The band patterns of twelve D 98/AH-2 marker chromosomes and their use for identification of intraspecific cell hybrids

The chromosomal complement of the human cell line D98/AH-2 has been studied by quinacrine mustard and trypsin Giemsa banding techniques. The dispersion of chromosome counts has been shown to be due to non-random variation involving mainly a few particular chromosomes. — Twelve different marker chromosomes could be distinguished and the presumptive derivation of most of their chromosomal material from normal human chromosomes has been determined. Most cells in 6 different hybrid clones derived from fusion of D98/AH-2 cells with skin fibroblasts from a cystinotic patient contained a single copy of each marker chromosome.Supported by: united States Public Health Service Grant HD 04608, National Institute of General Medical Sciences Grant GM 17702 and American Heart Association Grant 71-981.Established Investigator of the American Heart Association.     

Altered secretion of kidney lysosomal enzymes in the mouse pigment mutants ruby-eye,ruby-eye-2-J,and maroon

Melanosomes and lysosomes share structural and biosynthetic properties. Three mouse pigment mutants, ruby-eye, ruby-eye-2-J, and maroon, have abnormally high concentrations of kidney lysosomal enzymes. Concentrations of kidney nonlysosomal enzymes and of liver and serum lysosomal enzymes are normal. By light microscopy the mutants have normal kidney lysosome morphology. It does not appear that the mutant genes cause an increased rate of production of lysosomes since the increased kidney -glucuronidase concentration is not accompanied by a corresponding increase in rate of synthesis. The common defect in all mutants is a decreased rate of secretion of lysosomal enzymes from kidney into urine. Eight mouse pigment mutants are now known which affect both melanosome and lysosome function. They should serve as useful models for the study of the biogenesis, structure, and processing of these and other subcellular organelles.This work was supported in part by United States Public Health Service Research Grant GM-19521 and by National Science Foundation Grant PCM77-24804. E. K. N. was supported in part by United States Public Health Service Grant GM07093-03. F. W. was a high school student in the summer program supported by National Science Foundation Grant SP177-26980.     

Rapid characterization of the binding property of HtrA2/Omi PDZ domain by validation screening of PDZ ligand library

HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mammalian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial homeostasis. PDZ domain is one of the most important protein-protein interaction modules and is involved in a variety of important cellular functions, such as signal transduction, degradation of proteins,and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding properties of HtrA2/Omi PDZ domain. Besides the reported Class Ⅱ PDZ motif, it also binds to Class Ⅰ and Class Ⅲ motifs, and exhibits restricted variability at P-3, which means that the P-3 residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi.Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.     

Distribution and tissue specificity of 4-aminobutyrate-2-oxoglutarate aminotransferase

A rapid and specific method for assaying 4-aminobutyrate-2-oxoglutarate aminotransferase was developed. The method was based on the selectivity of ion exchange resin and the speed of vacuum filtration. With this new method, the aminotransferase activity in various tissues has been determined as follows: brain, 10.2; spinal cord, 11.8; liver, 5.7; kidney, 4.6; heart, 0.5; lung, 0.4 nmol glutamate formed/min/mg. No activity could be detected in muscle preparations. When the aminotransferases were tested with the antibody, against the purified 4-aminobutyrate aminotransferase from brain, no difference could be detected among brain, spinal cord, and kidney preparations as judged from the results of immunodiffusion, inhibition of enzyme activity by antibody, and microcomplement fixation. It is concluded that 4-aminobutyrate aminotransferases from various tissues of the mouse are probably identical or closely related.This work was supported in part by Grant No. NS 13224 and P01 NS 12116 from the National Institutes of Health, U.S.A. and Grant from Huntington's Chorea Foundation.All correspondence should be addressed to Dr. Wu.     

Mutagenicity of 4-nitroquinoline N-oxide after its complexation with copper(II) 2-chlorophenoxyacetate

Using the Ames test withSalmonella typhimurium strains TA97, TA100 and TA102 it was found that the mutagenicity of 4-nitroquinoline N-oxide was much lower than that of the tetrakis[μ-2-chlorophenoxyacetato(1−)-O,O′]bis(4-nitroquinoline N-oxide-O)dicopper(II) complex. This work was supported by theMinistry of Education of the Slovak Republic in Grant no. 95/5195/416.     

