Inhibition of human angiogenin by DNA aptamers: nuclear colocalization of an angiogenin-inhibitor complex

Specific ligands (aptamers) for angiogenin were selected from a 72-mer oligodeoxynucleotide library consisting of 28 randomized positions flanked by two constant regions of 22 residues each. From a starting pool of approximately 10(14) molecules, 19 angiogenin-binding ligands were obtained. Among them, two oligonucleotides showed significant inhibition of the ribonucleolytic activity of angiogenin with apparent Kis of 0.65 and 0.60 micro M, respectively. One of them was shortened on the basis of its secondary structure to provide a 45-mer oligonucleotide that retained much of the inhibitory properties of the parent molecule. It inhibits both the angiogenic and cell proliferative activities of angiogenin but does not interfere with its nuclear translocation in human endothelial cells. Importantly, the inhibitor is cotranslocated to the nucleus with angiogenin in a approximately 1:1 stoichiometric ratio. These results demonstrate that the inhibition of angiogenin-induced cell proliferation and angiogenesis by the oligonucleotide is due to suppression of the ribonucleolytic activity of angiogenin, an event that occurs most likely within the cell nucleus.     

Inhibition of fos-jun-DNA complex formation by dihydroguaiaretic acid and in vitro cytotoxic effects on cancer cells

The main cause of acquired inguinal hernia is weakness of Fruchaud's deep muscolofascial floor, following metabolically-determined collagen disorders. A technique for the anterior reinforcement of this structure with polypropylene mesh is described here. Following intermuscular decollement, the mesh is placed in direct contact with the surface formed by the transversalis fascia and the transversus abdominis muscle and stretched as extensively as possible. Because the posterior aspect of the inguinal canal is the true barrier to abdominal pressure, the author believe that its direct reinforcement, without interposition of the internal oblique muscle, constitutes the most correct anatomo-surgical approach to hernia repair. This is the case for both indirect hernias, in which the internal ring is reconstructed at a deeper level, and for direct hernias, in which the "tent effect" of the prosthesis is prevented. Ninety-two primary inguinal hernias (56 indirect, 29 direct and 7 direct and indirect) in 87 patients were repaired with this technique. Seventy-nine patients were followed up from 2 to 24 months. Early complications included: 7 ecchymosis, 3 seromas, 2 subcutaneous infections, 3 testicular swellings. Incision and testicular pain for longer than 6 months occurred in 2 cases. No prosthetic infections or recurrences have been detected up to the present.     

Inhibition of acid sialidase by inorganic sulfate

Sulfated glycosaminoglycans are known to inhibit mammalian acid-active sialidase. Although the inhibition depends clearly on the presence of sulfate groups on these macromolecules, there was no information on the intrinsic inhibitory potency of inorganic sulfate. In this study, we demonstrate that inorganic sulfates inhibit acid-active Mu-Neu5Ac sialidase of U937 cells. This inhibition was found to be reversible and it appeared to be of the mixed competitive type. Sulfate-induced inhibition was also observed in other cells as well as with other substrates such as sialyl lactose and bovine mixed brain gangliosides. We conclude that the intrinsic inhibitory potency of sulfate groups may be significantly involved in the inhibition of acid-active sialidase by sulfated glycosaminoglycans. In addition, inorganic sulfate by its apparent potency to selectively inhibit acid sialidases might constitute an interesting tool for the characterisation of the minor forms of sialidases occurring in mammalian cells.     

Pretreatment with intraventricular aurintricarboxylic acid decreases infarct size by inhibiting apoptosis following transient global ischemia in gerbils

The goal of this study was to determine whether aurintricarboxylic acid (ATA), an endonuclease inhibitor known to inhibit apoptosis, could ameliorate cell damage in a gerbil model of transient ischemia. Transient ischemia was induced in gerbils by bilateral carotid artery occlusion for a period of 5 minutes. Four micrograms of ATA was administered intraventricularly 1 hour before ischemia, and the brains were assessed histologically 1 week later to quantitate cell loss in the vulnerable CA-1 subsector of the hippocampus. In a separate set of experiments, 4 microg of ATA was administered intraventricularly 1 hour before ischemia and the brains were assessed for evidence of DNA fragmentation by the TUNEL method. There was only a 16% cell loss compared with nonischemic controls in animals pretreated with ATA that was significantly less (p < 0.05) than the 48% cell loss in animals pretreated with saline alone. TUNEL-positive cells were first evident at 3 days and were still present at 7 days subsequent to ischemia. Maximal staining occurred at 4 days. Pretreatment with ATA virtually eliminated TUNEL staining at 4 days. These results support the hypothesis that the delayed cell death secondary to transient ischemia is, in part, apoptotic. Furthermore, ATA afforded significant neuronal protection and prevented DNA fragmentation.     

