Prognostic relevance of the mitotic count and the amount of viable tumour after neoadjuvant chemotherapy for primary, localised, high-grade soft tissue sarcoma.

BACKGROUND: We sought to examine whether mitotic count (MC) and the amount of viable tumour (VT) following neoadjuvant systemic chemotherapy (SC) for primary, localised, high-grade soft tissue sarcoma (STS) correlate with prognosis. METHODS: Retrospective analysis of 57 patients who underwent SC involving a combination of an anthracycline and an alkylating agent, followed by surgical resection between 2001 and 2011. RESULTS: The amount of VT after chemotherapy was significantly associated with disease-specific survival (DSS) and event-free survival (EFS). Patients with <10% VT had a DSS of 94% at 5 years, compared with 61% for patients with ⩾10% VT (P=0.033); EFS was 75%, compared with 48% (P=0.030). Patients with an MC of ⩾20/10 high power fields (HPF) after chemotherapy had a significantly lower DSS (33% vs 84% at 5 years, P<0.001) and EFS (40% vs 63% at 5 years, P=0.019) than patients with an MC of <20/10 HPF. CONCLUSIONS: The MC and the amount of VT after neoadjuvant therapy for primary, localised, high-grade STS appear to correlate with prognosis. If these results are validated prospectively, then they could provide a rational for the design of neoadjuvant treatment modification/escalation studies, analogue to the EURAMOS-1 trial for bone sarcomas.

Phase I study of panobinostat and imatinib in patients with treatment-refractory metastatic gastrointestinal stromal tumors.

BACKGROUND: Panobinostat, a pan-deacetylase inhibitor, overcomes imatinib resistance in preclinical models of gastrointestinal stromal tumours (GIST). Here we determined the maximum tolerated dose (MTD) and dose-limiting toxicities (DLT) of panobinostat in combination with imatinib (IM) for treatment of patients with refractory GIST. METHODS: Following a 7-day run-in phase of IM (400 mg per day), escalating doses of panobinostat were added following a '3 plus 3' design. Twelve heavily pretreated GIST patients were enrolled in two dose levels. RESULTS: Most common adverse events were thrombocytopenia, anaemia, fatigue, creatinine elevation, nausea, emesis and diarrhoea. Twenty micrograms of panobinostat and 400 mg IM were declared the MTD. Pharmacologically active concentrations of panobinostat and IM were achieved as evidenced by histone H3 acetylation in blood mononuclear cells in vivo and inhibition of the IM-resistant KIT (D816) mutation in vitro. In FDG-PET-CT scans after IM run-in and following 3 weeks panobinostat treatment, 1 out of 11 evaluable patients showed a metabolic partial response, 7 patients were metabolically stable and 3 patients progressed. Longest treatment duration was 17 weeks (median 6). CONCLUSION: Panobinostat and IM can be administered at doses achieving target inhibition in vivo. Further clinical exploration of patients with treatment-refractory GIST is warranted. Correlative studies in this trial may help to optimise dosing schedules in GIST.

Interactions of protamine with the marine bacterium, Pseudoalteromonas sp. NCIMB 2021.

UNLABELLED: Pseudoalteromonas sp. NCIMB 2021 (NCIMB 2021) was grown in synthetic seawater (SSW) containing pyruvate, in the presence (SSW(++) ) and absence (SSW(-) ) of divalent cations. Cultures contained single cells. Addition of the cationic antibacterial peptide (CAP), protamine, did not inhibit, but rather increased, the growth of NCIMB 2021 in SSW(++) and caused the bacteria to grow in chains. Bacterial growth was assessed using turbidity, cell counts and the sodium salt of resazurin. In SSW(-) , NCIMB 2021 was no longer resistant to protamine. The minimum inhibitory concentration (MIC) was 5 mg ml(-1) . SIGNIFICANCE AND IMPACT OF THE STUDY: Protamine is a cationic antimicrobial peptide (CAP), which is active against a variety of bacteria. This is the first in-depth study of the interaction of protamine with a marine bacterium, Pseudoalteromonas sp. NCIMB 2021. Our results show that protamine is only active in seawater in the absence of divalent cations. In the presence of the divalent cations, Mg(2+) and Ca(2+) , protamine enhances the growth of Pseudoalteromonas sp. NCIMB 2021 and produces chains rather than individual cells. These are important considerations when deciding on applications for protamine and in terms of understanding its mechanism of action.

