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Biomedical subjects

M C Stuart

Publications and source records attributed to M C Stuart.

At least 19 recordsLinked to original sources

Gelation of liposome interior. A novel method for drug encapsulation.

Liposomes can be loaded with weak acids and bases, which exist in solutions in equilibrium with membrane permeable uncharged form, using various gradients across their membranes. Because in some cases the estimated drug concentration in the loaded liposomes exceeds their aqueous solubility we investigated the physical state of the liposome encapsulated anticancer drug Doxorubicin. X-Ray diffraction, electron microscopy, and test tube solubility experiments have shown that upon encapsulation the drug molecules form a gel-like phase.

Doxorubicin

Clustering of lipid-bound annexin V may explain its anticoagulant effect.

In 1985 we isolated a new vascular anticoagulant protein VAC alpha, now called annexin V, with a high binding affinity (Kd less than 10(-10) M) for phospholipids. Its anticoagulant effect was attributed to displacement of coagulation factors from the phospholipid membrane. The present study demonstrates that the inhibition of prothrombinase activity by annexin V strongly depends on the curvature of the membrane surface and on the calcium concentration. Half-maximal inhibition of prothrombinase on and binding of annexin V to small vesicles, composed of 20% phosphatidylserine and 80% phosphatidylcholine, requires 2-3 mM calcium. With large vesicles and planar bilayers considerably less calcium is required for inhibition of prothrombinase and for lipid binding. Half-maximal binding of annexin V to large vesicles and to planar bilayers occurs at 0.7 and 0.2 mM calcium, respectively. This seemingly confirms the displacement model. The displacement of coagulation factors, however, proved to be incomplete, with residual surface concentrations of factors Xa, Va, and prothrombin sufficient for effective production of thrombin. Cryoelectron microscopy revealed that annexin V binding to large vesicles caused planar facets, indicating the formation of large sheets of clustered annexin V. Apparently, the formation of these two-dimensional arrays is promoted by calcium and hampered by high surface curvature. It is speculated that the complete inhibition (greater than 99%) of prothrombinase activity by annexin V is caused by the reduced lateral mobility of prothrombin and factor Xa in rigid sheets of annexin V covering the membrane.

Annexin A5

Lipid polymorphism as observed by cryo-electron microscopy.

Lipid polymorphism was studied with the aim to gain more insight in bilayer to non-bilayer phase transitions, with particular emphasis on the development of cubic structures on one hand and inverted hexagonal structures on the other hand. Thin vitrified films prepared from aqueous lipid suspensions were used in this study. The entire hydrated contents of these films can be visualized in their two-dimensional projection by cryo-electron microscopy. As the starting material, unilamellar vesicles were prepared from mixtures of dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine and cholesterol. By heating of the suspension, vesicle fusion (Frederik et al. (1989) Biochim. Biophys. Acta 979, 275-278) and lipid polymorphism was induced. From these suspensions thin films were prepared at various temperatures, and vitrified for low temperature observation. In a parallel series of experiments samples were fast-frozen for freeze-fracture analysis. In vitrified thin films bilayer structures were often observed in coexistence with an inverted hexagonal structure. The bilayer areas were frequently of a complex structure because multiple contacts between stacked membranes were found. These contact points were variable in size and shape and usually had the form of a diabolo (when viewed side-on) giving the impression of a bilayer contact with an aqueous channel. This structure is compatible with the interlamellar attachment site (ILA) proposed by Siegel ((1986) Biophys. J. 49, 1155-1170). In some specimens ILA's seemed to merge into arrays. After thermal cycling of the suspension, arrays of packed globules were observed, which are likely the result of close packing of ILA's. The arrays probably represent a cubic structure. A comparison of freeze-fracture replicas and vitrified thin films indicated that both techniques may provide valuable structural information on lipid polymorphism. Most of the lipidic particles observed by freeze-fracturing probably correspond to the ILA's (fractured around their waist region) as observed in vitrified thin films. The results obtained with vitrified thin films were interpreted in relation to the principles of thin-film formation. Finally, we speculate that lipid structures occurring close to each other in space may represent a developmental series of structures occurring successively in time.

Cholesterol

Perspective and limitations of cryo-electron microscopy. From model systems to biological specimens.

