Wayne F. Flintoff

In vivo and in vitro models of demyelinating diseases: Tropism of the JHM strain of murine hepatitis virus for cells of glial origin

Abstract Infection of mice with the neurotropic JHM strain of murine hepatitis virus causes demyelinating lesions resulting from an infection of the oligodendroglia. This was most evident in mice inoculated intraperitoneally with JHM. Such CNS lesions were not observed in mice inoculated intraperitoneally with the MHV3 strain. An in vitro system is described in which the rat glial RN2 cell line functions as a discriminating host for the JHM virus. Shortly after inoculation, this virus establishes a persistent infection in which there is a cyclical rise and fall in titer with an accompanying cytopathology. Furthermore, this host cell confers a thermal lability which the virus does not demonstrate in the fully permissive host cell, L-2. By comparison, infection of RN2 cells with the prototype MHV3 is aborted immediately. In the persistent infection of RN2 cells with measles virus, Hallé strain, the cell again confers a temperature sensitivity which the virus does not possess when replicating in Vero cells. This appears to be the first instance in which a cloned cell line of glial origin determines the outcome of the infectious process, discriminating in favor of a neurotropic variant which possesses a tropism for the glia in vivo. Systems such as the one described here may now offer a specific screening procedure for selecting, identifying and characterizing the nature of neurotropic viruses.

Topological and functional analysis of the human reduced folate carrier by hemagglutinin epitope insertion.

The membrane topology of the human reduced folate carrier protein (591 amino acids) was assessed by single insertions of the hemagglutinin epitope into nine sites of the protein. Reduced folate carrier-deficient Chinese hamster ovary cells expressing each of these constructs were probed with anti-hemagglutinin epitope monoclonal antibodies to assess whether the insertion was exposed to the external environment or to the cytoplasm. The results are consistent with the 12-transmembrane topology predicted for this protein. The hemagglutinin epitope insertion mutants were also tested for their effects on the function of the reduced folate carrier. For these studies, each of the constructs had a carboxyl-terminal fusion of the enhanced green fluorescent protein to monitor and quantitate expression. Insertions into the external loop between transmembrane regions 7 and 8 (Pro-297), the cytoplasmic loop between transmembrane regions 6 and 7 (Ser-225), and near the cytoplasmic amino and carboxyl termini (Pro-20 and Gly-492, respectively) had minor effects on methotrexate binding and uptake. The insertion into the cytoplasmic loop between transmembrane regions 10 and 11 (Gln-385) greatly reduced both binding and uptake of methotrexate, whereas the insertion into the external loop between transmembrane regions 11 and 12 (Pro-427) selectively interfered with uptake but not binding.

In vivo and in vitro models of demyelinating diseases II. Persistence and host-regulated thermosensitivity in cells of neural derivation infected with mouse hepatitis and measles viruses

Abstract Following inoculation of continuous cell lines of neural and other derivations, persistent infections are established with facility by mouse hepatitis and measles viruses. This occurs equally with the prototype MHV3 and its neurotropic variant JHM as well as with the Edmonston vaccine and SSPE Hallé measles variants. In almost every instance that the infection becomes persistent at 32.5°, virus replication is found to be thermosensitive at 39.5°; however, progeny virus derived from such infections at 32.5° is itself thermostable when replicating in the indicator, fully permissive cell lines. The new data, therefore, reveal the existence of a host-conferred interrelationship between persistence and virus restriction at elevated temperature. They indicate that the two agents with neurotropic potential, when they become established as pathogens in the nervous system, could be under close host cell regulation involving as yet unknown mechanisms.

Transport of methotrexate in chinese hamster ovary cells: A mutant defective in methotrexate uptake and cell binding☆

The uptake of several folate compounds has been investigated in wild-type and one class of methotrexate-resistant Chinese hamster ovary cells. The wild-type cells can take up methotrexate, folic acid, and 5-methyl tetrahydrofolate. The uptake of methotrexate is characterized by a KT of 1.9 +/- 0.2 microM and a Vmax of 1 +/- 0.8 pmol/min/mg protein, is competitively inhibited by folic acid and 5-methyl tetrahydrofolate, and is sensitive to p-hydroxymercuriphenyl sulfonic acid. The resistant cells are unable to take up methotrexate, folic acid, and 5-methyl tetrahydrofolate. In addition, the resistant cells are unable to bind methotrexate specifically, whereas the wild-type cells bind the drug with an apparent KD of 2 +/- 0.4 microM and a Vmax of 1.3 +/- 0.3 pmol/mg protein. These data indicate that the resistant cells are resistant because of an inability to take up the drug resulting from a defective membrane-binding component. The data also suggest that both methotrexate and folic acid are transported by the same system in Chinese hamster ovary cells.

Cytoplasmic domains of the reduced folate carrier are essential for trafficking, but not function.

The reduced folate carrier (RFC) protein has a secondary structure consistent with the predicted 12 transmembrane (TM) domains, intracellular N- and C-termini and a large cytoplasmic loop between TM6 and TM7. In the present study, the role of the cytoplasmic domains in substrate transport and protein biogenesis were examined using an array of hamster RFC deletion mutants fused to enhanced green fluorescent protein and expressed in Chinese hamster ovary cells. The N- and C-terminal tails were removed both individually and together, or the large cytoplasmic loop was modified such that the domain size and role of conserved sequences could be examined. The loss of the N- or C-terminal tails did not appear to significantly disrupt protein function, although both termini appeared to have a role in the efficiency with which molecules exited the endoplasmic reticulum to localize at the plasma membrane. There appeared to be both size and sequence requirements for the intracellular loop, which are able to drastically affect protein stability and function unless met. Furthermore, there might be an indirect role for the loop in substrate translocation, since even moderate changes significantly reduced the V(max) for methotrexate transport. Although these cytoplasmic domains do not appear to be absolutely essential for substrate transport, each one is important for biogenesis and localization.