Differentiation of myxoid liposarcoma by magnetic resonance imaging: a histopathologic correlation.

BACKGROUND: Myxoid liposarcomas represent a heterogeneous group of soft tissue tumors in which prognosis is dependent on differentiation. PURPOSE: To identify magnetic resonance imaging (MRI) criteria to distinguish low-grade from high-grade myxoid liposarcomas. MATERIAL AND METHODS: MR images of 30 histologically proven myxoid liposarcomas were retrospectively reviewed. Tumors were evaluated according to size, localization, tumor border, and structure as well as tumor composition. These imaging criteria were correlated with histopathological findings. RESULTS: Nineteen myxoid liposarcomas were histologically classified as low-grade myxoid liposarcomas, whereas 11 were considered high-grade myxoid liposarcomas. Mean tumor volume of low-grade myxoid liposarcomas (710.1 ± 960.1 ccm) was significantly smaller as compared to high-grade myxoid liposarcomas (2737.0 ± 3423.7 ccm; P = 0.04). In addition to necrotic areas, three tumor components - fatty, myxoid, as well as contrast-enhancing non-fatty, non-myxoid - could be identified. The mean fraction of fatty tumor areas in low-grade myxoid liposarcomas was 10 ± 11% as compared to 6 ± 4% for high-grade myxoid liposarcomas (P = 0.66). Myxoid components accounted for 88 ± 16% in low-grade myxoid liposarcomas, but only for 45 ± 25% in high-grade myxoid liposarcomas (P < 0.0001). The non-fatty, non-myxoid tumor fraction was significantly higher in high-grade myxoid liposarcomas (50 ± 25%) as compared to low-grade myxoid liposarcomas (2 ± 9%; P < 0.0001). A proportion of > 5% of this tumor fraction was found to be a precise unique predictor for high-grade myxoid liposarcomas with a sensitivity of 100% and a specificity of 95%. CONCLUSION: Tumor components with contrast-enhancing non-fatty, non-myxoid imaging features were predominantly found in high-grade myxoid liposarcomas, which may histologically resemble round cell clusters.

Current unmet needs in locally advanced (unresectable) or metastatic dedifferentiated liposarcoma, the relevance of progression-free survival as clinical endpoint, and expectations for future clinical trial design: an international Delphi consensus report.

BACKGROUND: Locally advanced (unresectable) or metastatic dedifferentiated liposarcoma (DDLPS) is a common presentation of liposarcoma. Despite established diagnostic and treatment guidelines for DDLPS, critical clinical gaps remain driven by diagnostic challenges, symptom burden and the lack of targeted, safe and effective treatments. The objective of this study was to gather expert opinions from Europe and the United States on the management, unmet needs and expectations for clinical trial design as well as the value of progression-free survival (PFS) in this disease. Other aims included raising awareness and educate key stakeholders across healthcare systems. MATERIALS AND METHODS: An international panel of 12 sarcoma key opinion leaders (KOLs) was recruited. The study consisted of two rounds of surveys with pre-defined statements. Experts scored each statement on a 9-point Likert scale. Consensus agreement was defined as ≥75% of experts scoring a statement with ≥7. Revised statements were discussed in a consensus meeting. RESULTS: Consensus was reached on 43 of 55 pre-defined statements across disease burden, treatment paradigm, unmet needs, value of PFS and its association with overall survival (OS), and cross-over trial design. Twelve statements were deprioritised or merged with other statements. There were no statements where experts disagreed. CONCLUSION: This study constitutes the first international Delphi panel on DDLPS. It aimed to explore KOL perception of the disease burden and unmet need in DDLPS, the value of PFS, and its potential translation to OS benefit, as well as the relevance of a cross-over trial design for DDLPS therapies. Results indicate an alignment across Europe and the United States regarding DDLPS management, unmet needs, and expectations for clinical trials. Raising awareness of critical clinical gaps in relation to DDLPS can contribute to improving patient outcomes and supporting the development of innovative treatments.

