Asymmetric segregation of heat-shock proteins upon cell division in Caulobacter crescentus

Three Caulobacter crescentus heat-shock proteins were shown to be immunologically related to the Escherichia coli heat-shock proteins GroEL, Lon and DnaK. A fourth heat-shock protein was detected with antibody to the C. crescentus RNA polymerase. This 37,000 Mr heat-shock protein might be related to the E. coli 32,000 Mr heat-shock sigma subunit. The synthesis of the major C. crescentus RNA polymerase sigma factor was not induced by heat shock. The E. coli GroEL protein and the related protein from C. crescentus were also induced by treatment with hydrogen peroxide. Like some of the proteins in the heat-shock protein families of Drosophila and yeast, the four heat-shock proteins in C. crescentus were found to be regulated developmentally under normal conditions. All four proteins were synthesized in the predivisional cell, but the progeny showed cell type-specific bias in the level of enhanced synthesis after heat shock. The 92,000 Mr Lon homolog and the 37,000 Mr RNA polymerase subunit were preferentially synthesized in the stalked cell, whereas the synthesis of the 62,000 Mr GroEL homolog was enhanced in the progeny swarmer cell. Furthermore, the four heat-shock proteins synthesized in the predivisional cell were partitioned in a specific manner upon cell division. The stalked cell, which initiates chromosome replication immediately upon division, received the Lon homolog, the DnaK homolog and the 37,000 Mr RNA polymerase subunit. The GroEL homolog, however, was distributed equally to both the stalked cell and the swarmer cell. These results provide access to the functions of C. crescentus heat-shock proteins under both normal and stress conditions. They also allow an investigation of the regulatory signals that modulate the asymmetric distribution of proteins and their subsequent cell type-specific expression in the initial stages of a developmental program.     

A detailed study of the periodate oxidation of sialic acids in glycoproteins

Periodate oxidation of terminalN-acetyl- andN-glycoloylneuraminic acid residues in the mucins from edible bird nest substance and pig submandibular gland, respectively, can be carried out under conditions which exclusively give rise to the formation of the C-7 analogues of these sialic acids. In contrast, the C-8 compounds can be obtained in a maximum yield of about 40%. Under identical conditions,N-glycoloylneuraminic acid is oxidized about 1.5 times faster than theN-acetylated derivative. After release of the sialic acids by acid hydrolysis, the characterization of the oxidation products was carried out by TLC, by GLC and GLC-MS of the corresponding pertrimethylsilyl derivatives, and by 500-MHz¹H-NMR spectroscopy. In addition, molar response factors for GLC analysis and extinction coefficients in the orcinol/Fe³⁺/HCl assay were determined.     

Uptake and utilization of lipids by Paramecium tetraurelia

Uptake rates of a variety of 14C-labeled fatty acids and complex lipids by Paramecium tetraurelia during 48 h of log-phase growth varied. Fatty acid uptake was maximal during lag phase of growth when phagosome (food vacuole) formation was minimal. Food vacuole formation was shown to be suppressed by the presence of exogenous lipids and by starvation. The rates of uptake of lipids were significantly greater than those of small organic compounds such as amino acids, cyclitols, fatty acid precursors and metabolic intermediates. Significant amounts of radioactivity from 14C-labeled fatty acids were metabolized to 14CO2. The uptake rates of different saturated, straight-chain fatty acids of even carbon numbers were different and were not correlated with chain length, results suggesting that the primary mechanism for uptake of these compounds was neither by bulk transport nor simple diffusion and that carrier-mediated processes could possibly be involved.     

Inhibitory effect of nonviable preparations from human immunodeficiency virus 1 on inositol phospholipid metabolism

Previously it was established [Pahwa, S., Pahwa, R., Saxinger, C., Gallo, R. C. & Good, R. A. (1985) Proc. Natl. Acad. Sci. USA 82, 8198] that nonviable preparations of human immunodeficiency virus 1 (HIV-1) abolish the proliferative response of human lymphocytes to phytohemagglutinin A. Now we describe that this effect might be, at least partially, due to an impairment of the function of phospholipase C. It was found that addition of HIV-1 preparation to lymphocytes diminished the stimulation of phosphatidylinositol phosphorylation caused by phytohemagglutinin A. Moreover, this preparation completely abolished the phytohemagglutinin-A-stimulated release of inositol trisphosphate and prevented a translocation of protein kinase C from cytosol to membranes. From this data we conclude that nonviable HIV-1 preparations inhibit the intracellular signalling pathway, leading to a reduced mitogenic response to phytohemagglutinin A, at the level of protein kinase C.     

