Quantification of synapse formation and maintenance in vivo in the absence of synaptic release.

Outgrowing axons in the developing nervous system secrete neurotransmitters and neuromodulatory substances, which is considered to stimulate synaptogenesis. However, some synapses develop independent of presynaptic secretion. To investigate the role of secretion in synapse formation and maintenance in vivo, we quantified synapses and their morphology in the neocortical marginal zone of munc18-1 deficient mice which lack both evoked and spontaneous secretion [Science 287 (2000) 864]. Histochemical analyses at embryonic day 18 (E18) showed that the overall organization of the neocortex and the number of cells were similar in mutants and controls. Western blot analysis revealed equal concentrations of pre- and post-synaptic marker proteins in mutants and controls and immunocytochemical analyses indicated that these markers were targeted to the neuropil of the synaptic layer in the mutant neocortex. Electron microscopy revealed that at E16 immature synapses had formed both in mutants and controls. These synapses had a similar synapse diameter, active zone length and contained similar amounts of synaptic vesicles, which were immuno-positive for two synaptic vesicle markers. However, these synapses were three times less abundant in the mutant. Two days later, E18, synapses in the controls had more total and docked vesicles, but not in the mutant. Furthermore, synapses were now five times less abundant in the mutant. In both mutant and controls, synapse-like structures were observed with irregular shaped vesicles on both sides of the synaptic cleft. These 'multivesicular structures' were immuno-positive for synaptic vesicle markers and were four times more abundant in the mutant. We conclude that in the absence of presynaptic secretion immature synapses with a normal morphology form, but fewer in number. These secretion-deficient synapses might fail to mature and instead give rise to multivesicular structures. These two observations suggest that secretion of neurotransmitters and neuromodulatory substances is required for synapse maintenance, not for synaptogenesis. Multivesicular structures may develop out of unstable synapses.

Age-related changes in the ultrastructure of the resting follicle pool in human ovaries.

Age-related decline of fertility in women is the result of the decline in both quantity and quality of the resting ovarian follicle pool. The aim of the present study was to determine whether the decline of follicle quality with age is reflected by ultrastructural changes in the resting follicle pool. Ovarian biopsy specimens were obtained by laparoscopy from seven healthy women aged 25-32 yr (young group) and from 11 healthy women aged 38-45 yr (advanced-age group). A total of 182 resting follicles from the young group were compared with 81 resting follicles from the advanced-age group for signs of age-related changes by transmission-electron microscopy. The ooplasmic fraction of vacuoles was increased (P = 0.02), and the fraction of mitochondria decreased (P = 0.005), in the advanced-age group. Also, the density of the mitochondrial matrix (P < 0.001) and the frequency of dilated smooth endoplasmic reticulum (SER; P = 0.001) and Golgi complex (P = 0.02) were increased with age. The frequencies of ruptured mitochondrial membranes (P = 0.001) and dilated SER (P = 0.003) were increased with age in the granulosa cells. Overall follicle-quality scores, which should reflect atretic changes, were not different for the young and advanced-age groups. In conclusion, in resting follicles, the morphological changes with age are different from the changes seen in quality decline by atresia. The morphological changes with age specifically involved the mitochondria, the SER, and the Golgi complex, and they may be the cause of atresia on initiation of follicular growth because of the substantial increase in metabolic requirements.

Endosomal compartmentalization in three dimensions: implications for membrane fusion.

Endosomes are major sorting stations in the endocytic route that send proteins and lipids to multiple destinations in the cell, including the cell surface, Golgi complex, and lysosomes. They have an intricate architecture of internal membrane structures enclosed by an outer membrane. Recycling proteins remain on the outer membrane, whereas proteins that are destined for degradation in the lysosome are sorted to the interior. Recently, a retrograde pathway was discovered whereby molecules, like MHC class II of the immune system, return from the internal structures to the outer membrane, allowing their further transport to the cell surface for T cell activation. Whether this return involves back fusion of free vesicles with the outer membrane, or occurs via the continuity of the two membrane domains, is an unanswered question. By electron tomography of cryo-immobilized cells we now demonstrate that, in multivesicular endosomes of B-lymphocytes and dendritic cells, the inner membranes are free vesicles. Hence, protein transport from inner to outer membranes cannot occur laterally in the plane of the membrane, but requires fusion between the two membrane domains. This implies the existence of an intracellular machinery that mediates fusion between the exoplasmic leaflets of the membranes involved, which is opposite to regular intracellular fusion between cytoplasmic leaflets. In addition, our 3D reconstructions reveal the presence of clathrin-coated areas at the cytoplasmic face of the outer membrane, known to participate in protein sorting to the endosomal interior. Interestingly, profiles reminiscent of inward budding vesicles were often in close proximity to the coats.

Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography.

Cryoimmobilization is regarded as the most reliable method to preserve cellular ultrastructure for electron microscopic analysis, because it is both fast (milliseconds) and avoids the use of harmful chemicals on living cells. For immunolabelling studies samples have to be dehydrated by freeze-substitution and embedded in a resin. Strangely, although most of the lipids are maintained, intracellular membranes such as endoplasmic reticulum, Golgi and mitochondrial membranes are often poorly contrasted and hardly visible. By contrast, Tokuyasu cryosectioning, based on chemical fixation with aldehydes is the best established and generally most efficient method for localization of proteins by immunogold labelling. Despite the invasive character of the aldehyde fixation, the Tokuyasu method yields a reasonably good ultrastructural preservation in combination with excellent membrane contrast. In some cases, however, dramatic differences in cellular ultrastructure, especially of membranous structures, could be revealed by comparison of the chemical with the cryofixation method. To make use of the advantages of the two different approaches a more general and quantitative knowledge of the influence of aldehyde fixation on ultrastructure is needed. Therefore, we have measured the size and shape of endosomes and lysosomes in high-pressure frozen and aldehyde-fixed cells and found that aldehyde fixation causes a significant deformation and reduction of endosomal volume without affecting the membrane length. There was no considerable influence on the lysosomes. Ultrastructural changes caused by aldehyde fixation are most dramatic for endosomes with tubular extensions, as could be visualized with electron tomography. The implications for the interpretation of immunogold localization studies on chemically fixed cells are discussed.

Ultrastructure of the resting ovarian follicle pool in healthy young women.

In humans, follicle quantity and quality decline with age by atresia. In the present study we aimed to describe the quality of the follicle pool through an ultrastructural investigation of resting follicles in young healthy women. From ovarian biopsies of 7 women aged 25-32 yr, 182 small follicles were morphometrically assessed for various signs of atresia. Morphometric variables were analyzed by principal components analysis (PCA) to demonstrate correlations between variables and to construct an objective follicle score. One third of small follicles consisted of primordial follicles. Nucleus:cell ratios remained constant for oocytes and granulosa cells from primordial to primary follicles, suggesting that follicles up to primary stages belong to the resting pool. The distribution of follicle quality scores as derived from PCA showed that most follicles were of good quality and with little signs of atresia. Atresia in resting follicles appears to be a necrotic process, starting in the ooplasma. Early atresia was characterized by increasing numbers of multivesicular bodies and lipid droplets, dilation of smooth endoplasmic reticulum and Golgi, and irregular mitochondria with changed matrix density. In progressive atresia mitochondrial membranes ruptured, oocyte nuclear membranes were indented or ruptured, and the ooplasma showed extensive vacuolarization. The early involvement of mitochondria in this process suggests that damage is induced by oxygen radicals. PCA follicle quality scores can be reliably approximated using a reduced number of seven morphometric variables, which were selected by stepwise forward analysis. The algorithm to calculate these follicle scores is presented.

The ER to Golgi interface is the major concentration site of secretory proteins in the exocrine pancreatic cell.

