Cyclosporin A and FGF signaling support the proliferation/survival of mouse primordial germ cell-like cells in vitro†.

Primordial germ cells (PGCs) are the founding population of the germ cell lineage that undergo a multistep process to generate spermatozoa or oocytes. Establishing an appropriate culture system for PGCs is a key challenge in reproductive biology. By a chemical screening using mouse PGC-like cells (mPGCLCs), which were induced from mouse embryonic stem cells, we reported previously that forskolin and rolipram synergistically enhanced the proliferation/survival of mPGCLCs with an average expansion rate of ~20-fold. In the present study, we evaluated other chemicals or cytokines to see whether they would improve the current mPGCLC culture system. Among the chemicals and cytokines examined, in the presence of forskolin and rolipram, cyclosporin A (CsA) and fibroblast growth factors (FGFs: FGF2 and FGF10) effectively enhanced the expansion of mPGCLCs in vitro (~50-fold on average). During the expansion by CsA or FGFs, mPGCLCs comprehensively erased their DNA methylation to acquire a profile equivalent to that of gonadal germ cells in vivo, while maintaining their highly motile phenotype as well as their transcriptional properties as sexually uncommitted PGCs. Importantly, these mPGCLCs robustly contributed to spermatogenesis and produced fertile offspring. Furthermore, mouse PGCs (mPGCs) cultured with CsA ex vivo showed transcriptomes and DNA methylomes similar to those of cultured mPGCLCs. The improved culture system for mPGCLCs/mPGCs would be instructive for addressing key questions in PGC biology, including the mechanisms for germ cell migration, epigenetic reprogramming, and sex determination of the germline.

Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp.

A novel monoclonal antibody (MAb) against oxytetracycline (OTC) was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay (ELISA)-based detection system. An OTC-bovine serum albumin (BSA) conjugate was prepared and used in the immunization of mice. A conventional somatic cell fusion technique was used to generate MAb-secreting hybridomas denoted 2-4F, 7-3G, and 11-11A. An indirect competitive ELISA (icELISA) was applied to measure the sensitivity and specificity of each MAb in terms of its 50% inhibitory concentration (IC50) and percentage of cross-reactivity, respectively. MAb 2-4F exhibited the highest sensitivity, with an IC50 of 7.01 ng/ml. This MAb showed strong cross-reactivity to rolitetracycline, but no cross-reactivity to other unrelated antibiotics. When MAb 2-4F was used to detect OTC from shrimp samples, the recoveries were in the range of 82%-118% for an intra-assay and 96%-113% for an inter-assay. The coefficients of variation of the assays were 3.9%-13.9% and 5.5%-14.9%, respectively.

Unraveling the energetics and mode of the recognition of antibiotics tetracycline and rolitetracycline by bovine serum albumin.

An understanding of the detailed energetics and mechanism of the binding of drugs with target proteins is essential for devising guidelines to synthesize new drugs. Binding of the antibiotic drugs tetracycline and rolitetracycline with serum albumin has been studied by a combination of isothermal titration calorimetry, differential scanning calorimetry, steady-state and time-resolved fluorescence, and circular dichroism spectroscopies. Both tetracycline and rolitetracycline bind to bovine serum albumin in a sequential manner with first binding being the major binding event with an association constant of the order of 10(4) for tetracycline and 10(3) for rolitetracycline, respectively. Ionic strength dependence and binding in the presence of tetrabutylammonium bromide and sucrose indicate involvement of a mix of hydrophobic, ionic, and hydrogen bonding interactions. The isothermal titration calorimetry results for the binding of these drugs to bovine serum albumin in the presence of warfarin and in the presence of each other indicate that both these drugs share binding site 2 on bovine serum albumin. The differential scanning calorimetry results provide quantitative information on the effect of drugs on the stability of bovine serum albumin. A comparison of isothermal titration calorimetry and fluorescence results demonstrates that the former technique has been able to explain the sequential binding events that can be missed by the fluorescence measurements.

