Radiolabeling of EGCG with <Superscript>131</Superscript>I and biodistribution in rats

Epigallocatechin gallate (EGCG), is the most abundant and widely studied catechin in green tea (Camellia sinensis Theaceae). The inhibitory effects of EGCG and green tea extract on carcinogenesis in various organs in rodents have now been demonstrated over the past decade. The aim of study was to label EGCG with I-131, to determinate its structure and to evaluate its biodistribution in Wistar rats. Radiolabeling was carried out by direct electrophilic iodination method (iodogen) and yield was determined by radio thin layer chromatography (RTLC). Radiolabelling yield is determined as 89 ± 1.0%. Besides, determination of structure of iodinated molecule, serum stability, and partition coefficient experiments was performed. The structure analysis of synthesized cold ¹²⁷I-EGCG complex was assessed with LC–MS–MS and ¹H-NMR. ¹H-NMR and LC–MS–MS results of iodinated EGCG (¹²⁷I-EGCG) show that oxidize iodine reacts electrophilic with aromatic ring. Serum stability results showed that in vitro stability of ¹³¹I-EGCG was quite high. It is observed that labeling percentage decreased 83 ± 2% at 24th, Partition coefficient results show that the partition coefficient of EGCG was calculated as theoretical partition coefficient = 2.04 ± 0.42 and the experimental partition coefficient of ¹³¹I-EGCG was found as 1.46 ± 0.2. The biodistribution data shown that the maximum uptake of the radioiodinated EGCG was seen in lung and pancreas at 30 min. The blocking assay results indicated that the uptake of ¹³¹I-EGCG in lung was not significantly change (0.25, 0.23, and 0.22%ID/g at 30, 60, and 150 min, respectively). Biodistribution data showed no significant uptake in a specific organ of the rat. Hence radiolabeled EGCG is seen in some organs (lung, liver, pancreas, kidney, etc.).     

Sodium <Superscript>131</Superscript>i-mercaptododecaborate biodistribution in B-16 melanoma in mice

The biodistribution of sodium mercaptododecaborate labeled with radioactive iodine (¹³¹I-BSH) in melanoma B-16 and surrounding tissues in mice has been studied for administration by various methods including intraperitoneal, single and double intratumor injection, and introduction under tumor bed. It is shown that a high content of ¹³¹I-BSH in the tumor is reached in all cases. The maximum accumulation of the boron compound is observed 1 h after administration. In this case the ratio of radioactivities in melanoma B-16 and surrounding tissues in most animals was greater than 3. A study of ¹³¹I-BSH uptake in tumor cells showed that a considerably greater accumulation of the compound is observed 3 and 6 h after intraperitoneal administration in the intercellular space (65.3% and 63.0%, respectively) in comparison to the cellular content (34.7% and 37.0%, respectively). The levels of accumulation become identical in about 12 h after administration. Approximately equal uptake of the boron compound in the intercellular space and tumor cells was observed over the entire period of investigation (0.5–2 h) after a single intratumor administration of ¹³¹I-BSH. The results of this study suggest that the intratumor administration of boron compounds is promising for neutron capture therapy, especially in combination with neutron teletherapy.     

Examination of the structure of agaricinic acid using <Superscript>1</Superscript>H and <Superscript>13</Superscript>C NMR spectroscopy

Agaricinic acid was extracted from the carpophore of Fomitopsis officinalis (Vill. Bond. et Sing). Its structure was established by ¹H and ¹³C NMR spectroscopy and comparison to a standard sample (Sigma—Aldrich). The extraction was carried out using ethanol. The obtained extract was evaporated, cooled (−5°C), and purified by ether. The structure of unbranched alkyl radicals was determined using their proton spectrum. The ¹³C NMR spectrum and two-dimensional {¹³C,¹H} correlation diagram showed the presence of a hydroxyl (second quaternary C atom) and three types of carboxy groups (first, second, and third C atoms). The spectrum of the extracted sample is identical to that of the standard sample of agaricinic acid.     

