Ran/TC4: a small nuclear GTP-binding protein that regulates DNA synthesis

Ran/TC4, first identified as a well-conserved gene distantly related to H-RAS, encodes a protein which has recently been shown in yeast and mammalian systems to interact with RCC1, a protein whose function is required for the normal coupling of the completion of DNA synthesis and the initiation of mitosis. Here, we present data indicating that the nuclear localization of Ran/TC4 requires the presence of RCC1. Transient expression of a Ran/TC4 protein with mutations expected to perturb GTP hydrolysis disrupts host cell DNA synthesis. These results suggest that Ran/TC4 and RCC1 are components of a GTPase switch that monitors the progress of DNA synthesis and couples the completion of DNA synthesis to the onset of mitosis.     

Nucleus-associated pools of Rna1p, the Saccharomyces cerevisiae Ran/TC4 GTPse activating protein involved in nucleus/cytosol transit

Rna1p is the GTPase activating enzyme for Ran/TC4, a Ras-like GTPase necessary for nuclear/cytosolic exchange. Although most wild-type Rna1p is located in the cytosol, we found that the vast majority of the mutant Rna1-1p and, under appropriate physiological conditions, a small portion of the wild-type Rna1p cofractionate with yeast nuclei. Subnuclear fractionation studies show that most of the Rna1p is tightly associated with nuclear components, and that a portion of the active protein can be solubilized by treatments that fail to solubilize inactive Rna1-1p. To learn the precise nuclear locations of the Rna1 proteins, we studied their subcellular distributions in HeLa cells. By indirect immuno-fluorescence we show that wild-type Rna1p has three subcellular locations. The majority of the protein is distributed throughout the cytosol, but a portion of the protein is nucleus-associated, located at both the cytosolic surface and within the nucleoplasm. Mutant Rna1-1p is found at the outer nuclear surface and in the cytosol. We propose that a small pool of the wild-type Rna1p is located in the nuclear interior, supporting the model that the same components of the Ran/TC4 GTPase cycle exist on both sides of the nuclear membrane.     

Blockage of T-cell costimulation inhibits T-cell action in celiac disease

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the alpha-amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known alpha-amylases of various origins with a surprisingly greatest similarity to mammalian alpha-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent alpha-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted alpha-amylase.     

Functional analysis of the DNA-packaging/terminase protein gp17 from bacteriophage T4

In bacteriophage T4, the terminase complex constituted by the large subunit gp17 (69 kDa) and the small subunit gp16 (18 kDa) is a critical component of the ATP-driven DNA-packaging pump that translocates DNA into an empty capsid shell. Evidence suggests that the large subunit gp17 is the critical component and consists of a number of the functional sites required for DNA-packaging. It exhibits a terminase activity that introduces non-specific cuts into DNA, a portal vertex binding site that allows linkage of cleaved DNA to an empty prohead, an in vitro DNA-packaging activity, and an ATPase activity. In addition, a consensus metal-binding motif and two consensus ATP-binding sites have been identified by sequence analysis. In order to understand the mechanism of action of the multifunctional gp17, we developed an expression-based selection strategy to select for mutants that are defective in terminase function. Characterization of one of the mutants revealed a unique phenotype in which a single H436R mutation resulted in a dramatic loss of both the terminase and the DNA-packaging functions. Indeed, in vivo substitution of H436 with any of the 12 amino acids for which a suppressor is available was lethal to T4 development. According to one hypothesis, H436 is part of a metal-binding motif that is essential for gp17 function. This hypothesis was tested by introducing mutations at each of the three histidine pairs, the H382-X2-H385 pair, the H411-X2-H414 pair and the H430-X5-H436 pair, which constitute the histidine-rich region near the C terminus of gp17. A mutation at either the H411 pair or the H430 pair resulted in a loss of gp17 function, whereas a mutation at the H382 pair had no effect. In addition to the putative metal-binding motif, substitutions at residue K166 within the putative N terminus-proximal ATP-binding site also resulted in a loss of gp17 function. We propose that a metal-binding motif involving the histidine residues within the sequence H411-X2-H414-X15-H430-X5-H436 is essential for gp17 function. Metal-terminase interactions may be required for structural alignment and stabilization of functional sites in phage T4 terminase and other double-stranded DNA phage terminases.     

