Bacillus subtilis Cells Lacking Penicillin-Binding Protein 1 Require Increased Levels of Divalent Cations for Growth

Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg²⁺ or Ca²⁺ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of ponA cells in a medium low in Mg²⁺ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg²⁺ to growth media eliminated these phenotypes of a ponA mutant. In contrast to the effects of divalent cations, NaCl did not restore ponA cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg²⁺ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.     

Identification of a Novel, Putative Rho-specific GDP/GTP Exchange Factor and a RhoA-binding Protein: Control of Neuronal Morphology

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein–coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116^Rip, that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116^Rip stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116^Rip fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein–coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116^Rip inhibits RhoA-stimulated contractility and promotes neurite outgrowth.     

Outgrowth of nerve fibres from Drosophila central nervous system cultured in vitro

Explants of the central nervous system of Drosophila have been shown to produce nerve fibres in vitro. The effects of various culture conditions on fibre outgrowth have been examined. Nervous tissue could form nerve fibres in vitro when the explants were obtained from mid-embryonic or early- and mid-pupal stages, but not when they were obtained from larvae or late-pupae. The effect of the temperature-sensitive mutation shibire^ts has been investigated by placing mutant explants into culture at permissive (17°C) or restrictive (28°C) temperatures. No differences in the extent of fibre outgrowth between wild-type and shibire^ts were observed, regardless of the temperature of cultivation.     

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues¹ and survival factors² located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons³. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons⁴. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro^5,6.Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol⁷) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival⁸.     

ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA

Background

Neurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized.

Methodology/Principal Findings

Here we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR^−/− mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR^−/−) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR^−/− neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action.

Conclusions

proBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.     

The Absence of CD47 Promotes Nerve Fiber Growth from Cultured Ventral Mesencephalic Dopamine Neurons

In ventral mesencephalic organotypic tissue cultures, two timely separated sequences of nerve fiber growth have been observed. The first appearing nerve fiber pattern is a long-distance outgrowth that occurs before astrocytes start to proliferate and migrate to form an astrocytic monolayer that finally surrounds the tissue slice. These long-distance growing nerve fibers are retracted as the astrocytes migrate, and are followed by a secondary outgrowth. The secondary outgrowth is persistent in time but reaches short distances, comparable with outgrowth seen from a dopaminergic graft implanted to the brain. The present study was focused on the interaction between the astrocytes and the long-distance growing non-glial associated nerve fibers. Cross talk between astroglia and neurite formation might occur through the integrin-associated protein CD47. CD47 serves as a ligand for signal regulatory protein (SIRP) α and as a receptor for the extracellular matrix protein thrombospondin-1 (TSP-1). Embryonic day 14 ventral mesencephalic tissue from CD47^+/+ and CD47^−/− mice was used to investigate astrocytic migration and the tyrosine hydroxylase (TH) –positive outgrowth that occurred remote from the astrocytes. TH-immunohistochemistry demonstrated that the non-glial-associated nerve fiber outgrowth in CD47^−/− cultures reached significantly longer distances and higher density compared to nerve fibers formed in CD47^+/+ cultures at 14 days in vitro. These nerve fibers often had a dotted appearance in CD47^+/+ cultures. No difference in the astrocytic migration was observed. Further investigations revealed that the presence of CD47 in control culture did neither hamper non-glial-associated growth through SIRPα nor through TSP-1 since similar outgrowth was found in SIRPα mutant cultures and in CD47^+/+ cultures treated with blocking antibodies against the TSP-1, respectively, as in the control cultures. In conclusion, long-distance growing nerve fiber formation is promoted by the absence of CD47, even though the presence of astrocytes is not inhibited.     

