Optical detection of triplet-state magnetic resonance studies on individual tryptophan residues of serum albumin: correlation between phosphorescence and zero-field splittings

Cyanogen bromide cleavage of bovine serum albumin (BSA) yields two fragments, N (1-183) and C (184-582), containing 183 and 399 amino acid residues, respectively. Each in each fragment are characterized in this study by phosphorescence and optically detected magnetic resonance spectroscopy, and the results are compared with those of the intact albumin. Trp-134 in fragment N is located in a hydrophobic environment in the interior of the protein, as reflected by its red-shifted phosphorescence and characteristic zero-field splittings. The spectral properties of Trp-212 in fragment C suggest its location in a partially buried, inhomogeneous environment. They show great similarity to those of human serum albumin, which contains a single Trp at position 214. The Trp phosphorescence 0,0-bands of fragments C and N are fitted with Gaussian functions by computer, and their relative contributions to the phosphoresence 0,0-band of BSA are adjusted to fit the observed BSA 0,0-band. The wavelength dependence of the [D[-[E[ transition frequencies of fragments N and C is then weighted by their 0,0-band intensity, taking into account differences in spin alignment, and summed to predict the peak frequency of the [D[-[E[ band profile as a function of phosphorescence wavelength for the intact BSA. Good agreement between predicted and observed behavior of [D[-[E[ vs. wavelength for the intact protein provides strong evidence for the additivity of the phosphorescence and ODMR spectra of the individual Trp sites in BSA. We find that Trp-134 and Trp-212 have wavelength-independent and wavelength-dependent zero-field splittings, respectively.     

Domain II + III of bovine serum albumin: Isolation and its characterization

Some properties of a fragment of bovine serum albumin containing residues 184–582 of the protein sequence, produced by cyanogen bromide cleavage, have been reported. Urea-induced difference spectra of the fragment showed considerable exposure of aromatic chromophores by 8 M urea. Reversible unfolding of the fragment by urea, as followed by difference spectral measurements at 30°C, pH 7.0, occurred in two distinct steps involving at least 3 major conformational states, namely the native (N), intermediate (X) and completely denatured (D) states. The co-operativity values for the two transitions, N⇌X and X⇌Dwere found to be 4.0 and 16.4, respectively. Analysis of the data on bilirubin binding to bovine serum albumin and its fragment suggested that the fragment retains significant amount of its native structure. However, hydrodynamic parameters such as Stokes radius (3.f14 nm), diffusion coefficient (6.98 × 10⁻⁷cm²/s) and frictional ratio (1.32) obtained by analytical gel chromatography as well as intrinsic viscosity (4.31 ml/g) indicates some asymmetry in the fragment molecule.     

Direct evidence for the involvement of domain III in the N-F transition of bovine serum albumin.

The domain III of bovine serum albumin containing residues 377-582 of the protein sequence was isolated and its behaviour in acid solution was studied. The fragment was found to undergo structural transformations over the pH range 3.5-4.5 known to cause N-F transition in serum albumin. On the other hand, an albumin fragment that was devoid of domain III was unable to exhibit such a transition. These results were consistent with a mechanism where N-F transition involves the separation of domain III from the rest of the albumin starts at about pH 4.3 and is completed at pH 3.5.     

Equilibrium binding constants and facile dissociation of novel serum albumin-dicyanoaurate(I) complexes

