Sequence preferences of DNA interstrand crosslinking agents: quantitation of interstrand crosslink locations in DNA duplex fragments containing multiple crosslinkable sites.

A general approach to the quantitative study of the sequence specificity of DNA interstrand crosslinking agents in synthetic duplex DNA fragments is described. In the first step, a DNA fragment previously treated with an interstrand crosslinking agent is subjected to denaturing PAGE. Not only does this distinguish crosslinked from native or monoadducted DNA, it is shown herein that isomeric crosslinked DNAs differing in position of the crosslink can in some cases be separated. In the second stage, the now fractionated crosslinked DNAs isolated from denaturing PAGE are subjected to fragmentation using iron(II)/EDTA. For those fractions which are structurally homogeneous, analysis of the resulting fragment distribution has previously been shown to reveal the crosslink position at nucleotide resolution. It is shown herein that in fractions which are structurally heterogeneous due to differences in position of crosslink, this analysis quantifies the relative extent of crosslinking at distinct sites. Using this method it is shown that reductively activated mitomycin C crosslinks the duplex sequences 5'-GCGC and 5'-TCGA with 3 +/- 1:1 relative efficiency.     

Sites of ecdysteroid biosynthesis in female adults of Gryllus bimaculatus (Ensifera,Gryllidae)

Summary Ovaries from 4-day-old female adults of Gryllus bimaculatus produce about 5 ng of free and conjugated ecdysteroids per hour during a 16-h incubation in Grace's medium. During incubation of pieces of the abdominal integument together with the adjacent segmental fat body, a net synthesis of moulting hormones is observed (2.3 ng per hour per animal), similar to that in the ovary. Separate incubations of disunited abdominal epidermis and segmental fat body tissue result in much lower rates of ecdysteroid synthesis. Ecdysteroid synthesis in ovarian homogenates is about one-third of that in intact organs. This reduction is due to a lack of conjugate formation in homogenates. Homogenates of the abdominal integument complex are no longer capable of synthesizing ecdysteroids. For both tissues, a de novo synthesis of ecdysteroids is corroborated by following the in vitro incorporation of [¹⁴C]-label from cholesterol and [³H]-label from 2,22,25-trideoxyecdysone (5-ketodiol), respectively, into free ecdysone. The rate of incorporation into ecdysone is only 0.0014% for cholesterol but 0.48% for 5-ketodiol. Both tissues represent primary sources of ecdysteroids in female adult crickets.Abbreviations HPLC high performance liquid chromatography - IU international units - NP normal phase - RIA radioimmunoassay - RP reversed phase - SEM standard error of mean     

Nitrate reductase in needles,roots and trunk wood of spruce trees [Picea abies (L.) Karst.]

Summary Nitrate reductase (EC 1.6.6.2) activity (NRA), as measured by an in vivo assay, is present in needle leaves and mycorrhizal fine root tips of adult Norway spruce [Picea abies (L.) Karst.] in at least equal amounts on a fresh weight basis, in both adult and 5-year-old trees. NRA could also be demonstrated in trunk wood of deroted trees after fertilization with 5 mM , exhibiting a longitudinal profile in the trunk. Inducibility in needles can more efficiently be achieved by NO₂ (100 g·m^-3) than by 5 mM nitrate, which is effective only in root-amputated trees. A remarkably high level of needle-NRA in unfertilized trees, which are characterized by a very low level of nitrate in the xylem sap, suggests that NRA in spruce needles may in part be constitutive. Organic-N is a major nitrogen source for the needles even in root-amputated trees, indicating pronounced exchange processes between ray parenchyma and trunk xylem, which in turn are modified by the nitrogen source fed to the trunk stump. Intact trees exhibit a very similar amino acid composition of the xylem sap, regardless of whether or has been fed. The amino acid pattern of the needles is not thrown out of balance by flooding with and , which occurs in fertilized derooted trees. This indicates a distinct potential for homoeostasis of nitrogen entrance-metabolism (i.e. NRA and glutamine synthetase activity) in the needles. In the ectomycorrhiza/fine root-system (EMC), marked differences in NRA were observed depending on root-tip diameter and along the longitudinal profile of the fine roots. EMC-nitrate reductase is strongly enhanced by . Needle-NRA exhibits a circannual rhythm. An early summer maximum is followed by a December minimum. This activity pattern matches well the transitory increase of soluble nitrogen in spring and the total protein maximum in winter. In an indirect way assimilatory NRA may well contribute to nitrogen overfertilization (by consumption of NO_X) as one possible cause of the contemporary decline of spruce populations.     

Blood clotting factor IX. Loss of activity after cleavage of sialic acid residues

Enzymatic cleavage of sialic acid from human blood clotting factor IX results in a loss of factor IX clotting activity. The loss of clotting activity and the rate of release of sialic acid follow the same time courses. Control experiments have ruled out several explanations for the loss of factor IX activity: proteolytic degradation, inhibitory effects of free sialic acid, and non-specific inhibition of the clotting assays. Furthermore, no inhibition was seen when similar enzymatic cleavage was carried out on factor X and factor VIII. Therefore, we suggest that the loss of factor IX activity is the direct result of cleavage of sialic acid from the protein. Most of the inhibition appeared to be an effect on the activity of factor IXa itself, and thus far, little or no effect has been shown on the activation of factor IX to IXa. The structural basis for this unusual effect of sialic acid on protein function currently is being investigated.     

Influence of T-2 and HT-2 Toxin on the Blood-Brain Barrier In Vitro: New Experimental Hints for Neurotoxic Effects

The trichothecene mycotoxin T-2 toxin is a common contaminant of food and feed and is also present in processed cereal derived products. Cytotoxic effects of T-2 toxin and its main metabolite HT-2 toxin are already well described with apoptosis being a major mechanism of action. However, effects on the central nervous system were until now only reported rarely. In this study we investigated the effects of T-2 and HT-2 toxin on the blood-brain barrier (BBB) in vitro. Besides strong cytotoxic effects on the BBB as determined by the CCK-8 assay, impairment of the barrier function starting at low nanomolar concentrations were observed for T-2 toxin. HT-2 toxin, however, caused barrier disruption at higher concentrations compared to T-2 toxin. Further, the influence on the tight junction protein occludin was studied and permeability of both toxins across the BBB was detected when applied from the apical (blood) or the basolateral (brain) side respectively. These results clearly indicate the ability of both toxins to enter the brain via the BBB.     

Iberis amara Extract Induces Intracellular Formation of Reactive Oxygen Species and Inhibits Colon Cancer

Massively increasing global incidences of colorectal cancer require efficient treatment and prevention strategies. Here, we report unexpected anticancerogenic effects of hydroethanolic Iberis amara extract (IAE), which is known as a widely used phytomedical product for treating gastrointestinal complaints. IAE significantly inhibited the proliferation of HT-29 and T84 colon carcinoma cells with an inhibitory concentration (IC₅₀) of 6 and 9 μg/ml, respectively, and further generated inhibitory effects in PC-3 prostate and MCF7 breast cancer cells. Inhibition of proliferation in HT-29 cells was associated with a G2/M phase cell cycle arrest including reduced expression of various regulatory marker proteins. Notably, in HT-29 cells IAE further induced apoptosis by intracellular formation of reactive oxygen species (ROS). Consistent with predictions derived from our in vitro experiments, bidaily oral gavage of 50 mg/kg of IAE over 4 weeks resulted in significant inhibition of tumor growth in a mouse HT-29 tumor xenograft model. Taken together, Iberis amara extracts could become useful alternatives for preventing and treating the progression of colon cancer.