Partial reconstruction of the microvillus core bundle: characterization of villin as a Ca(++)-dependent, actin-bundling/depolymerizing protein

The brush border, isolated from chicken intestine epithelial cells, contains the 95,000 relative molecular mass (M(r)) polypeptide, villin. This report describes the purification and characterization of villin as a Ca(++)-dependent, actin bundling/depolymerizing protein. Then 100,000 g supernatant from a Ca(++) extract of isolated brush borders is composed of three polypeptides of 95,000 (villin), 68,000 (fimbrin), and 42,000 M(r) (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. Electron microscopy of negatively stained preparations of these villin-actin mixtures showed that filament bundles were present. This viscosity, sedimentability, and ultrastructural morphology of filament bundles are dependent on the villin:actin molar ratio, the pH, and the free Ca(++) concentration in solution. At low free Ca(++) (less than 10(-6) M), the amount of protein in bundles, when measured by sedimentation, increased as the villin: actin molar ratio increased and reached a plateau at approximately a 4:10 ratio. This behavior correlates with the conversion of single actin filaments into filament bundles as detected in the electron microscope. At high free Ca(++) (more than 10(-6) M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer. Furthermore, these Ca(++) effects were correlated with the loss of protein sedimented, the disappearance of filament bundles, and the appearance of short fragments of filaments. Bundle formation is also pH-sensitive, being favored at mildly acidic pH. A decrease in the pH from 7.6 to 6.6 results in an increase in sedimentable protein and also a transformation of loosly associated actin filaments into compact actin bundles. These results are consistent with the suggestions that villin is a bundling protein in the microvillus and is responsible for the Ca(++)-sensitive disassembly of the microvillar cytoskeleton. Thus villin may function in the cytoplasm as a major cytoskeletal element regulating microvillar shape.     

Reevaluation of brush border motility: calcium induces core filament solation and microvillar vesiculation

The report that microvillar cores of isolated, demembranated brush borders retract into the terminal web in the presence of Ca(++) and ATP has been widely cited as an example of Ca(++)-regulated nonmuscle cell motility. Because of recent findings that microvillar core actin filaments are cross-linked by villin which, in the presence of micromolar Ca(++), fragments actin filaments, we used the techniques of video enhanced differential interference contrast, immunofluorescence, and phase contrast microscopy and thin-section electron microscopy (EM) to reexamine the question of contraction of isolated intestinal cell brush borders. Analysis of video enhanced light microscopic images of Triton- demembranated brush borders treated with a buffered Ca(++) solution shows the cores disintegrating with the terminal web remaining intact; membranated brush borders show the microvilli to vesiculate with Ca(++). Using Ca(++)/EGTA buffers, it is found that micromolar free Ca(++) causes core filament dissolution in membranated or demembranated brush borders, Ca(++) causes microvillar core solation followed by complete vesiculation of the microvillar membrane. The lengths of microvilli cores and rootlets were measured in thin sections of membranated and demembranated controls, in Ca(++)-, Ca(++) + ATP-, and in ATP-treated brush borders. Results of these measurements show that Ca(++) alone causes the complete solation of the microvillar cores, yet the rootlets in the terminal web region remain of normal length. These results show that microvilli do not retract into the terminal web in response to Ca(++) and ATP but rather that the microvillar cores disintegrate. NBD-phallicidin localization of actin and fluorescent antibodies to myosin reveal a circumferential band of actin and myosin in mildly permeabilized cells in the region of the junctional complex. The presence of these contractile proteins in this region, where other studies have shown a circumferential band of thin filaments, is consistent with the hypothesis that brush borders may be motile through the circumferential constriction of this “contractile ring,” and is also consistent with the observations that ATP-treated brush borders become cup shaped as if there had been a circumferential constriction.     

The sensitivity of Afromontane tarns in the Maloti-Drakensberg region of South Africa and Lesotho to acidic deposition

Despite their remoteness from sources of atmospheric pollutant emissions, the Afromontane tarns in the Maloti-Drakensberg region are perfect candidates to study the negative effects of acidifying atmospheric pollution, because mountain lakes are widely recognised as sentinel ecosystems, unimpacted by direct human disturbance within their catchments. Thirty-four tarns were sampled in the Maloti-Drakensberg region and most were found to be extremely sensitive to acidic deposition, as indicated by their low acid neutralising capacity. There are very few studies of freshwater critical loads for any region within South Africa. The steady-state water chemistry model (SSWC) was adapted and used to determine critical loads, whereas exceedance was estimated relative to modelled regional deposition data, in order to understand the risk of harmful effects to aquatic ecosystems. Seventy-six percent of sampled sites across the Maloti-Drakensberg would exceed critical loads even at the lowest modelled deposition levels, but there are no current measured deposition data for the region. The sensitivity of the Maloti-Drakensberg tarns needs to be considered in future policy formulation regarding acceptable levels of acidifying atmospheric pollution from South Africa’s energy sector and indicates the need for assessing aquatic ecosystem impacts in other regions of South Africa.     

