Mouse Ltk Research Articles

The design of a new mutation model for active genes: expression of the Escherichia coli lac operon in mammalian cells.

The design of a novel transgenic mouse model is described that should allow analysis of mutations at a single cell level in all tissues of a model animal. The model is based on the correct regulation of the Escherichia coli lac operon in mammalian cells. Induction of a mutation in the lacI gene will result in the loss of transcriptional repression of the lacZ gene in mutated cells. Expression of beta-galactosidase can subsequently be detected at the single cell level. The model was first tested in vitro using transfection of mouse LTK- cells. LacZ expression was very heterogeneous in most of the stable transfectants and seemed to be subject to epigenetic inactivation. One clone (IIB1) was isolated that stably expressed lacZ in more than 99% of its cells. Subsequent introduction of the lacI gene into IIB1 cells resulted in correct transcriptional repression of the lacZ gene that could be alleviated by IPTG, an allosteric inducer of lacI repression. However, in time the extent of beta-galactosidase induction gradually declined suggesting that the prolonged repressed transcriptional state triggers epigenetic inactivation. Variegated expression of the lacZ gene was not confined to cultured cells since several transgenic lines also did not express the lacZ transgene. This study shows that while the susceptibility of the lacZ gene to inactivation processes poses a fundamental problem, correct regulation of the expression of a reporter gene by the lacI repressor protein is feasible in mammalian cells when assayed at the single cell level. Thus, the model can in principle be used for the detection of mutagenic events at the lacI locus. Targeting of the lacZ gene to an endogenous housekeeping gene might prevent epigenetic inactivation. Alternatively, with the use of another reporter gene in the mutation detection system the proposed transgenic mouse model could be realized.

Interactions of the nonsedating antihistamine loratadine with a Kv1.5-type potassium channel cloned from human heart.

The use of nonsedating antihistamines may, on rare occasions, be associated with cardiac arrhythmias. This could be due to blockade of voltage-dependent K+ channels in the heart, leading to a prolongation in repolarization in the human myocardium. For this reason, we examined the effects of the nonsedating antihistamine loratadine on a rapidly activating delayed-rectifier K+ channel (Kv1.5) cloned from human heart and stably expressed in HEK 293 cells or mouse Ltk- cells. Using patch-clamp electrophysiology, we found that loratadine blocked Kv1.5 current measured from inside-out membrane patches at concentrations of > or = 100 nM, resulting in an IC50 value of 808 nM at +50 mV. The drug enhanced the rate of Kv1.5 current decay, and block was enhanced at membrane potentials near threshold relative to higher potentials. Loratadine did not alter the kinetics of Kv1.5 current activation or deactivation. Unitary Kv1.5 currents were recorded in cell-attached patches. At the single-channel level, the main effect of loratadine was to reduce the mean probability of opening of Kv1.5. This effect of loratadine was achieved by a reduced number of openings in bursts and burst duration. Finally, loratadine (10 microM) failed to inhibit HERG K+ channel currents expressed in Xenopus laevis oocytes. It is concluded that loratadine is an effective blocker of Kv1.5 that interacts with an activated state or states of the channel. This interaction suggests a potential for loratadine to alter cardiac excitability in vivo.

Fine-resolution analysis of products of intrachromosomal homeologous recombination in mammalian cells.

Mouse Ltk- cell lines that contained a herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene with a 16-bp insertion mutation linked to either a defective HSV-2 tk gene or a hybrid tk sequence comprised of HSV-1 and HSV-2 tk sequences were constructed. HSV-1 and HSV-2 tk genes have 81% nucleotide identity and hence are homeologous. Correction of the insertion mutant HSV-1 tk gene via recombination with the hybrid tk sequence required an exchange between homeologous tk sequences, although recombination could initiate within a region of significant sequence identity. Seven cell lines containing linked HSV-1 and HSV-1-HSV-2 hybrid tk sequences gave rise to tk+ segregants at an average rate of 10(-8) events per cell division. DNA sequencing revealed that each recombinant from these lines displayed an apparent gene conversion which involved an accurate transfer of an uninterrupted block of information between homeologous tk sequences. Conversion tract lengths ranged from 35 to >330 bp. In contrast, cell lines containing linked HSV-1 and HSV-2 tk sequences with no significant stretches of sequence identity had an overall rate of homeologous recombination of <10(-9). One such cell line produced homeologous recombinants at a rate of 10(-8). Strikingly, all homeologous recombinants from this latter cell line were due to crossovers between the HSV-1 and HSV-2 tk genes. Our results, which provide the first detailed analysis of homeologous recombination within a mammalian genome, suggest that rearrangements in mammalian genomes are regulated by the degree of sequence divergence located at the site of recombination initiation.

