Accumulation Of Class Research Articles

Accumulation of class switched IgD-IgM- memory B cells in the cerebrospinal fluid during neuroinflammation

Accumulation of class switched IgD-IgM- memory B cells in the cerebrospinal fluid during neuroinflammation

Azetidine-induced Accumulation of Class I Small Heat Shock Proteins in the Soluble Fraction Provides Thermotolerance in Soybean Seedlings

Accumulation of class I small heat shock proteins (sHSPs) is induced by the proline analog, azetidine-2-carboxylic acid (Aze) in soybean seedlings to a level similar to that induced by exposure to 40 degrees C. However, only the treatment with 10 mM Aze for 6 h and subsequently with 10 mM proline for 24 h protected the seedlings from damage during subsequent exposure to 45 degrees C as assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. A chaperone activity assay showed that the purified class I sHSPs induced by Aze were functional in vitro and protected proteins from thermal denaturation. Amino acid composition analysis indicated that Aze was not incorporated into de novo synthesized class I sHSPs. Accumulation of class I sHSPs in the soluble post-ribosomal supernatant fraction was found to be important for acquisition of thermotolerance. We suggest that both the accumulation of class I sHSPs and their presence in the soluble fraction are important for establishment of thermotolerance.

Zinc ions differentially affect chitin synthase gene expression in an ericoid mycorrhizal fungus

Two chitin synthase (Chs) genes (class II and class IV, respectively) were identified in the mycelium of strain PSIV, a sterile ascomycete capable of establishing ericoid mycorrhiza. Both genes were constitutively expressed during mycelial growth in liquid medium. However, when the fungus was challenged with toxic amounts of zinc ions, the accumulation of class II, but not class IV, Chs mRNAs was inhibited. To our knowledge, this is the first report on the differential expression of distinct Chs mRNAs following exposure to heavy metals.

Developmentally and stress-induced small heat shock proteins in cork oak somatic embryos

The timing and tissue localization of small heat shock proteins (sHSPs) during cork oak somatic embryo development was investigated under normal growing culture conditions and in response to stress. Western blot analyses using polyclonal antibodies raised against cork oak recombinant HSP17 showed a transient accumulation of class I sHSPs during somatic embryo maturation and germination. Moreover, the amount of protein increased at all stages of embryo development in response to exogenous stress. The developmentally accumulated proteins localized to early differentiating, but not the highly dividing, regions of the root and shoot apical meristems. By contrast, these highly dividing regions were strongly immunostained after heat stress. Findings support the hypothesis of a distinct control for developmentally and stress-induced accumulation of class I sHSPs. The possible role of sHSPs is discussed in relation to their tissue specific localization.

Reduction of chilling injury and transcript accumulation of heat shock proteins in tomato fruit by methyl jasmonate and methyl salicylate

Changes in heat shock protein (HSP) gene expression induced by vapor application of methyl jasmonate (MeJA) and methyl salicylate (MeSA) in tomato fruit were investigated and compared to the well-described heat shock response. Northern hybridization experiments involving six cDNAs, encoding class I and II tomato small HSPs (sHSPs) and three members of HSP 70 family, showed that accumulation of class I and II sHSP mRNAs was increased significantly by MeJA and MeSA. When the treated fruits were transferred to low temperature, class I and II mRNA levels initially decreased, but then subsequently increased. Accumulation of HSP transcripts was also observed in non-treated fruit between 7 and 14 days at low-temperature storage, but all decreased to undetectable levels after 21 days. Following MeJA and MeSA treatments, the transcripts of HSP 70 family accumulated to higher levels than following the heat treatment. MeJA- and MeSA-treatments were clearly shown to alleviate chilling injury (CI), whereas tomato fruit stored at 5 °C without pretreatment developed typical symptoms and severe decay. These results demonstrated that MeJA and MeSA induced the accumulation of sHSP transcripts in tomato. The increased transcript abundance of HSPs, especially class II sHSPs, was correlated with protection against CI.

