Lipid and protein membrane components associated with cholesterol uptake by mycoplasmas

Membranes of Mycoplasma species take up 2–4 times more exogenous cholesterol than membranes of Acholeplasma species. To test whether the lower cholesterol uptake capacity of Acholeplasma is due to the high glycolipid content of their membranes, the phospholipids of Acholeplasma laidlawii and Mycoplasma capricolum membranes were hydrolyzed by phospholipase A². Digestion removed about 30% of the polar lipids of A. laidlawii, leaving the glycolipids and phospholglycolipids intact, and about 70% of the polar lipids of M. capricolum, the residue consisting mostly of sphingomyelin. Cholesterol uptake by the treated membranes from phosphatidylcholine/cholesterol vesicles decreased in rough proportion to the amount of polar lipid removed, indicating that the glycolipids in A. laidlawii membranes can participate in cholesterol uptake.Trypsin digestion of growing cells and isolated membranes of M. capricolum decreased cholesterol uptake by about one-half. Similar treatment of A. laidlawii cells and membranes had no effect on cholesterol uptake. These findings suggest the existence of protease-sensitive receptors on the cell surface of M. capricolum responsible for tighter contact with the cholesterol/phosphatidylcholine vesicles. It is proposed that the ability of Mycoplasma species to take up large quantities of exogenous cholesterol and phospholipids depends on the presence of protein receptors for cholesterol donors, receptors which are absent in Acholeplasma species.     

Identification of eleven human hemoglobin variants by high-performance liquid chromatography: Additional data on functional properties and clinical expression

Eleven abnormal hemoglobins were detected in the course of cord blood screening or in the evaluation of evident hematological problems in individual cases. Identification of the variant in each case was done by high-performance liquid chromatography (HPLC); HPLC provides a rapid, sensitive means for the examination of abnormal hemoglobins. Some of the 11 variants that were identified have been described repeatedly and are included to provide information on the HPLC behavior of tryptic peptides. Others are much rarer. Additional information is provided about the hematological and clinical expression as well as ethnic and geographical distribution of the abnormal hemoglobin.This investigation was supported in part by Grants HL-02558 and HL-15162 from the National Institutes of Health, U.S. Public Health Service.     

Efficient Learning Strategy of Chinese Characters Based on Network Approach

We develop an efficient learning strategy of Chinese characters based on the network of the hierarchical structural relations between Chinese characters. A more efficient strategy is that of learning the same number of useful Chinese characters in less effort or time. We construct a node-weighted network of Chinese characters, where character usage frequencies are used as node weights. Using this hierarchical node-weighted network, we propose a new learning method, the distributed node weight (DNW) strategy, which is based on a new measure of nodes'' importance that considers both the weight of the nodes and its location in the network hierarchical structure. Chinese character learning strategies, particularly their learning order, are analyzed as dynamical processes over the network. We compare the efficiency of three theoretical learning methods and two commonly used methods from mainstream Chinese textbooks, one for Chinese elementary school students and the other for students learning Chinese as a second language. We find that the DNW method significantly outperforms the others, implying that the efficiency of current learning methods of major textbooks can be greatly improved.     

Chimeric molecule IL-6/soluble IL-6 receptor is a potent mitogen for fetal hepatocytes

A novel recombinant molecule, termed IL-6c and consisting of a chimera of interleukin 6 (IL-6) and its soluble receptor is extremely potent in stimulating proliferation of hematopoietic progenitors. We investigated the effect of the IL-6c on the proliferation and differentiation of E14 fetal hepatocytes. IL-6c, in a dose-dependent manner, stimulated proliferation of E14 fetal rat hepatocytes. Adult hepatocyte mitogens together with IL-6c showed no further effect on proliferation. Hematopoietic stem cells mitogens SCF and flt3 ligand (FL) were also mitogenic for fetal hepatocytes, but did not further enhance the effect of IL-6c on cell proliferation. IL-6c decreased expression of fetal markers alpha-fetoprotein (AFP) and gamma-glutamyltranspeptidase, and induced expression of adult enzyme glucose-6-phosphatase (Gluc-6-P) in E14 hepatocytes. On the other hand, IL-6c strongly reduced, in a dose-dependant manner, expression of albumin and tyrosine aminotransferase (TAT). However, when the cells were grown for 3 days with IL-6c, and IL-6c was removed for the next 5 days, expression of albumin and TAT returned to levels found in control cultures. In conclusion, IL-6c stimulated proliferation and affected gene expression in fetal hepatocytes in culture.     

Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library

The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5′ and/or 3′ end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V_H families/segments was observed showing that ONCL did not introduce cloning biases for or against any V_H family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 × 10¹⁰ and by selecting five unique Fabs against GAPDH antigen.     

Caveolae as an additional route for influenza virus endocytosis in MDCK cells

Clathrin-mediated endocytosis has been described as the primary internalization pathway for many viruses, including the influenza virus. However, caveolae, an alternative clathrin-independent endocytotic pathway, has also been described as mediating the entry of some molecules, including viruses. To address the question of pathway selection by the influenza virus, we have investigated whether the virus is internalized via clathrin-coated pits and/or caveolae in Madin Darby canine kidney (MDCK) cells. By applying pharmacological manipulations to selectively disrupt the cell internalization pathways, we found that, in MDCK cells, the influenza virus may be internalized via caveolae in addition to entry by clathrin-mediated endocytosis. However, a small contribution by another mode of entry, as recently proposed, cannot be excluded.