Ultrastructure of rumen bacterial attachment to forage cell walls.

The degradation of forage cell walls by rumen bacteria was investigated with critical-point drying/scanning electron microscopy and ruthenium red staining/transmission electron microscopy. Differences were observed in the manner of attachment of different morphological types of rumen bacteria to plant cell walls during degradation. Cocci, constituting about 22% of the attached bacteria, appeared to be attached to degraded plant walls via capsule-like substances averaging 58 nm in width (range, 21 to 84 nm). Many bacilli appeared to adhere to forage substrates without distinct capsule-like material, although unattached bacteria with capsules were observed occasionally. Certain bacili appeared to be attached to degraded tissue via small amounts of extracellular material, but others apparently had no extracellular material. Bacilli with a distinct morphology due to an irregularly folded, electron-dense outer layer or layers (about 15 nm thick) and without fibrous extracellular material consituted about 37% of the attached bacteria and were observed to adhere so closely to degraded plant walls that the bacterial shape conformed to the shape of the degraded zone. In the rumen ecosystem, bacteria appeared to adhere to plant substrates during degradation by capsule-like material and by small amounts of extracellular material, as well as by the other means not observable by electron microscopy.     

Evaluation by electron microscopy and anaerobic culture of types of rumen bacteria associated with digestion of forage cell walls.

Different morphological types of rumen bacteria which degraded cell walls of forage grasses with various in vitro digestibilities were evaluated with electron microscopy. The majority of these bacteria (i.e., about 70% or more) consisted of two distinct types: (i) encapsulated cocci and (ii) irregularly shaped bacteria, resembling major fiber digesters found in the rumen. Each type was capable of degrading structurally intact cell walls. Differences (P less than or equal to 0.02) in the percent ratio of encapsulated cocci to irregularly shaped bacteria were observed between Bermuda grass and fescue; the ratio of encapsulated cocci to irregularly shaped bacteria between Bermuda grass and orchard grass was similar and variations were high. The proportion of irregularly shaped bacteria usually increased with increased time of digestion. Differences (P greater than 0.1) were not found in the percentage ratio of encapsulated cocci to irregularly shaped bacteria attached to specific tissue types in either Bermuda grass or fescue. However, encapsulated cocci tended to be more prevalent on sclerenchyma than other tissues in Bermuda grass, but less prevalent on sclerenchyma than other tissues in fescue. Transmission electron microscopy of tissue digestion of rapidly degraded orchard grass blades revealed that mesophyll, parenchyma bundle sheath, and parts of the epidermal cell wall apparently were degraded without direct attachment of bacteria although bacteria were near the cell walls undergoing digestion. Anaerobic growth studies showed that the total culturable bacteria developing on medium 10 and media containing carbohydrates similar to those in forage cell walls (i.e., pectin, xylan, and cellobiose) were 80% higher from rumen bacterial populations adapted in vitro to cell walls of orchard grass compared to those from Bermuda grass; the number of colonies from the orchard grass-adapted population was significantly (P less than or equal to 0.05) greater on the medium containing xylan. Filter paper tests showed that the cellulolytic activity of populations adapted to fescue was greater than that of orchard grass or Bermuda grass.     

Rumen bacterial degradation of forage cell walls investigated by electron microscopy

The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues.     

Use of negative stain and single-particle image processing to explore dynamic properties of flexible macromolecules

Flexible macromolecules pose special difficulties for structure determination by crystallography or NMR. Progress can be made by electron microscopy, but electron cryo-microscopy of unstained, hydrated specimens is limited to larger macromolecules because of the inherently low signal-to-noise ratio. For three-dimensional structure determination, the single particles must be invariant in structure. Here, we describe how we have used negative staining and single-particle image processing techniques to explore the structure and flexibility of single molecules of two motor proteins: myosin and dynein. Critical for the success of negative staining is a hydrophilic, thin carbon film, because it produces a low noise background around each molecule, and stabilises the molecule against damage by the stain. The strategy adopted for single-particle image processing exploits the flexibility available within the SPIDER software suite. We illustrate the benefits of successive rounds of image alignment and classification, and the use of whole molecule averages and movies to analyse and display both structure and flexibility within the dynein motor.     

