Histology of tendon and enthesis - suitable techniques for specific research questions.

The musculoskeletal system consists of different components comprising a wide range of tissue types, with tendons being one part. Tendon degeneration or rupture have a high prevalence in all age groups, often with poor outcomes of surgical treatment such as chronic pain and high re-tear rates. Therefore, much effort has been directed to further develop diagnostic and therapeutic methods as well as reconstruction techniques, including using adequate placeholders or implants. Diagnostic approaches and advanced stages of preclinical studies will inevitably include histological examination of the pathologically affected tissue. The present study presents adequate tendon-related, histological techniques, including the embedding of soft- and hard-tissue samples in different media. Consideration is also given to samples containing residual implant materials or having been subjected to standard staining protocols and immunohistochemical procedures. The study further examines cells and tendon structure to detect degenerative, fibrotic or inflammatory conditions and possible foreign-body responses to implanted materials. Infraspinatus tendons from preclinical studies carried on rat and sheep samples, as well as human biceps tendon samples, have been used as example materials.

Constrained tripolar liner in patients with high risk of dislocation - Analysis of incidence and risk of failure.

INTRODUCTION: The aim of this study was to determine number and type of failures and revisions after usage of a constrained tripolar acetabular liner in patients with high risk of dislocation. Potential correlations between these failures and the factors included were analyzed. MATERIALS AND METHODS: In this retrospective study 55 participants in 68 cases were included after treatment with constrained tripolar acetabular liner. Patient specific data as well as surgery and implant specific data were collected. Radiological images were assessed. Furthermore, the gluteal function was analyzed. The parameters were statistically verified with regard to their influence on the failure of the constrained tripolar liner. RESULTS: This study included 16 cases (in nine participants) of postoperative failure. This results in a survival rate of 76.5% regarding the number of cases after 17 months. The statistical analysis of the different parameters considered that the number of previous surgeries has a significant (p = 0.027) influence on the failure. CONCLUSIONS: This retrospective study shows that treatment with constrained tripolar acetabular liners is a satisfactory method of treatment in cases with a high risk of dislocation. However, in cases with an increasing number of previous surgeries, an increased risk of failure was found. Therefore, in such cases, this type of supply treatment should be treated critically.

Postimpoundment time course of increased mercury concentrations in fish in hydroelectric reservoirs of northern Manitoba, Canada.

Mercury (Hg) concentrations in fish in boreal reservoirs have been shown to be increased for up to 3 decades after impoundment. However, the time course of increased concentrations is not well known. The purpose of this study was to determine the evolution of Hg concentrations in fish in the boreal reservoirs of northern Manitoba, Canada, and its relationship with severity of flooding. We determined total Hg concentrations in three species of fish for up to 35 years after impoundment in 14 lakes and lake basins. Postimpoundment trends depended on fish species and reservoir. In the benthivorous lake whitefish (Coregonus clupeaformis), Hg concentrations increased after flooding to between 0.2 and 0.4 microg g(-1) wet weight compared with preimpoundment concentrations between 0.06 and 0.14 microg g(-1) and concentrations in natural lakes between 0.03 and 0.06 microg g(-1). Hg concentrations in lake whitefish were usually highest within 6 years after lake impoundment and took 10 to 20 years after impoundment to decrease to background concentrations in most reservoirs. Hg concentrations in predatory northern pike (Esox lucius) and walleye (Sander vitreus) were highest 2 to 8 years after flooding at 0.7 to 2.6 microg g(-1) compared with preimpoundment concentrations of 0.19 to 0.47 microg g(-1) and concentrations in natural lakes of 0.35 to 0.47 microg g(-1). Hg concentrations in these predatory species decreased consistently in subsequent years and required 10 to 23 years to return to background levels. Thus, results demonstrate the effect of trophic level on Hg concentrations (biomagnification). Peak Hg concentrations depended on the amount of flooding (relative increase in lake surface area). Asymptotic concentrations of approximately 0.25 microg g(-1) for lake whitefish and 1.6 microg g(-1) for both walleye and northern pike were reached at approximately 100% flooding. Downstream effects were apparent because many reservoirs downstream of other impoundments had higher Hg concentrations in fish than would be expected on the basis of flooding amount.

