Young rats prenatally exposed to ethanol exhibit heightened responses to dopaminergie (DA) drugs, altered brain concentrations of dopamine, and its metabolite dihydroxyphenylacetic acid (DOPAC), and transient reductions in DA receptor binding. Adult mice exposed to ethanol prenatally also exhibit increased responses to DA drugs; however, brain concentrations of DA and DOPAC are unaltered. The effects of prenatal ethanol exposure on DA or DOPAC concentrations in young mice or on DA receptor binding in mice of any age are unknown. Therefore, to determine if the different effects of prenatal ethanol exposure on rats and mice are due to age at time of testing or species, we determined its effects on DA concentrations and turnover in young mice under conditions previously reported for adult offspring and on DA D1 and D2 receptor binding in both young and adult offspring. Consistent with our previous report for adult offspring, prenatal ethanol exposure did not alter DA concentrations or turnover. The treatment did, however, diminish periadolescent growth as previously reported and produced a transient increase in DA D1, but not DA D2 receptor binding. DA receptor binding was not altered in adult offspring. Although unrelated to prenatal ethanol exposure, the sexes differed on all of the DA measures. Combined with previous reports, the present study suggests that species rather than age is more likely to account for the different effects of prenatal ethanol exposure on DA systems, and that sex differences in DA systems should be further examined.

3,4-Dihydroxyphenylacetic Acid↗

PubMedDate not supplied

Why do we need a three-dimensional architecture of the ligand-binding domain of the nuclear 1alpha,25-dihydroxyvitamin D(3) receptor?

Highly specific binding of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) by vitamin D receptor (VDR), a nuclear transcriptional factor, activates a genomic mechanism that is manifested in the multiple biologic properties of 1alpha,25(OH)(2)D(3). Numerous synthetic analogs of 1alpha,25(OH)(2)D(3) have been employed to study the interaction between 1alpha,25(OH)(2)D(3) and VDR, and to identify structural markers in 1alpha,25(OH)(2)D(3) that are important for VDR-binding. On the other hand the three-dimensional structure of VDR remained elusive till very recently. In the present study we employed affinity labeling (by 1alpha,25-dihydroxyvitamin D(3)-3-bromoacetate, 1alpha,25(OH)(2)D(3)-3-BE) of VDR to identify C(288) as the anchoring residue for the 3-hydroxyl group of 1alpha,25(OH)(2)D(3) inside the ligand-binding domain of VDR (VDR-LBD). In addition we carried out mutation/hormone-binding analyses to determine the importance of M(284) and W(286) toward hormone binding. We incorporated this information with the three-dimensional structure of the LBD of progesterone receptor to develop a homology-extension model of VDR-LBD. This model identified several amino acid residues as ligand-contact points inside the LBD. Mutational and hormone-binding analyses of these residues verified the structure-functional authenticity of this model, in comparison with the crystal structure of VDR, bound to 1alpha,25(OH)(2)D(3).

Animals↗

PubMedDate not supplied

A micro carbon electrode for nitric oxide monitoring.

A nitric oxide (NO) probe, consisting of a micro carbon fiber working electrode, 10 microns diameter, a platinum counter electrode, and a silver/silver chloride (Ag/AgCl) reference electrode, has been developed. The carbon fiber working electrode is covered with a Nafion cation exchange membrane. Using differential pulse voltammetry (DPV), we found the NO to N2O reduction current peak at approximately -1.35 V versus Ag/AgCl. This has been reported by others. The DPV current outputs are linearly related to dissolved NO concentrations [NO] in the 2-10 microM range. Catecholamines were found not to interfere with the reduction signal. The Nafion membrane also prevents interference by NO2-, NO3-, and amino acids at normal physiologic pH (pH 7.4). The effects of O2 are accounted for through sampling and subtracting background currents from the peak current. To increase sensitivity and shorten response time, a method of integrated pulse amperometry (IPA) was used for the study. The IPA charge outputs (delta C) are linear to the dissolved [NO] in the 50-350 nM range. The carbon fiber electrode has the potential of being miniaturized to a smaller electrode, allowing detection of NO released from the subendothelial space.

Carbon↗

PubMedDate not supplied

A microchip glucose sensor.