Rumen fungi: morphological types from Georgia cattle and the attack on forage cell walls

Fungal colonies developing in anaerobic media from zoospores in rumen fluid from cows eating Cynodon dactylon or Medicago sativa included types showing monocentric and polycentric growth. High energy supplements added to diets of Sorghum bicolor silage increased fungal numbers in the rumen, but increases were also affected by the history and predisposition of the animal. Mixed fungal types in rumen fluid and pure cultures of isolates showing monocentric and polycentric growth degraded and weakened lignocellulosic tissues and penetrated the cuticle of C. dactylon leaf blades. By weakening or degrading recalcitrant structures in forages, rumen fungi may alter physical parameters of plants that influence utilization of fibre by ruminants.     

Nitrogen and potassium fertilization and environmental factors affecting the grass tetany hazard of wheat forage

Summary The effects of temperature and soil moisture levels on the chemical composition of wheat forage grown in growth chambers were studied. In addition to the environmental variables, K and N fertilization effects were studied. In all the studies, increasing levels of K fertilization depressed the Mg and Ca concentration of the shoots. Nitrogen fertilization increased the Mg concentration but had no effect on the Ca concentration of the plants. N fertilization depressed the K concentration in the soil moisture experiment, but had no effect on K concentration in the temperature experiment. Increasing the temperature from 10 to 20°C did not affect the Mg and Ca concentration of the shoots, but the K concentration declined due to dilution effects caused by the greater yield at the higher temperature. In the soil moisture level experiment the K, Mg and Ca concentration in wheat tended to decline with soil moisture level due to dilution effects. Calculations showed that uptake of K was regulated primarily by diffusion of K from the soil to the plant root and that the uptake of Mg was regulated by the uptake process of the plant root and not by the nutrient transport process through the soil.This study was part of the program of the Center for Root-Soil Research. Dept. of Agronomy paper #1532.     

Investigation of the enzymatic digestion of plant cell walls using reflectance Fourier Transform Infrared spectroscopy

A method has been developed to determine the reflectance Fourier Transform Infrared spectra of plant cells grown in vitro and of the protoplasts released from such cells by enzymatic digestion. It is demonstrated that there is a smooth and reproducible transition in spectral detail as enzymatic digestion procedes. Reflectance Fourier Transform Infrared spectroscopy has been used to monitor the progress of protoplast release during enzymatic digestion of cell wall material.Abbreviations FTIR Fourier Transform Infrared - SEM Scanning Electron Microscope     

A technique for cytological preparations following enzymatic digestion of pollen mother cell walls.

A technique is presented for making meiotic chromosome preparations with enzymatic digestion of pollen mother cell walls with cellulase. This technique has given excellent chromosome definition from early pachytene stages on. Such chromosome preparations were found useful for in situ nucleic acid hybridisation studies on plant chromosomes.     

Plant cell walls to ethanol

Conversion of plant cell walls to ethanol constitutes second generation bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation and separation. Ultimately, it is desirable to combine as many of the biochemical steps as possible in a single organism to achieve CBP (consolidated bioprocessing). A commercially ready CBP organism is currently unreported. Production of second generation bioethanol is hindered by economics, particularly in the cost of pretreatment (including waste management and solvent recovery), the cost of saccharification enzymes (particularly exocellulases and endocellulases displaying kcat ~1?s-1 on crystalline cellulose), and the inefficiency of co-fermentation of 5- and 6-carbon monosaccharides (owing in part to redox cofactor imbalances in Saccharomyces cerevisiae).     