Genomic characterization of the mouse homolog of the Saccharomyces cerevisiae recombination and double-strand break repair gene RAD52

The yeast Saccharomyces cerevisiae RAD52 gene is involved in recombination and DNA double-strand break repair. Recently, mouse and human homologs of the yeast RAD52 gene have been identified. Here we present the genomic organization of the mouse RAD52 gene. It consists of 12 exons ranging in size from 67 to 374 bp spread over a region of approximately 18 kb. The first ATG is located in exon 2. Analysis of the promoter region revealed no classical promoter elements such as CCAAT or TATA boxes. Transcriptional mapping analysis revealed one major transcription start point. Analogous to the situation in yeast, transcription of the RAD52 gene in human skin fibroblasts and mouse Ltk− cells was not induced by methyl methanesulfonate treatment. Furthermore, no specific alteration in human RAD52 expression levels throughout the cell cycle was observed.

Overexpression of full- or partial-length MAP4 stabilizes microtubules and alters cell growth.

To investigate the in vivo functions of MAP4, a microtubule-associated protein expressed almost ubiquitously in vertebrate cells, we prepared stably transfected clonal mouse Ltk- cell lines expressing full-length MAP4 (L-MAP4 cells) or its MT-binding domain (L-MTB cells). Although transfectants showed no dramatic defect in morphology, organellar distribution, or level of MT polymer, as compared to naive Ltk- cells or L-MOCK cells (transfected with vector alone), MTs in L-MAP4 and L-MTB cells showed greater stability than those in control cells, as monitored by the level of post-translationally detyrosinated alpha-tubulin and by a quantitative nocodazole-resistance assay. In vivo, the MT-binding domain of MAP4 stabilized MTs less potently than full-length MAP4, in contrast to the equivalent efficacy demonstrated in studies of in vitro MT polymerization (Aizawa et al. (1991), J. Biol. Chem. 266, 9841-9846), L-MAP4 and L-MTB cells grew significantly more slowly than control cells; this growth inhibition was not due to mitotic arrest or cell death. L-MAP4 and L-MTB cells also exhibited greater tolerance to the MT-depolymerizing agent, nocodazole, but not to the MT-polymerizing agent, Taxol. Our results demonstrate that MAP4 and its MT-binding domain are capable of MT stabilization in vivo, and that increasing the intracellular level of MAP4 affects cell growth parameters.

Identification of a surface glycoprotein on African green monkey kidney cells as a receptor for hepatitis A virus.

Very little is known about the mechanism of cell entry of hepatitis A virus (HAV), and the identification of cellular receptors for this picornavirus has been elusive. Here we describe the molecular cloning of a cellular receptor for HAV using protective monoclonal antibodies raised against susceptible African green monkey kidney (AGMK) cells as probes. Monoclonal antibodies 190/4, 235/4 and 263/6, which reacted against similar epitopes, specifically protected AGMK cells against HAV infection by blocking the binding of HAV. Expression cloning and nucleotide sequence analysis of the cDNA coding for epitope 190/4 revealed a novel mucin-like class I integral membrane glycoprotein of 451 amino acids, the HAV cellular receptor 1 (HAVcr-1). Immunofluorescence analysis indicated that mouse Ltk- cells transfected with HAVcr-1 cDNA gained limited susceptibility to HAV infection, which was blocked by treatment with monoclonal antibody 190/4. Our results demonstrate that the HAVcr-1 polypeptide is an attachment receptor for HAV and strongly suggest that it is also a functional receptor which mediates HAV infection. This report constitutes the first identification of a cellular receptor for HAV.

