Linoleamide, a brain lipid that induces sleep, increases cytosolic Ca2+ levels in MDCK renal tubular cells

Linoleamide is an endogenous lipid that has been shown to induce sleep in cats, rats and humans. However, its physiological function remains unclear. In this study the effect of linoleamide on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) tubular cells was examined, by using fura-2 as a Ca2+ probe. In a concentration-dependent manner, linoleamide induced increases in [Ca2+]i between 10-500 microM with an EC50 of 20 microM. The signal comprised a slow rise and a persistent phase, and was a result of internal Ca2+ release and external Ca2+ influx because it was partly inhibited by external Ca2+ removal. In Ca2+-free medium, depletion of the endoplasmic reticulum Ca2+ store with 1 microM thapsigargin abolished 100 microM linoleamide-induced internal Ca2+ release, and conversely, pretreatment with linoleamide prevented thapsigargin from releasing internal Ca2+. This demonstrates that the internal source of linoleamide-induced [Ca2+]i increase is located in the endoplasmic reticulum. This discharge of internal Ca2+ caused capacitative Ca2+ entry because after incubation with 100 microM linoleamide in Ca2+-free medium for 8 min readmission of 3 mM CaCl2 induced increases in [Ca2+]i. After the formation of inositol-1,4,5-trisphosphate (IP3) was blocked by the phospholipase C inhibitor U73122 (1 microM), linoleamide still induced an increase in [Ca2+]i but the shape of the increase was altered. Similar results were found for another sleep-inducing lipid 9,10-octadecenoamide. Together, the present study shows that the endogenous sleep-inducing lipid linoleamide was able to cause significant increases in [Ca2+]i in renal tubular cells, by releasing the endoplasmic reticulum Ca2+ store and triggering capacitative Ca2+ entry in a manner independent of IP3.     

Zur Problematik der baktericiden Wirkung von Antibiotica bei tiefen Temperaturen

Zusammenfassung Es wurde der Einfluß von Penicillin und Streptomycin auf das Absterben von E. coli und M. pyogenes unter Kältewirkung untersucht und mittels spezifischer Inaktivation der Antibiotica während des Auftauens bewiesen, daß deren zusätzliche baktericide Wirkung nicht in der Frostperiode, sondern erst beim Auftauen einsetzt. Die Abhängigkeit der zusätzlichen Abtötung von der Gefriertemperatur und Antibioticumkonzentration wird mit dem Maß des ausgefrorenen Zellwassers, bzw. der aufgesaugten Antibioticamenge während der Quellung erklärt. Einige auf die Ergebnisse quantitativer Studien des Kältetodes von Mikroben sich störend auswirkende Erscheinungen, insbesondere das durch Eisbildung eingeleitete Auseinandersprengen der Zellverbände werden erläutert.     

Population dynamics of an introduced bacterium degrading chlorinated benzenes in a soil column and in sewage sludge

The capacity of the -Proteobacterium Pseudomonas sp. strain P51, which degrades chlorinated benzenes, to metabolize 1,2,4-trichlorobenzene (TCB) under environmental conditions was tested by its release into two experimental systems. The first system consisted of laboratory scale microcosms which were operated with and without the addition of TCB and which were inoculated with sludge from a wastewater treatment plant. The second system consisted of a non sterile, water saturated soil column. We determined survival of strain P51 after its introduction and its ability to degrade TCB. The population dynamics was followed by selective plating and applying the polymerase chain reaction (PCR) to detect strain P51 and the chlorobenzene ( tcb) genes on catabolic plasmid pP51. The results showed a completely different behaviour of strain P51 in the two habitats under the applied conditions. In the soil column the P51 bacteria inoculated the entire area and their population reached 2 × 10⁶ cells/g soil. The population remained active since TCB was degraded to concentrations below the detection limit of 30 g/l. In the sludge microcosms, the number of strain P51 cells immediately decreased from 4 × 10⁷ cells/ml to 10⁵ cells/ml over a period of 2 days after inoculation, and then the strain disappeared to levels below our detection limit (10³–10⁴ cells/ml). In the reactor without TCB the population of P51 maintained a stable value of 10⁵ cells/ml during 8 days but then also decreased to levels below the detection limit. In addition, no significant TCB degradation was found in the sludge reactors. The influence of presence of TCB on maintenance of strain P51 in the two habitats is discussed. This work demonstrates the possibility to successfully apply preselected strains to degrade otherwise poorly degradable substances in complex mixed microbial communities. However, survival and activity may depend strongly on the type of system into which the strain is introduced.     

