Effect of cyclic AMP and cytochalasin B on tissue cultured melanophores of Xenopus laevis

The effects of cytochalasin B or low concentrations of adenosine 3′,5′-monophosphate (cyclic AMP) were tested on melanophores in hanging drop preparations of neural fold explants from Xenopus laevis embryos in Barths' solution. After one week in culture, the melanophores were punctate in this medium. Cyclic AMP at 5 mM consistently caused reversible morphological transformation of these cells to the stellate state, whether they were situated within an epithelial outgrowth or isolated on the surface of the coverglass. Only the isolated melanophores consistently responded to 1 mM cyclic AMP. Cytochalasin B at 1–10 μg/ml caused aggregation of melanin granules in stellate cells, but left long, narrow cell branches containing some melanosomes. Its effect was at least partially reversible and appeared to be dose dependent. At 1% concentration, dimethyl sulfoxide caused melanin dispersion.     

Characterization of epithelial cell cultures derived from human tracheal glands

Summary Cultures of normal human tracheal gland epithelial cells that exhibit functional differentiation have been propagated in serum-free medium supplemented with insulin (5 μg/ml), epidermal growth factor (10 ng/ml), hydrocortisone (0.5 μg/ml), and bovine pituitary extract (25 μg/ml). The cells retain many characteristics of epithelial cells including microvilli on cell surfaces, desmosomes between cells, and tonofilaments in the cytoplasm. In addition, they exhibit keratin-positive titers and react positively with Peanut agglutinin, which is specific for the disaccharide β-d-galactose-(l→ 3)N-acetyld-galactosamine, a major component of mucin glycoprotein. The cells also exhibit normal Cl⁻ channel activity which was enhanced by the cAMP agonist Forskolin. The major component of the cellular secretion was hyaluronic acid; approximately 10% of the void volume material was resistant to hyaluronidase and may contain material similar to mucin glycoprotein. Some of the cell cultures have been maintained in serum-free conditions for 6 to 7 passages. This model will be important to study regulation of ion-channel activities and mucous glycoprotein secretion and to compare such regulations with the tracheal mucosal epithelial cells already established. This research was supported by USPHS grants HL 41979 and HL 33142 from the National Heart, Lung and Blood Institutes.     

Inhibition of melanin biosynthesis by cerulenin in appressoria ofColletotrichum lagenarium

Cerulenin [(2S) (3R)2,3-epoxy-4-oxo-7,10-dodecadienoylamide], a polyketide synthesis inhibitor, inhibited appressorial pigmentation ofColletotrichum lagenarium at concentrations higher than 10 μg/ml. The inhibitory concentrations of cerulenin inhibited neither spore germination nor appressorium formation but did inhibit penetration of nitrocellulose membranes by penetrating hyphae from appressoria. The colorless appressoria germinated laterally on nitrocellulose membranes and rarely penetrated them. In the presence of cerulenin, treatment with scytalone, a melanin precursor for this fungus, restored appressorial pigmentation and also penetration by appressoria of nitrocellulose membranes. In Czapek liquid medium, mutant 8015 which is defective in the enzyme involved in the conversion of scytalone to 1,3,8-trihydroxynaphthalene in the melanin biosynthetic pathway produced scytalone after 9 days of incubation when hyphal growth reached the maximum level. Application of 10 μg/ml of cerulenin at 9 days of incubation inhibited further production of scytalone by mutant 8015. From these results, it is concluded that appressorial melanin ofC. lagenarium was synthesizedvia the polyketide pathway.     