Towards a general approach for the impurity profiling of drugs by micellar electrokinetic chromatography.

A general micellar electrokinetic chromatographic (MEKC) strategy for the impurity profiling of drugs was developed involving a sodium dodecyl sulfate (SDS) and a cetyltrimethylammonium bromide (CTAB) MEKC system. With this combination, in principle, each sample component passes the detector in at least one of the two MEKC systems provided that separation buffers of the same pH are used in both systems. In order to select the proper MEKC systems, the electroosmotic flow (EOF) and micelle migration time (t(mc)) were determined for separation buffers of several pH values, containing various amounts of surfactant and organic modifier. The selectivity of the MEKC systems was studied using a mixture of compounds with a wide range of physico-chemical properties. The final selection of two adequate MEKC systems for this approach was based on the requirements that the t(mc) (i.e., analysis time) of both systems was below 20 min and that the t(mc)/t(eof) ratio was above 3 or 2 for the SDS and CTAB system, respectively. Furthermore, the systems should provide high efficiency, exhibit differences in selectivity and use moderate concentrations of modifier and surfactant, so that, if needed, further optimization is possible. The selected MEKC systems contained 60 mM SDS or 10 mM CTAB, respectively, in a phosphate buffer (pH 7.5) with 10% acetonitrile. Some test compounds with extreme mobilities were used to demonstrate the suitability of the MEKC approach to detect each component of a sample. The potential of the proposed MEKC combination for impurity profiling was demonstrated by the analysis of fluvoxamine with several impurities at the 0.1% level.

Neurological disease associated with EHV-1-infection in a riding school: clinical and virological characteristics.

An outbreak of neurological disease caused by EHV-1 infection is described with emphasis on diagnosis and prognosis for recumbent horses. In April 1995, an outbreak of the neurological form of Equine herpesvirus type 1 (EHV-1) occurred in a well-managed riding school with 41 horses: 34 horses showed a temperature spike and 20 some degree of neurological signs, of which 10 were nursed intensively in the indoor arena of the riding school for 3 to 20 days, 8 having to be maintained in slings for 2-18 days, while 9 needed bladder catheterisation b.i.d. for 2-16 days. Within the first 3 days, one horse was subjected to euthanasia and another horse died. Postmortem examination revealed a mild vasculitis with perivascular mononuclear cuffing and axonal degeneration in the central nervous system. Clinical diagnosis was confirmed by serology and virology: 28 horses seroconverted in one or more tests during the outbreak, whereas 12 had already high CF and SN titres in the first sample, suggestive of recent infection. Virus was isolated from nasal swabs of 4 horses, and identified as EHV-1 with type-specific monoclonal antibodies. Restriction enzyme analysis revealed that the EHV-1 strains from this outbreak belonged to genome type EHV-1.IP. The electropherotypes were identical to those from another, epidemiologically unrelated, outbreak of neurological disease 2 months earlier. The timing of the temperature spikes and seroconversions indicated that the infection was probably introduced by a horse purchased 3 weeks before neurological signs occurred. At follow-up one year later, the 10 horses that showed mild neurological signs had recovered completely. Of the 8 horses that survived intensive care, 3 had returned to around their former performance level (2 of which had been in slings), while the other 5 had become pasture-sound. At follow-up 4 years later, all pasture-sound horses had been subjected to euthanasia because of persistent mild ataxia and incontinence. In conclusion, the prognosis for recumbent horses due to EHV-1 infection is grave. For virological diagnosis, extensive and strategic sampling of febrile in-contact horses is required, and the EHV-1-specific glycoprotein G (gG) ELISA is a valuable tool for specific serological diagnosis of EHV-1 infection causing neurological disease.

The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins.

