The Ruthenium Complex cis-(Dichloro) Tetraammineruthenium(III) Chloride Presents Immune Stimulatory Activity on Human Peripheral Blood Mononuclear Cells

Ruthenium compounds in general are well suited for medicinal applications. They have been investigated as immunosuppressants, nitric oxide scavengers, antimicrobial agents, and antimalarials. The aim of this study is to evaluate the immunomodulatory activity of cis-(dichloro) tetraammineruthenium(III) chloride (cis-[RuCl(2)(NH(3))(4)]Cl) on human peripheral blood mononuclear cells (PBMC). The cytotoxic studies performed here revealed that the ruthenium( III) complex presents a cytotoxic activity towards normal human PBMC, only at very high concentration. Results also showed that cis-[ RuCl(2)(NH(3))(4)] Cl presents a dual role on PBMC stimulating proliferation and interleukin-2 (IL-2) production at low concentration and inducing cytotoxicity, inability to proliferate, and inhibiting IL-2 production at high concentration. The noncytotoxic activity of cis-[RuCl(2)(NH(3))(4)] Cl at low concentration towards PBMC, which correlates with the small number of annexin V positive cells and also the absence of DNA fragmentation, suggest that this compound does not induce apoptosis on PBMC. For the first time, we show that, at low concentration (10-100 mu g L(-1)), the cis-[ RuCl(2)(NH(3))(4)] Cl compound induces peripheral blood lymphocytes proliferation and also stimulates them to IL-2 production. These results open a new potential applicability of ruthenium(III) complexes as a possible immune regulatory compound acting as immune suppressor at high concentration and as immune stimulator at low concentration.

Suramin inhibits macrophage activation by human group IIA phospholipase A(2), but does not affect bactericidal activity of the enzyme

Suramin is a polysulphonated napthylurea antiprotozoal and anthelminitic drug, which also presents inhibitory activity against a broad range of enzymes. Here we evaluate the effect of suramin on the hydrolytic and biological activities of secreted human group IIA phospholipase A(2) (hsPLA(2)GIIA). The hsPLA(2)GIIA was expressed in E. coli, and refolded from inclusion bodies. The hydrolytic activity of the recombinant enzyme was measured using mixed dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) liposomes. The activation of macrophage cell line RAW 264.7 by hsPLA(2) GIIA was monitored by NO release, and bactericidal activity against Micrococcus luteus was evaluated by colony counting and by flow cytometry using the fluorescent probe Sytox Green. The hydrolytic activity of the hsPLA(2) GIIA was inhibited by a concentration of 100 nM suramin and the activation of macrophages by hsPLA(2) GIIA was abolished at protein/suramin molar ratios where the hydrolytic activity of the enzyme was inhibited. In contrast, both the bactericidal activity of hsPLA(2) GIIA against Micrococcus luteus and permeabilization of the bacterial inner membrane were unaffected by suramin concentrations up to 50 mu M. These results demonstrate that suramin selectively inhibits the activity of the hsPLA(2) GIIA against macrophages, whilst leaving the anti-bacterial function unchanged.

Central and peripheral mediation of human force sensation following eccentric or concentric contractions

Fatigue was induced in the triceps brachii of the experimental arm by a regimen of either eccentric or concentric muscle actions. Estimates of force were assessed using a contralateral limb-matching procedure, in which target force levels (25 %, 50 % or 75 % of maximum) were defined by the unfatigued control arm. Maximum isometric force-generating capacity was reduced by 31 % immediately following eccentric contractions, and remained depressed at 24 (25 %) and 48 h (13 %) post-exercise. A less marked reduction (8.3 %) was observed immediately following concentric contractions. Those participants who performed prior eccentric contractions, consistently (at all force levels), and persistently (throughout the recovery period), overestimated the level of force applied by the experimental arm. In other words, they believed that they were generating more force than they actually achieved. When the forces applied by the experimental and the control arm, were each expressed as a proportion of the maximum force that could be attained at that time, the estimates matched extremely closely. This outcome is that which would be expected if the estimates of force were based on a sense of effort. Following eccentric exercise, the amplitude of the EMG activity recorded from the experimental arm was substantially greater than that recorded from the control arm. Cortically evoked potentials recorded from the triceps brachii (and extensor carpi radialis) of the experimental arm were also substantially larger than those elicited prior to exercise. The sense of effort was evidently not based upon a corollary of the central motor command. Rather, the relationship between the sense of effort and the motor command appears to have been altered as a result of the fatiguing eccentric contractions. It is proposed that the sense of effort is associated with activity in neural centres upstream of the motor cortex.

