Clathrin-dependent and clathrin-independent endocytosis are differentially sensitive to insertion of poly (ethylene glycol)-derivatized cholesterol in the plasma membrane.

We examined the effect of a cholesterol derivative, poly (ethylene glycol) cholesteryl ether on the structure/function of clathrin-coated pits and caveolae. Addition of the compound to cultured cells induced progressive smoothening of the surface. Markedly, when the incorporated amount exceeded 10% equivalent of the surface area, fluid pinocytosis, but not endocytosis of transferrin, became inhibited in K562 cells. In A431 cells, both clathrin-independent fluid phase uptake and the internalization of fluorescent cholera-toxin B through caveolae were inhibited with concomitant flattening of caveolae. In contrast, clathrin-mediated internalization of transferrin was not affected until the incorporated poly (ethylene glycol) cholesteryl ether exceeded 20% equivalent of the plasma membrane surface area, at which point opened clathrin-coated pits accumulated. The cells were ruptured upon further addition of poly (ethylene glycol) cholesteryl ether. We propose that the primary reason for the differential effect of poly (ethylene glycol) cholesteryl ether is that the bulk membrane phase and caveolae are both more elastic than the rigid clathrin-coated pits. We analyzed the results with the current mechanical model (Rauch and Farge, Biophys J 2000;78:3036-3047) and suggest here that the functional clathrin-lattice is much stiffer than typical phospholipid bilayers.

Endocytosis switch controlled by transmembrane osmotic pressure and phospholipid number asymmetry.

The dynamics of endocytosis in living K562 cells was investigated after the osmotic pressure of the external medium was decreased and the transmembrane phospholipid number asymmetry was increased. When the external pressure was decreased by a factor of 0.54, a sudden inhibition of endocytosis was observed. Under these conditions, the endocytosis suddenly recovered after the phospholipid number asymmetry was increased. The phospholipid asymmetry was generated by the addition of exogenous phosphatidylserine, which is translocated by the endogenous flippase activity to the inner layer of the membrane. The recovery of endocytosis is thus consistent with the view that the phospholipid number asymmetry can act as a budding force for endocytosis. Moreover, we quantitatively predict both the inhibition and recovery of endocytosis as first-order phase transitions, using a general model that assumes the existence of a transmembrane surface tension asymmetry as the budding driving force. In this model, the tension asymmetry is considered to be elastically generated by the activity of phospholipid pumping. We finally propose that cells may trigger genetic transcription responses after the internalization of cytokine-receptor complexes, which could be controlled by variations in the cytosolic or external pressure.

Enhancement of endocytosis due to aminophospholipid transport across the plasma membrane of living cells.

Formation of intracellular vesicles is initiated by membrane budding. Here we test the hypothesis that the plasma membrane surface area asymmetry could be a driving force for vesicle formation during endocytosis. The inner layer phospholipid number was therefore increased by adding exogenous aminophospholipids to living cells, which were then translocated from the outer to the inner layer of the membrane by the ubiquitous flippase. Addition of either phosphatidylserine or phosphatidylethanolamine led to an enhancement of endocytosis, showing that the observed acceleration does not depend on the lipid polar head group. Conversely, a closely related aminophospholipid that is not recognized by the flippase, lyso-alpha-phosphatidylserine, inhibited endocytosis, and similar results were obtained with a cholesterol derivative that also remains in the plasma membrane outer layer. Thus an increase of lipid concentration in the inner layer enhanced internalization, whereas an increase of the lipid concentration in the outer layer inhibited internalization. These experiments suggest that transient asymmetries in lipid concentration might contribute to the formation of endocytic vesicles.

Increased vesicle endocytosis due to an increase in the plasma membrane phosphatidylserine concentration.

Endocytosis vesiculation consists of local membrane invaginations, continuously generated on the plasma membrane surface of living cells. This vesiculation process was found to be activated in vivo by the generation of a transmembrane surface area asymmetry in the plasma membrane bilayer, after enhancement of transbilayer phospholipid translocation. The observed enhancement was shown to be in good quantitative agreement with a theoretical model of elastic equilibrium describing stabilization of 100-nm vesicles in response to phospholipid redistribution. Very rapid dynamic vesiculation and direct re-fusion of the vesicles, both dependent on the phospholipid translocation activity, were found on a time scale of seconds. Both vesiculation and re-fusion were shown to result in a steady-state population of internal vesicles at long time points. The plasma membrane appears to be a dynamic structure, oscillating between two distinct curvature states, the 10 microns-1 "vesicle" and the 0.1 micron-1 "plasma membrane" curvature states. This dynamic behavior is discussed in terms of an elastic control of the membranes curvature state by the phospholipid translocation activity.

