Fiber-optic biosensor to assess circulating phagocyte activity by chemiluminescence

We describe herein the construction of a novel computerized multi-sample temperature-controlled luminometer for a fiber array-based biosensor to monitor circulating phagocyte activity. It can perform simultaneously integral measurements of chemiluminescence emitted from up to six samples containing less than 0.5 microl whole blood while the samples and detector do not change their position during the measurement cycle. The optical fibers in this luminometer are used as both light guides and solid phase sample holders. The latter feature of the instrument design simplifies the assessment process of both the extra-cellular and the intra-cellular parts of the phagocyte-emitted chemiluminescence using the same system. We describe some examples or proof of principle for the use of the biosensor. This new technology may find use in a wide range of analytical luminescence applications in biology, biophysics, biochemistry, toxicology and clinical medicine.     

Different domains control the localization and mobility of LIKE HETEROCHROMATIN PROTEIN1 in Arabidopsis nuclei

Plants possess a single gene for the structurally related HETEROCHROMATIN PROTEIN1 (HP1), termed LIKE-HP1 (LHP1). We investigated the subnuclear localization, binding properties, and dynamics of LHP1 proteins in Arabidopsis thaliana cells. Transient expression assays showed that tomato (Solanum lycopersicum) LHP1 fused to green fluorescent protein (GFP; Sl LHP1-GFP) and Arabidopsis LHP1 (At LHP1-GFP) localized to heterochromatic chromocenters and showed punctuated distribution within the nucleus; tomato but not Arabidopsis LHP1 was also localized within the nucleolus. Mutations of aromatic cage residues that recognize methyl K9 of histone H3 abolished their punctuated distribution and localization to chromocenters. Sl LHP1-GFP plants displayed cell type-dependent subnuclear localization. The diverse localization pattern of tomato LHP1 did not require the chromo shadow domain (CSD), whereas the chromodomain alone was insufficient for localization to chromocenters; a nucleolar localization signal was identified within the hinge region. Fluorescence recovery after photobleaching showed that Sl LHP1 is a highly mobile protein whose localization and retention are controlled by distinct domains; retention at the nucleolus and chromocenters is conferred by the CSD. Our results imply that LHP1 recruitment to chromatin is mediated, at least in part, through interaction with methyl K9 and that LHP1 controls different nuclear processes via transient binding to its nuclear sites.     

Profilin 1 is required for abscission during late cytokinesis of chondrocytes

Profilins are key factors for dynamic rearrangements of the actin cytoskeleton. However, the functions of profilins in differentiated mammalian cells are uncertain because profilin deficiency is early embryonic lethal for higher eukaryotes. To examine profilin function in chondrocytes, we disrupted the profilin 1 gene in cartilage (Col2pfn1). Homozygous Col2pfn1 mice develop progressive chondrodysplasia caused by disorganization of the growth plate and defective chondrocyte cytokinesis, indicated by the appearance of binucleated cells. Surprisingly, Col2pfn1 chondrocytes assemble and contract actomyosin rings normally during cell division; however, they display defects during late cytokinesis as they frequently fail to complete abscission due to their inability to develop strong traction forces. This reduced force generation results from an impaired formation of lamellipodia, focal adhesions and stress fibres, which in part could be linked to an impaired mDia1‐mediated actin filament elongation. Neither an actin nor a poly‐proline binding‐deficient profilin 1 is able to rescue the defects. Taken together, our results demonstrate that profilin 1 is not required for actomyosin ring formation in dividing chondrocytes but necessary to generate sufficient force for abscission during late cytokinesis.     

Reconstitution of the mitochondrial Hsp70 (mortalin)-p53 interaction using purified proteins - Identification of additional interacting regions

Previous studies have shown that the mammalian mitochondrial 70 kDa heat-shock protein (mortalin) can also be detected in the cytosol. Cytosolic mortalin binds p53 and by doing so, prevents translocation of the tumor suppressor into the nucleus. In this study, we developed a novel binding assay, using purified proteins, for tracking the interaction between p53 and mortalin. Our results reveal that: (i) P53 binds to the peptide-binding site of mortalin which enhances the ability of the former to bind DNA. (ii) An additional previously unknown binding site for mortalin exists within the C-terminal domain of p53.

Structured summary

MINT-7557591: p53 (uniprotkb:P04637) binds (MI:0407) to DnaK (uniprotkb:P0A6Y8) by affinity chromatography technology (MI:0004)MINT-7557644: mortalin (uniprotkb:P38646) binds (MI:0407) to p53 (uniprotkb:P04637) by pull down (MI:0096)MINT-7557580, MINT-7557611: p53 (uniprotkb:P04637) binds (MI:0407) to mortalin (uniprotkb:P38646) by affinity chromatography technology (MI:0004)     

Rapid effects of progesterone on ciliary beat frequency in the mouse fallopian tube

Background  

The physiological regulation of ciliary beat frequency (CBF) within the fallopian tube is important for controlling the transport of gametes and the fertilized ovum. Progesterone influences gamete transport in the fallopian tube of several mammalian species. In fallopian tubes isolated from cows, treatment with 20 micromolar progesterone caused a rapid reduction of the tubal CBF. The aims of this study were to establish methodology for studying fallopian tube CBF in the mouse, as it is an important model species, and to investigate if progesterone rapidly affects the CBF of mice at nM concentrations.     

