Central helix role in the contraction-relaxation switching mechanisms of permeabilized skeletal and smooth muscles with genetic manipulation of calmodulin

A prominent common feature of calmodulin and troponin structures is the unusually long central helix which separates the two lobes, each containing two Ca2(+)-binding sites. To study the role of certain highly conserved residues in the helix in the contraction-relaxation switching mechanism in muscle, we measured the Ca2(+)-activated force of permeabilized skeletal and smooth muscles with three genetically manipulated forms of calmodulin. Mutated calmodulin was made to substitute for troponin-C in vertebrate skeletal fiber. The mutants had 1-4 deletions in the conserved cluster (positions 81-84) in the solvent-exposed region of the central helix, which also substantially shortened the helix. The force of the maximally activated fiber was found to be diminished only with the mutant in which the entire cluster Ser-81 to Glu-84 (CaM delta 81-84) was deleted. All such deletions were found to be completely ineffective in blocking the Ca2(+)-switching process in smooth muscle strips. The results show for the first time that at least a part of the highly conserved four-residue cluster in the central helix is critical for the contraction mechanism of striated muscle. Further, the possibility is raised that the reduced length of the central helix may be a determining factor in the Ca2(+)-switching mechanism in fast-twitch muscle. These findings combined with the results on smooth muscle indicate diversity in the structure-function specifications for the central helix of calmodulin for different target proteins.     

First attempt to apply whole-lake food-web manipulation on a large scale in The Netherlands

Lake Breukeleveen (180 ha, mean depth 1.45 m), a compartment of the eutrophic Loosdrecht lakes system, was selected to study the effects of whole-lake foodweb manipulation on a large scale. In Lake Loosdrecht (dominated by filamentous cyanobacteria), due to water management measures taken from 1970–1984 (sewerage systems, dephosphorization) the external P load has been reduced from 1.2 g m⁻² y⁻¹ to 0.35 g m⁻² y⁻¹. The water transparency (Secchi-depthca. 30 cm), however, has not improved. The aim of the food-web manipulation in Lake Breukeleveen was not only to improve the light climate of the lake, but also to study if the successfull effects observed in small lakes (a few ha) can be upscaled. In March 1989 the standing crop of planktivorous and bentivorous fish populations was reduced by intensive fishery, fromca. 150 kg ha⁻¹ toca. 57 kg ha⁻¹. The lake was made unaccessible to fish migrating from the other lakes and it was stocked with large-sized daphnids and 0⁺ pike. However, water transparency did not increase in the following summer and autumn 1989, which is in contrast with great improvement in the light conditions previously observed in smaller lakes. The main explanations for the negative outcome in Lake Breukeleveen are: 1) the rapid increase of the planktivorous fish biomass and carnivorous cladocerans, predating on the zooplankton community; 2) suppression of the large daphnids by the high concentrations of filamentous cyanobacteria; 3) high turbidity of the lake due to resuspension of bottom material induced by wind, unlike in smaller lakes, and thus inability of submerged macrophytes to develop and to stabilize the ecosystem.     

Characteristics of central binding sites for [3H] DAMGO in spontaneously hypertensive rats

The binding of [3H] DAMGO, a highly selective ligand for mu-opiate receptors, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. [3H] DAMGO bound to membranes of brain regions and spinal cord at a single high affinity site. The receptor density (Bmax value) and apparent dissociation constant (Kd value) of [3H] DAMGO to bind to membranes of hippocampus, corpus striatum, pons and medulla, cortex and spinal cord of WKY and SHR rats did not differ. The Bmax value of [3H] DAMGO in membranes of hypothalamus and midbrain of SHR rats was significantly higher than in WKY rats but the Kd values in the two strains did not differ. On the other hand, the Bmax value of [3H] DAMGO in membranes of amygdala of SHR rats was lower than that of WKY rats but the Kd values in the two strains were similar. It is concluded that SHR rats have higher density of mu-opiate receptors in hypothalamus and midbrain but lower density in amygdala in comparison with WKY rats, and that such differences in the distribution of mu-opiate receptors may be related to the elevated blood pressure in SHR rats.     

