Fragment A of diphtheria toxin causes pH-dependent lesions in model membranes

Fragment A of diphtheria toxin has been shown to insert into lipid bilayers at low pH (Montecucco, C., Schiavo, G., and Tomasi, M. (1985) Biochem. J. 231, 123-128; Zhao, J.-M., and London, E. (1988) J. Biol. Chem. 263, 15369-15377). In this report, evidence is provided which demonstrates that fragment A, like diphtheria toxin, can also cause the release of a fluorescent dye (calcein) from vesicles under acidic conditions and that this release parallels fragment A insertion into the membrane. Although the permeability changes are not as large as those obtained with whole toxin (Jiang, G.-S., Solow, R., and Hu, V. W. (1989) J. Biol. Chem. 264, 13424-13429), molecular sieving experiments indicate that the lesion induced by fragment A increases in size with decreasing pH and reaches an upper limit of 30 A at pH 4.0. In addition to size differences, the lesion induced by fragment A releases calcein in a graded manner, whereas diphtheria toxin causes an all-or-none release. One possible interpretation of this result is that the fragment A lesion is transient in comparison to that induced by whole toxin. Although the molecular bases for the observed differences are not understood, these data suggest that fragment A interaction with the lipid bilayer may play a significant role in mediating its own translocation across membranes and that fragment B may aid this process by initiating, enlarging, and stabilizing the lesion formed.     

Dose-dependent metabolic response of mammary carcinoma to photodynamic therapy

The metabolic response of mammary carcinoma in the C3H mouse to photodynamic therapy (PDT) was measured using in vivo 31P nuclear magnetic resonance (31P-NMR) spectroscopy and pH microelectrodes. Twenty-four hours after administration of Photofrin II (12.5 mg/kg), the tumor was subjected to photoactivation using an argon dye laser. Optical treatment doses were 200, 400, and 600 J/cm2 and corresponded to the following tumor control doses: TCD10/30, TCD50/30, and TCD90/30, respectively. In vivo 31P-NMR spectra and pH micro-electrode measurements were obtained prior to treatment and at 4, 24, 48, and 72 h and 1 week post-treatment. The data revealed a significant (P less than 0.0002) alkalosis as indicated by the pH measured by NMR compared to pH measured by microelectrodes at all treatment levels and time points. Spectral differences between treatment groups were apparent as early as 4 h after treatment. The ratio of beta-nucleoside triphosphate to inorganic phosphate at 4 h after treatment was significantly (P less than 0.01) smaller for 600 J/cm2 treatment than for 200 J/cm2 treatment. At curative (600 J/cm2) levels, from 48 h on, no phosphate resonances were detected in the spectra. The pH measured by NMR transiently decreased from pretreatment levels after 200 and 400 J/cm2 treatment (P less than 0.002, P less than 0.009, respectively), while no change in pH from pretreatment values was found after 600 J/cm2 treatment. The data suggest that the early metabolic response of mammary carcinoma to PDT, as indicated by 31P-NMR spectroscopy, is dose dependent, and may be a sensitive indicator of biological outcome to treatment.     

Minimal sequence requirement of thrombin receptor agonist peptide.

A site-specific proteolytically generated neoamino terminus of the thrombin receptor having a sequence SFLLRNPNDKYEPF- has been reported to be a functional ligand of the receptor. This discovery raises question on the precise structural requirements of the "tethered ligand" responsible for receptor activation and signal transduction. By examining the agonist activity of a panel of synthetic sequence analogues of thrombin receptor agonist peptides (TRAP) on human platelet aggregation, we determined that the minimal sequence of the human platelet thrombin receptor ligand is SFLL-amide (TRAP1-4, EC50 = 300 uM). An extension of TRAP1-4 by an additional Arg-Asn segment yielded the most potent agonist among the series (TRAP1-6, EC50 = 1.3 microM). Based on the structure-activity relationships, we hypothesized a model of the ligand-binding site of the human platelet thrombin receptor that accommodates a hexapeptide structure. TRAP1-6, when administered intravenously, induced marked intravascular platelet aggregation in the anesthetized guinea pigs.     

