Immunochemical characterisation of parathyroid hormone-related protein from tumor and non-tumor cells

The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtraton chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1–86 and 1–34) and bioactivity (1–34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19–22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10–16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1–34 or 37–67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24–25 kDa, consistently slighty larger than recombinant PTHRP(1–141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments.     

Tandem duplications of the lac region of the Escherichia coli chromosome

Tandem duplications are caused by unequal crossing over between homologous sequences. Duplications in the lac region of the Escherichia coli chromosome were isolated by two methods. Duplication frequency using a method involving P1 transduction increased from 0.4% with no UV to 2.0% following UV irradiation at 35 J/m2. Duplication frequency in lac using a second generalizable method that does not involve P1 transduction increased from 0.7 to 12% at 35 J/m2 UV. In both cases the duplication frequency began to plateau at UV doses of 12 J/m2 and 24 J/m2. According to segregation analysis of sixteen duplications there may be at least seven classes of duplications isolated by each method. Pulsed-field gel electrophoresis was used to measure the duplications isolated without P1 transduction. The minimum size of the duplications ranged from 30 to 320 kb but could be much larger.     

Biosynthesis and processing of a variant surface glycoprotein from Trypanosoma brucei brucei

Iowa trypanosome antigen type (IaTat) 1.2 variant surface glycoprotein (VSG) is synthesized in vitro as a Mr 54,000 preprotein that contains a 31-amino-acid signal peptide. Translation of mRNA in the presence of either dog pancreas or trypanosome microsomal membranes results in cotranslational cleavage of the signal peptide and addition of core oligosaccharide side chains to the protein. Analysis of these products on sodium dodecyl sulfate (SDS)-gels indicates that removal of the signal peptide (Mr 3200) is almost exactly compensated for by an increase in molecular weight due to carbohydrate addition. Pulse-chase experiments in cultures of isolated trypanosomes indicate that two IaTat 1.2 VSG species (Mr 58,000 and 60,000) occur in vivo. When glycosylation is inhibited by incubation of trypanosomes with tunicamycin, a single Mr 50,000 polypeptide is immunoprecipitated. The multiple protein species, therefore, arise from heterogeneity in carbohydrate side chains whose synthesis and transfer to the protein are tunicamycin sensitive. Sequence analysis verified that both species of VSG contain identical amino-terminal sequences. Further post-translational processing of IaTat 1.2 VSG includes addition of phosphate and myristic acid residues, both of which have been shown to be located in the immunologically cross-reactive determinant at the carboxyl terminus of the protein. Exposure of this attachment site requires post-translational proteolytic removal of a 17-amino-acid peptide from the carboxyl terminus of an intermediate form of VSG.     

Evidence for posttranslational O-glycosylation of fetuin

Fetuin, a major glycoprotein in the serum of fetal calves that contains three N-linked and three O-linked carbohydrate side chains, was found to be synthesized in the liver with an 18 amino acid signal peptide, Met-X-X-X-X-Leu-Leu-X-Cys-Leu-Ala-X-Leu-X-X-Cys-X-X, and to undergo cotranslational N-glycosylation. In order to examine O-glycosylation, fetuin peptidyl-tRNA was purified from liver and analyzed for O-linked carbohydrate by quantitating the released [3H]GalNAcitol produced after beta-elimination in the presence of NaB3H4. Within the limits of the assay, less than 1.3% of the O-linked chains had been initiated. Additionally, rough microsomes were used to program a cell-free protein synthesis system. A radiolabeled fetuin intermediate was isolated by immunoprecipitation and shown to contain N-linked carbohydrate by binding to concanavalin A and by susceptibility to cleavage by endoglycosidase H. However, this fetuin intermediate was not detectably bound (less than 1%) by GalNAc-specific lectins, which were shown to bind asialoagalactofetuin. These results suggest that O-glycosylation of fetuin is a posttranslational event.     

Differences in ovarian activity between booroola X merino ewes which were homozygous, heterozygous and non-carriers of a major gene influencing their ovulation rate

Differences in the function and composition of individual ovarian follicles were noted in Booroola Merino ewes which had previously been segregated on at least one ovulation rate record of greater than 5 (FF ewes, N = 15), 3-4 (F+ ewes, N = 18) or less than 3 (++ ewes, N = 18). Follicles in FF and F+ ewes produced oestradiol and reached maturity at a smaller diameter than in ++ ewes. In FF (N = 3), F+ (N = 3) and ++ (N = 3) ewes, the respective mean +/- s.e.m. diameters for the presumptive preovulatory follicles were 3.4 +/- 0.3, 4.1 +/- 0.2 and 6.8 +/- 0.3 mm and in each of these follicles the respective mean +/- s.e.m. numbers of granulosa cells (X 10(6)) were 1.8 +/- 0.3, 2.2 +/- 0.3 and 6.6 +/- 0.3. During a cloprostenol-induced follicular phase, the oestradiol secretion rates from FF ewes with 4.8 +/- 0.4 'oestrogenic' follicles, F+ ewes with 3.2 +/- 0.2 'oestrogenic' follicles and ++ ewes with 1.5 +/- 0.02 'oestrogenic' follicles were not significantly different from one another. Moreover, the mean total numbers of granulosa cells from the 'oestrogenic' follicles from each genotype were identical, namely 5.4 X 10(6) cells. Irrespective of genotype the mean weight of each corpus luteum was inversely correlated to the ovulation rate (R = 0.91, P less than 0.001). Collectively, these findings support the notion that the maturation of greater than or equal to 5 follicles in FF ewes and 3-4 follicles in F+ ewes may each be necessary to provide a follicular-cell mass capable of producing the same quantity of oestradiol as that from 1-2 preovulatory follicles in ++ ewes.     