 Dicyanoaurate(I), Au(CN)₂ ^–, an important metabolite of chrysotherapy agents (anti-arthritic gold drugs), contains two tightly bound cyanide ligands which render it relatively unreactive toward ligand exchange reactions with potential gold-binding ligands. The extent and nature of its binding to bovine serum albumin (BSA), which may modulare the in vivo activity of Au(CN)₂ ^–, were investigated to determine whether Au(CN)₂ ^– might be more bioavailable than other gold complexes. ¹³C NMR spectroscopy, radioisotope tracers, chromatography, ultrafiltration, and atomic spectroscopy, employing Au(¹³CN)₂ ^– or Au(¹⁴CN)₂ ^– as appropriate, revealed two distinct binding mechanisms. The dominant reaction is reversible association (non-specific binding) of intact Au(CN)₂ ^– ions to form BSA·[Au(CN)₂ ^–] _n adducts. Approximately one equivalent binds with an equilibrium binding constant (pH 7.4, 25  °C) of K ₁=5.5 (±1.1)×10⁴, and three additional equivalents bind with a constant of 7.0 (±0.1)×10³. Au(¹³CN)₂ ^– associated with albumin is characterized by a broad ¹³C NMR resonance at δ_C=154.7 ppm compared to the sharp resonance of the free complex at 156.4 ppm. The BSA·[Au(CN)₂ ^–] _n adducts readily dissociate during gel exclusion chromatography and are therefore underestimated, but are retained and accurately quantitated by ultrafiltration methods. The second binding mechanism is a ligand exchange reaction at Cys-34, to form AlbSAuCN, which accounts for only a small fraction (≤11%) of the bound gold. The small extent of the latter interaction differentiates Au(CN)₂ ^– from the gold drugs such as auranofin, aurothiomalate (Myochrysin) and aurothioglucose (Solganol), which undergo ligand exchange at Cys-34 of albumin to form tightly bound gold-protein complexes. The weak interaction at Cys-34 and the facile dissociation of bound, intact Au(CN)₂ ^– are consistent with its putative role as a gold metabolite that can be accumulated intracellularly. Received: 2 July 1997 / Accepted: 24 September 1997     

FTIR-monitored thermal titration reveals different mechanisms for the alkaline isomerization of tuna compared to horse and bovine cytochromes c

 Fourier transform infrared (FTIR) spectroscopy is used to compare the thermally induced conformational changes in horse, bovine and tuna ferricytochromes c in 50 mM phosphate/0.2 M KCl. Thermal titration in D₂O at pD 7.0 of the amide II intensity of the buried peptide NH protons reveals tertiary structural transitions at 54  °C in horse and at 57  °C in bovine c. These transitions, which occur well before loss of secondary structure, are associated with the alkaline isomerization involving Met80 heme-ligand exchange. In tuna c, the amide-II-monitored alkaline isomerization occurs at 35  °C, followed by a second amide II transition at 50  °C revealing a hitherto unreported conformational change in this cytochrome. Amide II transitions at 50  °C (tuna) and 54  °C (horse) are also observed during the thermal titration of the CN^–-ligated cytochromes (where CN^– displaces the Met80 ligand), but a well-defined 35  °C amide II transition is absent from the titration curve of the CN^–adduct of tuna c. The different mechanisms suggested by the FTIR data for the alkaline isomerization of tuna and the mammalian cytochromes c are discussed. After the alkaline isomerization, loss of secondary structure and protein aggregation occur within a 5  °C range with T _m values at 74  °C (bovine c), 70  °C (horse c) and 65  °C (tuna c), as monitored by changes in the amide I′ bands. The FTIR spectra were also used to compare the secondary structures of the ferricytochromes c at 25  °C. Curve fitting of the amide I (H₂O) and amide I′ (D₂O) bands reveals essentially identical secondary structure in horse and bovine c, whereas splitting of the α-helical absorption of tuna c indicates the presence of less-stable helical structures. CN^– adduct formation results in no FTIR-detectable changes in the secondary structures of either tuna or horse c, indicating that Met80 ligation does not influence the secondary structural elements in these cytochromes. The data provided here demonstrate for the first time that the selective thermal titration of the amide II intensity of buried peptide NH protons in D₂O is a powerful tool in protein conformational analysis. Received: 1 April 1999 / Accepted: 24 August 1999     

Homoplasy corrected estimation of genetic similarity from AFLP bands,and the effect of the number of bands on the precision of estimation

