The relationship between compositional phase separation and vesicle morphology: implications for the regulation of phospholipase A2 by membrane structure

The action of phospholipase A2 (PLA2) on bilayer substrates causes the accumulation of reaction products, lyso-phospholipid and fatty acid. These reaction products and the phospholipid substrate generate compositional heterogeneities and then apparently phase separate when a critical mole fraction of reaction product accumulates in the membrane. This putative phase separation drives an abrupt morphologic rearrangement of the vesicle, which may be in turn responsible for modulating the activity of PLA2. Here we examine the thermotropic properties of the phase-separated lipid system formed upon hydrating colyophilized reaction products (1:1 palmitic acid:1-palmitoyl-2-lyso-phosphatidylcholine) and substrate, dipalmitoylphosphatidylcholine. The mixture forms structures which are not canonical spherical vesicles and appear to be disks in the gel-state. The main gel-liquid transition of these structures is hysteretic. This hysteresis is apparent using several techniques, each selected for its sensitivity to different aspects of a lipid aggregate's structure. The thermotropic hysteresis reflects the coupling between phase separation and changes in vesicle morphology.     

Changes in the norms governing practices for the manufacture of pharmaceutical products: implications for the MERCOSUR

It is done a comparative study between the "Recommended rules for drug products manufacturing and inspection", approved in 1975 by the World Health Organization (and still in force in the MERCOSUR); and the standards published in 1992 by the WHO Expert Committee on Specifications for Pharmaceutical Preparations 32nd Report, named "Good Manufacturing Practices for pharmaceutical products". The correspondence between the regulation in force in the MERCOSUR and the Good Manufacturing Practices Inspection Guide for pharmaceutical industry, used by Health Authorities in the Common Market Member States, is analysed. It is noticed a disagreement between the rule in force and the instrument for verifying its fulfillment. The proposal of this article is the adoption by the Common Market Group, of the rules published by the WHO in 1992, and the establishment of an inspection guide which absolute agrees with it.     

Acute phase proteins

One hundred eleven patients with burns who were age 60 years or older were treated from January 1984 through December 1992. Twenty-nine patients had pulmonary failure defined as 7 or more days of ventilatory support from the day of burn. The mortality rate for these patients was 41%; only four were discharged to home. The mortality rate for patients without pulmonary failure was 11%. Billing information was analyzed for 102 of the 111 patients. Charges for patients without pulmonary failure were two to three times greater than reimbursement. Charges for patients with pulmonary failure were 4 to 14 times greater than reimbursement. Reimbursement for elderly patients with burns is inadequate. Altering the Diagnosis-Related Group (DRG) definition of extensive burn to reflect the severity of injury in the geriatric population is one step toward reimbursement reform. Patients who require 7 or more days of ventilatory support after burn injury should be reimbursed under a separate DRG category or should have a DRG modifier.     

Two-dimensional separation of erythrocyte membrane proteins

1). Erythrocyte membrane proteins eluted with Triton X-100 or dilute EDTA have been separated two-dimensionally by isoelectric focusing in polyacrylamide gels containing 1 percent Triton X-100 plus 8 M urea, followed by electrophoresis using sodium dodecyl sulfate. Characteristic patterns, consistent among 40 healthy donors, were obtained. 2. The resulting patterns contain at least 30 components. The "spectrin" components (sodium dodecyl sulfate Bands 1 and 2) focus in the same pH range. Other membrane components giving single bands in sodium dodecyl sulfate electrophoresis appear to be heterogeneous. 3. Triton X-100, but not EDTA, extracts the principal membrane glycoproteins and the major "intrinsic" protein. Otherwise, proteins preferentially eluted by EDTA extract poorly with Triton X-100 and vice versa. 4. Membrane glycoproteins migrate anodally during electrofocusing and can be purified in a simple, one-step procedure.     

Therapeutic delivery of proteins to macrophages: implications for treatment of Gaucher's disease

BACKGROUND: The primary defect in Gaucher's disease, a lysosomal disorder affecting macrophages, is in the activity of glucocerebrosidase. Treatment with exogenous enzyme (modified to increase its affinity for macrophage glycoprotein receptors) aims to restore this activity. However, the fate of the exogenous enzyme in vivo is unknown. We used radiolabelled enzyme to assess macrophage receptor activity for mannosylated ligands in vivo. METHODS: We examined the uptake and tissue distribution of radiolabelled enzyme molecules by gamma scintigraphy after bolus injection of iodine-123-labelled recombinant or placental enzyme (imiglucerase and alglucerase, respectively) in eight patients with type 1 Gaucher's disease, and in one healthy individual. The metabolism of the tracer enzyme was followed by scintigraphy and by analysis of blood, urine, and faeces. RESULTS: The tracer enzyme was rapidly cleared from blood (half-life 4.7 min [SD 1.0]). Concomitantly, there was avid uptake by the liver (about 30% of the injected dose), the spleen (about 15%), and the bone marrow. 40-55% of the tracer was cleared rapidly from the viscera (half-life 1-2 h) and 45-60% was cleared slowly (half-life 34-42 h). The half-life in the bone marrow was 14.1 h. Infusion of alglucerase at dose of 5 U/kg bodyweight normalised acid beta-glucosidase activity of splenic Gaucher's cells in vivo. When the enzyme was administered at a seven-fold higher dose (35 U/kg over 1 h), the receptor-mediated uptake in vivo was saturated, as shown by the increase in blood-clearance half-life of tracer enzyme from 4.5 min to 12 min. INTERPRETATION: Avid and saturable uptake of modified glucocerebrosidase was found, which indicates high-affinity targeting to the macrophage system in vivo. The rate of enzyme turnover suggests a rational basis for use of this therapy in treatment of Gaucher's disease.     

