Bis[platinum(II)] and bis[platinum(IV)] complexes with optically active bis(vicinal-1,2-diamines) and their interaction with DNA.

Bis[platinum(II)] [Cl2Pt(LL)PtCl2] complexes 2,5 and 8 with chiral non-racemic ligands: 1a-c (LL = (R,R), (S,S) and (R,S) N,N'-bis(3,4-diaminobutyl)hexanediamide); 4a,b (LL = (R,R) and (S,S) N,N'-bis[3,4-bis(diaminobutyl)] urea); 7a-d (LL' = (R,R), (S,S), (R,S) and (S,R) 4,5-diamino-N-(3,4-diaminobutyl) pentanamide) and bis[platinum(IV)] complex 10-13 with ligands 1a,b and 4a,b have been prepared and characterized by IR, 1H, 13C and 195Pt NMR spectra. The interactions of 2a-c, 5a, 5b, 8a-d and 10a with dsDNA were investigated with the goal of examining whether the chirality, the nature of the spacer and the oxidation state have an influence on platinum-DNA binding properties. All the bis[platinum(II)] complexes form with dsDNA intra- and interstrand crosslinks and crosslinks over sticky ends, whereas the bis[platinum(IV)] complex 10a only forms intra- and interstrand crosslinks. The platinum-DNA coordination sites were determined by the T4 DNA polymerase footprinting method. The results show that all investigated bis(platinum) complexes have high preference towards distinct purines. All isomeric bis(amide) 2a-c and mono(amide) 8a-d complexes exhibit nearly the same binding pattern, whereas the ureide complexes 5a and 5b have other coordination sites with higher sequence preference. Interestingly, the ureides 5a and 5b differ in their coordination sites not only in comparison to the bis(amides) 2a-c and mono(amides) 8a-d, but also between each other. The bis[platinum(IV)] complex 10a also differs in coordination sites in comparison to all the bis[platinum(II)] compounds.     

Calcium measurements in primates during and after hypokinesia in establishing calcium deficiency during prolonged hypokinesia

Hypokinesia (diminished movement) induces significant calcium (Ca) changes, but little is known about the effect of hypokinesia (HK) on Ca deficiency. Measuring Ca changes during and after HK the aim of this study was to determine Ca deficiency during prolonged HK. Studies were done on 12 male Macaca mulatta (rhesus monkeys) aged 3–5 yr (5.58–6.42 kg) during a 90-d pre-HK period, a 90-d HK period, and a 15-d post-HK period. Monkeys were equally divided into two groups: vivarium control monkeys (VCM) and hypokinetic monkeys (HKM). Hypokinetic monkeys were kept in small individual cages that restricted their movements in all directions without hindering food and water intakes. Urinary, fecal, and serum Ca, urinary and serum magnesium (Mg) and phosphate (P), serum intact parathyroid hormone (iPTH), and calcitonin (CT) concentration, body weight, food intake, fluid consumed and eliminated in urine were measured. During the HK period, fecal Ca loss, urinary Ca, P, and Mg excretion, fluid elimination, and serum P, Ca, and Mg concentration increased significantly (p≤0.01), whereas serum iPTH and CT concentration, food and fluid intakes, and body weight decreased significantly (p≤0.01) in the HKM group when compared with the VCM group. During the initial days of the post-HK period, serum Ca, Mg, and P concentration, fecal Ca loss, urinary Ca, Mg, and P excretion, and fluid elimination decreased significantly (p≤0.01), whereas fluid intake increased significantly (p≤0.01) in the HKM group when compared with the VCM group. Food intake, body weight, and serum iPTH and CT concentrations remained significantly (p≤0.01) depressed in the HKP group when compared with the VCM; however, they increased as the duration of the post-HK period increased. By contrast, the corresponding parameters remained stable in the VCM group when compared with the baseline control values. It was shown that fecal and urinary Ca loss and serum Ca concentration increases significantly during HK, whereas during post-HK fecal, urinary, and serum Ca decreases significantly. It was concluded that significant decrease of serum, urinary, and fecal Ca during post-HK may suggest the presence of Ca deficiency during prolonged HK.     