AFLP is a DNA fingerprinting technique, resulting in binary band presence–absence patterns, called profiles, with known or unknown band positions. We model AFLP as a sampling procedure of fragments, with lengths sampled from a distribution. Bands represent fragments of specific lengths. We focus on estimation of pairwise genetic similarity, defined as average fraction of common fragments, by AFLP. Usual estimators are Dice (D) or Jaccard coefficients. D overestimates genetic similarity, since identical bands in profile pairs may correspond to different fragments (homoplasy). Another complicating factor is the occurrence of different fragments of equal length within a profile, appearing as a single band, which we call collision. The bias of D increases with larger numbers of bands, and lower genetic similarity. We propose two homoplasy- and collision-corrected estimators of genetic similarity. The first is a modification of D, replacing band counts by estimated fragment counts. The second is a maximum likelihood estimator, only applicable if band positions are available. Properties of the estimators are studied by simulation. Standard errors and confidence intervals for the first are obtained by bootstrapping, and for the second by likelihood theory. The estimators are nearly unbiased, and have for most practical cases smaller standard error than D. The likelihood-based estimator generally gives the highest precision. The relationship between fragment counts and precision is studied using simulation. The usual range of band counts (50–100) appears nearly optimal. The methodology is illustrated using data from a phylogenetic study on lettuce.     

The clearance of human fibrinogen fragments D1, D2, D3 and fibrin fragment D1 dimer in mice

The clearance of human fibrinogen fragments D₁, D₂, D₃ and fibrin fragment D₁ dimer were studied in the mouse model. Clearance of these fragments is a complex process involving clearance from blood into three other compartments. The overall clearance of fragment D₁ and its dimer were essentially identical. Fragments D₂ and D₃ cleared at a progressively slower rate. Competition studies were performed between ¹²⁵I-labeled fragment D₁ and large molar excesses of unlabelled human fragments D₁, D₂, D₃, D₁ dimer, fragment E, fibrinogen, macroalbumin, mannan and asialooroscomucoid. Of these ligands only the fragment D variants competed for the clearance of ¹²⁵I-labeled fragment D₁. Cross-competition was observed when ¹²⁵I-labeled fragment D₁ dimer was cleared in the presence of large molar excesses of fragment D₁. Autopsies demonstrated that injected fragments D₁, D₂, D₃ and D₁ dimer cleared primarily in liver and kidneys. In some clearance studies, livers were perfused with tissue culture fluid, subjected to light microscopic autoradiography, and silver grain counts performed to localize cleared fragment D₁. These experiments indicated that 80% of the liver uptake was in hepatocytes. However, when silver grain counts were normalized for the number of parenchymal and nonparenchymal cells, the distribution of silver grains was essentially identical (1.8 and 1.6 grains per cell, respectively). It is concluded that fragments D₁, D₂, D₃ and D₁ dimer are recognized by a similar clearance pathway. Since neither fibrinogen nor fragment E competed for the clearance of fragment D₁, it is suggested that determinants present in the fragment D domain become exposed after plasmin attack on fibrinogen and are responsible for clearance.     

Summer/winter differences in the serum 25-hydroxyvitamin D3 and parathyroid hormone levels of Japanese women

Serum 25-hydroxyvitamin D₃ [25(OH)D₃] is produced in the skin in response to exposure to ultraviolet radiation, and is a good indicator of vitamin D nutritional status. The aim of this study was to determine summer/winter differences in serum 25(OH)D₃ and parathyroid hormone (PTH) in Japanese women and how the summer and winter values are related. The subjects were 122 healthy Japanese women aged 45–81 years (average age: 65.7 years). They were medically examined twice, in September 1997 and February 1999. Serum 25(OH)D₃ and intact PTH were determined by high-performance liquid chromatography and a two-site immunoradiometric assay respectively. Lifestyle information was obtained through an interview. The seasonal differences (winter minus summer) in 25(OH)D₃ [Δ25(OH)D₃] and intact PTH concentrations were –18.8 nmol/l (SD 19.2, P<0.0001) and 0.98pmol/l (SD 1.02, P<0.0001) respectively. The correlation coefficient between summer (x) and winter (y) 25(OH)D₃ levels was 0.462 (P<0.0001), with a linearly fitted line of y=0.42x+26.4. This relationship was interpreted as subjects with higher summer 25(OH)D₃ values having greater reductions in winter 25(OH)D₃ concentrations. There were inter-individual differences in Δ25(OH)D₃, although the summer and winter 25(OH)D₃ concentrations were well-correlated. Since Δ25(OH)D₃ was not associated with any of the lifestyle factors, seasonal differences in the 25(OH)D₃ concentrations of an individual appeared to reflect her ability to produce 25(OH)D₃ photochemically in the skin. Sun bathing would be a less effective means of attaining adequate vitamin D nutritional status in a person with a small seasonal difference in 25(OH)D₃, i.e., one with a low 25(OH)D₃ level. Received: 17 December 1999 / Revised: 24 April 2000 / Accepted: 10 May 2000     