Effects of gabapentin and pregabalin on K<Superscript>+</Superscript>-evoked <Superscript>3</Superscript>H-GABA and <Superscript>3</Superscript>H-glutamate release from human neocortical synaptosomes

One site of action of the anticonvulsant, analgesic, and anxiolytic drugs gabapentin and pregabalin is the α₂δ-subunit of voltage-sensitive Ca²⁺ channels (VSCC). We therefore analyzed the effects of gabapentin and pregabalin on K⁺-evoked release of ³H-γ-aminobutyric acid (GABA) and ³H-glutamate from superfused human neocortical synaptosomes. These neurotransmitters are released by Ca²⁺-dependent exocytosis and by Ca²⁺-independent uptake reversal. When a GABA transport inhibitor was present throughout superfusion to isolate exocytotic conditions, gabapentin and pregabalin (100 μM each) reduced K⁺-evoked ³H-GABA release by 39% and 47%, respectively. These effects were antagonized by the α₂δ-ligand l-isoleucine (1 μM) suggesting the α₂δ-subunit of terminal VSCC to mediate the reduction of exocytosis. Both drugs had no effect on exocytotic ³H-glutamate release and also failed to modulate the release of ³H-GABA and ³H-glutamate caused by reversed uptake in the absence of external Ca²⁺. Thus, an inhibition of glutamate release by gabapentin and pregabalin as main anticonvulsant principle is not supported by our experiments. An anticonvulsant mode of action of both drugs may be the reduction of a proconvulsant exocytotic GABA release.     

Synthesis and biological characterization of <Superscript>123</Superscript>I-labeled 15-(<Emphasis Type="Italic">p</Emphasis>-iodophenyl)-3-methylpentadecanoic acid

Experimental batches of iodine-123-labeled beta-methyl iodophenylpentadecanoic acid (¹²³I-BMIPP) with an activity of 180 MBq/mL have been produced based on ¹²³I extracted from enriched ¹²²Te targets irradiated by accelerated deuterons at the R-7M cyclotron. It is shown that the radiochemical purity of ¹²³I-BMIPP in EtOH solutions exceeds 96% at an acceptable content of the main chemical impurities (Cu, Te). The conditions of ¹²³I-BMIPP dilution in human serum albumin (10%) solution for injection in experimental animals are determined. The biological behavior of the preparation in rats has been studied. It is established that ¹²³I-BMIPP is characterized by pronounced accumulation in the heart. A qualitative scintigram of the rabbit heart was obtained and showed the possibility of external detection. It is established that it is possible to judge the viability of myocardium by measuring the amount of ¹²³I-BMIPP accumulated in the heart.     

Inhibitory effect of YM-244769, a novel Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger inhibitor on Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchange current in guinea pig cardiac ventricular myocytes

Recently, YM-244769 (N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide) has been reported as a new potent and selective Na⁺/Ca²⁺ exchange (NCX) inhibitor by using various cells transfected with NCX using the ⁴⁵Ca²⁺ fluorescent technique. However, the electrophysiological study of YM-244769 on NCX had not been performed in the mammalian heart. We examined the effects of YM-244769 on NCX current (I_NCX) in single cardiac ventricular myocytes of guinea pigs by using the whole-cell voltage clamp technique. YM-244769 suppressed the bidirectional I_NCX in a concentration-dependent manner. The IC₅₀ values of YM-244769 for the bidirectional outward and inward I_NCX were both about 0.1 μM. YM-244769 suppressed the unidirectional outward I_NCX (Ca²⁺ entry mode) with an IC₅₀ value of 0.05 μM. The effect on the unidirectional inward I_NCX (Ca²⁺ exit mode) was less potent, with 10 μM of YM-244769 resulting in the inhibition of only about 50 %. At 5 mM intracellular Na⁺ concentration, YM-244769 suppressed I_NCX more potently than it did at 0 mM [Na⁺]_i. Intracellular application of trypsin via the pipette solution did not change the blocking effect of YM-244769. In conclusion, YM-244769 inhibits the Ca²⁺ entry mode of NCX more potently than the Ca²⁺ exit mode, and inhibition by YM-244769 is [Na⁺]_i-dependent and trypsin-insensitive. These characteristics are similar to those of other benzyloxyphenyl derivative NCX inhibitors such as KB-R7943, SEA0400, and SN-6. The potency of YM-244769 as an NCX1 inhibitor is higher than those of KB-R7943 and SN-6 and is similar to that of SEA0400.     