Predicting how adaptation to climate change could affect ecological conservation: secondary impacts of shifting agricultural suitability

Aim:  Ecosystems face numerous well‐documented threats from climate change. The well‐being of people also is threatened by climate change, most prominently by reduced food security. Human adaptation to food scarcity, including shifting agricultural zones, will create new threats for natural ecosystems. We investigated how shifts in crop suitability because of climate change may overlap currently protected areas (PAs) and priority sites for PA expansion in South Africa. Predicting the locations of suitable climate conditions for crop growth will assist conservationists and decision‐makers in planning for climate change. Location:  South Africa. Methods:  We modelled climatic suitability in 2055 for maize and wheat cultivation, two extensively planted, staple crops, and overlaid projected changes with PAs and PA expansion priorities. Results:  Changes in winter climate could make an additional 2 million ha of land suitable for wheat cultivation, while changes in summer climate could expand maize suitability by up to 3.5 million ha. Conversely, 3 million ha of lands currently suitable for wheat production are predicted to become climatically unsuitable, along with 13 million ha for maize. At least 328 of 834 (39%) PAs are projected to be affected by altered wheat or maize suitability in their buffer zones. Main conclusions:  Reduced crop suitability and food scarcity in subsistence areas may lead to the exploitation of PAs for food and fuel. However, if reduced crop suitability leads to agricultural abandonment, this may afford opportunities for ecological restoration. Expanded crop suitability in PA buffer zones could lead to additional isolation of PAs if portions of newly suitable land are converted to agriculture. These results suggest that altered crop suitability will be widespread throughout South Africa, including within and around lands identified as conservation priorities. Assessing how climate change will affect crop suitability near PAs is a first step towards proactively identifying potential conflicts between human adaptation and conservation planning.     

Functional aspects of co-variant surface charges in an antibody fragment

A mutational analysis of three co-variant pairs of residues, located at the surface of a single-chain fragment, variable (scFv), remote from the antigen-binding site, was performed to investigate the tolerance of these positions to amino acid changes. The replacements consisted of the elimination or addition of charges, or in their replacement by a charge of opposite sign. As measured by Biacore, antigen-binding kinetics and specificity were essentially unaffected by the mutations. The purified scFvs remained mostly 100% active for 14 h, and their sensitivity to guanidinium-chloride denaturation was similar. These observations indicate that the mutations did not affect antigen-binding properties and that protein folding was conserved. However, the various scFvs differed greatly in half-life in periplasmic extracts (<4 h to >16 h at 25 degrees C). The deleterious effect on half-life produced by single mutations could be reversed by introducing a second mutation that restores the natural combination of amino acids in the co-variant pair, indicating that the consequence of charge modifications at these locations depends on the sequence context. We propose that the differences in half-life result from differences in aggregation propensities with other periplasmic proteins, related to the presence of charged patches at the surface of the scFvs. The practical implication is that changes in surface charge may drastically affect the level of active molecules in complex protein mixtures, a potentially important consideration in engineering scFvs for biotechnological or medical purposes.     

Purification and partial characterization of a murein hydrolase, millericin B, produced by Streptococcus milleri NMSCC 061

Streptococcus milleri NMSCC 061 was screened for antimicrobial substances and shown to produce a bacteriolytic cell wall hydrolase, termed millericin B. The enzyme was purified to homogeneity by a four-step purification procedure that consisted of ammonium sulfate precipitation followed by gel filtration, ultrafiltration, and ion-exchange chromatography. The yield following ion-exchange chromatography was 6.4%, with a greater-than-2,000-fold increase in specific activity. The molecular weight of the enzyme was 28,924 as determined by electrospray mass spectrometry. The amino acid sequences of both the N terminus of the enzyme (NH(2) SENDFSLAMVSN) and an internal fragment which was generated by cyanogen bromide cleavage (NH(2) SIQTNAPWGL) were determined by automated Edman degradation. Millericin B displayed a broad spectrum of activity against gram-positive bacteria but was not active against Bacillus subtilis W23 or Escherichia coli ATCC 486 or against the producer strain itself. N-Dinitrophenyl derivatization and hydrazine hydrolysis of free amino and free carboxyl groups liberated from peptidoglycan digested with millericin B followed by thin-layer chromatography showed millericin B to be an endopeptidase with multiple activities. It cleaves the stem peptide at the N terminus of glutamic acid as well as the N terminus of the last residue in the interpeptide cross-link of susceptible strains.