By using quantitative immuno-electron microscopy of two-sided labeled resin sections of rat exocrine pancreatic cells, we have established the relative concentrations of the secretory proteins amylase and chymotrypsinogen in the compartments of the secretory pathway. Their total concentration over the entire pathway was approximately 11 and approximately 460 times, respectively. Both proteins exhibited their largest increase in concentration between the endoplasmic reticulum and cis-Golgi, where they were concentrated 3-4 and 50-70 times, respectively. Over the further pathway, increases in concentration were moderate, albeit two times higher for chymotrypsinogen than for amylase. From trans-Golgi to secretory granules, where the main secretory protein concentration is often thought to occur, relatively small concentration increases were observed. Additional observations on a third secretory protein, procarboxypeptidase A, showed a concentration profile very similar to chymotrypsinogen. The relatively high concentration of amylase in the early compartments of the secretory route is consistent with its exceptionally slow intracellular transport. Our data demonstrate that secretory proteins undergo their main concentration between the endoplasmic reticulum and cis-Golgi, where we have previously found concentration activity associated with vesicular tubular clusters (Martínez-Menárguez JA, Geuze HJ, Slot JW, Klumperman J. Cell 1999; 98: 81-90).

Synthetic peptides derived from the beta2-beta3 loop of Raphanus sativus antifungal protein 2 that mimic the active site.

Rs-AFPs are antifungal proteins, isolated from radish (Raphanus sativus) seed or leaves, which consist of 50 or 51 amino acids and belong to the plant defensin family of proteins. Four highly homologous Rs-AFPs have been isolated (Rs-AFP1-4). The structure of Rs-AFP1 consists of three beta-strands and an alpha-helix, and is stabilized by four cystine bridges. Small peptides deduced from the native sequence, still having biological activity, are not only important tools to study structure-function relationships, but may also constitute a commercially interesting target. In an earlier study, we showed that the antifungal activity of Rs-AFP2 is concentrated mainly in the beta2-beta3 loop. In this study, we synthesized linear 19-mer peptides, spanning the entire beta2-beta3 loop, that were found to be almost as potent as Rs-AFP2. Cysteines, highly conserved in the native protein, are essential for maintaining the secondary structure of the protein. Surprisingly, in the 19-mer loop peptides, cysteines can be replaced by alpha-aminobutyric acid, which even improves the antifungal potency of the peptides. Analogous cyclic 19-mer peptides, forced to adopt a hairpin structure by the introduction of one or two non-native disulfide bridges, were also found to possess high antifungal activity. The synthetic 19-mer peptides, like Rs-AFP2 itself, cause increased Ca2+ influx in pregerminated fungal hyphae.

Immunoelectron microscopy of antigen processing in dendritic cells.

UNLABELLED: The cell biology of intracellular compartments and their interrelationships require detailed knowledge of the proteins that characterize the compartment and that are involved in the communication between them. To date, this can be best achieved by high resolution immunoelectron microscopy (IEM). Other methods, which make use of different embedding materials, such as EPON, Spurr's resin, LR white, or Lowicryls, also allow the detection of immunodeterminants. However, IEM is in many cases the optimum technique owing to better accessibility of the immunodeterminants to antibodies and the absence of denaturing solvents. In our laboratory for IEM we use immunogold labeling on cryosections. This technique combines optimal ultrastructure and good preservation of protein and/or lipid antigens. The ultrathin cryosections (50-100 nm) are prepared from small tissue blocks or cell pellets with a cryo-ultramicrotome. The sections are thawed, and labeled with antibodies, which are visualized with protein A-gold particles (PAG). We recommend the books by Larson (1) and Griffith (2), and chapters in Handbook of Experimental Immunology (3) and METHODS: a Companion to METHODS in Enzymology (4). The present chapter will describe the different aspects of IEM in detail, such as fixation procedures, the processing of samples, ultrathin cryosectioning, and immunogold labeling.

Subcellular distribution of CFTR in rat intestine supports a physiologic role for CFTR regulation by vesicle traffic.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel critical to intestinal anion secretion. In addition to phosphorylation, vesicle traffic regulates CFTR in some epithelial cells. Studies of cultured intestinal cells are conflicting regarding the role of cAMP-dependent vesicle traffic in regulating chloride transport. Whether CFTR is present in vesicular compartments within chloride secretory cells in the intestine is unknown and the role of cAMP-dependent vesicle insertion in regulating CFTR and intestinal fluid secretion remains unclear. The purpose of this study was to: (1) examine and quantify the subcellular distribution for CFTR in rat intestine, (2) further define the ultrastructure of the previously identified CFTR High Expresser (CHE) cell, and (3) examine the cellular distribution of CFTR following cAMP stimulation in vivo. Using the sensitive techniques of cryoimmunogold electron microscopy we identified CFTR in subapical vesicles and on the apical plasma membrane in crypt, Brunner glands, and CHE cells. cAMP stimulation in rat proximal small intestine produced a fluid secretory response and was associated with an apical redistribution of CFTR, supporting a physiologic role for cAMP-dependent CFTR vesicle insertion in regulating CFTR in the intestine.

