The effects of microtubule dissociating agents on the physiology and cytology of the sensory neuron in the femoral tactile spine of the cockroach, periplaneta americana L

Microtubules are prominent cellular components of the mechanosensory and chemosensory sensilla associated with the insect cuticle, and a range of hypotheses have been proposed to account for their role in sensory transduction. Chemical agents such as colchicine and vinblastine, which dissociate microtubules, also interfere with transduction in these sensilla, and this has been attributed to their anti-microtubule activity. We have now examined the dynamic properties of sensory transduction in the mechanosensitive neuron of the cockroach femoral tactile spine, after the application of colchicine, vinblastine and lumicolchicine. Concurrently we have examined the ultrastructure of the same sensory ending by transmission electron microscopy. All of the drugs reduced the mechanical sensitivity o the receptor. Colchicine and vinblastine achieved this reduction without altering the dynamic properties of the receptor but lumicolchicine changed the dynamic response, and increased the relative sensitivity to rapid movements. Conduction velocity, another measure of neuronal function, which relies upon ionic currents flowing through the membrane, was reduced by all three drugs. The effects of the drugs upon the ultrastructure of the sensory ending were also disparate. In the case of colchicine there was complete dissociation of microtubules in the tubular body and distal dendrite before a total loss of mechanical sensitivity. Vinblastine was less effective in dissociating microtubules, although more effective in the reduction of mechanical sensitivity. With lumicolchicine the dominant morphological effect was a severe disruption of the dendritic membrane. We conclude from these experiments that microtubules are not essential in the transduction of mechanical stimuli by cuticular receptors and that the effects of these drugs upon mechanosensitivity are not directly related to their dissociation of the microtubules in the tubular body, but are more likely to arise from actions upon the cell membrane. These actions could include effects upon tubulin in the membrane or upon other membrane components.     

Assaying Actual Hematein Content of Commercial Hematoxylins and Hemateins

Hematein-free hematoxylin (HFH) was prepared by a modification of the procedure of Palmer and Lillie (Histochemie, 5: 44-54, 1965). Fifty mg of HFH were dissolved in 5 mg of ethylene glycol and then 45 nil of an aqueous solution of 2.25 gm KAl(SO₄)₂. 12H₂O and 5.445 mg KIO₃ were added. Since this amount of KIO₃ would be sufficient to oxidize 25 mg of HFH to hematein we have termed this half-oxidized hematoxylin (HOH). The peak absorbance (560 nm) of this purple solution remained constant for at least a week. With omission of the KIO₃ the solution was colorless. A curve was constructed by plotting absorbance against concentration of hematein in HOH at various dilutions. For analyses of hematein content of commercial hematoxylins 50 mg of sample and 100 mg of hydroquinone were dissolved in 5 ml of ethylene glycol and then 45 ml of a 5% solution of KAl(SO₄)₂. 12H₂O were added. The addition of the hydroquinone stabilized the absorbance for about 5 min. The hematein content could then be calculated by comparing the observed absorbance with the standard curve. Eleven samples of hematoxylin certified by the Biological Stain Commission had hematein concentrations varying from 0.01 to 0.43%. For analyses of the available hematein content of commercial hemateins, 50 mg of sample were dissolved in 10 ml of ethylene glycol, then 45 ml of water and 45 ml of 5% KAI(SO₄)₂. 12H₂O added. The hematein content could then be calculated by comparing the observed absorbance with the standard curve. In 9 samples of hematein from 4 different sources the active hematein content varied from 19 to 97%.     

Leucine aminopeptidase in extracts of swine muscle

1. Leucine aminopeptidase (EC 3.4.1.1) has been demonstrated in swine muscle at a level of activity one-fifth that of the swine kidney. 2. The enzyme has been purified 110-fold by precipitation with ammonium sulphate, heat treatment and chromatography on Sephadex G-100. 3. The enzyme is heat-stable, but is rapidly inactivated below pH7. It requires Mg(2+) or Mn(2+) for activity. The Michaelis constant for leucine amide with Mg(2+)-activated enzyme is 5.0x10(-3)m. 4. Muscle leucine aminopeptidase is very similar to the kidney enzyme.     

Respiratory sinus arrhythmia in the denervated human heart

We performed this study to test whether the denervated human heart has the ability to manifest respiratory sinus arrhythmia (RSA). With the use of a highly sensitive spectral analysis technique (cross correlation) to define beat-to-beat coupling between respiratory frequency and heart rate period (R-R) and hence RSA, we compared the effects of patterned breathing at defined respiratory frequency and tidal volumes (VT), Valsalva and Mueller maneuvers, single deep breaths, and unpatterned spontaneous breathing on RSA in 12 normal volunteers and 8 cardiac allograft transplant recipients. In normal subjects R-R changes closely followed changes in respiratory frequency (P less than 0.001) but were little affected by changes in VT. On the R-R spectrum, an oscillation peak synchronous with respiration was found in heart transplant patients. However, the average magnitude of the respiration-related oscillations was 1.7-7.9% that seen in normal subjects and was proportionally more influenced by changes in VT. Changes in R-R induced by Valsalva and Mueller maneuvers were 3.8 and 4.9% of those seen in normal subjects, respectively, whereas changes in R-R induced by single deep breaths were 14.3% of those seen in normal subjects. The magnitude of RSA was not related to time since the heart transplantation, neither was it related to patient age or sex. Thus the heart has the intrinsic ability to vary heart rate in synchrony with ventilation, consistent with the hypothesis that changes, or rate of changes, in myocardial wall stretch might alter intrinsic heart rate independent of autonomic tone.     

Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY

The protein (Escherichia coli CheY) that controls the direction of flagellar rotation during bacterial chemotaxis has been shown to be phosphorylated on the aspartate 57 residue. The residue phosphorylated is present within a conserved sequence in every member of a family of bacterial regulatory proteins. The phosphorylation is transient, with a much shorter half-life than that expected of a simple acyl phosphate intermediate, indicating that the sequence and conformation of the protein is designed to achieve a rapid hydrolysis. The CheY-phosphate linkage can be reductively cleaved by sodium borohydride. High-performance tandem mass-spectrometric analysis of proteolytic peptides derived from [3H]borohydride-reduced phosphorylated CheY protein was used to identify the position of phosphorylation. Mutants with altered aspartate 57 exhibited no chemotaxis. When aspartate 13, another conserved residue, was changed, greatly reduced chemotaxis was observed, suggesting an important role for aspartate 13. The rate-determining step of chemotactic signaling is governed by the kinetics of formation and hydrolysis of the CheY protein phosphoaspartate bond. The CheY protein apparently functions as a protein phosphatase that possesses a transient covalent intermediate. Transient phosphorylation of an aspartate residue is an effective mechanism for producing a biochemical signal with a short concentration-independent half-life. The duration of the signal can be controlled by small structural elements within the phosphorylated protein.