从一名国内感染的艾滋病人分离人免疫缺陷病毒

从一名国内感染的艾滋病人采血,分离其外周血单核细胞。首先与正常的ＰＭＣｓ共培养,４周后检测其ＨＩＶ－１ｐ２４抗原达到峰值。用此时的细胞及其上清分别感染Ｊｕｒｋａｔ－ｔａｔ,ＣＥＭ,ＭＴ４细胞,可很快地在这三株细胞中检测到ＨＩＶ生长。ＨＩＶ在Ｊｕｒｋａｔ－ｔａｔ细胞中生长情况最好。同时用病人血清直接感染Ｊｕｒｋａｔ－ｔａｔ和ＭＴ４细胞,４周后检测其细胞上清ＨＩＶ－１Ｐ２４抗原为阳性,但ＯＤ值很低。用     

广东两株人免疫缺陷病毒的分离和鉴定

从我省两名经性途径感染艾滋病的病人中采血,分离外周血单核细胞（ＰＢＭＣｓ）,将其与正常人ＰＢＭＣｓ共培养分离人免疫缺陷病毒（ＨＩＶ）,３周后检测上清ＨＩＶ－１ｐ２４抗原（ＥＬＩＳＡ法）超过阈值。将共培养第四周细胞和上清分别感染Ｈ９细胞（Ｔ细胞淋巴瘤传代细胞）,一周后检测ＨＩＶ－１ ｐ２４怕,证明有病毒生长。用ＨＩＶ－１ ＥＮＶ基因引物的套式聚合酶链反应（Ｎｅｓｔｅｄ ＰＣＲ）证实,两株新分离的病毒     

从一名国内感染的艾滋病人分离人免疫缺陷病毒（HIV）

从一名国内感染的艾滋病人采血,分离其外周血单核细胞（ＰＭＣｓ）。首先与正常的ＰＭＣｓ共培养,４周后检测其ＨＩＶ－１ｐ２４抗原（ＥＬＩＳＡ）达到峰值。用此时的细胞及其上清分别感染Ｊｕｒｋａｔ－ｔａｔ、ＣＥＭ、ＭＴ４细胞,可很快地在这三株细胞中检测到ＨＩＶ生长,ＨＩＶ在Ｊｕｒｋａｔ－ｔａｔ细胞中生长情况最好,同时用病人血清直接感染Ｊｕｒｋａｔ－ｔａｔ和ＭＴ４细胞,４周后检测其细胞上清ＨＩＶ－１ｐ２４抗原（ＥＬＩＳＡ法）为阳性,但ＯＤ值很低（约为ＰＭＣ共培养组的一半）。用病人的少量全血与正常ＰＭＣｓ共培养,得到的结果与分离病人ＰＭＣｓ法相近。应用间接免疫荧光法（ＩＦＡ）、免疫酶法（ＩＥＡ）、蛋白印迹法及ＨＩＶ－１ＰＯｌ基因和Ｅｎｖ基因特异引物的聚合酶链反应（ＰＣＲ）等证实为ＨＩＶ－１病毒。分离的ＨＩＶ在Ｊｕｒｋａｔ－ｔａｔ细胞中连续传代,细胞被感染后２－３天即出现以大量融合细胞为主的细胞病变,感染后７－１０天细胞几乎全部死亡。病毒在连续传代过程中的生长特征及致细胞病变特征不变。此病毒命名为ＣＡ－２毒株。     

人免疫缺陷病毒1型Rev单链抗体细胞内免疫抗病毒基因治疗研究

应用抗ＨＩＶ－１Ｒｅｖ单链抗体细胞内免疫方法,研究在人Ｔ细胞和周围血淋巴单核细胞内抗病毒复制的效果,探讨细胞内免疫抗ＨＩＶ－１基因治疗的可行性。克隆抗ＨＩＶ－１Ｒｅｖ单链抗抗体（ｓＦｖ）基因,以逆转录病毒为基因载体,将名装后的含靶基因的逆转录病毒转导至人ＣＤ４阳性Ｔ－细胞株ＣＥＭ和ＳｕｐＴ１,以及ＨＩＶ－１阴性自愿者的周围血淋巴单核细胞（ＰＢＭＣ）,再分别用不同剂量（ＭＯＩ）的ＨＩＶ－１病毒株ＰＮ     

