Stimulation of Ca2+ influx in rat pituitary cells under exposure to a 50 Hz magnetic field

The effect of exposure of single rat pituitary cells to 50 Hz sine wave magnetic fields of various strengths on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) was studied by using dual-emission microfluorimetry, using indo-1 as probe. A 30 min exposure of the cells to vertical 50 μT peak magnetic field triggered a long-lasting increase in [Ca²⁺]_i from a basal value of about 185 ± 4 nM to 326 ± 41 nM (S.E.; n = 150). The vertical and horizontal components of the static magnetic field were 57 and 15 μT, respectively. The 50 Hz ambient magnetic field was always below 0.1 μT rms. The effect was observed both at 25 ± 2 °C and at 37 ± 2 °C. Responsive cells, for which [Ca²⁺]_i rose to values above 309 nM, were identified as lactotrophs and represented 29% of the total pituitaries. [Ca²⁺]_i increase, for the most part, was due to Ca²⁺ influx through voltage-dependent dihydropiridine-sensitive calcium channels inhibited by PN 200-110. However, neither Ca²⁺ channel blockers nor removal of Ca²⁺ from the external medium during exposure completely prevented the field-induced [Ca²⁺]_i increase. Additional experiments using an MTT colorimetric assay showed that alteration of Ca²⁺ homeostasis of lactotrophs was associated with impairment of some mitochondrial processes. © Wiley-Liss, Inc.     

Single-Step Purification of Two Functional Human Apolipoprotein E Variants Hyperexpressed inEscherichia coli

We have cloned, from total human liver RNA, the cDNA encoding apolipoprotein E3 (apoE3). Site-directed mutagenesis was used to obtain the cDNA encoding the apoE4 isoform, a major variant of this apolipoprotein in man. These two cDNAs were subcloned into the procaryotic expression vector pAHRS. A polyhistidine tag was added at the NH₂-termini of the recombinant proteins (apoE3 and apoE4) to enable rapid purification. The resulting plasmids (pAHRS-apoE3 and pAHRS-apoE4) were introduced into theEscherichia colistrain BL21(DE3). Recombinant strains were grown at 37°C in a Luria and Bertani medium and the addition of isopropyl β-thiogalactoside resulted in the expression of large amounts of apoE protein (40.5 kDa), representing at least 15% of cellular proteins. The recombinant apoE isoforms were purified, under denaturating conditions, in one step by affinity chromatography on a Ni-chelated agarose column, yielding to about 20 mg of 96% pure protein per liter of culture. Compared to plasma apoE3 purified from human very low density lipoproteins, the two renatured recombinant apoE isoforms have the same secondary structure content, as revealed by circular dichroism measurement. Moreover, the recombinant apoE3 isoform shares similar properties for the association with lipids, compared to the human protein, indicating that the addition of the amino-terminal polyhistidine peptide does not influence the structure and the lipid binding properties of this recombinant apoE isoform. No differences in the secondary structure of recombinant apoE4 were detected, whereas this isoform presents specific reactivity with lipids. This simple and rapid procedure for the expression and the purification of functional recombinant apoE should therefore enable structural and physiological studies requiring large amounts of these apolipoproteins.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Structure and activities of constrained analogues of human parathyroid hormone and parathyroid hormone-related peptide: implications for receptor-activating conformations of the hormones

Parathyroid hormone (PTH) has a helix-bend-helix structure in solution. Part of the C-terminal helix, residues 21-31, is amphiphilic and forms a critical receptor-binding region. Stabilization of this alpha-helix by lactam formation between residues spaced i, i + 4 on the polar face was previously reported to increase adenylyl cyclase-stimulating (AC) activity if between residues 22 and 26 but to diminish it if between residues 26 and 30 [Barbier et al. (1997) J. Med. Chem. 40, 1373-1380]. This work reports the effects of other cyclizations on the polar face, differing in ring size or position, on alpha-helix conformation, as measured by circular dichroism, and on AC-stimulating activity. All analogues cyclized between residues 22 and 26 had at least a 1. 5-fold increase in activity, suggesting an alpha-helical structure between about residues 21 and 26. Cyclization between residues 25 and 29 or residues 26 and 30 diminished activity by 20-30%, despite stabilizing alpha-helix, suggesting that residues 25-31 bind to the receptor in a helical, but not classical alpha-helical, conformation. Analogues cyclized between residues 13 and 17 had slightly increased activity. A bicyclic analogue, with lactams between residues 13 and 17 and residues 22 and 26, had about the same activity as that cyclized only between 22 and 26. Parathyroid hormone-related peptide (PTHrP) may bind in a manner similar to the common receptor, but hydrophobic moment calculations suggest that it must bind as a tighter helix in order to optimally present its hydrophobic residues to the receptor. Both PTHrP analogues cyclized between either residues 22 and 26 or residues 26 and 30 had more stable alpha-helices but reduced AC activities, consistent with this hypothesis.     

