Expression of Cre recombinase during transient phage infection permits efficient marker removal in Streptomyces

We report a system for the efficient removal of a marker flanked by two loxP sites in Streptomyces coelicolor, using a derivative of the temperate phage C31 that expresses Cre recombinase during a transient infection. As the test case for this recombinant phage (called Cre-phage), we present the construction of an in-frame deletion of a gene, pglW, required for phage growth limitation or Pgl in S.coelicolor. Cre-phage was also used for marker deletion in other strains of S.coelicolor.     

Biological effect of mono- and dinuclear alkylamine platinum(II) compounds on human lymphoma cells

The effects of the mononuclear chloro[meso-1,2-bis(4-fluorophenyl)ethylenediamine][hexylamine]platinum(II) chloride HACl and the dinuclear di[meso-1,2-bis(4-fluorophenyl)ethylenediamine]dichloro(mu-1,n-diaminoalkane-N:N')diplatinum(II)dichloride complexes DAHCl (alkane:hexane), DANCl (alkane:nonane) and DADCl (alkane:dodecane) with different alkyl chain length (n) were investigated on non-Hodgkin's lymphoma (NHL) and chronic myeloid leukemia (CML) cell lines. All compounds showed an antiproliferative effect on the NHL cell lines RAJI and U-937 accompanied in the case of DANCl, DAHCl, HACl and cisplatin by an increase in apoptosis. The growth of another NHL (JEKO-1) and one CML cell line (K-562) was decreased only by cisplatin. In contrast to HACl, DAHCl, DANCl and cisplatin, DADCl induced necrosis, suggesting toxicity because cell viability decreased. Similar effects were observed when bone marrow-derived lymphoma cells from a patient with high-grade B-NHL were incubated with the platinum complexes.     

Head and pelvic vertical displacement in dogs with induced swinging limb lameness: an experimental study

Background

Swinging limb lameness is defined as a motion disturbance ascribed to a limb in swing phase. Little is known about its biomechanics in dogs, particularly about the body motions that accompany it, such as vertical head and pelvic motion asymmetry. The aim of this study was to describe the changes in vertical head and pelvic motion asymmetry in dogs with induced swinging limb motion disturbance, mimicking a swinging limb lameness. Fore- and hind-limb lameness was induced in ten sound dogs by placing a weight (200 g) proximal to the carpus or tarsus, respectively. Marker-based motion capture by eight infrared light emitting video cameras recorded the dogs when trotting on a treadmill. Body symmetry parameters were calculated, including differences between the two highest positions of the head (HDmax) and pelvis (PDmax) and between the two lowest positions of the head (HDmin) and pelvis (PDmin), with a value of zero indicating perfect symmetry.

Results

Induction of swinging forelimb lameness showed significant changes in HDmax (median and range: sound 1.3 mm [??4.7 to 3.1], in the left side ??28.5 mm [??61.2 to ??17.9] and in the right side 20.1 mm [??4.4 to 47.5]) and, induction of swinging hind limb lameness showed significant changes in PDmax (sound 2.7 mm [??7.4 to 7.2], in the left side ??10.9 mm [??22.4 to 0.5] and in the right side 8.6 mm [??3 to 30]), as well as an increased hip movement asymmetry (sound 1.6 mm [??8.6 to 19.9], in the left side ??18.1 mm [??36.7 to 5.4] and in the right side 15 mm [??20.7 to 32.1]) (P?<?0.05).

Conclusions

Induced swinging fore- and hind limb lameness resulted in significant increased asymmetry of the maximal vertical displacement movement of the head and pelvis, due to decreased lifting of the head in forelimb lameness and of the pelvis in hind limb lameness. The results suggest that asymmetry of the maximal vertical displacement of the head and pelvis (i.e. lifting) is a key lameness sign to evaluate during examination of swinging limb lameness.