小鼠LAK细胞对红系祖细胞及多向造血祖细胞增殖的影响

小鼠脾细胞经重组人白细胞介素-2(rhIL-2)激活后对YAC-1,LP-3和WEHI-164等肿瘤细胞均有很强的杀伤活性。在CFU-E和BFU-E培养体系中,不同浓度LAK细胞与BMC直接加入或预温育4h后再培养,均能加强CFU-E和BFU-E增殖。低浓度LAK细胞(LAK/BMC为0.5)与BMC直接加入或预温育后再加入CFU-mix培养体系中,均能增强CFU-mix增殖,而高浓度LAK细胞和BMC(LAK/BMC=8.0)直接加入培养体系则抑制CFU-mix增殖;若共温育后再培养则非常明显地抑制CFU-mix增殖,CFU-mix仅为对照的17.6％。小鼠LAK细胞对造血祖细胞体外增殖具有调节作用,这种调节可能包括分泌某些细胞因子以及细胞间直接相互作用两种方式。     

家兔脊髓蛛网膜下腔注射乙酰胆碱对血压和心率的影响

将乙酰胆碱(ACh)注入麻醉家兔脊髓蛛网膜下腔,观察其对心血管活动的影响。结果表明:(1)脊髓蛛网膜下腔注射50～100μg ACh可使血压下降,心率减慢;(2)预先由脊髓蛛网膜下腔注射阿托品,可阻断ACh引起的降压和降心率作用;(3)脊髓蛛网膜下腔注射六甲双铵、酚妥拉明或心得安均不能阻断上述ACh的心血管反应;(4)切断两侧颈部迷走神经,ACh不再使心率减慢,但其降低血压的作用不受到任何影响。 脊髓中ACh水平升高可通过激活胆碱能M-受体引起血压下降和心率减慢。ACh的这种降压作用既没有中枢肾上腺素能受体活动参与,也不是通过迷走神经实现的,可能是由于脊髓交感血管中枢紧张性降低所造成的。     

健康青少年口腔正常菌群的初步研究

本文采用非选择性培养基对22名健康青少年的唾液、沟裂菌斑、龈上菌斑及龈下菌斑中的需氧菌、兼性厌氧菌及专性厌氧菌进行了分离培养,并计算其在不同标本中占可培养菌的百分比及检出率。结果共分离到包括18个菌属的35种细菌。其中,链球菌、放线菌、奈瑟氏球菌、二氧化碳噬纤维菌、类杆菌、梭杆菌,奴卡氏菌及棒状杆菌在口腔4个部位的检出率及所占比例均较高,是健康青少年口腔中的优势菌群.通过比较还发现,其中一些菌在口腔4个部位的分布存在一定差异.本文还采用刚果红负性染色涂片法,镜下观察龈上、龈下菌斑中的螺旋体,并计算其相对比例.结果龈下菌斑中螺旋体的相对比例明显高于龈上菌斑.     

Contractile properties of bronchial smooth muscle with and without cartilage

The majority of in vitro studies on airway smooth muscle have used the trachealis (TSM) as a convenient substitute for muscle from airways that constitute the flow-limiting segment. The latter are technically difficult to work with. However, because the site of maximum resistance to airflow is at the third to seventh generations of the bronchial tree, the trachealis preparation is of limited value. Length-tension and force-velocity properties were therefore studied at optimal length (lo) of canine bronchial smooth muscle (BSM) from which cartilage had been carefully removed. Normalized maximum isometric tension or stress (Po x 10(4) N/m2) for BSM was 7.1 +/- 0.19 (SE), which was similar to that of BSM with cartilage (BSM+C, 6.8 +/- 0.21) but lower than for TSM (18.2 +/- 0.81). At length greater than lo, the BSM+C was stiffer than the BSM. The values of maximum shortening capacity (delta Lmax), obtained directly from isotonic shortening at a load equal to the resting tension at lo, were 0.76 lo +/- 0.03, 0.41 lo +/- 0.02, and 0.24 +/- 0.02 lo for TSM, BSM, and BSM+C, respectively. The BSM and BSM+C delta Lmaxs were different (P less than 0.05). Maximal shortening velocities (Vo) for BSM, elicited at 2, 4, and 8 s by quick release in the course of an isometric contraction were significantly higher than for the BSM+C. Vos showed gradual decreases in all three groups in the later phase of contraction, suggesting the operation of latch bridges.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Epstein-Barr virus-encoded nuclear antigen EBNA-5 accumulates in PML-containing bodies.