The stability of salt bridges at high temperatures: implications for hyperthermophilic proteins

Salt bridges have been proposed to play a crucial role in promoting hyperthermostability in proteins, yet they appear to make little contribution to protein stability at room temperature. The latter point has been rationalized previously on the basis that the association of two charged molecules to form a salt bridge incurs a substantial desolvation penalty, which is seldom completely compensated by favourable interactions within the salt bridge and with the rest of the protein. Here a continuum solvation model is used to investigate how this same argument applies at temperatures more appropriate to hyperthermophiles. The solvation model employed was previously parameterised to reproduce the hydration free energies of neutral and charged amino acid side-chains in the temperature range from 5-100 degreesC. A key result of the previous work was that the hydration free energies of charged side-chains are more adversely affected by increasing temperature than are the hydration free energies of hydrophobic side-chains of identical size and shape (isosteres). As is shown here, a direct consequence of the temperature dependence of the hydration free energies is that at high temperatures the desolvation penalty for formation of a salt bridge is markedly reduced in magnitude. As a result, the argument that relative to hydrophobic isosteres, salt bridges destabilise proteins, may no longer be true at high temperatures. We demonstrate this point first in the setting of a small model system, but then also show that the same argument is likely to carry over to real proteins. We compare three hyperthermophilic proteins with their mesophilic homologues and find that hydration effects preferentially stabilise the hyperthermophiles at high temperatures. When the hydration effects are incorporated into a model for the free energy of folding of the proteins, it is found that in each case, the hyperthermophile is predicted to remain stable to a temperature 20-25 deg.C higher than the corresponding mesophile. Higher thermal stability for the hyperthermophile is obtained even if the mesophile is more stable at room temperature. The results obtained therefore suggest one possible way in which the apparently destabilising effects of salt bridges at room temperature can be reconciled with their increased abundance in hyperthermophilic proteins.     

Ordering and separation in phase diagrams

Solid-solution fields in the generally accepted type-IV phase diagrams are shown to exhibit a tendency toward separation, which causes the formation of separation microstructures. Numerous examples of separation in solid solutions are given; this separation occurs at temperatures both above and below the fields of chemical compounds. Separation and ordering fields are separated by metastable-solid-solution regions in phase diagrams. At the temperatures corresponding to these regions, an ordering-separation phase transition takes place. New versions of the Fe-Cr, Fe-Co, and Fe-V phase diagrams are presented.     

Electrophoretic separation of serum proteins from gray squirrels

Enterotoxin was obtained from the culture of E. coli O15 by salt precipitation and gel-chromatography. The toxic activity of the preparation increased during the isolation and purification: 60-fold according to the results of the method of ligated rabbit intestinal segment and 66-100-fold according to the skin test. The "plateau" and the second fraction obtained as a result of gel-chromatography were inactive according to the results of the method of ligated intestinal segment, but possessed PF-activity in the skin test. Two suppositions are put forward: 1) possibly the factor of vascular permeability and the diarrheal factor were two different substances ((molecules), or 2) the skin test was more sensitive for determination of the toxicity than the method of the ligated segment of rabbit intestine.     

Heat shock proteins and molecular chaperones: implications for adaptive responses in the skin

Recent advances in the biology of heat-shock proteins (hsps) are reviewed. These abundant and evolutionarily highly conserved proteins (also called stress proteins) act as molecular escorts. Hsps bind to other cellular proteins, help them to fold into their correct secondary structures, and prevent misfolding and aggregation during stress. Cytoplasmic hsp70 and hsp60 participate in complicated protein-folding pathways during the synthesis of new polypeptides. Close relatives of hsp70 and hsp60 assist in the transport and assembly of proteins inside intracellular organelles. Hsp90 may have a unique role, binding to the glucocorticoid receptor in a manner essential for proper steroid hormone action. Hsps may also be essential for thermotolerance and for prevention and repair of damage caused by ultraviolet B light. A unique class of T lymphocytes, the gamma delta T cells, exhibits a restricted specificity against hsps. These T cells may constitute a general, nonspecific immune mechanism directed against the hsps within invading organisms or against very similar hsps within invading organisms or against very similar hsps expressed by infected (stressed) keratinocytes. Immunologic cross-reactivity between hsps of foreign organisms and of the host may play a role in some autoimmune diseases. Although hsps are expressed in the skin, many questions remain about their role during injury, infection, and other types of cutaneous pathophysiology.