Primary Neuron Culture for Nerve Growth and Axon Guidance Studies in Zebrafish (Danio rerio)

Zebrafish (Danio rerio) is a widely used model organism in genetics and developmental biology research. Genetic screens have proven useful for studying embryonic development of the nervous system in vivo, but in vitro studies utilizing zebrafish have been limited. Here, we introduce a robust zebrafish primary neuron culture system for functional nerve growth and guidance assays. Distinct classes of central nervous system neurons from the spinal cord, hindbrain, forebrain, and retina from wild type zebrafish, and fluorescent motor neurons from transgenic reporter zebrafish lines, were dissociated and plated onto various biological and synthetic substrates to optimize conditions for axon outgrowth. Time-lapse microscopy revealed dynamically moving growth cones at the tips of extending axons. The mean rate of axon extension in vitro was 21.4±1.2 µm hr⁻¹ s.e.m. for spinal cord neurons, which corresponds to the typical ∼0.5 mm day⁻¹ growth rate of nerves in vivo. Fluorescence labeling and confocal microscopy demonstrated that bundled microtubules project along axons to the growth cone central domain, with filamentous actin enriched in the growth cone peripheral domain. Importantly, the growth cone surface membrane expresses receptors for chemotropic factors, as detected by immunofluorescence microscopy. Live-cell functional assays of axon extension and directional guidance demonstrated mammalian brain-derived neurotrophic factor (BDNF)-dependent stimulation of outgrowth and growth cone chemoattraction, whereas mammalian myelin-associated glycoprotein inhibited outgrowth. High-resolution live-cell Ca²⁺-imaging revealed local elevation of cytoplasmic Ca²⁺ concentration in the growth cone induced by BDNF application. Moreover, BDNF-induced axon outgrowth, but not basal outgrowth, was blocked by treatments to suppress cytoplasmic Ca²⁺ signals. Thus, this primary neuron culture model system may be useful for studies of neuronal development, chemotropic axon guidance, and mechanisms underlying inhibition of neural regeneration in vitro, and complement observations made in vivo.     

Cell Surface Sialylation and Fucosylation Are Regulated by L1 via Phospholipase Cγ and Cooperate to Modulate Neurite Outgrowth,Cell Survival and Migration

Background

Cell surface glycosylation patterns are markers of cell type and status. However, the mechanisms regulating surface glycosylation patterns remain unknown.

Methodology/Principal Findings

Using a panel of carbohydrate surface markers, we have shown that cell surface sialylation and fucosylation were downregulated in L1^−/y neurons versus L1^+/y neurons. Consistently, mRNA levels of sialyltransferase ST6Gal1, and fucosyltransferase FUT9 were significantly reduced in L1^−/y neurons. Moreover, treatment of L1^+/y neurons with L1 antibodies, triggering signal transduction downstream of L1, led to an increase in cell surface sialylation and fucosylation compared to rat IgG-treated cells. ShRNAs for both ST6Gal1 and FUT9 blocked L1 antibody-mediated enhancement of neurite outgrowth, cell survival and migration. A phospholipase Cγ (PLCγ) inhibitor and shRNA, as well as an Erk inhibitor, reduced ST6Gal1 and FUT9 mRNA levels and inhibited effects of L1 on neurite outgrowth and cell survival.

Conclusions

Neuronal surface sialylation and fucosylation are regulated via PLCγ by L1, modulating neurite outgrowth, cell survival and migration.     

The Bacillus cereus GerN and GerT protein homologs have distinct roles in spore germination and outgrowth, respectively

The GerT protein of Bacillus cereus shares 74% amino acid identity with its homolog GerN. The latter is a Na⁺/H⁺-K⁺ antiporter that is required for normal spore germination in inosine. The germination properties of single and double mutants of B. cereus ATCC 10876 reveal that unlike GerN, which is required for all germination responses that involve the GerI germinant receptor, the GerT protein does not have a significant role in germination, although it is required for the residual GerI-mediated inosine germination response of a gerN mutant. In contrast, GerT has a significant role in outgrowth; gerT mutant spores do not outgrow efficiently under alkaline conditions and outgrow more slowly than the wild type in the presence of high NaCl concentrations. The GerT protein in B. cereus therefore contributes to the success of spore outgrowth from the germinated state during alkaline or Na⁺ stress.     