Reversed-phase high-performance liquid chromatography coupled to ultraviolet and electrospray time-of-flight mass spectrometry on-line detection for the separation of eight tetracyclines in honey samples.

Eight antibiotics, chlortetracycline, demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, tetracycline and rolitetracycline, were separated and quantified in Spanish honey extracts of different floral origin using a commercial RP-C18 HPLC column and two different on-line detectors (diode array and electrospray time-of-flight mass spectrometry (ESI-TOF-MS) systems). Operating a linear gradient at a flow of 2 ml min(-1) the HPLC separation of the eight antibiotics was obtained within 10 min with good peak symmetry and an acceptable resolution (2.1) for the critical band pair rolitetracycline and oxytetracycline. Values of the numbers of theoretical plates (N) were comprised between 2328 and 19448 while the limits of detection in honey were within 0.02-1.03 microg kg(-1) in the case of UV detection and 0.05-0.76 microg kg(-1) for ESI-TOF-MS detection (operating in negative mode). A recovery study was carried out by preparing some quality control samples at four levels of concentration (10, 25, 50 and 100 microg kg(-1)) and percentages between 72% and 98% were attained.

Trichloroacetic acid in Norway spruce/soil-system. II. Distribution and degradation in the plant.

Independently from its origin, trichloroacetic acid (TCA) as a phytotoxic substance affects coniferous trees. Its uptake, distribution and degradation were thus investigated in the Norway spruce/soil-system using 14C labeling. TCA is distributed in the tree mainly by the transpiration stream. As in soil, TCA seems to be degraded microbially, presumably by phyllosphere microorganisms in spruce needles. Indication of TCA biodegradation in trees is shown using both antibiotics and axenic plants.

4'-iodo-4'-deoxydoxorubicin and tetracyclines disrupt transthyretin amyloid fibrils in vitro producing noncytotoxic species: screening for TTR fibril disrupters.

Transthyretin Leu55Pro is one of the most aggressive mutations in familial amyloidotic polyneuropathy, an autosomal dominant disorder characterized by extracellular deposition of fibrillar amyloid protein. This variant has the ability to form fibrils in vitro under physiological conditions (PBS, pH 7.4). We studied by transmission electron microscopy the effect of the drug 4'-iodo-4'-deoxydoxorubicin (I-DOX) on the in vitro assembly of TTR Leu55Pro fibrils by following fibril growth over a 15 day period. Our results showed that I-DOX at a concentration of 10-5 M/100 microg fibrils does not inhibit fibril formation in up to 10 days since fibrils identical to the ones present in the untreated sample were observed. However, after 15 days of treatment, only round particles, resembling soluble native TTR, were observed. We also tested the ability of tetracyclines and nitrophenols to interfere with amyloid fibril formation for 17 days; the group of compounds tested showed fibril disruption activity to different extents: doxycycline and 2,4-dinitrophenol resulted in complete disaggregation of fibrils. The species generated upon I-DOX and tetracyclines treatments were nontoxic, as revealed by the lack of significant caspase-3 activation on a Schwannoma cell line, making them potential therapeutic drugs in TTR-related and other amyloidosis.

Fast rolitetracycline stability determination by spectrophotometer variable-temperature kinetic experiments.

The pseudo-first order rate constant for the hydrolysis of rolitetracycline as a function of temperature was obtained by performing variable-temperature kinetic experiments. The results are in agreement with those obtained at constant-parameter kinetics but saving experimental time and chemicals.

Disruption of pathologic amyloid beta-protein fibril assembly on the surface of cultured human cerebrovascular smooth muscle cells.

