A base-triple structural domain in RNA.

An oligonucleotide modeled on a proposed base-triple domain of the Tetrahymena group I intron has been characterized by NMR. The oligonucleotide contains two double-helix regions with adjacent single-stranded nucleotides. The NMR data show that the two helices stack coaxially, although the rotation between the two helices is approximately twice as large as the rotation between normal base pairs. The rotation between the two helices allows the single-stranded nucleotides to form U.U.G and A.G.C base triples in the minor groove. The A.G.C base triple contains a hydrogen bond between the adenine N1 and a 2'-hydroxyl in the minor groove of the G.C pair. A similar hydrogen bond between an adenine and a 2'-hydroxyl in transfer RNA suggests that this could be a recurring tertiary interaction in RNA.     

The use of ambulatory EMG monitoring to measure compliance with lumbar strengthening exercise

This study validates the use of ambulatory EMG monitoring as a measure of exercise compliance. The model rehabilitative exercise used was the Prone Back Extension. Thirty-two undergraduate volunteers were videorecorded as they performed the exercise alone in a closed room. The correlation between a direct observation count of the number of repetitions and an independent EMG-based count was .95. EMG amplitude was examined by repetition and gender with regression and ANOVA. There were significant gender differences in the amplitude of EMG across repetitions. There were no significant differences by gender in the declining slope of amplitude across repetitions. This slope may represent a typical fatigue curve. Thus, not only the occurrence but also the intensity of exercise can be quantified.     

Action of protein synthesis inhibitors in blocking electrogenic h efflux from corn roots

The block in the electrogenic H⁺ efflux produced by protein synthesis inhibitors in corn root tissue can be released or by-passed by addition of fusicoccin or nigericin. The inhibition also lowers cell potential, and the release repolarizes. Associated with the inhibition of H⁺ efflux is inhibition of K⁺ influx and the growth of the root tip; fusicoccin partially relieves these inhibitions, but nigericin does not. The inhibition of H⁺ efflux which arises from blocking the proton channel of the ATPase by oligomycin or N,N′-dicyclohexylcarbodiimide can also be partially relieved by fusicoccin, but not by nigericin; the inhibition produced by diethylstilbestrol is not relieved by fusicoccin.     

Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

EFFECT OF VITAMIN E SUPPLEMENTATION AND PARTIAL SUBSTITUTION OF POLY- WITH MONO-UNSATURATED FATTY ACIDS IN PIG DIETS ON MUSCLE,AND MICROSOME EXTRACT a-TOCOPHEROL CONCENTRATION AND LIPID OXIDATION

The experiment was organized in a 3×2 factorial arrangement with three dietary fat blends and a basal (20 mg kg^?1 diet) or supplemented (220 mg kg^?1) level of α-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle α-tocopherol concentration (α-tocopherol [log μg g^?1]=0.18 (±0.105)+0.0034 (±0.0003)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.39 (±0.122)·dietary MUFA/PUFA (P<0.0036)). An interaction between dietary α-tocopherol and dietary MUFA/PUFA exists for microsome α-tocopherol concentration (α-tocopherol [log μg g^?1]=1.14 (±0.169) (P<0.0001)+0.0056 (±0.00099)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.54 (±0.206)·dietary MUFA/PUFA (P<0.0131)?0.0033 (±0.0011)·dietary α-tocopherol [mg kg^?1)]×dietary MUFA/PUFA (P<0.0067)), and hexanal concentration in meat (hexanal [ng·g^?1]=14807.9 (±1489.8)?28.8 (±10.6) dietary α-tocopherol [mg·kg^?1] (P<0.01)?8436.6 (±1701.6)·dietary MUFA/PUFA (P<0.001)+24.0 (±11.22)·dietary α-tocopherol·dietary MUFA/PUFA (P<0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of α-tocopherol in muscle and microsome extracts. An interaction between dietary α-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of α-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values.     