A novel expression vector for transfection of bovine MHC class I genes

A vector is described for the expression of genomic or cDNA copies of bovine major histocompatibility complex (MHC) class I genes in transfected mouse Ltk − cells. Class I gene fragments are amplified by the polymerase chain reaction, using primers in conserved parts of exon 2 and the 3′-untranslated region of the gene. Amplified class I gene fragments can then be subcloned into the expression vector, pBoLA-21, which contains the necessary 5′-and 3′-sequences for correct expression. The vector was tested by subcloning and expressing genomic and cDNA clones.

Electrophysiological properties of the hypokalaemic periodic paralysis mutation (R528H) of the skeletal muscle α1S subunit as expressed in mouse L cells

Hypokalaemic periodic paralysis (HypoPP) is an autosomal dominant muscle disease which has been linked to point mutations in the skeletal muscle L-type calcium channel α 1 subunit ( α 1 S ). Here, we have introduced one of the point mutations causing HypoPP (R528H) into cDNA of the rabbit α 1 S . Expression of either the wild-type α 1S or the mutant R528H α 1 S ( α 1 S− R528 H) subunit was obtained in mouse Ltk − cell using a selectable expression vector. The α 1 S− R528 H subunit led to the expression of functional L-type Ca 2+ channels. Corresponding whole-cell Ba 2+ currents exhibit very slow activation and inactivation kinetics, typical for recombinant skeletal Ca 2+ channel currents. Voltage-dependent activation and inactivation properties were similar for α 1 S -and α 1 S − R528 H , as well as their sensitivity to the dihydropyridine agonist Bay K 8644. Differences in α 1 S -and α 1 S− R528 H -directed channels reside in the Ba 2+ current density, which was significantly reduced 3.2 fold in cells expressing α 1 S − R528 H . It was concluded that the R528H mutation of α 1 S results in minor differences in the electrophysiological properties but significantly reduces the whole-cell Ca 2+ channel current in its amplitude.

Detection of protein kinase activity specifically activated at metaphase-anaphase transition.

We have previously reported that Ser13 and Ser34 on glial fibrillary acidic protein (GFAP) in the cleavage furrow of glioma cells are phosphorylated during late mitotic phase (Matsuoka, Y., K. Nishizawa, T. Yano, M. Shibata, S. Ando, T. Takahashi, and M. Inagaki. 1992, EMBO (Eur. Mol. Biol. Organ.) J. 11:2895-2902). This observation implies a possibility that there is a protein kinase specifically activated at metaphase-anaphase transition. To further analyze the cell cycle-dependent GFAP phosphorylation, we prepared monoclonal antibodies KT13 and KT34 which recognize the phosphorylation of GFAP at Ser13 and Ser34, respectively. Immunocytochemical studies with KT13 and KT34 revealed that the GFAP phosphorylation in the cleavage furrow during late mitotic phase occurred not only in glioma cells but also in human SW-13 and mouse Ltk- cells in which GFAP was ectopically expressed, thus the phosphorylation can be monitored in a wide range of cell types. Furthermore, we detected kinase activity which phosphorylates GFAP at Ser13 and Ser34 in the lysates of late mitotic cells but not in those of interphase cells or early mitotic cells. These results suggest that there exists a protein kinase which is specifically activated at the transition of metaphase to anaphase not only in GFAP-expressing cells but also in cells without GFAP.

Microencapsulation and transplantation of genetically engineered cells: a new approach to somatic gene therapy.