Cerebral amyloid beta-protein (Abeta) angiopathy (CAA) is a common pathological feature of Alzheimer's disease and several related disorders. In this condition, the accumulation offibrillar Abeta deposits is associated with degeneration of smooth muscle cells within the cerebral blood vessel wall. We have been using primary cultures of human cerebrovascular smooth muscle (HCSM) cells to investigate pathogenic mechanisms of Abeta in CAA. The specific assembly of Abeta fibrils on the surface of these cell types initiates several pathologic responses including increased expression and cell surface accumulation of the Abeta precursor protein (AbetaPP) and induction of apoptotic cell death. These pathologic responses are not observed with preparations of Abeta that are assembled into fibrils in solution, further underscoring the significance of the fibril assembly process on the cell surface. Since cell surface Abeta fibril assembly is the key initiator of the cerebrovascular cellular pathology that is observed in vitro, inhibition of this process remains an attractive therapeutic target for CAA. We have tested the efficacy of a variety of compounds that have been reported to inhibit Abeta fibril assembly in solution and block the neurotoxic properties of Abeta in vitro. The vast majority of these agents were ineffective in inhibiting the cell surface fibrillar assembly of Abeta and the subsequent pathologic responses in the cultured HCSM cells. This emphasizes the likely requirement of therapeutic compounds that are effective in disrupting cell surface-driven Abeta fibril assembly in the treatment of CAA.

Common structural features determine the effectiveness of carvedilol, daunomycin and rolitetracycline as inhibitors of Alzheimer beta-amyloid fibril formation.

One of the major pathological features of Alzheimer's disease is the deposition of beta-amyloid peptide (Abeta). Cellular toxicity has been shown to be associated with fibrillar forms of Abeta; preventing this fibril formation is therefore viewed as a possible method of slowing disease progression in Alzheimer's disease. With the use of a series of tetracyclic and carbazole-type compounds as inhibitors of Abeta fibril formation, we here describe a number of common structural features that seem to be associated with the inhibitory properties of these agents. Compounds such as carvedilol, rolitetracycline and daunomycin, which are shown to inhibit Abeta fibril formation, also prevent the formation of species of peptide that demonstrate biological activity in a human neuroblastoma cell line. Molecular modelling data suggest that these compounds have in common the ability to adopt a specific three-dimensional pharmacophore conformation that might be essential for binding to Abeta and preventing it from forming fibrils. Understanding such drug-peptide interactions might aid the development of disease-modifying agents.

[Effects some drugs of re-epithelialization in the early postoperative period after photorefraction keratectomy]. / Vliianie nekotorykh medikamentoznykh sredstv na reépitalizatsiiu v rannem posleoperatsionnom periode posle fotorefraktsionnoi keratoéktomii.

Terms of re-epithelialization, severity of the painful syndrome, intensity of corneal "crepe" (opacity) are assessed in myopic patients treated by maxitrol, eubetal, colbiocin ointments and maxitrol eyedrops in the early postoperative period after photorefraction keratectomy. The crepe intensity was assessed routinely according to a clinical score: 0) transparent cornea, 1) trace crepe; 2) moderate crepe; and 3) intensive crepe. Biomicroscopy on day 4 after photorefraction keratectomy showed complete epithelialization in 91.7% patients after colbiocin ointment, in 91.% after maxitrol eyedrops, 87% after eubetal ointment, and 82.6% after maxitrol ointment. The least corneal opacity (0 and 0-1) was observed after eubetal ointment and maxitrol eyedrops. The mean score for pain was virtually the same in all groups; in the colbiocin ointment group more patients complained of pain for more than 24 h in comparison with other groups.

[Eye ointments eubetal and colbiocin in the treatment of chlamydial conjunctivitis]. / Glaznye mazi "Eubetal" i "Kolbiotsin" v lechenii khlamidiinykh kon"iunktivitov.

The above ointments were used for local treatment of chlamydial conjunctivitis in 325 patients. The results demonstrated their high efficacy, good tolerance, advantages over tetracycline ointment, and absence to toxic allergic reactions.

Wound modulation via sclerotherapy and tissue adhesion. Observations and discussion.