乙型肝炎病毒包膜M蛋白在巴斯德毕德毕赤酵母中的表达

转化了乙肝病毒ｐｒｅＳ２－２基因的重组巴斯德毕赤酵母菌株经甘油培养基充分增殖,然后转移到甲醇培养基中进行诱导表达,破碎细胞并提胞内蛋白,经ＥＬＩＳＡ和ＷｅｓｔｅｎＢｌｏｔ检测证明有４株ＧＳ１１５／ＨＩ＾＋ＭＵＴ＾＋表达了ＨＢＶ Ｍ蛋白。     

以痘苗病毒天坛株为载体的表达单纯疱疹病毒Ⅰ型糖蛋白D的重组活疫苗的建立

从我国分离到的一株单纯疱疹病毒Ⅰ型（ＨＳＶ－１－１６８株）病毒基因组中,分离出含有糖蛋白Ｄ（ｇＤ）基因的１．２ｋｂ片段,插入带有痘苗病毒天坛株ＴＫ区的质粒ｐＪＳＢ１１７５Ｐ７．５ｋ启动子下游,转染无白血病鸡胚原代细胞,获得带有ＨＳＶ－１－１６８ｇＤ基因的重组痘苗病毒。此株重组病毒在感染细胞膜上表达ＨＳＶ－１－１６８ｇＤ糖蛋白抗原,能与特异性单克隆抗体反应。在感染细胞中表达的膜抗原经ＳＤＳ－ＰＡＧＥ分析,表达分子量为５４ｋＤ糖蛋白。用Ｓｏｕｔｈｅｒｎ杂交分析了重组病毒ＤＮＡ中特异的ｇＤ基因,对作为活疫苗的重组痘苗病毒株进行了一些微生物学活性、免疫原性和毒力等方面的研究。     

单纯疱疹病毒Ⅰ型扩增子质粒载体的构建及LacZ基因的表达

从单纯疱疹病毒Ⅰ型ＨＳＶ－１基因组中分离出其包装信号序列,与含ＨＳＶ－１复制起点及ＩＥ－６８基因启动子的ＤＮＡ序列、β半乳糖苷酶基因和大肠杆菌质粒骨架,构建了一种ＨＳＶ－１扩增子质粒载体ｐＨＳＬ,其中ＬａｃＺ基因置于ＩＥ－６８基因启动子控制下。将此质粒转染已感染了ＨＳＶ－１温度敏感株ｔｓＫ株的Ｖｅｒｏ细胞,ｐＨＳＬ可在ＨＳＶ－１ｔｓＫ的辅助下包装成假病毒颗粒,这样就可得到同时含有假病毒和ＨＳＶ－１ｔｓＫ的混合毒株ｄｖＨＳＬ。将此混合毒株感染传代细胞和神经细胞,可在其中表达ＬａｃＺ基因,而在生理温度（３７℃）下,混合毒株的复制受到抑制。提示这种缺陷型疱疹病毒载体有用于向神经系统基因转移的可能性。     

人单核细胞系U_（937）和T细胞系HSB_2细胞的HSV－1感染

用ＨＳＶ－１接种人单核细胞传代株Ｕ＿（９３７）和Ｔ淋巴细胞传代株ＨＳＢ＿２细胞,通过对病毒滴度、细胞增殖与病变、病毒抗原表达及病毒ＤＮＡ的动态观察,研究了ＨＳＶ－１感染两种细胞的特点。结果发现接种病毒后,Ｕ（９３７）细胞仅允许病毒短时间低滴度复制,培养上清中病毒滴度在１２天内降至测不出水平,细胞经过１～２周后增殖受抑、死亡增多,然后又逐渐恢复;用ＬＰＳ持续刺激则能提高病毒感染滴度,延长感染时间,形成短期持续感染。ＨＳＢ＿２细胞允许病毒复制至较高滴度,呈现急性杀细胞性感染;用ＰＨＡ持续刺激也不改变细胞感染的特点。     

