Calmodulin Acts as an intermediary for the effects of calcium on gap junctions from crayfish lateral axons

Summary Lateral axons from the abdominal nerve cord of cray-fish were internally perfused with the calcium receptor calmodulin (CaM) in solutions with low (pCa>7.0) or high (pCa 5.5) calcium concentrations and studied electrophysiologically and morphologically. Results from these experiments show that when the internal solution contains calcium-activated calmodulin (Ca²⁺-CaM) the junctional resistance between the axons increases from control values of about 60 to 500–600 k in 60 min. In contrast, axons perfused with calmodulin in low calcium solutions maintain their junctional resistance at control levels during the 60-min perfusion. Similar results are obtained when only one or both coupled axons are perfused.The morphological study shows that in the perfused axons the axoplasmic organelles are replaced or grossly perturbed by the perfusion solution up to the region of the synapses. Additionally, in axons perfused with Ca²⁺-CaM there are regions where the synaptic gap between the membranes decreases from a control 4–6 to 2–3 nm. Both electrophysiological and morphological results can be interpreted as indicating that calcium-activated calmodulin acts directly on the junctional channels to induce their closure.     

Three-dimensional model of stellacyanin and its implications for electron transfer reactivity

Experimental data were combined with computational methods in constructing a hypothetical three-dimensional model for the blue single copper protein Rhus stellacyanin (St). The known sequence of stellacyanin and its homology with plastocyanin (Pc) were used together with the results of spectroscopic studies of the protein that yielded the current assignment of two histidines, one cysteine and a disulfide sulfur as copper ligands in stellacyanin. By computer graphics and energy minimization the folding of the protein was predicted. The model structure is somewhat less regular than Pc as judged by surface area and energy comparisons, but it is a stable structure. Besides rotation of one imidazole ring the copper site undergoes no change even in the absence of the copper ion and the model shows that the site can be constructed with the four assumed copper ligands without forming a strained system. The structure also indicates that a carbonyl oxygen atom is near the copper, thus the site may have analogy to the Alcaligenes denitrificans azurin (Az) site, although the amino acid sequence is more homologous to that of Pc. The model indicates that aspartate 49, reductively labeled by Cr(III), is near the copper center and homologous to the site labeled by Cr(III) on Pc. Also homologous to Pc is a tyrosine residue adjacent to the aspartate. This tyrosine has been implicated in Pc electron transfer and thus is probably involved in electron transfer reactivity of St as well. The higher reactivity of St with small-molecule redox reagents compared to Az and Pc, may be due to the proximity of the above-mentioned aspartate 49 to the Cu, or the greater exposure of one of the Cu cysteine ligands, in the predicted structure as compared to that in the known Pc and Az structures.     

Brain metabolism of sarcoma 45-bearing rats undergoing radiation and the effect of hyperthermia on the tumor

Activity of hexokinase and acetylcholinesterase and pyridoxal co-enzyme content of brain subcellular fractions were studied in rats, bearing sarcoma 45, after local exposure of the tumor to 20 Gy X-radiation and microwave hyperthermia. The carbohydrate metabolism was sharply inhibited while the pyridoxal coenzyme content and acetylcholinesterase activity increased.     

Chest-wall reconstruction by contralateral latissimus dorsi musculocutaneous flap

Reconstruction of chest wall and axilla are performed in 11 patients using a contralateral latissimus dorsi musculocutaneous flap. The entire lattisimus dorsi muscle, including the fascial portion, safely carried an island of skin from the area of the lumbodorsal fascia to the contralateral axilla. The flap was transposed to the defect through a tunnel between the pectoralis major and minor muscles. Most patients who needed reconstruction of the chest wall and axilla had compromised ipsilateral vasculature that prohibited its use in a pedicled flap but had an intact contralateral chest wall, axilla, and thoracodorsal vessels. Therefore, this procedure was performed easily in comparison with a free flap or pedicled omental flap. This is a new, valuable application for the versatile latissimus dorsi musculocutaneous flap.     

Regulation of malic-acid metabolism in Crassulacean-acid-metabolism plants in the dark and light: In-vivo evidence from 13C-labeling patterns after 13CO2 fixation

