The formation of small, mineralized structures in incubated serum under standard cell culture conditions was first introduced in 1997 (Kajander et al., SPIE Proc. 3111: 420--8) and was attributed to a previously unknown microorganism that was subsequently assigned to the aalpha-2 subgroup of Proteobacteria. Despite extensive controversy surrounding the existence of these "nanobacteria", the possibility exists that these microorganisms might be involved in pathological calcification such as kidney stones. Using a combined morphological and biochemical approach, we have investigated further the formation of these mineralized structures in incubated serum. Transmission electron microscopy, X-ray microanalysis, high-resolution lattice imaging, and freeze-fracturing techniques were used to demonstrate at the ultrastructural level the crystalline nature and composition of these structures. Additional studies were performed to elucidate the relationship of nanoforms with mineralizing bone cell cultures. The formation of HA in incubated serum represents an otherwise unknown calcification process that offers a unique opportunity to study mineralization under laboratory conditions. In addition, information gained from understanding this process might be useful for bone tissue engineering where mineralization is desirable within synthetic bone substitutes, and within which mineral-binding bioactive factors may also be incorporated. (Abstract shortened by UMI.)

Over the years, epithelial cells of the male reproductive tract have been suggested to be involved in apocrine secretion. However, this method of secretion has not been fully accepted, nor is it well understood. In the present study, apocrine secretion was examined in the mouse epididymis and rat and human vas deferens. Throughout these regions of the duct, the apex of the epithelial principal cells often shows protrusions extending towards the lumen of the duct, referred to as apical blebs (ABs). Of different shapes and sizes, ABs appear to form at the apex of principal cells, and morphological and immunocytochemical evidence suggests that ABs detach from the cell surface whereupon they eventually undergo fragmentation in the lumen. ABs contain selective organelles suggesting a segregation of their contents. Numerous polysomes suggest the synthesis of nonglycosylated proteins which upon the fragmentation of ABs in the lumen could act directly or indirectly on sperm. The presence of spherical vesicles of various sizes (20--200mn) within ABs as well as within the lumen of the duct supports AB fragmentation in the lumen and may play a role in modifying the sperm surface. Taken together the data provide strong evidence for apocrine secretion that could play important roles in relation to sperm maturation, viability and protection.

Nuclear import of proteins that are too large to passively enter the nucleus requires soluble factors, energy, and a nuclear localization signal (NLS). Nuclear protein transport can be regulated, and different forms of stress affect nucleocytoplasmic trafficking. As such, import of proteins containing a classical NLS is inhibited in starving yeast cells. In contrast, the heat shock protein hsp70 Ssa4p concentrates in nuclei upon starvation. Nuclear concentration of Ssa4p in starving cells is reversible, and transfer of nutrient-depleted cells to fresh medium induces Ssa4p nuclear export. This export reaction represents an active process that is sensitive to oxidative stress. Upon starvation, the N-terminal domain of Ssa4p mediates Ssa4p nuclear accumulation, and a short hydrophobic sequence, termed Star (for starvation), is sufficient to localize the reporter proteins green fluorescent protein or beta-galactosidase to nuclei. To determine whether nuclear accumulation of Star-beta-galactosidase depends on a specific nuclear carrier, I have analyzed its distribution in mutant yeast strains that carry a deletion of a single beta-importin gene. With this assay I have identified Nmd5p as a beta-importin required to concentrate Star-beta-galactosidase in nuclei of stationary phase cells.

We examined the mechanical properties of oocytes and eggs from Xenopus laevis frogs, in relation to the cells' ability to maintain volume in dilute media. Elastic properties were assessed by measuring intracellular pressure (Pic) and cell volume (Vc) during osmotic swelling. Cytoplasmic rheology was determined by magnetometry after microinjection of magnetic iron microparticles. Both oocytes and eggs had a small but measurable Pic. Oocytes markedly increased both Pic and Vc during osmotic swelling. The relationship between Pic and Vc was not linear and showed hysteresis indicating that oocytes are viscoelastic. After removal of the vitelline envelope, the Pic-Vc curve remained qualitatively similar but the Pic rise was reduced by 70%. During osmotic swelling, eggs increased Vc to a lesser extent than did oocytes and Pic did not change. Neither oocytes nor eggs displayed a regulatory volume decrease in response to hypotonic shock and osmolyte loss did not appear to limit swelling. Since Pic was low in eggs and did not increase with swelling, increased stiffness of the vitelline envelope was ruled out as the explanation for their reduced water uptake. Rheologic data indicated that cytoplasmic apparent viscosity and elastic shear modulus were lower in eggs than in oocytes, an observation not compatible with our hypothesis that eggs took up less water because their cytoplasm was a gel. We now feel that Vc of eggs may be controlled by the cortical actin cytoskeleton.