CCR5的结构和功能：近两年的研究进展

ＣＣＲ５是趋化蛋白ＭＩＰ－１α,ＭＩＰ－１β、ＲＡＮＴＥＳ的特异性受体,主要表达于单核／巨细胞及Ｔ细胞膜上,具有Ｇ蛋白受体所特有的７个胯膜区。ＣＣＲ５与白细胞的趋经性,炎症反应,嗜Ｍφ型ＨＩＶ－１株对ＣＤ４细胞的感染密度相关。ＣＣＲ５的缺陷与个体对ＨＩＶ－１的抗性有关。本文就近两年在ＣＣＲ５结构特点,转录调控,信号传递,生物学功能等方面的研究进展作一综述。     

HIV—1 p24蛋白在大肠肝菌中的高效可溶性表达,一步纯化抗原性分析

合成引物扩增ＨＩＶ－１ ｐ２４基因,并将其克隆到ｐＱＥ－３０质粒中,使其在大肠杆菌Ｅ．ｃｏｌｉ Ｍ１５中以ＩＰＴＧＨ诱导高效它ＳＤＳ－ＰＡＧＥ分析,该表达产物约占菌体总蛋白２０％,并且以可溶蛋白的形式存在于细菌裂解液上清之中。经镍离子柱亲和层一步纯化,洗脱产物中ｐ２４蛋白纯度达９５％。ＥＬＩＳＡ分析表明,该蛋白可与ＨＩＶ感染者血肖发生物异改正 免疫反应。以此蛋白交联Ｓｅｐｈａｒｏｓｅ ４Ｂ,新和层     

Cytoplasmic assembly and accumulation of human immunodeficiency virus types 1 and 2 in recombinant human colony-stimulating factor-1-treated human monocytes: an ultrastructural study.

Recombinant human colony-stimulating factor-1-treated human peripheral blood-derived monocytes-macrophages are efficient host cells for recovery of the human immunodeficiency virus (HIV) from blood leukocytes of patients with acquired immunodeficiency syndrome. These cells can be maintained as viable monolayers for intervals exceeding 3 months. Infection with HIV resulted in virus-induced cytopathic effects, accompanied by relatively high levels of released progeny virus, followed by a prolonged low-level release of virus from morphologically normal cells. In both acutely and chronically infected monocytes, viral particles were seen budding into and accumulating within cytoplasmic vacuoles. The number of intravacuolar virions far exceeded those associated with the plasma membrane, especially in the chronic phase, and were concentrated in the perinuclear Golgi zone. In many instances, the vacuoles were identified as Golgi elements. Fusion of virus-laden vacuoles with primary lysosomes were rare. The pattern of cytoplasmic assembly of virus was observed with both HIV types 1 and 2 and in brain macrophages of an individual with acquired immunodeficiency syndrome encephalopathy. Immunoglobulin-coated gold beads added to acutely infected cultures were segregated from the vacuoles containing virus; relatively few beads and viral particles colocalized. The assembly of HIV virions within vacuoles of macrophages is in contrast to the exclusive surface assembly of HIV by T lymphocytes. Intracytoplasmic virus hidden from immune surveillance in monocytes-macrophages may explain, in part, the persistence of HIV in the infected human host.     

Immunocytochemical localization of cathepsin B in rat kidney. II. Electron microscopic study using the protein A-gold technique

