Acid-base properties and copper(II) complexes of dipeptides containing histidine and additional chelating bis(imidazol-2-yl) residues

Copper(II) complexes of dipeptides of histidine containing additional chelating bis(imidazol-2-yl) agent at the C-termini (PheHis-BIMA [N-phenylalanyl-histidyl-bis(imidazol-2-yl)methylamine] and HisPhe-BIMA [N-histidyl-phenylalanyl-bis(imidazol-2-yl)methylamine]) were studied by potentiometric, UV-Visible and Electron Paramagnetic Resonance (EPR) techniques. The imidazole nitrogen donor atoms of the bis(imidazol-2-yl)methyl group are described as the primary metal binding sites forming stable mono- and bis(ligand) complexes at acidic pH. The formation of a ligand-bridged dinuclear complex [Cu2L2]4+ is detected in equimolar solutions of copper(II) and HisPhe-BIMA. The coordination isomers of the dinuclear complex are described via the metal binding of the bis(imidazol-2-yl)methyl, amino-carbonyl and amino-imidazole(His) functions. In the case of the copper(II)-PheHis-BIMA system the [NH2, N-(amide), N(Im)] tridentate coordination of the ligand is favoured and results in the formation of di- and trinuclear complexes [Cu2H(-1)L]3+ and [Cu3H(-2)L2]4+ in equimolar solutions. The presence of these coordination modes shifts the formation of "tripeptide-like" ([NH2, N-, N-, N(Im)]-coordinated) [CuH(-2)L] complexes into alkaline pH range as compared to other dipeptide derivatives of bis(imidazol-2-yl) ligands. Although there are different types of imidazoles in these ligands, the deprotonation and coordination of the pyrrole-type N(1)H groups does not occur below pH 10.     

Maximum-entropy decomposition of fluorescence correlation spectroscopy data: application to liposome–human serum albumin association

Fluorescence correlation spectroscopy was used to measure the diffusion behavior of a mixture of DMPC or DMPC/DMPG liposomes with human serum albumin (HSA) and mesoporphyrin (MP), which was used as the fluorescent label for liposomes and HSA as well. For decomposing the fluorescence intensity autocorrelation function (ACF) into components corresponding to a liposome population, HSA and MP, we used a maximum entropy procedure that computes a distribution of diffusion times consistent with the ACF data. We found that a simple parametric non-linear fit with a discrete set of decay components did not converge to a stable parameter set. The distribution calculated with the maximum entropy method was stable and the average size of the particles calculated from the effective diffusion time was in good agreement with the data determined using the discrete-component fit.     

An antibiotic factory caught in action

The synthesis of aromatic polyketides, such as actinorhodin, tetracycline and doxorubicin, begins with the formation of a polyketide chain. In type II polyketide synthases (PKSs), chains are polymerized by the heterodimeric ketosynthase-chain length factor (KS-CLF). Here we present the 2.0-A structure of the actinorhodin KS-CLF, which shows polyketides being elongated inside an amphipathic tunnel approximately 17 A in length at the heterodimer interface. The structure resolves many of the questions about the roles of KS and CLF. Although CLF regulates chain length, it does not have an active site; KS must catalyze both chain initiation and elongation. We provide evidence that the first cyclization of the polyketide occurs within the KS-CLF tunnel. The mechanistic details of this central PKS polymerase could guide biosynthetic chemists in designing new pharmaceuticals and polymers.     

Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones

Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisin-like prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.     

Hyperosmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation

Hyperosmotic stress initiates adaptive responses, including phosphorylation of myosin light chain (MLC) and concomitant activation of Na+-K+-Cl- cotransporter (NKCC). Because the small GTPase Rho is a key regulator of MLC phosphorylation, we investigated 1) whether Rho is activated by hyperosmotic stress, and if so, what the triggering factors are, and 2) whether the Rho/Rho kinase (ROK) pathway is involved in MLC phosphorylation and NKCC activation. Rho activity was measured in tubular epithelial cells by affinity pulldown assay. Hyperosmolarity induced rapid (<1 min) and sustained (>20 min) Rho activation that was proportional to the osmotic concentration and reversed within minutes upon restoration of isotonicity. Both decreased cell volume at constant ionic strength and elevated total ionic strength at constant cell volume were capable of activating Rho. Changes in [Na+] and [K+] at normal total salinity failed to activate Rho, and Cl- depletion did not affect the hyperosmotic response. Thus alterations in cellular volume and ionic strength but not individual ion concentrations seem to be the critical triggering factors. Hyperosmolarity induced mono- and diphosphorylation of MLC, which was abrogated by the Rho-family blocker Clostridium toxin B. ROK inhibitor Y-27632 suppressed MLC phosphorylation under isotonic conditions and prevented its rise over isotonic levels in hypertonically stimulated cells. ML-7 had a smaller inhibitory effect. In contrast, it abolished the hypertonic activation of NKCC, whereas Y-27632 failed to inhibit this response. Thus hyperosmolarity activates Rho, and Rho/ROK pathway contributes to basal and hyperosmotic MLC phosphorylation. However, the hypertonic activation of NKCC is ROK independent, implying that the ROK-dependent component of MLC phosphorylation can be uncoupled from NKCC activation.     

