Interactions between plasma proteins and pulmonary surfactant: surface balance studies

The influence of human fibrinogen, alpha-globulin, and albumin on the properties of monolayers of pulmonary surfactant under dynamic compression and expansion has been studied at 37 degrees C. Each of the proteins altered some of the properties of the normal compression and expansion isotherms of surfactant such that characteristics deemed desirable for proper lung function were impaired. The order of potency of these effects was fibrinogen greater than globulin greater than albumin. The proteins (a) decreased the maximum surface pressure (equivalent to the minimum surface tension) which the surfactant monolayers attained on compression, (b) decreased the areas occupied per mole of lipid phosphorus when the monolayers were at surface tensions of 20 and 12 mN.m-1, (c) reduced the areas of the hysteresis between compression and expansion isotherms, and (d) decreased the rate of change of surface tension with area at the point of initial expansion of the monolayers. The proteins might compete with surfactant lipid for available space at the interface, especially at low film compression. They might also enhance the desorption of lipid from the monolayer. The findings are consistent with the loss of pulmonary function and presence of edema that occur in adult respiratory distress syndrome being contributed to by plasma proteins interfering with surfactant function.     

A TAXONOMIC STUDY OF THE GENUS PADINA (DICTYOTALES,PHAEOPHYCEAE) INCLUDING THE DESCRIPTIONS OF FOUR NEW SPECIES FROM JAPAN,HAWAII, AND THE ANDAMAN SEA1

A taxonomic study of the genus Padina from Japan, Southeast Asia, and Hawaii based on morphology and gene sequence data (rbcL and cox3) resulted in the recognition of four new species, that is, Padina macrophylla and Padina ishigakiensis from Ryukyu Islands, Japan; Padina maroensis from Hawaii; and Padina usoehtunii from Myanmar and Thailand. All species are bistratose and morphologically different from one another as well as from any known taxa by a combination of characters relating to degree of calcification; the structure, position, and arrangement of hairlines (HLs) and reproductive sori; and the presence or absence of rhizoid‐like groups of hairs and an indusium. Molecular phylogenetic analyses demonstrated a close relationship between P. ishigakiensis, P. macrophylla, P. maroensis, and Padina australis Hauck. The position of P. usoehtunii, however, was not fully resolved, being either sister to a clade comprising the other three new species and P. australis in the rbcL tree or more closely related to a clade comprising several other recently described species in the cox3 tree. The finding of the four new species demonstrates high species diversity particularly in southern Japan. The following characters were first recognized here to be useful for species delimitation: the presence or absence of small rhizoid‐like groups of hairs on the thallus surface, structure and arrangement of HLs on both surfaces either alternate or irregular, and arrangement of the alternating HLs between both surfaces in equal or unequal distance. The evolutionary trajectory of these and six other morphological characters used in species delineation was traced on the phylogenetic tree.     

Identification of random amplified polymorphic DNA (RAPD) marker for differentiating male from female and sporophytic thalli of <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Rhodophyta)

There have been limited reports on molecular sex markers for macroalgae. We report the use of random amplified polymorphic DNA analysis (RAPD) to identify molecular sex markers for Gracilaria changii (Xia et Abbott) Abbott, Zhang et Xia. Two DNA extraction methods were used: a modified CTAB and phenol-chloroform combination method and the DNeasy Plant Mini Kit. The CTAB and phenol-chloroform method gave the best yield of DNA in quality and quantity and is suitable for larger-sized specimens like G. changii. Sixty-nine RAPD primers were screened to search for sex-linked DNA markers for G. changii, and only one sex-linked marker (716 bp) was identified using OPA 18. RAPD was also used to investigate the molecular characteristics of the three life-stages (male, female, tetrasporophyte) of G. changii. Seven (OPA7, OPA18, S14, S61, S64, S75 and S76) out of the 69 primers showed polymorphism and were selected for interpopulation analysis for DNA isolated from 23 samples collected from Morib and Sungai Pulai in Malaysia. The combination of data produced by the seven primers generated a dendrogram that grouped the specimens into different clades according to their sex and life-stage using the unweighted pair group and arithmetic averages (UPGMA) method. It showed that RAPD was able to differentiate tetrasporophytes, females, and males. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Use of RAPD in differentiation of selected species of <Emphasis Type="Italic">Sargassum</Emphasis> (Sargassaceae,Phaeophyta)

