Target-sensitive immunoliposomes: preparation and characterization

A novel target-sensitive immunoliposome was prepared and characterized. In this design, target-specific binding of antibody-coated liposomes was sufficient to induce bilayer destabilization, resulting in a site-specific release of liposome contents. Unilamellar liposomes were prepared by using a small quantity of palmitoyl-immunoglobulin G (pIgG) to stabilize the bilayer phase of the unsaturated dioleoylphosphatidylethanolamine (PE) which by itself does not form stable liposomes. A mouse monoclonal IgG antibody to the glycoprotein D of Herpes simplex virus (HSV) and PE were used in this study. A minimal coupling stoichiometry of 2.2 palmitic acids per IgG was essential for the stabilization activity of pIgG. In addition, the minimal pIgG to PE molar ratio for stable liposomes was 2.5 X 10(-4). PE immunoliposomes bound with HSV-infected mouse L929 cells with an apparent Kd of 1.00 X 10(-8) M which was approximately the same as that of the native antibody. When 50 mM calcein was encapsulated in the PE immunoliposomes as an aqueous marker, binding of the liposomes to HSV-infected cells resulted in a cell concentration dependent lysis of the liposomes as detected by the release of the encapsulated calcein. Neither uninfected nor Sendai virus infected cells caused a significant amount of calcein release. Therefore, the release of calcein from PE immunoliposomes was target specific. Dioleoylphosphatidylcholine immunoliposomes were not lysed upon contact with infected cells under the same conditions, indicating that PE was essential for the target-specific liposome destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and partial characterization of genomic clones coding for a human pro-alpha 1 (II) collagen chain and demonstration of restriction fragment length polymorphism at the 3' end of the gene

We have isolated two clones containing 19 kilobases (kb) of the human gene coding for a pro-alpha 1 (II) collagen chain from human lambda genomic DNA libraries. A 3' clone, HC2A, was selected by cross-hybridization with a cDNA clone containing sequences coding for the carboxy propeptide of chick type II procollagen. A second clone, HC2B, was obtained by screening the library with the 5' part of HC2A. The sequence analysis of exon 3 corresponding to the C propeptide reveals the presence of stretches of conserved nucleotides between the human and the chick type II procollagen genes. On Northern blots, the human collagen clone hybridizes strongly to a 5.5-kb RNA for the rat type II procollagen chain. Finally, studies of genomic DNAs from normal individuals reveal the presence of a HindIII and a BamHI polymorphic site at the 3' end of the gene.     

Massive scrotal edema as a complication of abdominoplasty

Massive scrotal edema is an unreported complication of abdominoplasty. This patient's postoperative decompensation of medial thigh and scrotal lymphatic return may well have been due to an occult lymphedema tarda or previously compromised lymphatics from the fibrosis of venous stasis disease and obesity.     

The thiol groups of mouse immunoglobulin A. Incomplete formation of the C alpha 1-domain disulphide bridge.

The BALB/c IgA (immunoglobulin A) myeloma protein M167 contained on average 5.7 free SH groups per IgA dimer. These groups were preponderantly on the heavy chains and comprised two distinct populations: 3.3 exposed SH groups per dimer in the Fc region, and 2.4 buried SH groups per dimer in the Fd region, detectable o only after denaturation. To locate the cysteine residues involved, labelled peptides were purified from thermolysin digests of radioalkylated IgA by high-performance liquid chromatography. From the amino acid compositions of the peptides, the exposed thiol groups were assigned to Cys-307 in the C alpha 2 domain, which thus existed in the reduced form to an extent exceeding 80%. This residue may allow attachment of secretory component to dimer IgA in the mouse to proceed via thiol-disulphide exchange. The buried thiol groups were assigned to Cys-150 and Cys-208, in the C alpha 1 domain, each being in the reduced form to the extent of approx. 30%. This pair of residues would normally give rise to the characteristic intradomain disulphide bridge. It appears that disulphide formation is not a crucial event during folding of the C alpha 1 domain in IgA biosynthesis. The sequence in the region 140-151 was re-investigated, and residue 142 was shown to be serine, not cysteine, helping explain the lack of heavy-chain-light chain bonding in BALB/c mouse IgA. A disulphide-bond model for mouse IgA is proposed on the basis of these assignments and other features of the mouse alpha-chain sequence.     