Effect of melanins from black yeast fungi on proliferation and differentiation of cultivated human keratinocytes and fibroblasts

The effects of melanin preparations from black yeast fungi (BYF) on the proliferation and differentiation of normal cultivated human skin keratinocytes and embryonic pulmonary fibroblasts have been investigated. Melanin preparations in the range of 5-0.1 microg/ml were optimally active, with a more pronounced effect on keratinocyte than on fibroblast proliferation. Of 17 dihydroxynaphthalene (DHN) natural melanin preparations and two commercial dihydroxyphenylalanine (DOPA) melanin preparations, only one preparation--DOPA melanin (of animal origin) significantly stimulated proliferation of keratinocytes at 5 microg/ml; four preparations (DHN melanin from BYF) significantly inhibited proliferation of these cells at 5 or 1 microg/ml. The remaining preparations had no significant effect. Similarly, of the 17 preparations of DHN melanin from BYF, one preparation significantly stimulated fibroblast proliferation, and four significantly inhibited proliferation at 5 microg/ml, one at all the concentrations, and three from 1 down to 0.1 microg/ml. These melanin preparations were also shown to affect the in vitro differentiation of keratinocytes.     

Role of Melanin as a Scavenger of Active Oxygen Species

The protective role of melanin, either synthetic or derived from a metastatic lung melanoma nodule, was studied in terms of its ability to interact with active oxygen species (O₂, H₂O₂, RO, ROO, etc.). Both melanins showed the ability to react with O₂. The superoxide dismutase-like activity corresponds to 21 and 10 U/mg for synthetic and tumor melanin, respectively. The latter value accounts for about 8% of the superoxide dismutase activity of cultured melanoma cells. Neither type of melanin showed catalase-like or glutathione peroxidase-like activity. Both types of melanin reacted with RO and ROO radicals as determined by inhibition of the lipid peroxidation reaction of rat liver homogenates. The spontaneous lipid peroxidation of rat liver homogenate was inhibited up to 90% and 80% by synthetic and tumor melanin with half-maximal effects at 2.5 and 5.5 μg melanin/ml, respectively. The 2,2-azobis-(2 amidino propane) (AAPH)-initiated lipid peroxidation of rat liver homogenate was inhibited up to 3% and 20% by synthetic and tumor melanin, with half maximal effect at 120 and 500 μg melanin/ml, respectively. Both types of melanin were able to protect the in vitro inactivation of glucose oxidase, which occurs in the presence of AAPH-generated radicals.     

Simultaneous Preparation of a DNA Ligase and Restriction Endonucleases from Bacillus amyloliquefaciens N

An N-acetylgalactosamine (GalNAc)-specific Ca²⁺-dependent lectin (C-type lectin), isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), CEL-I, showed potent mitogenic activity toward normal mouse spleen cells. The mitogenic activity of CEL-I, which reached a maximum at 100 μg/ml, was inhibited by GalNAc in a concentration-dependent manner. The mitogenic effect of CEL-I at 10 μg/ml on T cell- enriched splenocytes was at a similar level due to a well-known T cell mitogen, concanavalin A (Con A), at 10 μg/ml. Furthermore, CEL-I evoked a mitogenic response from nude mouse spleen cells, while no significant effects of Con A on this cell population were observed over a wide range of concentrations. These results suggest that CEL-I is a potent mitogenic lectin with the ability to stimulate both T and B cells.     

Puncturevine (Tribulus terrestris L.) affects the proliferation,apoptosis, and ghrelin response of ovarian cells

The objective of our study was to examine the direct effects of the medicinal plant Tribulus terrestris L. (puncturevine) on the basic functions of ovarian cells, including their proliferation, apoptosis, and response to the physiological hormonal stimulator ghrelin. In the first series of experiments, porcine ovarian granulosa cells were cultured with or without puncturevine extracts at concentrations of 0, 1, 10, or 100 μg/ml. In the second series of experiments, these cells were cultured with ghrelin at concentrations of 0, 1, 10, or 100 ng/ml, either alone or in combination with puncturevine (10 μg/ml). The expression levels of the proliferation marker PCNA and the apoptosis marker bax were analyzed via quantitative immunocytochemical methods. Puncturevine was found to stimulate the accumulation of both proliferation and apoptotic markers. Additionally, ghrelin alone could promote the proliferation and apoptosis of ovarian cells. The presence of puncturevine reversed ghrelin-stimulated apoptosis and instead induced apoptotic inhibition. However, puncturevine did not modify the proliferation-inducing effect of ghrelin. These observations demonstrated that (1) puncturevine directly promotes cell proliferation and apoptosis, turnover, of ovarian cells; (2) ghrelin is involved in the regulation of ovarian cell apoptosis and proliferation, consistent with existing evidence; (3) puncturevine antagonizes and even reverses the effects of the hormonal regulator, ghrelin, on ovarian cell apoptosis, but not proliferation; and (4) puncturevine affects not only the basic functions of ovarian cells but also their responses to upstream hormonal regulators.     