Comparison of single-, double- or triple-exposure protocols for the rodent bone marrow/peripheral blood micronucleus assay using 4-aminobiphenyl and treosulphan

Two human carcinogens, 4-aminobiphenyl (4AB) and treosulphan (Treo), were tested in male B6C3F1 mice for the induction of micronuclei in bone marrow and peripheral blood cells by 1-, 2- and 3-exposure protocols. Both compounds tested positive. The magnitude of response with respect to the incidence of micronucleated polychromatic erythrocytes by 2- and 3-exposure protocols was considerably higher than by the single-exposure protocol. The peripheral blood results for Treo were as typically seen with a 24-h delay when compared to the bone marrow. The peripheral blood results for 4AB, however, differed from those expected. The incidence of MN-PCE in peripheral blood of animals exposed to 4AB was significantly greater than seen in the bone marrow in 2- and 3-exposure protocols. There was also an increase in the % PCE at the 60 mg/kg dose level as a function of time. Based on these studies, it is concluded that a step-wise scoring scheme may be the best protocol for rodent micronucleus assay, involving a 3-exposure protocol with single sampling of bone marrow (24 h after the last treatment) and two samplings of peripheral blood (24 h and 48 h after the first treatment). This approach is cost-effective, it limits the number of animals required and provides maximum sensitivity.     

Culture conditions effecting plant regeneration from cotyledons of Vigna radiata (L.) Wilczek

Conditions for plant regeneration from excised cotyledons of Vigna radiata were studied. Complete plant developed from the uncallused proximal ends of cotyledons on Murashige & Skoog's (MS), Gamborg's (B₅) and C (MS salts + B₅ vitamins) basal media. The basal medium C was found to be best for plant regeneration. Regeneration frequency, however, varied with genotype, size, orientation and age of explant and the different plant growth regulators combination in the medium. Addition of cytokinins induced callusing at the proximal ends of cotyledons followed by multiple shoot formation. Out of 6-benzyl aminopurine (BAP), kinetin (KIN), N (^–2 isopentyl) adenine (2iP) and adenine sulphate (AS), only BAP and KIN were found to be more effective in enhancing the frequency of shoot regeneration. BAP at 1×10^-1M induced maximum (60%) shoot regeneration whereas maximum number of shoots (8 to 9 shoots) per explant was observed with 5×10^-6M BAP. Cotyledons excised from two-day old seedlings were most regenerative. The regenerative response of cotyledons decreased when sliced into two equal parts either longitudinally or transversely. Callusing and organogenic differentiation occured only if the petiolar end of cotyledons was in contact with medium. None of the tested treatments were effective in inducing shoot bud differentiation from subcultured callus. Well developed shoots rooted when incubated on half strength MS, MS and MS basal medium supplemented with IAA (5×10^-6M). The rooted plants were transferred to pots and later established in the field with 60% success.Abbreviations AS adenine sulphate - BAP 6-benzylaminopurine - B₅ medium after Gamborg et al. [6], - C Medium with MS salts + B₅ vitamins - 2iP N (^–2 isopentyl) adenine - IAA indole-3-acetic acid - KIN Kinetin - MS medium after Murashige & Skoog [21] - NAA 1-napthaleneacetic acid     

Functional delineation of the Ca(2+)-deficient EF-hand in cardiac muscle, with genetically engineered cardiac-skeletal chimeric troponin C.

