A method for delineating structurally homogeneous regions in protein sequences

A homogeneous region in a protein sequence is a set of contiguousresidues that share common features, concerning physsico-chemical,structural and mutational information. This paper presents amethod for identifying such homogeneous regions. From a profiledescribing a given type of biological information along thesequence, the algorithm allows the segmentation of the sequenceby optimizing a criterion characterized by two user-definedcontrol parameters: the ‘homogenizing degree’ ofthe regions and the ‘site neighbourhood’ size. Weapply the method to the envelope proteins of the human immunodeficiencyvirus HIV-1, for the identification of homogeneous regions ina hydrophobicity profile and the delineation of variable andconserved regions in a variability profile.     

Distinct modes of blockade in cardiac ATP-sensitive K+ channels suggest multiple targets for inhibitory drug molecules

Elementary K⁺ currents were recorded at 19°C in inside-out patches from cultured neonatal rat cardiocytes to elucidate the block phenomenology in cardiac ATP-sensitive K⁺ channels when inhibitory drug molecules, such as the sulfonylurea glibenclamide, the phenylalkylamine verapamil or sulfonamide derivatives (HE 93 and sotalol), are interacting in an attempt to stress the hypothesis of multiple channel-associated drug targets.Similar to their adult relatives, neonatal cardiac K_(ATP) channels are characterized by very individual open state kinetics, even in cytoplasmically well-controlled, cell-free conditions; at –7 mV, _open(1) ranged from 0.7 to 4.9 msec in more than 200 patches and _open(2) from 10 to 64 msec—an argument for a heterogeneous channel population. Nevertheless, a common response to drugs was observed. Glibenclamide and the other inhibitory molecules caused long-lasting interruptions of channel activity, after cytoplasmic application, as if drug occupancy trapped cardiac K_(ATP) channels in a very stable, nonconducting configuration. The resultant NP₀ depression was strongest with glibenclamide (apparent IC₅₀ 13 nmol/liter) and much weaker with verapamil (apparent IC₅₀ 9 mol/liter), HE 93 (apparent IC₅₀ 29 mol/liter) and sotalol (apparent IC₅₀ 43 mol/ liter) and may have resulted from the occupancy of a single site with drug-specific affinity or of two sites, the high affinity glibenclamide target and a distinct nonglibenclamide, low affinity target.Changes in open state kinetics, particularly in the transition between the O₁ state and the O₂ state, are other manifestations of drug occupancy of the channel. Any inhibitory drug molecule reduced the likelihood of attaining the O₂ state, consistent with a critical reduction of the forward rate constant governing the O₁-O₁ transition. But only HE 93 (10 mol/liter) associated (with an apparent association rate constant of 2.3 × 10⁶ mol^–1 sec^–1) to shorten significantly _open(2) to 60.6 ± 6% of the predrug value, not the expected result when the entrance in and the exit from the O₂ state would be drug-unspecifically nfluenced. Sotalol found yet another and definitely distinctly located binding site to interfere with K⁺ permeation; both enantiomers associated with a rate close to 5×10⁵ mol^–1 sec^–1 with the open pore thereby flicker-blocking cardiac K_(ATP) channels. Clearly, these channels accommodate more than one drug-binding domain.     

A device for simultaneously controlling multiple treatment levels of dissolved oxygen in laboratory experiments

Hypoxia is common in freshwater, estuarine, and shallow or enclosed marine systems. Research on the sub-lethal effects of hypoxia on feeding and growth includes both field and laboratory studies. Extended regulation of dissolved oxygen (DO) is difficult to precisely control in the laboratory. Here we describe a device that is capable of simultaneously monitoring and controlling DO at multiple treatment levels, in replicate experimental tanks, for indefinite periods of time. Each treatment level may be static or fluctuate on a diel cycle. The device consists of a series of independent aquarium systems, each of which is monitored and adjusted by a computer program. A data file and chart of DO levels in each treatment level is recorded. A computer program that repeatedly measures and adjusts DO controls the device. Because this apparatus is software-controlled, the user has the flexibility of altering treatment parameters at any time without interrupting experiments.     

