The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import

Oxysterol binding proteins (OSBPs) comprise a large conserved family of proteins in eukaryotes. Their ubiquity notwithstanding, the functional activities of these proteins remain unknown. Kes1p, one of seven members of the yeast OSBP family, negatively regulates Golgi complex secretory functions that are dependent on the action of the major yeast phosphatidylinositol/phosphatidylcholine Sec14p. We now demonstrate that Kes1p is a peripheral membrane protein of the yeast Golgi complex, that localization to the Golgi complex is required for Kes1p function in vivo, and that targeting of Kes1p to the Golgi complex requires binding to a phosphoinositide pool generated via the action of the Pik1p, but not the Stt4p, PtdIns 4-kinase. Localization of Kes1p to yeast Golgi region also requires function of a conserved motif found in all members of the OSBP family. Finally, we present evidence to suggest that Kes1p may regulate adenosine diphosphate-ribosylation factor (ARF) function in yeast, and that it may be through altered regulation of ARF that Kes1p interfaces with Sec14p in controlling Golgi region secretory function.     

Importance of sequence specificity for predicting protein folding pathways: perturbed Gaussian chain model

Recent experimental and theoretical studies suggest that rates and pathways of protein folding are largely decided by topology of the native structures, at least for small proteins. However, some exceptions are known; for example, protein L and protein G have the same topology, but exhibit different characteristics of the TSE. Thus, folding pathways of some proteins are critically affected by detailed information on amino acid sequences. To investigate the sequence specificity, we calculate folding pathways of 20 small proteins using the perturbed Gaussian chain model developed by Portman et al. (Phys Rev Lett 1998;81:5237-5240; J Chem Phys 2001;114:5069-5081). Characteristics of the TSE predicted by the model are in good agreement with experimental phi-value data for many proteins at coarse-grained level. Especially, estimation of folding TSE for protein G and protein L based on both topology and additional sequence information are consistent with experimental phi-value data. With only topology information, however, the model predicts the TSE of protein G incorrectly. Moreover, the model that uses topology and sequence information describes free energy profiles of two-state and three-state folders consistently with experiment, whereas the topology only model predicts free energy profiles of some proteins incorrectly. This indicates that sequence specificity also has critical roles in determining the folding pathways for some proteins.     

Subunit E of mitochondrial ATP synthase: a bioinformatic analysis reveals a phosphopeptide binding motif supporting a multifunctional regulatory role and identifies a related human brain protein with the same motif

The mitochondrial adenosine triphosphate (ATP) synthase is located in the inner membrane and consists of at least 16 subunit types in animals, one of which is subunit e, the function of which is not clearly defined. A highly homologous protein is located in the nucleus and named progesterone receptor binding protein (RBF), to designate its role in this organelle. In addition, the expression level of subunit e in mammalian cells fluctuates greatly and is induced by certain carcinogens and elevated in liver cancers. Because these previous observations suggested to us that subunit e may play multifunctional regulatory roles, we employed a bioinformatic approach to test this view. First, from sequence alignment studies, secondary structure analyses, and basic local alignment search tool (BLAST) searches, we concluded that mitochondrial subunit e and the homologous nuclear protein RBF are most likely the same protein. Second, we examined the known sequence and structure of one of the most common multifunctional cell regulatory proteins, the 14-3-3 protein, involved in phosphopeptide binding, and deduced that it has an apparent binding motif (-KX(6)R---RY-). Third, from careful examination of the conserved residues within all subunit e sequences in the database, we discovered that this protein has a comparable binding motif (-RY---KX(6)R-). Finally, in a BLAST search for additional homologs of subunit e, we found a human brain protein, KIAA1578, the C-terminal 30 amino acids of which are identical to those of human subunit e. This protein also contains a potential phosphopeptide binding motif. In summary, these studies provide support for the view that subunit e is a multifunctional cell regulator involved in cell signaling, and implicate the involvement of the KIAA1578 protein in cell signaling as well. These studies suggest also that, while functioning as a subunit of mitochondrial ATP synthases, subunit e may help regulate these complexes by binding to phosphopeptides within one or more of the other subunit types.     