An eye banking program for selecting donor corneas for surgical distribution.

Since fewer donated corneas have become available for surgery, we sought to chart the reasons to exclude them for surgical use over time. Those excluded from surgical use (1991-1994) were plotted using an algorithm based on the reasons for exclusion. Four general categories (universal contraindications [UC], national/local medical criteria [NLMC], serology, and morphology) yielded 13 possible areas. UC and NLMC exclusions for 1993-1994 were higher compared with 1991 and 1992 (p < 0.001). The proportion of corneas excluded for serological reasons decreased (p < 0.001) from 1991 to 1994. Exclusions due to morphology remained the same for all 4 years (p = NS). NLMC eliminate older donors but also exclude younger donors before the tissue reaches the eye bank (p > 0.001). Three of four of the youngest tissues ( < 30 years) are used for surgery, whereas one of five of the oldest ( > 70 years) is used. A quality control algorithm provides a heuristic and logical paradigm for noting changes from year to year. Heightened regulation has counteracted many gains in corneal donation fostered by favorable laws.

The impact of state legislation on eye banking.

Corneal transplantation, the most common transplantation procedure done in the United States, requires access to a sufficient number of donor eyes. We examined how laws governing tissue donation affect availability of corneal tissue by reviewing records of the Lions Eye Bank of Texas, Houston, from 1961 through 1990 (43,696 eyes from 21,898 donors). Relevant Texas statutes included the Uniform Anatomical Gift Act of 1970, the Justice of the Peace/Medical Examiner Law of 1977, and the Routine Inquiry Law of 1988. Before 1970, the mean (+/- SD) number of donated corneas was 72 +/- 38 per year; enactment of each statute above was associated with increased mean annual donations of 215 +/- 87, 1329 +/- 562, and 1958 +/- 33 corneas, respectively. The Justice of the Peace/Medical Examiner Law yielded significantly younger donors (who died of trauma), and the Routine Inquiry Law increased the number of hospitalized donors. Data from this eye bank were compared with current state laws nationwide. Effective legislation is a means to meet national ophthalmic surgical needs.

Shape changes of giant liposomes induced by an asymmetric transmembrane distribution of phospholipids.

The influence of a phospholipid transmembrane redistribution on the shape of nonspherical flaccid vesicles was investigated at a fixed temperature by optical microscopy. In a first series of experiments, a transmembrane pH gradient was imposed on egg phosphatidylcholine (EPC)-egg phosphatidylglycerol (EPG) (100:1) giant vesicles. The delta pH induced an asymmetric distribution of EPG. Simultaneously, discoid vesicles were transformed into tubular or a series of connected small vesicles. The fraction of phospholipid transfer necessary for a shape change from discoid to two connected vesicles was of the order of 0.1% of the total phospholipids. Additional lipid redistribution was accompanied by a sequence of shape changes. In a second series of experiments, lyso phosphatidylcholine (L-PC) was added to, or subtracted from, the external leaflet of giant EPC vesicles. The addition of L-PC induced a change from discoid to a two-vesicle state without further evolution, suggesting that lipid transfer and lipid addition are not equivalent. L-PC depletion from the outer leaflet generated stomatocyte-like vesicles. Whenever possible, we have determined whether the giant vesicles undergoing shape changes were unilamellar or multilamellar by measuring the elastic area compressibility modulus, K, by the micropipette assay (Kwok and Evans, 1981). Shape transformations triggered by phospholipid modification of the most external bilayer were indeed influenced by the presence of other underlying membranes that played a role comparable to that of a passive cytoskeleton layer. It appears that in real cells, invaginations of the plasma membrane or budding of organelles could be triggered by a phospholipid transfer from one leaflet to the other caused, for instance, by the aminophospholipid translocase which is present in eukaryotic membranes.

Biomembrane elastic response to intercalation of amphiphiles.

A model of the elastic behavior of a biomembrane in response to intercalation of amphiphiles into the bilayer is developed. This model takes into account the bilayer couple hypothesis (Sheetz and Singer 1974), and assumes that incorporation of amphiphiles into one layer of the membrane exerts mechanical work on the elastic biomembrane. The model accounts for an apparent experimental discordance noted by several authors: the variation in area observed upon incorporating amphiphiles is smaller by a factor of about 2 than the variation expected using previous models.

Morphologic changes of K-Sol preserved human corneas.