The N-terminal 17% of apolipoprotein B (apoB-17) readily associates with dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV) to form large (240-A diameter) discoidal particles. Because apoB is normally secreted with triacylglycerol (TAG)-rich lipoproteins, we studied the binding of apoB-17 to triolein-rich emulsions modeling nascent TAG-rich very low density-like lipoproteins. Emulsions with the following composition (by weight) were prepared: 85--89% triolein, 1.1--1.4% cholesterol, and 10--14% phosphatidylcholines (PC) including either egg yolk (EY)-, dimyristoyl (DM)-, or dipalmitoyl (DP)-PC representing (at 25 degrees C), respectively, a fluid surface, a surface at transition, and a mainly solid surface. The respective sizes were 1,260 +/- 500, 1,070 +/- 450, and 830 +/- 300 A mean diameter. The emulsions were incubated with conditioned medium containing apoB-17, and then reisolated by ultracentrifugation. Analysis of the emulsion-bound proteins by gel electrophoresis showed that all three emulsions bound primarily apoB-17. The DPPC emulsions bound more apoB-17 than EYPC or DMPC emulsions. Immunoaffinity-purified apoB-17 exhibited saturable, high affinity binding to EYPC and DPPC emulsions. The respective K(d) values were 32 +/- 23 and 85 +/- 27 nM and capacities (N) were 10 and 58 molecules of apoB-17 per particle. When apoB-17 bound to emulsions was incubated with DMPC MLV at 26 degrees C for 18 h, it remained bound to the emulsions, indicating that once bound to these emulsions it is unable to exchange off and solubilize DMPC into discs. In contrast, apoE-3 bound to emulsions dissociated from the emulsions when incubated with DMPC MLV and formed discs.Thus, apoB-17 binds strongly and irreversibly to emulsions modeling nascent lipoproteins. It therefore may play an important role in the stabilization of nascent VLDL and chylomicrons.- Herscovitz, H., A. Derksen, M. T. Walsh, C. J. McKnight, D. L. Gantz, M. Hadzopoulou-Cladaras, V. Zannis, C. Curry, and D. M. Small. The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins. J. Lipid Res. 2001. 42: 51;-59.

Calorimetry of apolipoprotein-A1 binding to phosphatidylcholine-triolein-cholesterol emulsions.

The thermotropic properties of triolein-rich, low-cholesterol dipalmitoyl phosphatidylcholine (DPPC) emulsion particles with well-defined chemical compositions (approximately 88% triolein, 1% cholesterol, 11% diacyl phosphatidylcholine) and particle size distributions (mean diameter, approximately 1000-1100 A) were studied in the absence and presence of apolipoprotein-A1 by a combination of differential scanning and titration calorimetry. The results are compared to egg yolk PC emulsions of similar composition and size. Isothermal titration calorimetry at 30 degrees C was used to saturate the emulsion surface with apo-A1 and rapidly quantitate the binding constants (affinity Ka = 11.1 +/- 3.5 x 10(6) M-1 and capacity N = 1.0 +/- 0.09 apo-A1 per 1000 DPPC) and heats of binding (enthalpy H = -940 +/- 35 kcal mol-1 apo-A1 or -0.92 +/- 0.12 kcal mol-1 DPPC). The entropy of association is -3070 cal deg-1 mol-1 protein or -3 cal deg-1 mol-1 DPPC. Without protein on the surface, the differential scanning calorimetry heating curve of the emulsion showed three endothermic transitions at 24.3 degrees C, 33.0 degrees C, and 40.0 degrees C with a combined enthalpy of 1.53 +/- 0.2 kcal mol-1 DPPC. With apo-A1 on the surface, the heating curve showed the three transitions more clearly, in particular, the second transition became more prominent by significant increases in both the calorimetric and Van't Hoff enthalpies. The combined enthalpy was 2.70 +/- 0.12 kcal mol-1 DPPC and remained constant upon repeated heating and cooling. Indicating that the newly formed DPPC emulsion-Apo-A1 complex is thermally reversible during calorimetry. Thus there is an increase in delta H of 1.17 kcal mol-1 DPPC after apo-A1 is bound, which is roughly balanced by the heat released during binding (-0.92 kcal) of apo-A1. The melting entropy increase, +3.8 cal deg-1 mol-1 DPPC of the three transitions after apo-A1 binds, also roughly balances the entropy (-3 cal deg-1 mol-1 DPPC) of association of apo-A1. These changes indicate that apo-A1 increases the amount of ordered gel-like phase on the surface of DPPC emulsions when added at 30 degrees C. From the stoichiometry of the emulsions we calculate that the mean area of DPPC at the triolein/DPPC interface is 54.5 A2 at 41 degrees C and 54.2 A2 at 30 degrees C. The binding of apo-A1 at 30 degrees C to the emulsion reduces the surface area per DPPC molecule from 54.2 A2 to 50.8 A2. At 30 degrees apo-A1 binds with high affinity and low capacity to the surface of DPPC emulsions and increases the packing density of the lipid domain to which it binds. Apo-A1 was also titrated onto DPPC emulsions at 45 degrees C. This temperature is above the gel liquid crystal transition. No heat was released or adsorbed. Furthermore, egg yolk phosphatidylcholine emulsions of nearly identical composition were also titrated at 30 degrees C with apo-A1 and were euthermic. Association constants were previously measured using a classical centrifugation assay and were used to calculate the entropy of apo-A1 binding (+28 cal deg-1 mol-1 apo-A1). This value indicates that apo-A1 binding to a fluid surface like egg yolk phosphatidylcholine or probably DPPC at 45 degrees C is hydrophobic and is consistent with hydrocarbon lipid or protein moities coming together and excluding water. Thus the binding of apo-A1 to partly crystalline surfaces is entropically negative and increases the order of the already partly ordered phases, whereas binding to liquid surfaces is mainly an entropically driven hydrophobic process.

