A nondestructive method for peptide bond conjugation of antigenic haptens to a diphtheria toxoid carrier, exemplified by two antisera specific to acetolactate synthase.

A method for the preparation of an activated protein carrier is described: Protein carboxyl groups are transformed into N-hydroxysulfosuccinimide esters, a structure that will react with primary amino groups under amide bond formation. Although the activated ester is unstable under aqueous conditions, a significant amount of hapten molecules can be bound covalently to the carrier under very mild conditions. Ligands can be peptides or other molecules possessing a primary amino group. The method avoids the risk of ligand polymerization and no derivatization of the ligand prior to conjugation is needed. Residual unreacted ligand molecules can therefore be recovered in their native form by size exclusion chromatography. The method was used to conjugate two synthetic sugar beet acetolactate synthetase (E.C. 4.1.3.18) peptides to diphtheria toxoid. Antibodies were raised against both of the conjugates. The specificity of these antibodies against sugar beet acetolactate synthetase was verified using immunoblotting, ELISA, and immunoprecipitation.     

Determination of the three-dimensional solution structure of barnase using nuclear magnetic resonance spectroscopy

The solution conformation of the ribonuclease barnase has been determined by using 1H nuclear magnetic resonance (NMR) spectroscopy. The 20 structures were calculated by using 853 interproton distance restraints obtained from analyses of two-dimensional nuclear Overhauser spectra, 72 phi and 53 chi 1 torsion angle restraints, and 17 hydrogen-bond distance restraints. The calculated structures contain two alpha-helices (residues 6-18 and 26-34) and a five-stranded antiparallel beta-sheet (residues 50-55, 70-75, 85-91, 94-101, and 105-108). The core of the protein is formed by the packing of one of the alpha-helices (residues 6-18) onto the beta-sheet. The average RMS deviation between the calculated structures and the mean structure is 1.11 A for the backbone atoms and 1.75 A for all atoms. The protein is least well-defined in the N-terminal region and in three large loops. When these regions are excluded, the average RMS deviation between the calculated structures and the mean structure for residues 5-34, 50-56, 71-76, 85-109 is 0.62 A for the backbone atoms and 1.0 A for all atoms. The NMR-derived structure has been compared with the crystal structure of barnase [Mauguen et al. (1982) Nature (London) 297, 162-164].     

The membrane fraction of homogenized rat kidney contains an enzyme that releases epidermal growth factor from the kidney membranes

High levels of epidermal growth factor (EGF) are excreted in the urine and high levels of mRNA for the EGF-precursor have been demonstrated in the kidney. The EGF-precursor is a membrane bound peptide in the kidney, but little is known about the renal processing of the precursor. The present study shows that the membrane fraction of homogenized rat kidney contains an enzyme that releases immuno and receptor reactive EGF from the kidney membranes when incubated at 37 degrees C. Gel filtration shows that the EGF reactivity released from the membranes is similar to the EGF reactivity in rat urine. The EGF releasing enzyme is inhibited by the serine proteinase inhibitor aprotinin and by low temperatures (4 degrees C). The pH optimum of the reaction is pH 7.5-8.0.     

A model for population regulation with density- and frequency-dependent selection

The life-cycle of a species with separate generations is divided into a reproduction phase and a growing-up phase. In the reproduction phase we assume random mating and selection due to genotype differences in fecundity of the parents and viability of the offspring. During the growing-up phase we assume a (deterministic) death process in continuous time with death rates for the genotypes which increase linearly with the genotype population sizes.In the absence of genotype differences the model gives logistic population regulation. With genotype differences the model generalizes the usual separate generations selection patterns. In addition to these we exhibit cases with three polymorphic equilibria or with a stable cycle.     

SDS removal from protein by polystyrene beads

The applications of sodium dodecyl sulfate (SDS) to molecular weight determination (1,2) and for the separation of protein subunits (3) have been of immense value in biochemical studies [see Waehneldt (4) for a general review]. The tight stoichiometric binding of SDS to polypeptide chains has proven to be a nuisance if one desires to recover the activity of the isolated polypeptides. Removal of the SDS has been affected by the use of anion exchangers in the presence (5,6) and absence of urea (6). However, the residual levels of SDS or urea are often quite unsatisfactory for further protein studies.We have attempted to adapt the procedure of Holloway for the removal of Triton X-100 by Bio-Beads to the removal of SDS. We chose bovine serum albumin as a test protein since it has well-established strong binding properties for linear-chain fatty acids (7).     

Renal uptake and excretion of epidermal growth factor from plasma in the rat

The rat excretes around 2 nmol epidermal growth factor (EGF) in the urine per 24 h. The urinary EGF might be derived from plasma and/or might be synthesized in the kidneys. We have used the rat to study the renal uptake and excretion of homologous EGF from plasma. I.v. injected 125I-EGF was removed from the circulation within a few minutes. 5 min after the injection, the kidneys contained 12% of the 125I-EGF. The kidneys seemed to degrade most of the 125I-EGF which they accumulated from blood, as only 4% of the injected label was excreted as intact 125I-EGF in the urine. The amount of endogenous EGF in plasma was under the detection limit of our enzyme-linked immunosorbent assay (0.03 nmol/l) and it remained so after bilateral nephrectomy. Even if plasma EGF was 0.03 nmol/l excretion of EGF from plasma could account for less than 5% of the urinary EGF. This study shows that the kidneys are able to accumulate EGF from plasma and excrete a part of it as intact EGF in the urine. However, excretion of immunoreactive EGF from plasma can only account for a minor part of the urinary EGF.     

PEATmoss (Physcomitrella Expression Atlas Tool): a unified gene expression atlas for the model plant Physcomitrella patens

Physcomitrella patens is a bryophyte model plant that is often used to study plant evolution and development. Its resources are of great importance for comparative genomics and evo‐devo approaches. However, expression data from Physcomitrella patens were so far generated using different gene annotation versions and three different platforms: CombiMatrix and NimbleGen expression microarrays and RNA sequencing. The currently available P. patens expression data are distributed across three tools with different visualization methods to access the data. Here, we introduce an interactive expression atlas, Physcomitrella Expression Atlas Tool (PEATmoss), that unifies publicly available expression data for P. patens and provides multiple visualization methods to query the data in a single web‐based tool. Moreover, PEATmoss includes 35 expression experiments not previously available in any other expression atlas. To facilitate gene expression queries across different gene annotation versions, and to access P. patens annotations and related resources, a lookup database and web tool linked to PEATmoss was implemented. PEATmoss can be accessed at https://peatmoss.online.uni-marburg.de     

Comparing the binding properties of peptides mimicking the Envelope protein of SARS‐CoV and SARS‐CoV‐2 to the PDZ domain of the tight junction‐associated PALS1 protein

The Envelope protein (E) is one of the four structural proteins encoded by the genome of SARS‐CoV and SARS‐CoV‐2 Coronaviruses. It is an integral membrane protein, highly expressed in the host cell, which is known to have an important role in Coronaviruses maturation, assembly and virulence. The E protein presents a PDZ‐binding motif at its C‐terminus. One of the key interactors of the E protein in the intracellular environment is the PDZ containing protein PALS1. This interaction is known to play a key role in the SARS‐CoV pathology and suspected to affect the integrity of the lung epithelia. In this paper we measured and compared the affinity of peptides mimicking the E protein from SARS‐CoV and SARS‐CoV‐2 for the PDZ domain of PALS1, through equilibrium and kinetic binding experiments. Our results support the hypothesis that the increased virulence of SARS‐CoV‐2 compared to SARS‐CoV may rely on the increased affinity of its Envelope protein for PALS1.