Cell shape changes during transition of basal keratinocytes to mature enucleate-cornified envelopes: modulation of terminal differentiation by fibronectin.

Normal human keratinocytes (NHK) in submerged culture were used to assess mechanisms associated with in vitro exposure to known stimulators (sodium butyrate; NaB) and inhibitors (fibronectin; FN) of NHK maturation. A multiparameter approach was used to define cell types generated under a range of growth conditions. Differentiation induced in response to NaB occurred through a series of morphologically distinct phenotypes and culminated in the formation of enucleate-cornified envelopes. Two-dimensional electrophoresis provided a limited database to evaluate global changes in cellular protein composition as a function of induced differentiation. Proteins were identified that characterized predominantly basal cell cultures, NaB-treated cells, and fully differentiated NHKs. Growth of NHKs on FN suppressed both spontaneous and NaB-directed maturation and inhibited maximal expression of protein changes associated with the differentiated state. Thus, the composition of the extracellular matrix can modulate (at both the morphologic and protein levels) the response of basal NHKs to a potent differentiation-inducing agent. Abrogation of NHK maturation by FN was not due to adverse effects on cellular metabolism, abortive differentiation, or altered timing of induced differentiation. FN appears to exert its suppressive effect by either maintaining an early stem cell phenotype which is poorly competent for terminal maturation or attenuating an as yet unknown aspect of the NaB-initiated differentiation cascade.     

The effect of the charge of aromatic amino acid, peptide and protein molecules on the rate of radical formation during UV-irradiation

It has been shown that the rate of radical formation under UV-irradiation of some aromatic amino acids, peptides and proteins abruptly increases with pH rise in the region 9.5-11. This effect is observed only in the case when the total charge of the molecule after ionization remains negative.     

Chemical mechanism to account for artifactual formation of shortened peptides with free alpha-amino groups in solid phase peptide synthesis

The formation of terminated peptides with free alpha-amino groups has often been observed in stepwise solid phase peptide synthesis. This has been attributed to variable accessibility in regions of the swollen crosslinked resin supports. It is now shown that impurities in the amino acid reagents are responsible for these by-products. Thus, sec.-butyloxycarbonylamino acids were isolated from tert.-butyloxycarbonylamino acids after treatment with trifluoroacetic acid under standard deprotection conditions for the removal of the tert.-butyloxycarbonyl (Boc) group. Direct reverse phase HPLC analysis of Boc-amino acids from commercial sources also showed the sec.-Boc-amino acids as impurities present at varying levels. The sec.-Boc group was stable to treatment at room temperature with trifluoroacetic acid in dichloromethane (1:1, v/v) (half-life 7 years), but was removed by HF-anisole under the standard conditions of cleavage and deprotection of assembled peptides. In model syntheses, the level of terminated free peptides corresponded to the level of preexisting sec.-Boc-amino acid impurities present in the Boc-amino acid reagents. Use of Boc-amino acids with no detectable sec.-Boc resulted in negligible levels (less than 0.05%) of terminated peptides. The problem is thus readily overcome by the use of pure Boc-amino acid starting materials and is not a reflection of a shortcoming inherent to the polymer supported nature of solid phase syntheses as has been previously suggested.     

Temperature-sensitive mutants of Saccharomyces cerevisiae variable in the methionine content of their protein.

Temperature-sensitive mutants were derived from Saccharomyces cerevisiae Y5alpha by ethyl methane sulfonate mutagenesis, in a search for mutants that would produce methionine-rich protein at the nonpermissive temperature. A total of 132 mutant strains were selected which showed adequate growth on minimal medium at 25 degrees C but little or no growth on the same medium supplemented with a high concentration (2 mg/ml) of l-methionine at 37 degrees C. Several of these mutants were found to increase the proportion of methionine in their protein to much higher levels than that of the wild-type parent after a temperature shift from 25 to 37 degrees C. Two strains, 476 and 438, which were temperature sensitive only in the presence of methionine, produced cellular protein with methionine contents as high as 3.6 and 4.3%, respectively, when incubated in the presence of methionine. The former strain contained 2.5% methionine even when incubated at 37 degrees C in the absence of methionine. Wild strain Y5alpha, on the other hand, had 1.75% methionine under all conditions tested. Most temperature-sensitive mutants isolated had the same methionine content as the wild strain. It is concluded that the proportion of a specific amino acid, such as methionine, in S. cerevisiae protein can be altered by culturing certain temperature-sensitive mutants at an elevated temperature.     

