Golgin-84 is a rab1 binding partner involved in Golgi structure

Members of the golgin family of coiled-coil proteins have been implicated in the tethering of vesicles to Golgi membranes and cisternal membranes to each other. Many also bind to rab GTPases. Golgin-84 is a membrane-anchored golgin that we now show binds preferentially to the GTP form of the rab1 GTPase. It is also present throughout the Golgi stack by immuno-EM. Antibodies to golgin-84 inhibit stacking of cisternal membranes in a cell-free assay for Golgi reassembly, whereas the cytoplasmic domain of golgin-84 stimulates stacking and increases the length of re-assembled stacks. Transient expression of golgin-84 in NRK cells helps prevent the disassembly of the Golgi apparatus normally triggered by treatment with brefeldin A. Together these data suggest that golgin-84 is involved in generating and maintaining the architecture of the Golgi apparatus.     

Global nutritional profiling for mutant and chemical mode-of-action analysis in filamentous fungi

We describe a method for gene function discovery and chemical mode-of-action analysis via nutrient utilization using a high throughput Nutritional Profiling platform suitable for filamentous microorganisms. We have optimized the growth conditions for each fungal species to produce reproducible optical density growth measurements in microtiter plates. We validated the Nutritional Profiling platform using a nitrogen source utilization assay to analyze 21 Aspergillus nidulans strains with mutations in the master nitrogen regulatory gene, areA. Analysis of these data accurately reproduced expected results and provided new data to demonstrate that this platform is suitable for fine level phenotyping of filamentous fungi. Next, we analyzed the differential responses of two fungal species to a glutamine synthetase inhibitor, illustrating chemical mode-of-action analysis. Finally, a comparative phenotypic study was performed to characterize carbon catabolite repression in four fungal species using a carbon source utilization assay. The results demonstrate differentiation between two Aspergillus species and two diverse plant pathogens and provide a wealth of new data on fungal nutrient utilization. Thus, these assays can be used for gene function and chemical mode-of-action analysis at the whole organism level as well as interspecies comparisons in a variety of filamentous fungi. Additionally, because uniform distribution of growth within wells is maintained, comparisons between yeast and filamentous forms of a single organism can be performed.Electronic Supplementary Material Supplementary material is available in the online version of this article at The revised version of the PDF file was published online in January 2004. The figures are now in color.An erratum to this article can be found at     

Infection of human urethral epithelium with Neisseria gonorrhoeae elicits an upregulation of host anti-apoptotic factors and protects cells from staurosporine-induced apoptosis

In order to better understand the host response to an infection with Neisseria gonorrhoeae, microarray technology was used to analyse the gene expression profile between uninfected and infected human urethral epithelium. The anti-apoptotic genes bfl-1, cox-2 and c-IAP-2 were identified to be upregulated approximately eight-, four- or twofold, respectively, following infection. Subsequent assays including RT-PCR, real time RT-PCR and RNase protection confirmed the increased expression of these apoptotic regulators, and identified that a fourth anti-apoptotic factor, mcl-1, is also upregulated. RT-PCR and RNase protection also showed that key pro-apoptotic factors including bax, bad and bak do not change in expression. Furthermore, our studies demonstrated that infection with the gonococcus partially protects urethral epithelium from apoptosis induced by the protein kinase inhibitor, staurosporine (STS). This work shows that following infection with Neisseria gonorrhoeae, several host anti-apoptotic factors are upregulated. In addition, a gonococcal infection protects host cells from subsequent STS-induced death. The regulation of host cell death by the gonococcus may represent a mechanism employed by this pathogen to survive and proliferate in host epithelium.     

Understanding the Hydraulics of Porous Pipes: Tradeoffs Between Water Uptake and Root Length Utilization

The water uptake region in roots is several hundred times longer than the root diameter. The distributed nature of the uptake zone requires that the hydraulic design of roots be understood by analogy to flow through a porous pipe. Here we present results of an analytical and experimental investigation that allowed an in-depth analysis of root hydraulic properties. Measurements on nodal maize roots confirm the nonlinear distribution of water uptake predicted by the porous pipe model. The major design parameter governing the distribution of water uptake along a porous pipe is the ratio between its axial and radial hydraulic resistance. However, total flow is proportional to the pipe's overall resistance. These results suggest the existence of a tradeoff between the effective utilization of root length and the total capacity for water uptake.     