Sentinel node biopsy in soft tissue sarcoma subtypes with a high propensity for regional lymphatic spread--results of a large prospective trial.

BACKGROUND: The role of sentinel lymph node biopsy (SLNB) in soft tissue sarcoma patients has yet to be determined. We sought to evaluate the role of SLNB in the treatment of patients with clear cell sarcoma (CCS), synovial sarcoma (SS), epithelioid sarcoma (ES) and rhabdomyosarcoma (RMS). PATIENTS AND METHODS: Sixty-two consecutive patients without history of regional lymphatic spread or evidence of distant metastases underwent SLNB. RESULTS: Positive sentinel nodes were identified in 2 out of 42 patients with SS and in 6 out of 12 patients with CCS. Only two CCS patients had further metastatic nodes in regional dissection. Both of these patients, along with another CCS patient, developed distant metastases and ultimately died of disease. The remaining three CCS patients are disease-free in follow-up. One patient with SS and another with ES developed regional lymph node metastases following a negative SLNB, while a further patient with RMS developed distant metastases followed by a local recurrence with regional metastases shortly after. CONCLUSIONS: SLNB is an important diagnostic tool for patients with CCS, who appear to have a high rate of clinically occult regional lymph node metastases at diagnosis. For SS patients, SLNB appears to be of very little relevance.

Comparison of RECIST and Choi criteria for computed tomographic response evaluation in patients with advanced gastrointestinal stromal tumor treated with sunitinib.

BACKGROUND: Controversies exist about computed tomography (CT) response evaluation criteria for patients with gastrointestinal stromal tumor (GIST). PATIENTS AND METHODS: Fifty-one patients with advanced GIST treated second line with sunitinib were evaluated with contrast-enhanced CT every 3 months. Response was rated according to RECIST and Choi criteria. Progression-free survival (PFS) and overall survival (OS) were estimated by Kaplan-Meier analysis. RESULTS: According to RECIST, patients were categorized as complete response (CR; n = 0; 0%), partial remission (PR; n = 1; 2.0%), stable disease (SD; n = 37; 72.5%), and progressive disease (PD; n = 13; 25.5%) at 3 months. When Choi criteria were applied responses were CR (n = 0; 0%), PR (n = 16; 31.4%), SD (n = 21; 41.1%), and PD (n = 14; 27.5%). Despite these discrepancies, patients rated as SD with RECIST and PR as well as SD according to Choi criteria displayed similar PFS (41.3, 40.7, and 41.3 weeks, respectively) and OS (100.4, 91.6, and 108.0 weeks, respectively). Patients with PD had significantly shorter PFS (10.1 weeks for both criteria) and OS (29.1 weeks for RECIST; 28.9 weeks for Choi) regardless of the response classification applied. CONCLUSION: In contrast to absence of progression, discrimination of PR from SD with Choi criteria was of no predictive value.

[Systemic therapy of soft tissue sarcomas]. / Systemische Therapie von Weichgewebssarkomen.

The gold standard for the treatment of primary, resectable, high-grade soft tissue sarcomas is complete surgical removal followed by radiotherapy. In cases where preservation of function is not possible, preoperative treatment options should be considered. Systemic therapy is the treatment of choice for metastatic soft tissue sarcomas. The most active single agents include the anthracyclines doxorubicin and epirubicin, as well as ifosfamide. While combination chemotherapy yields higher response rates, this is at the cost of increased toxicity with no evidence of prolonged overall survival. Current treatment strategies focus on the development of specific treatments for well defined soft tissue sarcoma subtypes. The first and highly successful targeted therapy was seen with the introduction of imatinib in the treatment of gastrointestinal stromal tumors.