Development of <Superscript>67</Superscript>Ga-labeled streptokinase

Radiolabeled streptokinase can be used in the imaging of thrombi in many cardiovascular diseases. Streptokinase was successively labeled with [⁶⁷Ga]-gallium chloride using cyclic DTPA-dianhydride followed by biodistribution studies in wild-type and thrombosis-induced mice using scarification and SPECT. The radiochemical purity if the tracer was checked using HPLC >95 and RTLC >99% followed by SDS-PAGE to check the protein integrity. The biodistribution studies were performed in normal (up to 167 h) and thrombosis-earing rats (2 h) using scarification and preliminary SPECT studies (up to 2 h). ID/g% and SPECT techniques demonstrated the specific binding of the tracer in the heart and aorta 2 h post-injection. The use of Ga-68 radiosiotope can lead to superior imaging results with respect to the fast accumulation of the tracer and ⁶⁸Ga half-life (68 min).     

Effects of trimebutine maleate on colonic motility through Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channels and L-type Ca<Superscript>2+</Superscript> channels

The effects of trimebutine maleate (TM) on spontaneous contractions of colonic longitudinal muscle were investigated in guinea pigs. The contractile responses of smooth muscle strips were recorded by an isometric force transducer. Membrane and action potentials were detected by an intracellular microelectrode technique. The whole-cell patch clamp recording technique was used to record the changes in large conductance Ca²⁺-activated K⁺ (BK_ca) and L-type Ca²⁺ currents in colonic smooth muscle cells. At high concentrations (30, 100, and 300 μM), TM inhibited the amplitude of spontaneous contractions. At low concentrations (1 and 10 μM), TM attenuated the frequency and tone of smooth muscle strips, whereas TM had no influence on the amplitude of spontaneous contractions. TM depolarized the membrane potentials, but decreased the amplitude and frequency of action potentials at high concentrations. TM inhibited BK_ca and L-type Ca²⁺ currents in a dose-dependent manner. In the presence of the BK_ca channel opener, NS1619, TM also inhibited BK_ca currents. Bayk8644, a L-type Ca²⁺ channel opener, increased L-type Ca²⁺ currents. This augmentation was also attenuated by TM. These results suggest that TM attenuates intestinal motility through inhibition of L-type Ca²⁺ currents, and depolarizes membrane potentials by reducing BK_ca currents. Thus, TM may be a multiple-ion channel regulator in the gastrointestinal tract.     

Differential modulation of K<Superscript>+</Superscript>-evoked <Superscript>3</Superscript>H-neurotransmitter release from human neocortex by gabapentin and pregabalin

Anticonvulsant, analgesic, and anxiolytic effects have been observed both in preclinical and clinical studies with gabapentin (GBP) and pregabalin (PGB). These drugs appear to act by binding to the α₂δ subunit of voltage-sensitive Ca²⁺ channels (VSCC), resulting in the inhibition of neurotransmitter release. In this study, we examined the effects of GBP and PGB (mostly 100 μM, corresponding to relatively high preclinical/clinical plasma levels) on the release of neurotransmitters in human neocortical slices. These slices were prelabeled with ³H-dopamine (³H-DA), ³H-choline (to release ³H-acetylcholine (³H-ACh)), ³H-noradrenaline (³H-NA), and ³H-serotonin (³H-5-HT), and stimulated twice in superfusion experiments by elevation of extracellular K⁺ in the presence and absence of GBP and PGB. The α₂δ ligands produced significant inhibitions of K⁺-evoked ³H-ACh, ³H-NA, and ³H-5-HT release between 22% and 56% without affecting ³H-DA release. Neither drug reduced ³H-NA release in the presence of l-isoleucine, a putative α₂δ antagonist. Interestingly, this antagonism did not occur using the enantiomer, d-isoleucine. These results suggest that GBP and PGB are not general inhibitors of VSCC and neurotransmitter release. Such α₂δ ligands appear to be selective modulators of the release of certain, but not all, neurotransmitters. This differential modulation of neurotransmission presumably contributes to their clinical profile. A preliminary report of this work was presented at the German Society for Experimental and Clinical Pharmacology and Toxicology, Mainz, Germany, March 13–15, 2007 (Brawek et al. 2007).     

