Possible explanation of the disparity between the in vitro and in vivo measurements of Rubisco activity: a study in loblolly pine grown in elevated pCO2.

Rubisco activity can be measured using gas exchange (in vivo) or using in vitro methods. Commonly in vitro methods yield activities that are less than those obtained in vivo. Rubisco activity was measured both in vivo and in vitro using a spectrophotometric technique in mature Pinus taeda L. (loblolly pine) trees grown using free-air CO2 enrichment in elevated (56 Pa) and current (36 Pa) pCO2. In addition, for studies where both in vivo and in vitro values of Rubisco activity were reported net CO2 uptake rate (A) was modelled based on the in vivo and in vitro values of Rubisco activity reported in the literature. Both the modelling exercise and the experimental data showed that the in vitro values of Rubisco activity were insufficient to account for the observed values of A. A trichloroacetic acid (TCA) precipitation of the protein from samples taken in parallel with those used for activity analysis was co-electrophoresed with the extract used for determining in vitro Rubisco activity. There was significantly more Rubisco present in the TCA precipitated samples, suggesting that the underestimation of Rubisco activity in vitro was attributable to an insufficient extraction of Rubisco protein prior to activity analysis. Correction of in vitro values to account for the under-represented Rubisco yielded mechanistically valid values for Rubisco activity. However, despite the low absolute values for Rubisco activity determined in vitro, the trends reported with CO2 treatment concurred with, and were of equal magnitude to, those observed in Rubisco activity measured in vivo.     

Catalysis by dienelactone hydrolase: A variation on the protease mechanism

Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.     

Systematic Screening of Drosophila Deficiency Mutations for Embryonic Phenotypes and Orphan Receptor Ligands

This paper defines a collection of Drosophila deletion mutations (deficiencies) that can be systematically screened for embryonic phenotypes, orphan receptor ligands, and genes affecting protein localization. It reports the results of deficiency screens we have conducted that have revealed new axon guidance phenotypes in the central nervous system and neuromuscular system and permitted a quantitative assessment of the number of potential genes involved in regulating guidance of specific motor axon branches. Deficiency “kits” that cover the genome with a minimum number of lines have been established to facilitate gene mapping. These kits cannot be systematically analyzed for phenotypes, however, since embryos homozygous for many deficiencies in these kits fail to develop due to the loss of key gene products encoded within the deficiency. To create new kits that can be screened for phenotype, we have examined the development of the nervous system in embryos homozygous for more than 700 distinct deficiency mutations. A kit of ∼400 deficiency lines for which homozygotes have a recognizable nervous system and intact body walls encompasses >80% of the genome. Here we show examples of screens of this kit for orphan receptor ligands and neuronal antigen expression. It can also be used to find genes involved in expression, patterning, and subcellular localization of any protein that can be visualized by antibody staining. A subset kit of 233 deficiency lines, for which homozygotes develop relatively normally to late stage 16, covers ∼50% of the genome. We have screened it for axon guidance phenotypes, and we present examples of new phenotypes we have identified. The subset kit can be used to screen for phenotypes affecting all embryonic organs. In the future, these deficiency kits will allow Drosophila researchers to rapidly and efficiently execute genome-wide anatomical screens that require examination of individual embryos at high magnification.     

Genetic evidence that the putative receptor binding domain of apolipoprotein B (residues 3130 to 3630) is not the only region of the protein involved in interaction with the low density lipoprotein receptor

We have searched for sequence differences in the region of the apolipoprotein B (apo B) gene encoding amino acids 3130-3630 in eight individuals with reduced affinity of low density lipoprotein (LDL) for the normal LDL-receptor. All individuals were hypercholesterolaemic and were selected either on the basis of reduced fractional catabolic rate (FCR) of autologous LDL or substantially reduced binding of their LDL to normal LDL-receptors determined by an in vitro cell growth assay using the U937 macrophage-like cell line. Segments of the apo B gene were amplified by the polymerase chain reaction. Using a combination of cloning and sequencing the amplified fragment, together with chemical cleavage mismatch analysis, no sequence differences were identified in this region of the gene. We therefore conclude that variation outside the region of the apo B gene that codes for amino acids 3130-3630 must be responsible for the reduced LDL clearance in these patients.     

Detection of asymptomatic herpes simplex virus infections after vaccination.