Lysozyme distribution and conformation in a biodegradable polymer matrix as determined by FTIR techniques.

Lysozyme distribution and conformation in poly(lactic-co-glycolic acid)(PLGA) microspheres was determined using various infrared spectroscopic techniques. Infrared microscopy and confocal laser scanning microscopy indicated that the protein was homogeneously distributed inside the microspheres in small cavities resulting from the water-in-oil emulsification step. Part of the protein was observed at or near the cavity walls, while the rest was located within these cavities. Attenuated total reflectance (ATR) and photoacoustic spectroscopy (PAS) also showed that there is hardly any protein at the surface of the microspheres. Since this microsphere formulation gave a large burst release (ca. 50%), this burst release can not be caused by protein at the surface of the particles. Probably, the protein is rapidly released through pores in the PLGA matrix. Conformational analysis of lysozyme in the PLGA microspheres by KBr pellet transmission suffered from band shape distortion and baseline slope. Despite incomplete subtraction of the PLGA background, a characteristic band of non-covalent aggregates at 1625 cm(-1) was observed in the second derivative spectrum of the protein Amide I region. The other Fourier-transform infrared (FTIR) methods yielded similar results, indicating that the sample preparation procedure did not introduce artifacts. The observed aggregation signal may correspond to the protein adsorbed to the cavity walls inside the microspheres.

Effects of physicochemical characteristics of poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes on cellular association and internalization.

The cationic polymer poly(2-(dimethylamino)ethyl methacrylate) (p(DMAEMA)) is able to efficiently bind and condense DNA and to mediate transfection of a variety of cell types. In this study, fluorescence activated cell sorting (FACS), confocal laser fluorescence microscopy (CSLM) and electron microscopy (EM) techniques were used to investigate in vitro the cellular interaction of p(DMAEMA)-based polyplexes with human ovarian carcinoma cells (OVCAR-3). Cellular association and subsequent internalization only occurred when the polyplexes exhibited a positive zeta potential. Small-sized polyplexes have an advantage over large-sized complexes regarding cellular entry. The effect of the presence of tertiary amine groups versus the presence of quatenary amine groups was evaluated by comparing p(DMAEMA) with its quaternary ammonium analogue poly(2-(trimethylamino)ethyl methacrylate) (p(TMAEMA)). The combined cellular interaction and transfection results suggest that the latter polymer does not have an intrinsic endosomal escape property, in contrast to the 'proton sponge' effect proposed for p(DMAEMA). PEGylation of p(DMAEMA) effectively shielded the surface charge and yielded a notably lower degree of cellular interaction. Data on the effects of the presence of endocytosis inhibitors and an endosome-disruptive peptide in the culture medium on the cellular interaction and transfection activity of p(DMAEMA)-based polyplexes support endocytosis as being the principal pathway for intracellular delivery of plasmid. Both the CLSM and EM studies did not reveal the presence of polyplexes or plasmid outside the endocytic vesicles or within the nucleus, suggesting that intracellular trafficking from the endosomes to the nucleus is a very inefficient process.

Moraxella (Branhamella) catarrhalis BRO beta-lactamase: a lipoprotein of gram-positive origin?

In the past 20 years, BRO beta-lactamase-producing Moraxella catarrhalis strains have emerged. We show that BRO is expressed as a 33-kDa lipoprotein associated with the inner leaflet of the outer membrane. To our knowledge, this is the first description of a lipidated beta-lactamase in a gram-negative species.

Resistance to both complement activation and phagocytosis in type 3 pneumococci is mediated by the binding of complement regulatory protein factor H.

