Prenatal exposure to airborne polychlorinated biphenyl congeners and male reproductive health.

STUDY QUESTION: Is fetal exposure to lower-chlorinated polychlorinated biphenyls (LC-PCBs) in indoor air of private homes built with PCB-containing materials associated with semen characteristics and testicular volume in adult men? SUMMARY ANSWER: We observed only marginal and inconsistent associations between maternal exposure to PCBs in indoor air and semen quality, testicular size and reproductive hormones in the adult offspring. WHAT IS KNOWN ALREADY: Recent studies have shown LC-PCBs to exhibit endocrine-disrupting properties and increase the risk of cryptorchidism. Although exposure to LC-PCBs in indoor air is relatively common, the long-term impact of prenatal exposure on male reproductive health has not yet been investigated. STUDY DESIGN, SIZE, DURATION: In this cohort study, participants were men (18+ years) whose mothers carried them while living in one of two residential areas where indoor air had been contaminated by LC-PCB evaporating from building materials in subsets of the apartments. Men were considered prenatally exposed if their mother had lived in a PCB-contaminated apartment and unexposed if their mother had lived in an uncontaminated apartment for a minimum of 1 year during the 3.6 years before conception or during the first trimester. Mothers of prenatally unexposed men could not have lived in a contaminated apartment at any point. Recruitment lasted from 2017 to 2019. In total, 73 exposed and 111 unexposed men gave a blood and semen sample. PARTICIPANTS/MATERIALS, SETTING, METHODS: Percentage differences in semen volume, sperm concentration, total sperm count, morphologically normal spermatozoa, progressively motile spermatozoa and DNA fragmentation index (DFI) between prenatally exposed and unexposed men were estimated using negative binomial regression. Associations with total and calculated free testosterone (CFT), LH and FSH were modeled using the linear regression. Odds of small testicular volume was estimated with logistic regression. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, the results of this study were conflicting. No differences in semen volume, sperm concentration, testosterone and CFT were observed between the groups, but there were slight indications of lower total sperm count, increased FSH and risk of small testicles, alongside lower sperm DFI and a higher proportion of normal spermatozoa in men exposed to LCB-PCBs from indoor air during fetal life. There is no apparent biologically plausible explanation for the apparently improved measures of DNA fragmentation and morphology, and these findings may have occurred purely by chance. LIMITATIONS, REASONS FOR CAUTION: Owing to the indirect measure of exposure, lack of adjustment for paternal factors, the potential for self-selection due to known exposure status and fertility issues, inability to take time spent away from the residence, limited statistical power and lack of comparable literature, independent replication of the study in larger cohorts is warranted. WIDER IMPLICATIONS OF THE FINDINGS: While our findings may appear reassuring for the large number of people residing and/or working in buildings with indoor air contaminated with LC-PCBs, further efforts to understand the full range of health consequences of fetal LC-PCB exposure are needed. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by the Independent Research Fund Denmark (ref no. 6110-00085B), Bispebjerg Hospital, Landsbyggefonden, Realdania (ref. no. PRJ-2017-00176), Grundejernes Investeringsfond (ref. no. 18-58) and Helsefonden (ref. no. 16-B-01-22 and 21-B-0412). K.S.H. was supported by FFIKA, Focused Research Effort on Chemicals in the Working Environment, from the Danish Government. The authors declare that they have no financial, personal or professional competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Allergy and respiratory health effects of dampness and dampness-related agents in schools and homes: a cross-sectional study in Danish pupils.

Little is known about the health effects of school-related indoor dampness and microbial exposures. In this study, we investigated dampness and dampness-related agents in both homes and schools and their association with allergy and respiratory health effects in 330 Danish pupils. Classroom dampness was identified based on technical inspection and bedroom dampness on parents' self-report. Classroom and bedroom dust was analyzed for seven microbial components. Skin prick testing determined atopic sensitization. Lung function was expressed as z-scores for forced expiratory volume in one-second (zFEV1 ), forced vital capacity (zFVC) and the ratio zFEV1 /zFVC using GLI-2012 prediction equations. The parents reported children's allergies, airway symptoms, and doctor-diagnosed asthma. High classroom dampness, but not bedroom dampness, was negatively associated with zFEV1 (ß-coef. -0.71; 95% CI -1.17 to -0.23) and zFVC (ß-coef. -0.52; 95% CI -0.98 to -0.06) and positively with wheezing (OR 8.09; 95% CI 1.49 to 43.97). No consistent findings were found between any individual microbial components or combination of microbial components and health outcomes. Among other indoor risk factors, environmental tobacco smoke (ETS) decreased zFEV1 (ß-coef. -0.22; 95% CI -0.42 to -0.02) and zFEV1 /zFVCratio (ß-coef. -0.26; 95% CI -0.44 to -0.07) and increased upper airway symptoms (OR 1.66; 95% CI 1.03-2.66). In conclusion, dampness in classrooms may have adverse respiratory health effects in pupils, but microbial agents responsible for this effect remain unknown.

