Fusion of Madin-Darby canine kidney cells by HVJ (Sendai virus): absence of direct association of virus particles with the site of membrane fusion

The process of cell fusion of Madin-Darby canine kidney (MDCK) cells by HVJ (Sendai virus) was investigated to determine whether the HVJ particles were directly associated with the site of membrane fusion. Confluent monolayer cultures of MDCK cells are sealed together by tight junctions on the apices of their lateral membranes, so added virus particles can be adsorbed only to the apical surfaces of the cells. After incubation with HVJ at 37 degrees C for 30 min, the cells still appeared mononucleate and unfused by light microscopy, but electron microscopic examination showed that fusion at the lateral membranes had occurred below the tight junctions. Furthermore, when fluorescein isothiocyanate (FITC)-labeled macromolecules, which cannot pass across the gap junctions, were injected into the cells at this stage, labeled macromolecules were found to diffuse into the adjacent cells. These findings strongly suggest that cell fusion was initiated in the lateral membrane, a region distinct from the site of adsorbed HVJ particles. Thus, the virus particles were not directly associated with the fusion site, but induced fusion of the lateral membranes indirectly.     

Alteration in the arrangement of the keratin-type intermediate filaments during mitosis in cultured human keratinocytes

The behavior of the keratin-type intermediate filaments (KIFs) during mitosis was characterized in cultured human keratinocytes by immunofluorescence microscopy using polyclonal antibodies to keratin. The structural relationship of KIFs with microtubules (MTs) was also studied at the same time using a monoclonal antibody to alpha-tubulin. The KIFs and MTs showed similar but different cytoskeletal networks and underwent structural rearrangements independently during the cell cycle. KIFs in keratinocytes formed two different arrangements during meta- and anaphase: a global aggregation of filaments around the spindle and a fibrous array radiating from the central, global aggregation of filaments to the cell periphery where they were connected with those of the adjacent cells at desmosomal sites. These radiating fibrous portions of KIFs appeared to play a role in retaining the cell in its correct relationship to the surrounding cells during mitosis. This behavior of KIFs in normal keratinocytes was different from the KIF-alterations which had been previously described in SV40-transformed keratinocytes and other cells which expressed two different IFs (keratin and vimentin).     

The human loricrin gene.

Loricrin is the major protein component of the cornified cell envelope of terminally differentiated mammalian epidermal (stratum corneum) cells. Using a specific human cDNA clone, we have isolated and characterized the human loricrin gene. We show that it has a very simple structure of a single intron of 1188 base pairs (bp) in the 5'-untranslated region; there are no introns in coding sequences. By use of rodent-human somatic cell hybrids, followed by in situ hybridization with a biotin-labeled genomic DNA clone, the single-copy gene maps to chromosome location 1q21. Polymerase chain reaction analyses of genomic DNAs from different individuals show that human loricrin consists of two allelic size variants, due to sequence variations in its second glycine loop domain, and these variants segregate in the human population by normal Mendelian mechanisms. Furthermore, there are multiple sequence variants within these two size class alleles due to various deletions of 12 bp (4 amino acids) in the major loop of this glycine loop domain. By use of a specific loricrin antibody, we show by immunogold electron microscopy that loricrin initially appears in the granular layer of human epidermis and forms composite keratohyalin granules with profilaggrin, but localizes to the cell periphery (cell envelope) of fully differentiated stratum corneum cells.     

Permanent night workers´ sleep and psychosocial factors in hospital work. A comparison to day and shift work

ABSTRACT

We aimed to study whether permanent night workers sleep and psychosocial factors differ from day workers and shift workers. The participants (n = 9 312, 92% females, average age 45 years, most commonly nurses and departmental secretaries) were day workers (DW, n = 2 672), shift workers (SW, n = 6 486) and permanent night workers (PNW, n = 154). The Finnish Public Sector survey responses from six hospital districts from 2012 were combined to payroll data from 91 days preceding the survey. The data were analyzed using Pearson χ²-test, one-way ANOVA and multinomial logistic regression analysis. The PNWs reported slightly longer average sleep length than the SWs or the DWs (7:27 vs. 7:13 and 7:10 h, p < 0.001). The PNWs reported least often difficulties in maintaining sleep (p < 0.001) compared to the SWs and the DWs. The PNWs reported most often difficulties to fall asleep and fatigue during free-time (p-values <0.001). The DWs and PNWs experienced less often work-life conflict than the SWs (25 and 26 vs. 38%, p < 0.001). The PNWs were more often satisfied with autonomy at work and appreciation and fair treatment by colleagues than the DWs or the SWs (p < 0.001). The SWs and PNWs reported remarkably higher occurrence of verbal (p < 0.001, OR 3.71, 95% CI 3.23–4.27 and OR 7.67, 95% CI 5.35–10.99, respectively) and physical workplace violence (p < 0.001, OR 9.24, 95% CI 7.17–11.90 and OR 28.34, 95% CI 16.64–43.06, respectively) compared to DWs. Conclusively, PNWs reported contradictory differences in sleep quality compared to DWs and SWs. PNWs are more often satisfied with their colleagues and autonomy at work than DWs or SWs but face workplace violence remarkably more often.     