Probing possible egress channels for multiple ligands in human CYP3A4: A molecular modeling study

Human cytochrome P450 (CYP) 3A4 extensively contributes to metabolize 50% of the marketed drugs. Recently, a CYP3A4 structure with two molecules of ketoconazole (2KT) was identified. However, channels for egresses of these inhibitors are unexplored. Thus, we applied molecular dynamics simulations followed by channel analyses. Two simulations of empty and 2KT-bound CYP3A4 results revealed the multiple ligand-induced conformational changes in channel forming regions, which appear to be important for the regulation of channels. In addition, we observed that the channel-3 entrance is closed due to the large structural deviation of the key residues from Phe-cluster. F215 and F220 are known as entrance blockers of channel-2 in metyrapone-bound CYP3A4. Currently, F220 blocks the channel-3 along with F213 and F241. Therefore, it suggested that channel-1 and 2 could potentially serve as egress routes for 2KT. It is also supported by the results from MOLAxis analyses, in which the frequency of channel occurrence and bottleneck radius during simulation favor channel-1 and 2. Several bottleneck residues of these channels may have critical roles in 2KT egresses, especially S119. Our modeling study for multiple ligand-channeling of CYP3A4 could be very helpful to gain new insights into channel selectivity of CYP3A4.     

An improved method for detecting and delineating genomic regions with altered gene expression in cancer

Genomic regions with altered gene expression are a characteristic feature of cancer cells. We present a novel method for identifying such regions in gene expression maps. This method is based on total variation minimization, a classical signal restoration technique. In systematic evaluations, we show that our method combines top-notch detection performance with an ability to delineate relevant regions without excessive over-segmentation, making it a significant advance over existing methods. Software (Rendersome) is provided.     

A simplified HPLC method for simultaneously quantifying ribonucleotides and deoxyribonucleotides in cell extracts or frozen tissues

Agents and conditions that induce alterations in deoxyribonucleotide pools can have important regulatory effects on the rate of DNA synthesis as well as cell cycle progression. A simplified procedure for the separation of both ribonucleoside triphos-phates (NTP) and deoxyribonucleoside triphosphates (dNTP) is presented which utilizes reversed phase high-performance liquid chromatography coupled with diode array detection. The simultaneous resolution of NTP and dNTP peaks within the same cell extract effectively eliminates the need for post-extraction steps such as periodate oxidation and/or boronate affinity chromatography previously used to degrade or isolate co-eluting NTP from dNTP. The resolution of two nucleotides, dGTP and ADP, was found empirically to vary with the efficiency of the C18 column. High efficiency columns (>90 000 plates/m) provided good separation; however, less efficient columns resulted in co-elution of dGTP and ADP. These co-eluting nucleotides can be accurately quantified, if necessary, using diode array technology and a mathematical expression which incorporates molar peak coefficients and peak areas obtained by monitoring at dual wavelengths. Tissue samples or single cell suspensions were extracted with trichloroacetic acid and the neutralized extract was injected directly into the column without prior lyophilization. The per cent recovery of standards was .99% and replicate extractions within or between samples were highly reproducible (sd<5%). The single step method described minimizes potential losses associated with post-extraction manipulation and provides the capability to examine alterations in nucleotide precursor–product metabolism under various physiological and pharmacological conditions.     

A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes

A rapid procedure has been devised to count the numbers of outer trophectoderm (TE) and inner cell mass (ICM) cells of mouse blastocysts by differentially labelling their nuclei in situ with polynucleotide-specific fluorochromes. The TE nuclei were labelled with propidium iodide (PI) by permeabilising the cells using selective antibody-mediated complement lysis (Solter and Knowles, '75). The blastocysts were then fixed in ethanol and the ICM nuclei labelled with bisbenzimide. These two fluorochromes have widely different fluorescent spectra. Thus, by using fluorescence microscopy with appropriate filter combinations, the PI-labelled TE nuclei appeared pink or red; the bisbenzimide-labelled ICM nuclei, blue or unlabelled. The total numbers of blastocyst nuclei and the numbers of ICM nuclei counted by differential labelling were similar to the numbers detected after spreading the nuclei of intact blastocysts or immunosurgically isolated ICMs by air-drying (Tarkowski '66). Differential labelling of TE and ICM nuclei in situ has two important advantages--that the numbers of both these cell types can be determined for individual blastocysts and that spatial relationships are partially preserved so that regional interactions can be studied.     