Use of synthetic signal sequences to explore the protein export machinery

The information for correct localization of newly synthesized proteins in both prokaryotes and eukaryotes resides in self-contained, often transportable targeting sequences. Of these, signal sequences specify that a protein should be secreted from a cell or incorporated into the cytoplasmic membrane. A central puzzle is presented by the lack of primary structural homology among signal sequences, although they share common features in their sequences. Synthetic signal peptides have enabled a wide range of studies of how these "zipcodes" for protein secretion are decoded and used to target proteins to the protein machinery that facilitates their translocation across and integration into membranes. We review research on how the information in signal sequences enables their passenger proteins to be correctly and efficiently localized. Synthetic signal peptides have made possible binding and crosslinking studies to explore how selectivity is achieved in recognition by the signal sequence-binding receptors, signal recognition particle, or SRP, which functions in all organisms, and SecA, which functions in prokaryotes and some organelles of prokaryotic origins. While progress has been made, the absence of atomic resolution structures for complexes of signal peptides and their receptors has definitely left many questions to be answered in the future.     

Use of a receptor competition assay to explore the interaction of the T cell antigen-specific receptor with its ligands

The observation has previously been made that receptor-bearing cells in culture compete with each other for their ligand. As a result, at a fixed concentration of ligand, the fractional occupancy of the receptor will tend to fall as the number of cells is increased. We have demonstrated that T cells in culture also compete for their ligand, the combination of foreign antigen and the Ia molecule (antigen-Ia), and that this manifests itself as shifts in the antigen dose-response curves as the number of responding T cells is increased. Because of the complexity of T cell activation, modifications to the antigen that affected its stimulatory capacity (i.e., its potency) could come about by altering its interaction with either the T cell receptor or the Ia molecule. We could distinguish between these two possibilities by studying the extent to which the antigen dose-response curves shifted as the T cell number was increased. Amino acid substitutions in the antigen that affected the interaction with the T cell receptor caused changes in the dose-response curve shifts, whereas substitutions that decreased potency by other means did not cause such changes. Finally, two allelic forms of the Ia molecule that differed only slightly in their amino-terminal domain were used to present a single antigen to a T cell clone. Despite a difference in antigenic potency in the presence of these two Ia molecules, no difference was demonstrated in the avidity of the T cell receptor for either antigen-Ia combination. These results suggest that the antigen and the Ia molecule make physical contact during the process of antigen recognition, and that the potency of an antigen can vary as a result of its interaction with either the T cell receptor or the Ia molecule.     

Use of secondary ion mass spectrometry to image44calcium uptake in the cell walls of apple fruit

Summary Calcium, an important agent in regulating cell wall autolysis during fruit ripening, interacts with pectic acid polymers to form cross-bridges that influence cell separation. In the present study, secondary ion mass spectrometry (SIMS) was used to determine whether the cell walls of apple fruit were able to take up exogenously applied⁴⁴Ca, which was infiltrated into mature fruit. SIMS, which has the ability to discriminate between isotopes, allowed localization of the exogenously applied⁴⁴Ca and the native⁴⁰Ca. The results indicated that the total amount of calcium present in the cell walls was enriched with⁴⁴Ca and that heterogeneity of⁴⁴Ca distribution occurred in the pericarp. Isotope ratio images showed microdomains in the cell wall, particularly in the middle lamella intersects that oppose the intercellular spaces. These domains may be the key areas that control cell separation. These data suggest that exogenously applied calcium may influence cell wall autolysis.Abbreviations SIMS secondary ion mass spectrometry     

Adhesion of cellulose to cell walls to Trichoderma reesei

Carbohydrate-binding components were shown to be present at the surface of Listeria monocytogenes by means of a panel of neoglycoproteins using direct agglutination. These lectin-like components bind on neoglycoproteins bearing D-glucosamine, L-fucosylamine, or para-amino-phenyl-alpha-D-mannopyrannoside residues. The interactions were inhibited by the carbohydrate moieties specific to the neoglycoproteins. The protein nature of the lectin-like components of L. monocytogenes was ascertained by the loss of carbohydrate-binding capacity following protease treatment.     