Theoretical studies on 2-diazo-4,6-dinitrophenol derivatives aimed at finding superior propellants

In an attempt to find superior propellants, 2-diazo-4,6-dinitrophenol (DDNP) and its –NO₂, –NH₂, –CN, –NC, –ONO₂, and –NF₂ derivatives were studied at the B3LYP/6-311++G^** level of density functional theory (DFT). Sensitivity was evaluated using bond dissociation enthalpies (BDEs) and molecular surface electrostatic potentials. The C–NO₂ bond appears to be the trigger bond during the thermolysis process for these compounds, except for the –ONO₂ and –NF₂ derivatives. Electrostatic potential results show that electron-withdrawing substituents make the charge imbalance more anomalous, which may change the strength of the bond, especially the weakest trigger bond. Most of the DDNP derivatives have the impact sensitivities that are higher than that of DDNP, making them favorable for use as solid propellants in micro-rockets. The theoretical densities (ρ), heats of formation (HOFs), detonation energies (Q), detonation pressures (P), and detonation velocities (D) of the compounds were estimated. The effects of various substituent groups on ρ, HOF, Q, D, and P were investigated. Some derivatives exhibit perfect detonation properties. The calculated relative specific impulses (I _r,sp) of all compounds except for –NH₂ derivatives were higher than that of DDNP, and also meet the requirements of propellants.     

Evidence for the spontaneous formation of interspecies hybrid molecules of human, rat and bovine serum albumins

Utilizing electrophoretic and gel filtration techniques it was shown that a bovine C-terminal peptic fragment [residues 307-582] spontaneously forms interspecies hybrid molecules with three complementary N-terminal fragments derived from human [residues 1-308; 49-308] and rat [residues 1-308] albumins. The fragments associate with 1:1 stoichiometry to produce an albumin-like complex which has a molecular weight and electrophoretic mobility similar to intact albumin. These data demonstrate, for the first time, that albumin fragments derived from different species associate in a complementary fashion and provide direct evidence that the tertiary structure may be highly conserved.     

Diversity and genetic connectivity among populations of a threatened tree (<Emphasis Type="Italic">Dalbergia nigra</Emphasis>) in a recently fragmented landscape of the Brazilian Atlantic Forest

In this study we evaluated the influence of recent landscape fragmentation on the dynamics of remnant fragments from the Brazilian Atlantic Forest. This biome is one of the richest in the world and has been extensively deforested and fragmented. We sampled five populations of the threatened Dalbergia nigra, a tree endemic to the Brazilian Atlantic Forest, two located in a large reserve of continuous forest and three in fragments of different sizes and levels of disturbance. In order to assess historical changes, considering the longevity of the analyzed species, 119 adults and 116 saplings were genotyped for six microsatellite loci. Lower levels of genetic diversity were found in the most impacted fragments when compared to the most preserved population located inside the reserve, and there was significant genetic structure among the populations studied (pairwise F _ST = 0.031–0.152; pairwise D _EST = 0.039–0.301). However, genetic structure among saplings (F _ST = 0.056; D _EST = 0.231) was significantly lower than among adults (F _ST = 0.088; D _EST = 0.275). Estimates of contemporary gene flow based on assignment tests corroborated this result, suggesting that fragmentation led to an increase in gene flow. This connectivity among remnant fragments could mitigate the loss of genetic diversity through a metapopulation dynamic, but the high rate of habitat loss and the unknown long-term genetic effects add uncertainty. These results, taken together with the presence of private alleles in disturbed populations, highlight the importance of preserving the extant fragments.     