Exposure to 27 polychlorinated biphenyls in the indoor environment of a workplace: a controlled bio-monitoring study.

PURPOSE: To assess the exposure to a broad-spectrum of polychlorinated biphenyl congeners (PCBs) from the indoor environment through bio-monitoring of people working in a building with PCB-containing materials and elevated PCB levels in the indoor air. METHODS: A cross-sectional study comparing the plasma concentration of 27 PCB congeners in 15 people working in a PCB-contaminated building and 30 matched controls. RESULTS: Median concentration of eight low-chlorinated PCB congeners was significantly higher in the exposed than in the control group. The sum of median concentrations of tri + tetra-chlorinated PCB was almost ten times higher in the exposed group than in the unexposed, and sums of dioxin-like and non-dioxin-like PCB were both relatively increased by 60 % in the exposed group. CONCLUSIONS: The occupational indoor environment may significantly add to PCB exposure, especially to the lower-chlorinated congeners. Health effect from this little-acknowledged exposure has not yet been documented, but data supporting lack of effect are sparse and research generating information on effect of exposure to specific congeners including at levels relevant for the indoor environment should be encouraged.

Determinants of house dust, endotoxin, and ß-(1→3)-D-glucan in homes of Danish children.

Little is known about the geographic variation and determinants of bacterial endotoxin and ß-(1,3)-D-glucan in Danish house dust. In a population of 317 children, we: (i) described loads and concentrations of floor dust, endotoxin, and ß-(1→3)-D-glucan and (ii) their correlations and (iii) assessed their determinants; (iv) Finally, we compared our findings with previous European studies. Bedroom floor dust was analyzed for endotoxin content by the kinetic limulus amoebocyte lysate assay and for ß-(1→3)-D-glucan by the inhibition enzyme immunoassay. The parents answered questions regarding potential determinants. We found: geometric means (geometric standard deviations) 186 mg/m(2) (4.3) for dust; 5.46 × 10(3) EU/m(2) (8.0) and 31.1 × 10(3) EU/g (2.6) for endotoxin; and 142 µg/m(2) (14.3) and 0.71 × 10(3) µg/g (7.3) for ß-(1→3)-D-glucan. High correlations (r > 0.75) were found between floor dust and endotoxin and ß-(1→3)-D-glucan loads, while endotoxin and ß-(1→3)-D-glucan concentrations were moderately correlated (r = 0.36-0.41) with the dust load. Having a carpet was positively associated with dust load and with endotoxin and ß-(1→3)-D-glucan concentrations. Pet keeping, dwelling type, and dwelling location were determinants of endotoxin concentrations. No other determinants were associated with ß-(1→3)-D-glucan concentrations. Compared with other European studies, we found lower ß-(1→3)-D-glucan loads and concentrations but higher endotoxin loads and concentrations suggesting a geographically determined different composition of Danish floor dust compared with other European regions.

Building-related symptoms and stress indicators.

UNLABELLED: This study aims to study physiological stress indicators in relation to prevalence of building-related symptoms (BRS) among teachers employed in three selected schools in Copenhagen. Three hypotheses were studied: (i) Perceived psychosocial work environment was associated with BRS; (ii) Perceived psychosocial work environment (job strain) was associated with physiological strain; (iii) BRS was associated with physiological strain. We found a tendency among women of an association between job strain and being BRS positive. Also an association between job strain and physiological strain was observed among women. Being BRS positive was not associated with single physiological stress indicators with the exception of a higher level free testosterone in serum among BRS-positive women. PRACTICAL IMPLICATIONS: Including physiological stress indicators may be a supplementary measure to questionnaires when studying the association between the psychosocial work environment and building-related symptoms (BRS). In this study, job strain was associated with physiological strain among women. Being BRS positive was not associated with single physiological stress indicators with the exception of a higher level free testosterone in serum among BRS-positive women. This study should be regarded as a preliminary study because of its small number of participants.