In order to develop a model for gene therapy which avoids dependence on an autologous source of target cells and immunosuppressive therapy, mouse Ltk fibroblasts transfected with a human growth hormone (hGH) fusion gene were encapsulated in a semipermeable alginate-poly-L-lysine-alginate (APA) membrane. The encapsulated cells were cultured in vitro or transplanted intraperitoneally into mice to monitor cell viability, cell growth, and hGH secretion. The effect of Zn2+ ions on vector expression was also monitored in vitro and in vivo. Results indicate that: (1) the capsule environment is compatible with cell viability and cell growth; (2) the capsule limits cell growth; (3) the capsule membrane is permeable to the exit of hGH; (4) gene product expression may be stimulated by external means; (5) the novel gene product is delivered in vivo; and (6) encapsulated cells recovered from transplant recipients continue to secrete hGH in vitro. The results suggest therapeutic potential of this approach to somatic gene therapy.

Dominant negative expression of a cytoplasmically deleted mutant of XB/U-cadherin disturbs mesoderm migration during gastrulation in Xenopus laevis

XB/U-cadherin is a maternal Xenopus cadherin which mediates interblastomere adhesion in early embryogenesis. In order to explore its role in gastrulation, we expressed a cytoplasmic deletion mutant of XB/U-cadherin (XBΔc38) under the control of the CMV promoter in Xenopus embryos. This truncated XB-cadherin fails to form complexes with catenins and does not mediate cell-cell aggregation as shown by transfection of mouse Ltk − cells. Injections of the deletion for XB/U-cadherin into the dorsal-marginal region of four cell stage embryos resulted in a dominant negative expression of the cadherin mutant after MBT. Two different phenotypes were observed in a dose dependent manner: high doses (125–250 pg DNA) led to severe distortions of the gastrulation movement. Involution of the mesoderm was impaired, posterior mesoderm migrated laterally around the blastopore and formed two bands of axial tissue. Low doses (up to 50 pg DNA) resulted in embryos of a posteriorized phenotype with disorganized neural structures. Both phenotypes could be rescued by coinjection of cDNA constructs containing wild-type XB/U-cadherin. Injections of constructs encoding a XB/U-cadherin protein truncated both in its extracellular and cytoplasmic domains yielded normal phenotypes. These results suggest that a proper function of XB/U-cadherin is essential for mesoderm movements during gastrulation.

Growth and induction kinetics of inducible and autoinducible expression of heterologous protein in suspension cultures of recombinant mouse L cell lines.

Mouse Ltk- cells were transfected with four different plasmids for autoinducible and highly-inducible expression of the bacterial lacZ gene and cultivated in suspension. Two selection genes, thymidine kinase (tk) and neomycin resistance (neor), were used to select the clones in both cell lines. The resulting two cell lines, designated M4 and R2, differ in that the inducible MMTV promoter from mouse mammary tumor virus (MMTV) controls glucocorticoid receptor (gr) gene and lacZ gene expression in the M4 cell line ("autoinducible"), while the constitutive rous sarcoma virus (RSV) promoter controls gr gene expression and the MMTV promoter controls lacZ gene expression in the R2 cell line ("highly-inducible"). Both cell lines were stable with respect to reproducibility of growth rate in spinner flasks and inducibility of beta-galactosidase expression. The exponential growth rate of R2 cells was slower than that of M4 cells before induction because the R2 cell line continuously expressed gr genes under the constitutive RSV promoter, and the percent reduction of exponential growth rate mainly caused by gr gene expression was about 20%. The inducibility of the M4 cell line was greater than that of the R2 cell line because in the M4 cell line MMTV promoter controlled gr and lacZ gene expression autoinducibly. Maximum induction of the M4 cell line occurred after induction with the hormone dexamethasone (Dex) at 10(-7) M, and the final beta-galactosidase content increased 400-fold after induction. The optimum conditions for inducer concentration and induction time were determined, and the highest production of beta-galactosidase occurred when Dex was added after the cell concentration had reached its maximum in batch culture. Dex (10(-9) M) is a critical inducer concentration in view of inducibility between M4 and R2 cell lines. The inducibility of R2 cell line is higher than that of the M4 cell line from 0 to 10(-9) M Dex, but the inducibility of M4 was higher than that of the R2 cell line at Dex concentrations of more than 10(-9) M.

