Pathogenesis of primary localized cutaneous amyloidosis (PLCA) is unclear, but pathogenic relationship to keratinocyte apoptosis has been implicated. We have previously identified galectin-7, actin, and cytokeratins as the major constituents of PLCA. Determination of the amyloidogenetic potential of these proteins by thioflavin T (ThT) method demonstrated that galectin-7 molecule incubated at pH 2.0 was capable of binding to the dye, but failed to form amyloid fibrils. When a series of galectin-7 fragments containing ?-strand peptides were prepared to compare their amyloidogenesis, Ser(31)-Gln(67) and Arg(120)-Phe(136) were aggregated to form amyloid fibrils at pH 2.0. The rates of aggregation of Ser(31)-Gln(67) and Arg(120)-Phe(136) were dose-dependent with maximal ThT levels after 3 and 48 h, respectively. Their synthetic analogs, Phe(33)-Lys(65) and Leu(121)-Arg(134), which are both putative tryptic peptides, showed comparable amyloidogenesis. The addition of sonicated fibrous form of Ser(31)-Gln(67) or Phe(33)-Lys(65) to monomeric Ser(31)-Gln(67) or Phe(33)-Lys(65) solution, respectively, resulted in an increased rate of aggregation and extension of amyloid fibrils. Amyloidogenic potentials of Ser(31)-Gln(67) and Phe(33)-Lys(65) were inhibited by actin and cytokeratin fragments, whereas those of Arg(120)-Phe(136) and Leu(121)-Arg(134) were enhanced in the presence of Gly(84)-Arg(113), a putative tryptic peptide of galectin-7. Degraded fragments of the galectin-7 molecule produced by limited trypsin digestion, formed amyloid fibrils after incubation at pH 2.0. These results suggest that the tryptic peptides of galectin-7 released at neutral pH, may lead to amyloid fibril formation of PLCA in the intracellular acidified conditions during keratinocyte apoptosis via regulation by the galectin-7 peptide as well as actin and cytokeratins.
Lichen sclerosus et atrophicus (LSA) is histopathologically characterized by upper dermal hyalinization with vacuolar alteration, whereas no particular microscopic change in the mid to lower dermis has been described. The purpose of this study was to investigate any histopathologic changes involving elastic fibers in the mid to lower dermis in patients with LSA.
We compared the cytoarchitectural features used for the cytologic diagnosis of endometrial adenocarcinoma (EC) using ThinPrep® (TPS = ThinPrep Sample) and BD SurePath™ (SPS = SurePath Sample) preparations. In 20 patients, a direct endometrial sample using the Uterobrush was obtained. Nineteen cases of EA and one case of carcinosarcoma were studied. TPS and SPS were performed according to the manufacturers recommendations. Moreover, after the TPS preparation, the residual material was also used to prepare an SPS sample (TP-SPS = ThinPrep-Surepath sample). The following points were investigated in both preparations: (1) number of cell clumps; SPS had a significantly higher (20.9) than TPS (1.7) and TP-SPS (10.3); (2) long axis of clumps; SPS had a significantly higher (215.4) than TPS (146.0); (3) rate of cell clumps with longer axes than 200 ?m; SPS had a significantly higher (36.7) than TPS (15.2) and TP-SPS (24.2). TP-SPS showed higher values than TPS; (4) nuclear area; TPS had a significant higher (61.2) than SPS (40.8) and TP-SPS (38.6); (5) degree of overlapping nuclei; SPS (3.4) had a significantly higher number of overlapping nuclei than TPS (0.7) and TP-SPS (2.1); (6) nuclear chromatin pattern; no significant differences for the nuclear chromatin pattern were found in the three different methods. The poor performance of TPS versus SPS and TP-SPS was explained with the heavy blood contamination of the samples, and the absence of adhesive coating in the slides is used for TPS. Further investigation of technical differences in liquid-based cytology methodologies is needed.
Accumulated evidence shows that some phytochemicals provide beneficial effects for human health. Recently, a number of mechanistic studies have revealed that direct interactions between phytochemicals and functional proteins play significant roles in exhibiting their bioactivities. However, their binding selectivities to biological molecules are considered to be lower due to their small and simple structures. In this study, we found that zerumbone, a bioactive sesquiterpene, binds to numerous proteins with little selectivity. Similar to heat-denatured proteins, zerumbone-modified proteins were recognized by heat shock protein 90, a constitutive molecular chaperone, leading to heat shock factor 1-dependent heat shock protein induction in hepa1c1c7 mouse hepatoma cells. Furthermore, oral administration of this phytochemical up-regulated heat shock protein expressions in the livers of Sprague-Dawley rats. Interestingly, pretreatment with zerumbone conferred a thermoresistant phenotype to hepa1c1c7 cells as well as to the nematode Caenorhabditis elegans. It is also important to note that several phytochemicals with higher hydrophobicity or electrophilicity, including phenethyl isothiocyanate and curcumin, markedly induced heat shock proteins, whereas most of the tested nutrients did not. These results suggest that non-specific protein modifications by xenobiotic phytochemicals cause mild proteostress, thereby inducing heat shock response and leading to potentiation of protein quality control systems. We considered these bioactivities to be xenohormesis, an adaptation mechanism against xenobiotic chemical stresses. Heat shock response by phytochemicals may be a fundamental mechanism underlying their various bioactivities.
