Ultrastructural co-localization of TFF3-peptide and oxytocin in the neural lobe of the porcine pituitary

TFF-peptides (formerly P-domain peptides, trefoil factors) are typical secretory products of many mucous epithelial cells. TFF3 is also synthesized in oxytocinergic neurons in the paraventricular and supraoptic nuclei of the human hypothalamus. Here, TFF3 and oxytocin are shown to be co-localized within the same secretory vesicles in the neural (posterior) lobe of the procine pituitary by means of immunoelectron microscopy. Relatively large amounts of TFF3, but not TFF1 and TFF2, are present in the neural lobe of the porcine pituitary, where it is probably released into the bloodstream.     

Characterisation of the mob locus of Rhodobacter sphaeroides WS8: mobA is the only gene required for molybdopterin guanine dinucleotide synthesis

The mob genes of several bacteria have been implicated in the conversion of molybdopterin to molybdopterin guanine dinucleotide. The mob locus of Rhodobacter sphaeroides WS8 comprises three genes, mobABC. Chromosomal in-frame deletions in each of the mob genes have been constructed. The mobA mutant strain has inactive DMSO reductase and periplasmic nitrate reductase activities (both molybdopterin guanine dinucleotide-requiring enzymes), but the activity of xanthine dehydrogenase, a molybdopterin enzyme, is unaffected. The inability of a mobA mutant to synthesise molybdopterin guanine dinucleotide is confirmed by analysis of cell extracts of the mobA strain for molybdenum cofactor forms following iodine oxidation. Mutations in mobB and mobC are not impaired for molybdoenzyme activities and accumulate wild-type levels of molybdopterin and molybdopterin guanine dinucleotide, indicating they are not compromised in molybdenum cofactor synthesis. In the mobA mutant strain, the inactive DMSO reductase is found in the periplasm, suggesting that molybdenum cofactor insertion is not necessarily a pre-requisite for export.     

Golvesin-GFP fusions as distinct markers for Golgi and post-Golgi vesicles in Dictyostelium cells

Golvesin is a new protein associated with membranes of the Golgi apparatus and post-Golgi vesicles in Dictyostelium cells. An internal hydrophobic sequence of 24 amino-acid residues is responsible for anchoring golvesin to the membranes of these organelles. In an attempt to visualize organelle dynamics in vivo, we have used specific antibody and other labels to localize golvesin-green fluorescent protein (GFP) constructs to different cellular compartments. With a GFP tag at its N-terminus, golvesin shows the same localization as the untagged protein. It is transferred to two post-Golgi compartments, the endosomal and contractile vacuole systems. Endosomes are decorated with GFP-golvesin within less than 10 min of their internalisation, and keep the label during the acidic phase of the pathway. Blockage of the C-terminus with GFP causes entrapment of the protein in the Golgi apparatus, indicating that a free C-terminus is required for transfer of golvesin to any of the post-Golgi compartments. The C-terminally tagged golvesin proved to be a reliable Golgi marker in Dictyostelium cells revealing protrusion of Golgi tubules at peak velocities of 3 to 4 microm x s(-1). The fusion protein is retained in Golgi vesicles during mitosis, visualizing Golgi disassembly and reorganization in line with cytokinesis.     

A recessive C-terminal Jervell and Lange-Nielsen mutation of the KCNQ1 channel impairs subunit assembly

The LQT1 locus (KCNQ1) has been correlated with the most common form of inherited long QT (LQT) syndrome. LQT patients suffer from syncopal episodes and high risk of sudden death. The KCNQ1 gene encodes KvLQT1 alpha-subunits, which together with auxiliary IsK (KCNE1, minK) subunits form IK(s) K(+) channels. Mutant KvLQT1 subunits may be associated either with an autosomal dominant form of inherited LQT, Romano-Ward syndrome, or an autosomal recessive form, Jervell and Lange-Nielsen syndrome (JLNS). We have identified a small domain between residues 589 and 620 in the KvLQT1 C-terminus, which may function as an assembly domain for KvLQT1 subunits. KvLQT1 C-termini do not assemble and KvLQT1 subunits do not express functional K(+) channels without this domain. We showed that a JLN deletion-insertion mutation at KvLQT1 residue 544 eliminates important parts of the C-terminal assembly domain. Therefore, JLN mutants may be defective in KvLQT1 subunit assembly. The results provide a molecular basis for the clinical observation that heterozygous JLN carriers show slight cardiac dysfunctions and that the severe JLNS phenotype is characterized by the absence of KvLQT1 channel.     