Fluid accumulation in a 'dead space' after surgery or trauma predisposes the area involved to delayed healing. The fluid may promote inflammatory mediators and other wound-modulating factors, which encourages the continued existence of the dead space. Tissue adhesion by means of agents such as tetracyclines has long been described. With this concept, tissue adhesion has been accomplished with varying success in diverse clinical situations involving non-healing wounds and prophylactically in seroma-prone areas. A total of 69 patients were treated and monitored. The most impressive response to tetracycline tissue adhesion in this study was seen in intra-oral sites, with rapid closure of wounds. More complex wounds such as orocutaneous fistulas or wounds of a more chronic nature such as pressure sores did respond but took longer to resolve. The lateral thigh was found to be the most difficult and unresponsive area to treat (particularly after delayed presentation). Successful prophylactic use of tetracycline in seroma-prone areas (latissimus dorsi/parascapular flaps, etc.) is also described.

[The intraoperative visualization of the bile ducts by the use of fluorescent substances. A feasibility study]. / Visualizzazione intraoperatoria delle vie biliari mediante impiego di sostanze fluorescenti. Studio di fattibilità.

Iatrogenic bile duct injury during cholecystectomy is the most serious complication of this surgical procedure. Initial reports suggest that this complication is particularly problematic during laparoscopic cholecystectomy. Proper identification of biliary anatomy in the subhepatic region is the only way to avoid this severe complication. The potential benefit from a simple, reliable method for intraoperative delineation of biliary anatomy is self-evident. In this experimental work the Authors-study the possibility and the feasibility of intraoperative biliary tree imaging with two fluorescent molecules (rolitetracyclin and fluorescin).

Species specific pharmacokinetics of rolitetracycline.

Comparative studies on some selected pharmacokinetic parameters of rolitetracycline (effective antimicrobial activity levels) in sheep, pigs, rabbits, chickens and pigeons were carried out after intravenous administration of the drug at a dose of 5 mg/kg. The results revealed that a two-compartment open model was usually optimal. Interspecies differences in the rate (alpha) and the volume of distribution (Vda), the area under the serum concentration time curve (AUC) and the total body clearance (ClB) were small. The values of the elimination half-life (t1/2 beta) found in sheep, pigs, rabbits, chickens and pigeons were 1.51, 3.06, 2.18, 4.33 and 2.51 h respectively. These results and data in mice, rabbits and man taken from literature were analysed to determine any correlation to body mass (W). The log of the elimination half-life values (log t1/2 beta) of rolitetetracycline revealed a significant correlation with log values of (W) between animal species. This relationship was described by the equation t1/2 beta = 1.27.W0.29 (r = 0.93, P < 0.01, n = 5) for mammal species and t1/2 beta = 1.81.W0.24 (r = 0.78, P < 0.05, n = 7) for mammal and bird species.

[The effect of rolitetracycline on the level of nonesterified fatty acids, phospholipids, and triacylglycerols in ischemic damage to the myocardium]. / Vliianie rolitetratsiklina na uroven' neésterifitsirovannykh zhirnykh kislot, fosfolipidov i triatsilglitserinov pri ishemicheskom povrezhdenii miokarda.

Myocardial ischemia developed in rabbits after ligation of coronary artery within 60 min. Single dose of rolytetracycline 40 mg/kg was administered intravenously before or after ligation. Alterations in content of unesterified fatty acids and phospholipids were studied in blood serum as well as of unesterified fatty acids, phospholipids and triacyl glycerols--in the myocardium. After ligation of coronary artery content of unesterified fatty acids and phospholipids was increased 2-fold in blood serum as compared with control values, while in the ischemic myocardium these substances were increased more than 2-fold and triacyl glycerol--about 7-fold. Administration of rolytetracycline before ligation of the artery led to a decrease in content of unesterified fatty acids and phospholipids in blood serum down to control values; the ligation-induced high content of triacyl glycerols and phospholipids in the myocardium was also decreased, while alterations in unesterified fatty acids content were not similar. Rolytetracycline administered after the artery ligation caused a decrease in content of unesterified fatty acids but triacyl glycerols were markedly increased. The antibiotic did not affect the lipid content in nonischemized tissues of myocardium.