Effects of increased salinity on the diatom assemblage in Fonda Lake,Michigan

A salt storage facility has been located adjacent to Fonda Lake since 1953. In February 1981 a core was taken from the profundal sediments of the lake and analyzed to determine the effects of salt perturbation on the diatom community over a 32-year period. Diatom assemblages from different levels were compared using multivariate techniques including cluster analysis and principal component analysis. Shifts in diatom composition related to salinification were revealed most clearly by subdominant taxa. Five distinct groups of diatom taxa were found to correspond with 5 depth intervals. The diatom component of the lake up to 1960 included two groups of taxa which were alkaliphilous and chloride indifferent. A reduction in species diversity beginning in 1960 may indicate a salt effect. By 1968, when diversity reached a minimum, a variety of halophilic taxa (including Diatoma tenue, Navicula gregaria and Synedra fasciculata) attained their highest relative abundances. At the top of the core, diversity increased slightly and some halophilic taxa decreased in relative abundance, which suggests a possible decrease in salt loading to the lake.     

The subcellular distribution of a membrane-bound calmodulin-stimulated protein kinase

Incubation of subcellular fractions isolated from rat cerebral cortex with [-³²P]ATP results in the phosphorylation of a number of proteins including two with apparent molecular weights of approximately 50,000 and 60,000 daltons. These phosphoproteins were shown to be the autophosphorylated subunits of a calmodulin-stimulated protein kinase by a number of physicochemical criteria, including their mobility on non-equilibrium pH gradient electrophoresis, their phosphopeptide profiles and phosphorylation characteristics. When a crude membrane fraction obtained following osmotic lysis of a P2 fraction was labeled and subsequently fractionated on sucrose density gradients, approximately 80% of the autophosphorylated kinase was associated with fractions enriched in synaptic plasma membranes. Other substrates of calmodulin kinase(s) were similarly distributed. Detergent extraction of synaptic plasma membranes to produce synaptic junctions and post-synaptic densities indicated that the majority of the autophosphorylated kinase was solubilized, apparently as a holoenzyme. The major post synaptic density protein (mPSDp) was not readily extracted by detergents and was largely unlabeled under the conditions used for phosphorylation, and yet this protein is structurally closely related to the kinase subunit. It is possible that this lack of labeling is due to the mPSDp being attached to the PSD in a different way or being present there in a different isoenzymic form from that of the readily autophosphorylated enzyme subunit. Thus, the data suggest that, in vitro at least, a number of pools of calmodulin kinase exist in neuronal membranes.A preliminary account of part of this work has been published (1).     

Biosynthesis and processing of rat alpha 1-antitrypsin

Various biosynthetic forms of rat alpha 1-antitrypsin (alpha 1AT) have been isolated by immunoprecipitation of in vitro and in vivo synthesized products. Rat alpha 1AT is synthesized in a rabbit reticulocyte system as a 45,000-Da preprotein with a 23-amino acid signal sequence. The majority of the amino acids in the signal sequence have been identified and resemble the signal peptides of other secretory proteins with respect to the abundance and positions of hydrophobic amino acids. Evidence from the translation of rat liver RNA in the presence of dog pancreas microsomes, from the translation of rat liver polysomes, and from tunicamycin-treated rat hepatocytes established that cleavage of the signal peptide of pre-alpha 1AT results in the formation of a 42,000-Da protein, the polypeptide backbone of mature alpha 1AT. A 50,000-Da glycoprotein is immunoprecipitated from translations programmed with rat liver microsomes or with rat liver mRNA and dog pancreas microsomes. Cotranslational glycosylation of alpha 1AT appears to occur in a stepwise fashion since three glycosylated forms of alpha 1AT (approximately 45,000, 47,000, and 50,000 Da) can be detected in polysome translations. These proteins are susceptible to cleavage by endo-beta-N-acetylglucosaminidase H and are digested to the same product, indicating that they have identical polypeptide chains. Two intracellular forms of alpha 1AT were detected in cultured rat hepatocytes, a 50,000- and a 52,000-Da protein; only the larger protein was immunoprecipitated from the medium of these cells. Digestion with endo-beta-N-acetylglucosaminidase H indicated that the 50,000-Da protein is a core glycosylated processing intermediate, whereas the 52,000-Da protein, which comigrated with purified serum alpha 1AT, appears to contain complex carbohydrate sidechains. When glycosylation was inhibited by incubation of hepatocytes with tunicamycin, a nonglycosylated 42,000-Da protein was immunoprecipitated from the cells and the culture medium, indicating that glycosylation of alpha 1AT is not essential for its secretion.     

Continuous Intragastric Milk Feeds in Infants of Low Birth Weight

In a feeding trial 66 infants of low birth weight received continuous intragastric milk feeds from the fourth hour of life, starting with 60 ml/kg/24 hr and reaching a maximum of 300 ml/kg/24 hr on the ninth day. Each infant received only full-strength milk, which was either expressed human breast milk or SMA-S26 (a proprietary low-protein adapted cows'' milk) or half-cream Regal milk (partly-skimmed evaporated cows'' milk). For various reasons 10 babies had to be withdrawn, and the final assessment was made on the 56 who completed the trial successfully.Persistent vomiting was a problem in only four infants. In two of them the trial was continued after gastric lavage and in the other two vomiting stopped when the volume was reduced. Despite a careful search no evidence was found of aspiration of feeds in any infant. Continuous intragastric milk infusion was shown to be a safe method of feeding infants of low birth weight and SMA-S26 was almost as well tolerated as human milk. Because of the high-protein content of half-cream cows'' milk preparations and the resultant high plasma amino-acid levels when they are given in these large volumes they should be avoided for this type of feeding although they produce better weight gains in the first week of life.