STS markers linked to the Rf 1 fertility restorer gene of cotton

Marker-assisted selection (MAS) can accelerate the process of plant breeding, and sequence-tagged site (STS) markers are highly specific for regions of DNA being used for MAS. The objective of this research was to develop STS markers tightly linked with Rf₁, the fertility restoring gene for cytoplasmic male sterility (CMS) in cotton (Gossypium hirsutum L.). Bulked segregant analysis was employed to screen for Rf₁-linked RAPD markers in a backcross population. Four RAPD markers were identified, three of which co-segregated with Rf₁ (UBC147₁₄₀₀, UBC607₅₀₀, and UBC679₇₀₀). Another fragment, UBC169₈₀₀, co-segregated with the previously reported UBC169₇₀₀ in repulsion phase at a distance of 4.5 cM from Rf₁. A marker published by others (UBC659₁₅₀₀) mapped to 2.7 cM from Rf₁ and 1.8 cM from UBC169₈₀₀. Four sets of STS primer pairs were designed based on the RAPD fragment sequences. The primer pairs from the UBC147₁₄₀₀ and UBC607₅₀₀ fragments both amplified a single fragment specific to fertile plants. The UBC679₇₀₀ and UBC659₁₅₀₀ STS primer pairs each amplified one fragment specific to fertile plants and a monomorphic fragment. These four Rf₁-linked STS markers were also present in the Rf₁ donor species G. harknessii (D_2-2). The three primer pairs that produced co-segregating STS markers also amplified fragments from G. trilobum (D₈). However, the D₈ fragment amplified by the UBC147₁₄₀₀ STS primers was larger than that from D_2-2, and G. trilobum does not restore fertility to CMS-D2-2 lines. These STS markers will be useful in the development of restorer parental lines in cotton CMS breeding efforts.     

Diet and reproduction in the Australian butterfly ray Gymnura australis from northern and north‐eastern Australia

The diet of Gymnura australis was dominated by teleosts (99·8% index of relative importance). A wide‐ranging species, females matured at 446 mm disc width (W_D), had a single functional ovary and two functional uteri. Males matured at 377 mm W_D and had a single functional testis.     

Surface Rheology Parameters of Source-Specific Surfactant Films as Indicators of Organic Matter Dynamics

The paper contains the results of natural film experiments carried out on inland and coastal waters in the Dead Vistula catchment area and mouth during 2000–2002, using the integrated Langmuir trough–Wilhelmy plate system. The static film parameters result from the generalized scaling procedures applied to the surface pressure–area isotherms. They appear to reflect in a quantitative and sensitive way the film composition (A_lim, M_w, E_isoth), film solubility and the miscibility of its components (via R, ΔS_c and y factors), and surface concentration (π_eq, Γ_eq). The adsorption kinetics parameters: effective diffusion coefficient D_eff/D and activation energy barrier E_a/RT are derived from dynamic surface pressure. There is a reason to suggest that certain classes of film-forming components or ‘end-members’ may dominate the static and dynamic surface properties. Variation in the surface rheological parameters of source-specific biosurfactants is postulated to reflect organic matter dynamics in natural waters and were measured for the Dead Vistula river, its tributaries and the adjacent coastal area.     

