MF Olson  NG Pasteris  JL Gorski  A Hall 《Canadian Metallurgical Quarterly》1996,6(12):1628-1633

BACKGROUND: Dbl, a guanine nucleotide exchange factor (GEF) for members of the Rho family of small GTPases, is the prototype of a family of 15 related proteins. The majority of proteins that contain a DH (Dbl homology) domain were isolated as oncogenes in transfection assays, but two members of the DH family, FGD1 (the product of the faciogenital dysplasia or Aarskog-Scott syndrome locus) and Vav, have been shown to be essential for normal embryonic development. Mutations to the FGD1 gene result in a human developmental disorder affecting specific skeletal structures, including elements of the face, cervical vertebrae and distal extremities. Homozygous Vav-/- knockout mice embryos are not viable past the blastocyst stage, indicating an essential role of Vav in embryonic implantation. RESULTS: Here, we show that the microinjection of FGD1 and Vav into Swiss 3T3 fibroblasts induces the polymerization of actin and the assembly of clustered integrin complexes. FGD1 activates Cdc42, whereas Vav activates Rho, Rac and Cdc42. In addition, FGD1 and Vav stimulate the mitogen activated protein kinase cascade that leads to activation of the c-Jun kinase SAPK/JNK1. CONCLUSIONS: We conclude that FGD1 and Vav are regulators of the Rho GTPase family. Along with their target proteins Cdc42, Rac and Rho, FGD1 and Vav control essential signals required during embryonic development.  相似文献   

    

FJ Dilworth  GR Williams  AM Kissmeyer  JL Nielsen  E Binderup  MJ Calverley  HL Makin  G Jones 《Canadian Metallurgical Quarterly》1997,138(12):5485-5496

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C Poussin  M Foti  JL Carpentier  J Pugin 《Canadian Metallurgical Quarterly》1998,273(32):20285-20291

Gram-negative bacterial endotoxin (a lipopolysaccharide (LPS)) specifically binds to CD14, a glycosylphosphatidyl inositol (GPI)-anchored surface myeloid glycoprotein. This interaction leads to cell activation, but it also promotes LPS internalization and detoxification. In this work, we investigated the route of LPS and CD14 internalization and the relevance of CD14 GPI anchor in the endocytic pathway. In promonocytic THP-1 cells transfected with a GPI or a chimeric integral form of CD14, we showed by differential buoyancy in sucrose density gradients that these two forms of CD14 were sorted to different plasma membrane subdomains. However, both forms of CD14 associated preferentially with the same surface microfilament-enriched microvilli or ruffles. Electron microscopic studies indicated that CD14 internalized via macropinocytosis, a process resembling that of phagocytosis, different from \"classical\" receptor-mediated endocytic pathways, such as clathrin-coated pits or caveolae. With cell warming, the CD14-enriched ruffles fused and formed large vesicles. Later, these vacuoles made stacks and condensed into phago-lysosomes. CD14 was specifically associated with all of these structures. Radiolabeled LPS internalization paralleled CD14 internalization. Confocal microscopic studies confirmed the co-localization of LPS and CD14 both at the cell surface and in endosomal compartments. The microfilament-disrupting, macropinocytosis blocking agent cytochalasin D inhibited LPS and CD14 internalization but did not prevent LPS-dependent activation, indicating that these two processes are dissociated.  相似文献   

    

AA Zamani  T Moriarty  L Hsu  CS Winalski  JL Schaffer  H Isbister  JF Schenck  KW Rohling  F Jolesz 《Canadian Metallurgical Quarterly》1998,8(6):1329-1333

High-level penicillin resistance in pneumococci is due to alterations in penicillin-binding proteins (PBPs) 2X, 2B, and 1A. We have sequenced the penicillin-binding domain of PBP 1A from penicillin-resistant South African pneumococcal isolates and have identified amino acid substitutions which are common to all the resistant isolates analyzed. Site-directed mutagenesis was then used to determine whether particular amino acid substitutions at specific positions in PBP 1A mediate penicillin resistance. PCR was used to isolate PBP 2X, 2B, and 1A genes from clinical isolate 8303 (penicillin MIC, 4 micrograms/ml). These wild-type PBP genes were cloned into pGEM-3Zf and were used as the transforming DNA. Susceptible strain R6 (MIC, 0.015 microgram/ml) was first transformed with PBP 2X and 2B DNA, resulting in PBP 2X/2B-R6 transformants for which MICs were 0.25 microgram/ml. When further transformed with PBP 1A DNA, 2X/2B/1A-R6 transformants for which MICs were 1.5 micrograms/ml were obtained. Site-directed mutagenesis of the PBP 1A gene from isolate 8303 was then used to reverse particular amino acid substitutions, followed by transformation of PBP 2X/2B-R6 transformants with the mutagenized PBP 1A DNA. For PBP 2X/2B/1A-R6 transformants, the introduction of the reversal of Thr-371 by Ser or Ala in PBP 1A decreased the MIC from 1.5 to 0.5 micrograms/ml, whereas the reversal of four consecutive amino acid substitutions (Thr-574 by Asn, Ser-575 by Thr, Gln-576 by Gly, and Phe-577 by Tyr) decreased the MIC from 1.5 to 0.375 micrograms/ml. These data reveal that amino acid residue 371 and residues 574 to 577 of PBP 1A are important positions in PBP 1A with respect to the interaction with penicillin and the development of resistance.  相似文献   

