and pol active sites. Nevirapine is almost completely buried in a pocket near but not overlapping with the pol active site. Residues whose mutation results in drug resistance have been approximately located.


Overlapping but nonidentical binding sites on CD2 for CD58 and a second ligand CD59.

The interaction of the T cell glycoprotein CD2 with one ligand, CD58, contributes to T cell function. We have identified CD59, a glycoprotein with complement-inhibitory function, as a second physiological ligand for CD2. Antibodies to CD59 inhibit CD2-dependent T cell activation in murine T cell hybridomas expressing human CD2. In an in vitro binding assay with purified CD58 and CD59, CD2+ cells bind not only immobilized CD58 but also CD59. With two complementary approaches, it was demonstrated that the binding sites on CD2 for CD58 and CD59 are overlapping but nonidentical. These observations suggest that direct interactions between CD2 and both CD58 and CD59 contribute to T cell activation and adhesion.


Primary yolk sac tumor of the cerebellar vermis: case report.

A rare case of yolk sac tumor in the cerebellar vermis is reported. A 2-year-old boy developed headaches, vomiting, and an unsteady gait. Later a tumor was demonstrated in the medial part of the cerebellum by gadolinium-enhanced magnetic resonance imaging (MRI). The tumor was totally removed, and the surgery was followed by chemotherapy. Soon after surgery the elevated alphafetoprotein (AFP) levels in the serum and cerebrospinal fluid were observed to decrease to normal levels. Three months later enhanced MRI showed a lesion in the vermis without any elevation of AFP, and the lesion turned out to be a granuloma. Six months after the second surgery a tumor recurred that could not be totally removed. Cranial radiotherapy was given together with chemotherapy, which resulted in a decrease of AFP to the normal range. The patient is doing well without any elevation in AFP at 1 year 6 months after onset. Related problems in the diagnosis and treatment of yolk sac tumors are discussed.


Valproic acid-induced spina bifida: a mouse model.

Prenatal exposure to the antiepileptic drug valproic acid (VPA) has been associated with the formation of spina bifida aperta, meningocele, and meningomyelocele in the human. Until now, a direct relationship between VPA application and spina bifida has not been experimentally demonstrated. VPA was known only to induce exencephaly in mice, a defect of the anterior neural tube. Maximal sensitivity toward production of this defect was on day 8 of gestation (plug day = day 0). The closure of the posterior neuropore occurs later in the development of mice than the closure of the anterior neuropore. To investigate whether there is a direct relationship between VPA application during pregnancy and induction of spina bifida in mice, we administered various doses of the drug on day 9 of gestation, at three time intervals (at 0, 6, and 12 hr). This administration of VPA produced spina bifida aperta and spina bifida occulta in mice. High doses of VPA (3 x 450 and 3 x 500 mg/kg) induced a low rate of spina bifida aperta in the lumbosacral region. High incidences of spina bifida occulta, a less serious form of spina bifida, were induced with lower doses. This malformation was demonstrated in double-stained fetal skeletons by measurements of the distance between the cartilaginous ends of each vertebral arch. The occurrence of this defect and its localization was dose-dependent. The lumbar region was affected by all doses investigated (3 x 300, 3 x 350, 3 x 400, 3 x 450, and 3 x 500 mg/kg). The sacral/coccygeal region was affected additionally, but with higher doses (3 x 400, 3 x 450, and 3 x 500 mg/kg). A comparison of the results obtained with day 16 and 17 control fetuses showed that the pattern of gaps present in the lumbar and sacral region of the spinal cord in treated groups was drug-specific and not related to a developmental delay. Our results indicate that multiple administrations of VPA on day 9 of gestation in mice result in a low incidence of spina bifida aperta and a high incidence of spina bifida occulta, and provides a relevant model for the study of human spina bifida defects.