Molds in floor dust, building-related symptoms, and lung function among male and female schoolteachers.

UNLABELLED: Five hundred and twenty-two teachers from 15 public schools, eight 'water-damaged' schools, and seven 'non-damaged' schools with no visible water damage were included in a cross-sectional design. Mold growth was assessed by recording the amount of dust on the floor and in the air in classrooms and the content of a number of mold species in the dust (CFU/g dust). The evaluation of health symptoms included symptoms recorded by questionnaire and spirometry, bronchial challenge, and CO-diffusion capacity. Nasal lavage fluid was analyzed for IL-8 and ECP. Personal and psychosocial factors were included as confounders. In this study population mucus membrane irritation symptoms (MMI) and general symptoms were reported more frequently by women than by men with odds ratios ranging from 1.4 to 2.1. Women's reports of symptoms from mucous membranes and skin and general symptoms were positively associated with mold exposure. Odds ratio for 'difficult to concentrate' after adjustment for confounders was 11.2 (1.4-90.1, 95% CI) at high levels of mold exposure. None of the lung function tests performed in this study were associated with mold exposure, to the 'water damaged' vs. 'non-damaged' classification, or to the symptoms reported. IL-8 and ECP were not associated either. PRACTICAL IMPLICATIONS: Psychosocial and personal reasons dominate in MMI and general symptoms. Headache and difficulties to concentrate associated with indoor mold exposure, mainly for women. No lung function impairment associated with indoor mold exposure.

Molds in floor dust and building-related symptoms among adolescent school children: a problem for boys only?

UNLABELLED: In this stratified cross-sectional study in eight 'wet' and seven 'dry' schools, 1024 adolescent school children reported potentially building-related symptoms (BRS) in self-administrated questionnaires. From their classrooms dust samples were collected from floors, ventilation ducts, and air; settled dust was collected in cardboard boxes over a period of 5 months. Measurements of temperature, relative humidity and CO2 were performed. BRS were strongly associated with personal factors like recent airway infections, hay fever, asthma and psycho-social work load, but also to molds in floor dust and presence of mechanical ventilation. The association between molds in floor dust and BRS has in stratified analyses shown a strong association among adolescent school boys, and no association among adolescent school girls using multivariable analyses controlling for relevant confounders. In contrast to the menstruating school girls, the symptoms among the small group of not yet menstruating girls were associated with the levels of molds in floor dust. Their symptom prevalences were very similar to those of the boys. This finding makes us suggest a new hypothesis: The higher endogenous estrogen levels of sexually matured adolescent females seems to protect them from the effects of molds in dust, despite their overall higher symptom prevalence. PRACTICAL IMPLICATIONS: In this cross-sectional epidemiological study of adolescent school children we found independent significant positive associations between building-related symptoms and viable molds in floor dust in boys and non-menstruating girls. In contrast, no such associations were seen among menstruating girls. The identification of these two susceptible groups adds further support the relevance of minimizing sources of dust and mold exposure.

Double blind placebo controlled exposure to molds: exposure system and clinical results.

UNLABELLED: The objective was to develop an experimental setup for human exposure to mold spores, and to study the clinical effect of this exposure in sensitive subjects who had previously experienced potentially building-related symptoms (BRS) at work. From three water-damaged schools eight employees with a positive histamine release test to Penicillium chrysogenum were exposed double- blinded to either placebo, approximately 600,000 spores/m3 air of P. chrysogenum or approximately 350,000 spores/m3 of Trichoderma harzianum for 6 min on three separate days. A statistically significant rise in symptoms from mucous membranes appeared from the 9-graded symptom scale after exposure to T. harzianum or placebo. Dichotomizing the data, whether the participants experienced at least a two-step rise on the symptom scale or not, gave borderline increase in mucous membrane symptoms after exposure to P. chrysogenum. In conclusion this is, to our knowledge, the first study to successfully conduct a human exposure to a highly controlled dose of fungal material aerosolized directly from wet building materials. This short-term exposure to high concentrations of two different molds induced no more reactions than exposure to placebo in eight sensitive school employees. However, a statistical type II error cannot be excluded because of the small sample size. PRACTICAL IMPLICATIONS: In this double blind, placebo controlled study of mold exposure changes in symptoms, objective measurements and blood samples were small and mostly non-significant, and at the same level as after placebo exposure. The developed exposure system based on the Particle-Field and Laboratory Emission Cell (P-FLEC) makes it possible to deliver a precise and highly controlled dose of mold spores from water-damaged building materials, imitating realistic field exposure conditions. The present experiment is too small to rule out an effect of mold exposure; long-term experimental exposure studies on larger number of subjects are needed.

