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131.
Jeffery R. Cook Robert G. van Buskirk 《In vitro cellular & developmental biology. Animal》1996,32(5):300-306
Summary Laminin synthesis and deposition are concomitant with the development of a basal lamina between the human epidermis and the
underlying dermis. One of the challenges in tissue engineering of human epidermal models is to develop substrates and conditions
that encourage the development of a basement membrane. The purpose of this study was to determine if actin filaments and/or
microtubules are involved in the synthesis/secretion of laminin by normal human epidermal keratinocytes (NHEK)in vitro. NHEK synthesize and secrete laminin subunits B1, B2, and M but little, if any, of laminin subunit A. Data indicate that
disruption of microfilaments by the destabilizing agent, cytochalasin D, had no apparent effect on the relative synthesis
rates of most cytosolic proteins as, revealed by one-dimensional sodium dodecyl sulfate (SDS) gel electrophoresis. This drug,
however, increased laminin B2 synthesis several fold over untreated controls. This enhanced synthetic rate was independent
of the type of collagen, matrix on which the NHEK were grown. Similar increases in synthesis of the M and B1 laminin chains
were not observed. To determine if this increase in synthesis lead to increases in laminin B2 secretion, laminin B2 was immunoprecipitated
from both the apical and basal domains of NHEK cells grown on microporous membranes. While more laminin B1, B2, and M were
secreted basally than apically, an observation consistent with laminin’s role in basal lamina formation, cytochalasin D had
no apparent effect on either basal or apical laminin B2 secretion. Experiments with the microtubule destabilizer, nocodazole,
showed no similar effects on laminin synthesis and/or secretion. We conclude that (a) disruption of the actin network in NHEK
selectively increases the synthesis of laminin B2, (b) the secretion of laminin B2 from NHEK cells is not governed by either
the microfilamentous cytoskeleton or the amount of laminin synthesized by NHEK, and (c) disruption of the microtubular network
does not alter laminin synthesis or secretion. 相似文献
132.
The Hymenoptera (ants, bees, wasps and sawflies) display a great variety of social systems and sex ratios and have played a key role in the development and testing of many evolutionary models. Traditionally, considerable emphasis was placed on the fact that hymenopterans have haploid males and diploid females but it is now clear that many species also regularly produce sterile, diploid males. Recent studies explore the diverse ways in which production of these diploid males influences selection on mating systems, sex ratios and social behaviour. 相似文献
133.
Identification of Trans-Acting Genes Necessary for Centromere Function in Drosophila Melanogaster Using Centromere-Defective Minichromosomes
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Deletions in the Drosophila minichromosome Dp1187 were used to investigate the genetic interactions of trans-acting genes with the centromere. Mutations in several genes known to have a role in chromosome inheritance were shown to have dominant effects on the stability of minichromosomes with partially defective centromeres. Heterozygous mutations in the ncd and klp3A kinesin-like protein genes strongly reduced the transmission of minichromosomes missing portions of the genetically defined centromere, but had little effect on the transmission of minichromosomes with intact centromeres. Using this approach, ncd and klp3A were shown to require only the centromeric region of the chromosome for their roles in chromosome segregation. Increased gene dosage also affected minichromosome transmission and was used to demonstrate that the nod kinesin-like protein gene interacts genetically with the centromere, in addition to interacting with extracentromeric regions as demonstrated previously. The results presented in this study strongly suggest that dominant genetic interactions between mutations and centromere-defective minichromosomes could be used effectively to identify novel genes necessary for centromere function. 相似文献
134.
135.
Novel β-Secretase Cleavage of β-Amyloid Precursor Protein in the Endoplasmic Reticulum/Intermediate Compartment of NT2N Cells
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Abraham S.C. Chyung Barry D. Greenberg David G. Cook Robert W. Doms Virginia M.-Y. Lee 《The Journal of cell biology》1997,138(3):671-680
Previous studies have demonstrated that NT2N neurons derived from a human embryonal carcinoma cell line (NT2) constitutively process the endogenous wild-type β-amyloid precursor protein (APP) to amyloid β peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generated by intracellular processing must also be present in these cells. Here we show that the NH2-terminal fragment of APP generated by β-secretase cleavage (APPβ) is indeed produced from the endogenous full length APP (APPFL). Pulse–chase studies demonstrated a precursor–product relationship between APPFL and APPβ as well as intracellular and secreted APPβ fragments. In addition, trypsin digestion of intact NT2N cells at 4°C did not abolish APPβ recovered from the cell lysates. Furthermore, the production of intracellular APPβ from wild-type APP appears to be a unique characteristic of postmitotic neurons, since intracellular APPβ was not detected in several non-neuronal cell lines. Significantly, production of APPβ occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15°C, or by expression of exogenous APP bearing the dilysine ER retrieval motif. 相似文献
136.