Immunological relationships and post-translational modifications of human salivary amylase (Amy1) and pancreatic amylase (Amy2) isozymes

Electrophoretic phenotypes of human salivary amylase (Amy₁) and pancreatic amylase (Amy₂) consist of complex isozyme patterns which may result from post-translational modifications of the primary products of the amylase loci. Biochemical separation of the two molecular weight families of salivary amylase and development of a new electrophoretic system have allowed the identification of complete isozyme patterns corresponding to variant alleles in Amy ₁ and Amy₂ heterozygotes. Further, immunological studies show no nonidentities among salivary isozymes and among pancreatic isozymes, which is to be expected if each series is derived from a single gene product. Both results support the hypothesis that the primary products of the amylase loci undergo post-translational modifications. Salivary and pancreatic amylase appear to be immunologically identical.This investigation was supported in part by PHS Research Grant GM-19178.Supported by PHS Training Grant DE 119.Supported by PHS Training Grant GM 1056.     

A sex-limited serum protein variant in the mouse: Inheritance and association with the H-2 region

An alloantiserum produced in the mouse has been used to detect an antigen which is present only in male serum from certain inbred strains of mice, e.g., DBA/2J, A/J, and BALB/c. Genetic tests reveal that the presence of this antigen is controlled by a dominant autosomal gene which is expressed only in males of the proper genotype. Test crosses and analysis of congenic resistant strains indicate close linkage between the sex-limited protein (Slp) and the histocompatibility-2 (H-2) region of linkage group IX. Analysis of seven intra-H-2 recombinant strains is consistent with the placement of the genetic determinant for Slp within the H-2 region in the same position as the Ss (serum substance) determinant. Immunological evidence suggests that the Slp antigenic sites reflect structural variation in the Ss component of mouse serum.Supported by U.S.P.H.S. Research Grant GM-15419, U.S.P.H.S. Career Development Award K3-HE-24, 980 (D.C.S.), and U.S.P.H.S. Training Grant 2T01-GM-00071 (H.C.P.).     

Land grant and sea grant: Implications and limitations of the model

Abstract

The National Sea Grant College Program was created by Congress in 1966 to assist the development of marine resources through science and technology as well as through education, research, and advisory services. The structure of the program resulted from the explicit effort of early Sea Grant supporters to forge the system as an oceans equivalent of Land Grant colleges. This discussion provides a brief history of the creation of Sea Grant based on the Land Grant analogy; it compares the size, scope, and major concerns of the two systems, including criticisms of the Land Grant program and their relevance to Sea Grant; and it suggests way in which Sea Grant might avoid some of the conditions which led to these criticisms of the Land Grant network.     

Screening and breeding of high taxol producing fungi by genome shuffling

To apply the fundamental principles of genome shuffling in breeding of taxol-producing fungi, Nodulisporium sylviform was used as starting strain in this work. The procedures of protoplast fusion and genome shuffling were studied. Three hereditarily stable strains with high taxol production were obtained by four cycles of genome shuffling. The qualitative and quantitative analysis of taxol produced was confirmed using thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and LC-MS. A high taxol producing fungus, Nodulisporium sylviform F4-26, was obtained, which produced 516.37 μg/L taxol. This value is 64.41% higher than that of the starting strain NCEU-1 and 31.52%―44.72% higher than that of the parent strains.     

Mathematical analysis of optimal multichannel filtering for nerve signals

This paper extends recent analyses (Roberts and Hartline, 1975; Ouztöreli and Stein, 1977) of optimal linear filters for separating neural signals from more than one electrode site. Roberts and Hartline's result, using a matched filter criterion, represents one of a class of optimal filters with different, but symmetrical, output waveforms derived by Ouztöreli and Stein (1977). Another narrow bandwidth filter of this class will give the optimal results according to an energy criterion, but may be less useful in practical situations.This work was partially supported by the Medical Research Council of Canada (Grant MRC-MT3307), the Technical Research Council of Denmark (Grant 516-6703 E406) and the National Research Council of Canada (Grant NRC A-4345)     