Stereoselective block of a human cardiac potassium channel (Kv1.5) by bupivacaine enantiomers

Stereoselective drug-channel interactions may help to elucidate the molecular basis of voltage-gated potassium channel block by local anesthetic drugs. We studied the effects of the enantiomers of bupivacaine on a cloned human cardiac potassium channel (hKv1.5). This channel was stably expressed in a mouse Ltk- cell line and studied using the whole-cell configuration of the patch-clamp technique. Both enantiomers modified the time course of this delayed rectifier current. Exposure to 20 microM of either S(-)-bupivacaine or R(+)-bupivacaine did not modify the activation time constant of the current, but reduced the peak outward current and induced a subsequent exponential decline of current with time constants of 18.7 +/- 1.1 and 10.0 +/- 0.9 ms, respectively. Steady-state levels of block (assessed with 250-ms depolarizing pulses to +60 mV) averaged 30.8 +/- 2.5% (n = 6) and 79.5 +/- 3.2% (n = 6) (p < 0.001), for S(-)- and R(+)-bupivacaine, respectively. The concentration dependence of hKv1.5 inhibition revealed apparent KD values of 27.3 +/- 2.8 and 4.1 +/- 0.7 microM for S(-)-bupivacaine and R(+)-bupivacaine, respectively, with Hill coefficients close to unity, suggesting that binding of one enantiomer molecule per channel was sufficient to block potassium permeation. Analysis of the rate constants of association (k) and dissociation (l) yielded similar values for l (24.9 s-1 vs. 23.6 s-1 for S(-)- and R(+)-bupivacaine, respectively) but different association rate constants (1.0 x 10(6) vs. 4.7 x 10(6) M-1 s-1 for S(-)- and R(+)-bupivacaine, respectively). Block induced by either enantiomer displayed a shallow voltage dependence in the voltage range positive to 0 mV, i.e., where the channel is fully open, consistent with an equivalent electrical distance delta of 0.16 +/- 0.01. This suggested that at the binding site, both enantiomers of bupivacaine experienced 16% of the applied transmembrane electrical field, referenced to the inner surface. Both bupivacaine enantiomers reduced the tail current amplitude recorded on return to -40 mV and slowed their time course relative to control, resulting in a "crossover" phenomenon. These data indicate 1) the charged form of both bupivacaine enantiomers block the hKv1.5 channel after it opens, 2) binding occurs within the transmembrane electrical field, 3) unbinding is required before the channel can close, 4) block of hKv1.5 channels by bupivacaine is markedly stereoselective, with the R(+)-enantiomer being the more potent one, 5) this stereoselective block was associated with a 1.11 -kcal/mol difference in binding energy between both enantiomers, and 6) the stereoselectivity derives mainly from a difference in the association rate constants, suggesting that the S(-)-enantiomer is less likely to access the binding site in an optimal configuration.

Coexpression of Skeletal Muscle Voltage-Dependent Calcium Channel α1 and β cDNAs in Mouse Ltk− Cells Increases the Amount of α1 Protein in the Cell Membrane

Coexpression of α1 and β subunits for all tissue-specific isotypes of calcium channels results in an increase in Ca2+ channel current density and drug receptor function. The quantification of the photoaffinity labeled skeletal muscle α1 subunit polypeptide by two alternative immunoadsorption techniques showed that 3 times higher amount of calcium channel α1 protein appears in the cell membrane when α1 and β subunits were coexpressed compared to α1 alone. Using Western blotting and immunodetection techniques, we identified two polypeptide bands of α1 with 210 kD and 170 kD molecular mass, respectively. The combined intensity of these bands was 7-9 times higher when α1 and β were coexpressed compared to α1 alone. The data provide evidence that coexpression of α1 and β results in an increased amount of α1 protein in the cell membrane.

Localization of dopamine D1A receptor protein in rat kidneys.