    

HB Goldman  RS Israeli  JL Lerner  RS Hollabaugh  MS Steiner 《Canadian Metallurgical Quarterly》1998,52(6):1073-1078

In this investigation, the presence of NKA-immunoreactive substances was determined in pineal glands from intact, castrated and castrated, testosterone-treated male rats. The effect of environmental light, melatonin treatment and superior cervical ganglionectomy on pineal NKA-immunoreactive substances was also investigated. The results obtained show that NKA is present in measurable amounts in the rat pineal, and NPK is probably also present, Orchidectomy was followed by an increase in the content of NKA-immunoreactive substances in the pineal gland. The replacement treatment with testosterone propionate in castrated rats blocked this effect. NKA-immunoreactive substances were not significantly different quantitatively in pineals from rats killed under light or under darkness. The removal of the superior cervical ganglia was followed by a significant increase in the NKA-immunoreactive substance content in the pineal gland of male rats. These results indicate that NKA and other tachykinins are present in the pineal gland of the male rat, and they seem to be regulated by gonadal hormones and the innervation originated from the superior cervical ganglia.  相似文献   

    

van Ruitenbeek JM  van Deursen AP  HW Myron  AJ Arko  JL Smith 《Canadian Metallurgical Quarterly》1986,34(12):8507-8511

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C Clark  D Bast  AM Sharp  PM St Hilaire  R Agha  PE Stein  EJ Toone  RJ Read  JL Brunton 《Canadian Metallurgical Quarterly》1996,19(4):891-899

The homopentameric B subunit of verotoxin 1 (VT1) binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). We produced mutants with alanine substitutions for residues found near the cleft between adjacent subunits. Substitution of alanine for phenylalanine 30 (Phe-30) resulted in a fourfold reduction in B subunit binding affinity for Gb3 and a 10-fold reduction in receptor density in a solid-phase binding assay. The interaction of wild-type and mutant B subunits with Pk trisaccharide in solution was examined by titration microcalorimetry. The carbohydrate binding of the mutant was markedly impaired compared with that of the wild type and was too weak to allow calculation of a binding constant. These results demonstrate that the mutation significantly impaired the carbohydrate-binding function of the B subunit. To ensure that the mutation had not caused a significant change in structure, the mutant B subunit was crystallized and its structure was determined by X-ray diffraction. Difference Fourier analysis showed that its structure was identical to that of the wild type, except for the substitution of alanine for Phe-30. The mutation was also produced in the VT1 operon, and mutant holotoxin was purified to homogeneity. The cytotoxicity of the mutant holotoxin was reduced by a factor of 10(5) compared to that of the wild type in the Vero cell cytotoxicity assay. The results suggest that the aromatic ring of Phe-30 plays a major role in binding of the B subunit to the Galalpha1-4Galbeta1-4Glc trisaccharide portion of Gb3. Examination of the VT1 B crystal structure suggests two potential carbohydrate-binding sites which lie on either side of Phe-30.  相似文献   

    

A Alonso  JL Martínez 《Canadian Metallurgical Quarterly》1997,41(5):1140-1142

A cryptic multidrug resistance (MDR) system in Stenotrophomonas maltophilia, the expression of which is selectable by tetracycline, is described. Tetracycline resistance was the consequence of active efflux of the antibiotic, and it was associated with resistance to quinolones and chloramphenicol, but not to aminoglycosides or beta-lactam antibiotics. MDR is linked to the expression of an outer membrane protein (OMP54) both in a model system and in multidrug-resistant clinical isolates.  相似文献   

    

JP McHale  R Kuersten  A Lauretti 《Canadian Metallurgical Quarterly》1996,(74):5-26

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XZ Zhang  MP Donovan  BT Williams  JL Mohler 《Canadian Metallurgical Quarterly》1996,47(3):402-404

OBJECTIVES: To determine whether subcapsular orchiectomy provides suboptimal treatment of metastatic prostate cancer when used to avoid the psychologic consequences of the empty scrotum that results from total orchiectomy. METHODS: We compared testosterone and prostate-specific antigen levels and survival of 37 patients who underwent total orchiectomy and 37 patients who underwent subcapsular orchiectomy for metastatic prostate cancer. RESULTS: The two groups of 37 patients were similar by clinical parameters. Postoperatively, testosterone levels were 21 +/- 11 ng/dL for subcapsular versus 21 +/- 9 ng/dL for total orchiectomy patients. Tumor response was similar in the two groups when assessed by prostate-specific antigen measured 3 weeks, 6 months, and 1, 2, and 3 years postoperatively. Survival was similar when assessed using Kaplan-Meier analysis (P = 0.76). CONCLUSIONS: Subcapsular orchiectomy is a viable option for treatment of metastatic prostate cancer.  相似文献