Fine mapping of the region on wheat chromosome 7D controlling grain weight

We report the fine mapping of the previously described quantitative trait loci (QTL) for grain weight QTgw.ipk-7D associated with microsatellite marker Xgwm1002-7D by using introgression lines (ILs) carrying introgressions of the synthetic wheat W-7984 in the genetic background of the German winter wheat variety ‘Prinz’. The BC₄F₃ ILs had a 10% increased thousand grain weight compared to the control group and the recurrent parent ‘Prinz’, and 84.7% of the phenotypic variance could be explained by the segregation of marker Xgwm1002-7D, suggesting the presence of a gene modulating grain weight, which was preliminarily designated gw1. It was possible to delimit the QTL QTgw.ipk-7D to the interval Xgwm295–Xgwm1002, which is located in the most telomeric bin 7DS4-0.61-1.00 in the physical map of wheat chromosome arm 7DS. Furthermore, our data suggest the presence of a novel plant height-reducing locus Rht on chromosome arm 7DS of ‘Prinz’. Larger grain and increased plant height may reflect the pleiotropic action of one gene or may be caused by two linked genes. In general, our data support the concept of using nearly isogenic ILs for validating and dissecting QTLs into single Mendelian genes and open the gateway for map-based cloning of a grain-weight QTL in wheat.     

Analysis of hybrids of durum wheat with Thinopyrum junceiforme using RAPD markers

 The objective of this study was to detect the presence of alien chromatin in intergeneric hybrids of durum wheat (Triticum turgidum, 2n=4x=28; AABB genomes) with the perennial grass Thinopyrum junceiforme (2n=4x=28; J₁J₁J₂J₂) using RAPD markers. The first step was to identify amplification of species-specific DNA markers in the parental grass species and durum wheat cultivars. Initially, the genomic DNA of five grass species (Thinopyrum junceiforme, Th. bessarabicum, Lophopyrum elongatum, Leymus karataviensis and Elytrigia pycnantha) and selected durum cultivars (‘Langdon’, ‘Durox’, ‘Lloyd’, ‘Monroe’, and ‘Medora’) was screened with 40 oligonucleotide primers (nano-mers). Three oligonucleotides that amplified DNA fragments specific to a grass species or to a durum cultivar were identified. Primer PR21 amplified DNA fragments specific to each of the five durum cultivars, and primers PR22 and PR23 amplified fragments specific to each of the grass species. Intergeneric hybrids between the durum cultivars ‘Langdon’, ‘Lloyd’ and ‘Durox’ and Th. junceiforme, and their backcross (BC) progeny were screened with all 40 primers. Six primers amplified parent-specific DNA fragments in the F₁ hybrids and their BC₁ progeny. Three primers, PR22, PR23 and PR41, that amplified Th. junceiforme DNA fragments in both F₁ and BC₁ were further analyzed. The presence of an amplified 1.7-kb Th. junceiforme DNA fragment in the F₁ hybrids and BC₁ progeny was confirmed using Southern analysis by hybridization with both Th. junceiforme genomic DNA and Th. junceiforme DNA amplified with primer PR41. With the exception of line BC₁F₂ no. 5, five selfed progeny of BC₁ and a BC₂ of line 3 (BC₁F₂ no. 3בLloyd’) from a cross of ‘Lloyd’×Th. junceiforme showed the presence of the 1.7-kb DNA fragment. All selfed BC₁ and BC₂ lines retained the 600-bp fragment that was confirmed after hybridization with Th. junceiforme DNA amplified with primer PR22. Other experiments using RFLP markers also showed the presence of up to seven Th. junceiforme DNA fragments in the F₁ hybrids and their BC progeny after hybridization with Th. junceiforme DNA amplified with primer PR41. These studies show the usefulness of molecular markers in detecting alien chromatin/DNA fragments in intergeneric hybrids with durum wheat. Received: 21 November 1996 / Accepted: 21 March 1997     

QM/MM investigation into binding of square-planar platinum complexes to DNA fragments

A series of calculations employing hybrid quantum mechanics/molecular mechanics methods to explore the binding of square-planar complexes to fragments of DNA are reported. Methylated analogues of the parent compound cis-[Pt(en)Cl₂], where en is ethylenediamine, show considerable variation in in vitro cytotoxicity depending on the number and position of methyl groups. Calculations reveal variations in the structure and the binding energy of adducts to single- and double-stranded fragments of DNA. Most such variations are relatively small, but the introduction of three or four methyls on nitrogen of ethylenediamine significantly changes the structure and reduces the binding energy, owing to replacement of strong N–H···O interactions by much weaker C–H···O contacts. Close correlation between measured activity and binding energy is observed, whereas little or no correlation is found with structural parameters or with estimates of the octanol/water partition coefficient, either alone or in combination via multiple linear regression.     