The dopamine D1A receptor subtype was identified in rat kidney with both light microscopic immunohistochemistry and electron microscopic immunocytochemistry. Antipeptide polyclonal antisera were directed to both extracellular and intracellular regions of the native receptor. The use of such receptor-subtype-selective antibodies allows for the identification of specific dopamine receptor subtype clones that are not distinguished by current pharmacological or receptor-ligand binding technology. Selectivity of the antipeptide antisera was validated by their ability to recognize native receptor protein expressed in permanently transfected mouse LTK- cells. In the rat kidney, D1A receptor protein was localized to the juxtaglomerular apparatus (JGA), proximal tubule, distal tubule, cortical collecting duct, and renal vasculature. In the JGA, the receptor was predominantly located in the arteriolar smooth muscle layer within cytoplasmic granules previously shown to contain renin. In the proximal tubules, staining was localized both on the brush-border and basolateral membranes. The D1A receptor, which is present in the central nervous system, is now identified in the rat kidney at those sites previously labeled as DA1 receptor sites on the basis of pharmacological binding studies. These results suggest that at least some of the renal dopamine DA1 receptors correspond structurally to the central dopamine D1A receptor.

Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain.

The present study reports the isolation of a cDNA clone that encodes a second member of the corticotropin-releasing factor (CRF) receptor family, designated as the CRF2 receptor. The cDNA was identified using oligonucleotides of degenerate sequence in a PCR paradigm. A PCR fragment obtained from rat brain was utilized to isolate a full-length cDNA from a rat hypothalamus cDNA library that encoded a 411-amino acid protein with approximately 70% identity to the known CRF1 receptor over the entire coding region. When expressed in mouse Ltk- cells, this receptor stimulates cAMP production in response to CRF and known CRF-like agonists. CRF and the nonmammalian CRF-related peptides sauvagine and urotensin I stimulate adenylate cyclase activity in a dose-dependent manner with a rank order of potency different from that of the CRF1 receptor: sauvagine > urotensin > or = rat/human CRF > ovine CRF. Tissue distribution analysis of the mRNAs by reverse transcriptase-PCR shows CRF2 receptor mRNA is present in rat brain and detectable in lung and heart. In situ hybridization studies indicate specific expression within the brain in the ventromedial nuclei of the hypothalamus, the lateral septum, the amygdala, and entorhinal cortex, but there is unremarkable expression in the pituitary. An additional splice variant of the CRF2 receptor with a different N-terminal domain has been identified by PCR, encoding a putative protein of 431 amino acids. Thus, the data demonstrate the presence of another functional CRF receptor, with significant differences in the pharmacological profile and tissue distribution from the CRF1 receptor, which would predict important functional differences between the two receptors.

Benzodiazepine Treatment Causes Uncoupling of Recombinant GABAA Receptors Expressed in Stably Transfected Cells

GABAA and benzodiazepine receptors are allosterically coupled, and occupation of either receptor site increases the affinity of the other. Chronic exposure of primary neuronal cultures to benzodiazepine agonists reduces these allosteric interactions. Neurons express multiple GABAA receptor subunits, and it has been suggested that uncoupling is due to changes in the subunit composition of the receptor. To determine if uncoupling could be observed with expression of defined subunits, mouse Ltk- cells stably transfected with GABAA receptors (bovine alpha 1, beta 1, and gamma 2L subunits) were treated with flunitrazepam (Flu) or clonazepam. The increase in [3H]Flu binding affinity caused by GABA (GABA shift or coupling) was significantly reduced in cells treated chronically with the benzodiazepines, whereas the KD and Bmax of [3H]Flu binding were unaffected. The uncoupling caused by clonazepam treatment occurred rapidly with a t1/2 of approximately 30 min. The EC50 for clonazepam treatment was approximately 0.3 microM, and cotreatment with the benzodiazepine antagonist Ro 15-1788 (5.6 microM) prevented the effect of clonazepam. The uncoupling observed in this system was not accompanied by receptor internalization, is unlikely to be due to changes in receptor subunit composition, and probably represents post-translational changes. The rapid regulation of allosteric coupling by benzodiazepine treatment of the stably transfected cells should provide insights to the mechanisms of coupling between GABAA and benzodiazepine receptors as well as benzodiazepine tolerance.