Ethanol treatment triggers a heat shock‐like response but no thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings

ABSTRACTNon‐lethal heat‐shock (HS) treatment has previously been shown to induce thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings. This acquired thermotolerance correlates with the de novo synthesis of heat‐shock proteins (HSPs). Interestingly, we found that ethanol treatments also elicited HS‐like responses in aetiolated soybean seedlings at their normal growth temperature of 28 °C. Northern blot analyses revealed that the expression of HS genes hsp17.5, hsp70 and hsc 70 was induced by ethanol. Radioactive amino acids were preferentially incorporated into high molecular weight (HMW) HSPs rather than class I low molecular weight (LMW) HSPs during non‐lethal ethanol treatments. Immunoblot analysis confirmed that no accumulation of class I LMW HSPs occurred after non‐lethal ethanol treatment. Pre‐treatment with a non‐lethal dose of ethanol did not provide thermotolerance, as the aetiolated soybean seedlings could not survive a subsequent heat shock of 45 °C for 2 h. In contrast, non‐lethal HS pre‐treatment, 40 °C for 2 h, conferred tolerance on aetiolated soybean seedlings to otherwise lethal treatments of 7·5% ethanol for 8 h or 10% ethanol for 4 h. These results suggest that plant class I LMW HSPs may play important roles in providing both thermotolerance and ethanol tolerance.

Accumulation of class I mutant p53 and apoptosis induced by carboplatin in a human glioma cell line.

Following DNA damage, wild-type p53 increases and mediates the multiple cellular responses for the repair of DNA damage or apoptosis. Inactivation of p53 by single-amino-acid substitutions contributes to the malignant phenotype and confers resistance to therapy. Among tumor-derived p53 mutants, class I mutants still retain a native-like three-dimensional structure, whereas class II mutants have unfolded DNA-binding domains. Sequencing analysis demonstrated that a human glioma cell line (U-373MG) had only a class I mutant form of p53 of His273, which targets an Arg273 that contacts DNA but retains the native structure. In this study, we investigated the metabolic alteration of the class I mutant p53 in apoptosis of U-373MG. The cell cycle progression of U-373MG cells was affected by the addition of carboplatin, while the amount of mutant p53 also increased in their nuclei. The treated cells underwent apoptosis 48h after exposure to 50 microg/ml carboplatin. Although the exact mechanism of the class I mutant p53 in the process of apoptosis has not yet been clarified, the fact that accumulation of the activated mutant p53 in the nucleus of U-373MG is concomitant with apoptosis, just as wild-type p53 does, implies that the class I mutant p53 might retain the ability to participate in apoptosis.

Binding of H-2Kb-specific Peptides to TAP and Major Histocompatibility Complex Class I in Microsomes from Wild-type, TAP1, and β2-Microglobulin Mutant Mice

Major histocompatibility complex (MHC) class I molecules are trimolecular complexes consisting of a heavy chain (HC), beta2-microglobulin (beta2m), and a short peptide. Assembly of MHC class I molecules is thought to take place early during biosynthesis. Deficiency in either beta2m or the transporter associated with antigen processing (TAP) results in accumulation of class I molecules in the endoplasmic reticulum (ER). In this study, we have assessed peptide binding to TAP and MHC class I in purified microsomes derived from wild-type, TAP1(-/-), beta2m-/-, and TAP1/beta2m-/- mice using a cross-linkable H-2Kb-binding peptide. This enabled us to study the influence of an intact TAP complex and beta2m on peptide binding to MHC class I and to analyze the stepwise interaction of peptide with TAP and MHC class I molecules. Peptide bound both immature and mature (terminally glycosylated) class I molecules in intact as well as permeabilized microsomes from wild-type mice. Efficient peptide binding to immature class I molecules was also detected in permeabilized microsomes from TAP1(-/-) mice. In contrast, no peptide binding to beta2m-free HC was detected in permeabilized microsomes from beta2m-/- and TAP1/beta2m-/- mice. However, the addition of exogenous beta2m allowed peptide binding to class I in permeabilized beta2m-/- and TAP1/beta2m-/- microsomes. These results demonstrate that a preformed class I HC middle dotbeta2m heterodimer is necessary for efficient peptide binding under physiological conditions. The observed peptide binding to class I in permeabilized TAP1(-/-) microsomes further suggests that TAP1 is not required for peptide binding to class I in the ER. Finally, kinetic studies allowed the demonstration of a stepwise binding of peptide to TAP, subsequent translocation across the ER membrane, a step that required ATP hydrolysis, and binding of peptide to preformed class I HC.beta2m heterodimers.