We investigated the possibility of vitrifying temperature-sensitive lipid phases as well as (small) biological specimens. From a suspension of unilamellar vesicles, prepared from dipalmitoyl-phosphatidylcholine (DPPC), thin aqueous films were formed at various temperatures. With cryo-electron microscopy vesicles were found to be smooth, rippled and faceted or faceted only, depending on the temperature of thin-film formation (318, 312 and 296 K respectively). The morphology and the electron diffraction patterns indicate that membranes can by physically fixed by vitrification in their high-temperature configuration and studied at low temperature by cryo-electron microscopy. This finding suggests that it may also be possible to preserve, in their original state, the more complex membrane systems found in living organisms by initiating rapid-cooling at a physiological temperature. This was explored by vitrification of thin films formed on specimen grids with (human) blood platelets adhering to collagen fibres. Low-temperature observation with an acceleration voltage of 120 kV revealed subcellular details, More details were observed when using higher accelerating voltages (200 and 300 kV) of the electron beam. The results presented in this paper illustrate the great potential of cryo-electron microscopy in the study of membrane dynamics, both in relatively simple model membrane systems and in more complex biological membrane systems.

Blood Platelets

Influence of nutritional status on growth hormone-dependent circulating somatomedin-C activity in mature sheep.

The effect of daily administration of ovine GH for a period of 4 weeks on somatomedin-C biological activity in plasma was investigated in mature Merino sheep fed a maintenance energy intake (low plane; LP) or 1.6 times this amount (high plane; HP). The GH treatment resulted in a significant (P less than 0.05) increase in plasma GH levels in blood samples collected 23.5 h after each daily injection in both LP and HP groups. Plasma concentrations of somatomedin-C activity and insulin were significantly stimulated to a maximum level by the third GH injection and remained at this level for 7 days. Subsequently, circulating levels of both hormones fell to 40-50% of the peak response to GH and returned to basal levels within 48 h of the cessation of GH injections. In the HP group the response of plasma insulin and somatomedin-C activity to GH injection was greater than in the LP group.

Animals

The ultrastructure of cryo-sections and intact vitrified cells--the effects of cryoprotectants and acceleration voltage on beam induced bubbling.

Chemically fixed pancreas was infiltrated with various cryoprotectants to obtain homogeneously vitrified samples upon cooling. The suitability of these samples for cryoultramicrotomy was tested. Contrast was hardly detectable initially in thin cryo-sections but increased upon irradiation, irrespective of the cryoprotectant (glycerol, propylene glycol, methanol) used. Contrast and beam damage were analyzed in vitrified thin films from collagen, phospholipid vesicles and various concentrations of glycerol. Glycerol increased the beam sensitivity of both collagen and phospholipid vesicles, but diminished the contrast between matrix and lipid vesicles or collagen fibers. The effects of glycerol as observed in thin films explain some of the effects of cryoprotectants in thin cryo-sections. To reduce beam damage in vitrified specimens two approaches are proposed. Firstly, when vitrified films are prepared, dilute suspensions should be used without cryoprotectant. In some cases, such as (thin) intact cells, the composition of the suspended material can only be marginally influenced. Then a second approach can be used involving the application of higher accelerating voltages (e.g. 300 kV). This has two advantages; the increase in mean free path-length of the electrons causes less beam damage on one hand and allows better resolution of thick specimens on the other hand. Micrographs from E. coli bacteria vitrified from suspension illustrate some of the potentials of "intermediate voltage" cryo-electron microscopy.

Animals

Effect of biosynthetic human growth hormone on insulin action in individual tissues of the rat in vivo.