Protein interactions with lipid bilayers: The channels of kidney plasma membrane proteolipids

Summary Proteolipids extracted from bovine kidney plasma membrane induce irreversible changes in the electrical properties of lipid bilayers formed from diphytanoyl phosphatidylcholine. The interaction with the proteolipid produces channels which are cation selective. At low protein concentrations (i.e., <0.6 g/ml), the single-channel conductance is approximately 10 pS in 100mm KCl and 3 pS in 100mm NaCl. In the presence of protein concentrations above 1 g/ml, another population of channels appears. These channels have a conductance of about 100 pS in 100mm KCl and 30 pS in 100mm NaCl. Further, these channels are voltage dependent in KCl, closing when the voltage is clamped at values 30 mV. The steady-state membrane conductance, measured at low voltages, was found to increase proportional to a high power (2–3) of the proteolipid concentration present in one of the aqueous phases. In 100mm NaCl, the conductance increases at protein concentrations above 5 g/ml, whereas in 100mm KCl in increases at protein concentrations above 0.6 g/ml. These measurements indicate that the higher steady-state conductance observed in KCl at a given proteolipid concentration in a multi-channel membrane presumably results because more channels incorporate in the presence of KCl than in the presence of NaCl.The two major fractions which comprise the proteolipid complex were also tested on bilayers. It was found that both fractions are required to produce the effects described.     

Melanin decreases clastogenic effects of ionizing radiation in human and mouse somatic cells and modifies the radioadaptive response

Melanin’s influence on the chromosome aberration frequency induced by radiation in human lymphocytes and mouse bone marrow cells has been studied. We revealed earlier that melanin significantly decreases the frequencies of different radiation-induced mutations in animal germ cells. Melanin protection in somatic cells has been found to be less effective. The melanin effect in somatic cells depends on radiation dose: the lower the damage level, the better the melanin protection. In order to determine the influence of melanin at low radiation doses, the adaptive response was investigated in mouse bone marrow cells in vivo. The level of chromosome aberrations in these cells after fractionated irradiation of 0.2 Gy+1.5 Gy with a 4-h interval was about half that after a single dose of 1.7 Gy. If melanin was injected prior to irradiation, the aberration level decreased by a factor of about two in both cases. This observed result may be due to the potential radioprotective effect of melanin and to the absence of any adaptive response, whereas in the case of melanin application between the priming and challenge doses, the combined effect of the adaptive response as well as melanin protection resulted in a 4-fold decrease of chromosome aberrations. These results allow us to draw the following conclusions: adaptive response can be prevented by a radioprotector such as melanin, and melanin is capable of completely removing low-dose radiation effects. Received: 2 December 1998 / Accepted in revised form: 15 September 1999     

Action of two juvenile hormone antagonists on lepidopteran epidermis

The juvenile hormone antagonist ETB (ethyl-4-2(t-butylcarbonyloxy)-butoxybenzoate) caused formation of precocious larval-pupal intermediates after the 4th (penultimate)-larval instar of the tobacco hornworm, Manduca sexta, when 50 μg were applied to any 3rd stage larvae or to 4th stage larvae within 12 hr after ecdysis. This dose was most effective within 12 hr after ecdysis to the 3rd stage. In the black mutant larval assay for juvenile hormone, ETB had activity, 0.75 μg per larva giving half-maximal score. In vitro ETB acted as a juvenile hormone to prevent the ecdysteroid-induced change in commitment at concentrations above 0.1 μg/ml with an ED₅₀ at 2.8 μg/ml and as a partial juvenile hormone antagonist to 0.1 μg/ml juvenile hormone I at concentrations between 10^?3 and 10^?2 μg/ml. By contrast, EMD (ethyl-E-3-methyl-2-dodecenoate) had little juvenile hormone-like activity in vitro up to its limits of solubility (100 μg/ml) and exhibited sporadic partial juvenile hormone antagonistic activity in vitro at concentrations between 1 and 100 μg/ml. Since these concentrations were 10–1000 times that of juvenile hormone I in the medium, EMD apparently is not an efficient competitor.     