Safety and efficacy of treatment with platelet GPIIb/IIIa receptor blockade in unstable angina patients awaiting PTCA at a referring clinic

BACKGROUND: Although balloon angioplasty has assumed an important role in the management of refractory unstable angina (UA), that is, UA that does not respond to conventional therapy, it is limited by complications related to thrombosis and acute coronary occlusion. The complication rate is higher in patients with UA than in those whose condition is stable. Preprocedural use of abciximab, a monoclonal platelet glycoprotein IIb/IIIa receptor blocker, has been used effectively in patients with UA, but its acceptance may be limited by safety concerns and economic constraints. The current trial investigated a protocol for abciximab pretreatment in patients with UA awaiting transfer from referring hospitals to a site of intervention (the 'drip and ship' protocol). AIMS: This observational study was conducted to evaluate whether a prophylactic, preprocedural regimen of abciximab can be safely and effectively administered to UA patients in referring hospitals while awaiting coronary angioplasty at the interventional clinic. METHODS: From April 1996 to December 1998, 168 consecutive patients with refractory UA (Braunwald class II or III) received abciximab prospectively at the referring clinic before undergoing PTCA or stent implantation at the interventional clinic. The following cost-conscious protocol was used: a 0.25 mg/kg bolus of abciximab followed by 10 micro g/min intravenously for 16 hours, in addition to intravenous nitrates, heparin and aspirin therapy. Patients were then transferred to a facility with PTCA capability via high-speed ambulance transport. No specific alterations of routine-transfer protocol were needed. Platelet aggregation studies were conducted during abciximab infusion. All interventions were performed while abciximab was given. Procedural and clinical success and long-term outcomes also were assessed. RESULTS: The primary angiographic success rate (patients with post-PTCA diameter stenosis < 50%) was 98%, and the in-hospital clinical success rate (angiographic success without major complications) was 98%. No major bleeding complications occurred during the abciximab pretreatment period. Platelet aggregation findings in the study patients showed a stable inhibition of >80% at the time of angioplasty. At 30-day follow-up, all patients were alive and 91% were free of major adverse events. Outcomes of balloon angioplasty and stenting were equally favorable, indicating no device-specific effect. Event-free survival at six months was 89% with a target vessel revascularization rate of 10%. CONCLUSION: Abciximab was administered safely and effectively to angioplasty patients with refractory UA awaiting transfer from a noninterventional setting to the site of angioplasty. These results extend the current knowledge base that has been established in randomized trials performed in interventional centers. The study protocol potentially could make abciximab therapy more feasible economically, and therefore more widely available to patients who are most likely to benefit from prophylactic administration.     

Evolution of Mhc–DRB Introns: Implications for the Origin of Primates

Introns are generally believed to evolve too rapidly and too erratically to be of much use in phylogenetic reconstructions. Few phylogenetically informative intron sequences are available, however, to ascertain the validity of this supposition. In the present study the supposition was tested on the example of the mammalian class II major histocompatibility complex (Mhc) genes of the DRB family. Since the Mhc genes evolve under balancing selection and are believed to recombine or rearrange frequently, the evolution of their introns could be expected to be particularly rapid and subject to scrambling. Sequences of intron 4 and 5 DRB genes were obtained from polymerase chain reaction-amplified fragments of genomic DNA from representatives of six eutherian orders—Primates, Scandentia, Chiroptera, Dermoptera, Lagomorpha, and Insectivora. Although short stretches of the introns have indeed proved to be unalignable, the bulk of the intron sequences from all six orders, spanning >85 million years (my) of evolution, could be aligned and used in a study of the tempo and mode of intron evolution. The analysis has revealed the Mhc introns to evolve at a rate similar to that of other genes and of synonymous sites of non-Mhc genes. No evidence of homogenization or large-scale scrambling of the intron sequences could be found. The Mhc introns apparently evolve largely by point mutations and insertions/deletions. The phylogenetic signals contained in the intron sequences could be used to identify Scandentia as the sister group of Primates, to support the existence of the Archonta superorder, and to confirm the monophyly of the Chiroptera. Received: 26 October 1998 / Accepted: 21 December 1998     

Cef1p is a component of the Prp19p-associated complex and essential for pre-mRNA splicing