Community nursing in Belgium, Germany and The Netherlands.

This paper contains a comparative study on community nursing in the Netherlands, Belgium and Germany, carried out in the region around Maastricht, where the borders of the three countries meet. The well-known problem of comparative studies (the incomparability of concepts and data) has been solved by using the same measuring instruments in the three countries. The comparison between the countries was on three aspects: the level of care dependency of the patients, the type and number of services provided and the nurses' job interpretation and job satisfaction. During 1 week in June or September 1991, 89 community nurses made records of all their home visits and nursing activities. In total the community nurses paid 5165 home visits to provide care to 1796 patients. The results indicate that both the level of care dependency of the patients as well as the type of care provided differs between the three countries. Belgian community nurses have the highest number of patients with a high level of care dependency. Curative services like technical nursing care and domestic care are most frequently provided by the German and Belgian community nurses. Informing, educating and supporting care is most frequently provided by the Dutch community nurses. The German community nurses spend less time on administration activities. With respect to job interpretation and job satisfaction the following results were found. The Dutch community nurses mentioned in their job interpretation many more preventive tasks, whereas the German community nurses more often mentioned domestic tasks. Concerning the hygienic and technical tasks, no significant differences were found between the three countries. Finally, job satisfaction is lowest in the Netherlands. Dutch community nurses are less satisfied with the work organization and the possibilities of autonomy and professionalization than the German and Belgian community nurses.

Expression of the Phytophthora infestans ipiB and ipiO genes in planta and in vitro.

The ipiB and ipiO genes of the potato late blight fungus Phytophthora infestans (Mont.) de Bary were isolated from a genomic library in a screen for genes induced in planta. Expression of these genes was studied during pathogenesis on various host tissues and different host plants, some of which show specific resistance against P. infestans infection. During pathogenesis on leaves and tubers of the fully susceptible potato cultivar (cv.) Ajax and on leaves of the fully susceptible tomato cv. Moneymaker, the P. infestans ipiB and ipiO genes show a transient expression pattern with highest mRNA levels in the early stages of infection. During the interaction with leaves of the partially resistant potato cv. Pimpernel, the expression is also transient but accumulation and disappearance of the mRNAs is delayed. Also in P. infestans inoculated onto a race-specific resistant potato cultivar and onto the nonhost Solanum nigrum, ipiB and ipiO mRNA is detectable during the initial stages of infection. Apparently, the expression of the ipiB and the ipiO genes is activated in compatible, incompatible and nonhost interactions. In encysted zoospores, ipiB and ipiO mRNA accumulation was not detectable, but during cyst germination and appressorium formation on an artificial surface the genes are highly expressed. Expression studies in mycelium grown in vitro revealed that during nutrient starvation the expression of the ipiB and ipiO genes is induced. For ipiO gene expression, carbon deprivation appeared to be sufficient. The ipiO gene promoters contain a sequence motif that functions as a glucose repression element in yeast and this motif might be involved in the regulation of ipiO gene expression.

