A metachromatic dye-ATPase method for the simultaneous identification of skeletal muscle fiber types I, IIA, IIB and IIC.

The histochemical demonstration of quantitative differences in myofibrillar ATPase activity at the selective pH optima of the various types of human skeletal muscle fibers is the most widely used technique for their differentiation. The basis of the reaction is the deposition of insoluble salts of inorganic phosphate cleaved from ATP by myofibrillar ATPase(s) followed by substitution of the phosphates with less soluble chromogenic salts. Doriguzzi and associates reported using metachromatic dyes to demonstrate quantitative differences in phosphate deposition among different fiber types. Following routine ATPase histochemistry and staining with either azure A or toluidine blue, fibers with low ATPase activity (and low phosphate content) were stained metachromatically while fibers with high ATPase activity (and high phosphate content) were orthochromatic with the intensity of color proportional to the content of insoluble phosphate. The metachromasia was readily lost after immoderate washing in aqueous solutions or routine dehydration in ethanol, with consequent diminished fiber type distinction. A critical modification of this technique is reported in which incubation of frozen sections of human skeletal muscle in ATP-containing medium is carried out at room temperature (22-24 C), rather than the usual 37 C, followed by a revised washing and dehydration protocol. With these modifications, the four human skeletal muscle fiber types (types I, IIA, IIB, and IIC) can be identified rapidly and reliably in single sections, obviating the need for examination of serial sections. The tinctorial differentiation allows fiber type identification even in black and white photographs.     

Comparative effects of vasodilator drugs on flow distribution and venous return

Systemic vascular effects of hydralazine, prazosin, captopril, and nifedipine were studied in 115 anesthetized dogs. Blood flow (Q) and right atrial pressure (Pra) were independently controlled by a right heart bypass. Transient changes in central blood volume after an acute reduction in Pra at a constant Q showed that blood was draining from two vascular compartments with different time constants, one fast and the other slow. At three dose levels producing comparable reductions in systemic arterial pressure (30-40% at the highest dose), these drugs had different effects on flow distribution and venous return. Hydralazine and prazosin had parallel and balanced effects on arterial resistance of the two vascular compartments, and flow distribution was unaltered. Captopril preferentially reduced arterial resistance of the compartment with a slow time constant for venous return (-26 +/- 6%, -30 +/- 6%, -50 +/- 5% at 0.02, 0.10, and 0.50 mg X kg-1 X h-1, respectively; means +/- SEM) without altering arterial resistance of the fast time-constant compartment. Blood flow to the slow time-constant compartment was increased 43 +/- 14% at the highest dose, and central blood volume was reduced 108 +/- 15 mL. In contrast, nifedipine had a balanced effect on arterial resistance with the lowest dose (0.025 mg/kg) but caused a preferential reduction in arterial resistance of the fast time-constant compartment at higher doses (-38 +/- 4% and -55 +/- 2% at 0.05 and 0.10 mg/kg, respectively). Blood flow to the slow time-constant compartment was reduced 36 +/- 5% at the highest dose of nifedipine, and central blood volume was increased 66 +/- 12 mL. Total systemic venous compliance was unaltered or slightly reduced by each of the four drugs. These results add further evidence to the hypothesis that peripheral blood flow distribution is a major determinant of venous return to the heart.     

Modification of guanine bases by nucleoside phosphoramidite reagents during the solid phase synthesis of oligonucleotides.

Nucleoside 3'-phosphoramidite and chlorophosphite reagents have been found to react with the lactam function of guanine. This reaction caused unsatisfactory results when oligodeoxyribonucleotides containing a large number of guanine bases were prepared in an automated solid phase synthesizer. The guanine modification is unstable, and leads to depurination and chain cleavage. This side reaction can be eliminated by protecting the O6-position. A new O6-p-nitrophenylethyldeoxyguanosine phosphoramidite derivative, 8, was used to prepare sequences containing up to 24 guanine bases with greatly improved results. A hexatriacontanucleotide, d(CGCGGGGTGGAGCAGCCTGGTAGCTCGTCGGGCTCA), was also prepared using O6-protected deoxyguanosine nucleosides.     

Identification and field testing of floral odorants that attract the rove beetle <Emphasis Type="Italic">Pelecomalium testaceum</Emphasis> (Mannerheim) to skunk cabbage, <Emphasis Type="Italic">Lysichiton americanus</Emphasis> (L.)

Western skunk cabbage, Lysichiton americanus (Araceae), is pollinated mainly by the rove beetle Pelecomalium testaceum (Staphylinidae). Our objective was to determine the floral semiochemical(s) of L. americanus that attract(s) P. testaceum. Porapak Q headspace volatile extracts of L. americanus inflorescences were analyzed by gas chromatographic–electroantennographic detection (GC–EAD) and GC–mass spectrometry. In GC–EAD analyses, three floral odorants [(E)-4 nonene, (E)-5-undecene, indole] elicited consistent responses from the antennae of female P. testaceum. In field experiments, traps baited with a blend of these three components (“3-CB”) captured significantly more P. testaceum than unbaited control traps. Traps baited with the 3-CB, the two hydrocarbons, or indole, each captured significantly more beetles than unbaited control traps, indicating redundancy in the semiochemical blend. Moreover, traps baited with indole captured significantly more beetles than traps baited with either the 3-CB, or the hydrocarbons, indicating that indole is a key floral attractant for P. testaceum. Many necrophilous and coprophilous insects respond to indole in search of carrion or feces, but P. testaceum has never been associated with these types of resources. Both electrophysiological and behavioral responses of P. testaceum to two hydrocarbon semiochemicals, which are not signature odorants of carrion or feces, may indicate that the beetles recognize the odor of L. americanus as an honest signal, seek and pollinate its inflorescences, and get rewarded with pollen and on-plant mating opportunities.     

