Errors in Conventional and Input-Output—based Life—Cycle Inventories

Conventional process-analysis-type techniques for compiling life-cycle inventories suffer from a truncation error, which is caused by the omission of resource requirements or pollutant releases of higher-order upstream stages of the production process. The magnitude of this truncation error varies with the type of product or process considered, but can be on the order of 50%. One way to avoid such significant errors is to incorporate input-output analysis into the assessment framework, resulting in a hybrid life-cycle inventory method. Using Monte-Carlo simulations, it can be shown that uncertainties of input-output– based life-cycle assessments are often lower than truncation errors in even extensive, third-order process analyses.     

Human parathyroid hormone as a secretory peptide in milk of transgenic mice

In a transgenic mouse model we have targeted the expression of recombinant human parathyroid hormone (hPTH) to the mammary gland yielding hPTH as a secretory, soluble peptide in milk. A 2.5 kb upstream regulatory sequence of the murine whey acidic protein (WAP) directed the expression of the hPTH cDNA in a fusion gene construct (WAPPTHSV2) containing the SV40 small t-antigen intron and polyadenylation site in the 3′ end. Established lines of transgenic mice secreted hPTH to milk in concentrations up to 415 ng/ml. Recombinant hPTH recovered from the milk was purified by HPLC and shown to be identical to hPTH standard as analyzed by SDS-PAGE followed by immunoblotting. Expression of the WAPPTHSV2 was limited to the mammary gland as analyzed by polymerase chain reaction (PCR) and Southern blot of reversed transcribed mRNA from different tissues. hPTH is an important bone anabolic hormone and may be a potentially important pharmaceutical for treatment of demineralization disorders such as osteoporosis. We present the transgenic animal as a possible production system for hPTH. © 1995 Wiley-Liss, Inc.     

Transamination of 3-phenylpyruvate in pancreatic B-cell mitochondria

High aminotransferase activities catalyzing the reaction between L-glutamate and the aromatic ketomonocarboxylic acid, 3-phenylpyruvate, were observed in the mitochondria from pancreatic B-cells. At very low concentrations of 3-phenylpyruvate, L-glutamine was an effective amino group donor. The aminotransferase activities for the aliphatic ketomonocarboxylic acids, pyruvate and 2-ketoisovalerate, were lower in B-cell mitochondria. High rates of transamination of 2-ketoisocaproate with L-glutamine were observed and may be an important prerequisite for the insulin secretory potency of this 2-keto acid. Since B-cell mitochondria are well supplied with L-glutamine and L-glutamate, 3-phenylpyruvate-induced 2-ketoglutarate production may explain the insulin secretory potency of 3-phenylpyruvate which is not a fuel for pancreatic islet cells.     

Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus beta-galactosidase sequences

The lacZ gene from Streptococcus thermophilus A054, a commercial yogurt strain, was cloned on a 7.2 kb PstI fragment in Escherichia coli and compared with the previously cloned lacZ gene from S. thermophilus ATCC 19258. Using the dideoxy chain termination method, the DNA sequences of both lacZ structural genes were determined and found to be 3071 bp in length. When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons. Only three bases separated the termination codon for the lacS gene from the initiation codon for lacZ, suggesting that the lactose utilization genes are organized as an operon. The amino acid sequence of the beta-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da. Comparison of the S. thermophilus amino acid sequences with those from Lactobacillus bulgaricus, E. coli and Klebsiella pneumoniae showed 48, 35 and 32.5% identity respectively. Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid sequence were identified when the beta-galactosidase proteins from S. thermophilus, E. coli and L. bulgaricus were compared.     