     

Novel B-ring modified allocolchicinoids of the NCME series: design,synthesis, antimicrotubule activity and cytotoxicity

Two new series of allocolchicinoids mimicking the structure of (-)-N-acetylcolchinol O-methyl ether (2, NCME) were synthesized and evaluated for their abilities to inhibit tubulin assembly. Possible antitumor properties resulting thereof were evaluated in vitro on the human MCF-7 breast cancer cell line. The first series of NCME-derivatives was brought about by extending the seven membered B-ring to novel semisynthetic variations with a nitrogen containing eight-membered B-ring similar, for example, to the artificial, potent steganacin aza-analogue 3. In the second series the seven-membered B-ring of NCME (2) was modified by annulation with a heterocyclic ring system. The racemic ketone 7a serving as key precursor involved in the syntheses of all the target NCME variants 9-13 and 15, 16 was easily transformed into the eight-membered B-ring lactams 9 and 10 via a Beckmann rearrangement of the corresponding E-oxime 8. The tetrazole annulated congener 11 was prepared via azidotrimethylsilane-mediated Schmidt rearrangement. Treatment of educt 7a with Bredereck's reagent led to the enamino ketone 14, which was easily converted into the pyrazole- or pyrimidine-annulated allocolchicinoids 15 and 16. Remarkably, all the allocolchicinoids 9-13 with an azocin-B-ring affected the tubulin/microtubule equilibrium only moderately. In contrast, the novel heterocycle annulated seven membered B-ring variants 15 and 16 proved to be highly potent tubulin-inhibitory, antimitotic agents. Interaction with tubulin occured at concentrations similar to those observed for colchicine (1) or the lead NCME (2). In all cases the antiproliferative effects correlated roughly with the inhibition of tubulin assembly.     

Antiglioma activity of GoPI-sugar,a novel gold(I)–phosphole inhibitor: Chemical synthesis,mechanistic studies,and effectiveness in vivo

Glioblastoma, an aggressive brain tumor, has a poor prognosis and a high risk of recurrence. An improved chemotherapeutic approach is required to complement radiation therapy. Gold(I) complexes bearing phosphole ligands are promising agents in the treatment of cancer and disturb the redox balance and proliferation of cancer cells by inhibiting disulfide reductases. Here, we report on the antitumor properties of the gold(I) complex 1-phenyl-bis(2-pyridyl)phosphole gold chloride thio-β-d-glucose tetraacetate (GoPI-sugar), which exhibits antiproliferative effects on human (NCH82, NCH89) and rat (C6) glioma cell lines. Compared to carmustine (BCNU), an established nitrosourea compound for the treatment of glioblastomas that inhibits the proliferation of these glioma cell lines with an IC₅₀ of 430 μM, GoPI-sugar is more effective by two orders of magnitude. Moreover, GoPI-sugar inhibits malignant glioma growth in vivo in a C6 glioma rat model and significantly reduces tumor volume while being well tolerated. Both the gold(I) chloro- and thiosugar-substituted phospholes interact with DNA albeit more weakly for the latter. Furthermore, GoPI-sugar irreversibly and potently inhibits thioredoxin reductase (IC₅₀ 4.3 nM) and human glutathione reductase (IC₅₀ 88.5 nM). However, treatment with GoPI-sugar did not significantly alter redox parameters in the brain tissue of treated animals. This might be due to compensatory upregulation of redox-related enzymes but might also indicate that the antiproliferative effects of GoPI-sugar in vivo are rather based on DNA interaction and inhibition of topoisomerase I than on the disturbance of redox equilibrium. Since GoPI-sugar is highly effective against glioblastomas and well tolerated, it represents a most promising lead for drug development. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.     

Recognition of envelope and tat protein synthetic peptide analogs by HIV positive sera or plasma

A series of synthetic peptides corresponding to segments of HIV encoded proteins were selected using criteria described by Welling et al. [(1985) FEBS Lett. 188, 215]. Synthetic peptide analogs to gp120 (2-13), (55-65), gp41 (582-596) (659-670) and tatIII (71-83) were recognized by 41-67% of sera or plasma from individuals known to be infected with HIV on the basis of virus isolation or Western blot screening. The peptide which reacted with most sera or plasma was gp41 (582-596), a conserved region in the transmembrane glycoprotein. An extended peptide analog, gp41 (579-599), tested against the same samples showed almost 100% reactivity, confirming independent studies identifying a highly immunodominant region of gp41. There was an unexpected high prevalence of antibodies (25%) to the tatIII peptide.     