Differential recognition of altered peptide ligands distinguishes two functionally discordant (arthritogenic and nonarthritogenic) autoreactive T cell hybridoma clones

Intravenous injection of a cartilage proteoglycan (aggrecan)-specific Th1 hybridoma clone 5/4E8 induced joint lesions similar to those seen in either primary or adoptively transferred arthritis in BALB/c mice. A sister clone, TA20, recognizing the same peptide epitope of human aggrecan and using the same Vbeta4 and Valpha1 segments, failed to induce joint inflammation. This study examines the fine epitope specificities of these two clones. Both 5/4E8 and TA20 hybridomas were generated using T cells from the same arthritic animal that has been immunized with human aggrecan, and both clones recognized peptides containing a consensus GRVRVNSAY sequence. However, flanking regions outside this nonapeptide sequence region had differential impact on peptide recognition by the two clones. Similarly, when single amino acid substitutions were introduced to the consensus sequence, significant differences were detected in the epitope recognition patterns of the T cell hybridomas. The 5/4E8 hybridoma showed greater flexibility in recognition, including a higher responsiveness to the corresponding self (mouse) aggrecan peptide, and produced more inflammatory cytokines (IFN-gamma and TNF-alpha), whereas hybridoma TA20 produced IL-5 in response to either human or mouse self peptide stimulation. These results demonstrate that, within the pool of immunodominant (foreign) peptide-activated lymphocytes, marked individual differences of degeneracy exist in T cell recognition, with possible implications to autopathogenic T cell functions.     

Expression and purification of enzymatically active forms of the human lysyl oxidase-like protein 4

The lysyl oxidase-like protein 4 (LOXL4) is the latest member of the emerging family of lysyl oxidases, several of which were shown to function as copper-dependent amine oxidases catalyzing lysine-derived cross-links in extracellular matrix proteins. LOXL4 contains four scavenger receptor cysteine-rich domains in addition to the characteristic domains of the LOX family, including the copper-binding domain, the cytokine receptor-like domain, and the residues of the lysyl-tyrosyl quinone cofactor. In an effort to assess its amine oxidase activity, we expressed LOXL4 as recombinant forms attached with hexa-histidine residues at the carboxyl terminus by using an Escherichia coli expression system. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis. The purified LOXL4 proteins showed beta-aminopropionitrile-inhibitable activity of 0.022-0.032 units/mg toward a nonpeptidyl substrate, benzylamine. These results indicate that LOXL4, with the four scavenger receptor cysteine rich domains, may also function as an active amine oxidase. Availability of the pure and active forms of LOXL4 will be significantly helpful in functional studies related to substrate specificity and crystal structure of this amine oxidase, which should provide significant insights into functional differences within the LOX family members.     

Alpha-adrenergic blockade: a possible mechanism of tocolytic action of certain benzodiazepines in a postpartum rat model in vivo

Benzodiazepines are frequently used for the treatment of maternal psychiatric disorders during pregnancy. Besides their anxiolytic effect, they are reported to exert a direct relaxing action on several smooth muscle preparations, including the uterus. In the present study, the possibility of the involvement of alpha(1)-adrenergic receptors in this peripheral effect is investigated. The tocolytic potencies of diazepam, midazolam and nitrazepam are assessed in vivo in a postpartum rat model, together with other drugs known to bind to alpha-adrenoceptors (e.g. alpha(1)-antagonists, tricyclic compounds and droperidol). The interactions of some benzodiazepines and norepinephrine were also examined in an isolated in vitro system. The affinities of these agents for the receptor in question were additionally tested by radioligand displacement assay. A correlation was found between the tocolytic potencies and inhibition constants of the tested drugs, suggesting that the smooth muscle-relaxing effect of these benzodiazepines is mediated through modulation of the alpha(1)-adrenergic receptors.     

Seasonal home range shift of red deer in a forest-agriculture area in southern Hungary

A long-term radiotelemetry study on red deerCervus elaphus Linnaeus, 1758 was carried out in a lowland forest-agriculture area in Hungary between 1993–2000. Previous observations suggested seasonal changes in population distribution between forested and agricultural habitat. Red deer concentrated in the forest during winter, but they appeared in the agricultural field during the vegetational period. We investigated the ranging behaviour, testing two alternative hypotheses: home range expansion and home range shift. Weekly radiotelemetry localisations revealed that 9 of 28 hinds showed a clear home range shift from the forest to the agricultural area for a prolonged time during the vegetational period. The remaining portion of the animals used a home range within the forest throughout the entire year. Diet composition analysis using indicator plant species showed that neither daily passages between habitats, nor home range expansion exists. These ranging behaviours were stable hence, if an animal shifted one year it shifted again in consecutive years andvice versa. Our results could be useful for the successful management of red deer populations in such complex habitats and to decrease agricultural damage.