Discovery of evocalcet,a next-generation calcium-sensing receptor agonist for the treatment of hyperparathyroidism

The calcium-sensing receptor (CaSR) plays an important role in sensing extracellular calcium ions and regulating parathyroid hormone secretion by parathyroid gland cells, and the receptor is a suitable target for the treatment of hyperparathyroidism. Cinacalcet hydrochloride is a representative CaSR agonist which widely used for the hyperparathyroidism. However, it has several issues to clinical use, such as nausea/vomiting and strong inhibition of CYP2D6. We tried to improve these issues of cinacalcet for a new pharmaceutical agent as a preferable CaSR agonist. Optimization from cinacalcet resulted in the identification of pyrrolidine compounds and successfully led to the discovery of evocalcet as an oral allosteric CaSR agonist. Evocalcet, which exhibited highly favorable profiles such as CaSR agonistic activity and good DMPK profiles, will provide a novel therapeutic option for secondary hyperparathyroidism.     

Co-precipitation molecules hemopexin and transferrin may be key molecules for fibrillogenesis in TTR V30M amyloidogenesis

The disease model of familial amyloidotic polyneuropathy—7.2-hMet30 mice—manifests amyloid deposition that consists of a human amyloidogenic mutant transthyretin (TTR) (TTR V30M). Our previous study found amyloid deposits in 14 of 27 7.2-hMet30 mice at 21–24 months of age. In addition, non-fibrillar TTR deposits were found in amyloid-negative 7.2hMet30 mice. These results suggested that TTR amyloidogenesis required not only mutant TTR but also an additional factor (or factors) as an etiologic molecule. To determine the differences in serum proteome in amyloid-positive and amyloid-negative mice in the 7.2-hMet30 model, we used proteomic analyses and studied serum samples obtained from these mice. Hemopexin (HPX) and transferrin (Tf) were detected in the serum samples from amyloid-positive mice and were also found in amyloid deposits via immunohistochemistry, but serum samples from amyloid-negative mice did not contain HPX and Tf. These two proteins were also not detected in non-fibrillar TTR deposits. In addition, in silico analyses suggested that HPX and Tf facilitate destabilization of TTR secondary structures and misfolding of TTR. These results suggest that HPX and Tf may be associated with TTR amyloidogenesis after fibrillogenesis in vivo.     

Partial purification and characterization of a protein kinase that is activated by nuclear localization signal peptides

A nuclear localization signal (NLS) is required for the transport of karyophilic proteins from the cytoplasm to the nucleus. In this study, NLS was examined in terms of its effect on diverse cellular functions such as protein phosphorylation reactions. When synthetic peptides containing the NLS of SV40 T-antigen were injected into the cytoplasm of Xenopus oocytes, and the oocytes incubated with [³²P]phosphorus-containing medium, a 32 kDa protein was found to be preferentially phosphorylated in an NLS-dependent manner. The incubation of fractionated cytosolic extracts prepared from mouse Ehrlich ascites tumor cells with [γ-³²P]ATP in the presence of the NLS peptides, results in the stimulation of the phosphorylation of several proteins. Similar in vitro stimulation was observed by other functional NLS peptides such as those of polyoma virus T-antigen and nucleoplasmin. Little or no stimulation, however, was detected for peptides of mutant type and reverse type NLS of SV40 T-antigen, and the C-terminal portion of lamin B. Using an in vitro assay, the phosphorylation activity was fractionated chromatographically and a fraction was obtained which contained a high level of activity. The fraction was found to contain three major proteins having molecular masses of 95, 70, and 43 kDa. The in vivo and in vitro results are consistent with the existence of a protein kinase, called NLS kinase, that is specifically activated by NLS peptides.     

Role of heat shock cognate 70 protein in import of ornithine transcarbamylase precursor into mammalian mitochondria.