The effects of viscous loading of the human forearm flexors on the stability of coordination

This experiment investigated whether the stability of rhythmic unimanual movements is primarily a function of perceptual/spatial orientation or neuro-mechanical in nature. Eight participants performed rhythmic flexion and extension movements of the left wrist for 30 s at a frequency of 2.25 Hz paced by an auditory metronome. Each participant performed 8 flex-on-the-beat trials and 8 extend-on-the-beat trials in one of two load conditions, loaded and unload. In the loaded condition, a servo-controlled torque motor was used to apply a small viscous load that resisted the flexion phase of the movement only. Both the amplitude and frequency of the movement generated in the loaded and unloaded conditions were statistically equivalent. However, in the loaded condition movements in which participants were required to flex-on-the-beat became less stable (more variable) while extend-on-the-beat movements remained unchanged compared with the unload condition. The small alteration in required muscle force was sufficient to result in reliable changes in movement stability even a situation where the movement kinematics were identical. These findings support the notion that muscular constraints, independent of spatial dependencies, can be sufficiently strong to reliably influence coordination in a simple unimanual task.

Contraction of the human diaphragm during rapid postural adjustments

1. The response of the diaphragm to the postural perturbation produced by rapid flexion of the shoulder to a visual stimulus was evaluated in standing subjects. Gastric, oesophageal and transdiaphragmatic pressures were measured together with intramuscular and oesophageal recordings of electromyographic activity (EMG) in the diaphragm. To assess the mechanics of contraction of the diaphragm, dynamic changes in the length of the diaphragm were measured with ultrasonography. 2. With rapid flexion of the shoulder in response to a visual stimulus, EMG-activity in the costal and crural diaphragm occurred about 20 ms prior to the onset of deltoid EMG. This anticipatory contraction occurred irrespective of the phase of respiration in which arm movement began. The onset of diaphragm EMG-coincided with that of transversus abdominis. 3. Gastric and transdiaphragmatic pressures increased in association with the rapid arm flexion by 13.8 +/- 1.9 (mean +/- S.E.M.) and 13.5 +/- 1.8 cmH(2)O, respectively. The increases occurred 49 +/- 4 ms after the onset of diaphragm EMG, but preceded the onset of movement of the limb by 63 +/- 7 ms. 4. Ultrasonographic measurements revealed that the costal diaphragm shortened and then lengthened progressively during the increase in transdiaphragmatic pressure. 5. This study provides definitive evidence that the human diaphragm is involved in the control of postural stability during sudden voluntary movement of the limbs.

Functional polymorphism of the human arylamine N-acetyltransferase type 1 gene caused by (CT)-T-190 and G(560)A mutations

Human N-acetyltransferase type 1 (NAT1) catalyses the N- or O-acetylation of various arylamine and heterocyclic amine substrates and is able to bioactivate several known carcinogens. Despite wide inter-individual variability in activity, historically, NAT1 was considered to be monomorphic in nature. However, recent reports of allelic variation at the NAT1 locus suggest that it may be a polymorphically expressed enzyme. In the present study, peripheral blood mononuclear cell NAT1 activity in 85 individuals was found to be bimodally distributed with approximately 8% of the population being slow acetylators. Subsequent sequencing of the individuals having slow acetylator status showed all to have either a (CT)-T-190 or G(560)A base substitution located in the protein encoding region of the NAT1 gene. The (CT)-T-190 base substitution changed a highly conserved Arg(64), which others have shown to be essential for fully functional NAT1 protein. The (CT)-T-190 mutation has not been reported previously and we have named it NAT1*17. The G(560)A mutation is associated with the base substitutions previously observed in the NAT1*10 allele and this variant (NAT1*14) encodes for a protein with reduced acetylation capacity. A novel method using linear PCR and dideoxy terminators was developed for the detection of NAT1*14 and NAT1*17. Neither of these variants was found in the rapid acetylator population. We conclude that both the (CT)-T-190 (NAT1*17) and G(560)A (NAT1*14) NAT1 structural variants are involved in a distinct NAT1 polymorphism. Because NAT1 can bioactivate several carcinogens, this polymorphism may have implications for cancer risk in individual subjects. (C) 1998 Chapman & Hall Ltd.

