Co-existence of immunoreactivity to anti-dopamine,anti-serotonin and anti-vasotocin in the cerebral giant neuron of the pond snail Lymnaea stagnalis

Summary Consecutive sections of certain neurons in the central ganglia of the pond snail Lymnaea stagnalis appear to be immunoreactive to anti-dopamine and anti-serotonin. The Cerebral Giant Neurons stain in addition with antivasotocin. The observations indicate the presence of two biogenic amines within the same neuron and in addition their co-existence with a biologically active peptide.     

The role of the carboxy-terminal membrane-spanning fragment in the biogenesis of Escherichia coli K12 outer membrane protein PhoE

Summary PhoE protein of Escherichia coli K12 is an outer membrane protein which is supposed to span the membrane sixteen times. By creating a deletion which removes the last membrane-spanning fragment and studying the localization of the truncated PhoE, we show that this fragment is indispensable for trimerization and outer membrane localization. In addition, circumstantial evidence for the proposed topology model of the protein was obtained. An insertion mutation in a region supposed to be cell surface-exposed, interferes with the binding of a monoclonal antibody which recognizes a cell surface-exposed epitope of the protein.     

Functional morphology of the neuroendocrine sodium influx-stimulating peptide system of the pond snail,Lymnaea stagnalis,studied by in situ hybridization and immunocytochemistry

Summary The functional morphology of the neuroendocrine system producing sodium influx-stimulating (SIS) peptide in the pond snail, Lymnaea stagnalis, was studied by in situ hybridization and immunocytochemistry. The SIS-peptide, which is 76 amino acids long, stimulates sodium uptake from the ambient medium. Two synthetic DNA probes were used for in situ hybridization. The nucleotide sequences were chosen from the cDNA structure; they encode amino acids 8–17 and 64–73, respectively. SIS-peptide sequences 10–20 and 67–76 were synthesized and antibodies were raised to them and affinity-purified. In addition to these antibodies, a monoclonal antibody raised to a bioactive, high-pressure liquid chromatography (HPLC)-purified brain extract was used for immunocytochemistry. Paraffin sections of central nervous systems and of whole snails were studied. The SIS-peptide system could be identified as the previously described yellow cell (YC) system by comparing alternate sections treated with the DNA probes, stained with the antibodies, or stained with alcian blue-alcian yellow. SIS-peptide neurons (45) occur in the ganglia of the visceral ring and in the proximal parts of visceral nerves. Axons run in the nerves of these and in several nerves of other ganglia. Numerous axon branches penetrate the perineurium forming a vast central neurohemal area. The SIS-peptide system innervates the pericardium, the nephridial gland, the reno-pericardial canal, the ureter, the spermoviduct and gonadal acini, the anterior aorta, the ventral buccal artery, and the penis protractor muscle. The morphology of the system is discussed in relation to the process of sodium ion uptake from the ambient medium and from pro-urine, and to that of regulating blood pressure. In the central nervous system and other organs, neurons and axons not labeled with the DNA probes, but immunoreactive to one or two of the antibodies, were observed. It seems unlikely that these elements are functionally related to the SIS-peptide system.     

Influence of Temperature of Incubation and Type of Growth Medium on Pigmentation in Serratia marcescens

Maximal amounts of prodigiosin were synthesized in either minimal or complete medium after incubation of cultures at 27 C for 7 days. Biosynthesis of prodigiosin began earlier and the range of temperature for formation was greater in complete medium. No prodigiosin was formed in either medium when cultures were incubated at 38 C; however, after a shift to 27 C, pigmentation ensued, provided the period of incubation at 38 C was not longer than 36 hr for minimal medium or 48 hr for complete medium. Washed, nonpigmented cells grown in either medium at 38 C for 72 hr could synthesize prodigiosin when suspended in saline at 27 C when casein hydrolysate was added. These suspensions produced less prodigiosin at a slower rate than did cultures growing in casein hydrolysate at 27 C without prior incubation at 38 C. Optimal concentration of casein hydrolysate for pigment formation by suspensions was 0.4%; optimal temperature was 27 C. Anaerobic incubation, shift back to 38 C, killing cells by heating, or chloramphenicol (25 mug/ml) inhibited pigmentation. Suspensions of washed cells forming pigment reached pH 8.0 to 8.3 rapidly and maintained this pH throughout incubation for 7 days. Measurements of viable count and of protein, plus other data, indicated that cellular multiplication did not occur in suspensions of washed cells during pigment formation. By this procedure utilizing a shift down in temperature, biosynthesis of prodigiosin by washed cells could be separated from multiplication of bacteria.     