Excessive endogenous production or exogenous administration of human growth hormone (hGH) causes insulin resistance at both the hepatic and extrahepatic levels. However, which extrahepatic tissues are involved have not been defined. We have examined the diabetogenic action of authentic biosynthetic hGH on whole body glucose disposal, hepatic glucose output, and glucose metabolism in individual peripheral tissues. The use of a highly purified preparation of the hormone allowed us to examine the isolated effects of 22K hGH. The euglycemic hyperinsulinemic (approximately 100 mU/L) clamp plus 3H-2-deoxyglucose technique was used to quantitate the effects of hGH on insulin action in vivo. Administration of biosynthetic hGH at a dose of 10 IU/kg/24 h for 48 hours in male Wistar rats (approximately 340 g) produced a highly significant decrease in the steady state clamp glucose infusion rate (GIR) when compared with controls (8.1 +/- 0.6 v 18.7 +/- 0.7 mg/kg/min, P less than .001), reduced insulin-mediated suppression of hepatic glucose output (Ra) (3.9 +/- 0.6 v 0.7 +/- 0.3 mg/kg/min, P less than .05) and a decreased clamp glucose disposal rate (Rd) (12.0 +/- 0.4 v 18.10 +/- 1.1 mg/kg/min, P less than .001). There was a significant decrease in insulin-mediated glucose uptake as indicated by tissue accumulation of [3H]-2-deoxyglucose phosphorylation in diaphragm and hindlimb muscles. Insulin action was more substantially reduced in muscles (approximately 50%) than in adipose tissues (approximately 20%). These studies confirm that the diabetogenic action of hGH in the rat is due to a combination of inhibition of insulin suppression of hepatic glucose output and inhibition of the uptake and subsequent utilization of glucose in skeletal muscles.

Adipose Tissue

Somatomedin-C, physical fitness, and bone density.

Somatomedin-C (Sm-C) or insulin-like growth factor-I, GH and physical fitness decline with age. Physical fitness and muscle strength are important determinants of bone density, and the age-related decline in bone density may be related in part to a decline in fitness and muscle strength. Also, Sm-C has been shown to stimulate osteoblasts in vitro and may effect skeletal muscle mass. We postulated that the age-related decline in GH and Sm-C levels may be related to an age-related decline in physical fitness and/or muscle strength, and the effect of physical fitness and muscle strength on bone may be mediated by Sm-C. We, therefore, examined the relationship between circulating GH and Sm-C levels and physical fitness, as determined by predicted maximal oxygen uptake (VO2max) in 134 normal women, 34 of whom were postmenopausal. In a subgroup of 62 women overall muscle strength was estimated as the sum of the Z-sores for biceps, quadriceps, and grip strength. Overall muscle strength correlated with GH levels (r = 0.28; P less than 0.02), but not with Sm-C levels. There was a significant positive relationship between plasma Sm-C levels and VO2max in all women (r = 0.47; P less than 0.001) and in the postmenopausal group alone (r = 0.05; P less than 0.01). Although there was a significant negative relationship between Sm-C and age (r = -0.36; P = 0.001), VO2max was a better independent predictor than age (r = 0.47; P = 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult

Intermediary structures during membrane fusion as observed by cryo-electron microscopy.

Lipidic phases, containing 'lipidic particles' (dioleoylphosphatidylethanolamine/cholesterol/dioleoylphosphatidylcho lin e and cardiolipin/dimyristoylphosphatidylcholine in the presence of Ca2+) have been investigated by preparing thin films from a suspension of sonicated vesicles. These thin films were vitrified and observed 'directly' by cryo-electron microscopy in their hydrated form. The thin films show various fusion products and fusion intermediates such as lipidic particles.

Calcium

Specificity of two-site immunoassays.

When cross-reaction in two-site immunoassays was investigated both theoretically and experimentally it was found that such systems do not always result in enhanced specificity. Computer simulation studies indicated that substances which display negligible cross-reaction in a radioimmunoassay could produce an assay response identical to that of the analyte in a two-site immunoassay using excess antibody. Cross-reactivity in two differing two-site immunoassays was compared to that obtained in radioimmunoassays using the same monoclonal antibodies for human chorionic gonadotrophin. In addition to the effects of excess antibody, cross-reactivity was observed in one of the two-site immunoassays which could not have been predicted from the specificity of the antibodies or cross-reactivity in the radioimmunoassays. The unexpected cross-reaction of the beta subunit of human chorionic gonadotrophin in the assay resulted from an apparent alteration in the specificity of one of the antibodies following binding of the beta subunit to the second antibody. These studies emphasise the complexity of binding reactions in two-site immunoassays.

Antibodies, Monoclonal

Quantification of urinary growth hormone (GH) excretion by centrifugal ultrafiltration and radioimmunoassay: appraisal of the relationship between 24 h urinary GH and mean 24 h serum GH levels in normal and abnormal states of GH secretion.