Factors affecting the digestion of restriction endonucleases in situ.

The influence of incubation buffers and glycerol on the enzyme activity of naked DNA of lambda phage and mouse, and of mouse chromosomal DNA was investigated. The results obtained varied in part from previously known data, but confirmed the importance of these factors in determining the patterns of in situ restriction enzyme digestion so far attributed exclusively to endonuclease activity.     

Some factors affecting the response of muscle to insulin

1. The effect of various changes in the composition of the supporting medium on the capacity of isolated rat diaphragm to incorporate amino acids into its protein has been studied. 2. Replacement of most of the normal ionic constituents by sucrose is inhibitory towards protein synthesis, as is also substitution of choline or K(+) for Na(+). 3. The capacity of the tissue to respond to a stimulatory effect of insulin is impaired in the sucrose media and under certain conditions in the absence of Na(+), particularly when Na(+) is replaced by K(+) and the (14)C-labelled amino acid is presented at a relatively high concentration. 4. Cutting of the tissue before incubation also decreases incorporating capacity and markedly decreases responsiveness to insulin. 5. In abnormal media the cellular content of ATP falls sharply. 6. The ATP content of the tissue also declines in the presence of 2-deoxyglucose. This change is prevented by the addition of glucose but not of pyruvate and succinate. 7. Although affecting the rate of amino acid incorporation the ATP content is not thought generally to limit sensitivity to insulin.     

Factors affecting the microbial digestion of an industrial seaweed-based residue

Commercial preparation of a seaweed extract from the brown alga Ascophyllum nodosum for use as fertiliser and soil improver produces a sludge residue which requires remediation. This residue is rich in nutrients and offers the potential for other value-added products. The residue composition was analysed, a microbial digestion process for the residue was developed, and several factors affecting the digestion process were studied. The residue showed an alkaline pH (8.61?±?0.39) and 16% (w/w) total solids, which comprised 40.6% mineral, 29.5% fibre, 24.3% lipid, 4.9% protein and 0.5% polyphenols. The optimised digestion system included a 3-day anaerobic phase to decrease pH (from 8.96?±?0.40 to 7.72?±?0.38), the addition of an inoculum, followed by a 10-day aerobic phase where the insoluble material was digested. Every 3 days, the solubilised material was decanted and replaced with water to delay metabolite inhibition. The rate of digestion (decrease in insoluble material of 28.6?±?14.2% over 13 days) was influenced by the initial insoluble (R ²?=?0.773) and soluble (R ²?=?0.672) matter, the pH at the beginning of the aerobic phase (R ²?=?0.528) and by the accumulation of solubilised digestion products. A compositional analysis of the insoluble material after digestion showed that the lipid content of the residue was 96% digested and that the proportion of protein increased by 82.4%. Inocula and metabolite inhibition were critical features of A. nodosum residue digestion. Similar organic residues require a carefully chosen inoculum and a minimum initial insoluble content (65–70%) and/or a maximum soluble content (25.30%).     

Factors affecting the digestion of moss DNA by restriction endonucleases

Abstract

The age of gametophytic tissues, de-starching, inclusion of PVP in the extraction medium, and column purification of isolated DNA have little or no effect upon the restriction of total DNA of Physcomitrella patens (Hedw.) Bruch, Schimp. & W.Gümbel. The relative longevity of restriction enzymes also appears unimportant. However, the extent of digestion of moss DNA by a given restriction endonuclease appears to correlate inversely with the number of cytosine residues in its recognition sequence that are susceptible to methylation in plant cells. Inclusion of spermidine in the restriction buffer slightly enhances restriction by a few specific endonucleases. This knowledge has practical significance when designing experiments in which it is desirable that restriction of isolated DNA samples is taken to completion.