Some properties of RAT liver mitochondrial RNA polymerase

Summary A rapid method suitable for purifying large amounts of mitochondria from rat liver using isopycnic zonal centrifugation is described. The RNA polymerase isolated from the purified mitochrondria was found associated with one peak when resolved by DEAE Sephadex chromatography. The enzyme was next fractionated on a phosphocellulose column followed by glycerol gradient centrifugation. A 600-fold purification was achieved when the enzyme was finally filtered through agarose gel. This final enzyme fraction consisted of one polypeptide chain as shown by polyacrylamide gel electrophoresis profiles. The enzyme has a greater preference for poly [d(A-T)] templates than for rat liver mitochondrial DNA. Inhibition of the enzyme activity required high concentrations of the inhibitors. The resistance of the enzyme to-amanitin indicated that there was no contamination from nuclear RNA polymerase II. The conclusion is drawn that the mitochondrial RNA polymerase activity is associated with a single polypeptide.This work was supported in part by Institutional Grant IN-4ON to the University of Michigan from the American Cancer Society, Grant 360133 from the Rackham School of Graduate Studies and Grant 121010 from the Institute of Science and Technology of the University of Michigan, Grant 340879 from the Michigan Heart Association, and Grants AMO5474 and RRO5641 from the National Institutes of Health.     

Murine liver arylsulfatase B processing influenced by region on chromosome 17

SM/J liver arylsulfatase B has a more rapid electrophoretic mobility and occurs as a series of more acidic isozymes following electrofocusing in narrow pH gradients than the liver enzyme from C57BL/6J mice. The SM/J and C57BL/6J electrofocusing patterns were both converted to a single isozyme with similar isoelectric points by pretreatment with neuraminidase, suggesting that the SM/J and C57BL/6J isozymes differed with respect to their sialic acid content. Arylsulfatase B electrofocusing and thermostability phenotypes segregated independently among progeny of SM/J×C57BL/6J crosses, suggesting that the electrofocusing phenotypes were not determined by different alleles at As-1, the putative structural locus for arylsulfatase B. Comparison of the joint segregation of hepatic acid phosphatase electrophoretic patterns and liver arylsulfatase B electrofocusing profiles revealed that the electrofocusing profiles may be determined by a region on chromosome 17 near or identical to Apl. Kidney, brain, and spleen arylsulfatase B electrofocusing patterns did not appear to differ between SM/J and C57BL/6J mice.This research was supported in part by Biomedical Sciences Research Support Grant RR-07030, by NIGMS Grant 1-RO1GM27707-01, and by Grant 1–570 from the National Foundation/March of Dimes.     

Differing effects of isoleucine deficiency on the toxicity of MNNG for 10T1/2 and CHO cells

Summary Isoleucine deficiency sensitizes C3H 10T1/2 cells to the cytotoxic effects of MNNG. Synchrony of proliferation is not required for this effect since it occurs prior to full growth arrest and subsequent establishment of synchronous proliferation after refeeding in complete medium. Furthermore, confluence arrest of proliferation of 10T1/2 cells does not affect their cytotoxic response to MNNG, although they proliferate synchronously after replating at low density. In contrast, the toxicity of MNNG for CHO cells is not altered by isoleucine deficiency, even though these cells are readily synchronized by refeeding in complete medium after transient isoleucine deficiency. This research was supported by Grant BC-142 from the American Cancer Society and Grants CA-16086 and CA-17973 from the National Cancer Institute.     

Gonadotroph-rich cell lines derived from pituitary clonal cells (2A8) grafted under the kidney capsule

Summary Gonadotroph-rich cell lines were established from multipotential pituitary clonal cells (2A8) which were implanted under kidney capsule of hypophysectomized female rats. These cell lines secrete gonadotrophins (FSH and LH) continuously over two months after establishment; LHRH stimulated the secretion of hormones into the culture medium. Many of the cells reacted immunohistochemically to antiserum to FSH or LH, while a small number reacted to antiserum to prolactin or TSH. They did not contain normal secretory granules such as those of gonadotrophs in vivo.Supported by USPHS Grant HD 11826 and NIH Grant P30 HD 10202. The authors wish to thank James Chambers (Immunocytochemistry), and Pat Koym and John Rhode (Radioimmunoassay) for their excellent technical assistance. We also express our thanks to NIAMDD for providing pituitary hormones     