We have applied a simple method for the quantification of 24 h urinary GH excretion (24 h UGH), combining centrifugal ultrafiltration and radio-immunoassay (RIA), to an appraisal of the relationship between 24 h UGH and mean 24 h serum GH levels in normal and abnormal states of GH secretion. Forty-four subjects, comprising 13 normal adults, 12 short-statured subjects and 19 subjects with active acromegaly, underwent blood sampling at 20-min intervals and concurrent urine collection for 24 h. Mean 24 h serum GH and 24 h UGH were also determined in four post-menopausal women before and during cyclical oestrogen replacement therapy, and 24 h UGH was measured in six normal men prior to and following the subcutaneous administration of biosynthetic GH (0.2 IU/kg). Each subject's mean 24 h serum GH level was determined by assaying a 'pooled' sample, derived from equal aliquots of the 73 serum samples obtained during the 24 h study. The method for quantification of 24 h UGH employs centrifugal microconcentrators, and involves a 50-fold concentration of urine, followed by dialysis and RIA. Surface adsorptive hormone loss during sample collection and ultrafiltration is minimized by the prior addition of bovine serum albumin to the urine collection container. Immunoreactive GH in ultrafiltered urine dilutes in parallel with the RIA standard curve. GH is stable in urine stored at - 20 degrees C for at least 12 months. There was a highly significant correlation between 24 h UGH and mean 24 h serum GH values obtained from the combined population of normal adults (including the post-menopausal women during oestrogen therapy) and short-statured subjects (r = 0.69, P less than 0.0001). A significant correlation was also found in short-statured subjects alone (r = 0.63, P less than 0.05). In contrast, there was no significant correlation between 24 h UGH and mean 24 h serum GH in subjects with active acromegaly, and their 24 h UGH values were not distinguishable from those of the 'non-acromegalic' subjects. A significant increase in 24 h UGH occurred in the post-menopausal women given cyclical oestrogen replacement therapy (9.7 +/- 2.6 (mean +/- SE) to 24.6 +/- 1.0 muIU/mmol creatinine, P less than 0.02), reflecting the increase in their mean 24 h serum GH levels (0.8 +/- 0.3 to 5.3 +/- 0.7 mIU/l, P less than 0.01). Twenty-four hour UGH increased from 4.6 +/- 0.6 to 17.1 +/- 2.1 muIU/mmol creatinine (P less than 0.002) in the men given biosynthetic GH. Twenty-four hour UGH measurements reflect mean 24 h serum GH levels in normal adults and short-statured subjects. While the measurement of 24 h UGH shows promise as an investigative tool, our results cast doubt on its use in the diagnosis of acromegaly.

Acromegaly

The pharmacokinetics, safety and endocrine effects of authentic biosynthetic human growth hormone in normal subjects.

The pharmacokinetics, safety and endocrine effects of an authentic human growth hormone (bio-hGH), produced by the expression of genomic hGH in a mammalian cell line, were studied in six healthy young men who were administered 0.2 U/kg/day subcutaneously for five consecutive days. Changes in sodium balance and in thyroid function were studied during the week of bio-hGH administration and safety parameters were monitored over a 3-week period. Growth hormone levels reached a mean (+/- SD) peak of 106 +/- 10 mIU/l at 3.3 +/- 0.5 h following the first dose and resulted in a significant rise of somatomedin C. free fatty acids, fasting blood glucose and insulin concentrations. Bio-hGH administration resulted in a significant increase in body weight (80.0 +/- 4.5 to 81.1 +/- 4.3 kg; P less than 0.01) which was associated with a marked reduction in urinary sodium excretion (196 +/- 38 to 45 +/- 20 mmol/day; P less than 0.025). Serum T3 increased during bio-hGH administration and was associated with reciprocal changes in free thyroxine and TSH concentrations. Cardiac, hepatic, renal, biochemical, haematological, endocrinological and immunological functions remained normal throughout the study. No antibodies to hGH or to host cell protein developed during the study. The results show that bio-hGH is safe in the short term, well tolerated, possesses pharmacokinetic and biological properties similar to pituitary hGH, and has distinct effects on sodium balance and on thyroid function. This study stresses the need to monitor patients for effects on sodium retention, carbohydrate metabolism and thyroid function when using hGH doses of 1.0 U/kg/week (40 U/m2/week) or more in patients with GH responsive short stature.

