Atomic force microscopy and scanning near-field optical microscopy studies on the characterization of human metaphase chromosomes

A better knowledge of biochemical and structural properties of human chromosomes is important for cytogenetic investigations and diagnostics. Fluorescence in situ hybridization (FISH) is a commonly used technique for the visualization of chromosomal details. Localizing specific gene probes by FISH combined with conventional fluorescence microscopy has reached its limit. Also, microdissecting DNA from G-banded human metaphase chromosomes by either a glass tip or by laser capture needs further improvement. By both atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), local information from G-bands and chromosomal probes can be obtained. The final resolution allows a more precise localization compared to standard techniques, and the extraction of very small amounts of chromosomal DNA by the scanning probe is possible. Besides new strategies towards a better G-band and fluorescent probe detection, this study is focused on the combination of biochemical and nanomanipulation techniques which enable both nanodissection and nanoextraction of chromosomal DNA.     

Glutamine cycling in isolated working rat heart

To what extent does glutamine turnover keep pace with oxidative metabolism in the rat heart? To address this question, the following groups of substrates were presented to the isolated, working rat heart: 1) glucose (5 mM), insulin (40 microU/ml), and [2-13C]acetate (5 mM; high workload, n = 5); 2) pyruvate (2.5 mM) and [2-13C]acetate (5 mM; normal workload, n = 5); or 3) propionate (1 mM) and [2-13C]acetate (2.5 mM; normal workload, n = 3). In a subset of these experiments, the exchange of glutamate and glutamine was quantified by separation with ion exchange chromatography and analysis by GC-MS. There was an apparent equilibration of mass isotopomers of glutamate and glutamine after 50 min of perfusion, although the extent of equilibration was not determined. The fractional enrichment in glutamine was 31% of the enrichment of glutamate with the three different perfusates. From high-resolution nuclear magnetic resonance spectra, we found a ratio of glutamine to glutamate content of 94.1, 53.4, and 96.9%, respectively, for each experimental group. In experiments for which l-[1-13C]glutamine (5 mM) was included in the perfusate of group 2, [1-13C]glutamine was detected in the heart, but transfer of 13C from glutamine to glutamate was not detected (n = 4). We conclude that, in the perfused working heart, production of glutamine by amidation of glutamate takes place and can be detected, whereas the reverse process, generation of glutamate from glutamine, remains undetected.     

Sensory and biomechanical responses to ramp-controlled distension of the human duodenum

The aim of this study was to develop a new method for investigation of the relationship among the mechanical stimulus, the biomechanical properties, and the visceral perception evoked by volume/ramp-controlled distension in the human duodenum in vivo. An impedance planimetric probe for balloon distension was placed in the third part of the duodenum in seven healthy volunteers. Distension of the duodenum was done at infusion rates of 10, 25, and 50 ml/min. The pump was reversed when level 7 was reached on a visual analog scale ranging from 0 to 10. Distensions were done with and without the administration of the antimuscarinic drug butylscopolamine. The total circumferential tension (T(total)) and the passive circumferential tension (T(passive)) were determined from the distension tests without and with the administration of butylscopolamine, respectively. T(total) and T(passive) showed an exponential behavior as a function of strain (a measure of deformation). The active circumferential tension (T(active)) was computed as T(total)-T(passive) and showed a bell-shaped behavior as a function of strain. At low distension intensities, the intensity of sensation at 10 ml/min was significantly higher than that obtained at 25 and 50 ml/min. The coefficient of variation at the pain threshold for circumferential strain (average 4.34) was closer to zero compared with those for volume (8.72), pressure (31.22), and circumferential tension (31.55). This suggests that the mechanoreceptors in the gastrointestinal wall depend primarily on circumferential strain. The stimulus-response functions provided evidence for the existence of low- and high-threshold mechanoreceptors in the human duodenum. Furthermore, the data suggest that high-threshold receptors are nonadapting.     