Movement in the normal visual hemifield induces a percept in the 'blind' hemifield of a human hemianope.

We have investigated visual responses to moving stimuli presented to the normal hemifield of a hemianope, GY, who exhibits residual visual function in his right, ''blind'' hemifield. Preliminary experiments established that his perception of moving stimuli localized in his ''blind'' hemifield is retained when a similar stimulus is presented simultaneously in the normal hemifield. In response to a grating stimulus moving horizontally towards fixation in the non-foveal region of the normal, left hemifield, he perceives in addition to a normal motion percept in the left hemifield, a sensation of movement localized in the right hemifield. Qualitatively, this latter is indistinguishable from responses elicited by direct stimulation localized within his ''blind'' hemifield by moving stimuli. We have investigated the characteristics of the mechanisms which induce the ''blind'' field component of GY''s responses to stimulation of the normal hemifield. We show that GY''s sensitivity for detection of movement localized within his ''blind'' hemifield is dependent on the direction of movement, the contrast and the velocity of a grating presented to the normal hemifield. No induced effects were recorded in response to colour or to non-moving, flickering stimuli. We examine the possible contribution of scattered light to our observations, and eliminate this factor by consideration of our experimental results. We discuss the neural mechanisms which may be involved in this response.     

Prevention of chain cleavage in the chemical synthesis of 2''-silylated oligoribonucleotides.

Strong aqueous ammonium hydroxide used to remove N-acyl protecting groups from synthetic oligoribonucleotides causes removal of some alkylsilyl protecting groups from 2'-hydroxyls and leads to chain cleavage. This problem is most severe when 30% ammonium hydroxide is used and substantially reduced but still detectable when 3:1 ammonium hydroxide-ethanol is used. We have virtually eliminated this unwanted cleavage by incorporating the labile phenoxyacetyl amino protecting group on adenosine and guanosine. The N-benzoyl protecting group remains adequate for cytidine nucleosides. Synthetic oligoribonucleotides containing these N-acylated nucleosides and 2'-t-butyldimethylsilyl or 2'-triisopropylsilyl protecting groups can be deacylated by room temperature treatment in saturated anhydrous methanolic ammonia (8-12 h) without causing any detectable chain cleavage.     

Compartmental vascular changes in dogs after nephrectomy, DOCA, and saline: effect of nifedipine

Hypertension (mean arterial pressure, (MAP) 131 +/- 3 mmHg) developed in 18 dogs 4 weeks after left nephrectomy, deoxycorticosterone acetate (DOCA), 5 mg/kg sc twice weekly), and 0.5% NaCl drinking solution. This can be compared with MAP (95 +/- 7 mmHg) of 13 dogs with nephrectomy alone and MAP (86 +/- 4 mmHg) of dogs without nephrectomy. The two-compartment model of the circulation revealed no differences in systemic vascular compliance, compartmental compliance, or flow distribution to the compartments. However, the time constant for venous return for the compartment with the rapid time constant was increased from 0.05 +/- 0.004 min in control animals to 0.07 +/- 0.006 min in the nephrectomy alone group and 0.09 +/- 0.008 min in the hypertensive group (p less than 0.001), as a result of an increase in venous resistance. Arteriolar resistance in this compartment was also increased in the hypertensive animals, as was the mean circulatory filling pressure and overall resistance to venous return. Nifedipine (0.025-0.05 mg/kg) reduced MAP by 15% in the nephrectomy alone group and by 22% in the hypertensive group, with reduction in arteriolar resistance only in the fast time constant compartment. In the slow time constant compartment, arteriolar resistance was increased by more than 100% and flow decreased by more than 50% after nifedipine. Unilateral nephrectomy, DOCA, plus NaCl resulted in hypertension by increasing arteriolar resistance in a vascular compartment with a fast time constant for venous return. Nifedipine countered this effect by inducing arteriolar vasodilation in this compartment. In addition, nifedipine reduced the mean circulatory filling pressure and overall resistance to venous return.     

The chemical synthesis of oligoribonucleotides VII. A comparison of condensing agents in the coupling of silylated ribonucleosides.

The t-butyldimethylsilyl group is shown to be an ideal protecting group for the 2T-hydroxyl function of ribonucleosides during the synthesis of ribonucleotides using any of nine commonly used condensing agents. The phosphite coupling procedure compares favorably with all of the widely used condensing agents and provides a most convenient route to the key intermediates in the "modified" triester strategy.     

Indications for admission to a coronary care unit in patients with unstable angina.

One hundred cases with an admission diagnosis of acute coronary insufficiency or unstable angina were reviewed to establish criteria for admission to a coronary care unit. Myocardial infarction was subsequently diagnosed in 20 of the patients. Ventricular tachycardia occurred in 16 patients and ventricular fibrillation in 1 patient. Clinical features found to predict an increased risk of myocardial infarction included chest pain for more than 30 minutes within 24 hours prior to admission, new nonspecific electrocardiographic abnormalities consistent with ischemia, and diaphoresis. All patients with ventricular tachydysrhythmias had presented with both prolonged chest pain prior to admission and new electrocardiographic changes. The sensitivity, specificity and predictive value of various clinical criteria for identifying patients likely to have a myocardial infarction were calculated, and criteria with very high (greater than 90%) sensitivity were identified. These could be used to establish which patients are at increased risk of myocardial infarction and therefore require admission to a coronary care unit.