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.

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.

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.

[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.

Efeitos da N(Pirrolidino-Metil)-Tetraciclina sobre o germe dentário do incisivo superior da preá (Cavia Aperea Aperea) Erxleben - 1777 (Rodentia)* / Effects of the tetracydine about the tooth germ of the upper incisor in the preas

Na presente pesquisa foram estudados os efeitos da tetraciclina sobre o germe dentário do incisivo superior nos filhotes de preás, logo após o nascimento e com três dias de idade. Administrou-se a droga durante a prenhez, em diversos períodos, bem como em filhotes com três dias de idade

[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.

In vitro and in vivo effects of combinations of cefotaxime or other beta-lactams with rolitetracycline on methicillin-resistant Staphylococcus aureus.

Combinations of cefotaxime (CTX) or other five beta-lactams with rolitetracycline (RTC) were examined using the checkerboard method for their synergistic effects against 27 strains of methicillin-resistant Staphylococcus aureus (MRSA), and the combination of CTX and RTC was further evaluated for its synergistic effect in vitro and in vivo against 1 or 2 strains. Synergy occurred against 44% of the strains when RTC was combined with CTX, 22 to 30% with cefazolin, methicillin, and ceftizoxime, and 4 to 11% with latamoxef and cefmetazole. No antagonism was found with any combinations tested. Killing curve studies also showed that CTX/RTC was synergistic between 3 and 24 hours after the beginning of exposure, and the synergy was especially strong at 24 hours and potencies of combined bactericidal effect determined at 24 hours were in the following order: the 2 antibiotics given simultaneously, CTX given 2 hours before RTC, and CTX given 2 hours after RTC. In addition, the 2 drugs in combination synergistically inhibited (a) mortality in mice infected intraperitoneally with MRSA and (b) formation of subcutaneous abscess induced by MRSA in mice. The results of our study indicate that beta-lactams, especially CTX, had synergistic effects in vitro when combined with RTC against MRSA and that the combination of CTX and RTC was also synergistic in vivo.

[Effect of tetracyclines on protein metabolism in cultured hepatocytes].

The purpose of the present study was to investigate the effect of Tetracyclines (TCs); Minocycline (MINO), Tetracycline (TC), Demethylchlortetracycline (DMTC), Pyrrolidinomethyltetracycline (PMTC), on amino acid transport and incorporation in hepatocytes. By the use of liver cell culture, this study was done without complex interactions taking place in whole animals. MINO inhibited most strongly both uptake and incorporation into cellular protein of 3H-L-leucine in cultures of a clonal strain of rat hepatoma cells. This inhibitory effect of MINO was proved to be dose-dependent and reversible. The order of the inhibitory effect of TCs was follows; MINO, DMTC, TC, PMTC. But this inhibitory effect showed no significant correlation with the intracellular content of TCs. Almost the same degree as the inhibition by TCs of 3H-L-leucine incorporation into protein was noted when measuring total intracellular 3H-L-leucine reduction by TCs. These results suggest that the TCs-induced liver dysfunction is probably due to the inhibitory effect of TCs on amino acids uptake followed by inhibition of protein synthesis.

Combined effects of high doses of rolitetracycline and testosterone on the liver of mice.

The effects of rolitetracycline (50 micrograms/g i.v.) and testosterone proprionate (4 X 1 micrograms/g s.c.) singly and combined on liver weight, serum GPT, SDH and bromsulfophthalein clearance have been measured in young mice. Pretreatment with testosterone tended to enhance the effects of rolitetracycline on the serum enzymes but was otherwise not effective.

[Morphological changes induced by the addition of various antibiotics to lens epithelium cultures]. / Morphologische Alterationen durch Zusatz verschiedener Antibiotika zu Linsenepithelkulturen.

Morphological and biochemical characteristics of surviving and cultured bovine lens epithelia in TCM 199 with 20% calf serum have been described several times in the authors previous publications. The injurious action of different substances in vitro was observed: gentamicin, penicillin and tetracycline. Even in common concentrations there were side effects of various types and degrees. This could be important in washing donor material or the anterior chamber.

Comparative evaluation of the effects of tetracycline, rolitetracycline and doxycycline on some blood parameters related to liver function.

A comparative study was made on the effects of tetracycline (TC), rolitetracycline (RTC), and doxycycline (DC) at doses of 10-100 micrograms/g i.v. on serum GOT, GPT, bilirubin and urea levels of male and female mice. All tetracyclines at doses of 50-100 micrograms/g caused an increase of serum GOT and GPT activities in females after 2-8 h. This effect was less pronounced in males. The serum urea levels were markedly raised by TC and DC (50-100 micrograms/g) and RTC (10-100 micrograms/g). This effect was produced only in females. It was long-lasting (up to 8 h) after TC and RTC and more transient after DC. Likewise the tetracyclines affected the serum bilirubin only in females but not in males. All tetracyclines (25-100 micrograms/g) increased the amount of unconjugated and total bilirubin. This effect was most pronounced after TC, which also reduced the level of conjugated bilirubin.