Hyaluronidase activity of macaque sperm assessed by an in vitro cumulus penetration assay

A model system consisting of cynomolgus macaque sperm and ovulated hamster ova-cumulus complexes (OCCs) was utilized to study the role of the sperm protein PH-20 in cumulus penetration. The hyaluronidase activity of solubilized macaque sperm PH-20 was evaluated using an ELISA-like microplate assay prior to and following the addition of the hyaluronidase inhibitors heparin (0–100 μg/ml) and apigenin (250 μM), as well as the Ig fraction of a polyclonal antibody raised against purified recombinant macaque PH-20 (R10; 10–400 μg/ml). Sperm motility following exposure to enzyme inhibitors was evaluated using computer-aided sperm motility analysis. Macaque sperm were labeled with the permeant fluorescent nuclear dye, Hoechst 33342, and were coincubated with ovulated hamster OCCs for 30 min at 37°C. The addition of heparin, apigenin, or R10 antibody to solubilized sperm extracts resulted in a linear dose-dependent decrease in hyaluronidase activity (P < .01). In the heterologous cumulus penetration assay, fluorescently labeled macaque sperm that were pretreated with heparin (1–100 μg/ml), apigenin (250 μM), or R10 antibody (Ig fraction, 10–400 μg/ml) demonstrated a dose-dependent decrease in the ability to penetrate hamster OCCs (P < 0.01), in the absence of effects on sperm motility. In the homologous assay, experiments using macaque OCCs and fluorescently labeled macaque sperm confirmed that the same concentrations of heparin and R10 antibody similarly suppressed spermatozoal cumulus penetration (P < .01). These results suggest that macaque sperm PH-20-derived hyaluronidase participates in cumulus penetration in this species, and that this model system is useful for further studies into primate gamete interaction. Mol. Reprod. Dev. 46:392–400, 1997. © 1997 Wiley-Liss, Inc.     

阿魏酸钠对人动脉平滑肌细胞和内皮细胞作用的机制研究

目的:研究阿魏酸钠(SF)对人主动脉平滑肌(HASMC)和内皮细胞(HAEC)的影响,探讨SF成为抑制支架内再狭窄药物的机制。方法:HASMC和HAEC经SF处理后(0-1000μg/ml),用CCK-8试剂和划痕愈合试验检测不同药物浓度对两种生长和细胞迁移能力的影响;采用免疫细胞化学和Western blot检测HAECs中FoxM1和VEGF的表达。结果:SF对两种细胞的作用呈剂量依赖性,SF在10-1000μg/ml浓度时,HASMC的生长活力明显降低,在0.1-100μg/mlHAECs生长活力显著增强(P0.05)。在1-1000μg/ml浓度下HASMCs迁移能力受到抑制,HAECs的迁移能力明显增加(0.1-100μg/ml)(P0.05)。同时HAECs内FoxM1和VEGF表达明显增高(P0.05),程度与SF浓度有剂量依赖关系。结论:阿魏酸钠能抑制血管平滑肌的增值和迁移;同时增加内皮细胞FoxM1和VEGF的表达,促进内皮细胞的增值和迁移,这些特征使其可能成为抑制支架内再狭窄的药物。     

