Walter D. Sierralta

Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter

A colony morphology type is described in which cells of Salmonella typhimurium form a rigid multicellular network with expression of thin aggregative fimbriae that mediate tight intercellular bonds. Surface translocation of cells on plates and adherence to glass and polystyrene surfaces in biofilm assays are further characteristics of the morphotype. This morphotype (rdar) is normally expressed only at low temperature. However, in two unrelated S. typhimurium strains, spontaneous mutants were found forming rdar colonies independent of temperature. Allelic replacement proved a single point mutation in the promoter region of PagfD in each of the two mutants to be responsible for the constitutive phenotype of a multicellular behaviour. Transcription levels of the two divergently transcribed agf operons required for biogenesis of thin aggregative fimbriae by Northern blot analysis with gene probes for agfA and agfD as well as expression levels of AgfA by Western blotting were compared in the wild type, the constitutive mutants and their respective ompR− and rpoS− derivatives. In the wild type the rdar morphotype and expression of thin aggregative fimbriae are restricted to low temperature on plates containing rich medium of low osmolarity, but biogenesis of thin aggregative fimbriae occurs upon iron starvation at 37°C. In the upregulated mutants biogenesis of thin aggregative fimbriae is only abolished at high osmolarity at 37°C and in the exponential phase in broth culture. Control of expression of thin aggregative fimbriae and rdar morphology takes place at the transcriptional level at the agfD promoter. A functional ompR allele is required, however an rpoS mutation abolishes transcription only in the wild type, but has no influence on expression of thin aggregative fimbriae in the constitutive mutants.

Cyclic temperature treatments of dark-grown pea seedlings induce a rise in specific transcript levels of light-regulated genes related to photomorphogenesis

SummaryDark-grown pea seedlings exposed to cyclic heat shocks or daily temperature changes undergo a morphogenetic development similar to that induced by far red light. The morphological changes observed include expansion of the leaves, shortening of the stems and opening of the hooks. Compared with control etioplasts, plastids of heat-treated seedlings are as large as fully mature chloroplasts and contain well developed, unstacked membranes. These morphogenetic changes correlate with elevated levels of SSU and LHCP mRNAs which, under these conditions, fluctuate in a circadian manner. In contrast, the ELIP mRNA remains under strict light control and shows circadian fluctuations only if the plants are exposed to a short period of illumination. We propose that periodic temperature changes, like light treatment, might serve as a ‘Zeitgeber’ signal for circadian rhythm. The data indicate a correlation between the existence of circadian oscillations and morphogenetic development.

Activation of transcription-regulating proteins by steroids☆

Abstract The intracellular proteins, which bind steriod hormones with high affinity and specificity have been generally considered as instruments of hormone action. A reversal of assignments might seem a merely semantic exercise, but is indeed in better agreement with experimental evidence identifying ‘receptors’ as transcription-regulating proteins. The series of events in the presence of hormone are: 1. attachment of the steroid to the ‘receptor’ which undergoes a major conformational change when ‘enveloping’ the steroid, 2. dimerization to steroid-receptor: steroid-receptor 3. translocation of the dimer into the nucleus, 4. enhancement of transcription. One product of the latter is ‘receptor’ mRNA, the translation of which initiates within 60–90 min after pulse-administration of steroid. In the absence of hormone, ‘receptor translocation’, degradation and biosynthesis continue to proceed but at a much slower rate. Although these results have been primarily obtained with the estradiol-‘receptor’ system, all other systems seem to follow the same pattern. The molecular mechanism by which enhancement of transcription is achieved is as yet unknown. Its specificity must be quite particular since several steroid-‘receptor‘ systems occur simultaneously within the same cell.

MECHANISMS INVOLVED IN THE REGULATION OF STEROID RECEPTOR LEVELS

Abstract The turnover of steroid receptors comprises: synthesis in the cytoplasm, translocation into the cell nucleus and degradation at a still unknown site. From studies on the oestradiol/receptor system, the following conclusions can be drawn. 1. The in-vivo uptake of oestradiol by target cell nuclei is receptordependent. Steroid and receptor are translocated from the cytoplasm in a 1:1 ratio. A recycling of receptor is undetectable after pulse administration of oestradiol. Receptor replenishment in the cytoplasm is accomplished by synthesis. 2. The nuclear uptake of receptor-in contrast-proceeds also in the absence of oestradiol. Both forms of receptor, monomer and “activated” dimer are present in oestrogen-free nuclei. 3. Oestradiol enhances the “nucleotropy” and the turnover rate of receptor. 4. Oestrogenicity and antioestrogenicity are apparently linked to effects exerted on the “nucleotropy” of receptor and its ability to interact with the relevant nuclear structures.

Binding and distribution of Na2B12H11SH on cellular and subcellular level in tumor tissue of glioma patients in boron neutron capture therapy

To determine binding and distribution of Na2B12H11SH (BSH)in glioma tissue in case of boron neutroncapture therapy, an antibody to this compound wasproduced and used in immunohistochemical investigations. It ispossible to trace BSH in immunohistochemistry, because BSHis firmly bound to the glioma tissue. Theantibody against BSH is specific for that antigen,as tumor tissue from patients without BSH administrationdid not stain. In areas of healthy brainfrom BSH infused patients, no staining of tissuewas detectable. In tumor tissues, BSH is presentingas a strong staining in cytoplasm and nucleusareas.