Pattern of recurrence in patients with ruptured primary gastrointestinal stromal tumour.

BACKGROUND: This study assessed the outcomes of patients with a gastrointestinal stromal tumour (GIST) that ruptured before or during resection. METHODS: The records of 23 patients (8 women, 15 men; median age 54 years) with ruptured primary non-metastatic GIST were retrieved from a database of 554 patients. The written surgical and pathology reports were analysed. Review pathology was performed in all 23 cases, and mutational analysis of KIT and platelet-derived growth factor α (PDGFRA) genes was performed in 21 patients. Median follow-up was 52 months. RESULTS: Tumour rupture was spontaneous in 16 patients, following abdominal trauma in two and occurred during resection in five. Primary tumour location was the stomach in six patients, duodenum in one and small bowel in 16. Mean tumour size was 10·2 (range 4-28) cm. According to the Miettinen and Lasota risk classification, the distribution of very low-, low-, intermediate- and high-risk cases was one, two, five and 15 respectively. One patient remained disease-free at 83 months. Fifteen of 16 patients who did not receive adjuvant therapy developed tumour recurrence after a median of 19 months. Median recurrence-free survival in patients with KIT mutations involving codons 557-558 was 11 months. CONCLUSION: Patients with a rupture of GIST into the abdominal cavity have a risk of recurrence of nearly 100 per cent. In patients with deletion mutations involving codons 557-558, recurrence-free survival was less than 1 year. All patient groups are clear candidates for adjuvant drug therapy.

Quantification of local water and biomass in wild type PA01 biofilms by Confocal Raman Microspectroscopy.

Confocal Raman Microspectroscopy (CRM) can be used as a tool for the in situ evaluation of the chemical composition of living, fully submerged, unstained biofilms. In this study the estimation of the local water content in Pseudomonas aeruginosa PA01 biofilms is given as an example. The ratio of the area of the O-H stretching vibration band at 3450 cm(-1), (water), to that of the C-H stretching bands at 2950 cm(-1) (biomass), was used to estimate the relative biofilm water content. The quantification of biofilm water and biomass was based on calibration curves generated from protein solutions. Water/biomass ratios (W:BR) equivalent to that of a 30% (w/v) protein solution were observed within some biofilm colonies.

Phyllosphere microbiology with special reference to diversity and plant genotype.

The phyllosphere represents the habitat provided by the aboveground parts of plants, and on a global scale supports a large and complex microbial community. Microbial interactions in the phyllosphere can affect the fitness of plants in natural communities, the productivity of agricultural crops, and the safety of horticultural produce for human consumption. The structure of phyllosphere communities reflects immigration, survival and growth of microbial colonists, which is influenced by numerous environmental factors in addition to leaf physico-chemical properties. The recent use of culture-independent techniques has demonstrated considerable previously unrecognized diversity in phyllosphere bacterial communities. Furthermore, there is significant recent evidence that plant genotype can play a major role in determining the structure of phyllosphere microbial communities. The main aims of this review are: (i) to discuss the diversity of phyllosphere microbial populations; (ii) to consider the processes by which microbes colonize the phyllosphere; (iii) to address the leaf characteristics and environmental factors that determine the survival and growth of colonists; (iv) to discuss microbial adaptations that allow establishment in the phyllosphere habitat and (v) to evaluate evidence for plant genotypic control of phyllosphere communities. Finally, we suggest approaches and priority areas for future research on phyllosphere microbiology.

Exatecan in pretreated adult patients with advanced soft tissue sarcoma: results of a phase II--study of the EORTC Soft Tissue and Bone Sarcoma Group.