The levels and biologic action of the human neutrophil granule peptide HP-1 in lung tumors.

HP-1 is the most abundant human representative of a recently discovered class of neutrophil cystine- and arginine-rich peptides. These peptides have many potentially regulatory activities expressed at nanomolar concentrations. To establish the levels of HP-1 that can accumulate in human lung tumors and nondiseased lung fragments, tissues were extracted for their peptide content. The extracts were purified on reverse phase HPLC, and HP-1 and related peptides were identified by sequence analysis and their concentrations in the tissue quantitated by amino acid analysis. Immunohistochemistry was performed and strongly suggests that HP-1 is confined to granulocytes under most circumstances, and indicates that the levels of HP-1 measured in the tumors reflect the levels obtained when solid tissue is infiltrated by neutrophils. The maximum observed levels were 26 nanomoles per gram wet weight of tissue. Attempts were then made to correlate this level to the cytotoxic potential of HP-1 by performing in vitro cytotoxicity dose-response curves on several cell lines. Most cells were killed at between 1 and 8 microM, and the response depended on the growth conditions of the cells. The levels of HP-1 that accumulate in tumors can exceed the in vitro cytolytic concentrations. The levels are also considerably in excess of those required to exert in vitro regulatory actions.     

The occurrence of the cell type containing a specific monoamine in the taste bud of the rabbit's foliate papila.

The fluorescence histochemical examination on biogenic amines of the rabbit's foliate papilla revealed that a specific monoamine exhibiting an yellow fluorescence was present in a certain cell type of taste buds. The fluorescence had the emission maximum at 520 mmu and faded rapidly under the influence of the UV-irradiation. The green fluorescence of adrenergic nerve had the emission maximum at 480 mmu and was fairly stable upon the UV-irradiation. The yellow fluorescence disappeared completely following reserpine treatment, while it was markedly enhanced by nialamide treatment. From the observations, it is suggested that certain taste bud cells of the foliate papilla contain a biogenic monoamine, probably 5-hydroxytryptamine (serotonin).     

TRANSFECTION OF NORMAL HUMAN EPIDERMAL KERATINOCYTES WITH LIPID/DNA COMPLEXES IN VITRO

Highly proliferative normal human epidermal keratinocytes (NHK) were isolated from human foreskin biopsies, cultivated in serum-free medium and characterized by flow cytometry. The expression of cytokeratin 19, cytokeratin 14 and vimentin indicated that the suspension contained a high percentage of undifferentiated cells of the basal epidermal layer. The NHK were transfected in vitro with lipid/DNA complexes made of Effectene or Lipofectamine and different reporter genes. The transfection efficiency of Effectene/DNA complexes was 20fold higher compared to Lipofectamine. Transfected keratinocytes continued to grow and developed within 2 weeks a cellular multilayer (3-D culture). Areas of transfected cells were detected within this layer.     

块磷鹅膏的培养条件及所产Amanitins毒素的分析

采集并组织分离到块磷鹅膏Amanita spissa的纯培养菌丝。探讨了各种培养条件对块磷鹅膏菌丝生长的影响,实验结果表明,在28℃,pH 6.0,避光的条件下块磷鹅膏的菌丝体生长最好。液体反应器人工培养块磷鹅膏获得成功,其液体培养的菌丝体干重在摇瓶中为0.893g/L,在气升式反应器内可达到2.33g/L。固体斜面培养和气升式反应器液体培养的菌丝体的HPLC分析表明菌丝体内含有鹅膏毒肽,而不含有鬼笔毒肽。两种培养条件下菌丝体的-αamanitin毒素含量略有不同,固体斜面菌丝体为26.02μg/gDCW、反应器培养菌丝体为15.25μg/gDCW。通过抑芽法试验也证明固体和液体培养菌丝体中含有-αamanitin,且具有和子实体中-αamanitin相同的生物学活性。结果表明有可能通过液体大规模培养鹅膏菌丝体来生产鹅膏毒素。     

Presence of endothelin-1 and endothelin-3 in peripheral tissues and central nervous system of the pig.