The dustfall collector--a simple passive tool for long-term collection of airborne dust: a project under the Danish Mould in Buildings program (DAMIB).

UNLABELLED: A newly developed dustfall device that collects airborne dust by sedimentation has been evaluated in Danish schools. Dust collected over 140 days was compared with airborne dust sampled during single school days and settled floor dust sampled at the end of a school day. Measurements of culturable fungi in air samples corresponded well to those from the dustfall collector in mechanically but not naturally ventilated rooms. Levels of beta(1 --> 3)glucan and culturable fungi in dust from the dustfall collector and settled floor dust did not agree well. Up to 10 times more beta(1 --> 3)glucan per milligram dust was found in the dustfall collectors than in settled floor dust. Only in dust from the dustfall collector was the mass concentration of culturable fungi (cfu/mg) significantly correlated with the degree of dampness. The culturability of fungi in the 140-day dustfall samples tended to be lower than in other types of samples. Nonetheless, the results indicated that the dustfall collector can be used to estimate the median concentration of airborne culturable fungi, provided identical sampling periods are used for all samples. The collector is cost effective and simple to use. PRACTICAL IMPLICATIONS: Traditional ways of assessing exposure to microorganisms by sampling airborne dust or dust from floors have several disadvantages. In the present paper, a new sampler, the dustfall collector, which collects airborne dust by sedimentation over a period of months, is introduced. Tests of this collector indicate that it can be used to estimate the mean concentration of airborne culturable fungi but not beta(1 --> 3)glucan over a long period and that the method can differentiate between buildings with different levels of water damage.

Molds in floor dust and building-related symptoms in adolescent school children.

This stratified cross-sectional epidemiological study included 1053 school children aged 13-17 years. All pupils filled in a questionnaire on building-related symptoms and other relevant health aspects. The following exposure measurements were carried out: room temperature, CO2 level, and relative humidity; building characteristics including mold infestation were assessed, and dust was collected from floors, air, and ventilation ducts during a working day. Dust was examined for endotoxin level, and cultivated for viable molds. We did not find a positive association between building-related symptoms and extent of moisture and mold growth in the school buildings. Five of eight building-related symptoms were significantly and positively associated with the concentration of colony forming units of molds in floor dust: eye irritation, throat irritation, headache, concentration problems, and dizziness. After adjusting for different potentially confounding factors in separate analyses of each symptom, the above-mentioned associations between molds in dust and symptoms were still present, except for concentration problems. However, in none of the analyses was mold exposure the strongest covariate, being secondary to either asthma, hay fever, recent airway infection, or psychosocial factors.

Inflammatory potential of dust from schools and building related symptoms.

AIMS: To test whether the inflammatory potential of dust samples might be used to differentiate schools with high and low prevalence of building related symptoms (BRS) among the occupants. METHODS: Ten schools with high prevalence of BRS and 10 schools with low prevalence were selected. Dust collected from floors, horizontal surfaces, and exhaust outlets was tested at five concentrations on the lung epithelial cell line A549. The potency of the dust (PF) to stimulate IL-8 secretion was calculated from the initial linear part of the dose-response curves. The organic fraction of the dust samples was determined by incineration. RESULTS: The schools with low prevalence of symptoms had a BRS% of 4.4-11.0 and the schools with high prevalence a BRS% of 19.6-31.9. The PF of floor dust and surface dust correlated, and the PF was associated with the organic content of the dust. The schools with low prevalence of symptoms had a significantly lower PF than the schools with high prevalence. Using the cut point value of 4.5 ng IL-8/mg floor dust, significantly more high prevalence schools were found above the cut point than below. CONCLUSION: The PF of the floor dust samples correlated significantly with the prevalence of symptoms in the schools. The content of endotoxin and microorganisms did not seem to explain the inflammatory potential of the dust or BRS, and the substances in the dust causing the inflammatory potential are presently unknown.

Serum IgE specific to indoor moulds, measured by basophil histamine release, is associated with building-related symptoms in damp buildings.