Magnesium isoglycyrrhizinate inhibits L-type Ca<Superscript>2+</Superscript> channels,Ca<Superscript>2+</Superscript> transients,and contractility but not hERG K<Superscript>+</Superscript> channels

To explore the cardiovascular protective effects of Magnesium isoglycyrrhizinate (MI), especially the underlying cellular mechanisms related to L-type calcium channels and myocardial contractility, and to examine the effects of MI on hERG K⁺ current expressed in HEK293 cells. We used the whole-cell patch clamp technique, video-based edge detection and dual excitation fluorescence photomultiplier systems to explore the effect of MI on L-type Ca²⁺ currents (I_Ca-L) and cell contraction in rat cardiomyocytes. We also examined the rapidly activating delayed rectifier potassium current (I_Kr) expressed in HEK293 cells using a perforated patch clamp. MI inhibited I_Ca-L in a dose-dependent manner, with a half-maximal inhibitory concentration (IC₅₀) of 0.22 mg/ml, and the maximal inhibitory effect was 61.10 ± 0.59%. MI at a concentration of 0.3 mg/ml reduced cell shortening by 24.12 ± 3.97% and the peak value of the Ca²⁺ transient by 36.54 ± 4.96%. MI had no significant influence on hERG K⁺ channels expressed in HEK293 cells at all test potentials. MI exerts protective effects on the heart via the inhibition of I_Ca-L and cell shortening in rat cardiomyocytes. However, MI had no significant influence on I_Kr; thus, MI may exert cardioprotective effects without causing drug-induced long QT syndrome.     

Abnormal activation of Na<Superscript>+</Superscript>-K<Superscript>+</Superscript> pump in aortas from rats with endotoxaemia

A diminished reactivity to several vasoconstrictor agents is usually observed in blood vessels obtained from animals with endotoxic shock. The contractile state of vascular smooth muscle is influenced by the activity of the electrogenical sodium (Na⁺-K⁺) pump. Thus, we examined inhibitors and agonists of nitric oxide (NO)-guanosine 3':5'-cyclic monophosphate (cGMP) on contractions to phenylephrine (PE) and relaxations to potassium in isolated aortic segments from rats treated with bacterial endotoxin (lipopolysaccharide, LPS) for 6 h (i.e. to mimic a shock syndrome). Endotoxaemia for 6 h was associated with a severe hypotension and vascular hyporeactivity to noradrenaline and an increased plasma nitrate level in vivo. The PE-induced contraction was attenuated in aortic smooth muscle obtained from rats with endotoxic shock while the potassium-induced relaxation was greater in these preparations. Ouabain dose-dependently inhibited the potassium-induced relaxation in aortas from normal and endotoxaemic rats. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one enhanced the PE-induced contraction in endotoxaemic rats only, whereas it attenuated the difference of potassium-induced relaxation between normal and endotoxaemic rats. In contrast, in aortas obtained from normal rats, 8-bromo-cGMP reduced the PE-induced contraction and enhanced the potassium-induced relaxation to the level as seen in endotoxaemic animals. In aortas obtained from endotoxaemic rats, methylene blue further restored the PE-induced contraction to the normal and abolished the difference of potassium-induced relaxation between normal and endotoxaemic rats. These results suggest that the Na⁺-K⁺ pump in the vascular bed of animals with endotoxic shock is abnormally activated and this augmented activation is modulated by cGMP.     

