Incidence of gap junctions and microvilli in variant cell lines with altered capacity for metabolic co-operation

The metabolic cooperation-defective embryonal carcinoma cell variant R5/3 has a reduced area of gap junctions per unit cell volume and an increased surface area of microvilli per unit volume compared with its parent PC13TG8. Both changes are reversed in the cooperation-competent revertant H2T12. This provides strong evidence in support of the view that metabolic cooperation is mediated by gap junctions, and suggests that the altered properties of R5/3 may be due to a cytoskeletal defect.     

Isolation of a revertant clone from a variant embryonal carcinoma cell line deficient in metabolic co-operation

A revertant clone with restored capacity for metabolic cooperation has been isolated from the cooperation-defective variant embryonal carcinoma cell line R5/3. The properties of the revertant clone provide evidence that deficiencies shown by R5/3 in intercellular transfer of nucleotides and sodium ions are the result of a common genetic lesion which can be dissociated from a secondary lesion causing increased thioguanine resistance and from an increase in ploidy.     

A relationship between gap junction-mediated intercellular communication and the in vitro developmental capacity of murine embryonic stem cells.

Two metabolic cooperation-deficient variants, 1P9 and 2P2[1], have been isolated from the embryonic stem cell line B2B2. Characterization of these cell lines has shown that both variants are severely restricted with respect to embryoid body differentiation capacity while a revertant isolated from 1P9, designated 2H4, has its differentiation phenotype restored to a level comparable with that of the parent line. These results are interpreted as indicating that the metabolic cooperation deficiency and the restricted differentiation phenotype of 1P9 are causally related. A revertant isolated from 2P2[1], designated H19, remains severely restricted with regard to embryoid body differentiation, suggesting that in these lines a secondary event deleterious to differentiation, but unrelated to metabolic cooperation, has taken place.     

Intraspecies transfer via total cellular DNA of the gene for hypoxanthine phosphoribosyltransferase into cultured mouse cells

Summary Under selective growth conditions a revertant of mouse cells, defective in hypoxanthine phosphoribosyltransferase activity (HPRT, EC-No. 2.4.2.8), was isolated, which contained an electrophoretically abnormal form of HPRT activity. The specific HPRT activity in crude extracts of the revertant cells is about 30% of the level determined in normal wild type cells. The variant HPRT reacts with antiserum against normal mouse HPRT but the rate of heat inactivation of the variant activity is different from the wild type form. By isozyme and karyotype analyses of somatic cell hybrids between the revertant mouse cells and Chinese hamster cells we found that the abnormal HPRT activity is coded for by the mouse X-chromosome as expected for a mutation in the structural HPRT gene.DNA has been purified from the abnormal HPRT revertant cells and incubated with mouse A9 cells (HPRT^-). After growth in selective medium one clone was isolated which expressed the electrophoretically abnormal form of HPRT. Six clones showed the normal form of HPRT due to reversion of the defective HRRT locus in A9 cells. This result indicates DNA-mediated transfer of the mouse HPRT gene at a frequency of about 0.5×10^-7. A similar frequency has been found for transfer of the variant HPRT locus via isolated metaphase chromosomes to A9 recipient cells. When placed in non-selective media the DNA-mediated transferent cells gradually lost their ability to express the HPRT transgenome at a rate of about 6% per average cell generation.     

A promising genomic transfectant into Xeroderma pigmentosum group A with highly amplified mouse DNA and intermediate UV resistance turns revertant

Following transfection of genomic mouse DNA into an SV40 transformed fibroblast cell line from a patient with Xeroderma pigmentosum (complementation group A, XPA), a single UV resistant cell clone was isolated out of a total of 10(4) independent transfectants. The recipient XPA cell line has as yet not produced spontaneous revertants among 2.2 x 10(8) cells. The isolated cell clone contains 50-70 kb of mouse sequences which are heavily amplified (500-fold), and has acquired both intermediate resistance to UV killing and intermediate unscheduled DNA synthesis (UDS) capacity. By continued passage without selective pressure, cells were generated, which had lost both the dominant marker gene and repetitive mouse sequences. Single colonies of these cells were still intermediately resistant to UV suggesting that either undetected unique mouse DNA had segregated from the bulk of repetitive DNA, or, more likely, that the initially isolated transfectant was a spontaneous revertant. This documents that a persuasive clone isolated can still be a false positive (spontaneous revertant) and that an extremely laborious approach may lead into a dead end.     