Cell-specific properties of red cell and liver ferritin from bullfrog tadpoles probed by phosphorylation in vitro

Cell-specific differences occur in the primary structure of ferritin. For example, red cell and liver ferritin from bullfrog tadpoles differ by 1.5 times in serine content. To determine if cell-specific differences in ferritin primary structure are expressed in the tetraeicosomer, which thus might distinguish the proteins in a functional state, phosphorylation in vitro was employed as a probe using [gamma-32P]ATP and the catalytic subunit from the cAMP-dependent protein kinase of bovine skeletal muscle. Subunits of both proteins in the tetraeicosomers were phosphorylated. Based on tryptic peptide maps, five regions common to both red cell and liver apoferritin were phosphorylated, as confirmed for two peptides by amino acid analyses. [32P]Apoferritin from red cells yielded an additional four 32P-fragments by mapping, at least three of which were unique by amino acid analysis and, in one case, might represent a 32P-Fe complex bound by a fragment of the iron-binding site. One peptide appeared to be unique to liver apoferritin. High concentrations of ATP yielded one additional peptide common to liver and red cell and one red cell-specific peptide in the tryptic peptide maps. The maximum moles of 32P/molecule were 13 +/- 4 and 6 +/- 2, respectively, for red cell and liver apoferritin, which corresponded within experimental error to the number of 32P-tryptic peptides. The level of phosphorylation was, on the average, not more than one site/subunit. Furthermore, above certain levels of phosphorylation, some subunits in the assemblage of 24 appeared to be unavailable as substrates, possibly because of charge repulsion or conformational changes. The possibility that post-translational modifications of ferritin which amplify cell-specific structural features occur in vivo with cytoplasmic components, e.g. protein kinases, is considered in terms of the physiological availability of iron from different iron storage cells and developmental changes in iron storage.     

Gene amplification in Bacillus subtilis

A strain of Bacillus subtilis that carries in its genome a staphylococcal chloramphenicol acetyltransferase gene (from pC194) responds to growth at different concentrations of chloramphenicol by an alteration in the number of copies per genome of the sequences encoding the gene. Growth at 20 micrograms chloramphenicol ml-1 results in a 15-fold amplification of the sequences, whereas growth in the absence of chloramphenicol results in their loss. The mechanism of in situ amplification probably has much in common with that involved in 'R factor transitioning'. The hybridization procedures that have been used for accurately determining the number of copies of the amplified DNA sequences are potentially useful for plasmid copy number determination. The findings reported here also provide a potentially useful alternative to more conventional cloning strategies that are based on autonomous plasmids in B. subtilis. The particular advantages that can be envisaged include enhanced stability of the cloned sequences and control of the number of copies that are present.     

Control of respiratory pattern in conscious dog: effects of heat and CO2

We measured tidal volume (VT) and inspiratory (TI) and expiratory (TE) durations in five conscious tracheostomized dogs breathing air or 5% CO2 in air either at normal (20 degrees C) or elevated (30 degrees C) ambient temperatures. Respiratory frequency ranged between 16 and 333/min due to changes in both TI and TE. During panting TI exceeded TE. During air inhalation instantaneous ventilation (V) spontaneously ranged from 100 to 1,600 ml . kg-1 . min-1. Hypercapnia, heat stress, or both, increased this range of V by increasing maximum V, primarily due to increases in mean inspiratory flow. Under these conditions, changes in TI accounted for more of the spontaneous changes in breath duration. During inhalation of air and 5% CO2, a positive correlation between VT and TI was obtained for TI between 0.13 and 1.05 s; above 1.05 s VT decreased. Heat stress increased VT at a given TI. We suggest that either the decay rate or position of the inspiratory off-switch threshold curve (Clark and von Euler, J. Physiol. London 222: 267, 1972) varies in conscious dogs. Shifts in either the reset (onset) value or decay rate of the curve yield a positive correlation between VT and TI. This modification to the Clark-von Euler model implies that the primary effect of anesthesia on respiratory control is fixation of the inspiratory off-switch threshold curve.     

Glucosylation of Teichoic Acid: Solubilization and Partial Characterization of the Uridine Diphosphoglucose:Polyglycerolteichoic Acid Glucosyl Transferase from Membranes of Bacillus subtilis

Polyglycerolteichoic acid:glucosyl transferase (TAG transferase), one of the three enzymes involved in the pathway leading to the glucosylation of teichoic acid in Bacillus subtilis 168, was investigated. During the early stages of the growth of B. subtilis, TAG transferase is predominantly a soluble enzyme found in the cytoplasm. As growth proceeds, the amount of soluble enzyme decreases and the proportion of insoluble, membrane-bound TAG transferase increases, reaching a maximal value at the close of the logarithmic phase. Data are presented which suggest that these are two forms of the same enzyme, or have some common component. The effects of chaotropic agents, such as sodium trichloroacetate and sodium perchlorate, on the cytoplasmic membrane were also studied. These data show that such compounds can effectively remove the TAG transferase from the membrane in a water-soluble form. A study of some of the physical properties of this solubilized enzyme suggests that there is little difference between the two forms of the enzyme. Experiments are described which indicate that the glucosyl transfer by both the membrane-bound and soluble enzymes is not mediated by lipids.