Immunocytochemistry of myoepithelial cells in the salivary glands

MECs are distributed on the basal aspect of the intercalated duct and acinus of human and rat salivary glands. However, they do not occur in the acinus of rat parotid glands, and sometimes occur in the striated duct of human salivary glands. MECs, as the name implies, have structural features of both epithelial and smooth muscle cells. They contract by autonomic nervous stimulation, and are thought to assist the secretion by compressing and/or reinforcing the underlying parenchyma. MECs can be best observed by immunocytochemistry. There are three types of immunocytochemical markers of MECs in salivary glands. The first type includes smooth muscle protein markers such as -SMA, SMMHC, h-caldesmon and basic calponin, and these are expressed by MECs and the mesenchymal vasculature. The second type is expressed by MECs and the duct cells and includes keratins 14, 5 and 17, 1β1 integrin, and metallothionein. Vimentin is the third type and, in addition to MECs, is expressed by the mesenchymal cells and some duct cells. The same three types of markers are used for studying the developing gland.

Development of MECs starts after the establishment of an extensively branched system of cellular cords each of which terminates as a spherical cell mass, a terminal bud. The pluripotent stem cell generates the acinar progenitor in the terminal bud and the ductal progenitor in the cellular cord. The acinar progenitor differentiates into MECs, acinar cells and intercalated duct cells, whereas the ductal progenitor differentiates into the striated and excretory duct cells. Both in the terminal bud and in the cellular cord, the immediate precursors of all types of the epithelial cells appear to express vimentin. The first identifiable MECs are seen at the periphery of the terminal bud or the immature acinus (the direct progeny of the terminal bud) as somewhat flattened cells with a single cilium projecting toward them. They express vimentin and later -SMA and basic calponin. At the next developmental stage, MECs acquire cytoplasmic microfilaments and plasmalemmal caveolae but not as much as in the mature cell. They express SMMHC and, inconsistently, K14. This protein is consistently expressed in the mature cell. K14 is expressed by duct cells, and vimentin is expressed by both mesenchymal and epithelial cells.

After development, the acinar progenitor and the ductal progenitor appear to reside in the acinus/intercalated duct and the larger ducts, respectively, and to contribute to the tissue homeostasis. Under unusual conditions such as massive parenchymal destruction, the acinar progenitor contributes to the maintenance of the larger ducts that result in the occurrence of striated ducts with MECs. The acinar progenitor is the origin of salivary gland tumors containing MECs. MECs in salivary gland tumors are best identified by immunocytochemistry for -SMA. There are significant numbers of cells related to luminal tumor cells in the non-luminal tumor cells that have been believed to be neoplastic MECs.     

Significance of rooting depth in mire plants: Evidence from natural <Superscript>15</Superscript>N abundance

Variation in stable nitrogen isotope ratios (¹⁵N) was assessed for plants comprising two wetland communities, a bog-fen system and a flood plain, in central Japan. ¹⁵N of 12 species from the bog-fen system and six species from the flood plain were remarkably variable, ranging from –5.9 to +1.1 and from +3.1 to +8.7, respectively. Phragmites australis exhibited the highest ¹⁵N value at both sites. Rooting depth also differed greatly with plant species, ranging from 5cm to over 200cm in the bog-fen system. There was a tendency for plants having deeper root systems to exhibit higher ¹⁵N values; plant ¹⁵N was positively associated with rooting depth. Moreover, an increasing gradient of peat ¹⁵N was found along with depth. This evidence, together with the fact that inorganic nitrogen was depleted under a deep-rooted Phragmites australis stand, strongly suggests that deep-rooted plants actually absorb nitrogen from the deep peat layer. Thus, we successfully demonstrated the diverse traits of nitrogen nutrition among mire plants using stable isotope analysis. The ecological significance of deep rooting in mire plants is that it enables those plants to monopolize nutrients in deep substratum layers. This advantage should compensate for any consequential structural and/or physiological costs. Good evidence of the benefits of deep rooting is provided by the fact that Phragmites australis dominates as a tall mire grass.     