Bioactive polar lipids from Chroococcidiopsis sp. (Cyanobacteria)

Many studies indicate that various bioactive metabolites subsist in cyanobacteria. Glycolipids of cyanobacteria are reported as molecules that exert specific bioactivities. In this study, total lipids of Chroococcidiopsissp., a coccoid cyanobacterium isolated from a Greek cave, were separated into neutral and polar-lipids and the latter were further fractionated by high-performance liquid chromatography (HPLC). Each polar lipid fraction was tested in vitro for its ability to inhibit platelet-activating factor (PAF)- and thrombin-induced washed rabbit platelet aggregation and/or to cause platelet aggregation. The structures of the most active fractions were elucidated by biological assays and identified by electrospray mass spectrometry. One fraction was a potent inhibitor of PAF-induced platelet aggregation. Structural studies of this fraction indicated the existence of phospho-glyco analog of ceramide. Another fraction that was a potent inhibitor of PAF- as well as of thrombin-induced platelet aggregation was structurally elucidated as a phospho-acetylated glyco-analog of diglyceride. The fraction that induced platelet aggregation was identified as a phospho-acetylated-glyco analog of ceramide. These novel bioactive polar lipids in cyanobacteria in regard to the structure and biological activity may contribute to the allergic character of cyanobacteria.     

Water and electrolyte changes in skeletal and cardiac muscles of rats during prolonged hypokinesia

The objective of this study was to show that hypokinesia (diminished movement) could affect differently water and electrolyte content in muscles having minimum differences in their function and morphology. To this end, we studied water and electrolyte content in skeletal and cardiac muscles, fluid excretion, electrolyte absorption, and electrolyte levels in plasma, urine and feces of rats during prolonged hypokinesia (HK). Studies were conducted on one-hundred-twenty-six 13-weeks old male Wister rats during a pre-hypokinetic period and a hypokinesia period. Animals were equally divided into two groups: vivarium control rats (VCR) and hypokinetic rats (HKR). Hypokinetic animals were kept in small individual cages which restricted their movements in all directions without hindering food and water intake. Control rats were housed in individual cages under vivarium control conditions. Sodium (Na+) and potassium (K+) absorption, electrolyte and water content in cardiac muscles (right and left ventricle), thigh extensor (quadriceps femoris muscle) and long muscle of the back (biceps femoris muscle), urine volume, and electrolyte levels in plasma and urine and feces did not change in VCR when compared to their pre-hypokinetic levels. The absorption of Na+ and K+, water and electrolyte content in cardiac and skeletal muscles decreased significantly, while urine volume, plasma electrolyte levels and urine and fecal electrolyte excretion increased significantly in HKR compared with their pre-HK values and with their respective vivarium control (VCR). Water and electrolyte content decreased more significantly in skeletal than in cardiac muscles. Water and electrolyte levels decreased more in the thigh extensor and in the right ventricle than in the long muscle of the back, the left ventricle or the septum. Muscles suffering from higher water and electrolyte loss against the background of lower water and electrolyte content show lower water and electrolyte deposition. Lower electrolyte and water content in skeletal than in cardiac muscle shows that water and electrolyte content decreases more in skeletal than cardiac muscles. Skeletal muscle showed lower water and electrolyte content than cardiac muscle indicating that the risk for decreased muscle water and electrolyte content is inversely related to the muscle function and morphology, i.e., the more weight-bearing supporting function and morphology muscles have, the higher the risk for lower muscle water and electrolyte content. It was concluded that the greater muscle function and morphology, the lower electrolyte and water deposition, the higher water and electrolyte losses, and the lower water and electrolyte content.     

Electrolyte changes in plasma and urine of athletes during acute and rigorous bed rest and ambulatory conditions