Exclusion of allelism of Noonan syndrome and neurofibromatosis‐type 1 in a large family with Noonan syndrome‐neurofibromatosis association

A large four-generation family with Noonan syndrome (NS) and neurofibromatosis-type 1 (NF1) was studied for clinical association between the two diseases and for linkage analysis with polymorphic DNA markers of the NF1 region in 17q11.2. Nonrandom segregation between NS and NF1 phenotypes was observed. Neurofibromatosis was tightly linked to NF1 markers, whereas Noonan syndrome was found not be allelic to NF1. These results suggest that two mutations at two independent but closely linked loci are the cause of neurofibromatosis-Noonan syndrome (NF-NS) association in this family.

Mutations in the reduced-folate carrier affect protein localization and stability

The reduced-folate-carrier (rfc) gene has been shown to be functionally important for reduced-folate transport in mammalian cells. In the present paper we describe the identification of alterations in both alleles of the rfc gene in a mutant Chinese-hamster ovary cell line deficient in methotrexate transport. One allele of the rfc gene contains a point mutation resulting in a Gly(345)-->Arg substitution in the predicted amino acid sequence. In this case, a protein of similar size to the wild-type protein is produced, although it remains as an immature, core-glycosylated, form. The second allele contains a point mutation in the last base of intron 5 that results in the utilization of a cryptic splice site leading to a seven-base deletion in the mRNA. The use of an alternate splice site changes the reading frame to yield a truncated protein with 68 different C-terminal amino acids as compared with the wild-type. Both of these altered gene products were monitored by fusion with green fluorescent protein and found to be non-functional with an increased rate of turnover. The protein with the point mutation is trapped in the endoplasmic reticulum with subsequent degradation, whereas the product of the splice mutation is not membrane-associated and is partially degraded. Thus mutations in both alleles of the rfc gene in this resistant cell line account for the loss of reduced-folate transport. The observations made regarding the degradation of these mutant gene products also provide support for putative checkpoints in the endoplasmic reticulum.

Structural Organization of the Human Reduced Folate Carrier Gene: Evidence for 5′ Heterogeneity in Lymphoblast mRNA

The reduced folate carrier (rfc1) gene encodes a protein that is involved in the intracellular accumulation of folates. Point mutations in this gene and alterations resulting in the down regulation of its message are major factors involved in the resistance to antifolate chemotherapeutic compounds. As a framework for understanding the significance of such changes in relation to gene expression and function, in this report we describe the organization of the rfc gene from human lymphoblasts. The gene contains 5 exons (2 to 6) coding for protein. At least four 5′ exons, used in a mutually exclusive manner in the production of the rfc message from lymphoblast cells, are spliced to exon 2, which contains the translational start site. “Semi-quantitative” PCR indicates that exon 1 is preferentially used. The major transcriptional start site has been mapped by RACE and RNase protection to a region 109 to 135 base pairs 5′ to the start of exon 1. The 5′ region of the gene has no TATA box-like sequence but contains several consensus binding sites for transcriptional factors such as SP-1, MZF1, CREB, AP-1, ETS, GATA-1 and GATA-2. The overall organization of the human gene is similar to that of the hamster and mouse genes.

Structural organization of the reduced folate carrier gene in Chinese hamster ovary cells.

The reduced folate carrier gene (rfc) encodes a putative protein that is involved in the intracellular accumulation of folates. In this report, we describe the organization of the rfc gene from Chinese hamster ovary cells. The hamster rfc gene contains 7 exons and 6 introns, which span 15.3 kilobases. It codes for two alternatively spliced messenger RNAs, one that contains all 7 exons and one that lacks exon 2 but contains the remaining 6 exons. The transcriptional start of the gene has been mapped to six sites approximately 200 base pairs upstream of the putative ATG initiation codon. The promoter region has no TATA box-like sequence but contains a consensus Sp1 binding site. This is the first report of the genomic structure of the reduced folate carrier gene from any species.

Mutant Chinese hamster ovary cells with defective methotrexate uptake are distinguishable by reversion analysis

Chinese hamster ovary cells that are about 50× more resistant to the cytotoxic action of methotrexate than wild-type cells were deficient in the ability to take up methotrexate. In the absence of any exogeneous folates, these cells require 100–250× the level of folinic acid as wild-type cells to support growth at a similar level. Two classes of mutants were distinguishable by their revertability for growth on folinic acid. Revenants derived from one class were similar to wild-type cells in both their ability to grow in medium containing low levels of folinic acid and in their sensitivity to methotrexate. In contrast, partial revertants from a second class were able to grow in medium containing low or no folinic acid, but retained their methotrexate resistance. Furthermore, mutants of the first class were unable to take up folic acid while the second class of mutants accumulated folic acid to levels similar to that of wild-type cells. Somatic cell hybrids formed between these two classes of mutants were noncomplementing. These observations suggested that some, but not all, components may be shared between the transport systems mediating methotrexate and folic acid uptake.