Biochemical properties of a novel cell wall protein associated with elongation growth in higher plants

A monoclonal antibody of IgM-type (TIM-11B2) was screened froma hybridoma library. The antibody recognizes a 40 kDa glycoprotein,p40, with high specificity. This protein was detected in allplant species examined so far and was found to be located bothsolubly and ionically-bound within the primary cell wall. The strongest immunobiochemical signals of p40 were found intissues undergoing elongation growth, whereas in other tissuesonly a faint signal could be detected. Those included the non-elongatingparts of different seedlings, such as the apical part of monocotprimary leaves or the leaves of dicots grown in light. Inhibitionof pea epicotyl growth by white light irradiation resulted ina strong decrease of the immunostain signal. On the other hand,induction of rapid coleoptile growth in rice seedlings inducedby submergence resulted in a strong increase of the immunobiochemicalsignal of p40. Time-course studies on the expression of p40during protoplast regeneration revealed that p40 is apparentlynot involved in cell wall formation. The hypothesis that p40is characteristic for tissues with the ability for elongationgrowth is discussed. Comparison of biochemical data and location of p40 with proteinsdescribed up to now indicate that this glycoprotein has notbeen characterized before. Key words: Cell wall protein, elongation growth, monoclonal antibody     

The Regional Distribution of N-Acetylaspartylglutamate (NAAG) and Peptidase Activity Against NAAG in the Rat Nervous System

Abstract: N -Acetylaspartylglutamate (NAAG), a prevalent peptide in the vertebrate nervous system, may be hydrolyzed by extracellular peptidase activity to produce glutamate and N -acetylaspartate. Hydrolysis can be viewed as both inactivating the peptide after synaptic release and increasing synaptic levels of ambient glutamate. To test the hypothesis that NAAG and the peptidase activity that hydrolyzes it coexist as a unique, two-stage system of chemical neurotransmission, 50 discrete regions of the rat CNS were microdissected for assay. In each microregion, the concentration of NAAG was determined by radioimmunoassay and the peptidase activity was assayed using tritiated peptide as substrate. The NAAG concentration ranged from 2.4 nmol/mg of soluble protein in median eminence to 64 in thoracic spinal cord. Peptidase activity against NAAG ranged from 54 pmol of glutamate produced per milligram of membrane protein per minute in median eminence to 148 in superior colliculus. A linear relationship was observed between NAAG peptidase and NAAG concentration in 46 of the 50 areas, with a slope of 2.26 and a correlation coefficient of 0.45. These data support the hypothesis that hydrolysis of NAAG to glutamate and N -acetylaspartate is a consistent aspect of the physiology and metabolism of this peptide after synaptic release. The ratio of peptide concentration to peptidase activity was >0.3 in the following four areas: ventrolateral medulla and reticular formation where the peptide is concentrated in axons of passage, thoracic spinal cord, where NAAG is concentrated in ascending sensory tracts as well as motoneuron cell bodies, and ventroposterior thalamic nucleus.     

Combining industrial ecology tools to assess potential greenhouse gas reductions of a circular economy: Method development and application to Switzerland

Industrial ecology (IE) methodologies, such as input/output or material flow analysis and life cycle assessment (LCA), are often used for the environmental evaluation of circular economy strategies. Up to now, an approach that utilizes these methods in a systematic, integrated framework for a holistic assessment of a geographic region's sustainable circular economy potential has been lacking. The approach developed in this study (IE4CE approach) combines IE methodologies to determine the environmental impact mitigation potential of circular economy strategies for a defined geographic region. The approach foresees five steps. First, input/output analysis helps identify sectors with high environmental impacts. Second, a refined analysis is conducted using material flow and LCA. In step 3, circular strategies are used for scenario design and evaluated in step 4. In step 5, the assessment results are compiled and compared across sectors. The approach was applied to a case study of Switzerland, analyzing 8 sectors and more than 30 scenarios in depth. Carbon capture and storage (CCS) from waste incineration, biogas and cement production, food waste prevention in households, hospitality and production, and the increased recycling of plastics had the highest mitigation potential. Most of the scenarios do not influence each other. One exception is the CCS scenarios: waste avoidance scenarios decrease the reduction potential of CCS. A combination of scenarios from different sectors, including their impact on the CCS scenario potential, led to an environmental impact mitigation potential of 11.9 Mt CO₂-eq for 2050, which equals 14% of Switzerland's current consumption-based impacts.     

The agony of choice: Impact of the host animal species on the enzyme-linked immunosorbent assay performance for host cell protein quantification

Host cell proteins (HCPs) are inevitable process-related impurities in biotherapeutics commonly monitored by enzyme-linked immunosorbent assays (ELISAs). Of particular importance for their reliable detection are the anti-HCP polyclonal antibodies (pAbs), supposed to detect a broad range of HCPs. The present study focuses on the identification of suitable host animal species for the development of high-performance CHO-HCP ELISAs, assuming the generation of pAbs with adequate coverage and specificity. Hence, antibodies derived from immunization of sheep, goats, donkeys, rabbits, and chickens were compared concerning their amount of HCP-specific antibodies, coverage, and performance in a sandwich ELISA. Immunization of sheep, goats, donkeys, and rabbits met all test criteria, whereas the antibodies from chickens cannot be recommended based on the results of this study. Additionally, a mixture of antibodies from the five host species was prepared to assess if coverage and ELISA performance can be improved by a multispecies approach. Comparable results were obtained for the single- and multispecies ELISAs in different in-process samples, indicating no substantial improvement for the latter in ELISA performance while raising ethical and financial concerns.     

