Synthetic Defects Research Articles

Regulation of glycoinositol phospholipid anchor assembly in human lymphocytes. Absent mannolipid synthesis in affected T and natural killer cell lines from paroxysmal nocturnal hemoglobinuria patients.

Glycoinositol phospholipid (GPI) anchor structures derive from sequentially glycosylated inositol phospholipid precursors assembled in the endoplasmic reticulum. To characterize GPI biosynthesis in nontransformed human lymphocytes and to define the GPI synthetic defect underlying deficient expression of GPI-anchored proteins by paroxysmal nocturnal hemoglobinuria (PNH) cells, putative intracellular GPI intermediates were analyzed following [3H]Man labeling of normal and affected lymphocytes. In unstimulated normal peripheral blood lymphocytes, [3H]Man incorporation into GPIs was minimally detectable but after phytohemagglutinin (PHA), allogeneic cell, or anti-CD3 stimulation, assembly of [3H]Man-labeled GPIs markedly increased. Expression of GPIs by prestimulated quiescent PHA blasts could be efficiently induced by phorbol 12-myristate 13-acetate (PMA) and increased by the Ca2+ ionophore A23187 independently of new protein synthesis. Utilizing allogeneically stimulated cells in conjunction with PMA induction, products deriving from [3H]Man labeling of affected CD48- T and natural killer lymphocyte cell lines from five PNH patients were compared to those deriving from unaffected CD48+ cell lines from the same patients or controls. In contrast to unaffected paired control cells, affected cells of all of the patients exhibited a common abnormality in which they assembled dolichol-phosphoryl-Man but failed to express [3H]Man-containing GPIs. These data indicate that 1) significant GPI production in lymphocytes is dependent on prior stimulation of the cells, 2) exposure of lymphocytes to agents which activate protein kinase C induces GPI synthesis, and 3) in five PNH patients affected lymphocytes are uniformly defective in an early GPI biosynthetic step which undermines expression of GPI mannolipids.

Impaired Glycosylation of Glycosylphosphatidylinositol-Anchor Synthesis in Paroxysmal Nocturnal Hemoglobinuria Leukocytes

Metabolic labeling with [ 3H]sugars in vivo or [ 3H]sugar nucleotides in vitro of glycosylphosphatidylinositol (GPI)-anchor precursors in peripheral blood granulocytes and cultured T lymphocytes of paroxysmal nocturnal hemoglobinuria (PNH) patients showed a synthetic defect in the GPI-anchor. Among the GPI-anchor precursors, phosphatidylinositol (PI) was normally synthesized, while the synthesis of glucosaminylphosphatidylinositol (GlcN-PI) and subsequent mannosylation of GlcN-PI were inhibited in affected cells. The defect in the GPI-anchor synthesis in PNH is thus attributed to interrupted glycosylation at plural sites in the synthesis of the common carbohydrate structure of the anchor.

Suppressors of a spo0A missense mutation and their effects on sporulation in Bacillus subtilis

The spo0A gene product of Bacillus subtilis is a transcriptional regulator that is required for the initiation of sporulation. It has not been possible to isolate mutations that suppress the sporulation defect caused by s po0A null mutations. We describe the isolation and characterization of mutations that suppress the severe sporulation defect caused by a spo0A missense mutation ( spo0A9V). Two suppressor mutations, spa2 and spa4, have been characterized in combination with, and separated from, the spo0A9V mutation. Both were located in the carboxyl half of Spo0A, in the putative DNA binding, transcriptional activation region. spa2 was in codon 174, causing a leucine to arginine change ( spo0A174LR), and spa4 was in codon 162 (of 267), causing a histidine to arginine change ( spo0A162HR). spa2 and spa4 significantly restored sporulation to the spo0A9V mutant, however, the appearance of heat resistant spores was delayed relative to wild-type. When separated from spo0A9V, that is, as single mutations in spo0A, spa4 caused a delay in sporulation, while spa2 allowed apparently normal sporulation. The spa mutations caused interesting phenotypes when combined with other early sporulation mutations. spa2 suppressed the sporulation defect caused by spo0E11. This was most easily seen in spo0E11 abrB double mutants, which had a much more severe sporulation defect than the spo0E11 single mutant. That is, spo0E11 and abrB mutations caused ay a synthetic (synergistic) sporulation phenotype. Both the spa2 spo0A9V and the spa4 spo0A9V alleles greatly enhanced the sporulation defect caused by mutations in spo11J, spo0J and spo0K. The significance of these synthetic sporulation defects is discussed.

