Modulating cell surface immunoreactivity by metabolic induction of unnatural carbohydrate antigens.

BACKGROUND: Sialic acid is a component of many tumor-associated oligosaccharide antigens. The repertoire of sialic acids presented by cells can be expanded to include unnatural variants by intercepting the sialic acid biosynthetic pathway with unnatural precursors. We explored whether unnatural cell surface sialosides produced by metabolism can act as neo-antigens and modulate the immunogenicity of cells. RESULTS: Immunization of rabbits with synthetic conjugates of an unnatural sialic acid bound to keyhole limpet hemocyanin produced significant titers of antibodies that were specific for the structurally altered sialic acid. The antibodies recognized cells that were fed the unnatural biosynthetic precursor, and were capable of directing complement-mediated lysis. CONCLUSIONS: Structural alteration of sialic acids replaces a tolerized self-antigen with an antigenic determinant. Incorporation of unnatural sialosides into cell surface glycoconjugates through biosynthetic means can alter the immunoreactivity of cells, providing new possibilities for tumor immunotherapy.

Chemoselective ligation reactions with proteins, oligosaccharides and cells.

Traditional chemical synthesis does not lend itself to the easy, rapid construction of even moderately sized biomolecules, because it requires elaborate protection schemes. Furthermore, many biological studies would be aided by the ability to assemble biomolecules under physiological conditions. These challenges have motivated the development of chemoselective ligation, the selective covalent coupling of mutually and uniquely reactive functional groups under mild, aqueous conditions. This technique has attracted significant attention recently for the synthesis of biological macromolecules of defined homogeneous composition, the design of self-assembling drugs and the chemical remodeling of cell surfaces.

Role of bcl-X(L) in the control of apoptosis in murine myeloma cells.

The development of an increasing number of tumors has been shown to involve the deregulation of not only cell proliferation but also normal cell death by apoptosis. Expression of the bcl-2 proto-oncogene has been shown to inhibit the apoptotic cell death of many types of cells. Recent work also has revealed the existence of several bcl-2-related genes that also can inhibit (e.g., bcl-X(L) and Mcl-1) or activate (e.g., bax, bcl-X(s), bag, and bad) apoptosis in several systems. Myelomas are antibody-secreting tumor cells derived from terminally differentiated B lymphocytes, and previous work from our laboratory showed that murine SP2/0 myeloma cells and derived B-cell hybridomas were highly sensitive to apoptosis induction by a block of gene expression (cycloheximide). Additional work revealed that a related murine myeloma cell line, P3X63Ag8.653, was resistant to apoptosis induction in similar conditions. To understand the genetic basis of this differential susceptibility, we examined the expression of apoptosis-related genes in these cell lines. Northern blot experiments showed no significant difference in the expression of myc and bax apoptosis-promoting genes in susceptible (SP2/0 and D5) and resistant (P3X63) cell lines. Also, no significant expression of the bcl-2 gene could be detected in these cell lines. However, a much higher expression level of bcl-X(L) mRNA was observed in apoptosis-resistant P3X63Ag8.653 cells. The role of bcl-X(L) was supported by the finding that expression of bcl-X(L) cDNA in transfected, apoptosis-sensitive D5 cells increased the viability of these cells greatly and reduced DNA fragmentation following apoptosis induction. Significant bcl-X(L) but not bcl-2 expression was also detected in three other murine myeloma cell lines (MOPC 315, RPC 5.4, and J558) derived from different plasmacytoma tumors. These results indicate a predominant role of bcl-X(L) in preventing apoptosis in myeloma cells and suggest that the expression of bcl-2 or bcl-X(L) genes in B-cell tumors depends on the differentiation stage of the precursor normal cell.

The two alleles of the hapP gene in Physarum polycephalum code for different proteins.

Many mRNAs show cell-type specific expression in the acellular slime mold Physarum polycephalum. The most abundant plasmodial-specific mRNA (hapP) encodes a small hydrophobic protein of 187 amino acids that contains a potential signal peptide. Southern hybridizations using the hapP cDNA showed that the hapP gene is a single copy gene with two alleles, hapP1 and hapP2. The alleles have restriction enzyme polymorphisms. The nucleotide sequence of the coding region of the hapP1 allele was obtained from a genomic clone, and the nucleotide sequence of the hapP2 allele was obtained from a cDNA clone. The hapP1 and hapP2 alleles code for proteins that are 9.6% different in amino acid sequence. All differences are found in the central region of the protein. The nucleotide sequences of the first and last exons, which contain coding and non-coding regions, are identical. PCR amplification of cDNAs (RT-PCR) showed that both alleles are expressed in the same cell.