Thin sections of Lowicryl K4M-embedded materials were labeled with protein A-gold complex. Gold particles representing the antigen sites for cathepsin B were exclusively confined to lysosomes of each segment of the nephron. The heaviest labeling was noted in the lysosomes of the S1 segment of the proximal tubules. Labeling intensity varied considerably with the individual lysosomes. Lysosomes of the other tubular segments, such as the S2 and S3 segments of the proximal tubules, distal convoluted tubules, and collecting tubules were weakly labeled by gold particles. Quantitative analysis of labeling density also confirmed that lysosomes in the S1 segment have the highest labeling density and that approximately 65% of labeling in the whole renal segments, except for the glomerulus, was found in the S1 segment. These results indicate that in rat kidney the lysosomes of the S1 segment are a main location of cathepsin B. Further precise observations on lysosomes of the S1 segment revealed that apical vesicles, tubules, and vacuoles were devoid of gold particles, but when the vacuoles contained fine fibrillar materials, gold labeling was detectable in such vacuoles. As the lysosomal matrix becomes denser, the labeling density is increased. Some small vesicles around the Golgi complex were also labeled. These results indicate that the endocytotic apparatus including the apical vesicles, tubules, and vacuoles contains no cathepsin B. When the vacuoles develop into phagosomes, they acquire this enzyme to digest the absorbed proteins.     

Fine structural localization of thiamine pyrophosphatase and acid phosphatase activities in the mouse pancreatic acinar cell

The fine structural localization of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) was examined in pancreatic acinar cells of fasting and fed mice. The results were not affected by these conditions. TPPase activity was positive in two and sometimes three cisternae of the inner Golgi lamellae as well as in the condensing vacuoles of the trans area, but negative in the rigid lamellae and small vesicles of the trans area. AcPase activity was demonstrated in two and sometimes three cisternae of inner Golgi lamellae, condensing vacuoles, rigid lamellae, lysosomes and smooth or coated vesicles in the trans area. The inner Golgi lamellae and the condensing vacuoles were positive for both enzyme activities. From these facts, the lysosome is considered to be formed not only in the GERL system but also through the rough endoplasmic reticulum-Golgi apparatus route. It is reasonable to consider that Novikoff's GERL is not independent from the Golgi apparatus but represents a part of this organelle.     

Lectin-gold cytochemistry of the Golgi apparatus in rabbit luteal cells, with special emphasis on the formation of a lysosomal-type membrane

In rabbit luteal cells embedded in glycolmethacrylate and stained with PTA at low pH highly glycosylated membrane patches can be observed after vesiculation of the trans-Golgi network. As these membranes could be prelysosomal, their sialic acid content was investigated by post-embedding labeling with Limax flavus agglutinin (LFA)/fetuin-Au. Additional labeling of the Golgi apparatus was performed with Wheat germ agglutinin (WGA)/ovomucoid Au, Ricinus communis agglutininI (RCAI)/Au and Helix pomatia agglutinin (HPA)/Au. The sections were then counterstained with PTA at low pH, which allows a clear distinction between the elements of the trans-Golgi network (G2-G1) and the saccules of the stack (g). With WGA, LFA and RCAI the trans-Golgi network was observed to be clearly more reactive than the stack. After vesiculation most intense labeling was found over the highly glycosylated vacuolar membranes derived from the G2-element. The limiting membrane of lysosomes, the MvB's and the plasma membrane also reacted strongly. Colloidal gold particles were also found over the membranes of the vacuoles derived from G1. The Golgi stack showed a lower reactivity and label for all three lectins could be found over three to four saccules of the stack (g3-g4). The matrix of the lysosomes was slightly labeled. Labeling with HPA was absent from the trans saccules and was consistently found in the cis and cis-most (g4-g5) saccules of the stack. Some cytoplasmic vesicles near the cell border were also labeled. With our procedure the Golgi apparatus can easily be detected and it is apparent that in rabbit luteal cells the highest lectin reactivity is found in the trans-Golgi network.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine structural localization of thiamine pyrophosphatase and acid phosphatase activities in the mouse pancreatic acinar cell

Summary The fine structural localization of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) was examined in pancreatic acinar cells of fasting and fed mice. The results were not affected by these conditions. TPPase activity was positive in two and sometimes three cisternae of the inner Golgi lamellae as well as in the condensing vacuoles of the trans area, but negative in the rigid lamellae and small vesicles of the trans area. AcPase activity was demonstrated in two and sometimes three cisternae of inner Golgi lamellae, condensing vacuoles, rigid lamellae, lysosomes and smooth or coated vesicles in the trans area. The inner Golgi lamellae and the condensing vacuoles were positive for both enzyme activities. From these facts, the lysosome is considered to be formed not only in the GERL system but also through the rough endoplasmic reticulum-Golgi apparatus route. It is reasonable to consider that Novikoff's GERL is not independent from the Golgi apparatus but represents a part of this organelle.This study was supported by a grant from the Japan Educational Ministry     