Identification of diagnostic antigens from Trichinella spiralis.

The Western blotting technique was used to determine the antigens of Trichinella spiralis muscle larvae that were recognized by antibodies in sera from humans and pigs displaying T. spiralis infections. This resulted in the identification of several antigens that were recognized by all sera. Some of these antigens, notably those that were recognized during the early stage of infection, cross-reacted with antibodies to other parasites. This cross-reactivity was caused by the presence of phosphorylcholine on these antigens. A large portion of the antigens that were recognized by antibodies from infected humans and pigs were found to share a single Trichinella-specific determinant. The Trichinella-specific antigen population could be isolated from phosphorylcholine-containing antigens by a simple two-step affinity chromatography procedure using monoclonal antibodies to both determinants. The resulting preparation consisted primarily of a single antigen showing an apparent molecular weight of 45 kDa that corresponded to a major constituent of excretory-secretory (ES) products of muscle larvae. When tested in an enzyme-linked immunosorbent assay (ELISA), this antigen displayed diagnostic specificity that was comparable with the ES fraction and diagnostic sensitivity comparable with the crude muscle-larvae extract.

Plasma clearance of emulsified triolein in conscious rats: effects of phosphatidylcholine species, cholesterol content and emulsion surface physical state.

In vivo clearance of an emulsified triglyceride from rat plasma is strongly influenced by the phosphatidylcholine molecular species and the cholesterol content of the emulsion. Chylomicron-sized, glycerol-tri[9,10-3H]oleate (triolein)-sonicated emulsions containing 2-3 or 9-14 mol% unesterified cholesterol and specific phosphatidylcholines (egg, dimyristoyl, dipalmitoyl, distearoyl) were prepared above the phospholipid gel----liquid crystalline transition temperatures and injected intravenously into awake, non-fasting rats in bolus doses of 1-3 mg lipid. The emulsified triolein was cleared from plasma both by lipolysis during circulation and by uptake of emulsion particles or their remnants into tissues. Increasing the cholesterol content greatly accelerated particle clearance from plasma when emulsions were made with mixed-chain (egg) phosphatidylcholine, but prolonged their circulation time when the emulsions contained saturated phosphatidylcholines. The relative amounts cleared by reticuloendothelial versus liver parenchymal cells were influenced both by the cholesterol content and the phosphatidylcholine species of the emulsions. Significant triolein lipolysis in plasma occurred only with egg or dimyristoyl phosphatidylcholine emulsions containing 2-3% cholesterol and this lipolysis was blocked by increasing the cholesterol content. No lipolysis occurred when emulsions contained dipalmitoyl or distearoyl phosphatidylcholine, irrespective of cholesterol content.

Size and shape determination of fixed chylomicrons and emulsions with fluid or solid surfaces by three-dimensional analysis of shadows.

Chylomicrons and chylomicron-sized emulsions are spherical particles in suspension but their shape and apparent size may be distorted by electron microscopy processing. To assess adsorption to grids, flattening, and shrinkage, chylomicrons and emulsions were fixed with osmium tetroxide and together with polystyrene beads were shadowed with platinum. Vertical profiles projected from particle shadows indicated that the chylomicrons and emulsions were slightly shrunken, slightly truncated, oblate spheroids while the polystyrene beads were spheres. Particle diameters were corrected by assuming that volumes of oblate spheroids on the grid surface were equal to volumes of spheres in the original lipid suspension. Because of the compensating effects of shrinkage (decreases diameter) and flattening (increases diameter) the differences between the means of measured diameters and corrected diameters were less than or equal to 5%.

Oleic acid allows more apoprotein A-1 to bind with higher affinity to large emulsion particles saturated with cholesterol.

