Molecular cloning of a pair of human pepsinogen A genes which differ by a Glu→Lys mutation in the activation peptide

Summary Three human cosmid clones containing pepsinogen A (PGA) encoding sequences were isolated from a genomic bank derived from a single individual. One cosmid contains two PGA genes in tandem in a head-to-tail orientation, while the other two cosmids each contain a single PGA gene. The three cosmids were characterized by restriction mapping and sequence analysis (exons 1 and 2 and flanking regions). As judged from these data, three of the four PGA genes isolated appear to be nearly identical, but one of the tandem genes is clearly different from the other genes. The first exon of all four genes codes for the same amino acid sequence. However, in the second exon of one of the tandem genes we found a nucleotide substitution giving rise to a GluLys substitution of the 43rd amino acid residue of the activation peptide, leading to a charge difference of the corresponding isozymogens. The presence of two distinct PGA genes in the isolated gene pair conclusively proves the multigene structure of the PGA system. These genes might be responsible for at least part of the electrophoretic polymorphism at the protein level.     

Immunocytochemical applications in neuroanatomy

Summary In the present paper we review immunocytochemical methods for anterograde tracing with the lectin Phaseolus vulgaris-leucoagglutinin (PHA-L), combined PHA-L tracing — neurotransmitter immunocytochemistry, and the immunocytochemical localization of receptor proteins. These methods will be mainly illustrated by examples from tracing- and neurotransmitter studies on the cholinergic basal forebrain system. The morphology of PHA-L labeled neurons strongly resembles that of Golgi impregnated neurons. The complete axonal trajectories and patterns of presynaptic endings of PHA-L labeled neurons are visualized, both for light- and electron microscopic application.PHA-L-tracing can very well be combined with second immunocytochemical labeling procedures. In this way, traced pathways can be studied in their relation to chemically identified fiber systems or target neurons. Application of immunocytochemistry for the localization of the muscarinic acetylcholine receptor, albeit in its early stages, holds great promise for the near future.     

Repair of UV damage in plasmid DNA by human fibroblasts

Summary Plasmid DNA from Bacillus subtilis was introduced into monolayers of human fibroblasts by means of a modification of the calcium phosphate coprecipitation technique, comprising centrifugation of the coprecipitate onto the cells and treatment with polyethyleneglycol. The amount of DNA resistant to removal from the monolayers ranged from 10% to 15% of the input DNA. By determination of the biological activity of the plasmid DNA, re-extracted after various periods following entry into the fibroblasts and subsequently used as donor for B. subtilis protoplasts, it was shown that the activity of the plasmid DNA was gradually lost. When ultraviolet light-inactivated plasmid DNA was used as donor, reactivation of the plasmid was observed, which was completed within 2 h. The dose-dependent incorporation of [¹⁴C]-thymidine suggests that DNA repair processes were involved in reactivation of the plasmid DNA.     

Spatial Analysis of the Canopy of Kiwifruit Vines as it Relates to the Physical, Chemical and Postharvest Attributes of the Fruit

The position of individual fruit on kiwifruit vines (Actinidiadeliciosa var. deliciosa) grown on a horizontal trellis (pergola)and on a T-bar trellis was determined using a theodolite. Thephysical, chemical, and postharvest attributes of the fruitwere related to their position on the vine during development. Fruit from the pergola vines were more numerous, of lesser weight,with lower concentrations of most mineral nutrients, but greaterconcentrations of soluble solids, and similar flesh firmnessafter 12 weeks of storage at 0 °C, than fruit from the T-barvines. The position on the vine accounted for most of the variationin the attributes of the fruit. Differences between fruit ona single lateral accounted for 43-56% of the variation. Variationbetween vines was relatively small (< 4% of the total variance). The heavier fruit were located at the apical ends of the laterals,while greater concentrations of soluble solids were associatedwith fruit located closer to the cordon. The larger fruit fromthe pergola vines developed from the early opening flowers.A similar relationship existed initially for the T-bar vines,but a reduction in growth of fruit from the early opening flowers8 weeks after anthesis resulted in a more even distributionof fruit size at harvest. The strongest relationship between mineral composition and postharvestattributes of the fruit was with soluble solids concentration(29-46% of the variance). The relationship with flesh firmnesswas weak (r = -0·14 to -0·32). Individual elementscould not be considered in isolation but rather in groups ofelements. Nitrogen was grouped strongly with phosphorus, sulphur,potassium, and copper, while calcium was linked with a secondgroup which included manganese and zinc. These two groups werenegatively related to one another. The greatest proportion of fruit with superior characteristicswas located in the denser parts of the canopy. Fruit with lessdesirable attributes were from the extremities of the canopywhere the leaf area index was low.Copyright 1994, 1999 AcademicPress Actinidia deliciosa, kiwifruit, fruit position, fruit quality, within-vine variation     

Seasonal changes in photosynthetic capacity of leaves of kiwifruit (Actinidia deliciosa) vines

A simplified procedure for the assay and purification of an enzyme which activates a galactosyltransferase (EC 2.4.1.96) involved in volume regulation of the unicellular alga Poterioochromonas malhamensis (Peterfi) is described. The enzyme was extracted with water from membranes, followed by chromatography on DEAE-Sephacel, phenyl-Sepharose and fetuin-agarose. Its proteinase activity was demonstrated by cleavage of oxidized insulin A- and B-Chains. The predominant cleavage site of the oxidized A-chain is the peptide bond between ¹³Leu and ¹⁴Tyr whereas ¹⁶Leu-¹⁷Glu is also hydrolyzed with minor activity. Besides this chymotrypsin-like endopeptidase activity some carboxypeptidase activity was also observed.     