Amino acid uptake by fetal kidney slices

Kidney slices of 7-17-week-old human fetuses were incubated for 90 min or less in Krebs-Ringer solution containing aminoisobutyric acid (AIB). Cell to medium ratios of AIB decrease with increasing fetal age. Uptake rate is slower than for mature human tissue. Both water content and extracellular space is high in embryonic tissue as compared to adult.     

Inhibition of high affinity uptake of GABA by branched fatty acids

Several branched fatty acids including an antiepileptic agent nDPA were tested as potential inhibitors of high affinity uptake of GABA by brain slices and synaptosomes. Only three compounds (2-butyl-3-propylhexanoic acid, 5-propyloctanoic acid, 2-propylpenten-2-oic acid) were found to be relatively weak inhibitors of the uptake system. There was no correlation between anticonvulsant properties of the branched fatty acids and their potencies as inhibitors of high affinity uptake of GABA.     

Inhibition of uptake of transferrin-bound iron by human hepatoma cells by nontransferrin-bound iron

The liver acquires iron from transferrin by transferrin receptor-mediated (TR) and transferrin receptor-independent pathways (NTR) and from nontransferrin-bound iron (NTB-Fe). Iron uptake by the NTR processes involves an iron-carrier mediated step. Experiments, using human hepatoma cells (HuH7) transfected with TR antisense (sense for control) RNA expression vectors to suppress TR expression, were performed to examine the effect of unlabeled NTB-Fe as iron citrate on the uptake of 59Fe-125I-transferrin. This was to determine if the uptake of transferrin-bound iron (Tf-Fe) and NTB-Fe uptake is mediated by a common iron-carrier. Iron citrate inhibited the uptake of 59Fe-transferrin (2.5 micromol/L Fe) in a concentration-dependent manner with a maximum effect when the citrate-iron:Tf-Fe molar ratio was 10:1. Transferrin uptake was not affected. At a lower Tf-Fe concentration of (0.125 micromol/L) when uptake of iron is TR-mediated, a 10-fold molar excess of iron citrate had no effect on Tf-Fe uptake by HuH7 TR antisense and sense cells. However, at a higher Tf-Fe concentration (2.5 micromol/L), when uptake occurs mainly by the NTR-mediated process, there was a 40% reduction in the membrane-bound and intracellular uptake of iron. Iron citrate did not affect the maximum rate (Vmax) of Tf-Fe uptake but the Michaelis-Menten constant (Km) for Tf-Fe uptake by the NTR-mediated process was increased, indicating there was competitive inhibition of Tf-Fe uptake by iron citrate. These results suggest that the uptake of NTB-Fe and Tf-Fe by the NTR- mediated process occurs by the same cellular pathway, using a common iron-carrier.     

Inhibition of homologous recombination by the plasmid MucA'B complex

By its functional interaction with a RecA polymer, the mutagenic UmuD'C complex possesses an antirecombination activity. We show here that MucA'B, a functional homolog of the UmuD'C complex, inhibits homologous recombination as well. In F- recipients expressing MucA'B from a Ptac promoter, Hfr x F- recombination decreased with increasing MucA'B concentrations down to 50-fold. In damage-induced pKM101-containing cells expressing MucA'B from the native promoter, recombination between a UV-damaged F lac plasmid and homologous chromosomal DNA decreased 10-fold. Overexpression of MucA'B together with UmuD'C resulted in a synergistic inhibition of recombination. RecA[UmuR] proteins, which are resistant to UmuD'C inhibition of recombination, are inhibited by MucA'B while promoting MucA'B-promoted mutagenesis efficiently. The data suggest that MucA'B and UmuD'C contact a RecA polymer at distinct sites. The MucA'B complex was more active than UmuD'C in promoting UV mutagenesis, yet it did not inhibit recombination more than UmuD'C does. The enhanced mutagenic potential of MucA'B may result from its inherent superior capacity to assist DNA polymerase in trans-lesion synthesis. In the course of this work, we found that the natural plasmid pKM101 expresses around 45,000 MucA and 13,000 MucB molecules per lexA(Def) cell devoid of LexA. These molecular Muc concentrations are far above those of the chromosomally encoded Umu counterparts. Plasmid pKM101 belongs to a family of broad-host-range conjugative plasmids. The elevated levels of the Muc proteins might be required for successful installation of pKM101-like plasmids into a variety of host cells.     