Regulation of plastid photosynthetic psbK‐I‐D‐C gene expression by light in rice plants

The expression of the plastid photosynthetic psbK‐I‐D‐C gene cluster is closely regulated by light. The time courses of expression of the gene cluster in rice seedlings (Oryza sativa L. cv. Tainong 67) grown under various irradiation regimes were examined by northern and western blot analyses. The data indicated that the gene cluster is photoregulated at the transcriptional and post transcriptional levels. Blue light not only enhanced the accumulation of the overlapping transcripts present in the 7 to 8‐day dark‐grown seedlings, but also induced the accumulation of new class d transcripts encoding D2 protein and CP43. This observation confirms the conservation of blue‐light regulation of psbD‐psbC transcription in plants. In etiolated seedlings, the accumulation of class e transcript encoding CP43 increased most remarkably during development: irradiation further enhanced the transcript level. Although the transcripts encoding D2 protein and CP43 are present in etiolated plants, only D2 protein is accumulated, and CP43 was not detected unless the seedlings were exposed to light. The transcript levels of RNAs d1 and f2 in green seedlings grown under a 12‐h photoperiod exhibited fluctuating patterns over the 24‐h period. The levels o1 RNAs dl and f2 also exhibited significant fluctuation during the 36‐h exposure of the etiolated seedlings to light. These data imply that the circadian regulation of pshD‐psbC expression takes place at the transcriptional and post‐transcriptional levels.

Developmental, hormonal, and pathogenesis-related regulation of the tobacco class I beta-1,3-glucanase B promoter.

The class I beta-1,3-glucanases are antifungal vacuolar proteins implicated in plant defense that show developmental, hormonal, and pathogenesis-related regulation. The tobacco enzymes are encoded by a small gene family with members derived from ancestors related to the present-day species Nicotiana sylvestris and N. tomentosiformis. We studied the expression in transgenic tobacco plants of a chimeric beta-glucuronidase (GUS) reporter gene fused to 1.6 kb of upstream sequence of the tobacco class I beta-1,3-glucanase B (GLB) gene, which is of N. tomentosiformis origin. Expression of the GUS reporter gene and the accumulation of class I beta-1,3-glucanase and its mRNA showed very similar patterns of regulation. In young seedlings the reporter gene was expressed in the roots. In mature tobacco plants it was preferentially expressed in lower leaves and roots and was induced in leaves by ethylene treatment and by infection with tobacco mosaic virus (TMV). Furthermore, it was down-regulated in cultured leaf discs by combinations of the hormones auxin and cytokinin. Histological studies of GUS activity showed that the GLB promoter shows highly localized expression in roots of seedlings. It is also expressed in a ring of cells around necrotic lesions induced by TMV infection, but not in cells immediately adjacent to the lesions or in the lesions themselves. The results of deletion analyses suggest that multiple positive and negative elements in the GLB promoter regulate its activity. The region from -1452 to -1193 containing two copies of the heptanucleotide AGCCGCC, which is highly conserved in plant-stress and defense-related genes, is necessary for high level expression in leaves. Additional regions important for organ-specific and regulated expression were: -568 to -402 for ethylene induction of leaves; -402 to -211 for expression in lower leaves and cultured leaf discs and for TMV induction of leaves; and -211 to -60 for expression in roots.

Sequences of acetyl CoA carboxylase promoter for tumour necrosis factor action

Tumour necrosis factor (TNF) inhibits the accumulation of acetyl CoA carboxylase (ACC) mRNA by decreasing the rate of ACC gene transcription. The ACC mRNA species found in 30A5 cells are generated from promoter II and TNF inhibits the accumulation of class 2 type mRNAs. By using 5' deletion mutants of promoter II fused to the bacterial chloramphenicol acetyltransferase (CAT) gene, the DNA mobility shift assay and the DNase I footprinting assay, the authors have identified the 30 bp from −389 to −359 as the TNF responsive element in promoter II. TNF treatment causes a decrease in the binding activity of nuclear protein(s) specific to the TNF responsive element. When the fragment containing the TNF responsive element was incorporated into the thymidine kinase promoter, the chimeric gene exhibited TNF induced inhibition of expression.