Vegetative axillary bud dormancy induced by shade and defoliation signals in the grasses

Vegetative axillary bud dormancy and outgrowth is regulated by several hormonal and environmental signals. In perennials, the dormancy induced by hormonal and environmental signals has been categorized as eco-, endo- or para-dormancy. Over the past several decades para-dormancy has primarily been investigated in eudicot annuals. Recently, we initiated a study using the monoculm phyB mutant (phyB-1) and the freely branching near isogenic wild type (WT) sorghum (Sorghum bicolor) to identify molecular mechanisms and signaling pathways regulating dormancy and outgrowth of axillary buds in the grasses. In a paper published in the January 2010 issue of Plant Cell and Environment, we reported the role of branching genes in the inhibition of bud outgrowth by phyB, shade and defoliation signals. Here we present a model that depicts the molecular mechanisms and pathways regulating axillary bud dormancy induced by shade and defoliation signals in the grasses.Key words: axillary bud, dormancy, shade, phytochrome, defoliation, shoot branching, teosinte branched1, MAX2, cell cycle, sorghumThe dormancy and outgrowth of axillary buds is regulated by several plant hormones such as auxin, cytokinins, abscisic acid and strigolactones, and by environmental factors such as light quality, quantity and duration as well as water, temperature and nutrient status.^1–3 Since the fate of an axillary bud is regulated by such diverse hormonal and environmental signals and their interactions, the type of dormancy induced varies. In perennials, three types of bud dormancy have been identified.^4,5 Dormancy mediated by factors within the bud is known as endo-dormancy; while dormancy induced by factors within the plant but outside the bud is called paradormancy or correlative inhibition; the best known example being apical dominance. Dormancy induced due to unfavorable environmental conditions is known as eco-dormancy. Although there is an indepth knowledge about para-dormancy in annuals,⁶ few studies have been conducted on eco-dormancy. Similarly, studies of endo-dormancy have largely been restricted to low-temperature mediated growth-cessation of axillary buds of perennial plants.^7,8 To understand the regulation of dormancy and outgrowth of axillary buds in monocots, we initiated a study on the molecular mechanisms inhibiting bud outgrowth by shade and defoliation signals in sorghum. Our results published in the January 2010 issue of Plant, Cell & Environment indicate that different types of dormancy may be induced in axillary buds of annual grasses by various signals and there may be overlapping and independent molecular mechanisms mediating induction of axillary bud dormancy.     

Characterization of yhcN,a new forespore-specific gene of Bacillus subtilis

A new Bacillus subtilis sporulation-specific gene, yhcN, has been identified, the expression of which is dependent on the forespore-specific sigma factor σ^G and to a much lesser extent on σ^F. A translational yhcN-lacZ fusion is expressed at a very high level in the forespore, and the protein encoded by yhcN was detected in the inner spore membrane. A yhcN mutant sporulates normally and yhcN spores have identical resistance properties to wild-type spores. However, the outgrowth of yhcN spores is slower than that of wild-type spores.     

Developmental changes in the regulation of calcium-dependent neurite outgrowth

Intracellular calcium ions (Ca²⁺) have an essential role in the regulation of neurite outgrowth, but how outgrowth is controlled remains largely unknown. In this study, we examined how the mechanisms of neurite outgrowth change during development in chick and mouse dorsal root ganglion neurons. 2APB, a potent inhibitor of inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R), inhibited neurite outgrowth at early developmental stages, but not at later stages. In contrast, pharmacological inhibition with Ni²⁺, Cd²⁺, or dantrolene revealed that ryanodine receptor (RyR)-mediated Ca²⁺-induced Ca²⁺ release (CICR) was involved in neurite outgrowth at later stage, but not at early stages. The distribution of IP₃R and RyR in growth cones also changed during development. Furthermore, pharmacological inhibition of the Ca²⁺-calmodulin-dependent phosphatase calcineurin with FK506 reduced neurite outgrowth only at early stages. These data suggest that the calcium signaling that regulates neurite outgrowth may change during development from an IP₃R-mediated pathway to a RyR-mediated pathway.     