Inhibition of 3H-1-norepinephrine uptake by ouabain is species dependent

Tissue slice to medium ratios of 3H-1-norepinephrine (3H-1-NE) were used to study the effect of ouabain on uptake of norepinephrine. The effects of ouabain were studied in slices of heart and spleen from three different species: rat, guinea pig, and dog. The drug produced a species as well as a concentration dependent inhibition of norepinephrine uptake in both types of tissue. In order of decreasing sensitivity, the following relationship between species was observed: dogs greater than guinea pigs greater than rats. Since 3H-1-norepinephrine uptake under the present experimental conditions represents uptake into sympathetic nerve terminals, it was concluded that Na+-K+-ATPases of sympathetic nerve terminals have species dependent differences in ouabain sensitivity similar to those of myocardium.     

Inhibition of the corrosion of steel by acid dihydrazides

The corrosion inhibitory properties of homologous series of acid dihydrazides were studied. Gasometry, potentiodynamic polarization and impedance measurements were recorded for mild steel specimens immersed in 1.0 M H₂SO₄ or a mixture of 1.0 M H₂SO₄ + 1.0 M Na₂SO₄ having the same ionic strength with a pH range from zero to 7. Effect of presence of different concentrations of oxalic, malonic, succinic or pimelic dihydrazide on the corrosion rate of steel in the above solutions were investigated. The results indicated that the protection efficiency of the acid dihydrazides increased with the increase of the number of the methylene groups in the acid dihydrazide molecule. The results are discussed on the basis of the change of each, of the electron density of the donating atoms, of the inhibitor molecules, and the orientation of the inhibitor at the metal surface with the molecular structure of the inhibitor.     

Inhibition of irritation and contact hypersensitivity by ethacrynic acid

The immunosuppressive effect of topical ethacrynic acid (ECA) was tested on both the induction and elicitation phases of contact sensitization in a mouse model. ECA (0.5% in vehicle) reduced the sensitization response by >50% when the sensitizer was either dinitrochlorobenzene (DNCB), oxazalone (OX) or para-phenylenediamine (PPD), and was applied 1 day later to the ECA-pretreated skin site. The immunosuppressive effect of combining ECA with either hydrocortisone or with cis-urocanic acid was also tested. An additive suppressive effect was observed with ECA in both combinations. The effect of ECA (1% in vehicle) on blocking the elicitation phase was also examined in a mouse ear edema assay. ECA was highly effective in preventing the challenge response in mice previously sensitized to either DNCB, OX or PPD. ECA (1% in vehicle) was also tested for its ability to inhibit contact irritation. ECA (1% in vehicle) was highly effective in preventing ear edema due to topically applied skin irritants including arachidonic acid, capsaicin, lactic acid, phorbol myristate acetate, trans-retinoic acid, and sodium lauryl sulfate. ECA may be useful for both prophylaxis and therapeutic treatment of diverse skin conditions including contact dermatitis, eczema, and other related allergic skin disorders.     

Inhibition of herpes simplex virus replication by retinoic acid

Superior laryngeal nerve (SLN) stimulation can activate the brainstem swallowing mechanism to produce a complete swallowing sequence consisting of oropharyngeal, oesophageal and lower oesophageal sphincter (LOS) components. However, little is known of the effect of SLN stimulation (peripheral-sensory input from the pharynx) on the characteristics of oesophageal motor activity, especially in the smooth muscle portion. The present study examined the effect of varying stimulus train length and frequency on each of the three components of the reflex. Acute studies were performed in urethane anaesthetized cats. Oesophageal motility was monitored using conventional manometric techniques, and oropharyngeal swallowing by the mylohyoid electromyogram. SLN stimulus train length (1-10 sec) and frequency (5-30 Hz) were varied independently. Increased train length or frequency resulted in (1) an increase in oropharyngeal swallowing and incidence of the complete swallowing response, (2) an increase in latency to onset of the oesophageal peristaltic wave, (3) reduction of the amplitude of the evoked peristaltic contraction in the smooth muscle portion, without altering its velocity, (4) increased LOS relaxation, and increased LOS after-contraction. The LOS contraction was abolished by atropine (100 micrograms kg-1). Therefore, increased SLN stimulation not only results in excitation of the central swallowing program and the oropharyngeal stage of swallowing, but has major effects on the oesophageal and LOS stages of swallowing. Afferent SLN stimuli can impact on the control mechanisms for each stage, to inhibit or excite the stages in different ways.     

Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates

Leucine-rich nuclear export signals (NESs) are recognized by the NES receptor exportin 1 and are central to the export of multiple shuttling proteins and RNAs. The export of messenger RNA in vertebrates was, however, thought to occur by a different pathway, because inhibition by injection of a synthetic Rev NES conjugate could not be demonstrated. Here we find that peptide conjugates composed of the NES of either protein kinase A inhibitor protein (PKI) or the HIV-1 Rev protein, when coupled to human serum albumin, are potent inhibitors of mRNA and small nuclear RNA export. These results provide direct evidence that mRNA export in vertebrates depends on interactions between an NES and its cognate NES receptors. PKI NES conjugates are significantly more efficient at inhibiting RNA export than are REV NES conjugates, indicating that different NESs may have different abilities to promote protein and RNA export. Surprisingly, an expected control conjugate containing the mutant Rev NES sequence M10 strongly inhibited the export of intronless dihydrofolate reductase mRNA. Nuclear injection of NES peptide conjugates led to mislocalization to the nucleus of 10-20% of the cytoplasmic Ran GTPase-binding protein (RanBP1) indicating that RanBP1 shuttles between the nucleus and the cytoplasm via an NES pathway. These results demonstrate that in vertebrates the export of mRNA, like that of small nuclear RNA, 5S rRNA, and transport factors such as RanBP1, employs NES-mediated molecular machinery.     

Inhibition of platelet serotonin uptake by cytochrome P450 inhibitors miconazole and econazole

Serotonin uptake in human platelets was inhibited by cytochrome P450 inhibitors such as miconazole and econazole but not clotrimazole. There was a correlation between inhibition of serotonin uptake and inhibition of imipramine binding, suggesting that these P450 inhibitors may inhibit serotonin uptake via direct binding to the transporter. P450 inhibitor effects on serotonin uptake did not seem to be related to the effects of these compounds on intracellular calcium mobilization. Additionally, nitric oxide pathway stimulation does not appear to be involved.     

Mechanism of valproic acid uptake by isolated rat brain microvessels

In an effort to characterize putative transport systems of valproic acid (VPA) at the blood-brain barrier, the effects of various substrates and inhibitors of known anion transporters on the equilibrium vessel-to-medium concentration (vessel/medium) ratio of VPA were investigated using isolated rat brain microvessels. The equilibrium vessel/medium ratio of VPA was decreased by the presence of high millimolar concentration of unlabeled VPA, indicating that a saturable transport system was involved in VPA transport from medium to microvessels. Short-chain monocarboxylates such as propionic acid, pyruvic acid, and L-lactic acid did not alter the vessel/medium ratio, whereas medium-chain fatty acids and unsaturated metabolites of VPA significantly inhibited the net transport of VPA. Dicarboxylates, tricarboxylate, and p-aminohippuric acid did not affect VPA accumulation in the brain microvessels. Several anionic drugs including salicylic acid, penicillin G, cefazolin, and probenecid significantly reduced the vessel/medium ratio of VPA. In addition, disulfonate inhibitors of inorganic anion exchangers, SH-group modifying reagent, and metabolic inhibitor showed remarkable inhibitory effects on the net transport of VPA between brain microvessels and medium. These results suggest that VPA may be actively transported through the antiluminal membrane via a carrier-mediated system shared by other anionic drugs.     

Inhibition in the human urinary bladder by gamma-amino-butyric acid

OBJECTIVE: To investigate the effects of gamma-amino-butyric acid (GABA) on detrusor activity in man to determine whether it has any inhibitory effect on detrusor contraction. The inhibitory neurotransmitter GABA has been found in mammalian urinary bladders and the effects of GABA on detrusor activity in the rabbit bladder has previously been described [1]. MATERIALS AND METHODS: Human detrusor muscle strips, obtained at cystectomy, were made to contract by electrical stimulation of their autonomic nerves or by the addition of carbachol in a superfusion apparatus. GABA and its analogues were added to the superfusion chamber and any changes in the responses were measured. RESULTS: The electrically evoked nerve-mediated contractions in human bladder muscle were exclusively cholinergic. GABA inhibited nerve-mediated contractions in human detrusor muscle-strips by the activation of the GABAB receptor, since baclofen (a GABAB receptor agonist) produced similar inhibition and muscimol (a GABAA receptor agonist) did not. There was no inhibition of carbachol-mediated contractions by GABA. CONCLUSION: This in vitro study shows that GABA has a peripherally mediated inhibitory effect on excitatory neurotransmission in human detrusor muscle. The site of action is on the post-ganglionic nerves and appears to be mediated via the GABAB receptor.