Detecting Allergens From Black Tiger Shrimp Penaeus monodon That Can Bind and Cross-link IgE by ELISA, Western Blot, and a Humanized Rat Basophilic Leukemia Reporter Cell Line RS-ATL8

BACKGROUND: Black tiger shrimp Penaeus monodon is one of the common causes of shellfish allergy that is increasing worldwide. One of the important problems in the management of shellfish allergy is the lack of accurate diagnostic assay because the biological and immunological properties of allergens in black tiger shrimp have not been well characterized. This study aims to detect proteins with the ability to bind and cross-link immunoglobulin E (IgE) from black tiger shrimp by enzyme-linked immunosorbent assay (ELISA), Western blot, and a humanized rat basophilic leukemia reporter cell line RS-ATL8. METHODS: Sera from shrimp allergic subjects were subjected to ELISA and Western blots using raw or cooked shrimp extract as antigens. Pooled sera were used to sensitize the RS-ATL8 reporter cell line and cells were activated by shrimp extract. Eluted protein extracts separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were tested on the RS-ATL8 cell line and subjected to mass spectrometry to identify potential candidate allergens. RESULTS: Allergic sera reacted stronger to raw shrimp extract than cooked shrimp extract (P=0.009). Western blot demonstrated that major IgE reactivity protein bands were at 32–39 kDa and 91–230 kDa in both raw and cooked shrimp extracts. The eluted protein bands at the molecular weight of 38 and 115 kDa from raw shrimp extract induced IgE cross-linking as assayed by the RS-ATL8 cell line. These protein bands were subjected to mass spectrometry for analysis. Ubiquitin-activating enzyme and crustacyanin were identified as potential candidate novel shrimp allergens. CONCLUSIONS: The RS-ATL8 reporter cell line can be used to identify potential new shrimp allergens that can functionally cross-link IgE and induce mast cell degranulation.

Atg7 in development and disease: panacea or Pandora's Box?

Macroautophagy is an evolutionarily conserved intracellular degradation system used by life ranging from yeasts to mammals. The core autophagic machinery is composed of ATG (autophagy-related) protein constituents. One particular member of the ATG protein family, Atg7, has been the focus of recent research. Atg7 acts as an E1-like activating enzyme facilitating both microtubule-associated protein light chain 3 (LC3)-phosphatidylethanolamine and ATG12 conjugation. Thus, Atg7 stands at the hub of these two ubiquitin-like systems involving LC3 and Atg12 in autophagic vesicle expansion. In this review, I focus on the pleiotropic function of Atg7 in development, maintenance of health, and alternations of such control in disease.

Cellular response to altered autophagy activity in human fibroblast cells overexpressing Ha-RasV12 / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of oncogenic Ras overexpression on autophagic activity in human fibroblast cells in vitro.</p><p><b>METHODS</b>BJ cells were transfected with H-RasV12 or control vector and treated with chloroquine, small interfering RNA (siRNA) for ATG7, or rapamycin. The cellular responses were analyzed by monitoring the parameters and biomarkers for cell growth, senescence and cell death.</p><p><b>RESULTS</b>In BJ cells overexpressing H-RasV12, chloroquine treatment resulted in more prominent cell senescence and a significantly increased cell death rate. Suppression of ATG7 mediated by siRNA also promoted cell senescence. Rapamycin treatment also caused an increased cell death rate but attenuated senescence in surviving cells. In control BJ cells, the cellular response to chloroquine included senescence and cell death, which occurred slowly. Rapamycin treatment and siRNA suppression of ATG7 had no obvious effect on control BJ cells.</p><p><b>CONCLUSION</b>Stable cellular overexpression of oncogenic Ras causes tightly controlled suppression of the autophagic activity of human fibroblast cells, and such changes produce significant effect on cell senescence and survival.</p>

The quantitative analysis of Ub and UbE1 in focus of traumatic epilepsy / 法医学杂志

OBJECTIVE@#To study the expression of ubiquitin proteasome system (UPS) in the traumatic epilepsy pathogenesis and its value in traumatic epilepsy by quantitative analysis.@*METHODS@#Fifteen specimens from human epileptic temporal cortex from PTE were collected as the PTE group. Fifteen specimens from non-PTE were collected as the non-PTE group. Fifteen normal cerebral cortex specimens died from acute traffic accident were collected as the control group. The expression of mRNA and protein of Ub and UbE1 were detected by RT-PCR and Western blot. Statistical analysis was used to compare the data between three groups.@*RESULTS@#The expression of mRNA and protein of Ub and UbE1 were the following order: PTE group(high), non-PTE group(middle) and control group(low).@*CONCLUSION@#The study confirms that UPS is up-regulated in the epilepsy's focus, especially in traumatic epilepsy. The activation of UPS may be an important pathological change in neurons in pathogenesis of traumatic epilepsy.

Morphology changes of ubiquitin-proteasome system in traumatic epilepsy / 法医学杂志

OBJECTIVE@#To investigate the value of ubiquitin(Ub) and ubiquitin-activating enzymel(UbE1) for the appraisement of post traumatic epilepsy (PTE).@*METHODS@#Fifteen specimens from human epileptic temporal cortex originating from PTE were collected as the PTE group. Fifteen specimens from non-PTE were collected as the non-PTE group. Meanwhile, 15 normal cerebral cortex specimens from people dead from acute traffic accident were collected as the control groups. Observe morphology changes of each group with HE, then with immunohistochemistry of Ub and UbE1.@*RESULTS@#Compared to the control group, morphology changes of neuron quantity reduction, neuron denaturation and so on were observed both in the PTE group and the non-PTE group under HE, especially in the PTE group. Ub and UbE1 mainly expressed in the nucleus and cytoplasm of the neurons in epilepsy spot without extracellular expression. The expression of Ub and UbE1 is PTE group > non-PTE group > control group (P < 0.05).@*CONCLUSIONS@#The neuron denaturation are one of the main pathology changes of epilepsy, and it is more obvious in the PTE group. Immunohistochemistry of Ub and UbE1 may be more helpful to distinguish PTE and non-PTE than HE staining.

Identification and characterization of a spermatogenesis-related gene Ube1 in rat testis / 生理学报

A gene that could be potentially involved in spermatogenesis was identified and characterized by using suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE) with total RNA from type A spermatogonia and pachytene spermatocytes of rat. This gene consists of 3 433 base pairs (bp) with a complete open reading frame (ORF) of 3 171 bp and encodes a putative protein containing 1057 amino acids. The nucleotide sequence displays a 93% identity to mouse ubiquitin-activating enzyme E1, Chr Y 1 (Ube1y1) and an 82% identity to human ubiquitin-activating enzyme E1 (UBE1). The putative protein of this gene contains an ubiquitin-activating enzyme signature 1 and an ubiquitin-activating enzyme active site, which are also existed in mouse ubiquitin-activating enzyme E1, human ubiquitin-activating enzyme E1 et al. So we named this gene as Rattus norvegicus ubiquitin-activating enzyme E1 (Ube1). The sequence of Ube1 was submitted to GenBank and the accession number is EF690356. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that Ube1 was specifically expressed in testis, while its expression was not detected in heart, brain, spleen, lung, liver, muscle, kidney and ovary. Comparison of the expression of Ube1 in different developmental stages of testis and Sertoli cells (real-time PCR) indicated that Ube1 was expressed more highly in spermatogonia than in spermatocytes, spermatids and Sertoli cells. In conclusion, Ube1 is a gene encoding rat ubiquitin-activating enzyme E1 and specifically expressed in testis, which might play a key role in ubiquitin system and influence spermatogenesis.