The PASDORO method for simultaneously demonstrating DNA and lipids in the brain

Conclusions and summary The PASDORO method has the advantage of allowing DNA and globular lipid to be demonstrated in frozen sections of formalin-fixed tissue. Its disadvantage is that, in most tissues, basement membrane staining interferes with the selective demonstration of DNA. However, in the central nervous system it is advantageous to be, able to trace capillary basement membrane. In this way cells related to the capillary endothelium can be easily identified. The addition of staining with Oil Red O to the basic PASD technique permits lipid-laden microglia to be readily identified around necrotic and demyelinating lesions.     

A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage

Structural information on RNA, emerging more and more as a major regulator in gene expression, dramatically lags behind compared with information on proteins. Although NMR spectroscopy has proven to be an excellent tool to solve RNA structures, it is hampered by the severe spectral resonances overlap found in RNA, limiting its use for large RNA molecules. Segmental isotope labeling of RNA or ligation of a chemically synthesized RNA containing modified nucleotides with an unmodified RNA fragment have proven to have high potential in overcoming current limitations in obtaining structural information on RNA. However, low yields, cumbersome preparations and sequence requirements have limited its broader application in structural biology. Here we present a fast and efficient approach to generate multiple segmentally labeled RNAs with virtually no sequence requirements with very high yields (up to 10-fold higher than previously reported). We expect this approach to open new avenues in structural biology of RNA.     

A new method for the isolation of histatins 1, 3, and 5 from parotid secretion using zinc precipitation

Histatins, a family of small-molecular-weight, histidine-rich cationic salivary proteins, have been difficult to isolate in an efficient way by conventional procedures due to their anomalous interactions with chromatographic resins. In the present study we explored the possibility of developing a new isolation procedure based on recent observations that histatins associate with various metal ions, including zinc. Since solubility studies showed that histatin 5 forms precipitates with zinc under alkaline conditions, we investigated whether this characteristic could be exploited for the preparative isolation of histatins from salivary secretions. A fast and efficient two-step procedure was developed using zinc precipitation of histatins from human parotid secretion followed by final purification using reversed-phase high-performance liquid chromatography (HPLC). Analysis of zinc precipitates by Tricine-SDS-PAGE, cationic PAGE, HPLC, and mass spectrometry revealed the presence of the three major histatins, 1, 3, and 5, as well as statherin. The histatin yield obtained by the precipitation step was approximately 90%. Therefore, zinc precipitation of histatins from glandular salivary secretions is a novel, rapid, and effective means for the isolation of these proteins.     

A noninvasive, direct real-time PCR method for sex determination in multiple avian species

Polymerase chain reaction (PCR)-based methods to determine the sex of birds are well established and have seen few modifications since they were first introduced in the 1990s. Although these methods allowed for sex determination in species that were previously difficult to analyse, they were not conducive to high-throughput analysis because of the laboriousness of DNA extraction and gel electrophoresis. We developed a high-throughput real-time PCR-based method for analysis of sex in birds, which uses noninvasive sample collection and avoids DNA extraction and gel electrophoresis.     

A rapid,sensitive method for the determination of 3-methylhistidine levels in urine and plasma using high-pressure liquid chromatography

A method is presented for the precolumn derivatization and subsequent high-pressure liquid chromatographic separation of 3-methylhistidine from urine and plasma. The solvent system is 10 mm sodium phosphate (pH 7.5) and acetonitrile. The elution can be performed isocratically and requires less than 10 min. Both fluorescent and ultraviolet detection may be utilized. This method is at least 10³ times more sensitive than conventional ion-exchange chromatography using ninhydrin. 3-Methylhistidine determinations performed on plasma and urine samples from normal volunteers correlated well with published literature values.     

A general method for cloning DNA fragments in multiple copies

A general method for the cloning of DNA fragments in tandem arrays is presented. Synthetic directional adapters are attached to the fragment ends to establish complete control over fragment orientation during ligation. Use of these directional adapters thereby ensures the production of direct repeats, an arrangement essential for clone stability. The technique involves a protocol that is both less complex and less time-consuming than previous methods. Using this technique, a 10-min ligation has yielded a plasmid containing 20 copies of a 151-bp fragment.