To study the role of surface-associated proteins in the virulence of Streptococcus pneumoniae, we used two serotype 3 strains, ATCC 6303 and WU2, and two PspA-negative mutants of WU2, an encapsulated one, JY1123 (Caps(+)/PspA(-)), and an unencapsulated one, DW3.8 (Caps(-)/PspA(-)). ATCC 6303 and WU2 were highly virulent in mice, while the virulence of JY1123 was slightly decreased (50% lethal doses [LD(50)s], 24, 6, and 147 CFU/mouse, respectively); DW3.8 was avirulent (LD(50), 2 x 10(8) CFU). In vitro, ATCC 6303, WU2, and JY1123 (Caps(+)/PspA(-)) strongly resisted complement activation and complement-dependent opsonophagocytosis, whereas DW3.8 (Caps(-)/PspA(-)) was easily phagocytized in fresh serum. Trypsin treatment of ATCC 6303, WU2, and JY1123 (Caps(+)/PspA(-)) resulted in enhanced complement activation and complement-dependent opsonophagocytosis. Trypsin had no deleterious effect on the polysaccharide capsule. In addition, trypsin pretreatment of ATCC 6303 strongly reduced virulence upon intraperitoneal challenge in mice. This indicated that surface proteins play a role in the resistance to complement activation and opsonophagocytosis and contribute to the virulence of type 3 pneumococci. In subsequent experiments, we could show that the modulation of complement activation was associated with surface components that bind complement regulator factor H; binding is trypsin sensitive and independent of prior complement activation. Immunoblotting of cell wall proteins of the virulent strain ATCC 6303 with anti-human factor H antibody revealed three factor H-binding proteins of 88, 150, and 196 kDa. Immunogold electron microscopy showed a close association of factor H-binding components with the outer surface of the cell wall. The role of these factor H-binding surface proteins in the virulence of pneumococci is interesting and warrants further investigation.

Extensive islet amyloid formation is induced by development of Type II diabetes mellitus and contributes to its progression: pathogenesis of diabetes in a mouse model.

AIMS/HYPOTHESIS: Type II (non-insulin-dependent) diabetes mellitus is a multifactorial disease in which pancreatic islet amyloid is a characteristic histopathological finding. Islet amyloid fibrils consist of the beta-cell protein "islet amyloid polypeptide" (IAPP)/"amylin". Unlike human IAPP (hIAPP), mouse IAPP cannot form amyloid. In previously generated transgenic mice, high expression of hIAPP as such did not induce islet amyloid formation. To further explore the potential diabetogenic role of amyloidogenic IAPP, we introduced a diabetogenic trait ("ob" mutation) in hIAPP transgenic mice. METHODS: Plasma concentrations of IAPP, insulin and glucose were determined at 3.5 (t1), 6 (t2), and 16-19 months of age (t3). At t3, the mice were killed and the pancreas was analysed (immuno)histochemically. RESULTS: In non-transgenic ob/ob mice, insulin resistance caused a compensatory increase in insulin production, normalizing the initial hyperglycaemia. In transgenic ob/ob mice, concurrent increase in hIAPP production resulted in extensive islet amyloid formation (more often and more extensive than in transgenic non-ob/ob mice), insulin insufficiency and persistent hyperglycaemia: At t3, plasma insulin levels in transgenic ob/ob mice with amyloid were fourfold lower than in non-transgenic ob/ob mice (p < 0.05), and plasma glucose concentrations in transgenic ob/ ob mice were almost twofold higher (p < 0.05). In addition, the degree of islet amyloid formation in ob/ob mice was positively correlated to the glucose:insulin ratio (r(s) = 0.53, p < 0.05). CONCLUSION/INTERPRETATION: Islet amyloid is a secondary diabetogenic factor which can be both a consequence of insulin resistance and a cause of insulin insufficiency. [Diabetol

Intestinal carbamoyl phosphate synthase I in human and rat. Expression during development shows species differences and mosaic expression in duodenum of both species.