A major problem in development of a glucose sensor for use in an implantable artificial pancreas is the lack of reproducibility in signals from sensor to sensor. Each glucose sensor fabricated with currently used methods has a unique response to varying levels of glucose concentration and thus needs to be individually calibrated before use. We have adapted microchip manufacturing techniques for the fabrication of electrochemically based glucose sensors with standardized and reproducible function. Scanning electron microscopic study of the resulting electrode surfaces shows them to be smooth and featureless at all levels of magnification. X-ray diffraction analysis of the electrodes indicates preferential exposure of the [1,1,1] crystal interface. Cyclic voltammetry evaluation of initial sensor response to varying glucose concentrations shows excellent sensor to sensor reproducibility for all sensors made with the same underlayment. Sensors made with titanium underlayment appear to be more differentiated and thus more sensitive to variations in glucose concentration than are sensors with chromium underlayment. Although the initial response of microchip glucose sensors appears to be standardized and reproducible, additional development of an appropriate electrical insulation material is required before long-term study of signal stability is feasible.

Biomedical Engineering↗

PubMedDate not supplied

A nitric oxide sensor using reduction current.

Nitric oxide (NO) has a wide range of biologic activity. Methods commonly used for the detection of biologically derived NO are indirect and measure only the amount of NO released during an interval of time. An electrochemical method available is capable of being direct and continuous but is subject to interference. The recent explosion of scientific research into NO activity requires better methods of NO detection. This article reports a new NO electrochemical sensing method and sensor design. The tip of the sensor is covered with a hydrophobic membrane and contains an internal electrolyte. Platinum is used for the working and counter electrodes and silver/silver bromide (Ag/AgBr) for the reference electrode. The components of the internal electrolyte are potassium bromide and sulfuric acid. The NO that diffuses to the working electrode is first oxidized to NO+; the NO+ is reduced to NO; and the reduction current is determined. An integrated pulsed amperometric method is used to achieve the redox of NO and the measurement and integration of the reduction current. The results show that the NO sensor is sensitive and has a rapid response and less interference.

Biosensing Techniques↗

PubMedDate not supplied

Permanent grafting of living skin substitutes: surgical parameters to control for successful results.

Autologous mesh grafting, widely used in the treatment of severe burns, remains the most conventional approach for permanent skin replacement. However, during the last decade several types of skin substitutes were reported as suitable alternatives for full-thickness burn wound coverage. The clinical use of such dressings requires new surgical skills to maintain the integrity of the grafts and favor their permanent implantation in vivo. This article reports observations made on nude mice grafted with cultured human skin equivalents. Some parameters such as the quality of adhesion between the implant and the graft bed, the size, the stability and the thickness of the graft, the humidity of the chamber, and the protocol of antibiotic administration were identified as crucial for the success of the surgery. The grafting procedures are described in this paper. These results should be taken into consideration in all transplantations of skin grafts in vivo.

Animals↗

PubMedDate not supplied

The dynein heavy chain gene family in Tetrahymena thermophila.

The dynein ATPases are a family of motor enzymes that drive microtubule sliding in cilia and flagella and contribute to microtubule-based transport inside cells. The multi-dynein hypothesis makes two predictions: 1) Axonemes contain multiple dynein heavy chain (DHC) isoforms, each encoded by a different gene; 2) Each isoform performs a specific role in ciliary beating. We used PCR-based techniques to clone thirteen different DHC sequences from Tetrahymena genomic DNA. All thirteen genes appeared to be expressed in growing cells. Comparisons of the deduced amino acid sequences of the thirteen DHCs with other known DHCs suggested that we have cloned three outer arm DHCs, two cytoplasmic DHCs, and eight inner arm DHCs.

Animals↗

PubMedDate not supplied

Shape memory alloy fixator system for suturing tissue in minimal access surgery.

