A sequence from drosophila DNA cross-hybridizing with a mouse class I H-2 Gene: absence of relevant nucleic acid or amino acid sequence homology

Following serological data (1) showing cross-reactivity of drosophila surface antigens with anti-H-2 and mouse beta 2-microglobulin (beta 2-m) antisera, we have looked for homologous sequences in the drosophila genome by low stringency hybridization with mouse H-2 and beta 2-m probes. A 240 bp drosophila DNA segment cross-hybridizing with a H-2 probe, was isolated and sequenced. No homology at the nucleotide or amino acid levels was found with the mouse probe which was used, except for a perfectly matching 16-mer containing 11 G-C, which might be responsible for the cross-hybridization. Therefore, our present data do not support the existence of class I H-2 and/or beta 2-m related gene sequences in the drosophila genome.     

H-2 I alloantigens and recall of memory cytotoxic responses

AQR mice were immunized with H-2K and H-2 I encoded alloantigens presented by (Ax6R)F₁ splenocytes. Spleen cells from these alloimmune mice were subsequently restimulated in vitro with B10.A lymphocytes and/or B10.T(6R) lymphocytes, thus presenting them with the immunizing H-2K and H-2 I alloantigens independently. When stimulated with B10.A lymphocytes, alloimmune lymphocytes develop significant cytotoxicity against the immunizing H-2K target antigens. When stimulated with a similar number of B10.T(6R) spleen cells, alloimmune lymphocytes undergo a prominant proliferative response, but develop little, if any, cytotoxicity against the immunizing H-2 K target antigens. The most efficient restimulation of cytotoxicity occurs when the alloimmune spleen cells are simultaneously restimulated by B10.A and B10.T(6R) lymphocytes. Stimulation with the immunizing H-2 I alloantigens alone is not sufficient for regeneration of detectable cytotoxic responses from alloimmune spleen populations. Stimulation with the immunizing H-2K alloantigens alone appears to be both necessary and sufficient to stimulate alloimmune cytotoxic responses. Although the immunizing H-2 I alloantigens are apparently not required to generate alloimmune cytotoxic responses, they markedly potentiate the cytotoxic responses induced by the immunizing H-2K alloantigens.     

Coumarins from the Malagasy Cedrelopsis rakotozafyi Cheek and Lescot (Rutaceae)

A phytochemical analysis of the bark of the Malagasy Cedrelopsis rakotozafyi Cheek and Lescot (Rutaceae) yielded the novel 8-hydroxy-7-methoxy-6-(2R-hydroxy-3-methylbut-3-enoxy)-2H-1-benzopyran-2-one, and 7-hydroxy-6-(2R-hydroxy-3-methylbut-3-enoxy)-2H-1-benzopyran-2-one, 5,6,7-trimethoxy-2H-1-benzopyran-2-one, scoparone, scopoletin, lupeol and β-amyrin. The placement of Cedrelopsis within the Rutaceae is supported phytochemically by the typically Rutaceous coumarins isolated.     

Lymphocyte recognition of H-2 antigen in liposomes

Liposomes containing purified H-2K^k will specifically stimulate generation of a secondary allogeneic cytolytic T lymphocyte response. Effective recognition was found to depend on the structure of the liposomes. Including detergent-insoluble plasma membrane matrix during formation resulted in liposomes having two- to fourfold more activity than those prepared using just lipid and H-2.     

Biochemical analysis of an MHC-linked hematopoietic cell surface antigen,Qa-2

The region of the murine 17th chromosome telomeric to H-2D encodes a group of serologically defined cell surface antigens termed Qa-1-5. These antigens are of interest because their expression is restricted to hematopoietic cells. In addition, the molecular weight and subunit structure (ie, association with β-2 microglobulin) of Qa-2 molecules are similar to H-2 and TL antigens. In the present studies, we have prepared isotopically labeled Qa-2 and H-2 molecules from mitogen-stimulated C57BL/6 spleen cells. Comparative peptide mapping of tryptic peptides from Qa-2 and H-2 molecules (K^b, D^bK^k, D^d) reveal that Qa-2 has a unique primary structure. However, considerable homology is indicated since 30–40% of the Qa-2 peptides cochromatograph with peptides derived from H-2K^b, H-2D^b, H-2K^k, and H-2D^d. Studies by other investigators have demonstrated that similar levels of structural homology are observed when H-2K, H-2D, and H-2L tryptic peptides are analyzed. We conclude from these studies that the Qa-2 alloantigen is structurally related to a class of cell surface molecules (ie, H-2) that play critical roles in immune recognition processes. These data further suggest that the genes encoding Qa-2 and H-2 molecules have arisen from a common primordial gene.     