Placental protein 14 functions as a direct T-cell inhibitor

Morphological changes in the myocardium after left ventricular hypertrophy, due to chronic experimental hypertension, require an understanding of the quantitative relationship between myocyte and nonmyocyte compartments forming the structural framework of the myocardium. Hypertension was induced by long-term low-dosage inhibition of nitric oxide synthesis by NG-nitro-L-arginine methyl ester (L-NAME) in rats. L-NAME (12 mg/kg) was given to animals in water ad libitum during 15 weeks. After this period, systolic blood pressure increased almost 50% as compared with that in the control group. Morphological changes in control and L-NAME animals were investigated with stereology and immunohistochemistry. Comparing control and L-NAME animals, the surface density of myocytes decreased 73.7% while the mean cross-sectional area increased 97.6% in L-NAME rats. The volume density of myocytes decreased 45.9% and the volume density of the interstitium increased 71.7% in L-NAME rats. No stereological difference was found in blood vessels comparing the two groups. Remodeling of the cardiac interstitium occurred with increased deposition of both fibronectin and type III collagen. Fibronectin was seen in both early and latter responses to infarction while type III collagen was seen mainly in areas of incomplete healing among myocytes and around intramyocardial branches of the coronary arteries. The long-term low-dosage administration of an inhibitor of the NO synthase such as L-NAME causes myocyte hypertrophy and early interstitial and perivascular fibrosis without important quantitative changes in microcirculation.     

CD19 as a membrane-anchored adaptor protein of B lymphocytes: costimulation of lipid and protein kinases by recruitment of Vav

Germ cells are distinct from somatic cells in their immortality, totipotency, and ability to undergo meiosis. Candidates for components that guide the unique germline program are the distinctive granules observed in germ cells of many species. We show that a component of germ granules is essential for fertility in C. elegans and that its primary function is in germline proliferation. This role has been revealed by molecular and genetic analyses of pgl-1. PGL-1 is a predicted RNA-binding protein that is present on germ granules at all stages of development. Elimination of PGL-1 results in defective germ granules and sterility. Interestingly, PGL-1 function is required for fertility only at elevated temperatures, suggesting that germline development is inherently sensitive to temperature.     

CD28/B7 costimulation: a review

The current model of T cell activation requires two signals. The first signal is specific, requiring T cell receptor recognition and binding to MHC/Antigen presented by an antigen-presenting cell. The second signal is nonspecific, resulting from the binding of B7 ligand on the antigen-presenting cell with its receptor, CD28, on the T cell. If both signals are provided, the T cell will proliferate and secrete cytokines. Recently, it has been shown that CTLA4, another receptor for B7 that is upregulated following T cell after activation, can deliver an inhibitory signal, downregulating T cell proliferation. The B7 family of ligands has two family members, B7-1 and B7-2. They both bind to CD28 and CTLA4, but they differ in their binding affinity, structure, and temporal expression. Considerable research has been done on the CD28/B7 costimulatory pathway. Different ways of manipulating this pathway could provide insights into the mechanism and treatment of opposing pathological states. Blocking the CD28/B7 pathway could result in immunosuppression, with implications for the treatment of autoimmune diseases, organ transplantation, and graft vs. host disease. Activating the CD28/B7 pathway could be useful for including the immune system to recognize and eliminate tumors that evade the immune system. Finally, the CD28/B7 pathway could be involved with maintaining immune tolerance, as recent studies suggest the preferential binding of the B7-CTLA4 pathway results in the down-regulation of the responding T cells. Thus, the B7/CD28/CTLA4 pathway has the ability to both positively and negatively regulate immune responses.     

Characterization of a unique T-cell clone established from a patient with HAM/TSP which recognized HTLV-I-infected T-cell antigens as well as spinal cord tissue antigens

PURPOSE: To describe our technique of vitrectomy and silicone oil tamponade for managing retinal detachment and to report the last results according to the posterior and anterior proliferative vitreoretinopathy. METHODS: A retrospective study was conducted in 108 patients who underwent vitreoretinal surgery and silicone oil tamponade for proliferative vitreo-retinopathy, 64% patients had already been operated without success and 42% underwent vitrectomy with SF6 or C3F8. Diffuse posterior proliferative vitreo-retinopathy (grade C3-D) was present in 64% patients and anterior proliferative vitreoretinopathy was present in 43.5%. Silicone oil was removed in 79% patients after a mean duration of 6.3 months. It was replaced by 16% C3F8. All patients were followed for a minimum of 6 months. RESULTS: Before silicone oil removal, 55% of the retinas were reattached posterior to the scleral buckle with one operation, 78% after 2 operations and 88% after 3 or 4 operations. An average of 2.1 vitrectomy surgeries were performed. Reproliferation was correlated with the anterior proliferative vitreo-retinopathy (p < 0.001), posterior proliferative vitreo-retinopathy (p < 0.01) or previous vitrectomy (p < 0.05). The final visual acuity was 1/20 or better in 61% of the eyes and 2/10 or better in 30.5%. After silicone oil removal, 8% retinas redetached, 21% of the eyes had hypertony, 7.5% had hypotony and 7% of the corneas had dystrophy. CONCLUSION: Silicone oil tamponade was effective for the treatment of retinal detachments with proliferative vitreo-retinopathy. Since the main complications were hypertony and corneal dystrophy, silicone oil should be reserved for severe proliferative vitreo-retinopathy cases.     