Effects on haemolytic activity of single proline substitutions in the <Emphasis Type="Italic">Bordetella pertussis</Emphasis> CyaA pore-forming fragment

The recombinant Bordetella pertussis CyaA pore-forming (CyaA-PF) fragment was previously shown to be expressed separately in Escherichia coli as a soluble precursor that can be in vivo palmitoylated to exert haemolytic activity. In this study, PCR-based mutagenesis was employed to investigate the contributions to haemolysis of five predicted helices within the N-terminal hydrophobic region of the CyaA-PF fragment. Single proline substitutions were made for alanine near the centre of each predicted helix as a means of disrupting local secondary structure. All mutant proteins were over-expressed in E. coli as a 126-kDa soluble protein at levels comparable to the wild-type. Marked reductions in haemolytic activity against sheep erythrocytes of mutants, A510P, A538P, A583P and A687P pertaining to the putative helices 1_500–522, 2_529–550, 3_571–593 and 5_678–698, respectively, were observed. However, a slight decrease in haemolytic activity was found for the proline replacement in the predicted helix 4_602–627 (A616P). MALDI–TOF–MS and LC–MS–MS analyses verified the palmitoylation at Lys⁹⁸³ of all five mutants as identical to that of the CyaA-PF wild-type protein, indicating that toxin modification via this acylation was not affected by the mutations. Altogether, these results suggest that structural integrity of the predicted helices 1, 2, 3 and 5, but not helix 4, is important for haemolytic activity, particularly for the putative transmembrane helices 2 and 3 that might conceivably be involved in pore formation of the CyaA-PF fragment.     

Hierarchic organization of globular proteins: Experimental studies on thermolysin

Previous studies from this laboratory have shown that the thermolysin fragment 121–316, comprising entirely the“all-α” COOH-terminal structural domain 158–316, as well as fragment 206–316 (fragment FII) are able to refold into a native-like, stable structure independently from the rest of the protein molecule. The present report describes conformational properties of fragments 228–316 and 255–316 obtained by chemical and enzymatic cleavage of fragment FII, respectively. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultra-violet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. Melting curves of the secondary structure of the fragments show cooperativity with a temperature of half-denaturationT _mof 65–66°C. The results of this study provide evidence that it is possible to isolate stable supersecondary structures (folding units) of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain 158–316 of thermolysin.     

Seeds and seedlings of two neotropical montane understory shrubs respond differently to anthropogenic edges and treefall gaps

Edges resulting from forest clear-cutting and treefall gaps can affect plant populations and consequently the distribution of species across landscapes. These two types of disturbance might interact to exacerbate or ameliorate “edge effects”, a rarely tested possibility. We focused on the effects of distance from forest edge (0–10, 30–40, 60–70, and 190–200 m) and habitat within forest fragments (treefall gaps and intact forest) on the early stages of development of Palicourea gibbosa and Faramea affinis, two common shrubs of montane forests in southwest Colombia. Seed germination and seedling growth did not change with distance from forest edge. Within forest fragments, however, seed germination and seedling growth were higher in treefall gaps than in intact forest understory for both species. In contrast, seed predation was influenced by distance from forest edge and in P gibbosa it depended on habitat. Seed predation was highest in the forest interior (190–200 m from forest edge) and in P. gibbosa this was true only in treefall gap habitats. These results suggest that animal mediated processes such as post-dispersal seed predation are more likely than physiological processes to be affected by anthropogenic edges. Our results provide some evidence that treefall gaps may interact with “edge effects”, however, they are inconclusive as to whether they exacerbate or ameliorate them. Received: 31 August 1998 / Accepted: 18 February 1999