OBJECTIVE: To study the relationship between basophil histamine release (HRT) to indoor moulds, indicating specific IgE, and building-related symptoms (BRS), asthma, and hay fever in individuals working in damp and mouldy buildings. METHODS: A cross-sectional study was performed among 86 school staff members, who on average had worked 143 months (range: 3-396) in moist buildings with mould growth in the constructions. A questionnaire concerning mucous membrane symptoms, facial skin symptoms, central nervous system symptoms, hay fever, and asthma was fulfilled by the participants, and blood samples were taken. Eight mould species growing on building constructions were identified and cultivated to obtain allergenic materials for testing. The presence in serum of IgE specific to moulds was verified by histamine release test (HRT) based on passive sensitization of basophil leukocytes. The validity of the method was confirmed by parallel testing of patients allergic to grass- and birch pollen and by the shift from positive to negative response after removal of serum IgE and by using sham sensitization. RESULTS: The prevalence of most BRS was between 32% and 62%. Positive HRT, showing serum IgE specific to one or more of the moulds, was observed in 37% of the individuals. The highest frequency of positive HRT was found to Penicillium chrysogenum and then to Aspergillus species, Cladosporium sphaerospermum and Stachybotrys chartarum. A significant association was found between most BRS and positive HRT, whereas no association was observed between positive HRT to moulds and self reported hay fever or asthma. CONCLUSION: Positive HRT to indoor moulds, showing the presence in serum of IgE specific to the fungi, was found to be related to BRS in individuals working in damp and mouldy buildings. Whether the association is of causal character is a question for further studies. The test may be useful in the evaluation and study of possible mould induced BRS.

Freeze-fracture studies on lipids and membranes.

Freeze-fracture electron microscopy is especially useful for investigation of lipid structures by the advantageous fracture course within hydrophobic zones. Freezing is, on the other hand, a restriction because the structures of lamellar and non-lamellar phase states with disordered acyl chains (L(alpha), H(II,) cubic) are difficult to preserve. An important aspect of this method is therefore the lipid structure of phase states with ordered acyl chains (crystal, gel), and with a different degree of hydration. Freeze-fracture of pure lipid systems creates a valid representation of the structure of non-lamellar phases and of the general structure of the "lamellar" lipid bilayer, and lamellar phases with characteristic deformations (ripples, curvatures, plane sectors) can be identified. Fracture through the hydrophobic bilayer centre of biological membranes reveals characteristic protein components, the intramembraneous particles (IMPs). The lateral distribution of the IMPs is a helpful marker for fluid and rigid phase states, also without deformation of the lamella. The overall history and the present state of knowledge concerning the different structures revealed by the freeze-fracture and freeze-etch techniques in lipid systems, and to a limited extent in biological membranes, is reviewed, taking into account studies from our own laboratory.

Exposure to cold and draught, alcohol consumption, and the NS-phenotype are associated with chronic bronchitis: an epidemiological investigation of 3387 men aged 53-75 years: the Copenhagen Male Study.

OBJECTIVES: This study was performed to estimate the strength of association between chronic bronchitis and lifetime exposure to occupational factors, current lifestyle, and the NS-phenotype in the MNS blood group among middle aged and elderly men. METHODS: The study was carried out within the frameworks of the Copenhagen Male Study. Of 3387 men 3331 men with a mean age of 63 (range 53-75) years could be classified by prevalence of chronic bronchitis. As well as the completion of a large questionnaire on health, lifestyle, and working conditions, all participants had a thorough examination, including measurements of height and weight and blood pressure and a venous blood sample was taken for the measurement of serum cotinine and MNS typing; 16.5% of the men had the NS-phenotype. Chronic bronchitis was defined as cough and phlegm lasting 3 months or more for at least 2 years; 14.6% had chronic bronchitis. RESULTS: Smoking and smoke inhalation were the factors most strongly associated with prevalence of chronic bronchitis. There were three major new findings: (a) long term (>5 years) occupational exposure to cold and draught was associated with a significantly increased prevalence of chronic bronchitis; compared with others, and adjusted for confounders, the odds ratio (OR) with 95% confidence interval (95% CI) was 1.4 (1.1 to 1.7), p=0.004; (b) a significant J shaped association existed between alcohol use and bronchitis, p<0.001, with the lowest prevalence found among moderate users; (c) a significant gene by environment association existed between smoking and the NS-phenotype in the MNS blood group; only among smokers was the NS-phenotype associated with a significantly decreased risk of chronic bronchitis, OR 0.67 (0.47-0.97), p=0.02. Other well known associations between dust, fumes, and even exposure to solvents and bronchitis were confirmed. CONCLUSION: The results emphasise the multifactorial nature of chronic bronchitis, and show some hitherto unrecognised associations between cold and draught exposure, alcohol consumption, and the NS-phenotype and chronic bronchitis.

The structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine codispersed with a branched-chain phosphatidylcholine.

The structure and thermotropic phase behaviour of a fully hydrated binary mixture of dipalmitoylphosphatidylcholine and a branched-chain phosphatidylcholine, 1, 2-di(4-dodecyl-palmitoyl)-sn-glycero-3-phosphocholine, were examined using differential scanning calorimetry, synchrotron X-ray diffraction and freeze-fracture electron microscopy. The branched-chain lipid forms a nonlamellar phase when dispersed alone in aqueous medium. Mixed aqueous dispersions of the two phospholipids containing less than 33 mol% of the branched-chain lipid form lamellar phases over the whole temperature range were studied (4 degrees C to 60 degrees C). When present in proportions greater than 33 mol% it induces a hexagonal phase in mixed aqueous dispersions with dipalmitoylphosphatidylcholine at temperatures above the fluid phase transition. At temperatures below 35 degrees C a hexagonal phase coexists with a gel bilayer phase. The lamellar<-->nonlamellar transition can be explained satisfactorily on the basis of the shape of the molecule expressed in terms of headgroup and chain cross-sectional areas. At temperatures below 35 degrees C macroscopic phase separation of two gel phases takes place. Freeze-fracture electron microscopy revealed that one gel phase consists of bilayers with a highly regular, periodic superstructure (macro-ripples) whereas the other phase forms flat, planar bilayers. The macro-ripple phase appears to represent a relaxation structure required to adapt to the packing constraints imposed by the incorporation of the branched-chain lipid into the dipalmitoylphosphatidylcholine host bilayer. The data suggest that structural changes that take place on cooling the mixed dispersion below the lamellar<-->nonlamellar phase transition temperature cannot be adequately described using the molecular form concept. Instead it is necessary to take into account the detailed molecular form of the guest lipid as well as its physical properties.

Hydration of DMPC and DPPC at 4 degrees C produces a novel subgel phase with convex-concave bilayer curvatures.

Hydration of dimyristoyl- and dipalmitoylphosphatidylcholines at 4 degrees C results in the formation of a characteristic subgel phase designated Pcc. Examination of the phase by freeze-fracture electron microscopy shows convex-concave deformations of the planar bilayer which are of two types. A smaller type with a radius of curvature of about 20 nm predominates in DMPC, and a larger type with about 70 nm radii of curvatures dominates in DPPC. The Pcc phase can also be formed in samples hydrated at temperatures above the main phase transition if the dispersion is frozen slowly and subsequently incubated at 4 degrees C for several days. The subgel Pcc phase was distinguished from the subgel Lc phase by the temperature of transition, packing of the acyl chains on the basis of wide-angle X-ray diffraction, and 2H-NMR spectra characteristic of a 'solid-ordered' phase. Vibrational spectra of the carbonyl and phosphate regions are consistent with a partially reduced hydration state. The origin of the convex-concave bilayer deformation is believed to result from constraints imposed by limiting hydration of the headgroup and a frustration arising from the spontaneous curvature of both monolayers.

Ultrastructural characterization of peptide-induced membrane fusion and peptide self-assembly in the lipid bilayer.

The peptide sequence B18, derived from the membrane-associated sea urchin sperm protein bindin, triggers fusion between lipid vesicles. It exhibits many similarities to viral fusion peptides and may have a corresponding function in fertilization. The lipid-peptide and peptide-peptide interactions of B18 are investigated here at the ultrastructural level by electron microscopy and x-ray diffraction. The histidine-rich peptide is shown to self-associate into two distinctly different supramolecular structures, depending on the presence of Zn(2+), which controls its fusogenic activity. In aqueous buffer the peptide per se assembles into beta-sheet amyloid fibrils, whereas in the presence of Zn(2+) it forms smooth globular clusters. When B18 per se is added to uncharged large unilamellar vesicles, they become visibly disrupted by the fibrils, but no genuine fusion is observed. Only in the presence of Zn(2+) does the peptide induce extensive fusion of vesicles, which is evident from their dramatic increase in size. Besides these morphological changes, we observed distinct fibrillar and particulate structures in the bilayer, which are attributed to B18 in either of its two self-assembled forms. We conclude that membrane fusion involves an alpha-helical peptide conformation, which can oligomerize further in the membrane. The role of Zn(2+) is to promote this local helical structure in B18 and to prevent its inactivation as beta-sheet fibrils.