Protein import into the mitochondria is a result of a series of sequential binding interactions between a mitochondrial targeting signal and the translocation machinery of both mitochondrial membranes. The targeting signals contained within protein of the outer membrane are distinct from those which target proteins to other subcompartments. The transmembrane domain of the yeast outer membrane receptor protein yTom70 is capable of both targeting and inserting the protein into the outer membrane. The efficiency of this process is increased by the addition of a positively-charged region preceding the transmembrane region. These two structural domains co-operate to form a signal-anchor sequence selective for the outer membrane, since this is the first membrane encountered by the targeting signal. / Consistent with this model, the signal-anchor sequence of the outer membrane protein yTom70 is also capable of importing into the inner membrane of mitochondria when the outer membrane is selectively removed. Import into the inner membrane requires the presence of an electrochemical potential across the lipid bilayer. Import proceeds in the absence of $ Delta Psi$ only when constructs are used which lack the positively-charged amino terminal region of the signal-anchor sequence. These results suggest that the positively-charged presequence leads the transmembrane domain into the import machinery and that $ Delta Psi$ is required to clear this region in order that the distal transmembrane region can enter the translocation pathway. / The charged N-terminal 10 residues of yTom70 are incapable of directing import into intact mammalian mitochondria, however, are able to efficiently direct import into the matrix of yeast mitochondria or mammalian mitoplasts. This potentially cryptic signal is excluded from intact mammalian mitochondria due to the presence of the receptor protein Tom20, since replacement of yeast Tom20 with mammalian Tom20 confers the mammalian phenotype onto yeast. These results suggest that receptor proteins may also have the ability to screen potentially cryptic signals from distal components of the outer and inner membrane translocation machinery.

The Golgi apparatus is organized into a characteristic differentiated stack in all eukaryotic cells. During mitosis, the Golgi apparatus disassembles into smaller clustered vesicles lacking recognizable cisternae whereupon they recombine to form typical stacks in each of the daughter cells (Lucocq et al., 1987). Paiment et al. (1989) have demonstrated that dispersed (unstacked) Golgi fragments will reconstitute into a functional stacked Golgi apparatus when microinjected into Xenopus laevis oocytes. Disrupted hepatic Golgi fractions (Gi) were isolated on discontinuous sucrose gradients and incubated at 37$ sp circ$C in the presence or absence of calf brain cytosol, ATP, and GTP$ gamma$S with or without pretreatment by N-ethylmaleimide. The reconstitution of saccule stacking was assayed using transmission electron microscopy on pelleted and filtered fractions. The role of cytosolically exposed Golgi membrane proteins in maintaining saccule was also addressed using controlled protease digests.

Amyloidosis describes a heterogenous collection of systemic diseases characterized by the extracellular deposition of proteinaceous amyloid fibrils derived from normally soluble proteins. With progressive tissue deposition of amyloid, death can occur through failure of the affected organ(s). However, the mechanism of amyloid fibril formation remains obscure. To understand this mechanism, a parasite (alveolar hydatid cyst, AHC)-mouse model of inflammation-associated AA amyloidosis, was used. AHC is a potent inducer of chronic inflammation, serum amyloid A (SAA) synthesis and AA amyloidosis. It has been proposed that reactive oxygen radicals (ROR) generated by inflammatory macrophages (mphi) and reticuloendothelial (RE) cells, which are also intimately involved in SAA clearance, could initiate intra-lysosomal AA fibril formation. ROS generated intracellularly could oxidize SAA fragments or other key cellular proteins, alter them structurally and thus render them resistant to enzymatic degradation and prone to intralysosomal nascent AA fibril formation. / The objective of this research was to identify oxidative stress (OS) markers (heme-oxygenase-1, HO-1; 4-hydroxy-2-nonenal, HNE; Nepsilon -(Carboxymethyl)lysine, CML) in peritoneal mphi and splenic/hepatic RE cells obtained from AHC-infected mice prior to and during AA amyloidosis. Histochemical and peroxidase-immunoperoxidase methods were used to detect the OS markers. High levels of HO-1, an antioxidant enzyme; HNE, a product of lipid peroxidation; and CML, an advanced glycation end product, were found in peritoneal mphi and splenic/hepatic RE cells proximal to AA fibril deposition. HNE and CML deposits were found in both the tissue interstitium and bound to AA amyloid deposits, indicating their possible role in the oxidative alteration of intracellular SAA. OS mediated changes in mphi/RE cells loaded with SAA may prove to be a prelude to nascent intracellular AA fibril formation.

