Possible ecological implications of floating microbial assemblages lifted from the lakebed on an Antarctic lake

Microbial assemblages can be found drifting/floating in lake water and being washed ashore in continental Antarctica. Two field studies in early and late January 2008 measured the light utilization properties and photosynthetic responses of these assemblages, which were then compared with those of pelagic and benthic microbial communities to evaluate the ecological implications of this phenomenon. The nutrient concentrations were low in the lake water, indicating oligotrophic conditions. Based on microscopic and pigment analysis, both the floating and benthic communities were mainly composed of Oedogonium sp. (Chlorophyceae), followed by cyanobacteria, diatoms, and dinoflagellates. Floating assemblages had a firmer and denser structure, and possessed more rich carotenoids than the benthic community. Measurements of photosynthesis conducted in early January indicated that the activities of the floating assemblages were considerably low. In late January almost all floating assemblages on the lakeshore turned white because of freezing and drying by the ambient temperature decrease, and had no photosynthetic signals. These results suggest that the floating assemblages could spontaneously lift off from the lakebed because of the bubbles created by photosynthesis and then repeatedly roll, flip, sink, or float depending on buoyancy. In addition, this phenomenon seemed to greatly change the cycling of matter by transporting the lake’s photosynthetic products to the surrounding ecosystems, then give the benthic subsurface communities in dark regions a chance to reactivate such as gap regeneration in the case of climax forest, and also allow the floating assemblages to restart photosynthesis at the top of the lakebed by resinking.     

Tissue-type plasminogen activator acts as a cytokine that triggers intracellular signal transduction and induces matrix metalloproteinase-9 gene expression

Tissue-type plasminogen activator (tPA), a serine protease well known for generating plasmin, has been demonstrated to induce matrix metalloproteinase-9 (MMP-9) gene expression and protein secretion in renal interstitial fibroblasts. However, exactly how tPA transduces its signal into the nucleus to control gene expression is unknown. This study investigated the mechanism by which tPA induces MMP-9 gene expression. Both wild-type and non-enzymatic mutant tPA were found to induce MMP-9 expression in rat kidney interstitial fibroblasts (NRK-49F), indicating that the actions of tPA are independent of its proteolytic activity. tPA bound to the low density lipoprotein receptor-related protein-1 (LRP-1) in NRK-49F cells, and this binding was competitively abrogated by the LRP-1 antagonist, the receptor-associated protein. In mouse embryonic fibroblasts (PEA-13) lacking LRP-1, tPA failed to induce MMP-9 expression. Furthermore, tPA induced rapid tyrosine phosphorylation on the beta subunit of LRP-1, which was followed by the activation of Mek1 and its downstream Erk-1 and -2. Blockade of Erk-1/2 activation by the Mek1 inhibitor abolished MMP-9 induction by tPA in NRK-49F cells. Conversely, overexpression of constitutively activated Mek1 induced Erk-1/2 phosphorylation and MMP-9 expression. In mouse obstructed kidney, tPA, LRP-1, and MMP-9 were concomitantly induced in the renal interstitium. Collectively, these results suggest that besides its classical proteolytic activity, tPA acts as a cytokine that binds to the cell membrane receptor LRP-1, induces its tyrosine phosphorylation, and triggers intracellular signal transduction, thereby inducing specific gene expression in renal interstitial fibroblasts.     

Cytocidal actions of parasporin-2, an anti-tumor crystal toxin from Bacillus thuringiensis

Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.     

Negative regulation of osteoclastogenesis by ectodomain shedding of receptor activator of NF-kappaB ligand

Receptor activator of NF-kappaB ligand (RANKL) is a transmembrane glycoprotein that has an essential role in the development of osteoclasts. The extracellular portion of RANKL is cleaved proteolytically to produce soluble RANKL, but definite RANKL sheddase(s) and the physiologic function of RANKL shedding have not yet been determined. In the present study, we found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 have strong RANKL shedding activity. In Western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in a reduction of both the lower and higher molecular weight products. Suppression of MMP14 in primary osteoblasts increased membrane-bound RANKL and promoted osteoclastogenesis in cocultures with macrophages. Soluble RANKL produced by osteoblasts from MMP14-deficient mice was markedly reduced, and their osteoclastogenic activity was promoted, consistent with the findings of increased osteoclastogenesis in vivo. RANKL shedding is an important process that down-regulates local osteoclastogenesis.     