Rates of development of stem nematode (Ditylenchus dipsaci) in white clover (Trifolium repens) seedlings were found to be linearly related to temperature. Basal developmental temperature (Tb) was 3 °C, and the thermal constant (S) for development of gravid adult females from freshly laid eggs was 270 accumulated day-degrees above the Tb. Only 12% at 20 °C and 4% at 4 °C of the gravid female nematodes inoculated into seedling axils successfully penetrated seedling epidermis. These nematodes slowly migrated within the seedling and after a lag of 5 days at 20 °C started to lay eggs. The maximal rate of egg production was temperature-dependent, being 0.8 and 3.1 eggs female⁻¹ day⁻¹ at 10 and 20 °C, respectively. Nematodes emigrated rapidly from infested stolons when they were immersed in water, with rates being highest at 25 °C and lowest at 4 °C. The sensitivity to temperature of many of the parameters that govern nematode population dynamics indicates that climatic changes will have a marked effect upon this host-parasite system. A study of infested stolons from the field indicated that nematode numbers increased up to 3,000 or more before tissue senesence, triggered by nematode damage, caused a mass emigration of nematodes from the stolon. 相似文献
137.
Stabilization of double-stranded oligonucleotides using backbone-linked disulfide bridges. 总被引:1,自引:1,他引:0
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A convenient, practical route to the synthesis of disulfide-bridged oligonucleotides has been developed. Aliphatic linkers with terminal thiol groups have been attached to the phosphodiester backbones of partially or fully complementary oligonucleotide sequences and oxidized to yield covalently closed oligonucleotides with disulfide bridges. This procedure has been used to prepare a duplex with disulfide bridges at both ends and stem-loop sequences with single disulfide bridges. Oxidation of a self-complementary duplex possessing terminal thiol groups produced both hairpin and duplex structures with disulfide bridges, the relative proportions of each being dependent upon the reaction conditions. These bridged hairpin and duplex structures were shown to be interconvertible by reduction and re-oxidation. The melting profiles of disulfide-bridged oligonucleotides were compared with the same sequences without bridges and with sequences possessing triethylene glycol bridges, and in all cases the introduction of disulfide bridges resulted in a considerable increase in thermal stability. EcoRI endonuclease was capable of cleaving a disulfide-bridged duplex possessing a recognition site for this enzyme, thus supporting a lack of distortion of the recognition site. The disulfide bridges could be cleaved using a large excess of DTT to regenerate the corresponding sulfhydryl compounds. A study of the serum stabilities of disulfide-bridged oligonucleotides showed that the bridged duplexes were much more stable than their unmodified counterparts, whereas the rate of degradation of the stem-loop structures was more dependent upon the size of the loop than the presence or absence of the disulfide bridge. In summary, we have described a novel methodology, employing commercially available reagents, for the stabilization of oligonucleotide duplexes or stem-loop structures by disulfide bridge formation. 相似文献
138.
139.
R A Levine G D Levine A P Healey D I Cook P W Kuchel J A Young 《Biochimica et biophysica acta》1984,804(3):324-330
1H-nuclear magnetic resonance spectroscopy (NMR) was adapted to isolated rabbit fundic glands and identification made of compounds responsible for several observed spectral resonances. A minimum gland concentration of 0.5 mg dry weight or 5 mg wet weight per 0.5 ml was needed for adequate signal-to-noise ratio. At physiological temperature and pH, the glands demonstrated reproducible spectra, stability for accumulation times greater than 30 min and responsiveness to histamine stimulation, as measured by oxygen consumption and aminopyrine uptake. The relatively anaerobic conditions favored use of proton compared to phosphorus NMR, since 1H-NMR allowed significantly shorter spectral accumulation times and therefore did not compromise glandular viability to the same extent as 31P-NMR. The most conspicuous resonance in the gland spectrum was assigned to the -N+(CH3)3 protons of choline and related compounds. In membrane-free lysates, several components of the signal were resolvable and assigned to choline, phosphatidylcholine, phosphocholine and L-alpha-glycerophosphocholine. Thin-layer chromatography verified that phosphatidylcholine and phosphatidylethanolamine were the major phospholipids present in gland lipid. Presumably, they represent the source of the surface-active phospholipids present in gastric juice, which may play a role in gastric cytoprotection. 相似文献
140.
The activity of the neutral, Mg2+-stimulated sphingomyelinase of cultured neuroblastoma cells (N1E-115) is enriched in the plasma membrane fraction and is reduced following treatment of intact or broken cells with trypsin, α-chymotrypsin, papain, and protease. Two protease-sensitive enzymes of the cell interior (lactate dehydrogenase and NADPH-cytochrome c reductase) are not affected by protease treatment of intact cells. These results indicate that the neutral, Mg2+-stimulated sphingomyelinase is oriented externally on the plasma membrane of the cultured neuroblastoma cell. 相似文献