Retention of xenobiotics along the phloem path

Detached leaves of Cyclamen persicum Mill. can be used as a simple source-sink system. Phloem transport in the excised material was monitored by the noninvasive ¹¹C-technique. Assimilate movement stopped immediately when the petiole was cut off. However, within 20 min a recovery of transport was observed. The translocation rate in the detached leaf was only 13% of that in the intact plant. ¹⁴C-Xenobiotics and [³H]sucrose were injected into the upper petiole parenchyma (source). They moved downstream by a symplastic route. The stump of the petiole was inserted into a buffer solution containing ethylenediaminetetraacetic acid (sink). After 3 h, the distribution of sucrose and xenobiotics was determined in five subsequent segments of the petiole (path). The retention coefficient (r) was calculated from the ratio of radioactivity in the vascular bundle to that in the petiole parenchyma. The distribution along the vascular path was given by a geometric progression, whereas its constant was the transport coefficient (q). Values of r and q corresponded with the degree of phloem mobility and ambimobility. Four groups of compounds were classified: (i) acidic substances with log K_ow = — 2 to — 2.4 (K_ow is the partition coefficient octanol/water) at pH 8 (pH of sieve tube sap), retained by ion trapping and exhibiting small lateral efflux (q0.7; maleic hydrazide, dalapon); (ii) acidic substances with log K_ow = — 0.7 to — 0.8 at pH 8, retained by ion trapping and subjected to a moderate lateral efflux (0.7>q> 0.5; 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, bromoxynil); (iii) nonionised substances retained by optimum permeability, exhibiting a considerable lateral leakage (q<0.5; glyphosate, amitrole); (iv) substances without basipetal transport in the phloem (atrazine, diuron). Retention of sucrose corresponded quantitatively with that shown in group (i). This classification was also supported by results of uptake and efflux experiments using the isolated conducting tissue. Theoretical translocation profiles were calculated from the determined transport coefficients (q).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - K_ow partition coefficient octanol/water - MCPA 2-methyl-4-chloro-phenoxyacetic acid - q transport coefficient in the vascular bundle - r retention coefficient in the vascular bundle The authors gratefully acknowledge the assistance of H. Fiedler and M. Neugebauer. We are particularly grateful to K. Dutschka, G. Hudepokl, and Dr. J. Knust for producing ¹¹CO₂.     

Immunohistochemical analysis of muscle cytochromec oxidase deficiency in children

Despite the demonstration of a clear biochemical defect, the genetic alterations causing childhood forms of cytochromec oxidase (COX) deficiency remain unknown. The double genetic origin (nuclear and mitochondrial DNA), and the complexity of COX enzyme structure and regulation, indicate the need for genetic iinvestigations of the molecular structure of individual COX subunits. In the present study a new monoclonal antibody, which reacts exclusively with heart-type human COX subunit VIIa (VIIa-H), and other monoclonal antibodies against human COX subunits, were used in the immunohistochemical analysis of skeletal muscle from children with different forms of mitochondrial myopathy with COX deficiency. By immunohistochemical investigation a normal reaction was seenn with antibodies to COX subunits IV, Va+Vb, and VIa+VIc in all four cases, and in two cases with antibodies to COX VIIa-H and VIIa+VIIb. In muscle from a fatal infantile case with cardiac and skeletal muscle involvement, no immunohistochemical reaction was seen with the monoclonal antibody against the tissue-specific subunit VIIa-H. In muscle from an 11-year-old boy with exclusive muscular symptoms and signs, immunohistological reactions were absent with COX subunit VIIa-H and COX subunits VIIa+VIIb, and slightly decreased with COX subunit II, thus demonstrating a different molecular mechanism in each case. It is concluded that the molecular basis of COX deficiency in childhood may vary greatly between patients.     

Autochthonous fungi are central components in microbial community structure in raw fermented sausages

Raw meat sausage represents a unique ecological niche rich in nutrients for microbial consumption, making it particularly vulnerable to microbial spoilage. Starter cultures are applied to improve product stability and safety as well as flavour characteristics. However, the influence of starter cultures on microbial community assembly and succession throughout the fermentation process is largely unknown. In particular the effect on the fungal community has not yet been explored. We evaluate the microbiological status of four different raw meat sausages using high-throughput 16S rRNA gene and ITS2 gene sequencing. The objective was to study temporal changes of microbial composition during the fermentation process and to identify potential keystone species that play an important role within the microbial community. Our results suggest that fungi assigned to the species Debaryomyces hansenii and Alternaria alternata play a key role in microbial community dynamics during fermentation. In addition, bacteria related to the starter culture Lactobacillus sakei and the spoilage-associated genera Acinetobacter, Pseudomonas and Psychrobacter are central components of the microbial ecosystem in raw fermented sausages. Elucidating the exact role and interactions of these microorganisms has the potential to have direct impacts on the quality and safety of fermented foods.