Regulation of transmembrane ion transport by reaction products of phospholipase A2. II. Effects of arachidonic acid and other fatty acids on mitochondrial Ca2+ transport

The effects of arachidonic acid and other fatty acids on mitochondrial Ca2+ transport were studied. Cis-unsaturated fatty acids generally strongly inhibited mitochondrial Ca2+ uptake, induced a net Ca2+ efflux, and thereby increased the extramitochondrial Ca2+ concentration, whereas trans-unsaturated fatty acids were ineffective. Saturated fatty acids exhibited slight activity at chain lengths from C(10) to C(14) only. The structure-activity relationship and the inability of some of the effective fatty acids such as palmitoleic and myristoleic acid to be metabolized to eicosanoids suggest that Ca2+ release was induced by the fatty acids themselves and resulted from changes in the mitochondrial membrane bilayer structure. There was a correlation between Ca2+-releasing potency and reduction of mitochondrial membrane potential, which is the main driving force for mitochondrial Ca2+ uptake. There were, however, considerable differences compared with the effects of lysophospholipids on the membrane potential. The mechanism of action of fatty acids may be that of a fluidizing effect on the hydrophobic core of the membrane, thereby modulating the activity of integral membrane proteins of the respiratory chain.     

Structure-function studies on gastrointestinal hormones: I. Synthesis of secretin analogs and their biological and immunological properties

Syntheses by conventional procedures of the three analogs corresponding to the porcine secretin sequence crossed at position 6 by the N-terminal hexapeptide sequences of VIP, GIP, and glucagon are described, viz., Ala⁴,Val⁵-, Tyr¹,Ala²,Glu³-, and Gln³-secretin (VIP-SN, GIP-SN, and GLU-SN). The analog Phe¹,Phe²,Trp³,Lys⁴-secretin (SOMA-SN), designed on the basis of the surprising homology of the sequence portions 10–13 of somatostatin and 5–8 of secretin, was also prepared. Finally, the synthesis of N^α-3-(4-hydroxyphenyl)propionyl-β-alanyl-secretin (DATA-SN), a tracer suitable for secretin radioimmunoassay and as an N-terminus modified secretin analog, is reported. The analogs are compared, in terms of their biological and immunological properties in different assay systems, with pure synthetic secretin.     

Molecular Cloning, Chromosomal Localization, and Cell Cycle-Dependent Subcellular Distribution of the A-Kinase Anchoring Protein, AKAP95

The cyclic AMP-dependent protein kinase (PKA) type II is directed to different subcellular loci through interaction of the RII subunits with A-kinase anchoring proteins (AKAPs). A full-length human clone encoding AKAP95 was identified and sequenced, and revealed a 692-amino acid open reading frame that was 89% homologous to the rat AKAP95 (V. M. Coghlan, L. K. Langeberg, A. Fernandez, N. J. Lamb, and J. D. Scott (1994)J. Biol. Chem.269, 7658–7665). The gene encoding AKAP95 was mapped to human chromosome 19p13.1-q12 using somatic cell hybrids and PCR. A fragment covering amino acids 414–692 of human AKAP95 was expressed inEscherichia coliand shown to bind RIIα. Competition with a peptide covering the RII-binding domain of AKAP Ht31 abolished RIIα binding to AKAP95. Immunofluorescence studies in quiescent human Hs-68 fibroblasts showed a nuclear localization of AKAP95, whereas RIIα was excluded from the nucleus. In contrast, during mitosis AKAP95 staining was markedly changed and appeared to be excluded from the condensed chromatin and localized outside the metaphase plate. Furthermore, the subcellular localizations of AKAP95 and RIIα overlapped in metaphase but started to segregate in anaphase and were again separated as AKAP95 reentered the nucleus in telophase. Finally, RIIα was coimmunoprecipitated with AKAP95 from HeLa cells arrested in mitosis, but not from interphase HeLa cells, demonstrating a physical association between these two molecules during mitosis. The results show a distinct redistribution of AKAP95 during mitosis, suggesting that the interaction between AKAP95 and RIIα may be cell cycle-dependent.     

A cDNA clone from barley encoding the precursor from the photosystem I polypeptide PSI-G: Sequence similarity to PSI-K