Increased avidity of mutant IgM antibodies caused by the absence of COOH-terminal glycosylation of the mu H chain.

We have previously described the isolation of two hybridoma variants secreting higher avidity IgM (D5 and 7F5), starting from the E11 hybridoma cell line, which produces an antibody specific for the A Ag of the ABO blood group system. In order to explain at the molecular level this increased reactivity, cDNA encoding the H and L chains of the E11, D5, and 7F5 mAb were cloned and sequenced. Comparison of the nucleotide sequences showed a single point mutation in each of the two mAb produced by the hybridoma variants. The mutations were both located in the H chain C region and caused a Ser to Phe substitution at position 565 in the D5 mAb and a Asn to Tyr substitution at position 563 in the 7F5 mAb. Both substitutions modified the consensus glycosylation sequence (Asn-X-Ser/Thr) located in the tail piece of the secretory mu-chain. The absence of glycosylation at this site was confirmed by CNBr cleavage of the [14C]mannose-labeled mAb. The two single point mutations were solely responsible for the increased avidity of the antibodies, as confirmed by site-directed mutagenesis of the E11 mu-chain and serologic analysis of the mutated E11 antibodies. We conclude that the absence of glycosylation at Asn 563 is responsible for the increased avidity of the mutant, possibly by altering the quaternary structure of the IgM polymer. To our knowledge, this is the first report that point mutations in the H chain C region can influence the reactivity of IgM mAb.

An unusual actin-encoding gene in Physarum polycephalum.

Actin is one of the most conserved proteins in eukaryotic organisms. In the present work, we cloned and determined the nucleotide sequence of an unusual actin-encoding gene, ardD, from the slime mold, Physarum polycephalum. The ardD gene encodes an ArdD protein containing 367 amino acids (aa) instead of the 375-376 aa found in a typical actin. The nine missing aa are accounted for by deletions of three aa in the first exon, five in the fifth exon and one in the sixth exon. These deletions in the coding sequence were observed in a polymerase chain reaction (PCR)-generated cDNA fragment, which excludes the possibility of a cloning artifact. In addition, ArdD contains numerous aa substitutions distributed throughout the protein. The ArdD aa sequence was compared with published actin sequences. The most identity is seen with the P. polycephalum ArdA, ArdB and ArdC (84%) and Acanthamoeba (82%) actins, while the least identity is found with Tetrahymena actin (67%). The expression of the ardD gene is developmentally regulated. The highest levels of ardD mRNA were found in spherules, less was seen in plasmodia and no detectable transcripts were observed in amoebae. The PCR amplification of an ardD cDNA from spherules confirmed the presence of mRNA in this developmental stage. The aa deletions and substitutions in the predicted ArdD aa sequence make it one of the most distinctive actins known.

Fission yeast promoter-probe vectors based on hygromycin resistance.

We have constructed fission yeast vectors that carry either complete or 5'-truncated alleles of the hph gene, encoding hygromycin B phosphotransferase. We show that plasmid-borne hph can be expressed in fission yeast to confer hygromycin resistance. The vectors permit selection or screening in fission yeast for promoter activity of DNA fragments from other species. We used the vectors to identify several genomic sequences from Physarum that provide promoter function in fission yeast.

Metabolic characteristics of cat kidney: failure to adapt to metabolic acidosis.

During studies performed on domestic cats made acidotic with ammonium chloride, it was found that the cat kidney is unable to adapt to metabolic acidosis. Renal proximal tubules do not increase their production of ammonia or glucose from glutamine during acidosis. During in vivo studies, the renal excretion of ammonia did not change much during acidosis. Other metabolic parameters in the cat were not very different from those found in other animals such as rat or dog. However, it was found that cats may show a relatively high plasma glucose concentration compared with other animals. Plasma insulin concentration was normal, and the animals showed no evidence of diabetes mellitus. It is not known whether limitation of ammoniagenesis and elevated plasma glucose concentration also characterize larger felidae such as panthers and cougars.

Cell-specific expression of a profilin gene family.

