Absence of fodrin (spectrin-like protein) under the pseudopod membrane in stimulated thyroid cells

A fodrin-like protein purified from porcine thyroid cells and characterized by its properties identical to those of pig brain spectrin (F. Regnouf et al., Eur. J. Biochem. 153, 313-319 (1985)) has been localized by immunofluorescence and electron immunocytochemistry in porcine and rat thyroid. Fodrin-like polypeptides were detected in subplasmalemmal meshworks of microfilaments attached to isolated or in situ plasma membranes. In resting cells, fodrin was found under apical and basolateral membrane domains, whereas it was always absent under the pseudopod membrane domain induced by acute TSH stimulation in vitro, using monolayers of porcine cultured cells attached to collagen permeable substrates, as well as in vivo, using rats intravenously treated with TSH. Thyroid fodrin could be involved in exocytosis and membrane stabilization which occurs during the formation of pseudopods induced by TSH stimulation.     

The three-dimensional structure of vascular tissues in Agrobacterium tumefaciens-induced crown galls and in the host stems of Ricinus communis L.

The three-dimensional pattern of phloem and xylem in 10-d-to two-month-old tumors induced by Agrobacterium tumefaciens (C58) and in adjacent Ricinus communis L. stem tissues was studied in thick sections by clearing with lactic acid and by staining with lacmoid. The crown galls contained two types of vascular strands: treelike branched bundles, which developed towards the tumor surface in fast-growing regions, and globular bundles in the slowly developing parts. Both types of vascular bundles contained xylem and phloem and were continuous with the vascular system of the host plant. The tumor bundles were interconnected by a dense net of phloem anastomoses, consisting of sieve tubes but no vessels. These vascular patterns reflect the apparent synthesis sites, concentration gradients and flow pathways of the plant hormones additionally produced in the tumors upon expression of the T-DNA-encoded genes. The A. tumefaciens-induced crown gall affected vascular differentiation in the host stem. In the basipetal direction, the tumor induced more xylem differentiation directly below it, where the crown-gall bundles joined the vascular system of the host. In the centripetal direction, the crown gall caused the development of pathologic xylem characterized by narrow vessels, giant rays and absence of fibers. On the other hand, most probably as a consequence of its gibberellic acid content, the host plant stimulated a local differentiation of regenerative phloem and xylem fibers with unique ramifications, only at the base of the tumor. However, fibers were absent from the main body of the crown gall. The study shows that A. tumefaciens-induced crown galls are characterized by a sophisticated network of vascular tissues in the tumor and are accompanied by a perturbated vessel system in the host. The hormonal mechanisms controlling vascular differentiation in the tumor and neighboring host tissues are discussed. In addition, the gall constriction hypothesis is proposed for explaining the mechanism which gives priority in water supply to the growing gall over the host shoot.We thank Dr. Zs. Koncz (Max-Planck-Institut für Züchtungsforschung, Köln, Germany) for Agrobacterium strains and the Deutsche Forschungsgemeinschaft (SFB 199) for financial support to C.I.U.     

The participation of annexin II (calpactin I) in calcium-evoked exocytosis requires protein kinase C

Permeabilized adrenal chromaffin cells secrete catecholamines by exocytosis in response to micromolar calcium concentrations. Recently, we have demonstrated that chromaffin cells permeabilized with digitonin progressively lose their capacity to secrete due to the release of certain cytosolic proteins essential for exocytosis (Sarafian T., D. Aunis, and M. F. Bader. 1987. J. Biol. Chem. 34:16671-16676). Here we show that one of the released proteins is calpactin I, a calcium-dependent phospholipid-binding protein known to promote in vitro aggregation of chromaffin granules at physiological micromolar calcium levels. The addition of calpactin I into digitonin- or streptolysin-O-permeabilized chromaffin cells with reduced secretory capacity as a result of the leakage of cytosolic proteins partially restores the calcium-dependent secretory activity. This effect is specific of calpactin I since other annexins (p32, p37, p67) do not stimulate secretion at similar or higher concentrations. Calpactin I requires the presence of Mg-ATP, suggesting that a phosphorylating step may regulate the activity of calpactin. Calpactin is unable to restore the secretory activity in cells which have completely lost their cytosolic protein kinase C or in cells having their protein kinase C inhibited by sphingosine or downregulated by long-term incubation with TPA. In contrast, calpactin I prephosphorylated in vitro by purified protein kinase C is able to reconstitute secretion in cells depleted of their protein kinase C activity. This stimulatory effect is also observed with thiophosphorylated calpactin I which is resistant to cellular phosphatases or with phosphorylated calpactin I introduced into cells in the presence of microcystin, a phosphatase inhibitor. These results suggest that calpactin I is involved in the exocytotic machinery by a mechanism which requires phosphorylation by protein kinase C.     

