HEAT SHOCK PROTEIN MICRO-ENCAPSULATION AS A DOUBLE TOOL FOR THE IMPROVEMENT OF NEW GENERATION VACCINES

ABSTRACT

The modern vaccinology encompasses the recombinant DNA technology, protein and carbohydrate chemistry to obtain safe molecularly defined vaccines. Nevertheless most of the vaccines are poorly immunogenic because a large number of antigens are membrane proteins and consequently they are not present in their active conformation in the vaccine. Others are not as potent because they contain only B epitopes and therefore, cannot stimulate cellular memory. We have been studying the characteristics of the recombinant heat shock protein 18kDa-hsp from Mycobacterium leprae as an alternative carrier protein with a T epitope source to enhance the activity of these second generation vaccines. Here we proved that the 18kDa-hsp acted as carrier, without masking the activity of the carried antigen, with similar immune stimulatory effect when compared with ODN1668. Supramolecular aggregates of 18kDa-hsp and Mice serum albumin (MSA) were obtained using glutaraldehyde as cross linker. The Neisseria meningitides serogroup C polysaccharide (PSC, a B epitope) and the carrier protein 18kDa-hsp were co-encapsulated within Soybean phosphatidylcholine liposomes (SPC: Cho : α-Toc, 22 : 5 : 0.18 molar ratio, respectively). These liposomes were prepared in MPB buffer (20 mM phosphate, 295 mM mannitol pH 7.2) in the presence or absence of the ODN1668, TCCATGACGTTCCTGATGCT. When mice were injected with 18kDa-hsp-MSA no antibody against the MSA was observed. This means that the 18kDa-hsp acted as carrier, without masking the carried protein immune activity. Stable liposomes of 150 nm were obtained using mannitol as a cryoprotector. Genetically selected mice when injected with liposomes containing PSC and 18kDa-hsp displayed an antibody titer of 12. In contrast, in those mice injected with free PSC there was no response. The 18kDa-hsp adjuvant effect on the PSC liposomal formulation was comparable to that observed when ODN1668 was co-encapsulated with PSC. Confirming our expectations we observed that the formulation containing 18kDa-hsp conferred a memory response to the carried antigen—the Neisseria meningitidis serogroup C polysaccharide.     

THE USE OF BEEF LIVER CATALASE AS A PROTEIN TRACER FOR ELECTRON MICROSCOPY

Beef liver catalase was injected intravenously into mice, and its distribution in the kidney, myocardium, and liver was studied with the electron microscope. A specific and relatively sensitive method was developed for its ultrastructural localization, based on the peroxidatic activity of catalase and employing a modified Graham and Karnovsky incubation medium. The main features of the medium were a higher concentration of diaminobenzidine, barium peroxide as the source of peroxide, and pH of 8.5. Ultrastructurally, the enzyme was seen to permeate the endothelial fenestrae and basement membranes of tubular and glomerular capillaries of the kidney. The urinary space and tubular lumina contained no reaction product. In the myocardial capillaries, the tracer filled the pinocytotic vesicles but did not diffuse across the intercellular clefts of the endothelium. In liver, uptake of catalase was seen both in hepatocytes and in Kupffer cells.     

REVISITING RUBISCO AS A PROTEIN SUBSTRATE FOR INSECT MIDGUT PROTEASES

Gene fragments encoding the large subunit (LS) of Rubisco (RBCL) were cloned from various species of host plants of phytophagous Lepidoptera and expressed as recombinant proteins in Escherichia coli. Recombinant RBCLs were compared among each other along with casein and native Rubisco as proteinaceous substrates for measuring total midgut protease activities of fourth instar larvae of Helicoverpa armigera feeding on casein, Pieris brassicae feeding on cauliflower, and Antheraea assamensis feeding on Litsea monopetala and Persea bombycina. Cognate rRBCL (from the pertinent host plant species) substrates performed similar to noncognate rRBCL reflecting the conserved nature of encoding genes and the versatile use of these recombinant proteins. Casein and recombinant RBCL generally outperformed native Rubisco as substrates, except where inclusion of a reducing agent in the enzyme assay likely unfolded the plant proteins. Levels of total midgut protease activities detected in A. assamensis larvae feeding on two primary host species were similar, suggesting that the suite(s) of digestive enzymes in these insects could hydrolyze a plant protein efficiently. Protease activities detected in the presence of protease inhibitors and the reducing agent dithiothreitol (DTT) suggested that recombinant RBCL was a suitable protein substrate for studying insect proteases using in vitro enzyme assays and substrate zymography.     