No standard treatment is established for patients with advanced soft tissue sarcoma after previous chemotherapy with anthracyclines and ifosfamide, given either in combination or sequentially. Exatecan (DX-8951f) is a totally synthetic analogue of the topoisomerase I-inhibitor camptothecin, which was synthesised to impart increased aqueous solubility, greater tumour efficacy, and less toxicity than camptothecin itself, topotecan or irinotecan. Since some activity against soft tissue sarcomas, especially leiomyosarcomas, has been reported for topoisomerase I-inhibitors, a study with a new and more potent agent seemed justified. We report on a prospective multicentre phase II study of Exatecan in adult soft tissue sarcomas failing 1 or 2 lines of chemotherapy in advanced phase, performed within the STBSG of EORTC. Thirty-nine patients (16 leiomyosarcomas and 23 other histologies) were included in two independent strata and received a total of 141 cycles (median 2). Median age was 61 years, range 25-76. Exatecan was given as i.v. infusion over 30 min at a dose of 0.5mg/m2 every day for five consecutive days, repeated every 21 days. Seventy-four percentage of cycles could be given without dose or schedule modification. The main toxicity was haematotoxicity with grade 3/4 neutropenia in 49%, grade 3/4 thrombocytopenia in 23%, and grade 3/4 anaemia in 15% of patients, respectively. Non-haematological toxicity consisted mainly of grade 2/3 dyspnoea in 36% of patients and grade 2/3 fatigue in 28%. One treatment-related toxic death due to septic shock was reported. Best overall response was no change with 60% in the leiomyosarcoma group and 53% in the non-leiomysarcoma group, respectively. The 3 months progression-free survival estimates are 56% for leiomysarcomas and 26% for other histologies, respectively. Using a two-step statistical design, the trial was stopped after the first step in both strata, due to lack of activity. In pretreated soft tissue sarcoma patients, Exatecan is well tolerated but does not achieve any objective responses. However, with respect to progression-free survival, Exatecan did show some activity in leiomyosarcomas.

Sheared edible oils studied using dissipative particle dynamics and ultra small angle X-ray scattering: TAGwood orientation aggregation and disaggregation.

Previous work on the computer simulation of edible fats and oils showed that triglyceride crystalline nanoplatelets (CNPs) aggregated into cylindrical structures dubbed "TAGwoods". This was experimentally verified using Ultra Small Angle X-ray Scattering experiments. In this paper, the aggregation of these TAGwoods was studied using the fluid simulation technique, Dissipative Particle Dynamics. The intent was to predict the TAGwood aggregation structures which arise via the application of a series of shear rates, [small gamma, Greek, dot above]. The effect of shear on TAGwood orientational order was also investigated. Three aggregation regimes were identified: At shear rates below a certain critical value, 0 < [small gamma, Greek, dot above] < [small gamma, Greek, dot above]t aggregation was enhanced. The value of the critical shear rate depended on the size of the CNPs. With large CNPs possessing a side length of ∼500 nm, the critical shear rate was [small gamma, Greek, dot above]t ≈ 0.6 s-1. However, if the CNPs were smaller with a side length of ∼100 nm, then [small gamma, Greek, dot above]t ≈ 75 s-1. For shear rates above the critical shear rate, [small gamma, Greek, dot above] > [small gamma, Greek, dot above]t aggregation was inhibited. The USAXS data was analyzed using the Unified Fit model and the observations were in accord with the simulation results. Three regimes were identified based on the values of the linear slope P2 of the USAXS data. P2 increased as [small gamma, Greek, dot above] increased, indicating increased aggregation of the TAGwoods as the shear rate was increased. P2 ceased increasing and began to decrease when [small gamma, Greek, dot above] ≈ [small gamma, Greek, dot above]t. With further increases in [small gamma, Greek, dot above], P2 decreased as [small gamma, Greek, dot above] increased further, which is indicative of a decrease in aggregation. The orientational quadrupole order parameter, S = 〈Q33〉 = 1/2〈cos2θ - 1〉, was computed, where θ is the angle between the axis of the TAGwood and the axis of flow, and showed that, for large [small gamma, Greek, dot above], it achieved a near-maximum value. This indicates that at high shear rates, the long axis of the cylindrical TAGwoods aligns in a direction parallel to that of the fluid flow.