The clinical importance of carbamoyl phosphate synthase I (CPSI) relates to its capacity to metabolize ammonia, because CPSI deficiencies cause lethal serum ammonia levels. Although some metabolic parameters concerning liver and intestinal CPSI have been reported, the extent to which enterocytes contribute to ammonia conversion remains unclear without a detailed description of its developmental and spatial expression patterns. Therefore, we determined the patterns of enterocytic CPSI mRNA and protein expression in human and rat intestine during embryonic and postnatal development, using in situ hybridization and immunohistochemistry. CPSI protein appeared during human embryogenesis in liver at 31-35 e. d. (embryonic days) before intestine (59 e.d.), whereas in rat CPSI detection in intestine (at 16 e.d.) preceded liver (20 e.d.). During all stages of development there was a good correlation between the expression of CPSI protein and mRNA in the intestinal epithelium. Strikingly, duodenal enterocytes in both species exhibited mosaic CPSI protein expression despite uniform CPSI mRNA expression in the epithelium and the presence of functional mitochondria in all epithelial cells. Unlike rat, CPSI in human embryos was expressed in liver before intestine. Although CPSI was primarily regulated at the transcriptional level, CPSI protein appeared mosaic in the duodenum of both species, possibly due to post-transcriptional regulation.

Site- and cell-specific alteration of lung copper/zinc and manganese superoxide dismutases by chronic ozone exposure.

The antioxidant enzymes copper/zinc (Cu-Zn) and manganese (Mn) superoxide dismutase (SOD) have been implicated in protection of the lungs from oxidant damage. Mn SOD in particular may be related to acquired tolerance in cells following chronic ozone exposure. In order to study these protective and adaptive phenomena in oxidant injury, the cellular location and relative abundance of Mn SOD and Cu-Zn SOD were examined in the lungs of Fischer 344 rats following exposure to 0.0 and 1.0 ppm ozone for up to 3 mo using immunolabeling and morphometric techniques. Cu-Zn SOD labeling was found to be markedly reduced following ozone exposure in epithelial cells within airways and parenchyma. In contrast, a significant increase was noted in Mn SOD labeling in the centriacinar regions of exposed lungs for both alveolar macrophages and epithelial type II cells. Mn SOD labeling per epithelial type II cell was significantly increased in alveoli 0-400 microm beyond the bronchiole-alveolar duct junction (BADJ), while type II cell Mn SOD labeling was similar to control values with greater distance down the alveolar duct. No induction of Mn SOD was noted in type I epithelial cells, fibroblasts, or Clara cells. Thus, alterations in Cu-Zn and Mn SOD are both site and cell specific in the lungs. The differential increase in Mn SOD in type II cells of the proximal alveolar duct may reflect the ability of these cells to acquire tolerance and to resist further injury to repeated ozone exposure.

Glucose transporter (GLUT-4) is targeted to secretory granules in rat atrial cardiomyocytes.

The insulin-responsive glucose transporter GLUT-4 is found in muscle and fat cells in the trans-Golgi reticulum (TGR) and in an intracellular tubulovesicular compartment, from where it undergoes insulin-dependent movement to the cell surface. To examine the relationship between these GLUT-4-containing compartments and the regulated secretory pathway we have localized GLUT-4 in atrial cardiomyocytes. This cell type secretes an antihypertensive hormone, referred to as the atrial natriuretic factor (ANF), in response to elevated blood pressure. We show that GLUT-4 is targeted in the atrial cell to the TGR and a tubulo-vesicular compartment, which is morphologically and functionally indistinguishable from the intracellular GLUT-4 compartment found in other types of myocytes and in fat cells, and in addition to the ANF secretory granules. Forming ANF granules are present throughout all Golgi cisternae but only become GLUT4 positive in the TGR. The inability of cyclohexamide treatment to effect the TGR localization of GLUT-4 indicates that GLUT-4 enters the ANF secretory granules at the TGR via the recycling pathway and not via the biosynthetic pathway. These data suggest that a large proportion of GLUT-4 must recycle via the TGR in insulin-sensitive cells. It will be important to determine if this is the pathway by which the insulin-regulatable tubulo-vesicular compartment is formed.

Antifungal activity of synthetic 15-mer peptides based on the Rs-AFP2 (Raphanus sativus antifungal protein 2) sequence.