Apoprotein (apo) A-1 binding to large triolein-rich emulsion particles saturated with cholesterol has been examined as a function of the oleic acid content. Six emulsion systems were formed containing 0.3-1.0% (by weight) oleic acid, 82.9-86.3% triolein, 10.6-7.2% egg yolk phosphatidylcholine, and 6.7-5.5% cholesterol. The average emulsion particle diameters calculated from these lipid compositions ranged between 84 and 116 nm. Negative stain electron microscopy of an emulsion containing 1% oleic acid showed a polydisperse population of only large spherical particles with a mean diameter of 116 +/- 54 nm. The calculated cholesterol concentrations of the particles surface and core for the six emulsions were 43.3 +/- 1.1 and 5.6 +/- 0.2 mol%, respectively, and were rather constant. Therefore, when the surface oleic acid concentrations increased from 2.6 to 10.1 mol%, the phospholipid concentration decreased from 55.1 to 45.9 mol%. In the core, oleic acid increased at the expense of triolein. In the range studied a nearly 4-fold increase in the surface oleic acid content produces a similar increase in the binding capacity (N) and reduces the dissociation constant (Kd). The changes in the Kd and N values were linearly dependent on the surface oleic acid concentration. These data show that oleic acid allows more apoA-1 to bind with higher affinity to large emulsion particles saturated with cholesterol.

Interaction of ApoA-1 and ApoE-3 with triglyceride-phospholipid emulsions containing increasing cholesterol concentrations. Model of triglyceride-rich nascent and remnant lipoproteins.

The cholesterol content of triglyceride-rich lipoproteins increases during their catabolism in circulation. We therefore studied the binding of the exchangeable apoprotein apoA-1 and apoE-3 to triolein-rich emulsions with increasing cholesterol content. Five emulsion systems containing 83.1-88.8% (w/w) triolein, 9.3-10.1% egg yolk phosphatidylcholine, and 1.1-7.3% cholesterol were isolated from sonicated lipid mixtures by flotation. Negative stain EM of emulsions containing 1.1 and 7.3% cholesterol showed polydisperse populations of large spherical particles with diameters of 106 +/- 39 and 108 +/- 57 nm. These values are similar to particle diameters calculated from the lipid composition data. No lamellar structures were observed by EM, even after addition of apoA-1 at a molar ratio to lecithin of 10(-2). Apolipoproteins apoA-1 and apoE-3 bound to the particles in a saturable manner without altering particle morphology. We found a dissociation constant Kd = 7.4 x 10(-7) M and a binding capacity N = 3.9 x 10(-3) proteins/lecithin for apoA-1 with particles containing 1.1% cholesterol; the Kd and N values for apoE-3 were very similar. When the emulsion particles were saturated with cholesterol at 7.3%, the protein binding capacity N sharply decreased to 0.6 x 10(-3) (apoA-1) and 0.7 x 10(-3) proteins/lecithin (apoE-3), but the Kd values were virtually unchanged. No change in N occurred when the particle cholesterol content was increased from 1.1 to 3.7%, which spans the normal physiological range. These results suggest that increases in lipoprotein cholesterol content above 3.7% may be responsible for impaired apoprotein redistribution and altered metabolism of remnants such as beta-VLDL.

Alteration of a lepidopteran peritrophic membrane by baculoviruses and enhancement of viral infectivity.

The peritrophic membrane (PM), which lines the midgut of many insect species, has several functions. In particular, it may serve as a mechanical barrier to invading microorganisms. The protein composition of the PM from healthy and baculovirus-treated Trichoplusia ni (cabbage looper) larvae was analyzed by polyacrylamide gel electrophoresis. A specific interaction took place between baculoviruses and the PM of susceptible T. ni larvae. A 68-kDa glycoprotein of the PM disappeared within 15 min postinoculation with occlusion bodies of either Autographa californica nuclear polyhedrosis virus (AcMNPV) or T. ni nuclear polyhedrosis virus (TnSNPV). In contrast, inoculation of larvae with a T. ni granulosis virus (TnGV) resulted in the disappearance of three distinct major glycoproteins with molecular weights of 253, 194, and 123 kDa. PMs of virus-treated larvae were very fragile compared with those of untreated controls, indicative of a physical/chemical change in their structure. T. ni larval bioassays showed that a factor, present in the TnGV granulin or AcMNPV polyhedrin, enhanced the infectivity of AcMNPV. These data showed that a factor present in the occlusion bodies of three distinct baculoviruses can cause specific biochemical and structural changes in the PM. The biological significance of these observations in relation to increased larval infection is not known at this time.

The partitioning of fatty acid and cholesterol between core and surfaces of phosphatidylcholine-triolein emulsions at pH 7.4.