槲皮素对人脑胶质瘤干细胞生物学行为及miR-29s家族的影响分析

目的探讨分析槲皮素对人脑胶质瘤干细胞（BGSCs）生物学行为及miR-29s家族的影响。 方法使用干细胞培养液对U87人脑胶质瘤细胞进行培养,采用CCK-8法检测槲皮素对BGSCs细胞增殖抑制率,采用流式细胞技术检测槲皮素对BGSCs细胞凋亡影响,并采用real-time PCR鉴定槲皮素对BGSCs细胞中miR-29a、miR-29b以及miR- 29c表达的影响。采用t检验以及方差分析进行统计学分析。 结果随着槲皮素浓度的增加,BGSCs细胞增殖抑制率增加24 h 0 μg/ml（0.00±0.12）%、10 μg/ml（1.36±0.38）%、20 μg/ml（15.33±3.01）%、40 μg/ ml（29.50±4.57）%、80 μg/ml（40.21±6.42）%、160 μg/ml（61.21±7.48）,F = 76.273,P < 0.05;48 h 0 μg/ml （0.09±0.05）%、10 μg/ml（9.84±2.17）%、20 μg/ml（28.57±3.84）%、40 μg/ ml（43.59±5.21）%、80 μg/ml（59.50±3.28）%、160 μg/ ml（70.21±9.48）%,F = 85.392,P < 0.05,且同浓度槲皮素作用48 h对BGSCs细胞增殖抑制率高于作用24 h（P < 0.05）。随着槲皮素浓度的升高,BGSCs细胞凋亡率升高[0 μg/ml（13.42±1.21）%、20 μg/ml（38.47±9.28）%、40 μg/ml（59.34±7.20）%、80 μg/ml（71.42±9.47）%,F = 57.493,P < 0.05]。不同浓度槲皮素处理BGSCs细胞后,可促进BGSCs细胞miR-29s家族miR- 29a/ b/ c相对表达量,且随着槲皮素浓度的增加,BGSCs细胞miR-29s家族相对表达量增加[miR-29a 0 μg/ml（1.04±0.08）、20 μg/ml（1.16±0.05）、40 μg/ml（1.30±0.10）、80 μg/ ml（1.41±0.09）,F = 19.281,P < 0.05;miR-29b 0 μg/ml（1.06±0.09）、20 μg/ml（1.13±0.05）、40 μg/ml（1.25±0.07）、80 μg/ml（1.30±0.09）,F = 13.427,P < 0.05;miR-29c 0 μg/ml（1.03±0.07）、20 μg/ml（1.15±0.03）、40 μg/ml（1.22±0.06）、80 μg/ml（1.31±0.08）,F = 14.502,P < 0.05]。 结论槲皮素可有效抑制人脑BGSCs增殖,促进人脑BGSCs凋亡,并促进人脑BGSCs中miR- 29s家族表达。     

Cytogenetic effects of a food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and its metabolite, 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), on human and Chinese hamster cells in vitro

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced structural chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) in human lymphocytes and human diploid fibroblasts (TIG-7) at concentrations above 12.5 μg/ml in the presence of rat S9 mix. PhIP also elevated the frequencies of SCEs in human lymphocytes in the presence of rat S9 at concentrations above 2.0 μg/ml with dose-dependency. A proximate form of metabolites of PhIP, 2-hydroxyamino-1-methyl -phenylimidazo[4,5-b]pyridine (N-OH-PhIP), caused CAs in human and Chinese hamster fi fibroblast cells in the absence of S9 mix at concentrations above 0.75 μg/ml and 1.25 μg/ml, respectively, which were 10 times lower than the effective concentration of PhIP. No marked differenceswere observed in the cytogenetic sensitivity to N-OH-PhIP between human and Chinese hamster cells, except between lymphocytes obtained from different donors.     

Influence of serum on in situ proliferation and genotoxicity in A549 human lung cells exposed to nanomaterials