Mouse subspecies differentiation and H-2 polymorphism

Four major groups of Mus musculus subspecies are analysed from the genetical view point. A number of genetic traits exhibited polymorphism. Most were common among domesticus, bactrianus and castaneus subspecies, while others were musculus subspecies-specific. This suggests that the earliest differentiation was between domesticus and musculus . Mouse class I MHC was compared in these two lines. Immunological and molecular analyses of H-2K molecules indicated that these two groups of mouse have several common haplotypes of class I molecules. Phenotype frequencies of these haplotypes are considerably different in each subspecies. "Overdominance selection" in class I H-2 is one of the possible explanations for this.     

两个NIH小鼠种群对不同种类的乙肝疫苗免疫应答效应的比较

目的比较两种不同的NIH小鼠种群基因组DNA中H-2q单倍型的百分比,对重组基因乙型肝炎疫苗（酿酒酵母）、重组基因乙型肝炎疫苗（CHO）、重组基因乙型肝炎疫苗（汉逊酵母）和治疗性乙型肝炎疫苗（乙克）免疫应答的影响。方法检测乙肝疫苗免疫效力用酶标法,测定乙肝疫苗的ED50值,再通过微量细胞毒法和PCR方法检测小鼠的H-2单倍型,计算不同品系小鼠H-2q的百分数,得出乙肝疫苗与不同动物种群的相关性。结果经验证,经1号NIH小鼠检测结果证实,重组基因乙型肝炎免疫效力依次为CHO〉汉逊〉酿酒,疗性乙型肝炎疫苗（乙克）效力检测合格,但经2号NIH小鼠检测结果显示除CHO疫苗合格外,其他三种疫苗检测结果均为不合格。基因检测结果为,1号种群NIH鼠含有H-2q型基因的百分比是96%,而2号种群NIH小鼠含有H-2q型基因基因百分比为30%,因此不同的q型基因百分比导致了不同的检测结果。结论根据本研究结果表明虽然NIH鼠的H-2单倍型中有q型基因,但不同来源的小鼠种群之间会存在差异,经不同的环境饲养后,小鼠的基因会发生不同的变化,因此在进行生物制品检定时,要注意不同品系小鼠及同一品系小鼠不同种群之间的差异对试验本身的影响。建立一种＂动物免疫＂的概念。     

血卟啉在H-22肝癌荷瘤小鼠体内的分布

目的探讨声敏剂血卟啉衍生物在肿瘤组织中的富集情况,以便在给药后超声结合血卟啉治疗肿瘤时选择最佳的超声处理时间。方法H-22肝癌荷瘤小鼠尾静脉注射HpD后,不同时间点取材,采用荧光分光光度法测定不同组织提取液中HpD的荧光强度,研究HpD在不同组织中的分布以及代谢变化。结果给药后2 h肿瘤组织以外的其他各组织内(血浆、肝、肾、皮肤、肌肉)血卟啉含量均达到其代谢过程的最高点,后逐渐下降;肿瘤组织中的HpD含量注射后不断上升,6 h时达到顶峰,随后又开始下降,10~24 h代谢比较缓慢,表现为肿瘤组织中对HpD的滞留作用,24 h时肿瘤组织中的HpD含量高于其他各组织,48~72 h趋于稳定在较低水平。结论提出了不同部位的肿瘤应选择各自适当的时间点进行超声处理。     

Genetic Control of Immune Responses to a Synthetic Fimbrial Antigen of Actinobacillus actinomycetemcomitans