Acetyl-coenzyme A carboxylase mRNA metabolism in the rat liver

The acetyl-coenzyme A carboxylase (ACC) gene contains two promoters (PI and PII), both of which are active in the liver. Various physiological stimuli affect one, or both of the promoters of the ACC gene, and result in the generation of two classes of ACC mRNAs which differ in the composition of their 5′ untranslated regions (5′ UTR). We have analyzed the amounts of the two major mRNAs species that are generated from each of these promoters in order to examine the regulation of ACC gene activity in the liver under different physiological conditions. Our findings can be summarized as follows: (1) In liver from normal animals, fed a complete laboratory chow ad libitum, the level of class 2 ACC mRNA species generated by PII is very low. These mRNA species disappear on starvation. Refeeding starved animals with a fat-free diet stimulates both PI and PII with different time courses of induction: PII responds quickly and PII gene products accumulate to maximum levels within 18 hours, while the PI response, as measured by the accumulation of class 1 mRNAs, shows a lag period of 6 hours before reaching maximal levels at the end of a 24-hour refeeding period. The half-lives estimated from the induction kinetics were 4.4 hours for class 2 mRNAs and 11.8 hours for class 1 mRNAs. Reinstatement of starvation causes an almost instantaneous disappearance of class 1 mRNA species, as compared with class 2 mRNA species. This rapid decay of PI transcripts suggests that factors stabilizing this class of ACC mRNAs contribute to the steady-state levels reached after the dietary induction. (2) In starved streptozotocin-diabetic rats, refeeding with a fat-free diet causes activation of both promoters, whereas refeeding with the standard diet has no effect. This indicates that activation of the promoters occurs in the absence of insulin. However, administration of insulin to these animals further activates both promoters, regardless of the type of diet given to the starved animals. There is no insulin effect in diabetic animals that are fed normally. These observations indicate that in the liver two ACC gene promoters are selectively regulated under different physiological conditions. Insulin is one of the factors that affect the ACC gene. However, the factor(s) that is responsible for the activation of the ACC gene under the lipogenic conditions created by administration of a fat-free diet is not insulin.

Accumulation kinetics of cotton late embryogenesis-abundant mRNAs and storage protein mRNAs: Coordinate regulation during embryogenesis and the role of abscisic acid

The accumulation of total RNA transcripts of 18 late embryo-abundant ( Lea) gene families, each encoding two closely related Lea mRNAs, was measured in cotyledon total RNA during embryogenesis and germination of Gossypium hirsutum L. by RNA dot hybridization. Transcript abundance of the three storage protein families was also followed. The Lea mRNAs belong to only two related groups of commonly regulated mRNAs. The transcript level of each of the 6 members of Class I has two transient maxima during early maturation and a maximum level at 3 days prior to desiccation. The transcript level of each of the 12 members of Class II increases abruptly in late maturation with a maximum concentration at 3 days before desiccation (Class IIA) or at desiccation (Class IIB). Several patterns of early accumulation also exist within Class II, some overlapping with those of storage protein or Lea Class I mRNAs. The concentrations of Lea mRNAs increase at least 10- to 1700-fold during embryogenesis and decline 15- to 220-fold during the first day of germination. Earlier studies indicated that most Lea mRNAs, but no storage protein mRNAs, are induced in excised embryos exposed to abscisic acid (ABA). Free (+)ABA was measured during embryogenesis using a monoclonal antibody-based ELISA. The ABA concentration shows maxima correlating with the maxima in early Class I Lea mRNA abundance. However, ABA declines during the accumulation of Class I and Class II Lea mRNAs in late embryogenesis. Consequently, ABA could be an endogenous regulator of Class I Lea mRNAs during early maturation but is not the primary regulator of Lea mRNAs during late maturation. In contrast, cotyledon water potential exhibits a decrease that correlates with the late induction of Lea mRNAs. These findings are consistent with the hypothesis that low water potential is the endogenous regulator of some ABA-inducible mRNAs. The cessation of vascular flow to the embryo may also be involved in the disappearance of storage protein mRNAs and the late induction of Lea mRNAs.