Roles of Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger isoforms NCX1 and NCX2 in motility in mouse ileum

The Na⁺/Ca²⁺ exchanger (NCX) is a plasma membrane transporter that is involved in regulating intracellular Ca²⁺ concentrations in various tissues. The physiological roles by which NCX influences gastrointestinal motility are incompletely understood, although its role in the heart, brain, and kidney has been widely investigated. In this study, we focused on the functions of the NCX isoforms, NCX1 and NCX2, in the motility of the ileum in the gastrointestinal tract. We investigated the response to electric field stimulation (EFS) in the longitudinal smooth muscle of the ileum obtained from wild-type mice (WT), NCX1-heterozygote knockout mice (NCX1 HET), NCX2 HET and smooth muscle-specific NCX1.3 transgenic mice (NCX1.3 Tg). EFS induced a phasic contraction that persisted during EFS and a tonic contraction that occurred after the end of EFS. We found that the amplitudes of the phasic and tonic contractions were significantly smaller in NCX2 HET, but not in NCX1 HET, compared to WT. Moreover, the magnitudes of acetylcholine (ACh)- and substance P (SP)-induced contractions of NCX2 HET, but not of NCX1 HET, were smaller compared to WT. In contrast, the amplitudes of the phasic and tonic contractions were greater in NCX1.3 Tg compared to WT. Similar to EFS, the magnitude of ACh-induced contraction was greater in NCX1.3 Tg than in WT. Taken together, our findings indicated that NCX1 and NCX2 play important roles in ileal motility and suggest that NCX1 and NCX2 regulate the motility in the ileum by controlling the sensitivity of smooth muscles to ACh and SP.     

Effect of infrared radiation on the pharmacokinetics of 131I-mercaptododecaborate in animals with model tumors

The influence of local IR irradiation of the experimental melanoma B-16 zone on the distribution of sodium mercaptododecaborate labeled with radioactive iodine (¹³¹I-BSH) in mice was studied for two regimes of thermal treatment (before and after administration of the labeled compound). It was established that a considerable increase in the ¹³¹I-BSH content in the model tumor was reached in the case of IR irradiation performed 1 h after drug administration. Based on these data, IR radiation can be considered an effective modifier of ¹³¹I-BSH uptake in tumors, increasing the efficacy of boron-enhanced neutron capture therapy. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 12, pp. 9–11, December, 2005.     

Pharmaceutical Policy and the Lay Public<Superscript>*</Superscript>

Almost every national and supranational health policy document accords high importance to the need to listen to and ‘empower’ patients. The relationship between pharmaceutical policy and the lay public is not direct but mediated by several actors, including health care workers, patient organisations, industry and, most recently, the media. Although the overall aim of health and pharmaceutical policy is to address the needs of all citizens, there are only a few, well organised groups who are actually consulted and involved in the policymaking process, often with the support of the industry. The reasons for this lack of citizen involvement in health and pharmaceutical policymaking are many, for example: there is no consensus about what public involvement means; there is a predominance of special interest groups with narrow, specific agendas; not all decision makers welcome lay participation; patients and professionals have different rationalities with regard to their views on medicine. Because the lay public and medicine users are not one entity, one of the many challenges facing policy makers today is to identify, incorporate and prioritise the many diverse needs. The authors recommend research which includes studies that look at: lay attitudes towards pharmaceutical policy; lay experiences of drug therapy and how it affects their daily lives; the problem of identifying lay representatives; the relationship between industry and the consumers; the effect of the media on medicine users and on pharmaceutical policy itself. The authors acknowledge that although lay involvement in policy is still in its infancy, some patient organisations have been successful and there are developments towards increased lay involvement in pharmaceutical policymaking.     

Gastroprotective effect of anti-cancer compound rohitukine: possible role of gastrin antagonism and H<Superscript>+</Superscript> K<Superscript>+</Superscript>-ATPase inhibition