Effect of rfaH (sfrB) and temperature on expression of rfa genes of Escherichia coli K-12.

In order to study the regulation of a large block of contiguous genes at the rfa locus of Escherichia coli K-12 which are involved in synthesis and modification of the lipopolysaccharide core, the transposon TnlacZ was used to generate in-frame lacZ fusions to the coding regions of five genes (rfaQ, -G, -P, -B and -J) within this block. The beta-galactosidase activity of strains in which these fusions had been crossed into the chromosomal rfa locus was significantly decreased when the rfaH11 (sfrB11) allele was introduced and was restored to wild-type levels when these strains were lysogenized with a lambda phage carrying wild-type rfaH. This indicates that the positive regulatory function encoded by rfaH is required throughout this block of genes. In addition, expression of the lacZ fusion to rfaJ was reduced by growth at 42 degrees C, and this correlated with a temperature-induced change in the electrophoretic profile of the core lipopolysaccharide.     

Biochemical Characterization of Annexins I and II Isolated from Pig Nervous Tissue

Five proteins having molecular masses of 90, 67, 37, 36, and 32 kDa (p90, p67, p37, p36, and p32, respectively) were identified in the particulate fractions of pig brain cortex and pig spinal cord prepared in the presence of 0.2 mM Ca2+ and further purified using a protocol previously described for the purification of calpactins. Proteins p90, p37, and p36 are related to annexins I and II. Annexin II, represented by p90, is found as an heterotetramer, composed of two heavy chains of 36 kDa and two light chains of 11 kDa, and as a monomer of 36 kDa. Protein p37, which differs immunologically from p36, is a monomer and could be related to annexin I. All three proteins are Ca(2+)-dependent phospholipid- and F-actin-binding proteins; they are phosphorylated on a serine and on a tyrosine residue by protein kinases associated with synaptic plasma membranes. Purified p36 monomer and p36 heterotetramer proteins bind to actin at millimolar Ca2+ concentrations. The stoichiometry of p36 binding to F-actin at saturation is 1:2, corresponding to one tetramer or monomer of calpactin for two actin monomers (KD, 3 x 10(-6) M). Synaptic plasma membranes supplemented with the monomeric or tetrameric forms of p36 phosphorylate the proteins on a serine residue. The monomer is phosphorylated on a serine residue by a Ca(2+)-independent protein kinase, whereas the heterotetramer is phosphorylated on a serine residue and a tyrosine residue by Ca(2+)-dependent protein kinases. Antibodies to brain p37 and p36 together with antibodies to lymphocytes lipocortins 1 and 2 were used to follow the distribution of these proteins in nervous tissues. Polypeptides of 37, 34, and 36 kDa cross-react with these antibodies. Anti-p37 and antilipocortin 1 cross-react on the same 37- and 34-kDa polypeptides; anti-p36 and antilipocortin 2 cross-react only on the 36-kDa polypeptides.     

Comparison of lipopolysaccharide biosynthesis genes rfaK, rfaL, rfaY, and rfaZ of Escherichia coli K-12 and Salmonella typhimurium.