Plant defensins are a class of cysteine-rich peptides of which several members have been shown to be potent inhibitors of fungal growth. A series of overlapping 15-mer peptides based on the amino acid sequence of the radish antifungal protein Rs-AFP2 have been synthesized. Peptides 6, 7, 8 and 9, comprising the region from cysteine 27 to cysteine 47 of Rs-AFP2 showed substantial antifungal activity against several fungal species (minimal inhibitory concentrations of 30-60 micrograms/mL), but no activity towards bacteria (except peptide 6 at 100 micrograms/mL). The active peptides were shown to be sensitive to the presence of cations in the medium and to the composition and pH of the medium. When present at a subinhibitory concentration (20 micrograms/mL), peptides 1, 7, 8 and 10 potentiated the activity of Rs-AFP2 from 2.3-fold to 2.8-fold. By mapping the characteristics of the active peptide on the structure of Rs-AFP2 as determined by nuclear magnetic resonance, the active region of the antifungal protein appears to involve beta-strands 2 and 3 in combination with the loop connecting those strands. A cyclized synthetic mimic of the loop, cysteine 36 to cysteine 45, was shown to have antifungal activity. Substitution of tyrosine 38 by alanine in the cyclic peptide substantially reduced the antifungal activity, indicating the importance of this residue for the activity of Rs-AFP2 as demonstrated carrier by mutational analysis.

Interventional thermal injury of the arterial wall: unfolding of von Willebrand factor and its increased binding to collagen after 55 degrees C heating.

PURPOSE: Thermal angioplasty alters the thrombogenicity of the arterial wall. In previous studies, platelet adhesion was found to increase after heating human subendothelium to 55 degrees C and decrease after heating to 90 degrees C. In the present electron microscopic study, the mechanism of this temperature-dependent platelet adhesion to the heated arterial wall is elucidated by investigating temperature-dependent conformational changes of von Willebrand factor (vWF) and collagen types I and III and the binding of vWF to heated collagen. METHODS: Purified vWF and/or collagen was applied to electron microscopic grids and heated by floating on a salt-solution of 37 degrees C, 55 degrees C or 90 degrees C for 15 s. After incubation with a polyclonal antibody against vWF and incubation with protein A/gold, the grids were examined by electron microscopy. RESULTS: At 37 degrees C, vWF was coiled. At 55 degrees C, vWF unfolded, whereas heating at 90 degrees C caused a reduction in antigenicity. Collagen fibers heated to 37 degrees C were 60.3 +/- 3.1 nm wide. Heating to 55 degrees C resulted in the unwinding of the fibers, increasing the width to 87.5 +/- 8.2 nm (p < 0.01). Heating to 90 degrees C resulted in denatured fibers with an enlarged width of 85.1 +/- 6.1 nm (p < 0.05). Heating of collagen to 55 degrees C resulted in an increased vWF binding as compared to collagen heated to 37 degrees C or to 90 degrees C. Incubation of collagen with vWF, prior to heating, resulted in a vWF binding after heating to 55 degrees C that was similar to the 37 degrees C binding and a decreased binding after 90 degrees C. CONCLUSIONS: After 55 degrees C heating, the von Willebrand factor molecule unfolds and collagen types I and III exhibit an increased adhesiveness for von Willebrand factor. Heating to 90 degrees C denatures von Willebrand factor and collagen. The conformation changes of von Willebrand factor and its altered binding to collagen type I and III may explain the increased and decreased platelet adhesion to subendothelium after 55 degrees C and 90 degrees C heating, respectively.

Endocytosis of GPI-linked membrane folate receptor-alpha.

GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform alpha in KB-cells. MFR-alpha was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-alpha was completely soluble in Triton X-100 at 0 degree C. In contrast, only 33% of the 36-38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37 degrees C or in the presence of saponin at 0 degree C. Similar solubilization characteristics were found when MFR-alpha at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinity-analog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-alpha could be separated from soluble MFR-alpha on sucrose flotation gradients. Only Triton X-100 soluble MFR-alpha was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100-resistant membrane domains. Internalized MFR-alpha recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0 degree C. Using immunoelectron microscopy, we found MFR-alpha along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-alpha bypasses caveolae.