The labeling patterns in malic acid from dark ¹³CO₂ fixation in seven species of succulent plants with Crassulacean acid metabolism were analysed by gas chromatography-mass spectrometry and ¹³C-nuclear magnetic resonance spectrometry. Only singly labeled malic-acid molecules were detected and on the average, after 12–14 h dark ¹³CO₂ fixation the ratio of [4-¹³C] to [1-¹³C] label was 2:1. However the 4-C carboxyl contained from 72 to 50% of the label depending on species and temperature. The ¹³C enrichment of malate and fumarate was similar. These data confirm those of W. Cockburn and A. McAuley (1975, Plant Physiol. 55, 87–89) and indicate fumarase randomization is responsible for movement of label to 1-C malic acid following carboxylation of phosphoenolpyruvate. The extent of randomization may depend on time and on the balance of malic-acid fluxes between mitochondria and vacuoles. The ratio of labeling in 4-C to 1-C of malic acid which accumulated following ¹³CO₂ fixation in the dark did not change during deacidification in the light and no doubly-labeled molecules of malic acid were detected. These results indicate that further fumarase randomization does not occur in the light, and futile cycling of decarboxylation products of [¹³C] malic acid (¹³CO₂ or [1-¹³C]pyruvate) through phosphoenolpyruvate carboxylase does not occur, presumably because malic acid inhibits this enzyme in the light in vivo. Short-term exposure to ¹³CO₂ in the light after deacidification leads to the synthesis of singly and multiply labeled malic acid in these species, as observed by E.W. Ritz et al. (1986, Planta 167, 284–291). In the shortest times, only singly-labeled [4-¹³C]malate was detected but this may be a consequence of the higher intensity and better detection statistics of this ion cluster during mass spectrometry. We conclude that both phosphoenolpyruvate carboxylase (EC 4.1.1.32) and ribulose-1,5-biphosphate carboxylase (EC 4.1.1.39) are active at this time.Abbreviations CAM Crassulacean acid metabolism - GCMS gas chromatography-mass spectrometry - MS mass spectrometry - NMR nuclear magnetic resonance spectrometry - PEP phosphoenolpyruvate - RuBP ribulose 1,5-bisphosphate     

Production of ethanol from cassava whey

Investigations were conducted into the potential use of enzyme hydrolysed cassava whey for ethanol production by Saccharomyces cerevisiae Aspergillus niger grown on whct bran was used as crude enzyme source to saccharify the whey starch. The whey with an initial HCN concentration of 54.0μg/ml was fermented at pH 4.5 and 30°C in a one-step process to produce ethanol. A maximum ethanol concentration of 4.5% (v/v) was obtained in 120 h with a decrease in HCN level to 4.0 μg/ml. In a two-stage fermentation, in which the raw whey was pre-hydrolysed and under the same fermentation conditions, the unsterilized hydrolysate yielded alcohol content of 5.5% (v/v), while the sterilized hydrolysate gave higher alcohol yield, 7.5% (v/v), in 48 h. No HCN was detected in the fermented liquour at the end of the two-stage process.     

Inhomogeneity during deflation of excised canine lungs. I. Alveolar pressures

Factors both intrinsic and extrinsic to the lung may cause inhomogeneity of alveolar pressures during deflation. Wilson et al. (J. Appl. Physiol. 59: 1924-1928, 1985) predicted that any such inhomogeneity would be limited by interdependence of regional expiratory flows. To test this hypothesis and to explore how the pleural pressure gradient might affect inhomogeneity of alveolar pressures, we deflated at submaximal flows excised canine lobes that first were suspended in air and then were immersed in foams that simulated the vertical gradient of pleural pressure. Interregional inhomogeneity of regional transpulmonary pressures was measured with use of an alveolar capsule technique. Flow-dependent inhomogeneity of alveolar pressures was present, with differences in alveolar pressure quickly relaxing to a constant limiting value at each flow. Foam immersion increased inhomogeneity at a given flow. We conclude that factors intrinsic to the lung cause significant inhomogeneity of alveolar pressures at submaximal expiratory flows and that this inhomogeneity is enhanced by the extrinsic gradient of pleural pressure. These observations are consistent with the interdependence of flow proposed by Wilson et al.     

A hormone-sensitive stage in the development of the retinal pigment epithelium in Hunter rats with hereditary retinal dystrophy

The sensitivity of the retinal pigment epithelium (RPE) to the melanotropic effects of alpha-MSH and dbcAMP was assayed in an organ culture of the eye scleral part in the Hunter rats with inherited retinal dystrophy. The melanin synthesis was estimated by liquid scintillation on the RPE isolated enzymatically after 48-h cultivation. One eye from every animal was cultivated in a medium without natural components and with the hormone or dbcAMP (experiment), while the other in a hormone-free medium (control). The melanin synthesis was estimated by 14C-thiouracil incorporation. The result was expressed as a cpm/microgram DNA (experiment) to cpm/microgram DNA (control) ratio. In addition, the index of labelled nuclei was determined using 3H-thymidine autoradiography in the central RPE zone of the eyes from young rats of the same litter. The experiments with alpha-MSH confirmed the earlier data according to which the RPE of the 3 day old Hunter rats were insensitive to melanotropic hormones. This was not due to defects in the cytoplasmic melanin-synthesizing system, since under the influence of dbcAMP the melanin synthesis in the RPE increases more than four-fold as compared with the control; dbcAMP stimulates also the total protein synthesis, as estimated by 3H-leucine incorporation. The RPE of the 4 day old rats proved to be sensitive to alpha-MSH: the melanin synthesis increases more than twice suggesting the healthy state of the RPE membrane melanotropic receptors. alpha-MSH also stimulates the total protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection by the use of silver ions of additional protein zones in gradient polyacrylamide gels stained with Coomassie

A simple method of revealing the additional zones of proteins in gradient polyacrylamide gels, preliminary dyed Coomassie by means of silver ions is described. The dyeing of Coomassie allows to avoid the time-consuming stages of preliminary treatment of gels as well to reveal more sensitive zones in gels. On the second stage of dyeing silver minor zones appear there which were not seen while Coomassie was dyed. The suggested method preserves high sensitivity characteristic of the methods of gel dyeing with silver.