Phase 1 European Organisation for Research and Treatment of Cancer study determining safety of pegylated liposomal doxorubicin (Caelyx) in combination with ifosfamide in previously untreated adult patients with advanced or metastatic soft tissue sarcomas.

This phase I study evaluated the toxicity of first-line combined pegylated liposomal doxorubicin (Caelyx) and ifosfamide in patients with advanced and/or metastatic soft tissue sarcomas. Five dose levels (L) were studied: Caelyx 30 mg/m2 (L1-4) or 40 mg/m2 (L5) 1-h infusion d 1 q 3 weeks + ifosfamide and mesna at X g/m2/4 h d 1-3 q 3 weeks at five doses: L1: X = 1.7 g; L2: X = 2 g; L3: X = 2.5 g; L4 and L5: X = 3 g. Cohorts of 3 patients were entered at each level unless a dose-limiting toxicity (DLT) occurred. In case of DLT in 1 of 3 patients a new cohort was added. Toxicity was evaluated by Common Toxicity Criteria (CTC). A total of 28 patients was included: 4 at dose L1, 8 at L2, 3 at L3, 6 at L4, and 7 at L5. Median age was 60 years (range 29-69 years). Male/female ratio was 12/16. Seventy-five percent of patients had a performance status of 1.0 and 36% had leiomyosarcomas. No DLT was observed at dose L1-4. Six patients developed a DLT at dose L5, and thus the recommended dose is level 4 (i.e. Caelyx 30 mg/m2/1 h d 1+ifosfamide at 3 g/m2/4 h d 1-3 q 3 weeks). Few haematological and biochemical events were observed and the principal toxicities were granulocytopaenia and leucopaenia. Five patients discontinued therapy because of toxicity, 4 of them at dose level 5. Non-haematological toxicities > grade 2 were also few. Palmar-plantar erythrodysesthesia (PPE) > grade 1 was not seen. Two patients obtained partial response (PR) and 13 stable disease (SD). Median overall survival (OS) was 333 d and median progression-free survival (PFS) 174 d. In conclusion, this seems to be a feasible combination in patients with advanced soft tissue sarcomas, allowing ifosfamide to be given in a dosage similar to that used when given alone. The recommended dose for future studies is Caelyx 30 mg/m2/1 h d 1+ifosfamide 3 g/m2/4 h d 1-3 q 3 weeks.

The lateral distribution of cholesterol in the plane of lipid multibilayers.

We consider three models of cholesterol distribution in the plane of a bilayer of DMPC. We analyse recent 2H-NMR data obtained from deuterated fluorescent probes and show that, on the characteristic time-scale of 2H-NMR, it is in accord with a random distribution of cholesterol in a fluid-like DMPC bilayer in a single phase at least for T greater than or approximately equal to 35 degrees C and for 0 less than or equal to c less than or equal to 0.42.

Peak locater for multicomponent spectra.

A sliding pivot technique capable of locating component peak maxima of multicomponent spectra is presented. The locations of peak maxima obtained in this way are shown to be the same as those of the minima in the second derivative. A major advantage over the second-derivative test is simply that derivative spectra are not needed. The sliding pivot technique requires only the original spectrum to locate the component peak maxima and consequently reduces the noise enhancement factor. The deconvoluted Fourier transform infrared spectrum of purple membrane is analyzed and compared to a Gaussian analysis and a second-derivative analysis. The sliding pivot technique identifies a band missed by second-derivative analysis.

Computer simulation of lipid diffusion in a two-component bilayer. The effect of adsorbing macromolecules.