Apolipoprotein synthesis in normal and abetalipoproteinemic intestinal mucosa

The genetic disease abetalipoproteinemia is characterized by a total absence of apolipoprotein B-containing lipoproteins from plasma. A presumed synthetic defect in apolipoprotein B synthesis was thought to be responsible for this disorder. The present study quantitates apoprotein B synthesis and apolipoprotein B messenger RNA levels in duodenal mucosa from normal patients and four patients with abetalipoproteinemia. After in vitro [3H]leucine incorporation, small intestinal biopsy specimens from three of four patients with abetalipoproteinemia synthesized immunoprecipitable apolipoprotein B of identical mobility (on sodium dodecyl sulfate gel electrophoresis) to normal apolipoprotein B. In abetalipoproteinemia, the apolipoprotein B content of intestinal mucosa by radioimmunoassay was 15% of normal mucosal values, whereas apolipoprotein B messenger RNA quantitation showed 3–20-fold increased levels compared with normal mucosa. In one patient, smaller-molecular-weight fragments of apolipoprotein B were immunoprecipitated from duodenal biopsy specimens. The synthesis rates and messenger RNA levels of two other chylomicron apoproteins (apolipoprotein A-I and apolipoprotein A-IV) were found to be reduced by 50%. These results show the synthesis of immunologically recognizable apolipoprotein B48 in abetalipoproteinemia. The significance of mucosal apolipoprotein B content in abetalipoproteinemia is discussed in terms of factors controlling apolipoprotein B synthesis in normal mucosa and in abetalipoproteinemia.

Cation and vacancy distribution in a synthetic defect spinel

Cation and vacancy distribution in a synthetic defect spinel

Genetic interactions at the FLA10 locus: suppressors and synthetic phenotypes that affect the cell cycle and flagellar function in Chlamydomonas reinhardtii.

Through the isolation of suppressors of temperature-sensitive flagellar assembly mutations at the FLA10 locus of Chlamydomonas reinhardtii, we have identified six other genes involved in flagellar assembly. Mutations at these suppressor loci, termed SUF1-SUF6, display allele specificity with respect to which fla10- mutant alleles they suppress. An additional mutation, apm1-122, which confers resistance to the plant herbicides amiprophos-methyl and oryzalin, was also found to interact with mutations at the FLA10 locus. The apm1-122 mutation in combination with three fla10- mutant alleles results in synthetic cold-sensitive cell division defects, and in combination with an additional pseudo-wild-type fla10- allele yields a synthetic temperature-sensitive flagellar motility phenotype. Based upon the genetic interactions of these loci, we propose that the FLA10 gene product interacts with multiple components of the flagellar apparatus and plays a role both in flagellar assembly and in the cell cycle.

Cation and vacancy distribution in a synthetic defect spinel

Determination de la structure cristalline d'un spinelle de composition Mg 0,388 Al 2,408 O 4 , synthetise a 2050 o C. A partir des occupations des sites et des longueurs des liaisons dans les sites octaedriques et tetraedriques, la distribution suivante est obtenue (Al 0,653 Mg 0,347 ) (Al 1,755 Mg 0,041 □0 ,204 )O 4 . Cette distribution est conservee apres chauffage du cristal a 1150 o C; elle serait donc stable sur une large gamme de temperature

The effects of oxygen free radicals on the carbohydrate moiety of IgG

The CH2-linked glycoform of rheumatoid IgG is abnormal in having a reduced galactose content. This has been postulated to be a synthetic defect due to a decrease in the level of rheumatoid B cell galactosyltransferase. However, more recent work has indicated that agalactosylation may be common to chronic inflammatory diseases. In this work we have investigated the effect of oxygen free radicals (OFRs), which are generated by activated phagocytic cells at inflammatory sites, on the carbohydrate moiety of IgG. Radiolytically generated peroxy (ROO •) and hydroxyl radicals (OH •) but not superoxide anion radicals (O— 2 −) were found to destroy galactose on IgG. After OH • attack, this was associated with an increase in the availability of N-acetylglucosamine, possibly due to its presence as a terminal residue. These results suggest that the agalactosylation associated with chronic inflammation may not only be synthetic in nature, but may also be a consequence of post-synthetic degradation by OFRs.