Intersubunit association induces unique allosteric dependence of the T127L CRP mutant on pH

The allosteric activation of the T127-->L mutant of 3',5'-cyclic adenosine monophosphate (cAMP) receptor protein (CRP) by cAMP changes from an exothermic, independent two-site binding mechanism at pH 7.0 to an endothermic, interacting two-site binding mechanism at pH 5.2, similar to that observed for CRP at pH 7.0 and 5.2. Since the T127-->L mutation at the subunit interface of the CRP dimer creates a more perfect leucine-zipper motif, it is believed to increase the intersubunit association and the stability of the CRP, as is observed by the higher thermal stability of the T127L mutant relative to that of CRP in differential scanning calorimetry (DSC) measurements. The DSC scans also exhibit a single thermal denaturation transition for CRP and a S128A mutant from pH 5.2 to 7. 0, while the broader transition peak of the T127L mutant becomes resolvable into two transitions below pH < or =5.2. Circular dichroism measurements on T127L and CRP at pH 7.0 and 5.2 show changes in the tertiary structure of both proteins with the exception of the tertiary structure around the two tryptophan residues in the amino-terminal domain. Although gel electrophoresis of the proteolysis (pH 5.2) products of T127L, CRP, and their cAMP- and cGMP-ligated complexes shows the subunit band and an amino-terminal domain fragment band, the fully allosterically activated complexes of T127L and CRP show the amino-terminal domain fragment band but not the subunit band. The results are interpreted in terms of the allosteric activation of CRP by cAMP by a conformational change from an "open" to a "closed" form of CRP, which involves changes in the tertiary structure of the carboxyl-terminal domains that are partially induced by an increase in the intersubunit association in T127L. While T127L retains its intersubunit association from pH 5.2 to 7.0, changes occur in the carboxyl-terminal domain so that the endothermic, allosteric activation mechanism of CRP by cAMP is restored in T127L at pH 5.2.     

Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, enteritidis, Dublin, Choleraesuis, Hadar and Saintpaul: construction and application of specific gene probes

A total of 65 epidemiologically unrelated tetracycline-resistant isolates of the six Salmonella enterica subsp. enterica (Salm.) serovars Dublin, Choleraesuis, Typhimurium, Enteritidis, Hadar and Saintpaul were investigated for the presence of tetracycline resistance genes. For this, specific gene probes of the tetracycline resistance genes (tet) of the hybridization classes A, B, C, D, E and G were constructed by cloning PCR-amplified internal segments of the respective tet structural genes. These gene probes were sequenced and used in hybridization experiments with plasmid DNA or endonuclease digested whole cell DNA as targets. Only tet(A) genes were detected on plasmids in all Salm. Dublin isolates as well as in single isolates of Salm. Choleraesuis and Salm. Typhimurium. Genes of the hybridization classes B, C, D and G, but also in some cases those of class A, were located in the chromosomal DNA of the corresponding Salmonella isolates. Restriction fragment length polymorphisms (RFLPs) of tet gene carrying fragments were detected in chromosomally tetracycline-resistant isolates. These RFLPs might represent valuable additional tools for the identification and characterization of tetracycline-resistant Salmonella isolates.     

Mode dynamics of interacting neural populations by bi-orthogonal spectral decomposition

A system of coupled bistable Hopf oscillators with an external periodic input source was used to model the ability of interacting neural populations to synchronize and desynchronize in response to variations of the input signal. We propose that, in biological systems, the settings of internal and external coupling strengths will affect the behaviour of the system to a greater degree than the input frequency. While input frequency and coupling strength were varied, the spatio-temporal dynamics of the network was examined by the bi-orthogonal decomposition technique. Within this method, effects of variation of input frequency and coupling strength were analyzed in terms of global, spatial and temporal mode entropy and energy, using the spatio-temporal data of the system. We observed a discontinuous evolution of spatio-temporal patterns depending sensitively on both the input frequency and the internal and external coupling strengths of the network. Received: 10 June 1998 / Accepted in revised form: 9 August 1999