Lysyl oxidase oxidizes basic fibroblast growth factor and inactivates its mitogenic potential

Lysyl oxidase (LO) plays a central role in the crosslinking of collagen and elastin in the extracellular matrix. Here we demonstrate that basic fibroblast growth factor (bFGF), a polypeptide which regulates proliferation, differentiation, and migration of a variety of cell types, is a substrate of LO. The oxidation of lysine residues in bFGF by LO resulted in the covalent crosslinking of bFGF monomers to form dimers and higher order oligomers and dramatically altered its biological properties. Both the mitogenic potential and the nuclear localization of bFGF were markedly inhibited in the Swiss 3T3 cells upon its oxidation by LO. NIH 3T3 IgBNM 6-1 cells (6-1 cells) overexpress bFGF which participates in an autocrine mechanism accounting for the transformation of these cells into a tumorigenic state. Exposure of the 6-1 cells to nanomolar concentrations of LO in culture oxidized lysine and generated crosslinkages in bFGF within the cell and markedly reduced proliferative rates. The lack of LO expression has been correlated with hyperproliferative cell growth, while this enzyme has been identified as a suppressor of ras-induced tumorigenesis. The present results illustrate a mechanism by which LO can depress normal and transformed cell growth.     

Gene expression databases and data mining

The DNA microarray technology has arguably caught the attention of the worldwide life science community and is now systematically supporting major discoveries in many fields of study. The majority of the initial technical challenges of conducting experiments are being resolved, only to be replaced with new informatics hurdles, including statistical analysis, data visualization, interpretation, and storage. Two systems of databases, one containing expression data and one containing annotation data are quickly becoming essential knowledge repositories of the research community. This present paper surveys several databases, which are considered "pillars" of research and important nodes in the network. This paper focuses on a generalized workflow scheme typical for microarray experiments using two examples related to cancer research. The workflow is used to reference appropriate databases and tools for each step in the process of array experimentation. Additionally, benefits and drawbacks of current array databases are addressed, and suggestions are made for their improvement.     

Identification and characterization of mouse SSX genes: a multigene family on the X chromosome with restricted cancer/testis expression

Human SSX was first identified as the gene involved in the t(X;18) translocation in synovial sarcoma. SSX is a multigene family, with 9 complete genes on chromosome Xp11. Normally expressed almost exclusively in testis, SSX mRNA is expressed in various human tumors, defining SSX as a cancer/testis antigen. We have now cloned the mouse ortholog of SSX. Mouse SSX genes can be divided into Ssxa and Ssxb subfamilies based on sequence homology. Ssxa has only one member, whereas 12 Ssxb genes, Ssxb1 to Ssxb12, were identified by cDNA cloning from mouse testis and mouse tumors. Both Ssxa and Ssxb are located on chromosome X and show tissue-restricted mRNA expression to testis among normal tissues. All putative human and mouse SSX proteins share conserved KRAB and SSX-RD domains. Mouse tumors were found to express some, but not all, Ssxb genes, similar to the SSX activation in human tumors.     

Na+/H+ exchanger-regulatory factor 1 mediates inhibition of phosphate transport by parathyroid hormone and second messengers by acting at multiple sites in opossum kidney cells

The opossum kidney (OK) line displays PTH-mediated activation of adenylyl cyclase and phospholipase C and inhibition of phosphate (Pi) uptake via regulation of the type IIa sodium-phosphate cotransporter, consistent with effects in vivo. OKH cells, a subclone of the OK cell line, robustly activates PTH-mediated activation of adenylyl cyclase, but is defective in PTH-mediated inhibition of sodium-phosphate cotransport and signaling via phospholipase C. Compared with wild-type OK cells, OKH cells express low levels of the Na+/H+ exchanger regulatory factor 1 (NHERF-1). Stable expression of NHERF-1 in OKH cells (OKH-N1) rescues the PTH-mediated inhibition of sodium-phosphate cotransport. NHERF-1 also restores the capacity of 8-bromo-cAMP and forskolin to inhibit Pi uptake, but the PTH dose-response for cAMP accumulation and inhibition of Pi uptake differ by 2 orders of magnitude. NHERF-1, in addition, modestly restores phorbol ester-mediated inhibition of Pi uptake, which is much weaker than that elicited by PTH. A poor correlation exists between the inhibition of Pi uptake mediated by PTH ( approximately 60%) and the inhibition mediated by phorbol 12-myristate 13-acetate ( approximately 30%) and the ability of these molecules to activate the protein kinase C-responsive reporter gene. Furthermore, we show that NHERF-1 directly interacts with type IIa cotransporter in OK cells. Although, PTH-mediated inhibition of Pi uptake in OK cells is largely NHERF-1 dependent, the signaling pathway(s) by which this occurs is still unclear. These pathways may involve cooperativity between cAMP- and protein kinase C-dependent pathways or activation/inhibition of an unrecognized NHERF-1-dependent pathway(s).