Adenovirus early region 4 is essential for normal stability of late nuclear RNAs.

H2dl808 is a deletion mutant of adenovirus type 2 lacking most of transcriptional early region E4. In most normal adenovirus host cells this virus displayed a complex mutant phenotype that included a dramatic reduction in the level of cytoplasmic late RNA, a corresponding defect in late protein synthesis, and a 5- to 10-fold defect in viral DNA accumulation. H5dl1004 is a deletion mutant of adenovirus type 5 that also lacks a portion of E4. It exhibited a reduction in levels of cytoplasmic late RNAs that was somewhat less severe than that of H2dl808 and a corresponding late protein synthetic defect but no defect in the production of viral DNA. In addition to the defect in the accumulation of late cytoplasmic mRNAs, HeLa cells infected by either H2dl808 or H5dl1004 showed substantially reduced levels of viral RNAs in their nuclei at late times after infection. Both mature mRNAs and apparent mRNA precursors were affected. The late transcription rates of the deletion mutant viruses were similar to that of wild-type virus. These results suggest that the underaccumulation of RNA in H2dl808- and H5dl1004-infected cells is caused by a reduction in the stability of viral RNA in the nucleus, and they implicate E4 products in a novel aspect of the regulation of viral gene expression.

Complementation of adenovirus E4 mutants by transient expression of E4 cDNA and deletion plasmids.

Human adenovirus mutants that carry a large deletion in early region 4 (E4) are severely defective in the synthesis of viral late proteins. Plasmids that carry intact E4 sequences can complement the late protein synthetic defect of such mutants when introduced into infected cells by transfection, presumably due to the transient expression of E4 products. Cells transfected with cDNA clones capable of expressing E4 open reading frame (ORF) 6, or deletion mutant clones expected to express either E4 ORF 6 or E4 ORF 3, also complement the mutants' defects. Thus, these E4 ORFs can individually satisfy the requirement for E4 products in viral late gene expression, and function effectively in the absence of other E4 products. Some E4 deletion mutants also exhibit a defect in the production of viral DNA. All of the clones that stimulate late gene expression also enhance one such mutant's ability to accumulate viral DNA. Thus, the ORF 3 and ORF 6 products are also individually sufficient to provide an E4 function necessary for normal viral DNA replication in the absence of other E4 products.

Early hypomethylation of 2'-O-ribose moieties in hepatocyte cytoplasmic ribosomal RNA underlies the protein synthetic defect produced by CCl4.

Carbon tetrachloride (CCl4) treatment of rats produces an early defect in methylation of hepatocyte ribosomal RNA, which occurs concurrently with a defect in the protein synthetic capacity of isolated ribosomes. The CCl4-induced methylation defect is specific for the 2'-O-ribose position, and a corresponding proportional increase in m7G base methylation occurs in vivo. Undermethylated ribosomal subunits (rRNA) from CCl4-treated preparations can be methylated in vitro to a much greater extent than those from control preparations, and in vitro methylation restores their functional capacity. In vitro methylation of treated ribosomal subunits (which restores functional capacity) occurs at 2'-O-ribose positions (largely G residues). In contrast, in vitro methylation of control ribosomal subunits (which does not affect functional activity) represents base methylation as m7G, sites which are apparently methylated in treated preparations in vivo. Methylation/demethylation of 2'-O-ribose sites in rRNA exposed on the surface of cytoplasmic ribosomal subunits may represent an important cellular mechanism for controlling protein synthesis in quiescent hepatocytes, and it appears that CCl4 disrupts protein synthesis by inhibiting this 2'-O-ribose methylation.

REFLECTIONS ON HEMATOLOGY

According to Homer, Odysseus wandered for ten years after the fall of Troy. On the occasion of my 20th anniversary as a pediatric hematologist-oncologist, join me in a reflective odyssey. The subspecialty has proliferated and differentiated amazingly during this period of explosive scientific discovery and, without question, where the action is today and will be in the extended future. Twenty years ago hematology and oncology were separated by much more than a hyphen. During my fellowship, the hematology and oncology services were totally separate. We devoted ourselves to hematologic clinical and laboratory pursuits. In those halcyon days, the red blood cell captured many young imaginations. Readily accessible and eager to surrender secrets, the glamorous erythrocyte was subjected to intense investigation. New laboratory techniques and genetic probes uncovered specific metabolic lesions and enzyme deficiencies, and structural and synthetic defects of the hemoglobin molecule were

Congenital genetic murine (ch) hydrocephalus. A structural model of cellular dysplasia and disorganization with the molecular locus of deficient proteoglycan synthesis.