Analysis of the sequence of a 4.3-kb region downstream of rfaJ revealed four genes. The first two of these, which encode proteins of 27,441 and 32,890 Da, were identified as rfaY and rfaZ by homology of the derived protein sequences of their products to the products of similar genes of Salmonella typhimurium. The amino acid sequences of proteins RfaY and RfaZ showed, respectively, 70 and 72% identity. Genes 3 and 4 were identified as rfaK and rfaL on the basis of size and position, but the derived amino acid sequences of the products of these genes showed very little similarity (about 12% identity) between Escherichia coli K-12 and S. typhimurium. The next gene in the cluster, rfaC, encodes a product which also shows strong protein sequence homology between E. coli K-12 and S. typhimurium, as do the rfaF and rfaD genes which lie beyond it. Thus, the rfa gene cluster appears to consist of two blocks of genes which are conserved flanking a central region of two genes which are not conserved between these species. Although the RfaL protein sequence is not conserved, hydropathy plots of the two RfaL species are nearly identical and indicate that this is a typical integral membrane protein with 10 or more potential transmembrane domains. We noted the similarity of the structure of the rfa gene cluster to that of the rfb gene cluster, which has now been sequenced in several Salmonella serovars. The rfb cluster also contains a gene which lies within a central nonconserved region and encodes an integral membrane protein similar to protein RfaL. We speculate that protein RfaL may interact in a strain- or species-specific way with one or more Rfb proteins in the expression of surface O antigen.     

125I derivatives of prostaglandins. A novel approach in prostaglandin analysis by radioimmunoassay.

We report the production of radioactive iodinated (125 I) derivatives of prostaglandins E1, E2, F2alpha and their use in radioimmunological assays. Histamine or tyramine was coupled to the prostaglandins carboxyl group and the iodination was accomplished using the chloramine T method. The high specific radioactivity of these tracers and the resolution of the purification procedure allowed the detection of 0.5 pg of prostaglandins. A comparison with tritiated prostaglandin was made and showed a 10-fold gain in sensitivity. Furthermore in the case of the prostaglandin E1 system using 125I-labelled histamine or tyramine as tracer the cross reaction curves obtained were different from those obtained with [3H]prostaglandin E1; we suggest that the blocking of the carboxyl group alters the prostaglandin E1 structure, modifying its immunoreactivity.     

A 4-vinylprotochlorophyllide complex accumulated by "phofil" mutant of Rhodopseudomonas spheroides. An authentic intermediate in the development of the photosynthetic apparatus.

A photosynthetically competent mutant strain of Rhodopseudomonas spheroides was isolated. In addition to bacteriochlorophyll, this organism produced particle-bound precursor 4-vinylprotochlorophyllide. The spectral characteristics of the pigment complexes(es) accumulated in the culture medium were very variable. The spectral form occurring within the bacteria was characterized from fluorescence data. Its particle weight, 130 000, was determined by Sephadex G200 filtration. The main components of the complex were protein, lipid and pigment (6.8:61, w/w). As indicated by qualitative analysis, the lipid components were characteristic constituents of the photosynthetic membrane. Kinetics of pigments synthesis showed that the total pigment synthesis was not affected by the mutation; bacteriochlorophyll content was always lower in the mutant than in the parent strain. The repigmentation process was followed by fluorescence emission. The results indicated that the mutation affected membrane component synthesis required for the bacteriochlorophyll(ide) incorporation. The pigment complex was concluded to be an authentic intermediate in photosynthetic apparatus morphogenesis. The reasons for its excretion are discussed.     

The tyrosyl residues in creatine kinase. Modification by iodine.

The effect of the iodination of tyrosyl residues in creatine kinase from rabbit muscle has been investigated at alkaline pH after reversible masking of the reactive thiol groups. The conversion of 4-5 tyrosyl residues to monoiodotyrosines as measured by spectrotitration and by radioactive iodine labelling resulted in almost total loss of enzymic activity. The modified enzyme was unable to bind its nucleotide substrates but no significant conformational change was revealed by optical rotatory dispersion or Stokes radius measurements. However, change in the reactivity of some non-essential thiol groups, presumably those located near the active thiol groups, was observed.