The present study was designed to evaluate the anti-ulcerogenic properties of an alkaloid chromane, rohitukine from Dysoxylum binectariferum. Anti-ulcer potential of rohitukine was assessed in cold restrained, pyloric ligated and ethanol induced ulcers in rats. In addition, rohitukine was tested in vitro for H⁺ K⁺-ATPase inhibitory activity in gastric microsomes. Moreover, we studied the role of rohitukine on the cytosolic concentration of Ca²⁺ in parietal cell-enriched cell suspension in order to ascertain its mechanism of action. Cytoprotective activity was evaluated through PGE₂ level. Rohitukine significantly attenuated the ulcers in cold restraint ulcer (CRU) model in a dose-related manner. Moreover, it significantly lowered the free acidity and pepsin activity in pyloric ligated rats while improved the depleted level of mucin. Furthermore, rohitukine significantly reversed the cold restrained-induced increase in gastrin level. Our in vitro study revealed that rohitukine moderately inhibited the microsomal H⁺ K⁺-ATPase activity with respect to positive control omeprazole. Furthermore, rohitukine potently antagonized the gastrin-elicited increase in cytosolic Ca²⁺ level in parietal cell-enriched suspension. In ethanol-induced gastric lesions in rats, rohitukine significantly inhibited the formation of erosions and increased PGE₂ content showing more potency than reference drug sucralfate. Our results thus suggest that rohitukine possess significant anti-ulcer and anti-gastrinic activity in rats. It is likely that gastro-protective influences of rohitukine are dependent partly on its acid-lowering potential and partly on cytoprotective property. The acid-reducing effect of rohitukine might be attributed to its lowering effect on gastrin production and/or antagonism of gastrin-evoked functional responses of parietal cells. Thus, rohitukine represent a useful agent in the treatment of peptic ulcer disease.     

Liquid Perfluorocarbons as Contrast Agents for Ultrasonography and <Superscript>19</Superscript>F-MRI

Perfluorocarbons (PFCs) are fluorinated compounds that have been used for many years in clinics mainly as gas/oxygen carriers and for liquid ventilation. Besides this main application, PFCs have also been tested as contrast agents for ultrasonography and magnetic resonance imaging since the end of the 1970s. However, most of the PFCs applied as contrast agents for imaging were gaseous. This class of PFCs has been recently substituted by liquid PFCs as ultrasound contrast agents. Additionally, liquid PFCs are being tested as contrast agents for ¹⁹F magnetic resonance imaging (MRI), to yield dual contrast agents for both ultrasonography and ¹⁹F MRI. This review focuses on the development and applications of the different contrast agents containing liquid perfluorocarbons for ultrasonography and/or MRI: large and small size emulsions (i.e. nanoemulsions) and nanocapsules.     

Activation of Antigen-Specific CD8<Superscript>+</Superscript> T Cells by Poly-DL-Lactide/Glycolide (PLGA) Nanoparticle-Primed Gr-1<Superscript>high</Superscript> Cells

Purpose

The aim of this study was to investigate the induction of antigen-specific T cell activation and cell cycle modulation by a poly-DL-lactide/glycolide (PLGA) nanoparticle (NP)-primed CD11b⁺Gr-1^high subset isolated from mouse bone marrow.

Methods

PLGA NPs containing the ovalbumin (OVA) antigen were prepared using the double emulsion and solvent evaporation method, and protein release rate and cell viability were determined. The Lin2^¯CD11b⁺Gr-1^highLy6c^low (Gr-1^high) subset was sorted from the bone marrow of C57BL/6 J mice by fluorescence-activated cell sorting (FACS) and co-cultured with OT-I CD8⁺ splenic T cells. Proliferation of OT-I CD8⁺ T cells was monitored, and cell cycles were determined by 5-bromo-2′-deoxyuridine (BrdU) labeling.

Results

Treatment of Gr-1^high cells with PLGA/OVA NPs upregulated expression of the SIINFEKL-H2K^b complex in the context of MHC I. Co-cultures of OT-I CD8⁺ T cells with the PLGA/OVA NP-primed Gr-1^high cells induced the proliferation of T cells in vitro and modulated cell division and morphology. Treatment of Gr-1^high cells with PLGA/OVA NPs also induced cell apoptosis and necrosis.

Conclusion

This study demonstrated the function of PLGA/OVA NPs in the activation of OT-I CD8⁺ T cells and the capability of cross-presentation via the Gr-1^high polymorphonuclear subset from mouse bone marrow.