[Penetration of antibiotics into the intervertebral disk]. / Penetrace antibiotika do intervertebrálního disku.

The role of antibiotics in the treatment and prevention of postoperative spondylodiscitis is still controversial. In a group of 15 patients operated on account of lumbar discopathy, the authors investigated the penetration of antibiotics into the intervertebral disc, in correlation to serum levels, when antibiotics were administered during operation. Despite high serum levels rolitetracycline was not detected in extirpated material of the disc, gentamycin reached 2-6% serum levels. The authors give an account of views on the role of antibiotics in the prophylaxis of postoperative spondylodiscitis and recommend their administration in particular in risk groups. They recommend antibiotics in case of early diagnosis of postoperative spondylodiscitis, in the later stages of the disease the role of antibiotics is controversial.

[The effect of age on pharmacokinetic parameters of oxytetracycline and rolitetracycline in rabbits]. / Vplyv veku králikov na niektoré farmakokinetické parametre oxytetracyklínu a rolitetracyklínu.

The effect of rabbit age in the Californian White breed was investigated on oxytetracycline and rolitetracycline concentrations in the blood serum and soft tissues, and on the biological half-life of the given antibiotics. The animals were at the age shortly before sexual maturity or they had reached sexual maturity. Oxytetracycline was administered in form of the preparation Oxymykoin inj. a. u. v. Spofa and roliteracycline nitrate in form of the preparation Rolitetracycline inj. Spofa. These tetracyclines were administered intramuscularly at a single dose of 20 mg per kg liveweight. In comparison with adult animals, in young rabbits the starting concentrations of the two tetracyclines in the blood (first hour) were statistically insignificantly higher; they were resorbed in the systemic circulation in a shorter time and were excreted faster from the blood (mainly oxytetracycline). The significantly lower serum concentrations were recorded in the 6th, 12th and 24th hour after oxytetracycline administration, and in the 12th and 24th hour after rolitetracycline administration. At the same time a decrease was observed in the concentrations of the two oxytetracyclines in the organs and muscles of young animals.

Sclerotherapy for hydroceles.

Sclerotherapy with 3% sodium tetradecyl sulfate and 3.5% rolitetracycline on an outpatient basis was applied to 55 hydroceles. The over-all cure rate was 96% with an average followup of 13 months. Of the patients 64% were cured after only 1 sclerosant instillation. A post-sclerotherapy operation was necessary in 4% of the patients. Pain of a significant degree occurred after sclerotherapy in only 29% of the patients. Sclerotherapy appears to be an effective, economical and safe form of outpatient therapy for hydroceles.

[The of effect of rolitetracycline on oxygen consumption in a suspension of E. coli]. / Vliv rolitetracyklinu na spotrebu kysliku suspenzi E. coli.

Authors studied the oxygen demand by E. coli suspensions (serotypes 023 and 026) modifying it with various concentration of Rolitetracyclin, and proceeded by comparison of both serotypes. Time dependence of oxygen demand has been determined using the direct Warburg's manometric method. The obtained results are summerized as following four points: 1. Tested Rolitetracyclin doses caused the decrease in oxygen demand of E. coli suspensions significantly for the majority of time intervals. Rolitetracyclin actioned similarly in both serotypes. 2. Rolitetracyclin 0.5 microgram concentration actioned bacteriostatically on both serotypes, and showed more pronounced action on E. coli 026 serotypes with higher metabolic activity. 3. Rolitetracyclin 5 and 50 micrograms bactericidal concentrations inhibited markedly the oxygen demand in both serotypes. 4. Using this method allowed to determine the difference in metabolic activity of both E. coli serotypes tested.