Osteoblastic and osteoclastic differentiation of mononuclear cells facing the resorbing surface of uncalcified cartilage in the tibia of embryonic chick

Summary The resorbing region of uncalcified cartilage in the tibia of embryonic chick was studied using ³H-proline autoradiography, histochemistry, and horseradish-peroxidase tracers.At the cartilage-bone marrow interface, two kinds of cells (A and B) were identified. Type-A cells were elongated, contacted the matrix of the uncalcified cartilage directly, and possessed extensive rough endoplasmic reticulum, one or two juxtanuclear Golgi apparatus and cell membranes exhibiting prominent alkaline phosphatase activity. Type-B cells were round to oval, mononucleate (occasionally binucleate), and contained abundant mitochondria, vacuoles and vesicles, well-developed Golgi apparatus, and lysosomes. The lysosomes and the majority of vacuoles and Golgi lamellae of these cells showed prominent acid phosphatase activity. Type-B cells accumulated more horseradish-peroxidase reaction product in their vacuoles and vesicles than type-A cells. Thick, banded collagen fibrils were occasionally found in the matrix of the resorbing surface. ³H-proline autoradiography revealed small numbers of grains at the cartilage-bone marrow interface.These findings suggest that type-A cells have osteoblastic and type-B cells osteoclastic properties and are precursor cells of osteoblasts and osteoclasts, respectively. The appearance of a mineral phase in the resorbing cartilage is probably important for the differentiation of these cells.     

Relationships between membranous organelles in amoebae studied by electron microscopic cytochemical staining

Summary The intracellular location of a variety of enzymes was studied in Amoeba proteus with the use of electron microscopic cytochemical methods, in an attempt to assess the relationships between different membranous organelles. One group of enzymes, including nucleoside diphosphatases (IDPase, UDPase, GDPase, ADPase), carbamoyl phosphatase, alkaline phosphatase, and BAXD oxidase was localized mainly in the rough endoplasmic reticulum, nuclear envelope, and convex side of the Golgi apparatus. Esterase activity had a similar localization except that the Golgi apparatus was "stained" throughout most of its extent. A second group of enzymes was found in Golgi cisternae and vesicles, and in some vacuoles. This group included acid phosphatase, thiamine pyrophosphatase, and aryl sulfatase. Some enzymes previously detected in cytoplasmic membranes of other cells, including glucose-6-phosphatase, showed little or no activity in amoebae. The results suggest that there are chemical similarities and probable functional relationships between the rough endoplasmic reticulum, the nuclear envelope, and the convex side of the Golgi apparatus. On the other hand, the concave pole of the Golgi apparatus, aggregates of smooth tubules and vesicles, and the cell surface appear more closely related to one another than to the endoplasmic reticulum and the convex side of the Golgi apparatus. The cytochemical similarity between the Golgi apparatus and certain vacuoles such as food vacuoles may reflect the role of the Golgi apparatus in the formation of lysosomes. The locations of reaction products of the various enzymes in amoebae are compared with observations reported for other cell types.Supported by a research grant (VC-169) from the American Cancer SocietyThe author is indebted for technical assistance to Mrs. Sue Thompson and Mrs. Christine Folsom-Kovarik     