Association of Tat protein and viral mRNA with nuclear matrix from HIV-1-infected H9 cells

The transactivating protein from human immunodeficiency virus type 1 (HIV-1), Tat, was found to bind to the nuclear matrix from uninfected and HIV-1-infected H9 cells. Addition of the Zn2+, Cd2+ and Cu2+ chelator o-phenanthroline destroyed the matrix fibrils and the binding affinity of Tat to the matrix. A sequential treatment of the matrix, first with o-phenanthroline and then with ZnCl2, partially restored the fibrillar-like matrix structure. Infection of H9 cells with HIV-1 resulted in a displacement of cellular mRNA by viral mRNA from the nuclear matrix. Both the matrix-bound host cell and HIV-1 mRNA were found to dissociate from the matrix in the presence of o-phenanthroline. This could be prevented by coincubation with Zn2+ or Cu2+ (but not Mg2+), which stabilize the mRNA containing nuclear matrix structure.     

Cytogenetic analysis of human solid tumors by in situ hybridization with a set of 12 chromosome-specific DNA probes

We have applied fluorescent in situ hybridization (FISH) to assess the presence of numerical chromosome aberrations in fresh specimens of human solid tumors of varying histology. For this purpose, a set of 12 biotinylated chromosome-specific, repetitive alpha-satellite DNA probes (for chromosomes 1, 6, 7, 9, 10, 11, 15, 16, 17, 18, X and Y) were hybridized directly to isolated interphase nuclei. Utilizing this approach, we found numerical chromosome changes in all tumors. FISH ploidy profiles were in accordance with flow cytometric DNA histograms of these tumor cells.     

Core substructure in Mastigocladus laminosus phycobilisomes

A native high molecular complex (M_r 850000) containing about 50% of the allphycocyanin of the phycobilisome but lacking allophycocyanin B was separated from isolated phycobilisomes by gel electrophoresis. It was designated AP_CM since the large linker polypeptide L_CM was exclusively localized in this complex. The complex exhibited a ?196°C fluorescence emission maximum at 673 nm (671 nm at 25°C). In addition, a core complex (designated AP_C, M_r≥1000000) consisting of both AP_CM and AP 680 was isolated by combined gel filtration and linear gradient centrifugation. At 25°C this complex showed dual emission peaks at 670 and 680 nm demonstrating functional independence of the terminal emitters. A complex similar to AP_CM can be isolated from phycobilisomes of Anabaena variabilis. This is evidence that AP_CM is the constitutive center of the tricylindrical core of hemidiscoidal cyanobacterial phycobilisomes. Two models summarizing the structural and functional consequences of the results are presented in the discussion.     

An improved method for the detection of Dcm methylation in DNA molecules

The intrinsic insensitivity of EcoRII recognition sites in RF DNAs of phage M13 and vector M13mp18 towards this restriction endonuclease can be overcome by adding site-specific oligodeoxyribonucleotide duplexes to the restriction sample. Since Dcm- DNA but not Dcm(+)-methylated DNA becomes susceptible under these conditions, this procedure constitutes an improvement of the Dcm methylation assay.     

Expression and function of the homoeotic genes Antennapedia and Sex combs reduced in the embryonic midgut of Drosophila

Drosophila homoeotic genes control the formation of external morphological features of the embryo and adult, and in addition affect differentiation of the nervous system. Here we describe the morphogenetic events in the midgut that are controlled by the homoeotic genes Sex combs reduced (Scr) and Antennapedia (Antp). The midgut is composed of two cell layers, an inner endoderm and an outer visceral mesoderm that surround the yolk. Scr and Antp are expressed in the visceral mesoderm but not in the endoderm. The two genes are required for different aspects of the midgut morphogenesis. In Scr null mutant embryos the gastric caeca fail to form. Scr is expressed in the visceral mesoderm cells posterior to the primordia of the gastric caeca and appears to be indirectly required for the formation of the caeca. Antp is expressed in visceral mesoderm cells that overlie a part of the midgut where a constriction will form, and Antp null mutant embryos fail to form this constriction. An ultrastructural analysis of the midgut reveals that the visceral mesoderm imposes the constriction on the endoderm and the yolk. The mesodermal tissue contracts within the constriction and thereby penetrates the layer of the midgut endoderm. Microtubules participate in the morphological changes of the visceral mesoderm cells. The analysis of the expression of Scr in Antp mutant embryos revealed a case of tissue-specific regulation of Scr expression by Antp. In the epidermis, Antp has been shown to negatively regulate Scr, but it positively regulates Scr in the visceral mesoderm.