The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the spliceosome during the splicing reaction. We have previously shown that Prp19p is not tightly associated with small nuclear ribonucleoprotein particles but is associated with a protein complex consisting of at least eight protein components. By sequencing components of the affinity-purified complex, we have identified Cef1p as a component of the Prp19p-associated complex, Ntc85p. Cef1p could directly interact with Prp19p and was required for pre-mRNA splicing both in vivo and in vitro. The c-Myb DNA binding motif at the amino terminus of Cef1p was required for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p self-interaction. We have identified a small region of 30 amino acid residues near the carboxyl terminus required for both cell viability and protein-protein interactions. Cef1p was associated with the spliceosome in the same manner as Prp19p, i.e. concomitant with or immediately after dissociation of U4. The anti-Cef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and at least three other components of the Prp19p-associated complex, suggesting that the Prp19p-associated complex is likely associated with the spliceosome and functions as an integral complex.     

Transmembrane structure of an inwardly rectifying potassium channel

Inwardly rectifying potassium channels (K(ir)), comprising four subunits each with two transmembrane domains, M1 and M2, regulate many important physiological processes. We employed a yeast genetic screen to identify functional channels from libraries of K(ir) 2.1 containing mutagenized M1 or M2 domains. Patterns in the allowed sequences indicate that M1 and M2 are helices. Protein-lipid and protein-water interaction surfaces identified by the patterns were verified by sequence minimization experiments. Second-site suppressor analyses of helix packing indicate that the M2 pore-lining inner helices are surrounded by the M1 lipid-facing outer helices, arranged such that the M1 helices participate in subunit-subunit interactions. This arrangement is distinctly different from the structure of a bacterial potassium channel with the same topology and identifies helix-packing residues as hallmark sequences common to all K(ir) superfamily members.     

Pseudanthocotyloides heterocotyle (van Beneden, 1871) Euzet & Prost, 1969 (Monogenea: Polyopisthocotylea: Mazocraeidae), a parasite of herring Clupea harengus L. and sprat Sprattus sprattus L. (Teleostei: Clupeidae)

A total of 690 herring Clupea harengus L. and 88 sprat Sprattus sprattus L. caught off the west coast of Sweden, in the North Sea and off the west and south coasts of the United Kingdom, were examined for gill parasites. The monogenean Pseudanthocotyloides heterocotyle (van Beneden, 1871) Euzet & Prost, 1969 was found in 38 (5.5%) herring and one (1.1%) sprat. The parasite was significantly (P>0.05) more common off the west coast of Sweden than elsewhere and most specimens (62.5%) were found on the pseudobranchs. Only the smaller herring were infected. P. heterocotyle is redescribed and its taxonomy discussed, together with the possibility of host and parasite misidentification in previous reports.     

A group of α-1,4-glucan lyase genes from the fungi Morchella costata,M. vulgaris and Peziza ostracoderma. Cloning,complete sequencing and heterologous expression

We here report genes encoding a newly discovered class of starch- and glycogen-degrading enzyme, -1,4-glucan lyase (EC 4.2.2.13), which degrades starch and glycogen to 1,5-anhydro-D-fructose. Two lyases were purified and partially sequenced from the macrofungi Morchella costata and M. vulgaris. The obtained lyase amino acid sequences were used to generate PCR primers, which were further used to probe the fungal genomic libraries. Two lyase genes (Agll1;Mo.cos and Agll1;Mo.vul) from the two fungi were fully sequenced and found to contain a coding region of 3201 bp and 3213 bp, respectively. A total of 13 small introns were found in each of the two genes with identical positions. The two lyase genes share 86% identity at the amino acid level. They encode mature lyases with 1066 and 1070 amino acids, respectively. The deduced molecular masses of 121530 and 121971 Da agree with the values found for the two purified lyases. A structure analysis of the promoter regions of the lyase genes revealed a number of putative regulatory DNA elements, such as the AREA and CREA sites, which are related to nitrogen and carbon metabolism, respectively, and the CCAAT/CAAT boxes, which are related to basal expression of genes. A third lyase gene (Agll1;Pe.ost) from the fungus Peziza ostracoderma was partially sequenced to 557 bp. The amino acid sequence deduced from this nucleotide fragment shares 76% identity with the M. costata lyase. Heterologous expression of the M. costata lyase gene was achieved intracellularly in Pichia pastoris and Aspergillus niger.