EBNA-5 is one of the Epstein-Barr virus (EBV)-encoded nuclear proteins required for immortalization of human B lymphocytes. In the nuclei of EBV-transformed lymphoblastoid cell lines EBNA-5 is preferentially targetted to distinct nuclear foci. Previously we have shown (W.Q. Jiang, L. Szekely, V. Wendel-Hansen, N. Ringertz, G. Klein, and A. Rosen, Exp. Cell Res. 197:314-318, 1991) that the same foci also contained the retinoblastoma (Rb) protein. Using a similar double immunofluorescence technique, we now show that these foci colocalize with nuclear bodies positive for PML, the promyelocytic leukemia-associated protein. Artificial spreading of the chromatin by exposure to the forces of fluid surface tension disrupts this colocalization gradually, suggesting that the bodies consist of at least two subcomponents. Heat shock or metabolic stress induced by high cell density leads to the release of EBNA-5 from the PML-positive nuclear bodies and induces it to translocate to the nucleoli. In addition to their presence in nuclear bodies, both proteins are occasionally present in nuclear aggregates and doughnut-like structures in which PML is concentrated in an outer shell. Nuclear bodies with prominent PML staining are seen in resting B lymphocytes. This staining pattern does not change upon EBV infection. In freshly infected cells EBNA-5 antigens are first distributed throughout the nucleoplasm. After a few days intensely staining foci develop. These foci coincide with PML-positive nuclear bodies. At a later stage and in established lymphoblastoid cell lines EBNA-5 is almost exclusively present in the PML-positive nuclear foci. The colocalization is restricted to EBV-infected human lymphoblasts. The data presented indicate that the distinct EBNA-5 foci are not newly formed structures but the result of translocation of the viral protein to a specialized domain present already in the nuclei of uninfected cells.     

Antixenotic resistance of subterranean clover cotyledons to redlegged earth mite, Halotydeus destructor

The short-term response of redlegged earth mite, Halotydeus destructor (Tucker) (Acarina: Penthaleidae) to cotyledons of different varieties of subterranean clover (Trifolium subterraneum subsp. subterraneum L.) was assessed by means of paired choice tests, and no-choice tests. H. destructor had lower numbers and fed less on detached cotyledons of resistant than susceptible varieties, yielding a correlation between the numbers of mites and feeding damage to the cotyledons during a three hour test period. For a number of resistant and susceptible varieties, there was a negative correlation between cotyledon deterrence in the three hour choice test and feeding damage to seedling after a two week period. Since the response of the mites to different subclover varieties occurred within three hours, it is concluded that the resistance is based on antixenosis.No evidence was obtained for antifeedant activity in organic solvent extracts from the variety DGI007 (resistant) in comparison with those from the variety Dalkeith (susceptible). Water soluble compounds from DGI007 cotyledons were preferred by mites, in feeding tests in terms of numbers, over those from Dalkeith (susceptible). Squeezed sap from the cotyledons of both varieties showed the same effects on mites as 5% glucose and were more phagostimulatory than water extracts. Mechanically damaged cotyledons of Dalkeith and DGI007 attracted more mites than the undamaged counterparts. The toughness of cotyledons in 17 varieties of T. subterraneum subsp. subterraneum was measured with a manual penetrometer. Results showed a negative correlation between toughness values and mite feeding damage scores (r²=0.752) for all varieties except S3615D (resistant). This implies a likely involvement of epidermal toughness as a contributor in the antixenotic resistance of these varieties. Other mechanisms may be involved in the resistance of S3615D.     

CWH41 encodes a novel endoplasmic reticulum membrane N-glycoprotein involved in beta 1,6-glucan assembly.

CWH41 encodes a novel type II integral membrane N-glycoprotein located in the endoplasmic reticulum. Disruption of the CWH41 gene leads to a K1 killer toxin-resistant phenotype and a 50% reduction in the cell wall beta 1,6-glucan level. CWH41 also displays strong genetic interactions with KRE1 and KRE6, two genes known to be involved in the beta 1,6-glucan biosynthetic pathway. The cwh41 delta kre6 delta double mutant is nonviable; and the cwh41 delta kre1 delta double mutation results in strong synergistic defects, with a severely slow-growth phenotype, a 75% reduction in beta 1,6-glucan level, and the secretion of a cell wall glucomannoprotein, Cwp1p. These results provide strong genetic evidence indicating that Cwh41p plays a functional role, possibly as a new synthetic component, in the assembly of cell wall beta 1,6-glucan.