Quirks of dye nomenclature. 5. Rhodamines

Rhodamines were first produced in the late 19^th century, when they constituted a new class of synthetic dyes. These compounds since have been used to color many things including cosmetics, inks, textiles, and in some countries, food products. Certain rhodamine dyes also have been used to stain biological specimens and currently are widely used as fluorescent probes for mitochondria in living cells. The early history and current biological applications are sketched briefly and an account of the ambiguities, complications and confusions concerning dye identification and nomenclature are discussed.     

Quirks of dye nomenclature. 6. Malachite green

Malachite green was discovered independently by two researchers in Germany in the 19^th century and found immediate employment as a dye and a pigment. Subsequently, other uses, such as staining biological specimens, emerged. A much later application was the control of fungal and protozoan infections in fish, for which the dye remains popular, although illegal in many countries owing to a variety of toxicity problems. In solution, malachite green can exist as five different species depending on the pH. The location of the positive charge of the colored cation on a carbon atom or a nitrogen atom is still debated. The original names of this dye, and their origins, are briefly surveyed.     

Lagging strand synthesis in coordinated DNA synthesis by bacteriophage t7 replication proteins

The proteins of bacteriophage T7 DNA replication mediate coordinated leading and lagging strand synthesis on a minicircle template. A distinguishing feature of the coordinated synthesis is the presence of a replication loop containing double and single-stranded DNA with a combined average length of 2600 nucleotides. Lagging strands consist of multiple Okazaki fragments, with an average length of 3000 nucleotides, suggesting that the replication loop dictates the frequency of initiation of Okazaki fragments. The size of Okazaki fragments is not affected by varying the components (T7 DNA polymerase, gene 4 helicase-primase, gene 2.5 single-stranded DNA binding protein, and rNTPs) of the reaction over a relatively wide range. Changes in the size of Okazaki fragments occurs only when leading and lagging strand synthesis is no longer coordinated. The synthesis of each Okazaki fragment is initiated by the synthesis of an RNA primer by the gene 4 primase at specific recognition sites. In the absence of a primase recognition site on the minicircle template no lagging strand synthesis occurs. The size of the Okazaki fragments is not affected by the number of recognition sites on the template.     

Further analysis of maize C(4) pyruvate,orthophosphate dikinase phosphorylation by its bifunctional regulatory protein using selective substitutions of the regulatory Thr-456 and catalytic His-458 residues

In C(4) plants such as maize, pyruvate,orthophosphate dikinase (PPDK) catalyzes the regeneration of the initial carboxylation substrate during C(4) photosynthesis. The primary catalytic residue, His-458 (maize C(4) PPDK), is involved in the ultimate transfer of the beta-phosphate from ATP to pyruvate. C(4) PPDK activity undergoes light-dark regulation in vivo by reversible phosphorylation of a nearby active-site residue (Thr-456) by a single bifunctional regulatory protein (RP). Using site-directed mutagenesis of maize recombinant C(4) dikinase, we made substitutions at the catalytic His residue (H458N) and at this regulatory target Thr (T456E, T456Y, T456F). Each of these affinity-purified mutant enzymes was assayed for changes in dikinase activity. As expected, substituting His-458 with Asn results in a catalytically incompetent enzyme. Substitutions of the Thr-456 residue with Tyr and Phe reduced activity by about 94 and 99%, respectively. Insertion of Glu at this position completely abolished activity, presumably by the introduction of negative charge proximal to the catalytic His. Furthermore, neither the T456Y nor inactive H458N mutant enzyme was phosphorylated in vitro by RP. The inability of the former to serve as a phosphorylation substrate indicates that RP is functionally a member of the Ser/Thr family of protein kinases rather than a "dual-specificity" Ser-Thr/Tyr kinase, since our previous work showed that RP effectively phosphorylated Ser inserted at position 456. The inability of RP to phosphorylate its native target Thr residue when Asn is substituted for His-458 documents that RP requires the His-P catalytic intermediate form of PPDK as its protein substrate. For these latter studies, synthetic phosphopeptide-directed antibodies specific for the Thr(456)-P form of maize C(4) PPDK were developed and characterized.