A cDNA clone encoding the photosystem I subunit, PSI-G was isolated from barley using an oligonucleotide specifying a partial amino acid sequence from a 9 kDa polypeptide of barley photosystem I. The 724 bp sequence contains an open reading frame encoding a precursor polypeptide of 15 107 kDa. Import studies using the in vitro expressed barley PsaG cDNA clone demonstrate that PSI-G migrates with an apparent molecular mass of 9 kDa on SDS-polyacrylamide gels together with PSI-C (subunit-VII). The previous assignment of the gene product of PsaG from spinach as subunit V (Steppuhn J, Hermans J, Nechushtai R, Ljungberg U, Thümmler F, Lottspeich F, Herrmann RG, FEBS Lett 237: 218–224, 1988) needs to be re-examined. The expression of the psaG gene is light-induced similar to other barley photosystem I genes. A significant sequence similarity to PSI-K from Chlamydomonas reinhardtii was discovered when a gene database was searched with the barley PSI-G amino acid sequence. Extensive sequence similarity between the nuclear-encoded photosystem I subunits has not previously been found. The observed sequence similarity between PSI-G and PSI-K suggests a symmetric location of these subunits in the photosystem I complex. The hydropathy plot of the barley PSI-G polypeptide indicates two membrane-spanning regions which are also found at the corresponding locations in the PSI-K polypeptide. PSI-G and PSI-K probably have evolved from a gene duplication of an ancestral gene.     

Effects of alloxan and ninhydrin on mitochondrial Ca2+ transport

Alloxan at millimolar concentrations slightly inhibited the velocity of Ca²⁺ uptake by isolated rat liver mitochondria irrespective of the free Ca²⁺ concentration between 1 and 10 µM and was an effective concentration-dependent stimulator of mitochondrial Ca²⁺ efflux. Ninhydrin also slightly inhibited the velocity of mitochondrial Ca²⁺ uptake but only at free Ca²⁺ concentrations above 5 µM. However, ninhydrin was a strong stimulator of mitochondrial Ca²⁺ efflux even at micromolar concentrations, 10–50 times more potent than alloxan. The mitochondrial membrane potential was reduced 10–20% at most by alloxan and ninhydrin. Alloxan and ninhydrin also stimulated Ca²⁺ efflux from isolated permeabilized liver cells. When isolated intact liver cells had been pre-incubated with alloxan or ninhydrin before permeabilization of the cells the ability of spermine to induce mitochondrial Ca²⁺ uptake was abolished. Glucose provided the typical protection against the effects of alloxan on mitochondrial Ca²⁺ transport only in experiments with intact cells but not in experiments with permeabilized cells or isolated mitochondria. Therefore glucose protection is apparently due to inhibition of alloxan uptake into the cell. Glucose provided no protection against effects of ninhydrin under any of the experimental conditions. Thus both alloxan and ninhydrin are potent stimulators of Ca²⁺ efflux by isolated mitochondria but very weak inhibitors of the velocity of mitochondrial Ca²⁺ uptake. The direct effects of ninhydrin on mitochondrial Ca²⁺ efflux may contribute to the cytotoxic action of this agent whereas the direct effects of alloxan on mitochondrial Ca²⁺ transport require concentrations which are too high to be of relevance for the induction of the typical pancreatic B-cell toxic effects of alloxan. However, the effects on mitochondrial Ca²⁺ transport during incubation of intact cells which may result from the generation of cytotoxic intermediates during alloxan xenobiotic metabolism may well contribute to the pancreatic B-cell toxic effect of alloxan. Mol Cell Biochem 118: 141–151, 1992)     

Structure et fonctionnement des organes de Kölliker chez les jeunes octopodes (Mollusca,Cephalopoda)

Summary The present study is a reinvestigation of the peculiar integumental differentiations described for the first time by Kölliker in 1844. These transitory organs of microscopic size are composed of two parts; one arises from the ectoderm and forms a sac adhering to the epidermis and enclosing a bundle of chitinous setae that are secreted by the microvilli of a specialized basal cell; the other, arising from the mesoderm, is a muscular apparatus that allows evagination and spreading of the bundle. This functioning was observed in fresh skin preparations of newly-hatched Eledone moschata. The Kölliker organs are present in the young of all ineirrate octopods known so far, except for two species of Octopus (O. briareus, O. maya).

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Abréviations C chromatophore - CB cellule basale - CG cellule glandulaire - CM cellule murale - CS cloison sous-cutanée - D dictyosome - E épiderme - F faisceau de soies - FC fibres conjonctives - FP fibre profonde - G golgi - I interdigitations - K organe de Kölliker - M muscle - MB membrane basale - MV microvillus - N noyau - NU nucléole - R réticulum endoplasmique - S soies du faisceau - VS vaisseau sanguin