We have modelled the effects of macromolecular adsorption upon lipid lateral diffusion in a two-component lipid bilayer or monolayer, which is at a temperature above both of the main transition temperatures. One set of lipids (binders, b) can bind to the macromolecules with a free energy of binding, FB, while the other set does not bind (non-binders, nb). We assumed that no phase separation of the lipids occurs in the absence of adsorbed macromolecules. We represented the lipid bilayer/monolayer by a triangular lattice, each site of which is occupied by a lipid molecule. Adsorbed macromolecules were represented by hexagons covering nH sites, and we defined a probability per unit of time, p, that a hexagon attempts to adsorb onto the lattice. We considered two sizes of hexagons, nH = 7 (Size-1) and nH = 19 (Size-2) and disallowed or permitted adsorbed hexagons to move laterally on the lattice. We calculate the lipid relative diffusion coefficients, Dnb, and Db, for three characteristic time-regimes, (i) tau c << tau a, (ii) tau c approximately tau a and (iii) tau c >> tau a, where tau c and tau a are the times for proteins to adsorb/desorb or for lipids to move from site to site, respectively. We obtain analytical expressions for Dnb and Db in the first case and calculate them using computer simulation in the other two cases. We found that (i) D alpha (iii) < or = D alpha (ii) < or = D alpha (i) (alpha = nb, b); (ii) D alpha could display a shoulder as a function of FB for low values of p; (iii) compared to cases in which lateral diffusion was disallowed, the lateral diffusion of absorbed hexagons appeared to have little effect on Dnb, but could cause Db to increase by 50%. (iv) Scatter in the calculated values of D via simulation appeared to be largest for Size-1 hexagons, and could be understood as a consequence of the large interfacial region between areas free of hexagons and areas 'covered' by hexagons. Our results suggest that it is advisable to measure Db, since Dnb might show little change from 1.0 for the values of F and p appropriate to the system being studied.

Line defects in gel phase lipid monolayers.

We investigate various models of the hydrolysis of gel-phase phosphatidylcholine monolayers by phospholipase A2 (Grainger et al. (1989) FEBS Lett. 252, 73-82). We assume that the probability of hydrolysis of a given lipid depends only upon how many of its nearest neighbour lipids have already been hydrolysed. We find that the experimental data are consistent with a model in which line defects exist in the gel phase and that lipids on such defects are more easily hydrolysed than the other gel-phase lipids. Based on this model, we calculate the course of hydrolysis of a gel-phase region possessing line defects, and we suggest how such a structure might be made and the model tested. An experiment, similar to that proposed by us, has been carried out by Grainger et al. (1990) Biochim. Biophys. Acta 1023, 365-379). We also calculate the fractal dimension, df, of the interface created by the hydrolytic process and show that a measurement of df might identify how this process proceeds.

Intersecting polymers in lipid bilayers: cliques, static order parameters and lateral diffusion.

We have modelled a macrolipid polymer composed of lipid molecules (monomers) embedded in a lipid bilayer or monolayer and polymerized via their polar groups. Because of fluctuations perpendicular to the plane of the bilayer, the polar region occupied by the polymer chain possesses sufficient space so that the polymer might exhibit 'self-intersection' if its conformational state is projected onto the plane of the bilayer/monolayer. We represent the plane of the bilayer/monolayer by a triangular lattice. Each site can be occupied by a monomer or be empty (and thus occupied by one of the unpolymerizable lipids which make up the bilayer/monolayer). A macrolipid is represented by a sequence of N monomers connected by N-1 bonds. Bonds may be either short (connecting nearest neighbour monomers) or long (between second neighbour monomers), in accord with the average properties of the spacers between the polymerized lipids. We have carried out computer simulation of this system using the Carmesin-Kremer bond stretching algorithm. Although no two monomers can occupy the same site, bonds may cross each other. We analyzed the dependence of and approximately N2vc and + approximately N2 sigma c, where Nsc and Nmc are the number of bond-crossings in the same macrolipid ('self-crossing') or in two different macrolipids ('mutual-crossing'). For single macrolipids, we confirmed that vc = 3/4 and have found that sigma c approximately 0.52, which we consider supports that sigma c = 1/2. For the dense case with monomer concentration, c = 0.72, we found that vc = 1/2 and that sigma c approximately 0.52 supports that sigma c = 1/2. In the semi-dilute regime (c = 0.2) we found crossover behaviour, although sigma c = 1/2. The total number of bond crossings thus scale like N, independent of concentration. We studied the connectivity of the system by calculating the weight averaged cluster, or 'clique', size. Cliques are defined as being composed of all macrolipids which exhibit at least one crossing bond with one other member of the clique. We found that while the average clique contains about two macrolipids at low concentrations, the clique size approaches the maximum possible value at high concentrations if the macrolipids are sufficiently long. In the latter case a transition appears to occur as the macrolipid length increases. This transition occurs at length = 40 when c = 0.72. These observations should have experimental consequences for the viscoelastic properties of the system.(ABSTRACT TRUNCATED AT 400 WORDS)