Profilin is a ubiquitous eukaryotic protein that inhibits actin polymerization. We cloned and sequenced the two profilin genes from the acellular slime mold Physarum polycephalum. The genes, proA and proP, each contain two introns. Primer extension experiments showed two possible transcription start sites in the profilin A gene and one start site in the profilin P gene. The profilin A mRNA has two polyadenylation sites, which yield mRNAs of about 600 and 500 nucleotides. The profilin P mRNA has a single polyadenylation site. The protein sequences were deduced from cDNA nucleotide sequencing. The profilin A and profilin P proteins contain 125 amino acids and are 66% identical in sequence. They show sequence similarity to Acanthamoeba (approximately 54%), yeast (approximately 46%), mouse (approximately 22%), calf (approximately 21%), and human (approximately 21%) profilins. The presence of the profilin A and profilin P mRNAs was studied throughout the life cycle. Profilin A mRNA was found in amebas, encysted amebas, and mature spores. The profilin P mRNA was present in plasmodia and spherulating plasmodia. Most cell types of P. polycephalum contain either the amebal or the plasmodial profilin mRNA, and no cell contains both mRNAs. This is the first evidence for developmental regulation of profilin isoforms.

Developmentally regulated late mRNAs in the encystment of Physarum polycephalum plasmodia.

Physarum polycephalum plasmodia survive adverse conditions by transforming into encysted cells called spherules. In this work we analysed the developmentally regulated mRNAs from the late stages of spherulation. A cDNA library was constructed and four abundant mRNAs were identified. One of the mRNAs was present in trace amounts in early spherules, while the other three were found only in late spherules. A cDNA clone for one of the late spherulation specific mRNAs was sequenced. It codes for a 332-amino-acid protein that did not show significant similarities with any known protein. Since the mRNA for this protein accumulates during spherulation, the protein was called spherulin 4. This protein has many features of a plasma membrane protein; it contains a signal peptide and a long hydrophobic region, which could serve as a transmembrane anchor. Another interesting feature is the presence of seven consecutive glycine residues in the N-terminal region. This is even more remarkable since the protein is not rich in glycine.

Selective gene expression during sporulation of Physarum polycephalum.

The two-dimensional gel electrophoresis of polypeptides synthesized in vitro from poly(A)+ RNA showed that mRNA populations change during sporulation of Physarum polycephalum. The differential hybridization of a cDNA library prepared from poly(A)+ RNA isolated from sporulating cells revealed that of 846 clones, 64 corresponded to sporulation-specific mRNAs. Further analysis demonstrated that these clones contained seven different sequences: three abundant sequences composing 3.2, 1.8, and 1.2% of the library and four other less abundant sequences. It is probable that all the major mRNAs specifically expressed in early stages of sporulation were identified. The most abundant mRNA from this group coded for a hydrophobic protein that contained a signal peptide. This protein is 47% similar to another Physarum protein, which was encoded by the most abundant plasmodium-specific mRNA. The plasmodial mRNA was degraded during sporulation and was replaced by the sporulation mRNA. These two proteins are thus encoded by members of a gene family whose expression is developmentally regulated.

Real importance of alanine in renal metabolism: in vitro studies in rat and dog.

In vitro studies were performed on cortical renal tubules to clarify possible differences between dog and rat with regard to alanine production and to define more precisely the role of alanine on ammonia and glucose production by the kidney. It was established that glutamate-pyruvate transaminase has an activity that is seven times lower in the rat than in the dog kidney. At the same time, alanine production from lactate, pyruvate, and glutamate is three times lower in the rat than in the dog kidney. The enzymatic reaction could be completely inhibited in a competitive fashion with aminooxyacetate. O2 consumption and CO2 production by the renal tubules were lower than that observed with glutamine. CO2 production in the rat was lowest. Production of ammonia and glucose by the kidney from alanine during acidosis averaged less than 20% of that produced with L-glutamine. Furthermore, during metabolic acidosis the production of ammonia and glucose from alanine was not augmented and failed to be influenced by increasing the concentration of alanine in the incubation medium.

Expression of the three unlinked isocoding actin genes of Physarum polycephalum.

The actin gene family in Physarum polycephalum contains four unlinked loci: ardA, ardB, ardC, and ardD. The ardA locus is complex and probably contains two genes which we designated ardA2-7 and ardA2-17. cDNA clones corresponding to the ardB and ardC loci were isolated. Nucleic acid sequencing showed that these two cDNAs coded for the only abundant form of Physarum actin, which is 96% homologous to human gamma-cytoplasmic actin. The ardA2-17 gene also codes for this same actin protein (Nader et al., Gene 48, 133-144, 1986). The coding regions of ardB and ardC differ by 15 nucleotides. A comparison of the ardB and ardC sequences with ardA2-17 showed 73 and 77 nucleotide substitutions, respectively, in the coding regions. The noncoding regions of these three sequences were not homologous to each other or to the noncoding regions of actin genes from other organisms. Southern genomic hybridizations indicated that the ardA2-7 and ardD genes have weak sequence similarities to the three isocoding actin genes and thus form a different subclass of the family. Northern hybridizations showed that the ardB and ardC transcripts varied in abundance but were present in all the developmental stages. No ardA2-17 transcripts were seen. The relative abundance of the ardB and ardC transcripts was measured in amoebae and plasmodia by S1 nuclease protection and dot hybridization assays. A ratio of approximately 3:1 for ardC versus ardB was found for both stages. P. polycephalum is the first organism shown to contain three unlinked isocoding actin genes, of which at least two are expressed.