Rapid evolution of major histocompatibility complex class I genes in primates generates new disease alleles in humans via hitchhiking diversity

A plausible explanation for many MHC-linked diseases is lacking. Sequencing of the MHC class I region (coding units or full contigs) in several human and nonhuman primate haplotypes allowed an analysis of single nucleotide variations (SNV) across this entire segment. This diversity was not evenly distributed. It was rather concentrated within two gene-rich clusters. These were each centered, but importantly not limited to, the antigen-presenting HLA-A and HLA-B/-C loci. Rapid evolution of MHC-I alleles, as evidenced by an unusually high number of haplotype-specific (hs) and hypervariable (hv) (which could not be traced to a single species or haplotype) SNVs within the classical MHC-I, seems to have not only hitchhiked alleles within nearby genes, but also hitchhiked deleterious mutations in these same unrelated loci. The overrepresentation of a fraction of these hvSNV (hv1SNV) along with hsSNV, as compared to those that appear to have been maintained throughout primate evolution (trans-species diversity; tsSNV; included within hv2SNV) tends to establish that the majority of the MHC polymorphism is de novo (species specific). This is most likely reminiscent of the fact that these hsSNV and hv1SNV have been selected in adaptation to the constantly evolving microbial antigenic repertoire.     

Molecular cloning and functional characterization of a prolactin-releasing peptide homolog from Xenopus laevis

Amino acid sequences for identified prolactin (PRL)-releasing peptides (PrRPs) were conserved in mammals (>90%) or teleost fishes (100%), but there were considerable differences between these classes in the sequence (<65%) as well as in the role of PrRP. In species other than fishes and mammals, we have identified frog PrRP. The cDNA encoding Xenopus laevis prepro-PrRP, which can generate putative PrRPs, was cloned and sequenced. Sequences for the coding region showed higher identity with teleost PrRPs than mammalian homologues, but suggested the occurrence of putative PrRPs of 20 and 31 residues as in mammals. The amino acid sequence of PrRP20 was only one residue different from teleost PrRP20, but shared 70% identity with mammalian PrRP20s. In primary cultures of bullfrog (Rana catesbeiana) pituitary cells, Xenopus PrRPs increased prolactin concentrations in culture medium to 130–160% of the control, but PrRPs was much less potent than thyrotropin-releasing hormone (TRH) causing a three- to four-fold increase in prolactin concentrations. PrRP mRNA levels in the developing Xenopus brain peak in early prometamorphosis, different from prolactin levels. PrRP may not be a major prolactin-releasing factor (PRF), at least in adult frogs, as in mammals.     

A Novel Endoplasmic Reticulum Export Signal: PROLINE AT THE +2-POSITION FROM THE SIGNAL PEPTIDE CLEAVAGE SITE*