Sargassum C. Agardh is one of the most common but little understood genera of Phaeophyta in Malaysia. The difficulty in species delineation is due to morphological plasticity. A combination of morphology and random amplified polymorphic DNA (RAPD) studies of selected Sargassum species was carried out to have a better understanding of the taxonomy. Primer OPA13 was found to be good for discriminating between Sargassum species. Sargassum binderi was shown to be different from S. oligocystum (S_D>0.5 = 14.11%), indicating the importance of the vesicle and receptacle in species differentiation. S. baccularia was clearly separated out from S. polycystum and S. stolonifolium using primer OPA13. RAPD analysis showed that the presence of the stolon is an important character for separating S. baccularia (no stolon) from S. polycystum (stolon) and S. stolonifolium (stolon). Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes

Establishing recordable channels in membranes of oocytes formed by expressing exogenous complementary DNA (cDNA) or messenger RNA (mRNA) has contributed greatly to understanding the molecular mechanisms of channel functions. Here, we report the extension of this semi-physiological system for monitoring the channel activity of preassembled membrane proteins in single cell oocytes by injecting reconstituted proteoliposomes along with substrates or regulatory molecules. We build on the observation that SecA from various bacteria forms active protein-conducting channels with injection of proteoliposomes, protein precursors, and ATP–Mg²⁺. Such activity was enhanced by reconstituted SecYEG–SecDF•YajC liposome complexes that could be monitored easily and efficiently, providing correlation of in vitro and intact cell functionality. In addition, inserting reconstituted gap junction Cx26 liposomes into the oocytes allowed the demonstration of intracellular/extracellular Ca²⁺-regulated hemi-channel activities. The channel activities can be detected rapidly after injection, can be monitored for various effectors, and are dependent on specific exogenous lipid compositions. This simple and effective functional system with low endogenous channel activity should have broad applications for monitoring the specific channel activities of complex interactions of purified membrane proteins with their effectors and regulatory molecules.     

The dispensability and requirement of SecA N-terminal aminoacyl residues for complementation,membrane binding,lipid-specific domains and channel activities

SecA is an essential multifunctional protein for the translocation of proteins across bacterial membranes. Though SecA is known to function in the membrane, the detailed mechanism for this process remains unclear. In this study we constructed a series of SecA N-terminal deletions and identified two specific domains crucial for initial SecA/membrane interactions. The first small helix, the linker and part of the second helix (Δ2-22) were found to be dispensable for SecA activity in complementing the growth of a SecA ts mutant. However, deletions of N-terminal aminoacyl residues 23–25 resulted in severe progressive retardation of growth. Moreover, a decrease of SecA activity caused by N-terminal deletions correlated to the loss of SecA membrane binding, formation of lipid-specific domains and channel activity. All together, the results indicate that the N-terminal aminoacyl residues 23–25 play a critical role for SecA binding to membranes and that the N-terminal limit of SecA for activity is at the 25th amino acid.     

SecAAA trimer is fully functional as SecAA dimer in the membrane: Existence of higher oligomers?

SecA is an essential ATPase in bacterial Sec-dependent protein translocation pathway, and equilibrates between monomers and dimers in solution. The question of whether SecA functions as monomers or dimers in membranes during the protein translocation is controversial. We previously constructed a tail-to-head SecAA tandem dimer, and showed it is fully functional by complementation in vivo and protein translocation in vitro, indicating that SecA can function at least as a dimer in the membrane without dissociating into monomers. In this study, we further constructed genetically a tail-to-head SecAAA trimer, which is functional in complementing a temperature-sensitive secA mutant. The purified SecAAA trimer per protomer is fully active as SecAA tandem dimers in ATPase activity, in protein translocation in vitro and in ion channel activities in the oocytes. With these functional tail-to-head trimer SecAAA and tandem SecAA, we examined their surface topology in the presence of liposomes using AFM. As expected, the soluble SecAAA without lipids are larger than SecAA. However, the ring/pore structures of SecAAA trimers were, surprisingly, almost identical to the SecA 2-monomers and SecAA dimers, raising the intriguing possibility that the SecA may exist and function as hexamer ring-structures in membranes. Cross-linking with formaldehyde showed that SecA, SecAA and SecAAA could form larger oligomers, including the hexamers. The molecular modeling simulation shows that both tail-to-head and tail-to-tail hexamers in the membranes are possible.