Rigorous bed rest (RBR) induces significant electrolyte changes, but little it is not known about the effect of acute bed rest (ABR) (i.e., abrupt confinement to a RBR). The aim of this study was to measure urinary and plasma electrolyte changes during ABR and RBR conditions. The studies were done during 3 d of a pre-bed-rest (BR) period and during 7 d of an ABR and RBR period. Thirty male trained athletes aged, 24.4 ± 6.6 yr were chosen as subjects. They were divided equally into three groups: unrestricted ambulatory control subjects (UACS), acute-bed-rested subjects (ABRS), and rigorous-bed-rested subjects (RBRS). The UACS group experienced no changes in professional training and daily activities. The ABRS were submitted abruptly to a RBR regimen and without having any prior knowledge of the exact date and time when they would be subjected to an RBR regimen. The RBRS were subjected to an RBR regime on a predetermined date and time known to them from the beginning of the study. Sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), and phosphate (P) in plasma and urine, plasma renin activity (PRA) and plasma aldosterone (PA), physical characteristics, peak oxygen uptake, and food and water intakes were measured. Urinary Na, K, Ca, Mg, and P excretion and plasma Na, K, Mg, Ca, and P concentration, PRA, and PA concentration increased significantly (p ≤ 0.01), whereas body weight, peak oxygen uptake, and food and water intakes decreased significantly in the ABRS and RBRS groups when compared with the UACS group. However, urinary and plasma Na, K, Mg, P, and Ca, PRA, and PA values increased much faster and were much greater in the ABRS group than in the RBRS group. Plasma and urinary Na, K, Ca, Mg, and P, PRA and PA levels, food and water intakes, body weight, and peak oxygen uptake did not change significantly in the UACS group when compared with its baseline control values. It was shown that RBR and ABR conditions induce significant increases in urinary and plasma electrolytes; however, urinary and plasma electrolyte changes appeared much faster and were much greater in the ABRS group than the RBRS group. It was concluded that the more abruptly motor activity is ended, the faster and the greater the urinary and plasma electrolyte change.     

From bench to bedside--preclinical and early clinical development of the anticancer agent indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A)

Indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A) is just the second ruthenium-based anticancer agent after NAMI-A which was developed to the stage of clinical trials. Important steps in the mode of action of KP1019 are thought to be the binding to the serum protein transferrin and the transport into the cell via the transferrin pathway. Additionally, the selective activation by reduction in the tumor might contribute to the low side effects observed in in vivo studies. Apoptosis is induced at non-toxic levels via the mitochondrial pathway. These features distinguish it from the established platinum anticancer drugs and suggest that different types of cancer might be treatable with this drug. Indeed, promising activity against certain types of tumors, which are not successfully treatable with cisplatin, and only a very low incidence of acquired resistance has been observed in in vitro and in vivo studies. Recently, a clinical phase I trial was finished in which none of the treated patients experienced serious side effects, while disease stabilization in five of six evaluable patients was achieved. In this review, the preclinical and early clinical development of KP1019 - from bench to bedside - is recapitulated.     

Active mammalian replication origins are associated with a high-density cluster of mCpG dinucleotides.

ori-beta is a well-characterized origin of bidirectional replication (OBR) located approximately 17 kb downstream of the dihydrofolate reductase gene in hamster cell chromosomes. The approximately 2-kb region of ori-beta that exhibits greatest replication initiation activity also contains 12 potential methylation sites in the form of CpG dinucleotides. To ascertain whether DNA methylation might play a role at mammalian replication origins, the methylation status of these sites was examined with bisulfite to chemically distinguish cytosine (C) from 5-methylcytosine (mC). All of the CpGs were methylated, and nine of them were located within 356 bp flanking the minimal OBR, creating a high-density cluster of mCpGs that was approximately 10 times greater than average for human DNA. However, the previously reported densely methylated island in which all cytosines were methylated regardless of their dinucleotide composition was not detected and appeared to be an experimental artifact. A second OBR, located at the 5' end of the RPS14 gene, exhibited a strikingly similar methylation pattern, and the organization of CpG dinucleotides at other mammalian origins revealed the potential for high-density CpG methylation. Moreover, analysis of bromodeoxyuridine-labeled nascent DNA confirmed that active replication origins were methylated. These results suggest that a high-density cluster of mCpG dinucleotides may play a role in either the establishment or the regulation of mammalian replication origins.     