     

<Emphasis Type="Italic">In vitro</Emphasis> Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase inhibitory activity and antimicrobial activity of sesquiterpenes isolated from <Emphasis Type="Italic">Thujopsis dolabrata</Emphasis>

A series of sesquiterpenes and hinokitiol-related compounds (1–15) was isolated from the essential oil of Thujopsis dolabrata Sieb. et Zucc. var. hondai Makino, and their structures were determined by combined spectroscopic analyses. The inhibitory effects of these compounds on microbial cell growth and Na⁺/K⁺-ATPase were evaluated in vitro. It was found that (−)-elema-1,3,11(13)-trien-12-ol (5), α,β,γ-costol (8), and chamigrenol (11) inhibit the activities of Na⁺/K⁺-ATPase, with IC₅₀ values of 11.2 ± 0.11, 12.2 ± 0.09, and 15.9 ± 0.54 μg/mL, respectively. Thujopsene (1), cedrol (9), γ-cuparenol (10), and chamigrenol (11) showed potent antibacterial activity, with MIC values in the range of 25–50 μg/mL, and β-thujaplicin (12) exhibited a broad spectrum of antibacterial and antifungal activity. These results indicate that these isolated compounds are promising candidates for the development of potent Na⁺/K⁺ ATPase inhibitors and antimicrobial agents.     

Dopamine Increases CD14<Superscript>+</Superscript>CD16<Superscript>+</Superscript> Monocyte Transmigration across the Blood Brain Barrier: Implications for Substance Abuse and HIV Neuropathogenesis

In human immunodeficiency virus-1 (HIV) infected individuals, substance abuse may accelerate the development and/or increase the severity of HIV associated neurocognitive disorders (HAND). It is proposed that CD14⁺CD16⁺ monocytes mediate HIV entry into the central nervous system (CNS) and that uninfected and infected CD14⁺CD16⁺ monocyte transmigration across the blood brain barrier (BBB) contributes to the establishment and propagation of CNS HIV viral reservoirs and chronic neuroinflammation, important factors in the development of HAND. The effects of substance abuse on the frequency of CD14⁺CD16⁺ monocytes in the peripheral circulation and on the entry of these cells into the CNS during HIV neuropathogenesis are not known. PBMC from HIV infected individuals were analyzed by flow cytometry and we demonstrate that the frequency of peripheral blood CD14⁺CD16⁺ monocytes in HIV infected substance abusers is increased when compared to those without active substance use. Since drug use elevates extracellular dopamine concentrations in the CNS, we examined the effects of dopamine on CD14⁺CD16⁺ monocyte transmigration across our in vitro model of the human BBB. The transmigration of this monocyte subpopulation is increased by dopamine and the dopamine receptor agonist, SKF 38393, implicating D1-like dopamine receptors in the increase in transmigration elicited by this neurotransmitter. Thus, elevated extracellular CNS dopamine may be a novel common mechanism by which active substance use increases uninfected and HIV infected CD14⁺CD16⁺ monocyte transmigration across the BBB. The influx of these cells into the CNS may increase viral seeding and neuroinflammation, contributing to the development of HIV associated neurocognitive impairments.     

<Emphasis Type="Italic">N</Emphasis><Superscript>4</Superscript>,<Emphasis Type="Italic">N</Emphasis><Superscript>9</Superscript>-Dioleoyl Spermine Is a Novel Nonviral Lipopolyamine Vector for Plasmid DNA Formulation

Purpose To study the effect of synthesized N⁴,N⁹-dioleoyl spermine on DNA condensation and then measure its transfection efficiency in cell culture.Methods The lipopolyamine was synthesized from the naturally occurring polyamine spermine. The ability of this novel compound to condense DNA was studied using ethidium bromide fluorescence quenching and light scattering assays. Transfection efficiency was studied in primary skin cells (FEK4) and in an immortalized cancer cell line (HtTA), and compared with the commercially available transfection formulations Lipofectin and Lipofectamine.Results The synthesized N⁴,N⁹-dioleoyl spermine formula is efficient at condensing calf thymus and circular plasmid DNA and effectively transfects both primary skin cells and cancer cell lines at low charge ratios of (+/– ammonium/phosphate) 2.5.Conclusions N⁴,N⁹-Dioleoyl spermine condenses DNA and achieves high transfection levels in cultured cells.