On T-cell recognition of nickel as a hapten

T-cells recognize antigens as peptides associated with self-molecules encoded by genes of the HLA region. In patients with contact allergy to nickel, T-cells that are specific for non-peptide haptens have been described. Previously, we have isolated HLA class II-restricted nickel-specific T-cell clones from patients with nickel sensitivity. In this paper, data on the fine specificity of a nickel-specific HLA-DR4-restricted clone have been reevaluated. Genomic tissue typing employing polymerase chain reaction and sequence-specific primers were used. Nickel was presented to the T-cell clone by all three subtypes of HLA-DR4 included in our panel. Two different DRB4*0404-positive cells presented nickel, whereas only 3 of the 7 DRB1*0401-positive and one of the 3 DRB1*0408-positive cells restimulated the T-cell clone. These findings are compatible with the notion that nickel interacts with endogenous peptides in the antigen-presenting groove of the HLA molecule, thereby changing these peptides' antigenicity rather than their ability to bind to the HLA molecule. Variations of the endogenous peptide in the antigen-presenting groove as well as differences of the HLA molecules give the DR4 specificity of the nickel-specific clone MCE2.     

TC4钛合金压坯的吸氢行为

利用管式氢处理炉研究了TC4钛合金压坯的温度-平衡氢分压关系以及吸氢动力学行为,测定了吸氢速率常数和激活能,并分析了吸氢反应各个阶段的反应机理.结果表明,随着吸氢温度的升高,达到平衡所需要的时间缩短,吸氢后的平衡氢分压升高.在350℃吸氢时,反应机制分别是形核长大、幂函数定律和三维扩散.在400℃吸氢时,反应机制分别是幂函数定律和三维扩散.在450~750℃吸氢时,反应机制均是三维扩散.TC4钛合金压坯吸氢反应的激活能为14.55kJ·mol^-1.     

添加微量B元素对铸造TC4钛合金显微组织和力学性能的影响

为细化铸造TC4钛合金的晶粒,改善合金的力学性能,在合金中加入了少量B元素.结果表明:随着B含量逐步增加至0.06％(质量分数,下同),ZTC4钛合金晶粒尺寸从添加B之前的4.5mm细化至0.36mm,硼化物的主要分布也随之由晶粒内部分布转变为晶界处分布.当B含量为0.03％时,ZTC4钛材料具有最高的延伸率,但当B含量超过0.06％时,ZTC4钛材料拉伸时接近脆性断裂.导致ZTC4-0.06B材料几乎脆断的原因可能是B含量过多导致B化物分布由主要在晶粒内部转向晶界处.从晶粒细化效果和材料力学性能综合考量,铸造TC4钛合金中B的最佳添加量为0.02％～0.04％.     

Functional analysis of the herpes simplex virus UL42 protein

The herpes simplex virus UL42 gene encodes a multifunctional polypeptide (UL42) that is essential for virus DNA replication. To further understand the relationship between the structure of UL42 and the role that it plays during virus replication, we analyzed an extensive set of mutant UL42 proteins for the ability to perform the three major biochemical functions ascribed to the protein:binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. Selected mutants were also assayed for their ability to complement the replication of a UL42 null virus. The results indicated that the N-terminal 340 amino acids of UL42 were sufficient for all three biochemical activities and could also support virus replication. Progressive C-terminal truncation resulted in the loss of detectable DNA-binding activity before Pol binding, while several mutations near the N terminus of the polypeptide resulted in an altered interaction with DNA but had no apparent affect on Pol binding. More dramatically, an insertion mutation at residue 160 destroyed the ability to bind Pol but had no effect on DNA binding. This altered polypeptide also failed to increase the length of DNA product synthesized by Pol, and the mutant gene could not complement the growth of a UL42 null virus, indicating that the specific interaction between Pol and UL42 is necessary for full Pol function and for virus replication. This study confirms the validity of the Pol-UL42 interaction as a target for the design of novel therapeutic agents.     

硼对TC4／B钛合金铸造组织与性能的影响

采用熔铸工艺法制备了含B量为0．2％-1．0％的TC4／B钛合金,分析并测试了合金的铸态组织和力学性能。研究结果表明：TiB相提高了TC4／B钛合金铸棒的弹性模量,B元素添加量为0．2％～1．0％范围内,合金弹性模量提高15％～30％;显微组织由片层α、晶界β及第二相（TiB）组成,第二相（TiB）聚集在卢晶界上呈链状分布,随B含量增加,显微组织细化效果越明显。由拉伸断口分析可知,随着B含量增加合金由典型的延性沿晶断裂向混合型断裂转变。     