Twenty-two volunteers seronegative for antibodies to herpes simplex virus (HSV) were enrolled in a trial to determine tolerance and immunogenicity of an HSV-2 glycoprotein subunit vaccine. Vaccine was administered at days 0, 28, and 140, and sera were obtained on days 0, 7, 14, 21, 28, 35, 49, 56, 140, 147, and 365 for determination of HSV neutralizing antibody activity and antibody-dependent cell cytotoxicity (ADCC). Sera were also tested by immunoprecipitation of radiolabeled HSV-2-infected cell proteins and polyacrylamide gel electrophoresis to identify the viral proteins which elicited antibody responses in vaccine recipients. After vaccination two male volunteers presented with atypical first-episode genital herpes: patient 1 with a culture-negative genital lesion at day 53 and patient 3 with urethritis at day 68. Seroconversion to wild-type viral proteins not present in the vaccine was detectable by radioimmunoprecipitation-polyacrylamide gel electrophoresis within 10 days in both patients. Two additional volunteers, one a sex contact of patient 1, seroconverted asymptomatically to nonvaccine proteins during the trial. All four vaccine breakthrough patients were indistinguishable from the other volunteers in the time required to develop neutralizing and ADCC antibodies, in the titer of these antibodies, and the time to seroconversion to gB and gD vaccine proteins. However, only one of the four breakthrough patients had antibodies to g80 (a complex of gC-2 and gE) after vaccination as compared with 15 of the other 18 volunteers (P = 0.05). Neither neutralizing antibody nor ADCC titers consistently identified acquisition of wild-type viral infection; therefore, protein-specific serologies were required to detect wild-type antibodies in these four patients. These data underscore the importance of using serologic assays which will distinguish naturally acquired infection from the immune response to vaccination.     

Nonhomologous synapsis of the XY during early pachynema in In(X)1H male mice

It has been previously supposed that meiotic synapsis is restricted to homology during early, but not late pachynema. The synaptic begavior of an inverted X chromosome, In(X)1H as reflected in the synaptonemal complexes of the sex chromosomes has been examined in microspread spermatocytes by electron microscopy and evidence of extensive nonhomologus synapsis between the X and Y during early pachynema has been obtained.     

The mouse homolog of the human amyloid beta protein (AD-AP) gene is located on the distal end of mouse chromosome 16: further extension of the homology between human chromosome 21 and mouse chromosome 16

The human amyloid beta protein is the major constituent of the brain amyloid plaques found in Alzheimer disease. The gene that encodes this protein is located on chromosome 21, and individuals with Down syndrome (trisomy 21) also exhibit an early onset form of Alzheimer disease. We have used the cloned human amyloid beta protein gene and a panel of somatic cell hybrids to map the location of the mouse homolog of this gene. We report here that the mouse gene is located on chromosome 16 within the region 16C3----ter, in common with three other genes which map within the Down syndrome region of human chromosome 21.     

Location of the redox-active thiols of ribonucleotide reductase: sequence similarity between the Escherichia coli and Lactobacillus leichmannii enzymes

The redox-active thiols of Escherichia coli ribonucleoside diphosphate reductase and of Lactobacillus leichmannii ribonucleoside triphosphate reductase have been located by a procedure involving (1) prereduction of enzyme with dithiothreitol, (2) specific oxidation of the redox-active thiols by treatment with substrate in the absence of exogenous reductant, (3) alkylation of other thiols with iodoacetamide, and (4) reduction of the disulfides with dithiothreitol and alkylation with [1-14C]iodoacetamide. The dithiothreitol-reduced E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of 14C. Sequencing of tryptic peptides shows that 2.8 equiv of 14C is on cysteines-752 and -757 at the C-terminus of B1, while 1.0-1.5 equiv of 14C is on cysteines-222 and -227. It thus appears that two sets of redox-active dithiols are involved in substrate reduction. The L. leichmannii reductase is able to convert 1.1 equiv of CTP to dCTP and is labeled with 2.1 equiv of 14C. Sequencing of tryptic peptides shows that 1.4 equiv of 14C is located on the two cysteines of C-E-G-G-A-C-P-I-K. This peptide shows remarkable and unexpected similarity to the thiol-containing region of the C-terminal peptide of E. coli B1, C-E-S-G-A-C-K-I.     

Morphological variation in diadromous and landlocked populations of the spotted galaxias,Galaxias truttaceus,in Tasmania,south-eastern Australia

Synopsis A comparison of a suite of morphometric measurements and meristic counts of individuals of two landlocked lacustrine and two diadromous riverine populations of Galaxias truttaceus was carried out utilising both univariate and canonical variate analyses. Lacustrine fish had fewer dorsal and anal fin rays than did riverine fish. Differences were not as clear for gill rakers and vertebrae. Comparisons of serial counts were made with two derived lacustrine species, G. auratus and G. tanycephalus, also from Tasmania. Lacustrine G. truttaceus varied in the same direction as the derived species, relative to riverine G. truttaceus. From an analysis of 12 body measurements, the first canonical variate clearly separated lacustrine fish from riverine fish largely based on measurements associated with fins (pre-anal fin length, length of anal base, pre-dorsal fin length, maximum length of dorsal fin and inter-orbital width). An overall value for the correct classification of fish into groups based on locality was 84%. The percentage of fish classified into the wrong habitat (lake or stream) was much less than the percentage classified between localities within habitats. Overall morphological variation was greater between than within habitats. It is suggested that the differences in water movement and food type may in part account for the differences shown and that selective pressures peculiar to the lacustrine environment may be causing the lake populations to diverge from the riverine populations.