Clerodane-type diterpenoids from Nannoglottis ravida

Chemical investigation of the roots of Nannoglottis ravida resulted in the characterization of two 5alpha,10alpha-cis-clerodane-type diterpenoides, ravidin A and B. Their structures and stereochemistry were established by spectroscopic methods, including X-ray crystallographic diffraction analysis of ravidin A. Their significance in terms of the chemotaxonomy of Nannoglottis is discussed.     

Independent expression of the two paralogous<Emphasis Type="Italic"> CCL4</Emphasis> genes in monocytes and B lymphocytes

The CCL4 chemokine is secreted by a variety of cells following stimulation. CCL4 affects several different types of cells that are important for acute inflammatory responses and are critical for the development of specific immune responses to foreign antigens. The human genome contains two genes for the CCL4 chemokine. Although highly homologous, the two genes encode slightly different proteins. We analyzed the mRNA expressed in monocytes and B lymphocytes and found that while monocytes express predominantly one CCL4 gene, known as ACT-2, peripheral blood B lymphocytes express a mixture of ACT-2 and the second CCL4 gene, lymphocyte activating gene-1 (LAG-1). Although peripheral blood B cells, CD27^– B cells, and CD27⁺ B cells all express a mixture of LAG-1 and ACT-2, the B-cell lines that were studied regulate the two genes independently. RL, SU-DHL-6, and REH cells predominantly express LAG-1. These studies demonstrate that monocytes and B cells utilize different mechanisms to regulate expression of the two CCL4 genes and suggest that the two genes may not have identical activities.     

Carnitine: transport and physiological functions in the brain

Carnitine (4-N-trimethylammonium-3-hydroxybutyric acid), a compound necessary for a transfer of fatty acids for their oxidation within the cell, accumulates in brain although β-oxidation of fatty acids is very low in neurons. Carnitine accumulates to lower extent in the brain than in peripheral tissues and the mechanism of its transport through the blood–brain barrier is discussed, with the involvement of two transporters, OCTN2 and B^0,+ being presented. A limitation by the blood–brain barrier of carnitine supply for the brain and the mechanism of its transport to neural cells by a protein belonging to neurotransmitters' transporters superfamily is further discussed.

Due to the beneficial effects of administration of acetylcarnitine in case of patients with dementia, the role of this acylcarnitine is presented in the context of neuronal cell metabolism and the role of acetylcarnitine in the synthesis of acetylcholine. The roles of long-chain acyl derivatives of carnitine, in particular palmitoylcarnitine, responsible for interaction with the membranes, lipids acylation and specific interactions with proteins have been summarized. Stimulation of protein palmitoylation and a possibility of changing the acylation status of G proteins is described, as well as interaction of palmitoylcarnitine with protein kinase C. Diminished interaction of the isoform δ of this kinase with GAP-43 (B-50, neuromodulin), whose expression increases upon accumulation of either carnitine or palmitoylcarnitine points to a possible regulation of differentiation by these compounds and their role in neuroregeneration.     

Solid-Phase Artificial Chaperone-Assisted Refolding using Insoluble β-Cyclodextrin–Acrylamide Copolymer Beads

Solid-phase refolding methods are advantageous since they facilitate both separation of solid additives from the refolded protein and recycling of the additives. -Cyclodextrin–acrylamide copolymer hydrogel beads were used as a matrix for detergents in solid-phase artificial chaperone-assisted refolding and improved the yield of lysozyme (up to 65%) and carbonic anhydrase B (up to 80%), compared with conventional solid host matrices.Revisions received 29 September 2004     

The evaluation of the oxidative state of native-LDL: three methods compared

LDL-oxidation is considered a contributing factor to the development of atherosclerotic lesions. However, to utilise the oxidative state of LDL as a marker of cardiovascular risk, reliable analytical methods for its detection must be defined. We have compared three methods for their capacity to evaluate the difference in the oxidation state of isolated LDL subjected to either dialysis (D-LDL) or gel filtration (F-LDL) to remove EDTA. Their susceptibility to oxidation promoted by Cu(2+) was monitored by following the time course of conjugated diene (CD) and lipid hydroperoxide (ROOH) accumulation. The relative electrophoretic mobility (REM) of the same LDL samples was evaluated by capillary electrophoresis. As measured by all three methods, F-LDL are less prone to oxidation than D-LDL when added with CuSO(4). REM of F-LDL and D-LDL significantly differs already before the addition of the metal catalyst, whereas CD and ROOH contents become significantly different only after it. Besides confirming that a rapid centrifugation followed by gel filtration is a more convenient procedure than dialysis to remove EDTA during LDL isolation, our study suggests the REM of isolated-LDL as the biochemical marker of choice in the evaluation of its oxidative state.     

Infectivity analysis of two variable DNA B components of<Emphasis Type="Italic">Mungbean yellow mosaic virus-Vigna</Emphasis> in<Emphasis Type="Italic">Vigna mungo</Emphasis> and<Emphasis Type="Italic">Vigna radiata</Emphasis>

Mungbean yellow mosaic virus-Vigna (MYMV-Vig), aBegomovirus that causes yellow mosaic disease, was cloned from field-infected blackgram (Vigna mungo). One DNA A clone (KA30) and five different DNA B clones (KA21, KA22, KA27, KA28 and KA34) were obtained. The sequence identity in the 150-nt common region (CR) between DNA A and DNA B was highest (95%) for KA22 DNA B and lowest (85·6%) for KA27 DNA B. The Rep-binding domain had three complete 11 -nt (5’-TGTATCGGTGT-3′) iterons in KA22 DNA B (and KA21, KA28 and KA34), while the first iteron in KA27 DNA B (5’-ATCGGTGT-3’) had a 3-nt deletion. KA27 DNA B, which exhibited 93·9% CR sequence identity to the mungbean-infecting MYMV, also shared the 3-nt deletion in the first iteron besides having an 18-nt insertion between the third iteron and the conserved nonanucleotide. MYMV was found to be closely related to KA27 DNA B in amino acid sequence identity of BV1 (94·1%) and BC1 (97·6%) proteins and in the organization of nuclear localization signal (NLS), nuclear export signal (NES) and phosphorylation sites. Agroinoculation of blackgram (V. mungo) and mungbean (V. radiata) with partial dimers of KA27 and KA22 DNA Bs along with DNA A caused distinctly different symptoms. KA22 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in blackgram. In contrast, KA27 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in mungbean. Thus, DNA B of MYMV-Vig is an important determinant of host-range betweenV. mungo andV. radiata.     

Unexpected intracellular localization of the AMD-associated cystatin C variant

Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26-amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age-related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full-length mutant (A25T) precursor cystatin C-enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild-type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild-type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.