A model of hydrogen bond formation in phosphatidylethanolamine bilayers.

We have modelled hydrogen bond formation in phospholipid bilayers formed, in excess water, from lipids with phosphatidylethanolamine (PE) headgroups. The hydrogen bonds are formed between the NH3+ group and either of the PO2- or the (sn2 chain) C=O groups. We used a model that represented the conformational states accessible to a PE headgroup by 17 states and modelled lipid dipole-dipole interactions using a non-local electrostatics theory to include the effects of hydrogen bonding in the aqueous medium. We used Monte-Carlo simulation to calculate equilibrium thermodynamic properties of bilayers in the fluid (T = 340 K) or gel (T = 300 K) phases of the bilayer. We defined Eh to be the difference in free energy between a hydrogen bond formed between a pair of lipid groups, and the energy of hydrogen bonds formed between water and those two groups, and we required its average value, [Eh], to be approximately -0.3kcal/mol (approximately -0.2 X 10(-13) erg) as reported by T.-B. Shin, R. Leventis, J.R. Silvius, Biochemistry 30 (1991) 7491. We found: (i) Eh = -0.9 X 10(-13) erg gave [Eh] = -0.21 X 10(-13) erg (gel phase) and [Eh] = -0.19 X 10(-13) erg (fluid phase). (ii) The relative number of C=O groups on the sn2 chain calculated to take part in interlipid hydrogen bonding in the fluid phase compared to the gel is 1.06 which compares well with the experimental ratio of approximately 1.25 (R.N.A.H. Lewis, R.N. McElhaney, Biophys. J. 64 (1993) 1081). The ratio of such groups taking part in interlipid hydrogen bonding compared to water hydrogen bonding in each phase was calculated to lie between 0.16 and 0.17. (iii) We calculated the distribution of positions of the headgroup moieties, P, O, CH2(alpha), CH2(beta) and N, and found that, in both phases, the O lay furthest from the hydrocarbon chain layer (average approximately 5.3A) with the PO2 and NH3 groups lying at approximately 5A. This results in the P-N dipole lying nearly parallel to the bilayer plane in both phases. The thickness of the headgroup layer underwent essentially no change on going from the gel to the fluid phase. The 2H NMR quadrupole splittings for the alpha and beta CH2 groups were 4.9 and 5.7kHz (fluid phase) and 7.1 and 7.3 kHz (gel phase), respectively, on the assumption of sufficiently rapid rotation around the z-axis. (iv) In both phases, the location of the NH3+ group exhibited a strong peak around 5.2 A into the aqueous medium, with much smaller peaks around 2.6 and 7.8 A, the two CH2 groups exhibited narrower, double-peaked distributions and the O and the PO2 each exhibited a narrow single peak. (v) PE headgroups, in a homogeneous gel phase, exhibited dipolar orientational long-range order in the plane of the bilayer. The distribution of orientation angles exhibited a full width at half height of between approximately 40 degrees and approximately 50 degrees. In a fluid phase no such order was observed. (vi) The number of hydrogen bonds did not differ substantially between the fluid and gel phases. This model is unlikely to display any significant effect of hydrogen bonding upon the "main" hydrocarbon chain melting phase transition at Tm, except, possibly, a broadening of any hysteresis, compared to the case of PC bilayers where interlipid hydrogen bonding is absent.