Amyloid fibrils derived from V-region together with C-region fragments from a lambda II-immunoglobulin light chain (HAR)

Amyloid fibril proteins were isolated from the spleen of a patient with IgD(lambda)-plasmocytoma by extraction and gel filtration in 5M guanidine hydrochloride. The molecular mass of the predominant polypeptide chain was approximately 5000 Da. Its complete amino-acid sequence was elucidated by stepwise automated degradation of the carboxymethylated polypeptide chain and by structural studies of tryptic and thermolysinolytic cleavage products. The length of the polypeptide chain was 58 to 59 residues and it was homologous to the amino acids in positions 8 through 65 of the variable part of an lambda-type immunoglobulin light chain, which was most closely related to the lambda II subgroup. The N-terminal sequence of this amyloid fibril protein proved to be heterogeneous, indicating cleavage after the amino acids in positions 7 and 8. Peptides from the constant part of the lambda-chain were unexpectedly found in the tryptic digest of the denatured amyloid protein HAR. One polypeptide derived from the constant region was separated from the main component by high performance liquid chromatography. Its amino-acid sequence commenced at position 111 and could be traced in 41 steps. In this case, at least two constant region fragments were shown to be constituents of the amyloid fibril protein. The association of fragments from the variable as well as the constant region is discussed with respect to amyloid formation.     

Identity of a cytosolic neutral cholesterol esterase in rat liver with the bile salt stimulated cholesterol esterase in pancreas

A neutral cholesterol esterase has been purified to homogeneity from the cytosolic fraction of rat liver. The 105,000 x g supernatant fraction of rat liver was applied to a DEAE-cellulose column to isolate a partially purified fraction of hepatic cholesterol esterase. Immunoblot analysis of the partially purified liver fraction with the anti-porcine pancreatic cholesterol esterase IgG demonstrated a single band with a molecular weight of 67,000. The hepatic protein was then isolated by immunoaffinity chromatography technique using a column constructed with antibodies prepared against the pancreatic cholesterol esterase. Characterization of the hepatic cholesterol esterase revealed that the hepatic enzyme shared antigenic epitopes with the pancreatic cholesterol esterase and was similarly activated by addition of bile salt such as taurocholate. Moreover, amino-terminal sequencing analysis of the hepatic cholesterol esterase showed an identical sequence with the pancreatic enzyme. Taken together, these results showed that the cholesterol esterases in the liver and the pancreas are very similar and possibly identical proteins.     

Amyloid kidney stones of uremic patients consist of beta 2-microglobulin fragments

Urinary stones with amyloid structure, obtained from uremic patients, were analyzed according to molecular weight, amino acid sequence, and antigenic content. A major protein of approximately 7 kD, designated AB protein, was isolated by size exclusion using HPLC in 60% formic acid. AB protein reacted in immunodiffusion only with an antiserum to beta 2-microglobulin, with beta 2m spurring over AB protein. N-terminal amino acid sequence analysis defined two fragments homologous to beta 2m. One fragment commenced with Ile at position 7 and the other with Ser at position 20, with a cleavage point subsequent to a lysyl residue in both. It is concluded that beta 2m is a precursor of urinary amyloid stones and intratubular concretions of patients with preterminal and terminal renal failure; limited proteolysis is involved in AB amyloid generation.     

Preferential expression of the maternally inherited X-linked phosphoglycerate kinase allele in human erythrocytes

Summary The stability of allelic gene expression of X-linked phosphoglycerate kinase was studied in seven carriers of a rare genetic variant named PGK München. The enzymatic activities in erythrocytes of five heterozygous females and three hemizygous males were determined repeatedly over a period of 10 years (1975–1984) and shown to remain constant. As the phosphoglycerate kinase activity is lower in cells expressing the PGK München allele, the ratio of the two cell types in all heterozygous females of the PGK München kindred could be calculated from the PGK activity and from the known allozyme activities in erythrocytes of homozygous wild type or hemizygous PGK München carriers. Since the maternal or paternal origin of both alleles is known from the pedigree, the quantitative expression of the maternally derived allozyme in heterozygous women could be determined. In heterozygous carriers the cell pool expressing the maternally inherited allele was significantly increased, independently, of the PGK allele linked to the maternal X chromosome (P<0.001). Our data show that inactivation of one of the two X chromosomes in human female erythropoietic stem cell precursors may be non-random, at least in the kindred and cell populations described here. The results are discussed in the context of random X chromosome inactivation (Lyon hypothesis).Dedicated to J.S., the senior of the family studied, on the occasion of her 80th birthday     

Uptake of U-14C-glucose by Streptococcus mutans in the presence of saccharin

The uptake of U-14C-glucose by resting cells of Streptococcus mutans OMZ-176 was studied in the presence of the artificial sweetener saccharin as well as sodium chloride. Glucose grown cells were resuspended in phosphate buffer (0.05 M, pH 7.8), and the uptake of U-14C-glucose was observed for 150 min in time intervals of 30 min, in the presence of 0.02 and 2.00 mg/ml of sodium saccharin as well as sodium chloride. As compared to the control and the sodium chloride treatments, sodium saccharin at the highest concentration range more than doubled the accumulation of radioactive labelled carbon within the cells.     