Energetics of proton transfer pathways in reaction centers from Rhodobacter sphaeroides. The Glu-H173 activated mutants

Electron transfer between the primary and secondary quinones (Q(A), Q(B)) in the bacterial photosynthetic reaction center (bRC) is coupled with proton uptake at Q(B). The protons are conducted from the cytoplasmic side, probably with the participation of two water channels. Mutations of titratable residues like Asp-L213 to Asn (inhibited mutant) or the double mutant Glu-L212 to Ala/Asp-L213 to Ala inhibit these electron transfer-coupled proton uptake events. The inhibition of the proton transfer (PT) process in the single mutant can be restored by a second mutation of Arg-M233 to Cys or Arg-H177 to His (revertant mutant). These revertant mutants shed light on the location of the main proton transfer pathway of wild type bRC. In contrast to the wild type and inhibited mutant bRC, the revertant mutant bRC showed notable proton uptake at Glu-H173 upon formation of the Q(B)- state. In all of these mutants, the pK(a) of Asp-M17 decreased by 1.4-2.4 units with respect to the wild type bRC, whereas a significant pK(a) upshift of up to 5.8 units was observed at Glu-H122, Asp-H170, Glu-H173, and Glu-H230 in the revertant mutants. These residues belonging to the main PT pathway are arranged along water channel P1 localized mainly in subunit H. bRC possesses subunit H, which has no counterpart in photosystem II. Thus, bRC may possess alternative PT pathways involving water channels in subunit H, which becomes active in case the main PT pathway is blocked.     

An intragenic revertant of a poliovirus 2C mutant has an uncoating defect.

A revertant was isolated from a temperature-sensitive poliovirus 2C mutant, 2C-31, which is defective in viral RNA synthesis. This revertant, called 2C-31R1, grew well at 39 degrees C and was not defective in RNA synthesis. However, in contrast to its parental mutant, 2C-31R1 was cold sensitive and could hardly grow at all at 32 degrees C. Analysis of a single-cycle growth revealed that 2C-31R1 was defective in virion uncoating at 32 degrees C, and a substantial amount (more than 30%) of input viruses could be recovered as infectious particles from an infected cell lysate up to 6 h postinfection. The uncoating defect and the inability to grow at cold temperatures could be overcome by a brief incubation at the permissive temperature (39 degrees C) before the infection was continued at 32 degrees C. cDNA cloning and mix-and-match recombination experiments indicated that the defect in uncoating was the result of two secondary point mutations, seven nucleotides apart, in the 2C-coding sequence downstream of the inserted linker which is the original mutation in the parental 2C-31 genome. Another revertant, 2C-31R3, isolated from the same 2C-31 stock, was not defective in uncoating and appeared to be a secondary revertant that contained an intragenic suppressor for the uncoating defect. The uncoating defect of 2C-31R1 could be complemented by type 2 poliovirus. These results suggested that protein 2C, in addition to its role in viral RNA synthesis, has a function in determining virion structure.     

Podophyllotoxin directly binds a hinge domain in E2 of HPV and inhibits an E2/E7 interaction in vitro