GRIN1 Regulates ��-Opioid Receptor Activities by Tethering the Receptor and G Protein in the Lipid Raft

The lipid raft location of μ-opioid receptor (MOR) determines the receptor activities. However, the manner in which MOR is anchored within the lipid rafts is undetermined. Using the targeted proteomic approach and mass spectrometry analyses, we have identified GRIN1 (G protein-regulated inducer of neurite outgrowth 1) can tether MOR with the G protein α-subunit and subsequently regulate the receptor distribution within the lipid rafts. Glutathione S-transferase fusion pulldown and receptor mutational analyses indicate that GRIN1-MOR interaction involves a receptor sequence ²⁶⁷GSKEK²⁷¹ within the MOR third intracellular loop that is not involved in Gα interaction. The GRIN1 domains involved in MOR interaction are also distinct from those involved in Gα interaction. Pertussis toxin pretreatment reduced the amount of GRIN1 co-immunoprecipitated with MOR but not the amount with Gα. Furthermore, overexpression of GRIN1 significantly enhanced the amount of MOR in lipid raft and the receptor signaling magnitude as measured by Src kinase activation. Such increase in MOR signaling was demonstrated further by determining the GRIN1-dependent pertussis toxin-sensitive neurite outgrowth. In contrast to minimal neurite outgrowth induced by etorphine in control neuroblastoma N2A cells, overexpression of GRIN1 resulted in the increase in etorphine- and non-morphine-induced neurite outgrowth in these cells. Knocking down endogenous GRIN1 by small interfering RNA attenuated the agonist-induced neurite outgrowth. Disrupting lipid raft by methyl-β-cyclodextrin also blocked neurite outgrowth. Hence, by tethering Gα with MOR, GRIN1 stabilizes the receptor within the lipid rafts and potentiates the receptor signaling in the neurite outgrowth processes.     

The QKI-6 RNA Binding Protein Regulates Actin-interacting Protein-1 mRNA Stability during Oligodendrocyte Differentiation

The quaking viable (qk^v) mice represent an animal model of dysmyelination. The absence of expression of the QKI-6 and QKI-7 cytoplasmic isoforms in oligodendrocytes (OLs) during CNS myelination causes the qk^v mouse phenotype. The QKI RNA-binding proteins are known to regulate RNA metabolism of cell cycle proteins and myelin components in OLs; however, little is known of their role in reorganizing the cytoskeleton or process outgrowth during OL maturation and differentiation. Here, we identify the actin-interacting protein (AIP)-1 mRNA as a target of QKI-6 by using two-dimensional differential gel electrophoresis. The AIP-1 mRNA contains a consensus QKI response element within its 3′-untranslated region that, when bound by QKI-6, decreases the half-life of the AIP-1 mRNA. Although the expression of QKI-6 is known to increase during OL differentiation and CNS myelination, we show that this increase is paralleled with a corresponding decrease in AIP-1 expression in rat brains. Furthermore, qk^v/qk^v mice that lack QKI-6 and QKI-7 within its OLs had an increased level of AIP-1 in OLs. Moreover, primary rat OL precursors harboring an AIP-1 small interfering RNA display defects in OL process outgrowth. Our findings suggest that the QKI RNA-binding proteins regulate OL differentiation by modulating the expression of AIP-1.     