Biological effects of ultrasound

When ultrasound is absorbed by tissue, heat is produced which may be destructive or, if controlled, can be therapeutic. In addition, ultrasound produces biological effects which cannot be explained on the basis of heating alone. Under certain exposure conditions production of ultrasonic lesions in brain tissue can be explained on a purely thermal basis. Examples of nonthermal effects of ultrasound include changes in growth, mitotic index and the production of chromosomal aberrations in plant roots.     

An optimized live bacterial delivery vehicle safely and efficaciously delivers bacterially transcribed therapeutic nucleic acids

There is an unmet need for delivery platforms that realize the full potential of next-generation nucleic acid therapeutics. The in vivo usefulness of current delivery systems is limited by numerous weaknesses, including poor targeting specificity, inefficient access to target cell cytoplasm, immune activation, off-target effects, small therapeutic windows, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we characterize the safety and efficacy of a delivery platform comprising engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli SVC1) for intracellular cargo delivery. SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to allow escape of their cargo from the phagosome, and to have minimal immunogenicity. We describe SVC1's ability to deliver short hairpin RNA (shRNA), localized SVC1 administration to various tissues, and its minimal immunogenicity. To validate the therapeutic potential of SVC1, we used it to deliver influenza-targeting antiviral shRNAs to respiratory tissues in vivo. These data are the first to establish the safety and efficacy of this bacteria-based delivery platform for use in multiple tissue types and as an antiviral in the mammalian respiratory tract. We expect that this optimized delivery platform will enable a variety of advanced therapeutic approaches.     

Persistent BK papovavirus infection of transformed human fetal brain cells. I. Episomal viral DNA in cloned lines deficient in T-antigen expression.

After infection of permissive human fetal brain cells by BK human papovavirus (BKV), the vast majority of the cells were killed by the virus, but rare survivors were recovered after frequent medium changes. These surviving cells grew and formed visible colonies after 5 to 6 weeks and were thereafter established as permanent cell lines. These cells, designated as BK-HFB cells, were persistently infected and shed BKV. Morphologically, they were small polygonal cells and had transformed growth properties. Their plating efficiency on solid substrates or in semisolid medium was high, and they were tumorigenic in athymic nude mice. Cloning experiments in medium containing BKV antiserum revealed that BKV did not persist in the cultures in a simple carrier state. All cloned cell lines were initially T-antigen negative and virus-free. However, every clone began to release BKV and again became persistently infected within 3 weeks after removal of BKV antiserum. After rigorous antibody treatment, four of seven clones still released virus spontaneously upon removal of antiserum; three clones have remained virus-free and are apparently cured. Although these cloned cell lines are T- and V-antigen negative when grown in antiserum-containing medium, they retain "free" or episomal BKV genomes; integrated viral DNA was not detected in any of the clones. These free genomes are indistinguishable from prototype BKV DNA and are found in much larger amounts in virus-shedding cell lines.     

Non-random X-chromosome expression early in mouse development

We have used a sensitive electrophoretic technique for estimating the activity, or ratio, of two allozymes of the X-chromosome-linked enzyme phosphoglycerate kinase (PGK-1), in order to investigate the randomness of X-chromosome expression in the derivatives of the three primary cell lineages of the early mouse conceptus. The maternally derived Pgk-1 allele is preferentially expressed in the derivatives of the primitive endoderm and trophectoderm lineages at 6 1/2 days post coitum in Pgk-1^a/Pgk-1^b heterozygous conceptuses, and in the one informative 5 1/2-day heterozygous conceptus analysed. This evidence for preferential expression of the maternally derived X chromosome (X^m), so soon after the time of X-chromosome inactivation, favors the possibility that the preferential expression of X^m is a consequence of primary non-random X-chromosome inactivation, rather than a secondary selection phenomenon. The majority of embryos analysed at 4 1/2 and 5 1/2 days pc produced only a single PGK-1 band, corresponding to the allozyme produced by the Pgk-1 allele on X^m, although 50% of these embryos should have been heterozygous females. Possible explanations are discussed.