Serum estradiol-17 beta, progesterone and respective uterine cytosol receptor concentrations in bitches with spontaneous pyometra

The role of serum estradiol-17 beta (E(2)) and progesterone (P(4)) in relation to uterine estrogen (ER) and progesterone receptors (PR) was investigated in canine cystic endometrial hyperplasia-pyometra (CEH-P). Blood and uterine samples were collected pre- and post-ovariohysterectomy, respectively, from 54 bitches presenting spontaneous CEH-P and 25 healthy control bitches. Competitive enzyme immunoassays (EIA) and enzyme ligand immunoassays (ELIA) were applied to estimate serum hormones and uterine cytosol active receptors, respectively. Animals were classified in the stages of first half of diestrus, second half of diestrus and early anestrus on the basis of reproductive history, clinical signs, uterine and ovarian macro- and microscopic inspection and serum P(4) concentration. Bitches with CEH-P, compared to their respective stage controls, exhibited (a) similar P(4) fluctuations, (b) higher E(2) concentrations, (c) lower PR concentrations during diestrus first and second half and (d) lower ER concentrations during diestrus first half and early anestrus. Negative correlation was detected between P(4) and ER within both CEH-P and control groups. It was concluded that P(4) was the main uterine receptor regulator for both PR and ER during diestrus and early anestrus in healthy and affected uteri. However, in CEH-P bitches, high P(4) levels in diestrus appeared to over-activate uterine PRs, leading to stronger PR self-down regulation and ER suppression. These findings indicate an increased sensitivity of CEH-P uterus to P(4) action. During early anestrus, a complementary role of endogenous E(2) was considered, since reduction of P(4) action appeared to permit uterine ER replenishment and activation by relatively high E(2) levels.     

Urinary excretion of microelements in endurance-trained volunteers during restriction of muscular activity and chronic rehydration

The objective of this investigation was to determine the effect of daily intake of fluid and salt supplementation (FSS) on increased urinary losses of microelements that developed during hypokinesia (decreased number of walking steps/d). The studies were performed on 30 endurance-trained male volunteers aged 23–26 yr, with an averaged maximum oxygen uptake of 65 mL/kg/min during 364 d of hypokinesia (HK). All volunteers were divided into three equal groups: Ten volunteers were placed continuously under an average of 10,000 running steps/d (14.2 km/d) (control subjects), ten volunteers subjected continuously to HK without the use of FSS (hypokinetic subjects), and ten volunteers were continuously submitted to HK and consumed daily FSS (hyperhydrated subjects). For the simulation of the hypokinetic effect the hypokinetic and hyperhydrated volunteers were kept under an average of 3,000 walking steps/d (2.7 km/d) for 364 d. Prior to their exposure to HK the volunteers were on an average of 10,000 running steps/d (14.2 km/d). During the prehypokinetic period of 60 d and during the hypokinetic period of 364 d were determined renal excretion of microelements responses of endurance-trained volunteers. In the hyperhydrated volunteers urinary excretion of iron, zinc, copper, manganese, cobalt, nickel, lead, tin, chromium, aluminum, molybdenum, and vanadium decreased, whereas in the hypokinetic volunteers it increased significantly. It was concluded that chronic hyperhydration may be used to attenuate urinary excretion of microelements in endurance-trained volunteers during prolonged restriction of muscular activity.     

Blood plasma concentrations of microelements in endurance trained volunteers during hypokinesia and chronic hyperhydration

The objective of this investigation was to determine the effect of daily intake of fluid and salt supplementation (FSS) on increased urinary losses of microelements that developed during hypokinesia (decreased number of walking steps/d). The studies were performed on 30 endurance-trained male volunteers aged 23–26 yr, with an averaged maximum oxygen uptake of 65 mL/kg/min during 364 d of hypokinesia (HK). All volunteers were divided into three equal groups: Ten volunteers were placed continuously under an average of 10,000 running steps/d (14.2 km/d) (control subjects), ten volunteers subjected continuously to HK without the use of FSS (hypokinetic subjects), and ten volunteers were continuously submitted to HK and consumed daily FSS (hyperhydrated subjects). For the simulation of the hypokinetic effect the hypokinetic and hyperhydrated volunteers were kept under an average of 3,000 walking steps/d (2.7 km/d) for 364 d. Prior to their exposure to HK the volunteers were on an average of 10,000 running steps/d (14.2 km/d). During the prehypokinetic period of 60 d and during the hypokinetic period of 364 d were determined renal excretion of microelements responses of endurance-trained volunteers. In the hyperhydrated volunteers urinary excretion of iron, zinc, copper, manganese, cobalt, nickel, lead, tin, chromium, aluminum, molybdenum, and vanadium decreased, whereas in the hypokinetic volunteers it increased significantly. It was concluded that chronic hyperhydration may be used to attenuate urinary excretion of microelements in endurance-trained volunteers during prolonged restriction of muscular activity.