The recessively inherited ch hydrocephalic mouse has been sporadically investigated over the past 40 years as a genetic murine model for congenital hydrocephalus. Since central nervous system anomalies are commonly associated with congenital anomalies in the musculoskeletal and urogenital systems, and since congenital hydrocephalus is also associated with other developmental abnormalities, an understanding of the pathophysiology at the cellular and molecular levels of the genetic defects of this hydrocephalic murine model has application to related human disorders associated with hydrocephalus. A unifying hypothesis is presented that can interrelate the multisystem developmental abnormalities. While the basic cellular defect is a failure of mesenchymal differentiation, the molecular locus is a synthetic defect in the production of the chondroitin sulfate proteoglycan. The primary end results of defective proteoglycan synthesis at a multiorgan-multisystem level includes chondrodysplasia, renal dysplasia, failure of endochondral ossification and gonadal development, and defective development of the adrenal medulla and the sympathetic ganglia. The development of the severe, communicating, congenital hydrocephalus in the ch mouse may be either a primary or secondary manifestation of the mesenchymal maldifferentiation.

Pathophysiology of hypoalbuminemia associated with carcinoid tumor

Decreased serum albumin levels are commonly observed in patients with carcinoid tumor, who also show several characteristic clinical and biochemical abnormalities. A large comparative study on a group of 96 carcinoid patients was performed with the purpose of identifying some of the mechanisms leading to hypoalbuminemia in patients with this form of cancer, and thereby to shed light on the cause of hypoalbuminemia of cancer in general. Serum albumin values were compared with a number of clinical parameters (including extent of liver metastases, severity of diarrhea, degree of right heart failure, and extent of gastrointestinal surgery) and of laboratory data (prothrombin time, BSP retention, serum transferrin concentration, hematocrit value, and daily urine excretion of 5-hydroxy-indoleacetic acid). In several patients the gastrointestinal protein loss was assessed by the 51Cr-albumin technique, whereas albumin renewal and distribution were evaluated by the use of 125I-albumin. The data obtained showed that the main factors in determining decreased serum albumin levels in patients with carcinoids are both reduced synthesis and increased loss of the protein. The hepatic synthetic defect appears to be related to a progressive decrease in the number of functioning liver cells; the origin of the gastrointestinal protein loss may be related to the obvious tumor involvement of the gut wall, as well as to the pharmacologically-induced diarrhea. Right heart failure occurring as a result of the carcinoid heart disease may be an additional cause for gastrointestinal protein loss in patients with carcinoid tumor.

Editorial: Synthetic defect in ganglioside metabolism.

Not so long ago, rare inborn errors of metabolism were of interest to only a few perverse types. They were worth studying only for academic one-upmanship or to foster promotion. They were about as exciting to the house staff as tsutsugamushi fever. The primary physician, working his 12-hour day and burdened by "information pollution," shared this view, and most still do. When another rare defect of ganglioside metabolism is reported, one predictable reaction is, "So, what else is new?"This reaction has been tempered somewhat by the practical spinoff generated by the above-mentioned perverse students of the rare. Even though . . .

A new syndrome of bile acid deficiency—a possible synthetic defect

A 3-month-old infant with chronic diarrhea, failure to thrive, and severe malabsorption of fat (50 per cent) was found to have low concentrations of bile acids in the duodenal fluid, with poor micellar incorporation of fatty acids. The deficiency of intraluminal bile acids was not due to (1) liver disease, (2) deconjugation of bile acids associated with bacterial overgrowth, (3) failure of intestinal reabsorption of bile acids, (4) failure of release of cholecystokinin, or (5) an excretion defect of bile acids. A trial of oral administration of bile acids produced increases in weight gain, absorption of fat, intraluminal micellar incorporation, and concentrations of intestinal bile acids. All of these returned to pretreatment values within a few days after discontinuance of administration of bile acids. A defect in the biosynthesis of bile acids, not secondary to liver disease, is suggested.