Allele-specific Characterization of Alanine: Glyoxylate Aminotransferase Variants Associated with Primary Hyperoxaluria

Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal recessive kidney stone disease caused by deficiency of the peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT), which is involved in glyoxylate detoxification. Over 75 different missense mutations in AGT have been found associated with PH1. While some of the mutations have been found to affect enzyme activity, stability, and/or localization, approximately half of these mutations are completely uncharacterized. In this study, we sought to systematically characterize AGT missense mutations associated with PH1. To facilitate analysis, we used two high-throughput yeast-based assays: one that assesses AGT specific activity, and one that assesses protein stability. Approximately 30% of PH1-associated missense mutations are found in conjunction with a minor allele polymorphic variant, which can interact to elicit complex effects on protein stability and trafficking. To better understand this allele interaction, we functionally characterized each of 34 mutants on both the major (wild-type) and minor allele backgrounds, identifying mutations that synergize with the minor allele. We classify these mutants into four distinct categories depending on activity/stability results in the different alleles. Twelve mutants were found to display reduced activity in combination with the minor allele, compared with the major allele background. When mapped on the AGT dimer structure, these mutants reveal localized regions of the protein that appear particularly sensitive to interactions with the minor allele variant. While the majority of the deleterious effects on activity in the minor allele can be attributed to synergistic interaction affecting protein stability, we identify one mutation, E274D, that appears to specifically affect activity when in combination with the minor allele.     

Phototoxic liposomes coupled to an antibody that alone cannot modulate its cell-surface antigen kill selected target cells

Summary Molecules such as antibodies that bind to cell surfaces can be used to deliver cytotoxic drugs to selected cells. To be effective the drug must usually be taken into the cells by endocytosis. In this study a T-cell line (CCRFCEM) was effectively killed by liposomes carrying a photosensitizer and bearing the antibody OKT4 (anti-CD4). The unconjugated antibody does not induce antigenic modulation in the target cells, an indication of the absence of endocytosis, and would therefore not normally have been selected as an agent for drug delivery. It cannot, however, be concluded with certainty that the conjugates act at the cell surface and several alternative explanations of their efficacy are offered.     

Evidence Against the Involvement of Ionically Bound Cell Wall Proteins in Pea Epicotyl Growth

Ionically bound cell wall proteins were extracted from 7 day old etiolated pea (Pisum sativum L. cv Alaska) epicotyls with 3 molar LiCl. Polyclonal antiserum was raised in rabbits against the cell wall proteins. Growth assays showed that treatment of growing region segments (5-7 millimeters) of peas with either dialyzed serum, serum globulin fraction, affinity purified immunoglobulin, or papain-cleaved antibody fragments had no effect on growth. Immunofluorescence microscopy confirmed antibody binding to cell walls and penetration of the antibodies into the tissues. Western blot analysis, immunoassay results, and affinity chromatography utilizing Sepharose-bound antibodies confirmed recognition of the protein preparation by the antibodies. Experiments employing in vitro extension as a screening measure indicated no effect upon extension by antibodies, by 50 millimolar LiCl perfusion of the apoplast or by 3 molar LiCl extraction. Addition of cell wall protein to protease pretreated segments did not restore extension nor did addition of cell wall protein to untreated segments increase extension. It is concluded that, although evidence suggests that protein is responsible for the process of extension, the class(es) of proteins which are extracted from pea cell walls with 3 molar LiCl are probably not involved in this process.     

Alternatively spliced RNAs encode several isoforms of CD46 (MCP), a regulator of complement activation

Five alternative cDNA clones were isolated for CD46, also known as the membrane cofactor protein (MCP) for the factor I-mediated cleavage of the complement convertases. One of these cDNA clones (a) was identical to an earlier MCP clone. The other four CD46 clones 3ontained the four NH₂-terminanl short consensus repeat (SCR) units of MCP, but differed at the region encoding the carboxyl-terminal of the protein which includes an extracellular segment rich in Ser, Thr, and Pro residues, a hydrophobic membrane-spanning domain, and a 33 amino acid cytoplasmic tail. The different CD46 cDNAs have variously: (b) inserted a 93 base pair (bp) exon resulting in a new cytoplasmic tail of 26 amino acids; (c) deleted a 42 bp exon from the extracellular Ser/Thr rich region; (d) used a cryptic splice acceptor sequence to delete 37 bp from an exon encoding transmembrane sequence; or (e) failed to splice the intron after the four SCR units. These were shown by northern blot and polymerase chain reaction to arise by alternative splicing of CD46 RNA. Forms (a), (b), and (c) of CD46 RNA are common in placental RNA, but (d) was rare, and (e) was incompletely processed and therefore aberrant. The polymerase chain reaction (PCR) was used to map the sites of the intron/exon junctions and demonstrate further possible splice variants of CD46. The alternative RNAs for CD46 may correlate to the different isoforms of CD46 found in different tissues, tumors, and in serum.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M58050. Address correspondence and offprint requests to: D. F. J. Purcell.     