Age dependency of rolitetracycline-induced hepatic steatosis.

A comparative study on the effect of rolitetracycline (50 micrograms/g i.v.) on the hepatic content of triglycerides and the release of esterified fatty acids from the liver into the blood was performed in young and adult NMRI mice of both sexes. Rolitetracycline caused an accumulation of triglycerides only in the livers of adult animals, the effect being much more pronounced in females than in males. In agreement with this a very strong inhibition of lipid release from the liver and a significant decrease of the esterified fatty acids of the serum was produced only in adult females. The results suggest that the livers of young animals of both sexes are relatively resistant to the steatotic effect of rolitetracycline.

[Antibacterial substances in tamponades after tympanoplasties - an "in-vitro"-study (author's transl)]. / Anwendung antimikrobieller Substanzen in Gehörgangstamponaden. Ein "in-vitro"-Studie.

This "in-vitro"-study is concerned with the efficiency of antibacterial agents used in packings after tympanoplasties. For this purpose, gel-foam-material was impregnated either with rolitetracyclin (mostly used in German ENT-clinics), or comparatively with a combination of bacitracin and neomycin or with antiseptics like phenylmercuriborate and povidon-iodine. It is tested, how these impregnated "packings" behave in the presence of pathogenic germs (Staph. aureus, Ps. aeruginosa). Antibacterial effects and alterations of the material were judged. It is shown, that in some regards the rolitetracyclin - very successful in clinical use - is superior to the other substances tested.

[On the circulation compatibility of doxycycline and rolitetracycline in conscious minipigs (author's transl)]. / Untersuchungen der Kreislaufverträglichkeit von Doxycyclin und Rolitetracyclin an wachen Zwergschweinen.

The effect of large doses of doxycycline (Vibravenös) and rolitetracycline on blood pressure, heart rate and ECG was investigated in 36 conscious minipigs. The antibiotics were infused into the v. tibialis at doses of 16, 24 and 32 mg/kg at injection periods of 4, 6 and 8 min. The dosage applied was about 10-20 times higher than the therapeutical single dose of doxycycline and about 4-8 times higher than the single dose of rolitetracycline. 16 mg doxycycline or rolitetracycline/kg resulted in a minor short-lasting increase of blood pressure and extension of the P-Q interval in the ECG. The definitely pathological P-Q value of 160 ms was not observed in any of the cases treated with doxycycline, whereas rotitetracycline led to clearly pathological P-Q values in 2/6 of the cases, and to AV block in 4/6 of the cases. Neither after doxycycline nor after rolitetracycline did the increased dose of 24 and 32 mg/kg result in an enhanced effect on circulation. However, both the blood pressure and the P-Q values following 32 mg rolitetracycline/kg significantly exceeded those following 32 mg doxycycline/kg. These trials suggest that with regard to circulatory tolerance there are no objections to increasing the usual therapeutical dose of intravenous doxycycline if the injection time is adequately extended.

[Tetracyclines: bacteriostatic or bactericidal drugs? In vitro studies with rolitetracycline, minocycline and doxycycline (author's transl)]. / Tetracycline: bakterizid oder bakteriostatisch? In vitro-Untersuchungen mit Rolitetracyclin, Minocyclin und Doxycyclin.

Minimal bacteriostatic and minimal bactericidal concentrations of rolitetracycline, minocycline and doxycycline on 20 different E. coli serotypes and 16 staphylococcus aureus strains have been compared in bouillon and serum. In addition growth curves of all strains in bouillon and serum without and with antibiotics in concentrations corresponding to the minimal inhibitory concentration of each strain have been followed for 24 hours. In E. coli minimal bactericidal concentrations of all 3 tetracyclines in bouillon on average were only twice as high as the minimal bacteriostatic concentrations of the drugs, tested. Minimal bacteriostatic and bactericidal concentrations in serum were significantly lower than in bouillon. 27 out of 20 E. coli strains were inhibited by less than 0.012 microgram/ml. In staphylococci minimal bactericidal concentrations were substantially higher than the minimal bacteriostatic concentrations (on average at least four times higher). In E. coli addition of serum increased the antimicrobial activity of all tetracyclines tested. Growth curves in bouillon tended to show some bactericidal shape at 3, 7 and 24 hours following addition of the tetracyclines at minimal bacteriostatic concentrations. Bactericidal activity of tetracyclines at the MIC was substantially improved by addition of serum. This was not true for staphylococci in which neither minimal bactericidal and bacteriostatic concentrations nor growth curves could be significantly improved by addition of serum.