Solid-phase extraction method with high-performance liquid chromatography and electrochemical detection for the quantitative analysis of oxycodone in human plasma

A sensitive and reproducible solid-phase extraction (SPE) method for the quantification of oxycodone in human plasma was developed. Varian Certify SPE cartridges containing both C-8 and benzoic acid functional groups were the most suitable for the extraction of oxycodone and codeine (internal standard), with consistently high (greater than or equal to 80%) and reproducible recoveries. The elution mobile phase consisted of 1.2 ml of butyl chloride-isopropanol (80:20, v/v) containing 2% ammonia. The quantification limit for oxycodone was 5.3 pmol on-column. Within-day and inter-day coefficients of variation were 1.2% and 6.8% respectively for 284 nM oxycodone and 9.5% and 6.2% respectively for 28.4 nM oxycodone using 0.5-ml plasma aliquots. (C) 1998 Elsevier Science BN. All rights reserved.

Human vascular to cardiac tissue selectivity of L- and T-type calcium channel antagonists

1 Voltage-operated calcium channel (VOCC) antagonists are effective antihypertensive and antianginal agents but they also depress myocardial contractility. 2 We compared four L-type calcium channel antagonists, felodipine, nifedipine, amlodipine and verapamil and a relatively T-type selective calcium channel antagonist, mibefradil, on human and rat isolated tissue assays to determine their functional vascular to cardiac tissue selectivity (V/C) ratio. 3 The V/C ratio was calculated as the ratio of the IC50 value of the antagonist that reduced (by 50%) submaximally contracted (K+ 62 mM) human small arteries from the aortic vasa vasorum (vascular, V) mounted in a myograph and the IC50 value of the antagonist that reduced (-)-isoprenaline (6 nM) submaximally stimulated human right atrial trabeculae muscle (cardiac, C) mounted in organ chambers. 4 The average pIC(50) Values (-log IC50 M) for the human vascular preparations were felodipine 8.30, nifedipine 7.78, amlodipine 6.64, verapamil 6.26 and mibefradil 6.22. The average pIC(50) values for the cardiac muscle were felodipine 7.21, nifedipine 6.95, verapamil 6.91, amlodipine 5.94, and mibefradil 4.61. 5 The V/C ratio calculated as antilog [pIC(50)V-pIC(50)C] is thus mibefradil 41, felodipine 12, nifedipine 7, amlodipine 5 and verapamil 0.2. 6 In rat small mesenteric arteries the pIC(50) values for the five drugs were similar to the values for human vasa vasorum arteries contracted by K+ 62 mM. However for methoxamine (10 mu M) contraction in the rat arteries the pIC(50) values were lower for felodipine 7.24 and nifedipine 6.23, but similar for verapamil 6.13, amlodipine 6.28 and mibefradil 5.91. 7 In conclusion in the human tissue assays, the putative T-channel antagonist mibefradil shows the highest vascular to cardiac selectivity ratio; some 3 fold higher than the dihydropyridine, felodipine, and some 200 fold more vascular selective than the phenylalkylamine, verapamil. This favourable vascular to cardiac selectivity for mibefradil, from a new chemical class of VOCC antagonist, may be explained by its putative T-channel selectivity.

Determination of pindolol enantiomers in human plasma and urine by simple liquid-liquid extraction and high-performance liquid chromatography

A simple method for the measurement of pindolol enantiomers by HPLC is presented. Alkalinized serum or urine is extracted with ethyl acetate and the residue remaining after evaporation of the organic layer is then derivatised with (S)-(-)-alpha-methylbenzyl isocyanate. The diastereoisomers of derivatised pindolol and metoprolol (internal standard) are separated by high-performance liquid chromatography (HPLC) using a C-18 silica column and detected using fluorescence (excitation lambda: 215 nm, emission lambda: 320 nm). The assay displays reproducible linearity for pindolol enantiomers with a correlation coefficient of r(2) greater than or equal to 0.998 over the concentration range 8-100 ng ml(-1) for plasma and 0.1-2.5 mu g ml(-1) for urine. The coefficient of variation for accuracy and precision of the quality control samples for both plasma and urine are consistently

Absorption of sunscreens across human skin: an evaluation of commercial products for children and adults

Aims Topical sunscreens are routinely applied to the skin by a large percentage of the population. This study assessed the extent of absorption of a number of common chemical sunscreen agents into and through human skin following application of commercially available products. Methods Sunscreen products were applied to excised human epidermis in Franz diffusion cells with the amount penetrating into and across the epidermis assessed by h.p.l.c. for 8 h following application. Results All sunscreen agents investigated penetrated into the skin (0.25 g m(-2) or 14% of applied dose), but only benzophenone-3 passed through the skin in significant amounts (0.08 g m(-2) or 10% of the applied dose). With one exception, suncreen agents in corresponding products marketed for adults and children had similar skin penetration profiles. Conclusions Whilst limited absorption across the skin was observed for the majority of the sunscreens tested, benzophenone-3 demonstrated sufficiently high penetration to warrant further investigation of its continued application.