The incidence of infection by Actinobacillus actinomycetemcomitans, one of the important pathogens in human periodontal diseases, has been reported to be associated with racial background and genetic factors. We attempted to determine the genetic regulation of immune responses to A. actinomycetemcomitans fimbriae, an attachment factor, using various inbred strains of mice. For this purpose, we synthesized an oligopeptide antigen using the amino acid sequence of the fimbriae and conjugated this antigen to branched lysine polymer resin beads. After immunization with the synthetic A. actinomycetemcomitans fimbrial antigen, serum antibody levels and the delayed-type hypersensitivity (DTH) reaction to the antigen were measured by enzyme-linked immunosorbent assay (ELISA) and footpad swelling responses, respectively. The strains of mice found to be high-IgG responders to the antigen were B10.HTT, B10.RIII, B10A (5R) and B10.S (9R). These results indicate that mice with E_β^s: E_α^k, E_β^r: E_α^r and E_β^b: E_α^k respond strongly to the synthetic peptide. All of the high-IgG responders showed a high DTH response. A cell transfer experiment confirmed that CD4 T cells mediated with a DTH response to the synthetic peptide. Thus, the results of this study demonstrate that the immune responses to A. actinomycetemcomitans fimbriae are genetically controlled.     

植物铁代谢及植物铁蛋白结构与功能研究进展

铁在生命过程中起着很重要的作用,植物缺铁后叶绿素合成受阻而导致的黄化症状已成为世界性植物营养失调问题。与之相随,人类铁营养的缺乏也极为严重,因此研究植物铁代谢并且开发安全、天然、高效的补铁因子具有重要的意义。到目前为止,在已经发现的植物中,只有豆科类植物是将其种子中～90％的铁储藏在铁蛋白中,所以来源于豆科类植物的铁蛋白是一个理想的补铁资源。与动物铁蛋白相比,植物铁蛋白具有两个显著的特点：首先,植物铁蛋白在其N端具有一个独特的EP肽段;其次,植物铁蛋白只含有H型亚基,且有两种不同的H型亚基组成。主要阐述有关植物铁代谢及铁蛋白的结构、功能的最新研究进展。     

Southern杂交方法用于近交系小鼠H-2基因检测

目的建立一种简便、快捷、准确的检测方法用于近交系小鼠的遗传检测。方法根据近交系小鼠的H-2基因序列设计相应的探针,并标记生物素,利用微孔板Southern杂交技术,使探针与模板DNA杂交,再加入亲和素标记的辣根过氧化物酶进行酶显色反应,通过酶标仪检测杂交结果,以确定近交系小鼠的基因型。结果57BL／6和C57B：／10为H-2^b型;DBA／2和Scid为H-2^d型;615和C3H为H-2^k型;NCPC／2、TA1、TA2和T739均为H-2^b型。结论通过Southern杂交检测可以确定近交系小鼠的基因型。该检测方法简便、易行,检测结果客观,可以应用于近交系小鼠的遗传检测。     

泡疹病毒特异性MHC限制的T辅助细胞杂交瘤的制备及其特性的初步研究

将HSV-I免疫过的小鼠淋巴结细胞在体外扩增,收集富集HSV-I抗原特异性的T细胞与BW 5147胸腺瘤细胞融合。对融合后产生的杂交瘤细胞进行抗原特异性及MHC识别限制选择。结果所得的T杂交瘤细胞具有在HSV-I特异刺激下,MHC一致时,有分泌IL-2的能力,并在体内耳廓病毒清除中发挥作用。它们是HSV-I抗原特异性,H-2~d限制的,具有辅助活性的T杂交瘤细胞。     

Developmental expression of insulin-like growth factor II receptor (IGF-IIR) in congenic mouse embryonic lungs: Correlation between IGF-IIR mRNA and protein levels and heterochronic lung development

Embryonic lung maturation in the H-2 congenic pair, B10.A and B10, proceeds at different rates. The dependence of this heterochronic development on maternal haplotype suggests the involvement of a parentally imprinted gene. Since B10.A (H-2^b) and B10 (H-2^b) mice are genetically identical except for a 3-18 cM region of chromosome 17 that includes the H-2 complex, we sought a promising candidate gene(s) involved in regulating the rate of lung development from genes encoded in this region. The best candidate is the gene encoding the type II insulin-like growth factor receptor (IGF-IIR), whose ligand is the growth factor IGF-II. Only the maternal copy of this gene is expressed in postimplantation embryos. This receptor does not appear to transduce mitogenic signals; instead, IGF-IIR appears to regulate the levels of its ligand available to the growth-promoting type I IGF receptor (IGF-IR). Using in situ hybridization and indirect immunofluorescence, we demonstrate that IGF-IIR mRNA and protein are localized throughout the pulmonary mesenchyme, as well as in branching epithelia of the pseudoglandular and canalicular stages. We also examined the levels of IGF-IIR mRNA and protein expression by RNase protection assay and ligand blotting during the embryonic period of lung development in B10.A and B10 mice, and found that there is a highly significant positive correlation of IGF IIR levels with progressive development in both strains. Further, slower developing B10.A lungs contain significantly higher levels of IGF-IIR mRNA and protein than the more rapidly developing B10 lungs. These results suggest that haplotype-dependent elevation of IGF-IIR levels reduces the available concentration of IGF-II, resulting in a decreased rate of morphogenesis in B10.A mice. Heterochronic lung maturation, then, appears consequent to variable extracellular levels of this important growth factor. These results may be of clinical importance to predicting susceptibility to Respiratory Distress Syndrome in prenatal newborns. © Wiley-Liss, Inc.     