Novel tetrahydroisoquinolines are histamine H3 antagonists and serotonin reuptake inhibitors

A series of novel 4-aryl-1,2,3,4-tetrahydroisoquinoline-based histamine H(3) ligands that also have serotonin reuptake transporter inhibitor activity is described. The synthesis, in vitro biological data, and select pharmacokinetic data for these novel compounds are discussed.     

Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase

Background  

Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure.     

Caulerpenyne from Caulerpa taxifolia has an antiproliferative activity on tumor cell line SK-N-SH and modifies the microtubule network.

Caulerpenyne, the major secondary metabolite synthesized by the green marine alga Caulerpa taxifolia, is cytotoxic against several cell lines. To identify possible targets of this toxin, we investigated the effect of caulerpenyne on the neuroblastoma SK-N-SH cell line. Caulerpenyne induced an inhibition of SK-N-SH cell proliferation with an IC50 of 10 +/- 2 microM after 2 hr of incubation.We observed no blockage in G2/M phase and an increase in cell death. On immunofluorescence microscopy, caulerpenyne affected the microtubule network in SK-N-SH cell line; we observed a loss of neurites and a compaction of the microtubule network at the cell periphery. In vitro, after 35 min of incubation, caulerpenyne inhibited the polymerization of pig brain purified tubulin or microtubule proteins, with an IC50 of 21 +/- 2 microM and 51 +/- 6 microM respectively. Analysis by electron microscopy indicated that caulerpenyne induced aggregation of tubulin, which may be responsible for inhibition of microtubule polymerization and bundling of residual microtubules.     

Sex differences in the rate of fatigue development and recovery

Background

Many musculoskeltal injuries in the workplace have been attributed to the repetitive loading of muscle and soft tissues. It is not disputed that muscular fatigue is a risk factor for musculoskeltal injury, however the disparity between gender with respect to muscular fatigability and rate of recovery is not well understood. Current health and safety guidelines do not account for sex differences in fatiguability and may be predisposing one gender to greater risk. The purpose of this study was to quantify the sex differences in fatigue development and recovery rate of lower and upper body musculature after repeated bouts of sustained isometric contractions.

Methods

Twenty-seven healthy males (n = 12) and females (n = 15) underwent bilateral localized fatigue of either the knee extensors (male: n = 8; female: n = 8), elbow flexors (male: n = 8; female: n = 10), or both muscle groups. The fatigue protocol consisted of ten 30-second sub-maximal isometric contractions. The changes in maximum voluntary contraction (MVC), electrically evoked twitches, and motor unit activation (MUA) were assessed along with the ability to control the sustained contractions (SLP) during the fatigue protocol using a mixed four-factor repeated measures ANOVA (gender × side × muscle × time) design with significance set at p < 0.05.

Results

There was a significant loss of MVC, MUA, and evoked twitch amplitude from pre- to post-fatigue in both the arms and legs. Males had greater relative loss of isometric force, a higher rate of fatigue development, and were less capable of maintaining the fatiguing contractions in the legs when compared to the females.

Conclusion

The nature of the induced fatigue was a combination of central and peripheral fatigue that did not fully recover over a 45-minute period. The results appear to reflect sex differences that are peripheral, and partially support the muscle mass hypothesis for explaining differences in muscular fatigue.

     

NMR solution structures of delta-conotoxin EVIA from Conus ermineus that selectively acts on vertebrate neuronal Na+ channels

Delta-conotoxin EVIA, from Conus ermineus, is a 32-residue polypeptide cross-linked by three disulfide bonds forming a four-loop framework. delta-Conotoxin EVIA is the first conotoxin known to inhibit sodium channel inactivation in neuronal membranes from amphibians and mammals (subtypes rNa(v)1.2a, rNa(v)1.3, and rNa(v)1.6), without affecting rat skeletal muscle (subtype rNa(v)1.4) and human cardiac muscle (subtype hNa(v)1.5) sodium channel (Barbier, J., Lamthanh, H., Le Gall, F., Favreau, P., Benoit, E., Chen, H., Gilles, N., Ilan, N., Heinemann, S. F., Gordon, D., Ménez, A., and Molgó, J. (2004) J. Biol. Chem. 279, 4680-4685). Its structure was solved by NMR and is characterized by a 1:1 cis/trans isomerism of the Leu(12)-Pro(13) peptide bond in slow exchange on the NMR time scale. The structure of both cis and trans isomers could be calculated separately. The isomerism occurs within a specific long disordered loop 2, including residues 11-19. These contribute to an important hydrophobic patch on the surface of the toxin. The rest of the structure matches the "inhibitor cystine-knot motif" of conotoxins from the "O superfamily" with a high structural order. To probe a possible functional role of the Leu(12)-Pro(13) cis/trans isomerism, a Pro(13) --> Ala delta-conotoxin EVIA was synthesized and shown to exist only as a trans isomer. P13A delta-conotoxin EVIA was estimated only two times less active than the wild-type EVIA in binding competition to rat brain synaptosomes and when injected intracerebroventricularly into mice.