TC4钛合金棒材缺陷分析

TC4棒在改锻时,超声波检验发现超限缺陷波。用金相显微镜观察了缺陷处微观组织形貌,用能谱仪测试了缺陷部位成分。结果表明：缺陷区Al、V含量尤其是V含量偏低,富氧含碳和少量的氮,组织为粗大的α相。由此得出缺陷区是冶金缺陷,是氧化的海绵钛熔化形成的富氧夹杂和低密度偏析。为了防止此类缺陷给材料带来危害,生产厂在熔炼时应挑选海绵钛,除去氧化硬块。     

Role of free protein S and C4b binding protein in regulating the coagulant response to Escherichia coli

Previous studies showed that infusion of C4b-binding protein with sublethal Escherichia coli (E. coli) in the primate produced a consumptive coagulopathy followed by microvascular thrombosis and renal failure. The first objective of this study was to characterize the pathophysiology and mechanism of this phenomena following infusion of both these agents with emphasis on defining the role of free protein S. The second objective was to examine the relevance of this model to the hemolytic uremic syndrome. Infusion of C4b-binding protein alone reduced free protein S and decreased platelet concentration to 20% of baseline, whereas infusion of the C4b-binding protein/protein S complex did not. There was no activation of other inflammatory or coagulant factors. Infusion of sublethal E coli alone produced a transient inflammatory response with no reduction of free protein S. However, coinfusion of C4b-binding protein with sublethal E coli reduced free protein S and produced a thrombocytopenia, anemia, and a microvascular thrombotic response, whereas infusion of the C4b-binding protein/protein S complex with sublethal E coli did not. Studies comparing the effects of neutralizing (S-163) and nonneutralizing (S-145) antibodies with protein S coinfused with sublethal E coli produced similar contrasting results. Therefore, we concluded that neutralization of free protein S, and not some other property of C4b-binding protein influenced by protein S, accounted for this microvascular thrombotic response. This response is similar to the hemolytic uremic syndrome characterized by thrombocytopenia, anemia, shistocytosis, and renal glomerular thrombosis with uremia. Comparison of the respective renal histopathologic appearance supports this conclusion. This raises the possibility that inhibition of protein S activity (possibly by one of the forms of C4b-binding proteins) might be one of the factors contributing to microvascular thrombotic disorder, such as the hemolytic uremic syndrome.     

QAl10-4-4/TC6双金属连接界面组织与力学性能分析

铜合金/钛合金双金属材料能发挥各自的性能优势,兼具轻质、耐磨、高强等优异性能。本文通过真空热压扩散法连接QAl10-4-4铝青铜和TC6钛合金,并采用显微组织观察和剪切强度测试等方法,研究了直接连接和添加AgCuZnCd连接的QAl10-4-4/TC6双金属的界面组织和力学性能,探究了连接参数与中间层对QAl10-4-4/TC6双金属连接质量的影响规律,分析了双金属界面过渡层形成机理,建立了连接工艺-界面组织-力学性能的内在关联。结果表明：直接扩散连接的QAl10-4-4/TC6双金属连接质量较差,生成的金属间化合物导致界面上生长了贯穿长裂纹,剪切强度仅有21 MPa;添加AgCuZnCd连接QAl10-4-4/TC6双金属后界面金属间化合物减少,当连接温度为850 ℃时,界面剪切强度最大为178.19 MPa,温度超过850 ℃时,双金属界面强度迅速降低。     

Functional analysis of human replication protein A in nucleotide excision repair

Human replication protein A (RPA) is a three-subunit protein complex (70-, 34-, and 11-kDa subunits) involved in DNA replication, repair, and recombination. Both the 70- (p70) and 34-kDa (p34) subunits interact with Xeroderma pigmentosum group A complementing protein (XPA), a key protein involved in nucleotide excision repair. Our deletion analysis indicated that no particular domain(s) of RPA p70 was essential for its interaction with XPA, whereas 33 amino acids from the C terminus of p34 (p34Delta33C) were necessary for the XPA interaction. Furthermore, mutant RPA lacking the p34 C terminus failed to interact with XPA, suggesting that p34, not p70, is primarily responsible for the interaction of RPA with XPA. RPA stimulated the interaction of XPA with UV-damaged DNA through an RPA-XPA complex on damaged DNA sites because (i) the RPA mutant lacking the C terminus of p34 failed to stimulate an XPA-DNA interaction, and (ii) the ssDNA binding domain of RPA (amino acids 296-458) was necessary for the stimulation of the XPA-DNA interaction. Two separate domains of p70, a single-stranded DNA binding domain and a zinc-finger domain, were necessary for RPA function in nucleotide excision repair. The mutant RPA (RPA:p34Delta33C), which lacks its stimulatory effect on the XPA-DNA interaction, also poorly supported nucleotide excision repair, suggesting that the XPA-RPA interaction on damaged DNA is necessary for DNA repair activity.