一例t(2；14)致自然流产报告

1病例报告　　患者女性,农民,4次反复自然流产,流产时间特殊。第一胎与第二胎均在60天左右不明原因的阴道流血,流血7到8天时自然流产,未去医院进行任何治疗。在怀孕第三胎时曾服中药治疗,怀孕10个月零20天时自然分娩一死胎女婴。第四胎怀孕时曾保胎治疗,11个月时自然分娩一死胎女婴。患者第三胎与第四胎均为过期妊娠死胎,但也未去任何医院检查。患者平时体弱易感冒,未曾接触任何有害物质,非近亲结婚,夫妇表型均正常,智力正常,患者身高1.60米,体重55公斤。　　对夫妇双方取外周血作细胞遗传学检查,常规培养,染色体制片,G显…     

Subcellular and developmental expression of alternatively spliced forms of fibroblast growth factor 14

Fibroblast growth factors (FGFs) 11–14 comprise a subfamily of FGFs with poorly defined biological function. Here we characterize two isoforms of FGF14 (FGF14-1a and FGF14-1b) that result from the alternative usage of two different first exons. We demonstrate that these isoforms have differential subcellular localization and that they are differentially expressed in various adult tissues. Using in situ hybridization we show that Fgf14 is widely expressed in brain, spinal cord, major arteries and thymus between 12.5 and 14.5 days of mouse embryonic development. We also show that during cerebellar development, Fgf14 is first observed at postnatal day 1 in post mitotic granule cells, and later in development, in migrating and post migratory granule cells. The developmental expression pattern of Fgf14 in the cerebellum is complementary to that of Math1, a marker for proliferating granule cells in the external germinal layer.     

Ornithine-containing lipids stimulate CD14-dependent TNF-alpha production from murine macrophage-like J774.1 and RAW 264.7 cells

The ornithine-containing lipids (OL)-induced cytokine production pattern in macrophage-like J774.1 and RAW 264.7 cells was different from that in the peritoneal macrophages previously reported. OLs, as well as lipopolysaccharide (LPS) of Escherichia coli, strongly induced tumor necrosis factor (TNF) alpha but not interleukin (IL)-1beta in J774.1 cells. In the RAW cells, IL-1beta, TNF-alpha and prostaglandin E(2) were strongly induced by the OLs and LPS. OL- and serine-glycine-containing lipid (SGL)-induced TNF-alpha production in J774.1 and RAW 264.7 cells required serum. However, in CD14-deficient LR-9 cells, TNF-alpha was not induced by the OLs in the presence or absence of serum. OLs and a SGL almost completely inhibited the binding of (125)I-LPS to J774.1 cells. These results suggested that OLs and SGL activate macrophages via the CD14-dependent pathway.     

Calprotectin (MRP8/14 protein complex) release during mycobacterial infection in vitro and in vivo

The calprotectin (MRP8/14) protein complex belongs to the S100 family of Ca2+ binding proteins and is expressed during myelomonocytic differentiation. MRP8/14 plasma levels were determined by ELISA in 35 patients with active pulmonary tuberculosis (TB) showing mild (n = 12), moderate (n = 11) or severe (n = 12) disease, 13 patients with active pulmonary sarcoidosis (SR) and 21 healthy controls. TB patients had significantly increased plasma levels of MRP8/14 in comparison with SR and controls, which significantly depended on the volume of lung tissue involved in the inflammatory process. In TB patients, there was no correlation between plasma levels of MRP8/14 and total white blood cell (WBC) count, and blood polymorphonuclear neutrophil (PMN) count. In SR patients, MRP8/14 plasma levels were twofold higher in comparison with controls, but were lower compared with mild TB, and correlated with PMN and WBC counts. Human monocytes infected and cultured for 7 days with Mycobacterium bovis bacillus Calmette-Guérin showed fivefold higher MRP8/14 levels in supernatants compared with unstimulated or purified protein derivative-stimulated cells. Human MRP8/14 significantly increased Mycobacterium tuberculosis H37Rv growth in liquid medium in a dose- and time-dependent manner. These findings suggest that MRP8/14 plays an important role in the immunopathogenesis of tuberculosis.