NUCB1 (nucleobindin 1) is a Golgi-localized soluble protein with a signal peptide and multiple functional domains. We reported recently that NUCB1 is a negative regulator of the unfolded protein response that activates various endoplasmic reticulum (ER)-originating signaling pathways. In that report, we also showed that Golgi localization of NUCB1 was essential to regulate the unfolded protein response. However, the localization mechanism of NUCB1 is still unknown. Here, we report that the proline residue at the +2-position (Pro⁺²) from the signal peptide cleavage site is the determinant of NUCB1 protein export from the ER and subsequent transport to the Golgi. Fusion of the N-terminal amino acids 1–35 peptide region, including both signal peptide (amino acids 1–26) and Pro⁺², was sufficient for enhanced green fluorescent protein to localize in the Golgi, whereas single amino acid mutation of Pro⁺² resulted in defective export from the ER without affecting the protein maturation process. Furthermore, we demonstrated that Pro⁺² was important for the enhanced green fluorescent protein fusion protein to concentrate at a transport vesicle formation site within the ER, often termed the ER exit site. Interestingly, such a Pro⁺² has also been functionally conserved in other Golgi-localized soluble proteins, Cab45 (Ca²⁺-binding protein of 45 kDa), reticulocalbin 1, and calumenin. Our findings indicate that Pro⁺² can function as a novel ER export signal of some Golgi proteins.NUCB1 (nucleobindin 1), also known as calnuc, was first identified as a soluble secretory 55-kDa protein (461 amino acids) in lupus-prone mice with the lymphoproliferation (lpr) mutation (1). NUCB1 has also been shown to be secreted in culture supernatant of a murine B cell line established from the mice (2). Later studies also demonstrated that NUCB1 is expressed ubiquitously and localizes in the Golgi apparatus of intact cells (3, 4). NUCB1 contains multiple putative functional domains, including an N-terminal endoplasmic reticulum (ER)² signal peptide, a DNA binding site, a leucine zipper domain, two EF-hand Ca²⁺-binding sites, a nuclear localization signal, and G-protein-binding and cyclooxygenase-binding domains (1, 5, 6). Consistently, NUCB1 has been reported to function in various cellular processes, including osteogenesis, inflammation, autoimmunity, intracellular signaling, and cancer (6–10).Newly synthesized, premature NUCB1 protein is first targeted into the ER via its N-terminal ER signal peptide. After removal of the signal peptide in the ER, a mature NUCB1 protein is transported to the Golgi apparatus and then secreted to the extracellular matrix (11). NUCB1 in the Golgi pool is probably involved in establishing the agonist-mobilizable Golgi Ca²⁺ store (3). Furthermore, the Golgi-localized NUCB1 regulates the unfolded protein response, which is a cellular stress response that triggers various events, such as ER-resident molecular chaperone induction, translational repression, and apoptosis under ER stress conditions (12). On the other hand, extracellular NUCB1 has been suggested to serve as a modulator of matrix maturation in bone, based on the observations that NUCB1 is secreted by osteoblasts and osteocytes and can, indeed, be detected in the osteoid extracellular matrix (7, 13). Thus, Golgi transport and subsequent secretion of NUCB1 seem to be important to exert the protein''s activity, but little is known about its transport regulation mechanism.In eukaryotic cells, a tremendous variety of soluble and membrane cargo proteins are packaged into transport vesicles at the ER. Vesicle formation on the ER membrane begins with the assembly of a coat protein complex II (COPII) (14). This COPII coat consists of Sar1, Sec23-Sec24, and Sec13-Sec31 complexes that are sequentially recruited to the ER membrane. Sar1 is a small GTPase that regulates coat assembly and disassembly. To assemble the COPII coat, Sar1-GTP transiently associates with an export cargo protein and then binds to Sec23-Sec24, which in turn attracts Sec13-Sec31 (14). Polymerization of the formed COPII coat, which occurs at the so-called ER exit site (ERES), triggers transport vesicle budding on the ER membrane (14, 15). Then the vesicles fuse with the VTC compartment (vesiculo-tubular clusters, also called ERGIC) that mediates further protein transport to the Golgi apparatus. Cargo proteins are then carried to their final destinations, such as organelle, cell surface membrane, and extracellular matrix (14).Recent studies reveal that some transmembrane cargoes contain specific motifs to be selectively concentrated in the transport vesicle within the ER. This sorting motif is called the ER export signal. Representative ER export signals are the diacidic motif (DXE), dihydrophobic (LL) motif, and diaromatic motif (FF, YY) that have been found in vesicular stomatitis virus glycoprotein, ERGIC-53, and Emp46p, respectively (16–18). These export signals are present in the cytoplasmic region of the cargo proteins and mediate their interaction with the COPII complex at the outer side of the ER membrane, resulting in concentration in the newly formed budding vesicle. On the other hand, the soluble type of cargo proteins require their cargo receptor to be sorted into the COPII vesicle, because they cannot interact directly with COPII complex, since these proteins have no cytoplasmic region. Although recent studies have reported the existence of the cargo receptor and functional ER export signals found in some soluble cargo proteins, little is known about many other cargo receptors and the export signals of soluble cargo proteins (19, 20).Here, we report that Pro²⁸, which is located at the +2-position (Pro⁺²) from the signal peptide cleavage site of the NUCB1 protein, is a determinant of its export from the ER. In fact, single amino acid substitution (P28A) led to predominant ER distribution and reduced the secretion of NUCB1 without affecting its maturation process in the ER. We also demonstrated that Pro⁺² is required for concentration at the ERES. It is important to note that Pro⁺² was also conserved functionally in other proteins. Our results indicate that Pro⁺² can function as a new ER export signal.     

Demonstration of aluminum in amyloid fibers in the cores of senile plaques in the brains of patients with Alzheimer’s disease

Aluminum (Al) exposure has been reported to be a risk factor for Alzheimer’s disease (senile dementia of Alzheimer type), although the role of Al in the etiology of Alzheimer’s disease remains controversial. We examined the presence of Al in the Alzheimer’s brain using energy-dispersive X-ray spectroscopy combined with transmission electron microscopy (TEM-EDX). TEM-EDX analysis allows simultaneous imaging of subcellular structures with high spatial resolution and analysis of small quantities of elements contained in the same subcellular structures. We identified senile plaques by observation using TEM and detected Al in amyloid fibers in the cores of senile plaques located in the hippocampus and the temporal lobe by EDX. Phosphorus and calcium were also present in the amyloid fibers. No Al could be detected in the extracellular space in senile plaques or in the cytoplasm of nerve cells. In this study, we demonstrated colocalization of Al and beta-amyloid (Abeta) peptides in amyloid fibers in the cores of senile plaques. The results support the following possibilities in the brains of patients with Alzheimer’s disease: Al could be involved in the aggregation of Abeta peptides to form toxic fibrils; Al might induce Abeta peptides into the beta-sheet structure; and Al might facilitate iron-mediated oxidative reactions, which cause severe damage to brain tissues.