Analysis of proximal femur DXA scans in growing children: Comparisons of different protocols for cross-sectional 8-month and 7-year longitudinal data

Dual-energy X-ray absorptiometry (DXA) is a widely used method for measuring bone mineral in the growing skeleton. Because scan analysis in children offers a number of challenges, we compared DXA results using six analysis methods at the total proximal femur (PF) and five methods at the femoral neck (FN), In total we assessed 50 scans (25 boys, 25 girls) from two separate studies for cross-sectional differences in bone area, bone mineral content (BMC), and areal bone mineral density (aBMD) and for percentage change over the short term (8 months) and long term (7 years). At the proximal femur for the short-term longitudinal analysis, there was an approximate 3.5% greater change in bone area and BMC when the global region of interest (ROI) was allowed to increase in size between years as compared with when the global ROI was held constant. Trend analysis showed a significant (p < 0.05) difference between scan analysis methods for bone area and BMC across 7 years. At the femoral neck, cross-sectional analysis using a narrower (from default) ROI, without change in location, resulted in a 12.9 and 12.6% smaller bone area and BMC, respectively (both p < 0.001), Changes in FN area and BMC over 8 months were significantly greater (2.3 %, p < 0.05) using a narrower FN rather than the default ROI, Similarly, the 7-year longitudinal data revealed that differences between scan analysis methods were greatest when the narrower FN ROI was maintained across all years (p < 0.001), For aBMD there were no significant differences in group means between analysis methods at either the PF or FN, Our findings show the need to standardize the analysis of proximal femur DXA scans in growing children.

Quantitative analysis of vascular to cardiac selectivity of L- and T-type voltage-operated calcium channel antagonists in human tissues

1. Classical L-type voltage-operated calcium channel (VOCC) antagonists dilate blood vessels, depress myocardial contractility and slow cardiac conduction. 2. We compared four L-type VOCC antagonists and a novel tetralol derivative, mibefradil, reportedly 10-fold more selective for T- (transient) over L-type VOCC in two in vitro assays of human tissue, namely isolated small arteries from the aortic vasa vasorum in a myograph and right atrial trabeculae muscle under isometric force conditions. 3. In arteries contracted with K+ (62 mmol/L), the relaxation pIC(50) values for the VOCC antagonists felodipine, nifedipine, amlodipine, verapamil and mibefradil were 8.30, 7.78, 6.64, 6.26 and 6.22, respectively. In atrial trabeculae, the pIC(50) values to inhibit the inotropic response to a submaximal concentration of isoprenaline (6 nmol/L) for felodipine, nifedipine, verapamil, amlodipine and mibefradil were 7.21, 6.95, 6.91, 5.94 and 4.61, respectively. 4. Taking the anti-log (pIC(50) vessel - pIC(50) atrium) the vascular relaxation to cardiac depression potency ratios for mibefradil, felodipine, nifedipine, amlodipine and verapamil were 41, 12, 7, 5 and 0.22, respectively. 5. We conclude that, in human tissue assays, perhaps T- over L-type VOCC selectivity confers the most favourable vascular selectivity on mibefradil. Alternatively, splice variants of L-type VOCC in the vasculature (CaV1.2b) may be more sensitive to mibefradil than the splice variants in the heart (CaV1.2a).

Human cyclophilin 40 is a heat shock protein that exhibits altered intracellular localization following heat shock