Adult

Phospholipid, nature's own slide and cover slip for cryo-electron microscopy.

Thin films of surface-active compounds, with or without particulate material, can be obtained by immersing and withdrawing a bare specimen grid from a solution/suspension of the compound. Immediately after withdrawing the grid, thinning of the film starts. Thinning is initially powered by gravity and capillary forces and will proceed in thin films (less than 100 nm) driven by intermolecular forces until the London-van der Waals attractive forces come to an equilibrium with electrostatic repulsion of similarly charged surfaces of the film. With small unilamellar vesicles prepared from the phospholipid dimyristoyl phosphatidyl choline (DMPC) the draining behaviour of these films was studied by cryo-electron microscopy. Small unilamellar vesicles were observed within the film as well as the coalescence of these vesicles into sheets ('leaky' membrane fusion). Sheets dominate the images when films are allowed to drain for longer periods (greater than 3 min). Thin films were formed on grids from catalase crystals suspended in a DMPC suspension and vitrified by cooling. High-resolution information was obtained by electron diffraction at low temperature and under low-dose conditions from catalase crystals surrounded by small vesicles as well as from catalase crystals surrounded by sheets of DMPC. In the latter case the water content drops from 99% (DMPC in small vesicles) to less than 30% (DMPC in sheets) during draining. Ferritin was added to a DMPC suspension and thin films were prepared and vitrified. After prolonged draining ferritin molecules were deposited in layers with a stepwise increase in thickness. Draining of thin films has thus a dehydrating effect as well as a sorting and ordering effect. These effects must be considered when using surface-active compounds at air-water interfaces as a slide and cover slip for electron microscopy.

Catalase

Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles.

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.

Animals

Heterophilic antibodies: a problem for all immunoassays.

We verified that antibody-binding substances in serum that interfere in two-site immunoassays involving murine antibodies are heterophilic antibodies. Incubation of serum containing heterophilic antibodies and a murine monoclonal antibody to human choriogonadotropin (hCG) leads to formation of a series of soluble immune complexes. We investigated the recognition of hCG by reagent antibody in the presence of heterophilic antibodies and found this recognition to be diminished. Consequently, about 30% of serum samples containing heterophilic antibodies falsely appear to contain increased concentrations of hCG. The effect on analyte recognition probably results from steric inhibition of hCG binding to complexed antibody. Heterophilic antibodies detected with a murine antibody also bound immunoglobulin from several other species but did not bind all of those tested.

Animals

Localization of human pituitary hormones by multiple immunoenzyme staining procedures using monoclonal and polyclonal antibodies.

Mouse monoclonal and rabbit polyclonal antibodies to human pituitary hormones were applied together to sections of normal and neoplastic human pituitary tissue. Binding sites were revealed with species-specific immune reagents combined with various enzymes (peroxidase, alkaline phosphatase, and beta-D-galactosidase). The enzymes were developed separately to give differently colored end-products. Where two hormones were present in the same cell, a mixed color was produced. Up to four hormones could be immunostained in a single section. Multiple immunoenzymatic staining has great potential for the analysis of plural antigen production by single cells and relationships between cells producing different antigens.

Animals

Covert cross reactants in a two-site immunoassay studied with monoclonal antibodies.

A one-step two-site immunoradiometric assay for the measurement of free beta subunit of human chorionic gonadotrophin (beta-hCG) was developed using monoclonal antibodies. The immobilized antibody was specific for free beta subunit and the radiolabeled antibody recognized both intact human chorionic gonadotrophin (hCG) and free beta subunit. Although the level of hCG "cross-reaction" was low when studied using conventional techniques, the apparent beta-hCG content of samples was found to be inversely proportional to the hCG level. From both experimental evidence and computer simulation studies this was found to be due to the binding of hCG to the limited amount of 125I-labeled antibody present. The term covert cross reactants has been introduced to describe substances which bind to only one of the antibodies in a two-site immunoassay. When establishing such an assay the effect of covert cross reactants on the response of an analyte should be investigated.

Antibodies, Monoclonal