Astroglial expression of human alpha(1)-antichymotrypsin enhances alzheimer-like pathology in amyloid protein precursor transgenic mice

Proteases and their inhibitors play key roles in physiological and pathological processes. Cerebral amyloid plaques are a pathological hallmark of Alzheimer's disease (AD). They contain amyloid-ss (Ass) peptides in tight association with the serine protease inhibitor alpha(1)-antichymotrypsin.(1,2) However, it is unknown whether the increased expression of alpha(1)-antichymotrypsin found in AD brains counteracts or contributes to the disease. We used regulatory sequences of the glial fibrillary acidic protein gene(3) to express human alpha(1)-antichymotrypsin (hACT) in astrocytes of transgenic mice. These mice were crossed with transgenic mice that produce human amyloid protein precursors (hAPP) and Ass in neurons.(4,5) No amyloid plaques were found in transgenic mice expressing hACT alone, whereas hAPP transgenic mice and hAPP/hACT doubly transgenic mice developed typical AD-like amyloid plaques in the hippocampus and neocortex around 6 to 8 months of age. Co-expression of hAPP and hACT significantly increased the plaque burden at 7 to 8, 14, and 20 months. Both hAPP and hAPP/hACT mice showed significant decreases in synaptophysin-immunoreactive presynaptic terminals in the dentate gyrus, compared with nontransgenic littermates. Our results demonstrate that hACT acts as an amyloidogenic co-factor in vivo and suggest that the role of hACT in AD is pathogenic.     

应用双色荧光原位杂交技术对转基因小鼠进行整合位点的染色体定位

目的 建立一种高度灵敏、特异的双色荧光原位杂交(dual-color fluorescence in situ hybridization,D-FISH)技术,对两种双阳性转基因小鼠外源基因进行整合位点的染色体定位.方法 对一只整合单纯疱疹病毒胸苷嘧啶激酶(herpes simplex virus thymidine kinase,HSV-tk)/增强型绿色荧光蛋白(enhanced green fluorescence protein,eGFP)的转基因小鼠以及两只整合RNA干扰载体(RNA interference,RNAi)的β654地中海贫血模型小鼠进行实验,脾脏细胞经培养后获得中期分裂相标本片,各加入适量生物素、地高辛标记探针混合液杂交,分别用罗丹明红色荧光抗体及FFIE绿色荧光抗体进行D-FISH检测.结果 两种转基因小鼠均能在同一个分裂相上同时检测到双色荧光信号.其中,HSV-tk/eGFP双阳性小鼠的分裂相上出现较强的绿色HSV-tk信号和红色eGFP信号,分别定位于染色体2E5-G3及8A2-A4;β654/RNAi双阳性小鼠检测到红色β654荧光信号及绿色RNAi荧光信号.经定位分析,β654均整合在染色体7D3-E2,RNAi病毒载体则是随机整合,其中一只鼠主要整合在1281位点,而另一只鼠主要整合在染色体1E2.3-1F、3A3两个位点.结论 用自行制备的DNA探针建立了高度灵敏、特异的D-FISH技术,同时结合G显带对双阳性转基因小鼠进行染色体基因定位.该技术平台的建立对于转基因动物和基因治疗动物模型的研究具有非常重要的意义.     

Hydrophobic interaction chromatography of human serum alpha1-antitrypsin and alpha1-acid glycoprotein

The relative hydrophobicity of human serum alpha1-antitrypsin (AAT) and alpha1-acid glycoprotein (AGP) in comparison to various reference proteins was determined by hydrophobic interaction chromatography (HIC). Apolar character of glycoproteins was generated using three different cosmotropic salts, ammonium sulfate, sodium sulfate, sodium citrate and isocratic, or reversed linear gradient elution techniques. Human serum AAT and AGP showed different apolar properties on Fractogel EMD phenyl and propyl columns modulated either by the type and concentration of cosmotropic salts, or by the pH of the mobile phase. According to its higher carbohydrate content AGP proved to be more polar than AAT. Human serum AAT and AGP were pre-separated by Cibacron Blue F3G-A dye ligand affinity chromatography and based on their different hydrophobicity were fractionated and purified by HIC.     