Opposite regulatory effects of TRPC1 and TRPC5 on neurite outgrowth in PC12 cells

The transient receptor potential (TRPC) family of Ca^2 + permeable, non-selective cation channels is abundantly expressed in the brain, and can function as store-operated (SOC) and store-independent channels depending on their interaction with the ER Ca^2 + sensor STIM1. TRPC1 and TRPC5 have critical roles in neurite outgrowth, however which of their functions regulate neurite outgrowth is unknown. In this study, we investigated the effects of TRPC channels and their STIM1-induced SOC activity on neurite outgrowth of PC12 cells. We report that PC12 cell differentiation down-regulates TRPC5 expression, whereas TRPC1 expression is retained. TRPC1 and TRPC5 interact with STIM1 through the STIM1 ERM domain. Transfection of TRPC1 and TRPC5 increased the receptor-activated Ca^2 + influx that was markedly augmented by the co-expression of STIM1. Topical expression of TRPC1 in PC12 cells markedly increased neurite outgrowth while that of TRPC5 suppressed neurite outgrowth. Suppression of neurite outgrowth by TRPC5 requires the channel function of TRPC5. However, strikingly, multiple lines of evidence show that the TRPC1-induced neurite outgrowth was independent of TRPC1-mediated Ca^2 + influx. Thus, a) TRPC1 and TRPC5 similarly increased Ca^2 + influx but only TRPC1 induced neurite outgrowth, b) the constitutively STIM1^D76A mutant that activates Ca^2 + influx by TRPC and Orai channels did not increase neurite outgrowth, c) co-expression of TRPC5 with TRPC1 suppressed the effect of TRPC1 on neurite outgrowth, d) and most notable, channel-dead pore mutant of TRPC1 increased neurite outgrowth to the same extent as TRPC1^WT. Suppression of TRPC1-induced neurite outgrowth by TRPC5 was due to a marked reduction in the surface expression of TRPC1. We conclude that the regulation of neurite outgrowth by TRPC1 is independent of Ca^2 + influx and TRPC1-promoted neurite outgrowth depends on the surface expression of TRPC1. It is likely that TRPC1 acts as a scaffold at the cell surface to assemble a signaling complex to stimulate neurite outgrowth.     

Nerve growth factor-mediated induction of tyrosine hydroxylase and of neurite outgrowth in cultures of bovine adrenal chromaffin cells: dependence on developmental stage

Adrenal medullary cells were cultured in a serum-free medium from fetal, neonatal (calves), and adult bovine animals. Neurite outgrowth in response to nerve growth factor (NGF) was observed in cells obtained from fetuses up to a gestational age of 3 months but not in cultures from older animals. The tyrosine hydroxylase (TH) specific activity was found to depend on the cell density and corresponded, at a density of 2 × 10⁵ cells/cm², to the specific activity found in vivo. The TH specific activity increased about sevenfold from fetuses to adult animals. Administration of NGF in vitro caused an increase of the TH specific activity in fetal cells by up to 140% and in calf cells typically by 70–100%. Cultures from adult animals showed no significant TH increase in response to NGF. Scatchard analysis and kinetic studies of the NGF binding at 0°C to intact adrenal medullary cells cultured from calves or from adult bovine animals revealed the presence of only one class of receptors, having a dissociation constant (K_D) of 1 × 10 ⁹, M. There are 16,000 binding sites per cell. The affinity of the reeptors in vivo (determined in crude membrane preparations) did not alter during development, whereas the receptor density decreased with increasing fetal age, but was the same for calves and adults. Whereas the loss of NGF-mediated fiber outgrowth during development might be related to the reduction of receptor density, the disappearance of the NGF-mediated TH induction does not correlate with changes in the binding characteristics of NGF to the adrenal chromaffin cells.     

Preliminary Evaluation of a Bunyavirus Vector for Cancer Immunotherapy

Replicon particles of Rift Valley fever virus, referred to as nonspreading Rift Valley fever virus (NSR), are intrinsically safe and highly immunogenic. Here, we demonstrate that NSR-infected human dendritic cells can activate CD8⁺ T cells in vitro and that prophylactic and therapeutic vaccinations of mice with NSR encoding a tumor-associated CD8 peptide can control the outgrowth of lymphoma cells in vivo. These results suggest that the NSR system holds promise for cancer immunotherapy.     

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts ¹. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis ². Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring ^3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.Portal fibroblasts have been isolated using various techniques including outgrowth ^{6, 7} and liver perfusion with enzymatic digestion followed by size selection ⁸. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff ⁸ for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.