Electron spectroscopic imaging of antigens by reaction with boronated antibodies.

Two small homogeneous markers for electron spectroscopic imaging (ESI) containing eight dodecaborane cages linked to a poly‐α,ε‐l‐lysine dendrimer were synthesized; one of these was made water soluble by the attachment of a polyether. The markers were coupled to the sulfhydryl group of (monovalent) antibody fragments (Fab′) by a homobifunctional cross‐linker. While the coupling ratios of the poorly water‐soluble compound did not exceed 20%, the polyether‐containing variant reacted quantitatively. Its suitability for immunolabelling was tested in a study of the mechanism of the transcellular transport of an administered heterologous protein (bovine serum albumin, BSA) through ileal enterocytes of newborn piglets by endocytotic vesicles in comparison to conventional immunogold reagents. The post‐embedding technique was employed. The boronated Fab′ gave rise to considerably higher tagging frequencies than seen with immunogold, as could be expected from its form‐ and size‐related physical advantages and the dense packing of BSA in the vesicles. The new probe, carrying the antigen‐combining cleft at one end and the boron clusters at the opposite end of the oval‐shaped conjugate, add to the potential of ESI‐based immunocytochemistry.

Immunogold labelling of estradiol receptor in MCF7 cells

The distribution of estradiol receptor in serial sections of estradiol-deprived and estradiol-stimulated MCF7 cells was studied by using mouse monoclonal antibodies reacting with different domains of the receptor and goat-antimouse IgG/6 nm gold. In the nucleus and the cytoplasm of estradiol-deprived cells, the receptor was detected by all three monoclonals (13H2, HT 65 and MA1-310). The antibodies 13H2 and MA1-310 detected receptor associated to the microfilament bundles in the cytoplasm. Higher densities of antireceptor attachment to the nuclear areas were accompanied by a reduction in the attachment to the cytoplasm after estradiol stimulation of the cells. The results confirm earlier observations on the presence of cytoplasmic estrogen receptor in estradiol-deprived cells and support the premise of an estradiol-induced translocation of this ligand-dependent transcription regulator.

Retrieval of estradiol receptor in paraffin sections of resting porcine uteri by microwave treatment. Immunostaining patterns obtained with different primary antibodies.

The unmasking of estradiol receptor in paraffin sections of Bouins-fixed uterine tissue from ovariectomized gilts was attained with microwave treatment. Immunocytochemistry of the receptor was performed using a polyclonal or five monoclonal antibodies, two of which are commercially available, reacting with different domains of the protein and an amplified-peroxidase system for detection. With five of the antibodies, a predominance of nuclear staining was observed in cells of endometrial glands, while one monoclonal antibody (13H2), reacting with the receptors domain E, showed a preference for the cytoplasmic receptor. In stroma, all antibodies detected more receptor in nuclei than in cytoplasm. In epithelium, the commercially available antibody H222, our monoclonals 13H2 and HT65, and the polyclonal antibody 402 demonstrated more receptor in cytoplasmic than in nuclear areas. In myometrium, the nuclei from longitudinal and ring muscles were definitely stained with the antibodies. We conclude that the accessibilities of the antibody epitopes of the receptor differ according to the functional uterine cell type.

Immunogold labelling of the cytoplasmic estradiol receptor in resting porcine endometrium

SummarySerial sections of resting porcine endometrium were analyzed with the monoclonal antibody 13H2 using goat antimouse IgG/5 nm gold as secondary reagent or with either polyclonal antibodies from goat #402 or the rat monoclonal antibody H222, both in combination with protein G/12 nm gold. A modestly higher labelling of nuclei than of cytoplasm was seen only with the monoclonal antibody H222. Polyclonal #402 and monoclonal 13H2 showed fewer attachments over nuclear than over cytoplasmic areas. The highest densities of attachment and of predominantly cytoplasmic labelling were obtained with the monoclonal antibody 13H2. The results confirm the earlier assumption of a restricted accessiblity of estradiol receptor in the cytoplasm of resting cells for immunoreagents.

The 23-kDa light-stress-regulated heat-shock protein of Chenopodium rubrum L. is located in the mitochondria

The 23-kDa nuclear-encoded heat-shock protein (HSP) of Chenopodium rubrum L. is regulated by light at the posttranslational level. Higher light intensities are more effective in inducing the accumulation of the mature protein under heat-shock conditions. Based on this and other properties the protein was considered to belong to the group of small chloroplastic HSPs. However, we have now obtained the following evidence that this 23-kDa HSP is localized in the mitochondria: (i) Immunogold-labelled protein was almost exclusively restricted to the mitochondria in electron microscope thin sections. (ii) Using purified, isolated mitochondria from potato tubers the in-vitro-synthesized translation product of 31 kDa was readily transported into mitochondria where it was processed to the 23-kDa product. (iii) The protein could be detected by Western blotting in a preparation of washed mitochondria of Chenopodium, while under the same conditions no signal could be obtained in a preparation of isolated chloroplasts. (iv) Finally, sequence comparison with the published sequences of mitochondrial proteins by Lenne et al. (1995, Biochem J 311:805–813) and LaFayette et al. (1996, Plant Mol Biol 30:159–169) showed clearly that the 23-kDa protein is considerably more similar to these two proteins than to the group of plastid small HSPs. From these data we infer that mitochondria are involved in the response of the plants to high light stress under heat-shock conditions.