The precursor protein of localized cutaneous amyloidosis (LCA) is believed to be cytokeratins on the basis of previous immunohistochemical studies. To identify the candidate amyloid protein biochemically, amyloid proteins were extracted with distilled water from lesional skin of LCA associated with Bowens disease. The proteins were resolved on one- or two-dimensional polyacrylamide gel electrophoresis followed by characterization with immunoblot analysis. The proteins with multiple molecular weights of 50-67 kDa and two proteins with 25 and 35 kDa were identified as keratins, serum amyloid P component and apolipoprotein E, respectively. The unknown 14-kDa (pI = 7.0) and 42-kDa (pI = 5.4) proteins reacted with the antibody against galectin-7 and actin, respectively. The protein with the molecular weight of 14 kDa was identified as galectin-7 by MALDI-TOF mass spectrometer. Their electrophoretic mobilities were identical with normal counterparts extracted from cultured normal human keratinocytes. Galectin-7 and actin were detected by immunoblot assay in the water-soluble fractions prepared from the lesional skins of two patients with primary LCA. Immunohistochemical studies of tumor-associated (n = 9) and primary (n = 10) LCA revealed various degrees of positive immunoreactivities with the antibodies for galectin-7 and F-actin. Galectin-7 and actin, which contain considerable amount of ?-sheet structure, may be candidate amyloidogenic proteins of primary and secondary LCA.
The photo-aged facial skin is characterized by various unique features such as dark spots, wrinkles, and sagging. Elderly people, particularly Asians, tend to show a yellowish skin color change with photo-aging. However, there has been no analytical study conducted on this unique skin color change of the aged facial skin.
Orally ingested collagen undergoes degradation to small di- or tripeptides, which are detected in circulating blood 2 h after ingestion. The influence of collagen-derived peptides on dermal extracellular matrix components and cell proliferation was studied using cultured human dermal fibroblasts. Of the various collagenous peptides tested here, the dipeptide proline-hydroxyproline (Pro-Hyp) enhanced cell proliferation (1.5-fold) and hyaluronic acid synthesis (3.8-fold) at a dose of 200 nmol/mL. This was concomitant with a 2.3-fold elevation of hyaluronan synthase 2 (HAS2) mRNA levels. Small interfering RNA (siRNA)-mediated knockdown of the HAS2 gene in human dermal fibroblasts inhibited Pro-Hyp-induced HAS2 mRNA transcription and cell mitotic activity. Addition of genistein or H7, a protein kinase inhibitor, abolished the Pro-Hyp-induced HAS2 mRNA stimulation. Pro-Hyp elevated phosphorylation of signal transducer and activator of transcription 3 (STAT3) within a short time period (60 min). These results suggest that Pro-Hyp stimulates both cell mitotic activity and hyaluronic acid synthesis, which is mediated by activation of HAS2 transcription.
To clarify the molecular mechanism underlying the transepidermal extrusion of dermal collagen in acquired perforating dermatosis (APD) associated with diabetes mellitus and renal failure, we studied the interaction between advanced glycation end product (AGE)-modified extracellular matrix proteins and keratinocytes (KCs) in a cell culture system. The expression of involucrin (INV) and keratin 10 was significantly enhanced in normal human KCs grown on AGE-modified collagen I or III compared with cells grown on unmodified collagen I or III. Glycated collagens I and III preferentially induced the expression of AGE receptor CD36, but not of other AGE receptors. KCs induced to terminal differentiation demonstrated markedly elevated CD36 expression. Glycated collagen I- and III-induced INV expression was partially blocked by the anti-CD36 antibody (Ab). These substrates also induced epidermal matrix metalloproteinase 9 (MMP-9) expression. Lesional skin from APD patients reacted moderately or strongly with the anti-CD36 Ab as well as the anti-MMP-9 Ab in the epidermal cells surrounding the collagenous materials being eliminated. These results suggest that exposing KCs to AGE-modified interstitial collagen (types I and III) by scratching induces terminal differentiation of KCs via the AGE receptor (CD36), leading to the upward movement of KCs together with glycated collagen.
Viral reverse genetics, particularly infectious cloning, is a valuable tool with applications to many areas of viral research including the generation of vaccine candidates. However, this technology is sometimes insufficient for the construction cDNA clones as the genome sequences and/or encoding proteins of some viral agents may be toxic to the host cells used for cloning. To circumvent this problem, we developed a polymerase chain reaction (PCR)-based protocol for generating a complete West Nile virus (WNV) cDNA. The fragmented cDNAs were synthesized from WNV RNA by reverse transcription-PCR, and subsequently cloned into plasmids for use as templates for WNV cDNA synthesis. The fragmented cDNAs were amplified and assembled by PCR to generate a full-length WNV cDNA. Using this cDNA as a template, WNV RNA was synthesized in vitro and transfected into mammalian cells. We also examined the generation of a mutant recombinant WNV containing a site-directed mutation within the viral genome sequence. Here, we discuss the possibility of developing a method for the generation of recombinant WNVs.
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