A gut-specific serine protease from the malaria vector Aanopheles gambiae is downregulated after blood ingestion

A chymotrypsin-like serine protease gene (AgChyL) was cloned from the mosquito Anopheles gambiae by a polymerase chain reaction (PCR)-based subtractive cDNA cloning strategy. AgChyL messenger RNA (mRNA) is abundant in the adult female gut prior to, and for 8 h following, a blood meal. During the peak of digestion, from 12 to 24 h following a blood meal, AgChyL mRNA abundance decreased to barely detectable levels. AgChyL mRNA was abundant again by 48 h following a blood meal. Recombinant pro-AgChyL was expressed in Escherichia coli. The pro-enzyme can be activated by trypsin. Activated AgChyL cleaves the synthetic chymotrypsin substrate succinyl-L-Ala-Ala-Pro-Phe-nitroanilide, but not two other synthetic chymotrypsin substrates or synthetic trypsin and elastase substrates. The potential role of AgChyL in the coordination of An. gambiae digestion is discussed.     

Storage and secretion of the peritrophic matrix protein Ag-Aper1 and trypsin in the midgut of Anopheles gambiae

The gene Ag-Aper1 encodes a peritrophic matrix (PM) protein from the mosquito Anopheles gambiae. Ag-Aper1 gene expression and protein localization in the mosquito midgut were studied during the course of a blood meal. Ag-Aper1 mRNA abundance does not change appreciably during the course of blood ingestion and digestion. Prior to a blood meal, the protein is stored in secretory vesicles of midgut epithelial cells. Moreover, Ag-Aper1 colocalizes to the same secretory vesicles as trypsin, indicating that these proteins use a common secretory pathway. Blood feeding triggers the secretion of vesicle contents into the midgut lumen, after which Ag-Aper1 is incorporated into the PM. Newly synthesized Ag-Aper1 protein was again detected within the midgut epithelial cells at 60 h after blood ingestion.     

Characterization of a carboxypeptidase A gene from the mosquito, Aedes aegypti

A gut-specific carboxypeptidase A gene (AeCPA) from the mosquito, Aedes aegypti, was cloned and characterized. The gene has an open reading frame that predicts a protein of 427 amino acids, 61% of which are identical to an Anopheles gambiae carboxypeptidase A sequence. AeCPA messenger RNA (mRNA) was not detected during larval and pupal development. In situ hybridization experiments indicated that AeCPA mRNA is expressed by posterior midgut epithelial cells. In sharp contrast to An. gambiae carboxypeptidase A gene expression, AeCPA mRNA accumulates to high levels only late ( approximately 16-24 h) after ingestion of a blood meal. The temporal profile of AeCPA gene induction is similar to that of Ae. aegypti late trypsin, suggesting the existence of common regulatory elements.     

Effects of malaria infection on vitellogenesis in Anopheles gambiae during two gonotrophic cycles

We report changes in the abundance of vitellogenin (Vg) mRNA, and concentration of haemolymph Vg and ovarian vitellin (Vn) in Anopheles gambiae following infection with Plasmodium yoelii nigeriensis. A parasite-induced reduction in Vg mRNA abundance was first detected 24 h after feeding on an infective blood meal, when ookinetes were invading the midgut. During a second gonotrophic cycle post-infection, developing oocysts reduced Vg mRNA abundance by up to 33% and the effect was detected from 2 h post blood meal. Concentrations of Vg were initially reduced by infection during the second cycle, as predicted from Vg mRNA measurements. However, after 24 h, excess Vg had accumulated in the haemolymph. This accumulation may be due to impaired uptake, since ovarian vitellin accumulation was significantly decreased by infection during both gonotrophic cycles.     

定量检测bcr-abl mRNA在慢性粒细胞白血病患者异基因造血干细胞移植后的意义

本研究确切了解CML患者异基因造血干细胞移植后bcr-abl mRNA的变化情况,为临床诊断早期复发提供实验依据。用实时荧光定量PCR方法检测15例慢性粒细胞白血病(CML)患者异基因造血干细胞移植前后78份外周血和骨髓标本的bcr-abl mRNA水平。结果表明,移植前患者的bcr-ablmRNA水平较高,中位数为29.303%;移植后1个月时检测bcr-abl mRNA水平较移植前大幅度降低,中位数为0;移植后1年内,连续多次检测bcr-abl mRNA水平,变化模式不一致,但6个月后的总体水平较6个月前明显降低,移植1年以上的受者绝大多数bcr-abl mRNA检测不到,或偶可检测到,但水平极低(0.007%、0.004%和0.021%),所检测受者的骨髓和外周血像均正常;同期骨髓与外周血bcr-abl水平无明显差异,且变化趋势一致。结论:CML患者移植后早期bcr-abl水平变化起伏较大,连续动态检测可明确其变化趋势,有助于判断移植效果、指导临床治疗,但6个月前检测到bcr-abl存在并不提示疾病复发;检测外周血bcr-abl或许更适于临床上对患者的随访。     

Distribution and kinetics of superantigen-induced cytokine gene expression in mouse spleen