Inactivation of urinary kallikrein by alpha1-antitrypsin

Summary Proteolytic activity, with azocasein as substrate in the presence and absence of 0.4 IU kallikrein (Padutin) was measured in the 24 h urine fractions of 100 ambulatory patients with hypertension, proteinuria or haematuria. Urinary protein and alpha₁-antitrypsin concentration have also been assayed. There was an inverse relationship between kallikrein activity and urinary alpha₁-antitrypsin concentration (r=0.84;y=39.2e ^–0.009x). Furthermore, kallikrein activity and 24 h urinary alpha₁-antitrypsin excretion were also inversely correlated (r=0.81;y=886.4e ^–0.011x). Our data suggest an inactivation of renal kallikrein by urinary alpha₁-antitrypsin.This work was supported by the Deutsche Forschungsgemeinschaft (Ho 781/3-2)     

Expression of the human α-1-antitrypsin gene in transgenic rats

A genetic-engineering construction is developed to provide expression of the full-size cDNA of human α-1-antitrypsin controlled by the promoter elements of the murine metallothionein-1 gene. Transgenic animals were obtained by microinjection of pAT-MT plasmid into fertilized rat oocytes. The genome of such animals contains the sequences of human α-1-antitrypsin-cDNA revealed by blot- and dot-hybridization. Integrated human α-1-antitrypsin-cDNA copies are found with a high frequency (9 out of 22 animals) in the second-generation offspring obtained after crossings of transgenic animals. A protein with antigenic specificity of human a-1-antitrypsin is found in the serum of transgenic rats by the immunoblotting method. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 7, pp. 81–83, July, 1995 Presented by A. N. Klimov, Member of the Russian Academy of Medical Sciences     

Human alpha 1-antitrypsin therapy induces fatal anaphylaxis in non-obese diabetic mice

Previous studies have shown that human alpha-1 antitrypsin (hAAT) gene delivery prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. Furthermore, hAAT protein administration prolongs acceptance of islet allografts. Therefore, we evaluated the use of purified hAAT protein therapy to prevent T1D in NOD mice. Female NOD, non-obese resistant (NOR), Balb/c and C57BL/6 mice were injected intraperitoneally with vehicle alone or vehicle containing hAAT, human albumin or mouse albumin (or mg/injection/mouse; 2x/week). Preparations of clinical-grade hAAT included API(R), Aralast, Prolastin and Zemaira. Surprisingly, hAAT administration was associated with a high rate of fatal anaphylaxis. In studies seeking T1D prevention at 4 weeks of age, 100% mice died after six injections of hAAT. When administrated at 8-10 weeks of age, most (80-100%) NOD mice died following the fourth injection of hAAT, while 0% of Balb/c and C57BL/6 mice and 10% of NOR mice died. Interestingly, repeated injections of human albumin, but not mouse albumin, also induced sudden death in NOD mice. Antibodies to hAAT were induced 2-3 weeks after hAAT administration and death was prevented by treatment with anti-platelet-activating factor along with anti-histamine. In studies of disease reversal in NOD mice, using the four pharmaceutical grade formulations of hAAT, anaphylactic deaths were observed with all hAAT preparations. The propensity for fatal anaphylaxis following antigenic administration appears to be NOD- but not hAAT-specific. The susceptibility of NOD mice to hypersensitivity provides a significant limitation for testing of hAAT. Development of strategies to avoid this unwanted response is required to use this promising therapeutic agent for T1D.     

生物素标记探针原位杂交法检测细胞中c-myc基因的表达

本文采用生物素标记的c-mye基因作探针，通过细胞原位分子杂交技术，对人舌癌、膀胱癌和正常细胞中c-myc基因的转录量进行了定性和半定量分析。结果表明与正常细胞相比，舌癌和膀胱癌细胞中c-myc基因异常高度表达。     

Complex chromosome rearrangement involving chromosomes 1, 4 and 16 revealed by fluorescence in situ hybridization

A 7-year-old boy with mental retardation had apparently balanced reciprocal translocations, involving the telomeric regions of chromosomes 1p and 4q, which was detected by routine chromosome analysis. Fluorescence in situ hybridization (FISH) was used and also revealed the telomeric region of chromosome 16p to be involved in a still apparently balanced translocation-complex, impossible to discover with classical cytogenetic analysis. We want to emphasize the importance of FISH in detecting small chromosomal aberrations. We discuss whether the abnormal phenotype is caused by unbalanced karyotype with cryptic undetected translocations or small deletions or mutations in the translocation-breakpoints.     

Differential expression of human telomerase catalytic subunit mRNA by In situ hybridization in pheochromocytomas

In pheochromocytomas, it is very difficult to predict malignant potential by conventional histology or immunohistochemical and molecular markers. We investigated the expression of human telomerase catalytic component (hTERT) mRNA, hTERT protein, Ki-67 antigen, and p27^kip1 in pheochromocytomas (27 benign, 7 suspected malignant, and 7 malignant), and evaluated the possibility of expressions of these proteins, and hTERT mRNA serve as diagnostic markers for predicting the biological behavior of these tumors. All tumors showed the classical histology and typical immunohistochemical pattern. By in situ hybridization, hTERT mRNA was expressed in 5/7 malignant tumors (defined as the presence of metastasis and/or extensive local invasion) as compared with 3/27 benign tumors. We examined the hTERT by immunohistochemistry to confirm the mRNA. hTERT mRNA expression was correlated with hTERT protein expression. All benign tumors exhibited no immunopositivity or <1% of cells stained for Ki-67 antigen. Six out of seven malignant tumors have shown either hTERT mRNA expression or Ki-67 immunoreactivity While no statistical difference in p27^kip1 expressions was observed among benign, malignant, and suspected malignant tumors, there was a statistical difference between the normal adrenal medulla samples and tumors (p<0.001). Thus, hTERT mRNA detection by in situ hybridization, hTERT expression, and Ki-67 antigen expression are all useful tools for differentiating malignant from benign pheochromocytomas.     