The parasitome of the phytonematode Heterodera glycines

Parasitism genes expressed in the esophageal gland cells of phytonematodes encode secretions that control the complex process of plant parasitism. In the soybean cyst nematode, Heterodera glycines, the parasitome, i.e., the secreted products of parasitism genes, facilitate nematode migration in soybean roots and mediate the modification of root cells into elaborate feeding cells required to support the growth and development of the nematode. With very few exceptions, the identities of these secretions are unknown, and the mechanisms of cyst nematode parasitism, therefore, remain obscure. The most direct and efficient approach for cloning parasitism genes and rapidly advancing our understanding of the molecular interactions during nematode parasitism of plants is to create gland cell-specific cDNA libraries using cytoplasm microaspirated from the esophageal gland cells of various parasitic stages. By combining expressed sequence tag analysis of a gland cell cDNA library with high throughput in situ expression localization of clones encoding secretory proteins, we obtained the first comprehensive parasitome profile for a parasitic nematode. We identified 51 new H. glycines gland-expressed candidate parasitism genes, of which 38 genes constitute completely novel sequences. Individual parasitome members showed distinct gland cell expression patterns throughout the parasitic cycle. The parasitome complexity discovered paints a more elaborate picture of host cellular events under specific control by the nematode parasite than previously hypothesized.     

Effects of CO2 concentrations on the freshwater microalgae, Chlamydomonas reinhardtii, Chlorella pyrenoidosa and Scenedesmus obliquus (Chlorophyta)

In order to investigate the possible impacts of increased atmospheric CO₂ levels on algal growth and photosynthesis, the influence of CO₂ concentration was tested on three planktonic algae (Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Scenedesmus obliquus). Increased CO₂ concentration enhanced significantly the growth rate of all three species. Specific growth rates reached maximal values at 30, 100, and 60 M CO₂ in C. reinhardtii, C. pyrenoidosa, and S. obliquus, respectively. Such significant enhancement of growth rate with enriched CO₂ was also confirmed at different levels of inorganic N and P, being more profound at limiting levels of N inC. pyrenoidosa and P in S. obliquus. The maximal rates of net photosynthesis, photosynthetic efficiency and light-saturating point increased significantly (p < 0.05) in high-CO₂-grown cells. Elevation of the CO₂ levels in cultures enhanced the photoinhibition of C. reinhardtii, but reduced that of C. pyrenoidosa and S. obliquus when exposed to high photon flux density. The photoinhibited cells recovered to some extent (from 71% to 99%) when placed under dim light or in darkness, with better recovery in high-CO₂-grownC. pyrenoidosa and S. obliquus. Although pH and pCO₂ effects cannot be distinguished from this study, it can be concluded that increased CO₂ concentrations with decreased pH could affect the growth rate and photosynthetic physiology of C. reinhardtii, C. pyrenoidosa, and S. obliquus.     

1H(C) and 1H(N) total NOE correlations in a single 3D NMR experiment. 15N and 13C time-sharing in t1 and t2 dimensions for simultaneous data acquisition

Simultaneous data acquisition in time-sharing (TS) multi-dimensional NMR experiments has been shown an effective means to reduce experimental time, and thus to accelerate structure determination of proteins. This has been accomplished by spin evolution time-sharing of the X and Y heteronuclei, such as ¹⁵N and ¹³C, in one of the time dimensions. In this work, we report a new 3D TS experiment, which allows simultaneous ¹³C and ¹⁵N spin labeling coherence in both t ₁ and t ₂ dimensions to give four NOESY spectra in a single 3D experiment. These spectra represent total NOE correlations between ¹H_N and ¹H_C resonances. This strategy of double time-sharing (2TS) results in an overall four-fold reduction in experimental time compared with its conventional counterpart. This 3D 2TS CN-CN-H HSQC-NOESY-HSQC pulse sequence also demonstrates improvements in water suppression, ¹⁵N spectral resolution and sensitivity, which were developed based on 2D TS CN-H HSQC and 3D TS H-CN-H NOESY-HSQC experiments. Combining the 3D TS and the 3D 2TS NOESY experiments, NOE assignment ambiguities and errors are considerably reduced. These results will be useful for rapid protein structure determination to complement the effort of discerning the functions of diverse genomic proteins.     