The distribution of endothelin (ET) peptides in the pig was studied in a variety of tissues using selective radioimmunoassays combined with reverse-phase high performance liquid chromatography (HPLC). The levels of ET-like immunoreactivity (LI) were overall relatively low. The highest levels of ET-LI were found in blood vessels, cerebral and coronary arteries containing 3190 +/- 910 and 1330 +/- 450 fmol/g, respectively. Veins generally contained higher levels of ET-LI per tissue weight than corresponding arteries. Peripheral sympathetic and sensory ganglia contained a higher concentration of ET-LI than the studied central nervous system (CNS) areas. In the CNS the highest concentration of ET-LI was found in a non-neuronal structure, the choroid plexus. The levels of ET-LI were also relatively high in the respiratory tract (100-400 fmol/g). In the heart, the endocardium contained the highest levels (190 +/- 44 fmol/g). In the kidney, the concentration of ET-LI was 3-fold higher in the medulla than in the cortex. In the gastrointestinal tract all levels were below 100 fmol/g, except for the colon which contained 120 +/- 50 fmol/g. The characterization of ET-LI in extracts of some of these tissues revealed that ET-1 dominated in the lung, spleen and hypothalamus while ET-3 and ET-1 were present in approximately equal amounts in renal medulla and thoracic spinal cord. The HPLC analysis provided no clear-cut evidence for significant presence of vasoactive intestinal contractor, ET-2 or big ET-1(1-39) in the lung, spleen, kidney, spinal cord or hypothalamus. It is concluded that mature ET-1 and ET-3 are the predominant ET peptides in peripheral tissues and CNS.     

Stimulation of ERAD of misfolded null Hong Kong alpha1-antitrypsin by Golgi alpha1,2-mannosidases

Terminally misfolded or unassembled proteins are degraded by the cytoplasmic ubiquitin-proteasome pathway in a process known as ERAD (endoplasmic reticulum-associated protein degradation). Overexpression of ER alpha1,2-mannosidase I and EDEMs target misfolded glycoproteins for ERAD, most likely due to trimming of N-glycans. Here we demonstrate that overexpression of Golgi alpha1,2-mannosidase IA, IB, and IC also accelerates ERAD of terminally misfolded human alpha1-antitrypsin variant null (Hong Kong) (NHK), and mannose trimming from the N-glycans on NHK in 293 cells. Although transfected NHK is primarily localized in the ER, some NHK also co-localizes with Golgi markers, suggesting that mannose trimming by Golgi alpha1,2-mannosidases can also contribute to NHK degradation.     

Identification of a peptide of the membrane protein of tobacco mosaic virus, cross-linked with intraviral RNA under the effect of UV-radiation

As reported previously, UV-irradiation induces crosslinking between tobacco mosaic virus (TMV) coat protein molecules and intraviral RNA nucleotides. We have irradiated [3H]-uridine labeled TMV and isolated TMV coat protein subunits with the attached nucleotide label. These TMV protein subunits were hydrolyzed with trypsin. The tryptic peptides were separated by high-performance liquid chromatography and [3H]-labeled peptides were identified. The UV-irradiation of TMV was found to result in crosslinking to intraviral RNA of the T8 tryptic peptide (residues 93-112) of TMV coat protein.     

The fate of p21 in cells surviving UV-irradiation

p21(CDKN1A) is a critical regulator of cell cycle progression in response to DNA damage. There are conflicting conclusions as to whether p21(CDKN1A) levels increase or decrease after ultraviolet (UV)-irradiation and recently it was even reported to disappear entirely following 2.5-30 Jm(-2) of UV-irradiation in the presence of growth medium. The latter would suggest an alternative mechanism for cell cycle arrest after UV-irradiation, since p21(CDKN1A) induction has been considered to be the major mediator of p53-mediated cell cycle arrest after DNA damage. Using physiological UV doses based on cell-killing, we previously observed and here verify that low doses (1.2-6 Jm(-2)) induce p21(CDKN1A) immediately after UV-irradiation, though higher doses cause a latency during which p21(CDKN1A) levels remain fairly constant before increasing. As expected, p53 induction preceded p21(CDKN1A) induction at all doses. Thus, p21(CDKN1A) levels after low doses of UV-irradiation may be controlled in a p53-dependent manner without severe reduction. We propose that physiological relevant UV doses should be determined for each target cell type prior to studying UV-induced responses and that p21(CDKN1A) is indeed critical for cell cycle arrest in cells that survive UV-irradiation.     