In this work in situ proliferation of A549 human lung epithelial carcinoma cells exposed to nanomaterials (NMs) was investigated in the presence or absence of 10% serum. NMs were selected based on chemical composition, size, charge and shape (Lys-SiO(2), TiO(2), ZnO, and multi walled carbon nanotubes, MWCNTs). Cells were treated with NMs and 4h later, cytochalasin-B was added. 36 h later, cell morphology was analyzed under a light microscope. Nuclearity was scored to determine the cytokinesis-block proliferation index (CBPI). CBPI, based on percentage of mono-, bi- and multi-nucleated cells, reflects cell toxicity and cell cycle delay. For some conditions depending on NM type (TiO(2) and MWCNT) and serum concentration (0%) scoring of CBPI was impossible due to overload of agglomerated NMs. Moreover, where heavy agglomeration occurs, micronuclei (MN) detection and scoring under microscope was prevented. A statistically significant decrease of CBPI was found for ZnO NM suspended in medium in the absence or presence of 10% serum at 25 μg/ml and 50 μg/ml, respectively and for Lys-SiO(2) NM at 3.5 μg/ml in 0% serum. Increase in MN frequency was observed in cells treated in 10% serum with 50 μg/ml ZnO. In 0% serum, the concentrations tested led to high toxicity. No genotoxic effects were induced by Lys-SiO(2) both in the absence or presence of serum up to 5 μg/ml. No toxicity was detected for TiO(2) and MWCNTs in both 10% and 0% serum, up to the dose of 250 μg/ml. Restoration of CBPI comparable to untreated control was shown for cells cultured without serum and treated with 5 μg/ml of Lys-SiO(2) NM pre-incubated in 100% serum. This observation confirms the protective effect of serum on Lys-SiO(2) NM cell toxicity. In conclusion in situ CBPI is proposed as a simple preliminary assay to assess both NMs induced cell toxicity and feasibility of MN scoring under microscope.     

Effects of the peptide toxin from Microcystis aeruginosa on intracellular calcium,pH and membrane integrity in mammalian cells

Extracts of water blooms of the toxic cyanobacterium Microcystis aeruginosa showed a range of toxicities not related to their ability to lyse mammalian red cells. The HPLC-purified heptapeptide toxin (mol. wt. 1035) from Microcystis did not lyse red cells at up to 500-fold higher concentrations than that required to kill mice. This toxin (LD₅₀ 110 μg/kg for male mice) was used to investigate in vitro effects on isolated thymocytes, hepatocytes, mammary alveolar cells, and cultured Swiss 3T3 fibroblasts. Thymocytes were stimulated to progressive Ca²⁺ entry by toxin (0.1–10 μg/ml), reaching a peak after approx. 5 min. No deformation, intracellular pH change, Trypan Blue entry or cell lysis was seen within 60 min at 37°C. Hepatocytes were grossly deformed by the toxin, with a dose/response relationship between 0.1 and 1.0 μg/ml. No progressive Ca²⁺ entry was observed on toxin addition, instead a rapid rise in intracellular Ca²⁺, presumably from intracellular sources. No change in intracellular pH, Trypan Blue exclusion or cell lysis was observed over 60 min. Mammary alveolar cells and 3T3 fibroblasts were unresponsive to toxin at the concentrations tested. No change in protein synthesis or nucleic acid synthesis in thymocytes was observed after culture with 0.5 or 5.0 μg/ml toxin. It was concluded that cytoskeletal changes in deformed hepatocytes (the target cells in vivo) demonstrated the most probable cellular basis for toxicity, rather than changes in membrane permeability or cell metabolism.     

Optimal conditions for blastogenic response of peripheral blood lymphocytes to T and B cell mitogens in cynomolgus monkeys

Optimal conditions for the mitogen-induced proliferation of T and B lymphocytes of cynomolgus monkeys were determined. The T cell mitogens concanavalin A and phytohemagglutinin, at concentrations of 1.25–10 μg/ml and 1.25–10 μg/ml, respectively, and the T and B cell mitogen pokeweed mitogen, at concentrations of 0.2–10 μg/ml, induced high lymphoproliferative responses, the average stimulation index (SI) being 34–93. Since suitable mitogens have not been reported for monkey B cells, we tested three types of lipopolysaccharide (LPS): two derived from Escherichia coli—one extracted with phenol and one extracted with trichloroacetic acid (TCA); and one derived from Salmonella typhimurium, extracted with phenol. All three LPS had a high mitogenic effect for monkey lymphocytes, with SI of 2.3–6.4. The highest response was observed for 25 μg/ml of Salmonella LPS, and the addition of trypsin to the culture augmented the proliferative response of low or non-responder lymphocytes. © 1994 Wiley-Liss, Inc.     