Supplementation of tissue culture medium with chondroitin sulfate has been shown to enhance donor corneal preservation. We assessed the efficacy of one of these chondroitin-supplemented media (K-Sol) in comparison with McCarey-Kaufman (MK) medium in maintaining corneal cellular morphology. Thirty-six human corneas, obtained within 8.6 h after death, were placed into K-Sol medium for up to 20 days preservation, and five paired control corneas were placed into MK medium for up to 6 days preservation. Specular photomicrographs were obtained every second to third day for a predetermined storage interval, then studied morphologically in a masked protocol by light microscopy, transmission electron microscopy, and scanning electron microscopy. Endothelial cell loss by specular microscopy averaged 5.8% after 1 week (6 to 8 days) and 7.4% after 13 days in K-Sol medium. Epithelial changes were erratic throughout the 20 day K-Sol preservation period. However, substantial keratocyte changes occurred after 10 days, and endothelial morphology uniformly deteriorated after 17 days. The morphologic data suggest that human corneas may be able to be preserved in K-Sol medium at 4 degrees C for up to 10 days but should be cautiously used thereafter.

Eye banking in Texas.

Texas is providing corneal tissue in the 1980s that is quite adequate to meet the needs of all Texas patients. Infrequent maldistribution is corrected by tissue-sharing between eye banks, facilitated by a toll-free number in Dallas (800-433-6667) for Texas eye banks to call when they cannot meet a local need. With distribution of tissue through eye banks, no patient need wait when tissue is abundantly available. The most important law aiding corneal tissue procurement has been the 1977 Medical Examiners/Justice of the Peace Act, that permits removal of corneas under controlled conditions. This has been implemented in almost all jurisdictions where medical examiners are active, although only a few counties have the services of a medical examiner. The implementation of this law, particularly in Austin, Dallas, El Paso, Fort Worth, Houston, and San Antonio, has increased previous donations by more than double.

Eye banking: 1944 to the present.

Through the efforts of Dr. Townley Paton, eye banks were founded in New York in the early 1940s. Since that time there has been steady growth in both the supply of and demand for donor material. A number of factors have contributed to the success of eye banks in the U.S.A., including improvements in eye banking techniques and administration, increase in corneal surgery, legislation facilitating corneal donations, and growing confidence of patients and ophthalmologists in the success of corneal tissue transplantation.

HIV antibody screening of corneal donors.

Because of the risk of transmitting the acquired immune deficiency syndrome through corneal transplantation, health officials have recommended donor screening. We prospectively studied the seropositivity rate for human immunodeficiency virus infection among ocular tissue donors at our eye bank during 1986. Of 1,517 corneal donors, 5 (0.3%) were repeatedly reactive by enzyme immunoassay. For comparison, 131 (0.06%) of 206,415 blood donors in Houston were similarly seropositive during this same 1-year period. Routine serological testing can be successfully implemented by eye banking personnel to potentially reduce the risk of viral transmission by keratoplasty.

Two treatments of eye bank donor corneas.

The many published studies on sterile procedures used by eye banks have included none until now on ways to optimize sterile handling of corneas procured in the morgues of forensic pathologists. The present study sought to discover if fewer positive cultures would be reported at the time of surgery from corneas which had been treated with an entire bottle of antibiotic (10 ml), as opposed to the more usual method of instilling 10 to 12 drops of antibiotic prior to the corneal excision. The result showed a marginal improvement of 3.1% fewer positive cultures when the entire bottle of antibiotic was used. The findings bolstered the theory that thorough and vigorous washing of the conjunctival sac prior to corneal removal is important and that positive cultures are no more likely when the cornea is shipped away from the eye bank than when used locally.

Evaluating the impact of a conference.

A comprehensive five-phase evaluation of the first Conference on Citizen CPR was implemented to measure its educational value and impact on attitudes regarding key issues in the lay CPR movement. Pre- and post-conference surveys of 480 members of the national CPR community and 165 conference participants along with an on-site evaluation of 152 participants indicated that participants felt that new and relevant information was presented and the conference resulted in little change in the attitudes of either participants or members of the national CPR community. The data reaffirm the educational value of conferences as new information was disseminated easily. However, the data raise questions about the impact conferences have upon attitudes, as only minor changes were reported. Weaknesses in the evaluation design are discussed, and recommendations for future conference evaluations are presented.

Indicators of response to a mass media CPR recruitment campaign.

Respondents to a mass media cardiopulmonary resuscitation (CPR) recruitment campaign in Harris County, Texas were more likely than non-respondents to be White, female, and under 45 years of age, to have had previous CPR training, experienced an incident in which knowledge of CPR might have been useful, or to have a friend or relative with a relevant medical history. The majority of the respondents were housewives, and professionals or technical workers, particularly in the health field. Findings can be used to identify audiences for future mass media CPR campaigns.