Morphological transitions of brain sphingomyelin are determined by the hydration protocol: ripples re-arrange in plane, and sponge-like networks disintegrate into small vesicles.

The phase transition of hydrated brain sphingomyelin occurs at around 35 degrees C, which is close to the physiological temperature. Freeze-fracture electron microscopy is used to characterize different gel state morphologies in terms of solid-ordered and liquid-ordered phase states, according to the occurrence of ripples and other higher-dimensional bilayer deformations. Evidently, the natural mixed-chain sphingomyelin does not assume the flat L beta, phase but instead the rippled P beta, phase, with symmetric and asymmetric ripples as well as macroripples and an egg-carton pattern, depending on the incubation conditions. An unexpected difference was observed between samples that are hydrated above and below the phase transition temperature. When the lipid is hydrated at low temperature, a sponge-like network of bilayers is formed in the gel state, next to some normal lamellae. The network loses its ripples during cold-incubation, which indicates the formation of a liquid-ordered (lo) gel phase. Ripples re-appear upon warming and the sponge-like network disintegrates spontaneously and irreversibly into small vesicles above the phase transition.

Membrane structure of caveolae and isolated caveolin-rich vesicles.

Caveolae are specialized invaginated domains of the plasma membrane. Using freeze-fracture electron microscopy, the shape of caveolae and the distribution of intramembrane particles (integral membrane proteins) were analyzed. The caveolar membrane is highly curved and forms flask-like invaginations with a diameter of 80-120 nm with an open porus of 30-50 nm in diameter. The fracture faces of caveolar membranes are nearly free of intramembrane particles. Protein particles in a circular arrangement surrounding the caveolar opening were found on plasma membrane fracture faces. For isolation of caveolin-enriched membrane vesicles, the method of Triton X-100 solubilization, as well as a detergent-free isolation method, was used. The caveolin-rich vesicles had an average size of between 100 and 200 nm. No striated coat could be detected on the surface of isolated caveolin-rich vesicles. Areas of clustered intramembrane particles were found frequently on membrane fracture faces of caveolin-rich vesicles. The shape of these membrane protein clusters is often ring-like with a diameter of 30-50 nm. Membrane openings were found to be present in the caveolin-rich membrane vesicles, mostly localized in the areas of the clustered membrane proteins. Immunogold labeling of caveolin showed that the protein is a component within the membrane protein clusters and is not randomly distributed on the membrane of caveolin-rich vesicles.

High-pressure freezing causes structural alterations in phospholipid model membranes.

The influence of high-pressure freezing (HPF) on the lipid arrangement in phospholipid model membranes has been investigated. Liposomes consisting of pure dipalmitoyl-phosphatidylcholine (DPPC) and of DPPC mixed with a branched-chain phosphocholine (1,2-di(4-dodecyl-palmitoyl)- sn-glycero-3-phosphocholine) have been analysed by freeze-fracture electron microscopy. The liposomes were frozen either by plunging into liquid propane or by HPF. The characteristic macroripple-phase of the two-component liposome system is drastically changed in its morphology when frozen under high-pressure conditions. The influence of ethanol which acts as pressure transfer medium was ruled out by control experiments. In contrast, no high-pressure alterations of the pure DPPC bilayer membrane have been observed. We assume that the modification of the binary system is due to a pressure-induced relaxation of a stressed and unstable lipid molecule packing configuration. HPF was performed with a newly designed sample holder, for using sandwiched copper platelets with the high-pressure freezing machine Balzers HPM010. The sandwich construction turned out to be superior to the original holder system with regard to freeze-fracturing of fluid samples. By inserting a spacer between the supports samples with a thickness of 20-100 microns can be high-pressure frozen. The sandwich holder is provided with a thermocouple to monitor cooling rates and allows exact sample temperature control. Despite a two-fold mass reduction compared to the original holder no HPF cooling rate improvement has been achieved (4000 degrees Cs-1). We conclude that the cooling process in high-pressure freezing is determined mainly by cryogen velocity.