Gene families encode the major encystment-specific proteins of Physarum polycephalum plasmodia.

The encystment of Physarum polycephalum plasmodia, also called spherulation, involves the synthesis of many specific mRNAs and proteins. Most of these molecules accumulate at the onset of the major morphological and physiological changes typical of this differentiation pathway and are not present during the other two transitions leading to dormancy in Physarum, namely sporulation and encystment of amoebae. The nucleotide sequences of apparently full-length cDNA copies of the four major encystment-specific mRNAs were determined. The four sequences included the entire coding regions and at least 26 nucleotides of the 5'-nontranscribed leaders. The encoded proteins were named spherulins. We found that spherulins 1a and 1b are 81% homologous and are thus members of a gene family. They both possess putative signal peptides and N-glycosylation sites, suggesting that they are cell-wall glycoproteins. Spherulin 2a and spherulin 3a are non-homologous proteins. The absence of signal peptides suggests that they are intracellular structural proteins. Low-stringency Southern hybridizations showed that each also belongs to a two-member gene family.

Renal tissue metabolism in the rat during chronic metabolic alkalosis: importance of glycolysis.

Chronic metabolic alkalosis was induced in rats drinking 0.3 M NaHCO3 and receiving 1 mg furosemide/100 g body weight per day intraperitoneally. Another group of animals received a potassium supplement in the form of 0.3 M KHCO3. In this group, hypokalemia did not develop and muscle potassium fell by only 18% versus 50% in those not receiving potassium. In vitro renal production of ammonia and uptake of glutamine fell by 40% with a decrease in the activity of glutaminase I and glutamate dehydrogenase. Activity of phosphofructokinase, a major enzyme of glycolysis, rose only in the kidney of animals receiving a potassium supplement. Fructose-1,6-diphosphatase fell as well as phosphoenolpyruvate carboxykinase. Malate dehydrogenase also fell. The activity of phosphofructokinase also rose in the liver, heart, and leg muscle. The major biochemical changes in the renal cortex were the following: glutamate, alpha-ketoglutarate, malate, lactate, pyruvate, alanine, aspartate, and citrate rose as well as calculated oxaloacetate. The concentration of intermediates like 2-phosphoglycerate, 3-phosphoglycerate, and glucose-6-phosphate fell. The cytosolic redox potential (NAD+/NADH) decreased. In addition to the fall in ammoniagenesis, it could be demonstrated in vitro that the renal tubules incubated with glutamine showed decreased glucose production and increased production of lactate and pyruvate. The concentration of lactate was elevated in all tissues examined including liver, heart, and leg muscle. This study confirms in the rat that decreased renal ammoniagenesis takes place following decreased uptake of glutamine in metabolic alkalosis. All other changes are accounted for by the process of increased glycolysis, which appears to take place in all tissues in metabolic alkalosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of mRNAs expressed specifically during spherulation of Physarum polycephalum.

A cDNA library was constructed using the poly(A)+ RNA extracted from spherulating Physarum polycephalum microplasmodia. This library (740 clones) was screened by differential hybridization with 32P-labeled poly(A)+ RNA from growing plasmodia and developing spherules. The results showed that at least 30% of the clones corresponded to mRNAs expressed specifically in spherulating plasmodia. The 35 spherulation-specific cDNA clones giving the strongest hybridization signals were analysed. From this group, four different sequences complementary to very abundant mRNAs were identified. They each accounted for 1.5% of 4.5% of all the clones in the library and probably represented the most abundant spherulation-specific mRNAs. In addition, four less abundant mRNAs were identified from stage-specific clones giving weaker hybridization signals. These sequences represented individually between 0.3% and 0.7% of the clones in the library. Northern blots showed that these eight different sequences were absent from plasmodia and were most abundant 24-36 h after the induction of spherulation. Similar results were also obtained when spherulation was induced by the addition of a sublethal concentration of ferrous iron ions to the growth medium. Hybridization of the spherule-specific clones to Southern blots of genomic DNA suggested the presence of one copy for each gene.