Global detection and characterization of hypothetical proteins in Shewanella oneidensis MR-1 using LC-MS based proteomics

The availability of whole genome sequences has enabled the application of powerful tools for assaying global expression patterns in environmentally relevant bacteria such as Shewanella oneidensis MR-1. A large number of genes in prokaryote genomes, including MR-1, have been annotated as hypothetical, indicating that no similar protein has yet been identified in other organisms. Using high-sensitivity MS coupled with accurate mass and time (AMT) tag methodology, 1078 tryptic peptides were collectively detected in MR-1 cultures, 671 of which were unique to their parent protein. Using only these unique tryptic peptides and a minimum of two peptides per protein, we identified, with high confidence, the expression of 258 hypothetical proteins. These proteins ranged from 3.5 to 139 kDa, with 47 being 100 amino acid residues or less. Using a combination of information including detection in cells grown under specific culture conditions, presence within a specific cell fraction, and predictive algorithms such as PSORT and PSORT-B, possible/plausible functions are proposed for some hypothetical proteins. Further, by applying this approach a number of proteins were found not only to be expressed, but only expressed under certain culturing conditions, thereby suggesting function while at the same time isolating several proteins to distinct locales of the cell. These results demonstrate the utility of the AMT tag methodology for comprehensive profiling of the microbial proteome while confirming the expression of a large number of hypothetical genes.     

The fate of p21CDKN1A in cells surviving UV-irradiation

p21(CDKN1A) is a critical regulator of cell cycle progression in response to DNA damage. There are conflicting conclusions as to whether p21(CDKN1A) levels increase or decrease after ultraviolet (UV)-irradiation and recently it was even reported to disappear entirely following 2.5-30 Jm(-2) of UV-irradiation in the presence of growth medium. The latter would suggest an alternative mechanism for cell cycle arrest after UV-irradiation, since p21(CDKN1A) induction has been considered to be the major mediator of p53-mediated cell cycle arrest after DNA damage. Using physiological UV doses based on cell-killing, we previously observed and here verify that low doses (1.2-6 Jm(-2)) induce p21(CDKN1A) immediately after UV-irradiation, though higher doses cause a latency during which p21(CDKN1A) levels remain fairly constant before increasing. As expected, p53 induction preceded p21(CDKN1A) induction at all doses. Thus, p21(CDKN1A) levels after low doses of UV-irradiation may be controlled in a p53-dependent manner without severe reduction. We propose that physiological relevant UV doses should be determined for each target cell type prior to studying UV-induced responses and that p21(CDKN1A) is indeed critical for cell cycle arrest in cells that survive UV-irradiation.     

β-Endorphin and ACTH related peptides in primary cultures of rat anterior pituitary cells: evidence for different intracellular pools

Acid extracts of rat anterior pituitary cells and cell-derived culture media were shown to contain three forms of β-endorphin immunoreactive peptides, corresponding in molecular size to the prohormone pro-opiomelanocortin (POMC), β-lipotropin and 3.5 kDa β-endorphin, and essentially two forms of adrenocorticotropin (ACTH) immunoreactivity, representing a 20 kDa intermediate fragment and 4.5 kDa ACTH. Under basal conditions the intracellular peptides contained a high proportion of the bioactive forms of β-endorphin and ACTH whereas the extracellular peptides contained a higher proportion of the inactive precursors. When the cells were incubated for 3 h in the presence of 10⁻⁸ M CRF, the levels of intracellular β-endorphin and ACTH immunoreactivity were reduced by 15–30% and there was a 4–5-fold increase in the level of the secreted peptides; furthermore, unlike the peptides released under basal conditions, the peptides secreted under the influence of CRF contained much higher proportions of 4.5 kDa ACTH and 3.5 kDa β-endorphin, reflecting the intracellular patterns of these peptides. Similar results were obtained when secretion was stimulated by 10⁻⁷ M epinephrine, which produced a 2-fold increase in peptide release. In the presence of 10⁻⁶ M dexamethasone the basal secretion of ACTH and β-endorphin related peptides, and the intracellular levels of these peptides, remained unaltered. The results point to the existence of different intracellular compartments from which peptides at different states of maturation can be released selectively.β-EndorphinACTHPituitary cell cultureProcessingCRFEpinephrine     