The roles of the 70-kDa cytosolic heat shock protein (hsp70) in import of precursor proteins into the mitochondria were postulated to be related to (i) unfolding of precursor proteins in the cytosol, (ii) maintenance of the import-competent state, and (iii) unfolding and transport of precursor proteins through contact sites, in cooperation with matrix hsp70. We examined roles of cytosolic hsp70 family members in import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria, using an in vitro import system and antibodies against hsp70. Immunoblot analysis using an hsc70 (70-kDa heat shock cognate protein)-specific monoclonal antibody and a polyclonal antibody that reacts with both hsc70 and hsp70 showed that hsc70 is the only or major form of hsp70 family members in the rabbit reticulocyte lysate. The hsc70 antibody did not inhibit pOTC import when added prior to import assay. However, when pOTC was synthesized in the presence of the antibody and then subjected to import assay, pOTC import was markedly decreased. pOTC import was also decreased when the precursor was synthesized in the lysate depleted for hsc70 by treatment with hsc70 antibody-conjugated Sepharose. This reduction was almost completely restored by readdition of purified mouse hsc70 during pOTC synthesis. The readdition of hsc70 after pOTC synthesis and only during the import assay was not effective. Thus, once import competence of pOTC was lost, hsc70 was ineffective for restoration. Newly synthesized pOTC lost import competence in the absence of hsc70 somewhat more rapidly than in its presence. These results indicate that hsc70 is required during pOTC synthesis and not during import into the mitochondria. hsc70 presumably binds to pOTC polypeptide and maintains it in an import-competent form.     

Hair cell regeneration in the chick inner ear following acoustic trauma: ultrastructural and immunohistochemical studies

The regeneration of hair cells in the chick inner ear following acoustic trauma was examined using transmission electron microscopy. In addition, the localization of proliferation cell nuclear antigen (PCNA) and basic fibroblast growth factor (b-FGF) was demonstrated immunohistochemically. The auditory sensory epithelium of the normal chick consists of short and tall hair cells and supporting cells. Immediately after noise exposure to a 1500-Hz pure tone at a sound pressure level of 120 decibels for 48 h, all the short hair cells disappeared in the middle region of the auditory epithelium. Twelve hours to 1 day after exposure, mitotic cells, binucleate cells and PCNA-positive supporting cells were observed, and b-FGF immunoreactivity was shown in the supporting cells and glial cells near the habenula perforata. Spindle-shaped hair cells with immature stereocilia and a kinocilium appeared 3 days after exposure; these cells had synaptic connections with the newly developed nerve endings. The spindle-shaped hair cell is considered to be a transitional cell in the lineage of the supporting cell to the mature short hair cell. These results indicate that, after acoustic trauma, the supporting cells divide and differentiate into new short hair cells via spindle-shaped hair cells. Furthermore, it is suggested that b-FGF is related to the proliferation of the supporting cells and the extension of the nerve fibers.     

Inhibition of [3H]MK-801 Binding by Ferrous (II) but Not Ferric (III) Ions in a Manner Different from That by Sodium Nitroprusside (II) in Rat Brain Synaptic Membranes

Abstract: The addition of sodium nitroprusside (SNP) significantly inhibited binding of (+)-5-[³H]methyl-10,11-dihydro-5 H -dibenzo[ a,d ]cyclohepten-5,10-imine ([³H]MK-801) to an ion channel associated with the N -methyl- d -aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of >1 µ M in rat brain synaptic membranes not extensively washed. However, neither S -nitroso- N -acetylpenicillamine nor S -nitroso- l -glutathione inhibited binding even at 100 µ M . Of the two compounds structurally related to SNP (II), similarly potent inhibition was induced by potassium ferrocyanide (II) but not by potassium ferricyanide (III). In addition, ferrous chloride (II) induced much more potent inhibition of binding than ferric chloride (III), at a similar concentration range. In contrast, iron chelators prevented the inhibition by ferrous chloride (II) without markedly affecting that by SNP (II) and potassium ferrocyanide (II). Pretreatment with ferrous chloride (II) also led to potent inhibition of [³H]MK-801 binding in a manner insensitive to subsequent addition of the iron chelators. Pretreatment with Triton X-100 resulted in significant potentiation of the ability of ferrous chloride (II) to inhibit [³H]MK-801 binding irrespective of the addition of agonists, moreover, although binding of other radioligands to the non-NMDA receptors was unaltered after pretreatment first with Triton X-100 and then with ferrous chloride (II). These results suggest that ferrous ions (II) may interfere selectively with opening processes of the NMDA channel through mechanisms entirely different from those underlying the inhibition by both SNP (II) and potassium ferrocyanide (II) in rat brain.