Fatty acids are important intermediate molecules in lipid metabolism. During lipolysis of intracellular lipid droplets or plasma triacylglycerol-rich lipoproteins, fatty acids are generated and may transiently accumulate. We therefore studied the distribution of both fatty acid and free cholesterol between the core and surface of phosphatidylcholine-triolein emulsions at pH 7.4. Nine emulsion systems containing 0.8 to 6.6% cholesterol and 0.16 to 1.02% oleic acid were formed, and core and surface phases were isolated. Phospholipid distributes only to the surface phase. The distribution coefficient of cholesterol surface to core was 23.9 +/- 3.6 S.D., i.e., there was approx. 24-times more cholesterol per unit mass in the surface than in the core phase. This distribution was unchanged by the presence of different quantities of fatty acid in the emulsion particles. The apparent distribution coefficient of fatty acid in surface to core was about 10 at low cholesterol contents and fell to about 7 at high cholesterol contents. However, when the apparent distribution coefficient of fatty acid was related only to the phospholipid component of the surface, the apparent distribution coefficient was constant at about 12.3 +/- 1.1 S.D. Since the fatty acid in the surface phase is about half ionized the true distribution coefficient of unionized fatty acids is about 6.2. The results indicate that fatty acids partition into the phospholipid domains of the surface and not into cholesterol domains and the distribution of fatty acids into surface phospholipid domain is not affected by cholesterol content.

Phosphatidylcholine composition of emulsions influences triacylglycerol lipolysis and clearance from plasma.

Sonicated emulsions containing triolein, a specific phosphatidylcholine and cholesterol were prepared. Bolus doses were injected intravenously into rats and plasma clearance kinetics and organ uptakes were determined. Emulsion triacylglycerol lipolysis by rat heart lipoprotein lipase was measured in vitro. Phosphatidylcholine molecular species influenced emulsion metabolism in vivo and in vitro. Emulsions containing saturated phosphatidylcholines at temperatures below their melting points were poor substrates for lipoprotein lipase, compared with those stabilized by mixed chain phosphatidylcholines. Distearoylphosphatidylcholine stimulated hepatic uptake compared with emulsions made with egg yolk phosphatidylcholine, which modeled chylomicrons closely. Emulsion populations with the same surface compositions but with mean diameters of 700-800 A and 1100-1300 A were metabolized similarly, suggesting that, within the normal chylomicron size range, size alone does not determine the disposition of triacylglycerol-rich emulsions or lipoproteins.

Replication of the Trichoplusia ni granulosis and nuclear polyhedrosis viruses in cell cultures.

Newly established Trichoplusia ni (cabbage looper) embryonic cell lines were infected with T. ni granulosis virus (TnGV) and T. ni nuclear polyhedrosis virus (TnSNPV). Infection of cultured cells with TnGV was ascertained by peroxidase-antiperoxidase staining, DNA slot-blot hybridization, and transmission electron microscopy. Initially, 15 cell lines supported TnGV replication, the percentage of infected cells ranging from 1 to 50%.However, susceptibility of the 15 cell lines to TnGV infection either decreased or was lost within 20 to 25 passages from the initial primary culture. Infection of cells with TnSNPV was determined by phase contrast and electron microscopy. TnSNPV infected 29 36 cell lines tested, the percentage of infected cells ranging from 1 to 60%.

Technetium-99m low density lipoproteins: preparation and biodistribution.

The focal uptake by human atherosclerotic lesions of 125I bound to low density lipoproteins (LDL) can be demonstrated by external imaging. However, 125I has poor imaging characteristics. Therefore, we have developed a technique for labeling LDL with technetium. To facilitate analysis, LDL was first labeled with 99mTc, by reduction of TcO4- with dithionite in the presence of the protein. The labeled LDL was stable to electrophoresis, ultracentrifugation, and passage in vivo. This technique was repeated with minor modification with 99mTc to prepare [99mTc] LDL for use as an imaging agent. Its biodistribution in 16 rabbits was similar to that of [125I] LDL and it allowed high resolution external imaging of LDL uptake by tissues, including the injured, healing, arterial wall, and the adrenal cortex.