LYSOSOMES AND GERL IN NORMAL AND CHROMATOLYTIC NEURONS OF THE RAT GANGLION NODOSUM

The rat ganglion nodosum was used to study chromatolysis following axon section. After fixation by aldehyde perfusion, frozen sections were incubated for enzyme activities used as markers for cytoplasmic organelles as follows: acid phosphatase for lysosomes and GERL (a Golgi-related region of smooth endoplasmic reticulum from which lysosomes appear to develop) (31–33); inosine diphosphatase for endoplasmic reticulum and Golgi apparatus; thiamine pyrophosphatase for Golgi apparatus; acetycholinesterase for Nissl substance (endoplasmic reticulum); NADH-tetra-Nitro BT reductase for mitochondria. All but the mitochondrial enzyme were studied by electron microscopy as well as light microscopy. In chromatolytic perikarya there occur disruption of the rough endoplasmic reticulum in the center of the cell and segregation of the remainder to the cell periphery. Golgi apparatus, GERL, mitochondria and lysosomes accumulate in the central region of the cell. GERL is prominent in both normal and operated perikarya. Electron microscopic images suggest that its smooth endoplasmic reticulum produces a variety of lysosomes in several ways: (a) coated vesicles that separate from the reticulum; (b) dense bodies that arise from focal areas dilated with granular or membranous material; (c) "multivesicular bodies" in which vesicles and other material are sequestered; (d) autophagic vacuoles containing endoplasmic reticulum and ribosomes, presumably derived from the Nissl material, and mitochondria. The number of autophagic vacuoles increases following operation.     

Ultrastructural study of synovial membrane from the antebrachiocarpal joint of calves

The synovial intima from the antebrachiocarpal joint of 4-month-old calves was between 1 and 3 cells in thickness and did not have a basal lamina. Numerous areas of the intimal matrix were in direct contact with the joint lumen. The synovial membrane was comprised mainly of A-type synoviocytes usually located adjacent to the joint lumen. These cells were characterized by numerous filopodia (or lamellipodia), large, empty-appearing vacuoles, numerous lysosomes, large vacuoles containing granular material separated from the vacuolar membrane by a radiolucent band, and coated micropinocytotic vesicles. Smooth micropinocytotic vesicles were seen only rarely in these cells. In contrast, B-type cells had few filopodia, numerous smooth micropinocytotic vesicles, few coated micropinocytotic vesicles, a well-developed Golgi apparatus and rough endoplasmic reticulum, mitochondria that were longer and had a denser matrix than that of A cell mitochondria, and surprisingly, only few maturing or fully formed secretory granules. A distinct intermediate (C or AB) type synoviocyte could not be unequivocally identified. Desmosome-like structures were present between synoviocytes, although it was considered questionable if these were true intercellular junctions. No other junctions were present.     

Different arrangements of phagolysosome membranes which depend upon the particles phagocytized : Observation with markers of the two sides of plasma membranes

Phagolysosome vesicles containing either polystyrene latex (PLP), or PLP plus bovine serum albumin (PLP-BSA), or paraffin oil emulsified with BSA (POE-BSA) were prepared from guinea pig polymorphonuclear leukocytes (gp-PMNL). They were compared using the specific markers of the two sides of the plasma membrane of gp-PMNL characterized previously [1, 2]. Anticell surface IgG (Anti-C-S IgG) which binds exclusively to the outer side of plasma membranes [1] was found to bind to the cytoplasmic side of all three types of phagolysosomes. Anti-100000 g sup IgG which binds exclusively to the cytoplasmic side of plasma membranes of gp-PMNL [1] did not bind to the cytoplasmic side of these phagolysosomes, as shown by immunoferritin technique. On the other hand, influenza virus, which is a marker of the outer surface of plasma membranes [1] bound to the cytoplasmic side of POE-BSA phagolysosomes but not PLP or PLP-BSA phagolysosomes, as demonstrated by electron microscopy. The content of myosin, another marker of the cytoplasmic side of plasma membranes [2], differed in these phagolysosomes, as shown by SDS-polyacrylamide gel electrophoresis: it was very low in PLP- and POE-BSA phagolysosomes but high in PLP-BSA phagolysosomes. Myosin was found on the cytoplasmic side of PLP-BSA phagolysosomes by the improved immunoferritin technique reported previously [3]. These phagolysosomes, containing different particles and having different arrangements of membrane components, showed different efficiencies of fusion with lysosomes. The possible mechanism of the specific fusion of phagosomes with lysosomes was discussed in relation to these different features of phagolysosomal membranes.