Development of a new transformant selection system for Penicillium chrysogenum: isolation and characterization of the P. chrysogenum acetyl-coenzyme A synthetase gene (facA) and its use as a homologous selection marker

A new transformation system for the filamentous fungus Penicillium chrysogenum is described, based on the use of the homologous acetyl-coenzyme A synthetase (facA) gene as a selection marker. Acetate-non-utilizing (Fac^–) strains of P. chrysogenum were obtained by positive selection for spontaneous resistance to fluoroacetate. Among these fac mutants putative facA strains were selected for a loss of acetyl-coenzyme A (CoA) synthetase activity. The facA gene, coding for the enzyme acetyl-CoA synthetase, was isolated from a P. chrysogenum genomic library using synthetic oligonucleotides derived from conserved regions from the corresponding genes of Aspergillus nidulans and Neurospora crassa. Vector pPC2-3, comprising a genomic 6.5 kb PstI fragment, was able to complement P. chrysogenum facA strains with frequencies up to 27 transformants·g^–1 DNA. Direct selection of transformants was accomplished using acetate and low amounts (0.001%) of glucose as carbon sources. About 50% of the transformants arose by integration of pPC2-3 DNA at the homologous facA locus and 50% by integration elsewhere in the genome. Determination of the nucleotide sequence of part of the cloned fragment showed the presence of an open reading frame of 2007 nucleotides, interrupted by five putative introns. Comparison of the nucleotide and the amino acid sequence of the facA gene of P. chrysogenum with the facA gene of A. nidulans reveals similarities of 80% and 89%, respectively. The putative introns present in the P. chrysogenum facA gene appear at identical positions as those in the A. nidulans facA gene, but show no significant sequence similarity. Correspondence to: R. F. M.van Gorcom     

Bacterial lipopolysaccharides form procollagen-endotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis

Introduction

We have previously reported that bacterial toxins, especially endotoxins such as lipopolysaccharides (LPS), might be important causative agents in the pathogenesis of rheumatoid arthritis (RA) in an in vitro model that simulates the potential effects of residing in damp buildings. Since numerous inflammatory processes are linked with the nuclear factor-κB (NF-κB), we investigated in detail the effects of LPS on the NF-κB pathway and the postulated formation of procollagen-endotoxin complexes.

Methods

An in vitro model of human chondrocytes was used to investigate LPS-mediated inflammatory signaling.

Results

Immunoelectron microscopy revealed that LPS physically interact with collagen type II in the extracellular matrix (ECM) and anti-collagen type II significantly reduced this interaction. BMS-345541 (a specific inhibitor of IκB kinase (IKK)) or wortmannin (a specific inhibitor of phosphatidylinositol 3-kinase (PI-3K)) inhibited the LPS-induced degradation of the ECM and apoptosis in chondrocytes. This effect was completely inhibited by combining BMS-345541 and wortmannin. Furthermore, BMS-345541 and/or wortmannin suppressed the LPS-induced upregulation of catabolic enzymes that mediate ECM degradation (matrix metalloproteinases-9, -13), cyclooxygenase-2 and apoptosis (activated caspase-3). These proteins are regulated by NF-κB, suggesting that the NF-κB and PI-3K pathways are involved in LPS-induced cartilage degradation. The induction of NF-κB correlated with activation of IκBα kinase, IκBα phosphorylation, IκBα degradation, p65 phosphorylation and p65 nuclear translocation. Further upstream, LPS induced the expression of Toll-like receptor 4 (TLR4) and bound with TLR4, indicating that LPS acts through TLR4.

Conclusion

These results suggest that molecular associations between LPS/TLR4/collagen type II in chondrocytes upregulate the NF-κB and PI-3K signaling pathways and activate proinflammatory activity.