Role of cadherins 5 and 13 in the aortic endothelial barrier

We investigated the role of the cadherins 5 and 13 in the solute barrier formed by aortic endothelial cells in vitro. In confluent monolayers of bovine aortic endothelial cells, immunofluorescence with antibodies to the external domain of cadherin 5 (Mab 9H7) or to cadherin 13 (Mab Ec6C10) found staining for both cadherins at endothelial cell borders. Western blotting with an antibody to the characteristic cadherin cytoplasmic tail or with an antibody to the extracellular domain of cadherin 5 revealed a single 125 kD protein band. A second larger band was found at 130 kD with the anti-cadherin 13 Mab which was not recognized by an antibody to the cadherin cytoplasmic tail. A calcium switch strategy was used to investigate the involvement of these cadherins in the endothelial barrier. Changes in the permeability of small solutes in an endothelial cell column produced by a decrease in calcium concentration followed by a return to normal calcium, with or without antibody, were recorded. We found that anti-cadherin 5 IgG (10 μg/ml) interfered with the reforming of interendothelial junctions after restoration of calcium at every time point tested for a total of 45 min after restoration of calcium. The anti-cadherin 13 IgG (10 μg/ml) did not block reforming of the endothelial barrier in a similar manner. The presence of this antibody delayed only by 15 min the restoration of the normal barrier. Without calcium switch, addition of either monoclonal antibody (10 μg/ml) to the endothelial cell column had no effect on solute permeability. These results suggest that cadherin 5 in bovine aortic endothelial cells has a major functional role in forming the calcium-sensitive endothelial junction in vitro and may play an important role in the normal structure and function of the in vivo barrier. J. Cell. Physiol. 171:243–251, 1997. © 1997 Wiley-Liss, Inc.     

Effects of β-bungarotoxin and taipoxin on contractions of canine airways caused by nerve stimulation

β-Bungarotoxin and taipoxin are snake venoms that have been reported to induce neuromuscular blockade exclusively in somatic motor neurons by a presynaptic mechanism. Taipoxin (1 μg/ml), but not β-bungarotoxin (1 μg/ml), depressed the contractile response of canine airway smooth muscle to electrical stimulation. Taipoxin (1 μg/ml) also slightly depressed the contractile activity of canine airways to two concentrations (5 × 10^?6M and 10^?5M) of exogenously administered acetylcholine. We conclude that taipoxin, but not β-bungarotoxin, induces a weak neuromuscular blockade in the parasympathetic fibers innervating canine airways. The sites of this inhibition are believed to be both presynaptic and postsynaptic.     

Efficacy of Novel Antimicrobials Against Clinical Isolates of Opportunistic Amebas

ABSTRACT We examined the effects of the macrolide antimicrobial agent azithromycin and phenothiazine compounds against clinical isolates of Acanthamoeba spp. and Balamuthia mandrillaris , opportunistic pathogens of human beings and other animals. Acanthamoeba growth was inhibited in vitro at 1,5, and 10 μg/ml of azithromycin, but not the macrolides, erythromycin, and clarithromycin. In experiments attempting to simulate in vivo conditions, azithromycin protected monolayers of rat glioma cells from destruction by Acanthamoeba at a concentration of 0.1 μg/ml, and delayed destruction at concentrations of 0.001 and 0.01 μg/ml. We concluded that the minimal inhibitory concentration of azithromycin was 0.1 μg/ml. Our results, however, suggested that the drug was amebastatic but not amebicidal, since ameba growth eventually resumed after drug removal. The phenothiazines (chlorpromazine, chlorprothixene, and triflupromazine) inhibited Acanthamoeba growth by 70-90% at 5 and 10 μg/ml, but some of these compounds were toxic for rat glioma cells at 10 μg/ml. Azithromycin was not very effective against B. mandrillaris in an in vitro setting, but was amebastatic in tissue culture monolayers at concentrations of 0.1 μg/ml and higher. Balamuthia amebas showed in vitro sensitivity to phenothiazines. Ameba growth was inhibited 30-45% at 5 μg/ml in vitro, but completely at 5 μg/ml in the rat glioma model. In spite of their potential as antiamebic drugs in Balamuthia infections, toxicity of phenothiazines limits their use in clinical settings.