Generation of an antibody to HLA-G in transgenic mice and demonstration of the tissue reactivity of this antibody

A method was devised to generate antibodies against the non-classical class I HLA-G antigen. This consisted of immunising HLA-A2/ β 2m double transgenic mice with HLA-G transfected into mouse Ltk − cells. A polyclonal antibody was obtained which appears to be specific for HLA-G. The staining pattern of this antibody was restricted solely to all populations of extravillous trophoblast. No fetal tissues reacted with this antibody, including those where HLA-G mRNA has been demonstrated, such as fetal eye, thymus and liver. This study confirms that HLA-G is a trophoblast-specific protein, although it remains a possibility that the technique of immunohistology is not sufficiently sensitive to detect low level HLA-G antigen expression in non-trophoblast tissues.

Structural requirements of procathepsin D activation and maturation.

Cathepsin D biosynthesis involves several proteolytic events; however, the enzymology and sequence of these events are not known. Procathepsin D undergoes a pH-dependent, intramolecular proteolysis in vitro which removes 26 residues yielding an active form that is intermediate in size between procathepsin D and single-chain cathepsin D. This form, designated pseudocathepsin D, has not been shown to be an in vivo intermediate. The N-terminal sequence of the light chain of cathepsin D, isolated from human placenta, showed that 42 residues were removed as compared with 44 residues predicted by comparison with porcine cathepsin D. Site-directed mutations were generated at both processing sites within the propeptide of procathepsin D. Mutation at the autocatalytic site prevented in vitro autoactivation, but, after transfection of mouse Ltk- cells, the mutant procathepsin D was transported to the lysosome and processed normally to the mature enzyme despite its inability to autoactivate in vitro. Mutation at the mature N terminus of cathepsin D prevented in vivo formation of the single-chain form of the enzyme; however, the protein was still processed to the two-chain form of human cathepsin D. This change at the mature N terminus did not prevent in vitro autoactivation. Procathepsin D with mutations at both cleavage sites was processed to the two-chain form despite the inability to undergo removal of the propeptide. These results indicated that stepwise autoactivation and propeptide removal were not necessary for later processing or delivery of human cathepsin D to the lysosome. The results also suggested that pseudocathepsin D was not a normal intermediate of procathepsin D processing in vivo.

Growth of recombinant fibroblasts in alginate microcapsules

To develop a novel strategy of nonautologous somatic gene therapy, we now demonstrate the feasibility of culturing genetically modified fibroblasts within an immunoprotective environment and the optimal conditions required for their continued survival in vitro. When mouse Ltk(-) fibroblasts transfected with the human growth hormone gene were enclosed within permselective microcapsules fabricated from alginate-polylysine-alginate, they continued to secrete human growth hormone at the same rates as the nonencapsulated cells. They also continued to proliferate in vitro for at least 1 month even though their viability gradually declined to about 50%. The viability can be improved by controlling for (a) temperature during encapsulation, (b) duration of treatment with polylysine, (c) duration of liquefying the core alginate with sodium citrate, and (d) cell density at the time of encapsulation. The best conditions leading to improved survival and maximum proliferation of cells within the microcapsules were obtained by encapsulating the cells at 4 to 10 degrees C instead of room temperature, coating the microspheres with polylysine for 6 to 10 min instead of 20 min, liquefying the core alginate by treating with citrate for 20 min instead of 6 to 10 min, and using a concentration of 2 x 10(6) cells/mL of alginate for encapsulation. Under such conditions, normally adherent and genetically engineered mouse fibroblasts survived and proliferated optimally within the microcapsule environment. The encapsulated fibroblasts maintained their level of transgene expression while recombinant gene products such as human growth hormone could diffuse through the microcapsule membrane without impediment. The demonstration that genetically modified fibroblasts can survive and continue to deliver recombinant gene products from within these microcapsules and the optimization for their maximal viability and growth within microcapsules should increase the potential for success in using such microencapsulated recombinant cells for somatic gene therapy. (c) 1994 John Wiley & Sons, Inc.