Boar acrosin digestion of the porcine egg coat, zona pellucida, and rearrangement of the zona proteins

Mechanically isolated, structurally intact porcine zonae pellucidae composed of three families of glycoproteins (PZP1-3) were digested with purified boar acrosin, and then the solublized and unsolubilized fractions were separately analyzed by HPLC and/or SDS-PAGE. In isotonic solution, PZP1 was first degraded and then PZP2, whereas PZP3 was quite resistant to acrosin, reflecting the organization of these families in the zona structure. As the proteolytic hydrolysis proceeded, high-molecular-weight products appeared in the unsolubilized fraction. These products disintegrated on treatment with beta-mercapto-ethanol. The zona materials solubilized by acrosin were separated into seven fractions by reverse phase HPLC. The total yield of the latter was only about 5% by weight. Thus, limited sites of the porcine zona were cleaved by the homologous sperm acrosin. Since five fractions contained peptides that were more hydrophilic than the original proteins, these peptides seemed likely to be present on the outer surface of the zona structure. SDS-PAGE of the unsolubilized fraction showed that acrosin cleaved the zona at many more sites in hypotonic solution than in isotonic solution. Thus, structural relaxation of the inner region of the zona was indicated to be induced under hypotonic conditions. However, no high-molecular-weight products were formed in hypotonic solution, suggesting that the native architecture of the zona is a prerequisite for their formation.     

Semi-conservative synthesis of DNA in UV-sensitive mutant cells of Chinese hamster after UV-irradiation

A study was made of the rate of semi-conservative DNA synthesis in asynchronous UV-resistant (clone V79) and UV-sensitive clones (VII and XII) of Chinese hamster cells after UV-irradiation. In all 3 clones studied, UV-irradiation (5-30 J/m2) induced a decrease in the rate of DNA synthesis during the subsequent 1-2 h. In the resistant clone (V79) recovery of DNA synthesis rate started after the first 2 h post-irradiation (5 J/m2) and by the 3rd hour reached its maximum value, which constituted 70% of that observed in control, non-irradiated cells. The UV-sensitive mutant clones VII and XII showed no recovery in the rate of DNA synthesis during 6-7 h post-irradiation. The results obtained show that the survival of cells is correlated with the ability of DNA synthesis to recover after UV-irradiation in 3 clones studied. The observed recovery of UV-inhibited DNA synthesis in mutant clones may be due to certain defects in DNA repair.     

Side chain contributions to the interconversion of the topological isomers of guanylin-like peptides.

The peptide hormones guanylin and uroguanylin are ligands of the intestinal guanylyl cyclase-C (GC-C) that is involved in the regulation of epithelial water and electrolyte transport. The small peptides contain 15 and 16 amino acids, respectively, and two disulfide bonds with a 1-3/2-4 connectivity. This structural feature causes the unique existence of two topological isoforms for each peptide in an approximate 3:2 ratio, with only one of the isoforms exhibiting GC-C-activating potential. The two uroguanylin isomers can be separated by HPLC and are of sufficient stability to be studied separately at ambient temperatures while the two guanylin isomers are rapidly interconverting even at low temperatures. Both isomers show clearly distinguishable (1)H chemical shifts. To investigate the influence of certain amino acid side chains on this isomerism and interconversion kinetics, derivatives of guanylin and uroguanylin (L-alanine scan and chimeric peptides) were designed and synthesized by Fmoc solid-phase chemistry and compared by HPLC and 2D (1)H NMR spectroscopy. Amino acid residues with the most significant effects on the interconversion kinetics were predominantly identified in the COOH-terminal part of both peptides, whereas amino acids in the central part of the peptides only moderately affected the interconversion. Thus, the conformational conversion among the isomers of both peptides is under the control of a COOH-terminal sterical hindrance, providing a detailed model for this dynamic isomerism. Our results demonstrate that kinetic control of the interconversion process can be achieved by the introduction of side chains with a defined sterical profile at suitable sequence positions. This is of potential impact for the future development of GC-C peptide agonists and antagonists.