Human immunoglobulin VH and VK repertoire revealed by in situ hybridization.

We report in this paper the first analysis of the expression pattern of Ig VH and VK families in human adult normal peripheral B lymphocytes, by in situ hybridization using specific VH1 to VH6 and VK1 to VK4 probes, which cover the known human V gene families reported to date. The major families were VH3 and VK1, with the respective gradient VH3 greater than VH4 greater than VH1 greater than VH5 greater than VH6 greater than VH2, and VK1 greater than VK3 greater than VK4 greater than VK2. Using a large sampling of EBV clones, we found that the pattern of VH and VK family usage was similar. The expression level correlated fairly with the estimated gene number for the VH, but diverged noticeably for the K chains. Taken together with the fact that the level of light chain expression (K + lambda) was about two-fold that of heavy chains, these results suggest that the VH and the VK repertoires are not regulated by a similar selective process.     

Characterization of alpha1-antitrypsin in the inclusion bodies from the liver in alpha 1-antitrypsin deficiency.

alpha1-antitrypsin was isolated from periodic acid-Schiff-positive inclusion bodies from the hepatocytes of patients with alpha1-antitrypsin deficiency and further purified to enable more detailed chemical analysis. Amino acid and cyanogen bromide fragmentation studies showed a close similarity between hepatic and serum (PiMM) antitrypsin in contrast to the carbohydrate analysis, which revealed markedly deficient glycosylation of hepatic antitrypsin. A complete lack of sialic acid and a relative deficiency of all other carbohydrate components could fully explain the difference of approximately 6000 daltons in molecular size between the two proteins. The accumulation of hepatic globules is probably related to the physical properties of the defective antitrypsin, which include marked insolubility and tendency toward aggregation. The results strongly suggest an abnormal amino acid sequence in the peptide chain of the deficient antitrypsin. The interference with glycosylation may be related to steric hindrance.     

Analysis of human pituitary tumors by in situ hybridization

A procedure for performing in situ hybridization histochemistry (ISH) on frozen and paraffin sections of human pituitary tissues is described. The use of oligonucleotide probes for hPRL and hGH labeled with 35S allowed detection of a specific messenger RNA in frozen and paraffin sections. This technique can be combined with immunochemistry to localize both the gene product and the hormone(s) produced by specific cells and should be very helpful in the characterization of normal and neoplastic human pituitary cells.     

Analysis of human IgA subclasses by in situ hybridization and combined in situ hybridization/immunohistochemistry.

In this report we describe a method for the analysis of the cellular expression of the two human IgA subclasses by in situ hybridization (ISH). The technique permits the detection of specific mRNA in individual cells using 35S-labeled oligonucleotide probes without any detectable cross-hybridization between the subclasses. This method was applicable both on cytospin preparation of peripheral blood mononuclear cells as well as in formalin-fixed, paraffin-embedded tissue sections. Furthermore, we describe a combined ISH/immunohistochemistry technique for the simultaneous detection of IgA subclass mRNA and protein at the single cell level. Examination of tissue sections from tonsils revealed a striking localization of labeled cells within the germinal center of some of the lymphoid follicles. The implications of this novel finding and the development of the method are discussed.     

Role of osteopontin in the rodent inner ear as revealed by in situ hybridization

Osteopontin (OPN) is considered to be a non-collagenous bone matrix protein that is involved in the ossification process. However, OPN has recently been observed in ectopic sites such as the kidney and nervous tissue. In the present study, the expression of OPN mRNA was examined in the rat and mouse inner ear by nonradioisotopic in situ hybridization. Signals of OPN mRNA were observed in the marginal cells of the stria vascularis, spiral ganglion cells, vestibular sensory hair cells, and vestibular dark cells. OPN protein was detected only in the otoconia by immunohistochemistry. The presence of OPN mRNA in the cochlea and vestibular dark cells may indicate that OPN is involved in the regulation of ions in the inner ear fluid. Findings in the saccule and utricle suggest that OPN is one of the protein components of the rodent otoconia and that the vestibular sensory hair cells are involved in the production of otoconia.