Podophyllotoxin (PT), a strong cytotoxic agent from berberidaceae, has been known to inhibit tubulin polymerization. Although PT has been used for developing anticancer drugs as one of seed compounds, clinical treatment by itself has been unsuccessful because of the side effects, except one example in the treatments of warts. In this study, we screened peptides binding to PT with T7 phage display clonings in order to obtain more information about molecular mechanism of the action. A selected phage clone has a specific amino acid sequence to be SVPSRRRPDGRTHRSSRG. A homology search by protein database BLAST showed that this sequence had a similarity to a hinge domain (HD) of E2 protein in human papillomavirus (HPV) type 1a which is known to cause plantar warts. Surface plasmon resonance (SPR) analysis showed that PT bound to a recombinant HPV 1a E2 protein giving a K(D)=24.1microM which has compared with those of other domains of E2 protein. Also we demonstrated whether PT inhibited HD interaction or not. E7 protein of HPV has been known to be an oncoprotein and was reported to interact with HD of E2 protein. We demonstrated that an E2/E7 interaction was inhibited by the addition of PT in this report. And we showed the bindings of PT to other types of HPV. Our results suggest that PT is potential as a tool for clarifying the molecular mechanism of HPV.     

Reversion in expression of hypoxanthine-guanine phosphoribosyltransferase in 6-thioguanine resistant neuroblastoma: evidence for reduced enzyme levels associated with unaltered catalytic activity.

Five clones of mouse neuroblastoma cells able to grow in hypoxanthine-aminopterin-thymidine containing medium were isolated from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8; IMP: pyrophosphate phosphoribosyltransferase) deficient cell line. These hypoxanthine-aminopterin-thymidine resistant revertant clone had 45-55% of wild-type cell HGPRT activity. Kinetic studies indicated that the HGPRT in revertant clones had a reduced maximal velocity as compared to wild type cells based on cell protein. Apparent Km values of HGPRT for hypoxanthine and 5-phosphoribosyl-1-pyrophosphate were similar in wild-type and revertant cells. Heat inactivation studies demonstrated a similar heat lability for HGPRT in revertant and wild-type cells. An antibody fraction prepared from serum of rabbits immunized with HGPRT partially purified from mouse liver was used to measure the amount of cross-reacting material in normal and revertant clones. The revertant clones had one-half the amounth of cross-reacting material present in wild-type cells, based on a given amount of cell protein. These data indicate that the revertant cells may contain fewer HGPRT molecules with unaltered catalytic activity.     

Reconstruction of tubular structures in three-dimensional collagen gel culture using proximal tubular epithelial cells voided in human urine

Human proximal tubular (PT) epithelial cells were isolated from urine and monoclonally cultured as monolayers for 1 wk, after which they were subcultured between two layers of collagen gel, designated a "collagen gel sandwich." Under these culture conditions, PT cells formed three-dimensional tubular structures exhibiting distinct polarized cell morphology. Scanning and transmission electron microscopic studies showed that they bore numerous microvilli at the apical surface and that they closely contacted the collagen gel at the basal surface. These studies indicate that PT cells exfoliated in urine still exhibit the potential to proliferate and form organized structures mimicking in vivo tubules. Because of the current lack of useful culture systems for human tubular epithelial cells originating from kidney tissue, we suggest that this unique culture system using voided PT cells in urine could open up new avenues to study not only the mechanisms of morphogenesis but also the physiology of human PT cells.     

The spread and antibiotic resistance of Salmonella enterica serotype typhimurium DT104 in Hungary

Comparison of phage types (PTs) determined by Felix and Callow's and Anderson's methods was performed testing 99 human strains of S. enterica serotype Typhimurium (S. typhimurium) isolated in Hungary. PT2 and PT2c--according to Felix-Callow--corresponded with Anderson's DT104 in case of 39 strains out of 40. Among 59 isolates belonging to other Felix-Callow's PTs only one strain was found which was DT 104. Similar unambiguous equalities could not be established between any other PTs comparing the two methods. The PTs of 17,877 human strains isolated between 1988 and 1999 were determined using Felix-Callow's method. On the basis of the above equality the emergence of DT104 could be followed retrospectively by means of the rate of PT2 and PT2c. The increase of DT104 began already in 1989, emerging first PT2c then PT2. It predominated since 1991 and it reached its maximum (78.3%) in 1999. The incidence of multiresistance among one of the groups of DT104 strains (Felix-Callow's PT2) was significantly higher in 1998 than the average of non-DT104 strains. The predominant R-type was ACST.     