Deletion of hypoxia-inducible factor prolyl 4-hydroxylase 2 in FoxD1-lineage mesenchymal cells leads to congenital truncal alopecia

Hypoxia-inducible factors (HIFs) induce numerous genes regulating oxygen homeostasis. As oxygen sensors of the cells, the HIF prolyl 4-hydroxylases (HIF-P4Hs) regulate the stability of HIFs in an oxygen-dependent manner. During hair follicle (HF) morphogenesis and cycling, the location of dermal papilla (DP) alternates between the dermis and hypodermis and results in varying oxygen levels for the DP cells. These cells are known to express hypoxia-inducible genes, but the role of the hypoxia response pathway in HF development and homeostasis has not been studied. Using conditional gene targeting and analysis of hair morphogenesis, we show here that lack of Hif-p4h-2 in Forkhead box D1 (FoxD1)-lineage mesodermal cells interferes with the normal HF development in mice. FoxD1-lineage cells were found to be mainly mesenchymal cells located in the dermis of truncal skin, including those cells composing the DP of HFs. We found that upon Hif-p4h-2 inactivation, HF development was disturbed during the first catagen leading to formation of epithelial-lined HF cysts filled by unorganized keratins, which eventually manifested as truncal alopecia. Furthermore, the depletion of Hif-p4h-2 led to HIF stabilization and dysregulation of multiple genes involved in keratin formation, HF differentiation, and HIF, transforming growth factor β (TGF-β), and Notch signaling. We hypothesize that the failure of HF cycling is likely to be mechanistically caused by disruption of the interplay of the HIF, TGF-β, and Notch pathways. In summary, we show here for the first time that HIF-P4H-2 function in FoxD1-lineage cells is essential for the normal development and homeostasis of HFs.     

Biochemical analysis of related,independently arising histocompatibility mutants: bm17 and KB-98 enlarge the “bg series” of H-2Kb mutants

The “bg” series of MHC mutations is the most prevalent type of mutations of K^b in C57BL/6 mice screened by reciprocal tail skin grafting. The basis for identification of this series of mutations is the incompatibility of grafts between the parental B6 and the mutant. This series takes the longest to reciprocally reject the skin grafts. The series can be subdivided into “bg 1” and “bg 2” groups based on K^b-restricted recognition of virus-infected mutant target cells. The biochemical basis for these mutations are amino acid substitutions at residues 116 and 121 of the K^b transplantation antigen. These substitutions do not alter monoclonal antibody binding sites. The structural basis of MAb binding and the genetic basis of the mutation are discussed. This study was supported in part by USPHS Grants AI-07289, AI-10702, NCI P30-CA-13330, American Cancer Society Grant IM-236, and American Cancer Society Fellowship PF-2126. Stanley G. Nathenson is a member of the Irvington House Institute for Medical Research.     