Biogenesis of chloroplast membranes X. Changes in the photosynthetic specific activity and the relationship between the light harvesting system and photosynthetic electron transfer chain during greening of Chlamydomonas reinhardi y-1 cells

Specific activities of photophosphorylation and light-dependent pH rise at different stages of the greening process, have been measured as a function of the illumination intensity.     

Structure of rough,smooth, stripped and reconstituted rough membranes derived from rat liver as visualized by the freeze fracture technique

The structure of purified fractions of rough, smooth, stripped rough and reconstituted rough membranes have been investigated by the freeze etching technique. Preparations of rough and reconstituted rough membranes, active in protein synthesis, show vesicles whose outer surface is covered with ribosome-like particles. The inner surface of these vesicles contains also numerous particles of the same size. The particles located on the outer surface are largely absent in the stripped rough membrane preparations which, however, retain the particles located on the inner face. Particles were not seen either on the outer nor on the inner face of the smooth membranes. The possibility is considered that the particles located on the inner face are specific to the rough membranes and might play a role in the specific binding of ribosomes to the membranes.     

Polypeptides of chloroplastic and cytoplastic origin required for development of Photosystem II activity,and chlorophyll-protein complexes,in Euglena gracilis Z chloroplast membranes

The development of photosynthetic activity and synthesis of chloroplast membrane polypeptides was studied during greening of Euglena gracilis Z in alternate light-dark-light cycles. The results show: (a) The development of both Photosystem II and Photosystem I can be dissociated from chlorophyll synthesis. (b) Most of the polypeptides required for development of Photosystem I are already synthesized during the initial light period (10–12 h); the further rise in Photosystem I activity in the dark is not inhibited by cycloheximide nor by chloramphenicol. (c) The development of Photosystem II requires continuous de novo synthesis of polypeptides and is inhibited by chloramphenicol. The water-splitting activity already present at the end of the first light period decays in the presence of chloramphenicol while that of 1,5-diphenylcarbazide oxidation is only partially retained. The activity can be repaired in the absence of chlorophyll synthesis and is correlated with the de novo synthesis of polypeptides of 50 000–60 000 daltons. The synthesis of these polypeptides and associated repair of Photosystem II activity is not inhibited by cycloheximide. (d) The chloroplast membranes can be resolved into about 40 distinct polypeptides, among them several in the molecular weight range 50 000–60 000, 20 000–35 000 and 10 000–15 000, which are major membrane constitutents. (e) The synthesis of two major polypeptides (M_r = 20 000–30 000) required for the formation of chlorophyll-protein complex(es) containing chlorophyll a and traces of chlorophyll b (CPII?) is light-dependent and cycloheximide-inhibited. It is concluded that the synthesis and addition to the growing membrane of chlorophyll and polypeptides required for the formation of Photosystem II and Photosystem I complexes can be dissociated in time. The H₂O-splitting enzyme(s) and possibly other components of Photosystem II complex are of chloroplastic origin and turn over in the dark while at least some of the chlorophyll binding polypeptides are of cytoplastic origin and their synthesis is light-controlled.     

A similar structure of the herbicide binding site in photosystem II of plants and cyanobacteria is demonstrated by site specific mutagenesis of the psbA gene

Many herbicides inhibit the photosynthetic electron transfer in photosystem II by binding to the polypeptide D1. A point mutation in the chloroplast gene psbA, which leads to a change of the amino acid residue 264 of D1 from serine to glycine, is responsible for atrazine resistance in higher plants. We have changed serine 264 to glycine in Synechococcus PCC7942 and compared its phenotype to a mutant with a serine to alanine shift in the same position. The results show that glycine at position 264 in D1 gives rise to a similar phenotype in cyanobacteria and in higher plants, indicating a similar structure of the binding site for herbicides and for the quinone Q_B in the two systems. A possible mode of binding of phenyl-urea herbicides to D1 is predicted from the difference in herbicidal cross-resistance between glycine and alanine substitutions of serine 264.Abbreviations DCPIP 2,6-dichlorophenolindophenol - I₅₀ concentration of herbicide giving 50% inhibition - K_b binding constant - kb kilobase - MES 2(N-morpholino)ethanesulfonic acid - PS II photosystem II     

From lamins to lamina: a structural perspective

Lamin proteins are the major constituents of the nuclear lamina, a proteinaceous network that lines the inner nuclear membrane. Primarily, the nuclear lamina provides structural support for the nucleus and the nuclear envelope; however, lamins and their associated proteins are also involved in most of the nuclear processes, including DNA replication and repair, regulation of gene expression, and signaling. Mutations in human lamin A and associated proteins were found to cause a large number of diseases, termed ‘laminopathies.’ These diseases include muscular dystrophies, lipodystrophies, neuropathies, and premature aging syndromes. Despite the growing number of studies on lamins and their associated proteins, the molecular organization of lamins in health and disease is still elusive. Likewise, there is no comprehensive view how mutations in lamins result in a plethora of diseases, selectively affecting different tissues. Here, we discuss some of the structural aspects of lamins and the nuclear lamina organization, in light of recent results.