Cardiac and fast skeletal isoforms of TnC each comprise four putative EF-hand (helix-loop-helix) motifs as potential Ca(2+)-binding sites (sites 1-4), except that site 1 in cardiac TnC is deficient in Ca2+ coordination. In skeletal TnC, the N-terminal sites 1 and 2 are both essential for the trigger mechanism of the contraction switch. However, the mechanism in cardiac muscle is unsettled; it is obscure whether the cardiac site 1 is functionally inert due to calcium deficiency and consequently site 2 is the lone trigger site, or whether sites 1 and 2 perform interactively despite the impairment. These possibilities were addressed by mutagenizing site 1 in skeletal TnC to mimic the cardiac response. In one mutant (STnC-1), two selected Ca(2+)-ligands were abolished. In another (C1/S chimera), 41 N-terminal residues from cardiac TnC were spliced to STnC. The Ca(2+)-binding capacities as well as skinned fiber responses were measured. The STnC-1 derivative failed to switch on contraction. In contrast, the chimeric construct expressed close to full contractile potential in myocardium (74 +/- 3% Po; Po = maximal tension) and also the manifest cardiac phenotype. By devising supplemental chimeric constructs, cardiac-type N-terminal overhang together with cardiac-type EF-hand for site 1 both were found essential for the phenotype. We conclude that cardiac TnC site 1 is actively engaged in the trigger mechanism and in fact dominates the phenotype despite the inability to chelate Ca2+. The N-terminal overhang also participates in this mechanism, which is a novel finding. The conclusion that a non-chelating site functions interactively with a proximal site in cardiac TnC may have wider significance, inasmuch as similar pairings of disparate EF-hands are of common occurrence.     

Methionine-enkephalin and beta-endorphin levels in spleen and thymus gland of morphine tolerant-dependent and abstinent rats

The effect of chronic administration of morphine and abrupt and naloxone-precipitated withdrawal on the levels of beta-endorphin and methionine-enkephalin in spleen, adrenals and thymus glands of Sprague-Dawley rats was determined. Rats were made tolerant to and dependent on morphine by subcutaneous implantation of 6 morphine pellets (75 mg morphine in each) during a 7-day period. The tolerant-dependent (with pellets intact) and abstinent (pellets removed 18 hours earlier) rats were sacrificed. In another group, rats with pellets intact were injected with naloxone and sacrificed 10 min later (precipitated abstinence). The weights of the tissues under any of the above treatments did not change nor did the levels of methionine-enkephalin and beta-endorphin in adrenals. The level of beta-endorphin was elevated in the spleen and thymus of morphine tolerant-dependent rats, while the levels of methionine-enkephalin in rats undergoing abrupt or naloxone-precipitated abstinence were significantly higher than in their respective placebo controls. The levels of methionine-enkephalin in the thymus gland of rats with placebo and morphine pellets left intact did not differ. It is concluded that in morphine tolerant-dependent rats the levels of beta-endorphin in spleen and thymus are elevated. During abrupt and naloxone-precipitated abstinence, the levels of methionine-enkephalin in the thymus gland are significantly elevated possibly due to an inhibition of their release. Since these opioid peptides have been implicated in immunomodulation, and alterations were seen in organs controlling immune function, the present results may be helpful in explaining altered immune function in morphine dependent and abstinent states.     

A laboratory study of feeding and assimilation in Euchlanis dilatata lucksiana

Feeding of Euchlanis dilatata lucksiana, a brachionid rotifer isolated from the Loosdrecht Lakes (The Netherlands), was examined in the laboratory using ¹⁴C-labelled food. The gut-filling time at a food concentration of 9.6 µg C ml^–1 was about 15 minutes. Animals which were fed on 3 size fractions of the lake seston (< 7 µm, 7–15 µm, and 15–33 µm) showed a clear preference (Jacobs' selectivity index) for the largest size fraction. This fraction was composed predominantly of filamentous cyanobacteria. For animals weighing 0.37–0.49 µm C ind.^–1 the daily ration (daily food consumption per unit body weight × 100) ranged from 50 to 100% at food levels of 2 mg C l^–1 and below, but increased to 150–250% at food concentrations of 5 mg C l^–1 and above. The assimilation efficiency was 100% up to 5 mg C l^–1 of food, but decreased to about 80% at higher food levels.