Breast cancer inhibiting diastereomeric diacetato[1,2-bis(4-fluorophenyl)ethylenediamine]platinum(II) derivatives: synthesis and studies on the relationship between reactivity and antitumor activity

Antitumor active [1,2-bis(4-fluorophenyl)ethylenediamine]platinum(II) diastereoisomers containing acetic acid derivatives as ‘leaving groups’ (acetate: meso/rac-4F-Pt(Ac)₂; monochloroacetate: meso/rac-4F-Pt(ClAc)₂; dichloroacetate: meso/rac-4F-Pt(Cl₂Ac)₂; trichloroacetate: meso/rac-4F-Pt(Cl₃Ac)₂; glycolate: meso/rac-4F-Pt(OHAc)₂; phenylacetate: meso/rac-4F-Pt(PhAc)₂) were synthesized and characterized by IR and ¹H NMR spectroscopy. In all complexes except meso/rac-4F-Pt(PhAc)₂, which exist as [meso/rac-4F-PtPhAc]⁺PhAc⁻, both carboxylic acid residues are coordinated to platinum. Kinetic studies on the reaction behavior of the title compounds with nucleophiles were performed by using iodide as nucleophile. The studies show that the new complexes react with nucleophiles predominantly via the ‘solvent path’ (i.e. via the reactive intermediates = Pt(X)(OH₂)⁺ and =Pt(OH₂)₂²⁺. Therefore the rates of reactions in which the reactive species are formed affect the antitumor activity of the complexes as well as their inactivation by bionucleophiles during the transport to the tumor. The extent of accumulation in the tumor cell, too, influences the antitumor activity of a complex. The rate constants are discussed in view of the activities of the respective complexes on the human MCF-7 breast cancer cell line. From the title compounds the Cl₂Ac and Cl₃Ac derivatives do not come close to the standard cisplatin, neither in chemical reactivity nor in biological activity. Meso/rac-4F-Pt(Ac)₂ and meso/rac-4F-Pt(ClAc)₂, respectively, show similar hydrolysis rates but lower antitumor activities than cisplatin, presumably due to a reduced drug uptake by the tumor cell. Meso/rac-4F-Pt(PhAc)₂ compare well with their standard carboplatin in respect to both properties. Other than the remaining, poorly water soluble title compounds, meso/rac-4F-Pt(OHAc)₂ equal their standard cisplatin in terms of water solubility and antitumor activity rac-4F-Pt(OHAc)₂ > meso-4F--Pt(OHAc)₂). However, they are markedly faster hydrolyzed than cisplatin. By use of rac-4F-Pt(Ac)₂ as an example it was confirmed that, in contrast to the parent compound rac-4F-PtCl₂, the new complex type is also active under in vivo conditions owing to its markedly lower reactivity (mainly due to the lack of a direct substitution by strong nucleophiles), which entails a reduced inactivation of the drug on its way to the tumor. The in vitro testing on tumor cell lines combined with the evaluation of the water solubility and with kinetic studies on the reaction with nucleophiles is a useful method for the preselection of potent platinum complexes deserving further thorough in vitro and in vivo investigations.     

Male dominance rank and offspring-initiated affiliative behaviors were not predictors of paternity in a captive group of pigtail macaques (Macaca nemestrina)

Paternity of 16 pigtail macaque offspring was determined using a DNA profile analysis and was based on two independent assays of the genome of each individual using multilocus DNA probes. The offspring were members of a group of 59 pigtail macaques, including 5 adult males, 1 subadult male, and 37 adult and subadult females. Rank was unrelated to paternity as the first ranking male sired 0, the second ranking male sired 3; the third ranking male sired 0, the fourth ranking male sired 8, and the fifth ranking male sired 2 offspring. The subadult male sired 0 offspring. The DNA analysis was effective in excluding possible sires of 3 offspring whose mothers had become pregnant by another male before being introduced to the study males. Subsequent semen evaluation revealed an absence of sperm in the semen of the alpha male, but revealed a sperm count within normal limits in the third ranking male, who also sired no offspring. Behavioral data focusing on male-offspring interactions found that offspring did not preferentially affiliate with their sire and that males did not affiliate with their offspring frequently enough for analysis. Thus, this study of one captive pigtail macaque group demonstrates that: (1) rank was not a predictor of reproductive success; and (2) there was no preferential attraction for one's own offspring by males or one's own sire by offspring.