Studies on the virulence and attenuation of Trypanosoma cruzi using immunodeficient animals

Tissue invasion and pathology by Trypanosoma cruzi result from an interaction between parasite virulence and host immunity. Successive in vivo generations of the parasite select populations with increasing ability to invade the host. Conversely, prolonged in vitro selection of the parasite produces attenuated sublines with low infectivity for mammals. One such subline (TCC clone) has been extensively used in our laboratory as experimental vaccine and tested in comparative experiments with its virulent ancestor (TUL). The experiments here reviewed aimed at the use of immunodeficient mice for testing the infectivity of TCC parasites. It has not been possible to obtain virulent, revertant sublines by prolonged passaged in such mice.     

Role of a membrane glycoprotein in Friend virus erythroleukemia: nucleotide sequences of nonleukemogenic mutant and spontaneous revertant viruses.

We previously isolated spontaneous env gene mutants of Friend spleen focus-forming virus that are nonleukemogenic in adult mice but form leukemogenic revertants in newborns; we found that the revertants contain secondary env mutations. To identify sites in the encoded membrane glycoprotein that are important for its pathogenic function, we molecularly cloned and partially sequenced the env genes of two mutant viruses (clone 63 and clone 4) and one revertant (clone 4REV). Clone 63 contained three noncontiguous point mutations that caused nonconservative amino acid substitutions of Gly-119----Arg-119, Cys-180----Tyr-180, and Gly-203----Arg-203 in the xenotropic-related domain of the env glycoprotein. These substitutions were presumably responsible for the altered electrophoretic and pathogenic properties of the mutant glycoprotein. The presence of these and several other G-A nucleotide substitutions at different sites in one spontaneous mutant provided striking evidence that error-rich proviruses can form during retroviral replication. Clone 4 contained a point mutation that generated a premature termination condon at amino acid residue 304 (Gln-304----Ochre-304). This termination codon was located immediately after the proposed xenotropic-ecotropic recombination site and eliminated the ecotropic-related domain, including the putative membrane anchor of the glycoprotein. Clone 4REV was a true revertant derived from clone 4 in which the premature termination codon had back-mutated to re-form the wild-type sequence. These results confirm an essential role for the env gene in Friend spleen focus-forming virus pathogenesis and suggest that the encoded membrane glycoprotein contains different domains that contribute to its pathogenic function.     

Isolation of a suppressor host bacterium in Staphylococcus aureus

A bacterial mutant of Staphylococcus aureus NCTC 8325 has been isolated which has the properties of a suppressor host mutant. The mutant was isolated as a one-step phenotypic revertant to wild type of a strain containing mutations in two unlinked markers concerned with metabolism of lactose via the phosphoenol pyruvate-dependent phosphotransferase system. The revertant (called sup1(+)) has been used to isolate seventy conditional lethal mutants of the phage O11. The phage mutants, which plate on sup1(+) but not on the original 8325 strain, have been assigned by complementation studies into 10 groups. It is probable that this technique for the isolation of suppressor hosts would be applicable to other Staphylococcus strains.     

Is caveolin involved in normal proximal tubule function? Presence in model PT systems but absence in situ

Kidney proximal tubule (PT) cells are specialized for the uptake and transport of ions, solutes, peptides, and proteins. These functions are often regulated by hormones that signal at the cell surface and are internalized by clathrin-mediated endocytosis. However, the caveolin/caveolae pathway has also been implicated in normal PT function, often based on data from isolated PTs or PT cells in culture. Although we reported previously that caveolae and caveolin 1 are not detectable in PTs in vivo, reports of caveolin expression and function in PT cells appear periodically in the literature. Therefore, we reexamined caveolin expression in PTs in vivo, in isolated "purified" PTs following collagenase digestion, and in cultured PT cells. Caveolin 1 and 2 protein, mRNA, or immunofluorescence was undetectable in PTs in vivo, but PT cell cultures expressed caveolin 1 and/or 2. Furthermore, caveolin 1 and 2 mRNAs were detected in isolated PTs along with the endothelial markers CD31 and ICAM1. In contrast, no caveolin or endothelial marker mRNAs were detectable in samples isolated from snap-frozen kidneys by laser cut microdissection, which eliminates contamination by other cell types. We conclude 1) caveolin 1 and 2 are not normally expressed by PT cells in situ, 2) caveolin expression is "activated" in cultured PT cells, 3) contamination with non-PT, caveolin-expressing cells is a potential source of caveolin 1 and 2 that must be taken into account when isolated PTs are used in studies to correlate expression of these proteins with PT function.     

CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS : I. Biologic, Virologic, and Chemical Properties

Two contact-inhibited "revertant" cell lines were isolated from an SV40-transformed mouse 3T3 cell line (SV-3T3) after exposure to 5-fluoro-2'-deoxyuridine. Revertant cells resembled 3T3 cells morphologically and grew to saturation densities which were similar to those of 3T3 cells; however, revertant cells readily formed both single and multinucleated giant cells in confluent cultures. SV40 virus was rescued from revertant cells by fusion with permissive monkey cells. The rescued virus transformed 3T3 cells with the same efficiency as wild type virus, and produced transformed colonies which were phenotypically similar to those produced by wild type virus. The revertant cells also resembled normal 3T3 cells in that they contained higher quantities of sialic acid than SV-3T3 cells. An inverse correlation was found between the saturation density of cells and their sialic acid content. Collagen content, however, of revertant cells was similar to that of SV-3T3 cells. The data presented suggest that the property of contact inhibition in revertant cells is related to the sialic acid content of the plasma membrane and that changes in sialic acid content of transformed cells are not directly specified by the viral genome.     

Dominance and independent segregation of metabolic cooperation-competence and pluripotency in an embryonal carcinoma cell hybrid

We report the isolation of a fusion hybrid, PR3, from a pluripotent embryonal carcinoma (EC) cell line, PSA4, which is metabolic cooperation-competent, and an EC line R5/3OA which has a reduced capacity for metabolic cooperation and a restricted developmental capacity. PR3 resembles its pluripotent parent PSA4 in its capacity for gap-junction-mediated transfer of uridine nucleotides and in its pluripotency both in embryoid bodies in vitro and in tumors in vivo. This enabled the relationship between pluripotency and metabolic cooperation to be examined by the selection of segregant lines. Cooperation-deficient lines were isolated from a thioguanine-resistant intermediate line (PR3Tg12) using "Kiss of Death" selection. A novel method was devised for the selection of differentiation-deficient segregants using feeder cell-conditioned medium which partially inhibits in vitro differentiation. It was found that communication-competence and in vitro pluripotency segregated independently, demonstrating that the loss of developmental capacity in R5/3OA cannot be attributed to its communication-deficiency.     

A Thy-1 − mutant defining a gene acting in trans position to regulate cell-surface Thy-1 glycoprotein expression and Thy-1 messenger RNA content

The Thy-1 glycoprotein is a differentiation antigen which exhibits tissue-specific regulation. A mutant of a Thy-1.1⁺ T-cell lymphoma has been isolated which does not express Thy-1 glycoprotein on the cell surface and does not accumulate Thy-1 mRNA in the cytoplasm. Hybrids between the mutant and a Thy-1.2⁺ T-cell lymphoma express 20–30-fold lower levels of Thy-1 glycoprotein on their cell surface compared to wild-type T-cell lymphomas, and they have correspondingly low levels of cytoplasmic Thy-1 mRNA. A revertant of one hybrid was isolated which expressed wild-type levels of both Thy-1 alleles on its surface and contained correspondingly increased levels of Thy-1 mRNA. A Thy-1⁺ revertant of the Thy-1 ^– mutant was isolated by cell sorting. A second generation Thy-1 ^– mutant could be isolated from this revertant which also did not accumulate Thy-1 mRNA and which behaved in a way similar to the first generation mutant when hybridized to a Thy-1.2⁺ lymphoma. No changes in the structure or copy number of the Thy-1 structural gene could be detected in this series of mutants and revertants. These properties are consistent with a mutation in one (or more) gene(s) which acts in trans position to regulate Thy-1 glycoprotein expression.