The stable bacteriocin release protein signal peptide, expressed as a separate entity, functions in the release of cloacin DF13

Abstract The pCloDF1S encoded bacteriocin release protein (BRP) plays a role in the release of the bacteriocin cloacin DF13. The BRP signal peptide is stable after cleavage, and accumulates in the cytoplasmic membrane. A BRP which is correctly targeted by the unstable murein lipoprotein signal peptide (Lpp-BRP) is not capable of inducing the release of cloacin DF13. To investigate the role of the stable BRP signal peptide in the release of cloacin DF13, the stable BRP signal peptide and the Lpp-BRP were expressed in trans in cells also producing cloacin DF13. Expression and release experiments indicate that the stable signal peptide can complement the Lpp-BRP in the release of cloacin DF13.     

Excretion of proteins by gram-negative bacteria: export of bacteriocins and fimbrial proteins byEscherichia coli

In gram-negative bacteria only few proteins are exported across both the cytoplasmic membrane and the outer membrane which forms an extra barrier for protein excretion. In this review we describe the mechanisms of production and export of two types of plasmid-encoded proteins inEscherichia coli. These proteins are the bacteriocin cloacin DF13 and the K88ab and K99 fimbrial subunits. Specific so-called helper proteins located at different positions in the cell envelope play an essential role in the export of these proteins. The genetic organization, subcellular location and functions of these helper proteins, as well as the effects of mutations and culture conditions on the export of the proteins are described. Models for the export mechanisms are presented and future application possibilities for engineering foreign protein excretion inE. coli with these export systems are discussed.     

An improved neutral landscape model for recreating real landscapes and generating landscape series for spatial ecological simulations

Many studies have assessed the effect of landscape patterns on spatial ecological processes by simulating these processes in computer‐generated landscapes with varying composition and configuration. To generate such landscapes, various neutral landscape models have been developed. However, the limited set of landscape‐level pattern variables included in these models is often inadequate to generate landscapes that reflect real landscapes. In order to achieve more flexibility and variability in the generated landscapes patterns, a more complete set of class‐ and patch‐level pattern variables should be implemented in these models. These enhancements have been implemented in Landscape Generator (LG), which is a software that uses optimization algorithms to generate landscapes that match user‐defined target values. Developed for participatory spatial planning at small scale, we enhanced the usability of LG and demonstrated how it can be used for larger scale ecological studies. First, we used LG to recreate landscape patterns from a real landscape (i.e., a mountainous region in Switzerland). Second, we generated landscape series with incrementally changing pattern variables, which could be used in ecological simulation studies. We found that LG was able to recreate landscape patterns that approximate those of real landscapes. Furthermore, we successfully generated landscape series that would not have been possible with traditional neutral landscape models. LG is a promising novel approach for generating neutral landscapes and enables testing of new hypotheses regarding the influence of landscape patterns on ecological processes. LG is freely available online.     

Crystal structures of native and inactivated cis-3-chloroacrylic acid dehalogenase. Structural basis for substrate specificity and inactivation by (R)-oxirane-2-carboxylate

The bacterial degradation pathways for the nematocide 1,3-dichloropropene rely on hydrolytic dehalogenation reactions catalyzed by cis- and trans-3-chloroacrylic acid dehalogenases (cis-CaaD and CaaD, respectively). X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by (R)-oxirane-2-carboxylate were elucidated. They locate four known catalytic residues (Pro-1, Arg-70, Arg-73, and Glu-114) and two previously unknown, potential catalytic residues (His-28 and Tyr-103'). The Y103F and H28A mutants of these latter two residues displayed reductions in cis-CaaD activity confirming their importance in catalysis. The structure of the inactivated enzyme shows covalent modification of the Pro-1 nitrogen atom by (R)-2-hydroxypropanoate at the C3 position. The interactions in the complex implicate Arg-70 or a water molecule bound to Arg-70 as the proton donor for the epoxide ring-opening reaction and Arg-73 and His-28 as primary binding contacts for the carboxylate group. This proposed binding mode places the (R)-enantiomer, but not the (S)-enantiomer, in position to covalently modify Pro-1. The absence of His-28 (or an equivalent) in CaaD could account for the fact that CaaD is not inactivated by either enantiomer. The cis-CaaD structures support a mechanism in which Glu-114 and Tyr-103' activate a water molecule for addition to C3 of the substrate and His-28, Arg-70, and Arg-73 interact with the C1 carboxylate group to assist in substrate binding and polarization. Pro-1 provides a proton at C2. The involvement of His-28 and Tyr-103' distinguishes the cis-CaaD mechanism from the otherwise parallel CaaD mechanism. The two mechanisms probably evolved independently as the result of an early gene duplication of a common ancestor.     

The two membrane segments of leader peptidase partition one by one into the lipid bilayer via a Sec/YidC interface

We have addressed the mechanism of insertion of both transmembrane segments (TMs) of leader peptidase, a double-spanning protein, into the Escherichia coli inner membrane. Using photo-crosslinking, the first TM (H1) was shown to insert at a Sec-translocon/YidC interface in a fixed orientation. H1 lost its contacts with the Sec-translocon and gained access to lipids near YidC soon after complete exposure outside the ribosome. Following lipid integration, it moved away from the Sec/YidC insertion site. The second TM (H2) inserted and interacted with SecY and YidC in a similar transient fashion. The data are consistent with a linear integration model in which the TMs of polytopic inner membrane proteins move one by one from a Sec/YidC insertion site into the lipid bilayer. We propose that YidC assists the lipid partitioning of single TMs.