The polyclonal stimulation of T cells by bacterial superantigens is involved in the pathogenesis of the toxic shock syndrome in certain staphylococcal and streptococcal infections. Here we describe the onset and kinetics of superantigen-induced cytokine production in situ in spleens of normal BALB/c mice monitored at the level of cytokine mRNA expression by in situ hybridization. Messenger RNAs for interleukin 2 (IL-2), interferon gamma, and tumor necrosis factors (TNF) alpha and beta were not expressed at detectable levels in spleens of unstimulated animals but became visible already 30 min after intraperitoneal application of 50 micrograms staphylococcal enterotoxin B. All mRNA levels showed peak expression approximately 3 h after injection and a slow decrease up to 24 h after injection. Expression of the mRNAs was restricted to the T cell-dependent area of the periarteriolar lymphatic sheets of the spleen. Interestingly, TNF-alpha mRNA showed a biphasic response, the early appearing mRNA had the same localization as the other mRNAs, whereas after 3 h TNF-alpha mRNA showed a broader distribution indicating a second cell population producing TNF-alpha. The expression of IL-2 and TNF proteins in the serum increased in parallel to the observed mRNA changes with a slight delay. The presence of macrophages was not required for the expression of the cytokine mRNAs in the spleen as the expression was unchanged in macrophage- depleted mice. Only the second phase of TNF-alpha mRNA expression was abrogated in such animals. The expression of all mRNAs was completely suppressed by prior administration of cyclosporin A. These data show that nonphagocytic cells are the essential superantigen-presenting cells in vivo and indicate that at least part of the pathogenetic TNF- alpha is T cell derived.     

Suppression of hepatic cytochrome p450-mediated drug metabolism during the late stage of sepsis in rats

The effects of polymicrobial sepsis on the activity and gene expression of hepatic microsomal cytochrome P450 (CYP) were examined. Rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). Liver and blood samples were taken 2, 6, and 24 h after CLP. The serum aminotransferase levels and lipid peroxidation increased 24 h after CLP. The hepatic concentrations of reduced glutathione and total CYP content decreased 24 h after CLP. The CYP1A1 activity and its protein level decreased 24 h after CLP. The CYP1A2 activity decreased 2 h and 24 h after CLP. Although the CYP2B1 mRNA expression level decreased 6 h and 24 h after CLP, the CYP2B1 activity and its protein level did not change in any of the experimental groups. The CYP2E1 activity and its protein level decreased 24 h after CLP. The CYP2E1 mRNA levels were lower at both 6 h and 24 h after CLP. The TNF-alpha mRNA expression level increased 2, 6, and 24 h after CLP. The iNOS mRNA expression level increased 24 h after CLP. These findings suggest that sepsis causes abnormalities in the microsomal drug-metabolizing function, particularly in the late stage, which is associated with higher level of oxidant stress and lipid peroxidation.     

Cecal ligation and double puncture impairs heat shock protein 70 (HSP-70) expression in the lungs of rats

Induction of the heat shock response may improve outcome from pathophysiological disturbances. This improvement is associated with and believed to result from expression of heat shock protein (HSP)-70. Therefore, we examined the temporal expression of HSP-70 in an animal model of acute respiratory distress syndrome (ARDS) secondary to fecal peritonitis. Specifically, we hypothesize that sepsis in rats impairs pulmonary HSP-70 expression. ARDS was induced in adolescent rats via cecal ligation and double puncture (2CLP). Sham-operated animals served as controls. Lung tissue was collected 0, 3, 6, 16, 24, and 48 h after 2CLP and sham operation. Northern blot hybridization analysis was performed to detect steady-state HSP-70 messenger ribonucleic (mRNA) levels. HSP-70 protein levels were determined via immunoblotting and immunohistochemistry. Mortality after 2CLP was 50% at 24 h and 75% at 48 h. Northern blot hybridization analysis revealed no significant change in steady-state HSP-70 mRNA levels in lung at any time after 2CLP. HSP-70 steady-state mRNA levels increased after sham operation and was higher than values in 2CLP at 6, 16, and 24 h. HSP-70 protein levels did not change over time in either group. Thus, the expression of HSP-70 does not change after 2CLP. Although lack of an increase in protein levels may be adaptive after sham operation, it is not appropriate after 2CLP. Therefore, failed HSP-70 expression represents a form of pulmonary epithelial dysfunction that may contribute to lung injury in sepsis.     

Hypocalcemia increases and hypercalcemia decreases the steady-state level of parathyroid hormone messenger RNA in the rat.

To examine the effects of serum calcium concentrations on PTH biosynthesis, rats were made hyper- (serum total calcium, approximately 3.5 mM) or hypocalcemic (approximately 1.25 mM) and steady-state levels of PTH mRNA in parathyroid cells were measured by the primer extension method using a 32P-labeled synthetic oligomer. PTH mRNA levels increased about twofold in the rats made slightly hypocalcemic by infusion of calcium-free solution and decreased slightly in those made hypercalcemic by CaCl2 infusion (120-150 mumol/h) compared with the levels present in nonfasting control rats. Infusion of calcitonin (0.5 U/h) or EGTA (90 mumol/h) with calcium-free solution increased PTH mRNA levels further (two- to sevenfold) above the levels present in animals infused with calcium-free solution alone. These changes in PTH mRNA levels were observed after 48- but not 24-h infusion, and there was an inverse correlation between PTH mRNA levels and serum calcium concentrations. The results suggest that changes in serum calcium concentrations in the near physiological range regulate the biosynthesis of PTH by affecting steady-state levels of PTH mRNA when hypercalcemia or hypocalcemia continues for a relatively long period.