Tissue transglutaminase (TG2) is a widely distributed, protein-cross-linking enzyme that creates high-molecular weight polymers from its substrate proteins. Its substrates in bone are mainly matricellular adhesion proteins: fibronectin (FN), osteopontin (OPN) and bone sialoprotein (BSP), which have been localized to the osteoid and to the pericellular matrix of osteocytes. The aim of this study was to compare in vitro the effects of monomeric versus polymeric FN, OPN and BSP on MC3T3-E1/C4 osteoblast adhesion, proliferation and differentiation. We showed that for each of the three TG2-mediated protein polymers tested, a significant increase in cell adhesion was observed. Also when observed by phase-contrast microscopy, the morphology of the cultured cells demonstrated increased cell spreading on polymerized protein. / Our results show that FN, OPN and BSP, when polymerized by TG2, significantly increase cell adhesion and decrease proliferation of MC3T3-E1/C4 osteoblasts without affecting their ability to differentiate. Decrease in proliferation appears to be modulated by beta1 integrin possible affecting beta 5 activity and its translocation to cell surface. We hypothesize that OPN and BSP polymers aid the maintenance of non-proliferatioe state of osteocytes in bone.

Mammalian prosomatostatin (PSST) yields two bioactive peptides SST-14 and SST-28 through C-terminal cleavage at basic residues by protein convertases (PCs). Additionally, a second cleavage, mediated by an unknown enzyme at a putative Lys13 residue at the NH2-termius generates PSST[1--10] (antrin). The function of the PSST prosegment is unknown, but its high degree of sequence homology throughout vertebrate evolution argues that it may harbor information for directing PSST towards the regulated secretory pathway (RSP). Therefore, the PSST NH2-terminal region has been extensively analyzed with the following questions asked: Does the NH2-terminus play a role in targeting of PSST to the RSP? Is PSST cleaved at the Lys13 site resulting in antrin production, and by the action of which enzyme? What is the identity of the putative receptor in the trans-Golgi involved in properly targeting PSST? To test the role of antrin in sorting function, alanine scanning and deletional mutagenesis were constructed and stably expressed in AtT-20 cells. The region Pro5 to Gln12 which constitutes an alpha-helix was identified as being important in precursor targeting with Leu7 and Leu11 being critical. Earlier morphological studies have implicated immature secretory granules as the site of prohormone processing however, blockade of PSST targeting through disruption of the helix did not hinder the ability of PSST to be processed at its C-terminus. Furthermore, I find that PSST NH2-terminal cleavage does not take place at a basic site but, most likely at the -R6-L 7-R8-Q9-F 10-L11↓ recognition motif. Overexpression experiments in COS-1 and HEK-293 cells implicate the recently cloned subtilase-kexin-isozyme 1 (SKI-1) in antrin production. Additionally, as proteins function exclusively by means of interaction with other molecules, using the PSST NH2-terminus as a bait in yeast two-hybrid screening assay I attempted to find specific components in the trans-Golgi for

Transcription factors encoded by the Hox gene family are conserved throughout the animal kingdom. Their differential spatial and temporal expression is crucial for axial patterning in early embryonic development. The affinity and specificity of DNA-binding by the homeodomain (HD)-containing HOX proteins is raised by interaction with cofactors. The three-amino-acid-loop-extension (TALE) class of HD-containing proteins comprises HOX-cofactors which are further categorized into subfamilies including the PBC family, the MEIS family and the PKNOX/PREP family. HOX-cofactors belonging to the TALE class have been shown to form dimeric or trimeric complexes with other TALE family members and with HOX proteins to regulate Hox target genes. It is the composition of these complexes that controls their stability, target gene selectivity and recruitment of coregulators, and finally determines Hox-dependent developmental programs. We used the proto-oncoprotein PBX, which belongs to the PBC family, to identify new PBX-interacting partners in a yeast-two-hybrid approach. In this process, we identified a novel member of the PREP family, which we designated PREP2. PREP2 is ubiquitously expressed during embryonic development and exists in multiple isoforms that localize to different subcellular compartments as revealed by two antibodies that we raised against the N and C-terminus of the protein. The subcellular distribution is regulated by several non-exclusive mechanisms including active nucleocytoplasmic transport, cytoplasmic retention, protein-protein interaction as well as phosphorylation. Most strikingly, the actin as well as the tubulin cytoskeleton seem to be involved in this regulatory process, hence representing a novel means of regulation for a TALE class protein. In an attempt to identify the various PREP2 isoforms, we cloned and characterized a splicing variant -PREP2/E12- that has the potential to act as a dominant negative. Interestingly, PREP