A simple method for synthesizing [γ-32P]nucleoside triphosphates using [32P]acetylphosphate and acetate kinase

A simple, rapid, and inexpensive method is described for the synthesis of γ-³²P-labeled ribo- or deoxyribonucleoside triphosphates. The procedure involves chemical synthesis of [³²P]acetylphosphate and subsequent phosphorylation of nucleoside diphosphates using acetate kinase (EC 2.7.2.1) and a final purification step. The entire procedure is performed 8 h or less.     

Albumin-binding PARACEST agents

Lanthanide complexes (Eu³⁺, Gd³⁺ and Yb³⁺) of two different 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tetraamide derivatives containing two (2) and four (3) O-benzyl-l-serine amide substituents were synthesized and their chemical exchange saturation transfer (CEST) and relaxometric properties were examined in the presence and absence of human serum albumin (HSA). Both Eu2 and Eu3 display a significant CEST effect from a single slowly exchanging Eu³⁺-bound water molecule, making these PARACEST complexes potentially useful as vascular MRI agents. Yb2 also showed a detectable CEST effect from both the Yb³⁺-bound water protons and the exchangeable NH amide protons, making it potentially useful as a vascular pH sensor. Fluorescence displacement studies using reporter molecules indicate that both Gd2 and Gd3 displace dansylsarcosine from site II of HSA with inhibition constants of 32 and 96 μM, respectively, but neither complex significantly displaces warfarin from site I. Water proton relaxation enhancements of 135 and 171% were observed upon binding of Gd2 and Gd3 to HSA, respectively, at 298 K and pH 7.4.     

Effects of Dichloroacetate on Brain Pyruvate Dehydrogenase

The action of dichloroacetate (DCA) on pyruvate dehydrogenase (PDH) activity of rat brain has been studied in vitro and in vivo. In a crude brain mitochondrial fraction, DCA inhibits PDH kinase and in rat brain slices this compound increases PDH activity and stimulates glucose oxidation. In the whole animal, intraperitoneal injection of DCA causes activation of brain PDH, indicating that this inhibitor crosses the blood-brain barrier. The same treatment with DCA also produced a large increase in heart PDH activity. Further studies of the effects of DCA on the CNS should lead to results of considerable importance.     

XYY chromosomal complement,proven by fluorescence,in a child with trisomy 21: 48,XYY,21+

Summary Clinical, cytogenetic, autoradiographic, fluorescence microscopic and dermatoglyphic data of a boy with 48,XYY,21+ karyotype are reported. The child had some clinical anomalies common in Down's syndrome. DNA replication of the 2 Y chromosomes was synchronous in all labelled metaphases. It was possible to distinguish the two Y's from the pairs No. 21-22 under the fluorescence microscope and single as well as double Y bodies were demonstrated in buccal mucosa cells. Dermatoglyphic investigation revealed some features typical for mongolism.     

Conserved structure of the chloroplast-DNA encoded D1 protein is essential for effective photoprotection via non-photochemical thermal dissipation in higher plants

Naturally selected atrazine-resistant (AR) weeds possessing a Ser₂₆₄ → Gly D1 protein encoded by a mutant psbA allele in the chloroplast-DNA have increased photosensitivity and lower fitness. The D1 mutant lines of S. nigrum revealed impaired regulation of photosystem II (PSII) activity as compared with the wild-type plants resulting in a less effective photochemical light utilization and in addition, a lower capacity of non-photochemical thermal dissipation (NPQ), one of the main photoprotective mechanisms in oxygenic photosynthetic organisms. In this work, comparative chlorophyll fluorescence analysis in attached leaves of wild-type and AR Solanum nigrum L. and in their reciprocal crosses has been used to establish how the lower NPQ is inherited. Both a 50% reduction in steady-state NPQ and a 60–70% reduction in the rapidly reversible, energy-dependent (qE) component of NPQ were common phenomena in the parent and hybrid lines of D1 mutant S. nigrum. The nuclear hybrid status of the F2 plant material was confirmed by morphological observations on fully developed leaves. No alteration was found in the nucleotide sequence and the deduced amino acid sequences of the nuclear psbS gene isolated from different biotypes of S. nigrum, and there were no differences in the expressions of both the PsbS and the D1 proteins. All things considered, co-inheritance of the lower photoprotective NPQ capacity and the Ser₂₆₄ → Gly D1 protein mutation was clearly observed, suggesting that the evolutionarily conserved D1 structure must be indispensable for the efficient NPQ process in higher plants.     

Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins

We developed a cost sensitive isotope labelling procedure using a fed-batch fermentation method and tested its efficiency producing the ¹⁵N-, ¹³C- and ¹⁵N/¹³C-labelled variants of an amyloidogenic miniprotein (E5: EEEAVRLYIQWLKEGGPSSGRPPPS). E5 is a surface active protein, which forms amyloids in solution. Here, we confirm, using both PM-IRRAS and AFM measurements, that the air–water interface triggers structural rearrangement and promotes the amyloid formation of E5, and thus it is a suitable test protein to work out efficient isotope labelling schemes even for such difficult sequences. E. coli cells expressing the recombinant, ubiquitin-fused miniprotein were grown in minimal media containing either unlabelled nutrients, or ¹⁵N-NH₄Cl and/or ¹³C-D-Glc. The consumption rates of NH₄Cl and D-Glc were quantitatively monitored during fermentation and their ratio was established to be 1:5 (for NH₄Cl: D-Glc). One- and two-step feeding schemes were custom-optimized to enhance isotope incorporation expressing five different E5 miniprotein variants. With the currently optimized protocols we could achieve a 1.5- to 5-fold increase of yields of several miniproteins coupled to a similar magnitude of cost reduction as compared to flask labelling protocols.     

Acetalation of 1,6-anhydro-1(6)-thio-D-glucitol

Acetalation of 1,6-anhydro-1(6)-thio-D-glucitol (1a) with acetone, formaldehyde, or benzaldehyde afforded 2,3:4,5-diacetals (2a, 2b, and 2c) whose structure, after desulfurization, was proved by mass spectrometry. Upon partial hydrolysis of 2a, one of the isopropylidene groups was split off, and the other migrated to O-3,O-4 to give 4b. In 4b, in the stable conformation, OH-2 occupies an equatorial position, whereas OH-5 is axially oriented. Accordingly, OH-2 reacts faster than OH-5 on methylation of 4b, giving 4e. Hydrolysis of the isopropylidene group of the 2,5-di-O-methyl derivative 4d and subsequent mesylation afforded the corresponding 3,4-di-O-mesyl compound 1c, which showed significant ulcerostatic activity.