THE ULTRASTRUCTURAL BASIS OF CAPILLARY PERMEABILITY STUDIED WITH PEROXIDASE AS A TRACER

The transendothelial passage of horseradish peroxidase, injected intravenously into mice, was studied at the ultrastructural level in capillaries of cardiac and skeletal muscle. Peroxidase appeared to permeate endothelial intercellular clefts and cell junctions. Abnormal peroxidase-induced vascular leakage was excluded. Neutral lanthanum tracer gave similar results. The endothelial cell junctions were considered to be maculae occludentes, with gaps of about 40 A in width between the maculae, rather than zonulae occludentes. Some observations in favor of concurrent vesicular transport of peroxidase were also made. It is concluded that the endothelial cell junctions are most likely to be the morphological equivalent of the small pore system proposed by physiologists for the passage of small, lipid-insoluble molecules across the endothelium.     

PTEN蛋白在子宫内膜癌中的表达及其临床意义

目的　探讨抑癌基因PTEN产物PTEN蛋白在由正常子宫内膜过渡至子宫内膜癌过程中的表达缺失情况及其临床意义。方法　用SP免疫组化法测定 73例子宫内膜癌、 2 5例子宫内膜非典型增生、 71例子宫内膜增殖症和 31例正常子宫内膜组织中PTEN蛋白的表达 ,并与组织学类型、组织学分级、肌层浸润深度和临床分期等生物学行为进行相关分析。结果　PTEN蛋白在子宫内膜腺癌和癌前病变中的表达缺失率分别为 6 6 6 7%和 76 0 0 % ,表达缺失率与组织学类型(P <0 0 0 5 )和组织学分级有关 (P <0 0 5 ) ,与子宫内膜癌肌层浸润深度和临床分期无关 (P >0 0 5 )。结论　PTEN表达缺失是子宫内膜癌发生的早期分子事件 ,PTEN蛋白表达缺失是早期诊断子宫内膜癌及癌前病变较好的生物标志。     

AUXIN-INDUCED GROWTH OF TUBER TISSUE OF JERUSALEM ARTICHOKE: II. THE RELATION TO PROTEIN AND NUCLEIC ACID METABOLISM

The following results were obtained using tissue slices excisedfrom cold-stored Jerusalem artichoke tuber. 1. Increase in protein content of the tissue was small duringthe washing (i.e. "aging"), and great in the growth phase, particularlyin washed tissue. 2. RNA content of tissue increased during the growth periodsimilarly in non-growing tissue (in water) and actively growingtissue (in 2,4-D plus KIN). 3. Both RNA and DNA increased during the washing, the increasebeing greater in RNA than in DNA. This RNA increase was enhancedby gibberellic acid. 4. 2-Thiouracil, 8-azaguanine, puromycin, and mitomycin C givenat the washing inhibited the subsequent growth. The effect ofthese inhibitors was not significant when they were given inthe growth period. 5. Mitomycin C reduced the basophilia of nuclei and made themswell, as did deoxyribonuclease. 6. The effect of inhibitors of nucleic acid metabolism was reversedto some extent by gibberellic acid and by kinetin. 7. Chloramphenicol inhibited the growth strongly if given inthe growing period, but not so strongly if given during thewashing. 8. An autoradiographic study using ³H-cytidine suggested thatRNA is synthesized in nucleus during the period of washing andis transferred to cytoplasm via nucleolus. It is conjectured that the RNA synthesized during the agingis responsible for the expansion growth to be caused later byauxin or auxin plus kinetin. (Received September 4, 1965; )     

急性热暴露大白鼠肝脏热休克蛋白形成的研究

本研究采用人工气候室模拟自然热环境对大白鼠进行急性热暴露实验。用苯酚法提取大白鼠肝脏总RNA;用 Oligo(dT)-纤维素亲和层析柱分离出Poly(A)⁺mRNA。将各条件下的大白鼠肝脏Poly(A)⁺mRNA在麦胚无细胞体外转译系统中表达。结果证明急性热暴露大白鼠肝脏同样能生成分子量分别为71kD、90kD、98kD和 110kD的一组热休克蛋白。     

用酵母双杂交体系确定病毒特异性蛋白与宿主蛋白的相互作用

用B型流行性感冒病毒BM2蛋白开放阅读框架片段,构建酵母双杂交体系中的"饵”载体,明确其在酵母细胞中的表达,排除自身激活作用,并用酵母双杂交法从人cDNA基因库中筛选出与BM2蛋白相互作用的蛋白.结果表明B型流感病毒BM2蛋白与宿主细胞的某一蛋白有相互作用.应用酵母双杂交体系,可以非常有效地从人cDNA基因库中确定与"饵”蛋白相互作用的蛋白基因.     

