The organization of the gene for the human cerebroside sulfate activator protein

The organization of 14 exons covering 97% of the cDNA sequence of human cerebroside sulfate activator protein precursor has been determined from two overlapping EMBL-4 human genomic clones extending over 17kb. All exons and exon/intron splice junctions and five introns were sequenced. Exon 8 consists of only 9 bp and is involved in alternative splicing which generates three different mRNAs of cerebroside sulfate activator precursor.     

Effect of canine surfactant protein (SP-A) on the respiratory burst of phagocytic cells

Cells obtained from bronchoalveolar lavage, or neutrophils of peripheral blood of dog, were incubated with the canine surfactant-associated protein A (SP-A). A significant decrease of the production of Superoxide anion was observed after subsequent stimulation with phorbol-12-myristate-13-acetate (PMA) as measured by the lucigenin-dependent chemiluminesence (CL). Several other proteins used for control experiments did not decrease lucigenin-dependent CL, indicating a specific effect of SP-A on phagocytes. Treatment of SP-A with collagenase prior to incubation with neutrophils destroyed the depleting effect on oxygen radical production of PMA-stimulated cells. We propose that SP-A acts as a regulatory factor of the respitatory burst of alveolar macrophages and neutrophils in the lungs. The inhibitory effect of SP-A is down-regulated by collagenase released from stimulated alveolar macrophages.     

On the phospholipid metabolism of glial cell primary cultures

Primary cultures prepared from newborn rat brain, consisted after 16 or 17 days mainly of astrocytes and of oligodendrocytes. 1-Alkenyl-sn-glycero-3-phosphoethanolamine (lysoplasmalogen) was used as substrate for studies on the metabolism of ethanolamine-glycerophospholipids. After 3 hr incubation two main products were observed: a) 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (=ethanolamine plasmalogen) and b) 1-alkenyl-2-acyl-sn-glycero-3-phosphocholine (=choline plasmalogen). The acylation rate reached saturation at about 10 nmol substrate/mg cell protein with aV _max of 30 nmol×mg cell protein^–1×3 hr^–1. This acylated compound amounted to almost 60% of all radioactivity internalized, whereas the second product, choline plasmalogen, came to 20%. Unchanged substrate was found within the cells only in small amounts, even at maximum substrate internalization. These results were discussed in comparison with those obtained with 1-alkyl-sn-glycero-3-phosphoethanolamine under the same conditions (25).     

LuTHy: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ?L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems.     

Studies on the side-chain hydroxylation of ifosfamide and its bromo analogue

Deutero-substituted (alpha,alpha,alpha',alpha'-tetradeuterated) derivatives of ifosfamide (IF-d(4)) and its bromo analogue were synthesised. In vitro metabolic studies showed that microsomal hydroxylation of IF-d(4) is slower than for unlabelled compound, suggesting that kinetic isotope effect operates during those transformations. At the same time deutero-substituted derivatives are more active against L1210 leukaemia in mice than unlabelled compounds, suggesting a negative role of side-chain hydroxylation metabolic pathways in the anticancer activity of ifosfamide and its analogues.     

Fast identification of folded human protein domains expressed in <Emphasis Type="Italic">E. coli</Emphasis> suitable for structural analysis

Background  

High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination.     

Functional morphology and neuronal innervation of the prothoracic defence gland in Timema

Timema is the most basal genus of Phasmatodea and the sister group to the remaining stick and leaf-insects (Euphasmatodea). An autapomorphy of all phasmids is the paired prothoracic exocrine defence glands. In this study, the anatomy and innervation of the defence glands in Timema petita and Timema chumash are described and compared with the data on Euphasmatodea. In all phasmids, the glands consist of a cuticular epithelium, a secretory epithelium and muscular fibres that compress the lumen. In Timematodea, the muscular part of the gland is less developed than in Euphasmatodea and the ejection of the defence secretion depends on the dorsal longitudinal neck muscles. On the neuroanatomical level, Timema petita and Timema chumash lack neurons that are involved in the independent contraction of the gland in euphasmids. In both studied species of Timema, neck muscles play an active role in the gland function which is not observed in any other phasmid. Considering the basal position of this genus, this supports the hypothesis that in euphasmids, the muscular part of the gland evolved from the dorsal longitudinal neck muscles. Additionally, the same nerves that innervate the dorsal longitudinal neck muscles in all Polyneoptera also innervate the defence glands in phasmids.