Genetic analysis of thirty-three platelet polypeptides detected in two-dimensional polyacrylamide gels.

Two-dimensional gel electrophoresis followed by silver-staining was utilized to visualize platelet polypeptides for genetic analysis. A subset of 33 polypeptides that were most suited for scoring was selected. Families consisting of father-mother-child trios were studied. Thirty-six polypeptides of a total of 1,413 scored in children's gels exhibited the combination of a normal and a variant polypeptide. The observed index of heterozygosity of 2.55% is comparable to our previously reported findings for red cell proteins.     

Effects of arginine-vasopressin and ACTH 4-10 on acquisition of active avoidance response in rats with alcohol pretreatment in prenatal and adult age

Alcohol administration in drinking water to mother rats during gestation resulted in a permanent learning deficit of the offspring when behavioural reactions were tested in active avoidance conditioned reflex situation in adult age. A similar deficit of learning capacity was observed in adult rats following alcohol administration for two weeks; the acquisition of active avoidance response was tested later. Administration of arginine-vasopressin and ACTH 4-10 during behavioural test led to a significant improvement of learning ability of the animals pretreated with alcohol. The observations indicate that the ethanol-induced deficit of learning capacity involves peptidergic mechanisms and the behavioural manifestations following chronic alcohol treatment are not irreversible processes.     

The reproductive biology of female Père David's deer (Elaphurus davidianus)

Data for this study came from breeding records of 27 Père David's (Elaphurus davidianus) hinds maintained in large pastures and from estrous records of four hand-reared nulliparous hinds. The mean estrous cycle length ranged from 17.5 to 19.6 days. Standing estrus resembled that of other cervids, except that a low, moaning vocalization was given in response to contact, and activity (as measured by pedometers) did not increase. Mean gestation length was 183.38 ± SD 6.11 days (n = 21), and nearly all females conceived in the second and third years. The median interbirth interval was 362 days. The median birth date was April 8, and 80% of the births occurred over a 9.5-week period. Multiparous hinds gave birth an average of 20.5 days earlier in the season than primiparous hinds. There was no dimorphism in birth weight. The results are discussed in light of comparative data for other species.     

Modulation of 5-Hydroxytryptamine1A Receptor Density by Nonhydrolyzable GTP Analogues

Co-incubation of rat cortical membranes with 10(-4) M GTP results in a competitive inhibition of 5-hydroxytryptamine1A (5-HT1A) receptor binding sites labeled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin [( 3H]8-OH-DPAT). Preincubation of cortical membranes with 10(-4) M GTP does not significantly change either KD or Bmax values, indicating that the effect of GTP is reversible. By contrast, GTP gamma S and 5'-guanylylimidodiphosphate (GppNHp) are nonhydrolyzable analogues of GTP which lengthen the time course of guanine nucleotide activation of guanine nucleotide binding proteins (G proteins) and thereby alter G protein-receptor interactions. These nonhydrolyzable GTP analogues were used to characterize the effects of persistent alterations in G proteins on [3H]8-OH-DPAT binding to 5-HT1A receptors. Co-incubation of rat cortical membranes with either 10(-4) M GTP gamma S or GppNHp results in a decrease in both the affinity and apparent density of 5-HT1A binding sites. Co-incubation with the nonhydrolyzable nucleotides reduces the affinity of [3H]8-OH-DPAT binding by 65-70% and lowers the density of the binding site by 53-61%. Similarly, preincubation of membranes with a 10(-4) M concentration of either GTP gamma S or GppNHp significantly increases the KD value and reduces the Bmax value of [3H]8-OH-DPAT binding. These results indicate that GTP gamma S and GppNHp induce persistent changes in 5-HT1A receptor-G protein interactions that are reflected as a decrease in the density of binding sites labeled by [3H]8-OH-DPAT.