Cloning and functional expression of alkaline alpha-galactosidase from melon fruit: similarity to plant SIP proteins uncovers a novel family of plant glycosyl hydrolases

Raffinose and stachyose are ubiquitous galactosyl-sucrose oligosaccharides in the plant kingdom which play major roles, second only to sucrose, in photoassimilate translocation and seed carbohydrate storage. These sugars are initially metabolised by alpha-galactosidases (alpha-gal). We report the cloning and functional expression of the first genes, CmAGA1 and CmAGA2, encoding for plant alpha-gals with alkaline pH optima from melon fruit (Cucumis melo L.), a raffinose and stachyose translocating species. The alkaline alpha-gal genes show very high sequence homology with a family of undefined 'seed imbibition proteins' (SIPs) which are present in a wide range of plant families. In order to confirm the function of SIP proteins, a representative SIP gene, from tomato, was expressed and shown to have alkaline alpha-gal activity. Phylogenetic analysis based on amino acid sequences shows that the family of alkaline alpha-gals shares little homology with the known prokaryotic and eukaryotic alpha-gals of glycosyl hydrolase families 27 and 36, with the exception of two cross-family conserved sequences containing aspartates which probably function in the catalytic step. This previously uncharacterised, plant-specific alpha-gal family of glycosyl hydrolases, with optimal activity at neutral-alkaline pH likely functions in key processes of galactosyl-oligosaccharide metabolism, such as during seed germination and translocation of RFO photosynthate.     

Design,synthesis and binding affinity of 3'-fluoro analogues of Cl-IB-MECA as adenosine A3 receptor ligands

Several 3'-fluoro analogues, 1a, 1b, and 1c of selective and potent adenosine A(3) receptor agonist, Cl-IB-MECA were synthesized from D-xylose via highly regioselective opening of lyxo-epoxides, 8a and 8b with fluoride anion. Compared to the high binding affinity of Cl-IB-MECA to the A(3) adenosine receptor, the corresponding 3'-fluoro derivative showed remarkably decreased binding affinity, indicating that 3'-hydroxyl group acts as hydrogen bonding acceptor, not hydrogen bonding donor like fluorine atom in binding to the A(3) adenosine receptor.     

Exploration of RNA 3′ terminal locations in rat ribosome

The locations of the 3 ends of RNAs in rat ribosome were studied by a fluorescencelabeling method combined with high hydrostatic pressure and agarose electrophoresis. Under physiological conditions, only the 3 ends of 28 S and 5.8 S RNA in 80 S ribosome could be labeled with a high sensitive fluorescent probe – fluorescein 5thiosemicarbazide (FTSC), indicating that the 3 termini of 28 S and 5.8 S RNA were located on or near the surface of 80 S ribosome. The 3 terminus of 5 S RNA could be attacked by FTSC only in the case of the dissociation of the 80 S ribosome into two subunits induced by high salt concentration (1 M KCl) or at high hydrostatic pressure, showing that the 3 end of 5 S RNA was located on the interface of two subunits. However, no fluorescencelabeled 18 S RNA could be detected under all the conditions studied, suggesting that the 3 end of 18 S RNA was either located deeply inside ribosome or on the surface but protected by proteins. It was interesting to note that modifications of the 3 ends of ribosomal RNAs including oxidation with NaIO₄, reduction with KBH₄ and labeling with fluorescent probe did not destroy the translation activity of ribosome, indicating that the 3 ends of RNAs were not involved in the translation activity of ribosome.