Discovery and identification of Serum Amyloid A pro-tein elevated in lung cancer serum

Two hundred and eighteen serum samples from 175 lung cancer patients and 43 healthy individuals were analyzed by using Surface Enhaced Laser Desorption/Ionization Time of Flight Mass Spectrome-try (SELDI-TOF-MS). The data analyzed by both Biomarker Wizard™ and Biomarker Patterns™ software showed that a protein peak with the molecular weight of 11.6 kDa significantly increased in lung cancer. Meanwhile,the level of this biomarker was progressively increased with the clinical stages of lung cancer. The candidate biomarker was then obtained from tricine one-dimensional sodium dodecyl sul-fate-polyacrylamide gel electrophoresis by matching the molecular weight with peaks on WCX2 chips and was identified as Serum Amyloid A protein (SAA) by MALDI/MS-MS and database searching. It was further validated in the same serum samples by immunoprecipitation with commercial SAA antibody. To confirm the SAA differential expression in lung cancer patients, the same set of serum samples was measured by ELISA assay. The result showed that at the cutoff point 0.446（OD value）on the Receiver Operating Characteristic (ROC) curve, SAA could better discriminate lung cancer from healthy indi-viduals with sensitivity of 84.1% and specificity of 80%. These findings demonstrated that SAA could be characterized as a biomarker related to pathological stages of lung cancer.     

Growth of preadipocyte cell lines and cell strains from rodents in serum-free hormone-supplemented medium

Summary Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin, transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described. This work was supported by the “Centre National de la Recherche Scientifique” (Grant 1208-Biochimie du Développement and Grant 4162-Endocrinologie), by the “Ministère de la Recherce et de la Technologie” (Grant 81-L-1322), by the “Fondation pour la Recherche Médicale,” by NATO (Grant 1704), and by the “Institut National de la Santé et de la Recherche Médicale” (Grant 827006).     

Interspecies transmission and host restriction of avian H5N1 influenza virus

Long-term endemicity of avian H5N1 influenza virus in poultry and continuous sporadic human infections in several countries has raised the concern of another potential pandemic influenza. Suspicion of the avian origin of the previous pandemics results in the close investigation of the mechanism of interspecies transmission. Entry and fusion is the first step for the H5N1 influenza virus to get into the host cells affecting the host ranges. Therefore receptor usage study has been a major focus for the last few years. We now know the difference of the sialic acid structures and distributions in different species, even in the different parts of the same host. Many host factors interacting with the influenza virus component proteins have been identified and their role in the host range expansion and interspecies transmission is under detailed scrutiny. Here we review current progress in the receptor usage and host factors. Supported by the National Basic Research Program of China (Grant Nos. 2005CB523001, 2005CB523002), National Key Technologies Research & Development Program (Grant 2006BAD06A01/2006BAD06A04); US National Institutes of Health (NIH) (Grant 3 U19 AI051915-05S1), the National Natural Science Foundation of China (Grant 30599434). GAO FG is a distinguished young investigator of the NSFC (Grant No. 30525010).     

Repair effect of diabetic ulcers with recombinant human epidermal growth factor loaded by sustained-release microspheres

In this study the w/o/w extraction–evaporation technique was adopted to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres loading recombinant human epidermal growth factor (rhEGF). The micro-spheres were characterized for morphology by transmission electron microscopy (TEM) and particle size distribution. The release performances, the proliferation effects and therapeutic effects of rhEGF-loaded PLGA microspheres were all studied. The results showed that these spherical micro-spheres had a narrow size distribution and a high drug encapsulation efficiency (85.6%). RhEGF-loaded microspheres enhanced the growth rate of fibroblasts and wound healing more efficiently than pure rhEGF. The number of the proliferating cell nuclear antigen (PCNA) in the epidermis layer with the mi-crosphere treatment was significantly larger than those of the control groups. Overall locally sustained delivery of rhEGF from biodegradable PLGA microspheres may serve as a novel therapeutic strategy for diabetic ulcer repair.     

Further analysis of genomic fragments fromSchizosaccharomyces pombe restoring glucose transport in a transport-deficient mutant

This work was supported by the NATO Linkage Grant HTECH.LG 930686 and by theDeutsche Forschungsgemeinschaft, grant no. Ho 555/16-1.