H-2Db四聚体的制备及其在检测LCMV特异性CD8+ T细胞中的应用

MHC四聚体技术是研究抗原特异性淋巴细胞应答的关键技术之一。为研究H-2Db基因型(如C57BL/6)小鼠的特异性CD8+ T细胞免疫应答, 需要建立H-2Db四聚体制备技术平台。首先以RT-PCR方法克隆H-2Db重链基因的cDNA, 进而构建H-2Db胞外域与生物素化酶BirA底物肽(BSP)融合蛋白的表达载体, 并在大肠杆菌中获得表达。在LCMV GP33-41抗原肽(KAVYNFATC, KAV)和人β2-微球蛋白存在时, 通过稀释法复性获得H-2Db/KAV单体。该单体经生物素化并纯化后与PE-链亲和素按4: 1的比例混合, 即形成四聚体。通过流式细胞术检测经KAV肽免疫的C57BL/6小鼠体内的LCMV特异性CD8+ T细胞的频率, 结果表明在外周血、引流淋巴结和脾脏中均可检测到一定频率的LCMV特异性CD8+ T细胞, 其中以对外周血标本染色的效果最佳。成功建立了小鼠H-2Db四聚体制备技术平台, 为监测及分析基于H-2Db基因型小鼠的实验性免疫治疗创造了条件。     

Effects of Protein Kinase Inhibitors on Morphology and Function of Cultured Bovine Adrenal Chromaffin Cells: KN-62 Inhibits Secretory Function by Blocking Stimulated Ca2+ Entry

Abstract: In cultured bovine adrenal chromaffin cells, a nonselective protein kinase inhibitor, staurosporine, inhibits secretory function and induces neurite outgrowth. In the present study, effects of other nonselective protein kinase inhibitors (K-252a, H-7, and H-8) and reportedly selective protein kinase inhibitors (KN-62 and chelerythrine chloride) were examined on bovine adrenal chromaffin cell morphology, secretory function, and ⁴⁵Ca²⁺ uptake. Treatment of chromaffin cells with 10 µ M K-252a, 50 µ M H-7, or 50 µ M H-8 induced changes in cell morphology within 3 h; these compounds also induced a time-dependent inhibition of stimulated catecholamine release. Chelerythrine chloride, a selective inhibitor of Ca²⁺/phospholipid-dependent protein kinase, did not induce outgrowth or inhibit secretory function under our treatment conditions. KN-62, a selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMK II), significantly inhibited stimulated catecholamine release (IC₅₀ value of 0.32 µ M ), but had no effect on cell morphology. The reduction of secretory function induced by 1 µ M KN-62 was significant within 5 min and rapidly reversible. Unlike H-7, H-8, and staurosporine, KN-62 significantly inhibited stimulated ⁴⁵Ca²⁺ uptake. KN-04, a structural analogue of KN-62 that does not inhibit CaMK II, inhibited stimulated ⁴⁵Ca²⁺ uptake and catecholamine release like KN-62. These studies indicate that KN-62 inhibits secretory function via the direct blockade of activated Ca²⁺ influx. The nonselective inhibitors, K-252a, H-7, H-8, and staurosporine, inhibit secretory function by another mechanism, perhaps one involving alterations in the cytoskeleton.     

Synthesis and Evaluation of Anticonvulsant Activity of Some Schiff Bases of 7‐Amino‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one

A variety of 1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one azomethines and 1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one benzamide were prepared, characterized and evaluated for the anticonvulsant activity in the rat using picrotoxin‐induced seizure model. The prepared 1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one azomethine derivatives emerged potentially anticonvulsant molecular scaffolds exemplified by compounds, 7‐{(E)‐[(4‐nitrophenyl)methylidene]amino}‐5‐phenyl‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one, 7‐[(E)‐{[4‐(dimethylamino)phenyl]methylidene}amino]‐5‐phenyl‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one, 7‐{(E)‐[(4‐bromo‐2,6‐difluorophenyl)methylidene]amino}‐5‐phenyl‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one and 7‐[(E)‐{[3‐(4‐fluorophenyl)‐1‐phenyl‐1H‐pyrazol‐4‐yl]methylidene}amino]‐5‐phenyl‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one. All these four compounds have shown substantial decrease in the wet dog shake numbers and grade of convulsions with respect to the standard drug diazepam. The most active compound, 7‐[(E)‐{[4‐(dimethylamino)phenyl]methylidene}amino]‐5‐phenyl‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one, exhibited 74 % protection against convulsion which was higher than the standard drug diazepam. Furthermore, to identify the binding mode of the interaction amongst the target analogs and binding site of the benzodiazepine receptor, molecular docking study and molecular dynamic simulation were carried out. Additionally, in silico pharmacokinetic and toxicity predictions of target compounds were carried out using AdmetSAR tool. Results of ADMET studies suggest that the pharmacokinetic parameters of all the target compounds were within the acceptable range to become a potential drug candidate as antiepileptic agents.