The unactivated steroid receptors are chaperoned into a conformation that is optimal for binding hormone by a number of heat shock proteins, including Hsp90, Hsp70, Hsp40, and the immunophilin, FKBP52 (Hsp56). Together with its partner cochaperones, cyclophilin 40 (CyP40) and FKBP51, FKBP52 belongs to a distinct group of structurally related immunophilins that modulate steroid receptor function through their association with Hsp90. Due to the structural similarity between the component immunophilins, FKBP52 and cyclophilin 40, we decided to investigate whether CyP40 is also a heat shock protein. Exposure of MCF-7 breast cancer cells to elevated temperatures (42 degreesC for 3 hours) resulted in a 75-fold increase in CyP40 mRNA levels, but no corresponding increase in CyP40 protein expression, even after 7 hours of heat stress. The use of cycloheximide to inhibit protein synthesis revealed that in comparison to MCF-7 cells cultured at 37 degreesC, those exposed to heat stress (42 degreesC for 3 hours) displayed an elevated rate of degradation of both CyP40 and FKBP52 proteins. Concomitantly, the half-life of the CyP40 protein was reduced from more than 24 hours to just over 8 hours following heat shock. As no alteration in CyP40 protein levels occurred in cells exposed to heat shock, an elevated rate of degradation would imply that CyP40 protein was synthesized at an increased rate. hence the designation of human CyP40 as a heat shock protein. Application of heat stress elicited a marked redistribution of CyP40 protein in MCF-7 cells from a predominantly nucleolar localization, with some nuclear and cytoplasmic staining, to a pattern characterized by a pronounced nuclear accumulation of CyP40, with no distinguishable nucleolar staining. This increase in nuclear CyP40 possibly resulted from a redistribution of cytoplasmic and nucleolar CyP40, as no net increase in CyP40 expression levels occurred in response to stress. Exposure of MCF-7 cells to actinomycin D for 4 hours resulted in the translocation of the nucleolar marker protein, B23, from the nucleolus, with only a small reduction in nucleolar CyP40 levels. Under normal growth conditions, MCF-7 cells exhibited an apparent colocalization of CyP40 and FKBP52 within the nucleolus.

Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of human melanoma is regulated by Smac/DIABLO release from mitochondria

In previous studies we have shown that the sensitivity of melanoma cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)induced apoptosis was determined largely by the level of expression of death receptor TRAIL receptor 2 on the cells. However, approximately one-third of melanoma cell lines were resistant to TRAIL, despite expression of high levels of TRAIL receptor 2. The present studies show that these cell lines had similar levels of TRAIL-induced activated caspase-3 as the TRAIL-sensitive lines, but the activated caspase-3 did not degrade substrates downstream of caspase-3 [inhibitor of caspase-activated DNase and poly(ADP-ribose) polymerase]. This appeared to be due to inhibition of caspase-3 by X-linked inhibitor of apoptosis (XIAP) because XIAP was bound to activated caspase-3, and transfection of XIAP into TRAIL-sensitive cell lines resulted in similar inhibition of TRAIL-induced apoptosis. Conversely, reduction of XIAP levels by overexpression of Smac/ DIABLO in the TRAIL-resistant melanoma cells was associated with the appearance of catalytic activity by caspase-3 and increased TRAIL-induced apoptosis. TRAIL was shown to cause release of Smac/DIABLO from mitochondria, but this release was greater in TRAIL-sensitive cell lines than in TRAIL-resistant cell lines and was associated with downregulation of XIAP levels. Furthermore, inhibition of Smac/DIABLO release by overexpression of Bcl-2 inhibited down-regulation of XIAP levels. These results suggest that Smac/DIABLO release from mitochondria and its binding to XIAP are an alternative pathway by which TRAIL induces apoptosis of melanoma, and this pathway is dependent on the release of activated caspase-3 from inhibition by XIAP and possibly other inhibitor of apoptosis family members.

Continuous reproduction and recruitment in the narrowback mud crab Panopeus americanus (Brachyura, Panopeidae) in a remnant human-impacted mangrove area

The population structure, reproductive period, and juvenile recruitment of Panopeus americanus were studied in order to enhance knowledge of its life cycle and reproductive strategy and promote the maintenance of its natural stocks in an impacted region. Specimens were collected in the remnant human-impacted mangrove at Araca, state of Sao Paulo, Brazil, at two-month intervals from January to November 2005, at low tide, with a capture effort by three people. The crabs were measured (carapace width, CW) and sexed. The total catch was 275 animals, including 132 males (48.0%); 127 females (46.2%), of which 39 were ovigerous (14.2% of total catch); and 16 individuals whose sex could not be identified (5.8%). No correlation was observed between water temperature and the number of collected individuals; however, there was a significant, positive correlation with salinity. Males were significantly larger than females. The size-frequency distribution was bimodal, reflecting the occurrende of more than one recruitment pulse and the differential abundance of adults during the period of study. The overall sex ratio was 1:0.97 favoring males, and was not significantly different from the expected value, i.e., this population fits the anomalous pattern of sex occurrence in size classes. Ovigerous females were captured in all sampled months, which explain the continuous recruitment observed. Expected low levels of individuals of different size classes in the population were not confirmed. All population aspects found here allowed us to infer that this population of P. americanus is well established in the impacted mangrove by virtue of its reproductive strategy.