A new technique for suturing human tissue is described in which tissue closure is achieved by means of small fixators made from shape memory alloy. The aim of the development is to provide an alternative to thread suturing in minimal access surgery, which is quicker and requires less skill to achieve the required suturing quality. The design of the fixators is described in terms of the thermal shape recovery of shape memory alloy and a novel form of finite element analysis, which uses a nonlinear elastic element for the material property. Thermal analysis of the fixators and surrounding tissue is used to predict the temperature distribution during and after the application of electric current heating. This was checked in an in vitro experiment, which confirmed that deployment caused no detectable collateral damage to surrounding tissue. In vivo animal studies on the use of the shape memory alloy fixator for suturing tissue are ongoing to establish safety and healing effects.

Alloys↗

PubMedDate not supplied

Variety of nonsense suppressor phenotypes associated with mutational changes at conserved sites in Escherichia coli ribosomal RNA.

To screen for ribosomal RNA mutants defective in peptide chain termination, we have been looking for rRNA mutants that exhibit different patterns of suppression of nonsense mutations and that do not suppress missense mutations at the same positions in the same reporter gene. The rRNA mutations were induced by segment-directed randomly mutagenic PCR treatment of a cloned rrnB operon, followed by subcloning of the mutagenesis products and transformation of strains containing different nonsense mutations in the Escherichia coli trpA gene. To date, we have repeatedly obtained only two small sets of mutations, one in the 3' domain of 16S rRNA, at five nucleotides out of the 610 mutagenized (two in helix 34 and three in helix 44), and the other in 23S rRNA at only four neighboring nucleotide positions (in a highly conserved hexanucleotide loop) within the 1.4 kb mutagenized segment. There is variety, however, in the suppression patterns of the mutants, ranging from suppression of UAG or UGA, through suppression of UAG and UGA, but not UAA, to suppression of all three termination codons. The two helices in 16S rRNA have previously been associated both physically and functionally with the decoding center of the ribosome. The 23S region is part of the binding site for the large subunit protein L11 and the antibiotic thiostrepton, both of which have been shown to affect peptide chain termination. Finally, we have demonstrated that the 23S mutant A1093, which suppresses trpA UGA mutations very efficiently, is lethal at temperatures above 36 degrees C (when highly expressed). This lethality is overcome by secondary 23S rRNA mutations in domain V. Our results suggest that specific regions of 16S and 23S rRNA are involved in peptide chain termination, that the lethality of A1093 is caused by high-level UGA suppression, and that intramolecular interaction between domains II and V of 23S rRNA may play a role in peptide chain termination at the UGA stop codon.

Codon, Nonsense↗

PubMedDate not supplied

Protein transfection of intact microvessels specifically modulates vasoreactivity and permeability.

Precise regulation of microvascular tone and barrier function is essential for proper coronary perfusion and performance. Agonist-induced alterations in either or both of these functions ultimately lead to microcirculatory dysfunction and cardiac insufficiency. Two important pathways involved in regulating vasomotor response and barrier function are the activation of nitric oxide synthase (NOS) and upregulation of protein kinase C (PKC). To date, studies of these two signaling proteins have relied mainly on pharmacological approaches. Unfortunately, the specificity of various inhibitors can be cause for concern. In order to address this problem, a protein transfection technique we developed for cultured endothelial cells has been modified and applied to isolated, intact coronary microvessels. Our results from green fluorescent protein transfection in arterioles and venules showed that this procedure could be used to introduce proteins into the microvascular wall. By transfecting inhibitor peptides against NOS and PKC into coronary arterioles and venules, we have been able to determine the specific roles of these two enzymes in vasodilation and hyperpermeability responses.

Animals↗

PubMedDate not supplied

Involvement of mu opioid receptors of periaqueductal gary (PAG) in acupuncture inhibition of noxious blood pressure response in rabbits.

Strong electric shock stimulation of the rabbit front paw elicited a pressor blood pressure response regarded as noxious response. Ligands of mu opioid receptors were microinjected into the PAG to observe their effects on acupunture inhibition of the pressor response. (1) Ohmefentanyl (OMF), a mu agonist, significantly attenuated the pressor response. Mu antagonist TCTAP greatly enhanced the pressor response. (2) Electroacupuncture (EA) significantly inhibited the pressor response, the inhibition being readily reversed by TCTAP. The response after TCTAP was significantly greater than that of the control before EA. The results suggest that noxious stimulation is able to activate the mu opioid receptor of the PAG to modulate the noxious response and EA is able to enhance the activation.

Animals↗