配体与受体相互作用诱导的膜蛋白构象变化WGA与GPA作用对膜蛋白构象的影响

用ＦＴＩＲ、ＣＤ、微量ＤＳＣ和ＳＴＭ、ＡＦＭ等研究重组脂质体、血影和完整红细胞在配体（ＷＧＡ）与膜受体（ＧＰＡ）相互作用下膜蛋白构象改变以及纳米水平上膜表面蛋白的形貌。结果表明ＷＧＡ可诱导重组脂质体膜、血影膜和完整红细胞膜蛋白发生一定的不同的构象变化、红细胞及其膜的热力学行为变更,以及红细胞膜表面蛋白的可能交联。     

配体与受体相互作用诱导的膜蛋白构象变化WGA与GPA作用对膜蛋白构象的影响

用ＦＴＩＲ、ＣＤ、微量ＤＳＣ和ＳＴＭ、ＡＦＭ等研究重组脂质体,血影和完整红细胞在配体与膜受体相互作用下膜蛋白构象改变以及纳米水平上膜表面蛋白的形貌、结果表明ＷＧＡ可诱导重组脂质体膜、血影膜和完整红细胞膜蛋白发生一定的不同的构象变化,红细胞及其膜的热力学行为变更,以及红细胞膜表面蛋白的可能交联。     

菜豆热激蛋白在生物膜上的定位

选用菜豆 Phaseolus vulgris L. 下胚轴 ,运用35S- Met标记放射自显影和二维电泳技术 ,研究热激蛋白 HSPs 的表达和在生物膜组分中的定位 .实验结果表明 ,盐溶蛋白中主要HSPs为 70 k D HSPs和小分子量 HSPs,而小分子量组 HSPs大量富集在质膜和液泡膜组分中 .     

蛋白质结构成对比较的新方法

介绍一种蛋白质三维结构的快速比较方法.此方法毋需初始联配,而能自动寻找和智能迭代.利用本程序对珠蛋白、丝氨酸蛋白酶、天冬氨酸蛋白酶、钙结合蛋白和溶菌酶作了系统的结构比较,取得了满意的结果.本文也讨论了衡量联配结果好坏的要素问题.     

THE COLOR VISION OF DICHROMATS : II. SATURATION AS THE BASIS FOR WAVELENGTH DISCRIMINATION AND COLOR MIXTURE

1. Wavelength discrimination for the colorblind is entirely determined by saturation differences in the spectrum. From the neutral point to the short-wave end, his spectrum may be completely matched by 440 mµ plus white; to the long-wave end by 650 plus white. The proportion of color to white, hence the relative saturation, changes rapidly in the region of small Δλ at the center, and slowly in regions of large Δλ at the ends. 2. The data of spectrum gauging with two primaries (color mixture) by the dichromat are shown to contain the saturation distribution in the spectrum for the dichromat. This is because each mixture of primaries may be considered as composed of a mixture which matches white and of an excess of one primary. The data when so computed yield saturation distributions almost identical with those found by direct measurement, and show that on each side of the neutral point the basis of color mixture for the colorblind lies in saturation and not in hue differences. 3. To judge by these measurements, the spectrum for the protanope and deuteranope is composed of only two hues, themselves probably of low saturation, situated one at each end. Toward the center these hues decrease still more in saturation until they completely disappear in the white of the neutral point.     

PHYTOCHROME CONTROLLED NYCTINASTY IN ALBIZZIA JULIBRISSIN. II. POTASSIUM FLUX AS A BASIS FOR LEAFLET MOVEMENT

Excised Albizzia leaflet pairs exposed to red (R) light close within 30–90 min after transfer to darkness. Interruption of darkness by far-red (FR) light at any time after R inhibits closure within ca. 10 min. Similarly, irradiation with R at any time after prior FR promotes closure within ca. 10 min, and the increased rate of closure is independent of the time lapse between the FR and R irradiations. Closure in the dark is inhibited by NaN₃ and DNP (5 X 10^–4 m ), by anaerobic conditions and by externally applied salts of monovalent cations, especially K; it is also temperature sensitive. Pulvinule cells are very high in K. Electron microprobe analysis of cryostated, lyophilized pulvinules reveals that during closure, K is lost from ventral cells and enters dorsal cells. FR before darkness inhibits the former but not the latter process. Thus, K flux appears to control the changes in volume of the pulvinule cells that control leaflet movement. While leaflet closure normally requires a dark period, salts of organic acids such as sodium acetate, propionate, and butyrate cause closure in the light.