The ovary of the desert locust, Schistocerca gregaria, contains multiple inhibitors of serine proteases. Five serine protease inhibitors, designated SGPI-1^5 (Schistocerca gregaria protease inhibitors) were purified from methanolic extracts of mature ovaries and analyzed by mass spectrometry and amino acid sequencing. The revealed primary structures display amino acid similarities and are related to the serine protease inhibitors identified in the hemolymph of Locusta migratoria. All inhibitors show an in vitro inhibiting activity towards K K-chymotrypsin. In addition, SGPI-1 displays in vitro inhibiting activity towards trypsin, and SGPI-2 is a potent pancreatic elastase inhibitor. Differences in inhibitory specificities towards the locust endogenous serine proteases can be readily attributed to the amino acid sequence within the active region and also to amino acid residues beyond the P1-PP1 bond. A difference in one or two amino acid residues around the reactive sites results in considerable alteration of the inhibitory specificity. The temporal and spatial distribution of SGPI-1^5 was studied by RP-HPLC analysis. All inhibitors occur in hemolymph, ovaries, testes and fat body of adults but are absent in the gut. They are also present in larval hemolymph and fat body. An antibody raised against SGPI-2 shows positive immunostaining in the ovarian follicle cells.z 1998 Federation of European Biochemical Societies.

The molecular functions and pathophysiologic role of the lymphocyte α-kinase gene (ALPK1) in gout are unknown. We aimed to examine ALPK1 expression in patients with gout and investigate its role in monosodium urate monohydrate (MSU)-induced inflammatory responses. Microarray data mining was performed with six datasets containing three clinical gout and three volunteer samples. Real-time quantitative polymerase chain reaction (qPCR) assay was used to profile ALPK1 mRNA expression in 62 independent samples. RNA interference for ALPK1 suppression in THP1 cells (human monocytic cell line) was used to scrutinize the functional role of ALPK1 in MSU-mediated inflammatory responses, and ALPK1 expression in MSU-treated THP1 cells was determined by qPCR and Western blot analysis. Cytokine mRNA expression in HEK293 cells after incubation with different concentrations of MSU crystals in the presence or absence of ALPK1 was also detected by qPCR, and ERK1/2, p38, and JNK expressions were investigated by Western blot analysis. ALPK1 mRNA was overexpressed in the clinical gout samples. MSU treatment promoted ALPK1 expression at the mRNA and protein levels. Furthermore, ALPK1 knockdown in THP1 cells resulted in a markedly decreased IL-1β, TNF-α, and IL-8 mRNA expression; plasmid ALPK1 transfection and MSU stimulation synergistically increased the mRNA expression of these cytokines in a concentration-dependent manner. The synergistic effect also led to ERK1/2 activation. ALPK1 is a gout-susceptible gene involved in MSU-induced inflammatory responses. It may contribute to the development of gout by enhancing the inflammatory responses via the mitogen-activated protein kinase pathway.

Metastatic renal cell carcinoma (RCC) is highly resistant to conventional systemic treatments, including chemotherapy, radiotherapy and hormonal therapies. Previous studies have shown over-expression of EGFR is associated with high grade tumors and a worse prognosis. Recent studies suggest anticancer therapies targeting the EGFR pathway have shown promising results in clinical trials of RCC patients. Therefore, characterization of the level and localization of EGFR expression in RCC is important for target-dependent therapy. In this study, we investigated the clinical significance of cellular localization of EGFR in human normal renal cortex and RCC. RCC and adjacent normal kidney tissues of 63 patients were obtained for characterization of EGFR expression. EGFR protein expression was assessed by immunohistochemistry on a scale from 0 to 300 (percentage of positive cells × staining intensity) and Western blotting. EGFR membranous staining was significantly stronger in RCC tumors than in normal tissues (P < 0.001). In contrast, EGFR cytoplasmic staining was significantly higher in normal than in tumor tissues (P < 0.001). The levels of membranous or cytoplasmic EGFR expression in RCC tissues were not correlated with sex, tumor grade, TNM stage or overall survival (P > 0.05). These results showed abundant expression of membranous EGFR in RCC, and abundant expression of cytoplasmic EGFR in normal tissues. EGFR expression in RCC was mostly located in the cell membrane, whereas the EGFR expression in normal renal tissues was chiefly seen in cytoplasm. Our results suggest different locations of EGFR expression may be associated with human renal tumorigenesis.

This study describes the cloning of two cDNAs encoding three serine-protease-inhibiting peptides, SGPI I, II and III, which were recently identified from ovarian extracts of the desert locust, Schistocerca gregaria. The first cDNA codes for the precursor polypeptides of SGPI I and SGPI II; the second encodes only a single inhibitor, SGPI III. Northern-blot analysis revealed an approximate length of 0.8 kb for SGPI-I/II mRNA and 0.6 kb for SGPI-III mRNA. The transcripts are present in several locust tissues, but they could not be detected in the midgut. The gene for SGPI-I/II is abundantly transcribed during all larval and adult stages, whereas SGPI-III mRNA is mainly present in adults. Northern-blot hybridization also revealed important changes in the SGPI-mRNA content during the molting cycle and during the adult reproductive cycle. Moreover, a differential hormonal control was observed in adult females which had been treated with precocene, juvenile hormone or ecdysone.

Non-small cell lung cancer (NSCLC) is a type of malignant cancer, and 85% of metastatic NSCLC patients have a poor prognosis. C2-ceramide induces G2/M phase arrest and cytotoxicity in NSCLC cells. In this study, the autophagy-inducing effect of C2-ceramide was demonstrated, and cotreatment with the autophagy inhibitor chloroquine (CQ) was investigated in NSCLC H460 and H1299 cells. The results suggested that C2-ceramide exhibited dose-dependent anticancer effects in H460 and H1299 cells and autophagy induction. Zebrafish-based acridine orange staining confirmed the combined effects in vivo. Importantly, the combination of a sublethal dose of C2-ceramide and CQ resulted in additive cytotoxicity and autophagy in both cell lines. Alterations of related signaling factors, including Src and SIRT1 inhibition and activation of the autophagic regulators LAMP2 and LC3-I/II, contributed to the autophagy-dependent apoptosis. We found that C2-ceramide continuously initiated autophagy; however, CQ inhibited autophagosome maturation and degradation during autophagy progression. Accumulated and non-degraded autophagosomes increased NSCLC cell stress, eventually leading to cell death. This study sheds light on improvements to NSCLC chemotherapy to reduce the chemotherapy dose and NSCLC patient burden.