DNA binding and I kappa B inhibition of the cloned p65 subunit of NF-kappa B, a rel-related polypeptide

The sequence and biochemical properties of the product of the cloned cDNA for the p65 subunit of nuclear factor kappa B (NF-kappa B) have been determined. The cDNA has an open reading frame of 549 amino acids capable of encoding a 60 kd protein. NF-kappa B p65 contains an amino-terminal region of 320 amino acids with extensive similarity to the oncogene c-rel and lesser similarity to NF-kappa B p50. In vitro translated p65 forms a DNA-binding complex with NF-kappa B p50, and the binding of this complex can be specifically inhibited by purified I kappa B. Progressive carboxy-terminal deletions of p65 show that, contrary to previous assumptions, p65 does include a DNA-binding domain that in vivo might become activated only through hetero-oligomerization with p50. DNA binding by truncated p65 is inhibited by I kappa B, thus mapping the I kappa B interaction domain to the rel-homologous region and suggesting that I kappa B exerts its inhibitory effect upon NF-kappa B primarily through interaction with p65.     

The acute effects of cigarette smoke exposure on experimental skin flaps

Random vascular patterned caudally based McFarlane-type skin flaps were elevated in groups of Fischer 344 rats. Groups of rats were then acutely exposed on an intermittent basis to smoke generated from well-characterized research filter cigarettes. Previously developed smoke inhalation exposure protocols were employed using a Maddox-ORNL inhalation exposure system. Rats that continued smoke exposure following surgery showed a significantly greater mean percent area of flap necrosis compared with sham-exposed groups or control groups not exposed. The possible pathogenesis of this observation as well as considerations and correlations with chronic human smokers are discussed. Increased risks of flap necrosis by smoking in the perioperative period are suggested by this study.     

Factors influencing survival and growth of mammalian cells exposed to hypothermia. I. Effects of temperature and membrane lipid perturbers

The Arrhenius plot of the rate of V79 Chinese hamster cell inactivation due to hypothermia has a "break" around 7-10 degrees C with optimum storage temperature for unprotected cells being about 10 degrees C. Addition of the membrane lipid perturber, butylated hydroxytoluene, improves survival of cells when compared to controls at temperatures below this break but not above. Arrhenius plots of growth rates of the cells show breaks at 30 and 40 degrees C. Measurements of membrane fluidity by electron spin resonance or membrane polarization anisotropy by fluorescence spectrophotometry techniques as a function of temperature in these cells also reveal "breaks" centered around 8 and 30 degrees C. Hence, the changes in the rate of cell inactivation and growth as a function of temperature may be related to membrane lipid phase changes.     

Correction of lower eyelid ptosis in the anophthalmic orbit: a long-term follow-up

We describe a 10-year review of 53 patients having had correction of lower eyelid ptosis using fascia lata sling suspension by the operation first described in 1973. The overall conclusion is that this continues to be a reliable procedure with a low complication rate. Four major changes relating to operative technique that create a better result are as follows: (1) the surgical correction must begin with a prosthesis that is ideal for the socket; (2) the fascial strip is narrower at 2 mm; (3) the lateral orbital rim burr hole is placed higher; and (4) the passage of the fascial strip is facilitated by the use of Wright's needle. The optimal sequence of operative procedures in the anophthalmic orbit syndrome is (1) correction of enophthalmos and superior sulcus depression, (2) correction of lower eyelid ptosis, and (3) correction of upper eyelid ptosis.     

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen     

Evolutionary study of multigenic families mapping close to the human MHC class I region

Summary During a search for novel coding sequences within the human MHC class I region (chromosome 6p21.3), we found an exon (named B30-2) coding for a 166-amino-acid peptide which is very similar to the C-terminal domain of several coding sequences: human 52-kD Sjögren's syndrome nuclear antigen A/Ro (SS-A/Ro) and ret finger protein (RFP), Xenopus nuclear factor 7 (XNF7), and bovine butyrophilin. The first three of these proteins share similarities over the whole length of the molecule whereas butyrophilin is similar in the C-terminal domain. The N-terminal domain of butyrophilin is similar to rat myelin/oligodendrocyte glycoprotein (MOG) and chicken B blood group system (B-G) protein. These domains are components of a new subfamily of the immunoglobulin superfamily (IgSF). Butyrophilin is thus a mosaic protein composed of the MOG/B-G Ig-like domain and the C-terminal domain of 52-kD SS-A/Ro, RFP, and XNF7 (1330-2-like domain). Moreover, in situ hybridization shows that RFP, butyrophilin, and MOG map to the human chromosome 6p2l.3-6p22 region and are thus close to the MHC class I genes. It is therefore possible that the butyrophilin gene is the product of an exon shuffling event which occurred between ancestors of the RFP and MOG genes. To our knowledge, this is the first example of the colocalization of a chimeric gene and its putative progenitors. Finally, regulatory protein T-lymphocyte 1 (Rpt-1) shares similarities with the N-terminal halves of RFP, 52-kD SS-A/Ro, and XNF7, but not with the B30-2-like domain. We show that the ancestral Rpt-l gene evolved by overprinting. Correspondence to: P. Pontarotti     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.     

Artificial induction of exocytosis in bull sperm.

We have investigated an exocytotic event, the acrosome reaction (AR), induced by treatment of bovine sperm with vesicles composed of dilauroyl phosphatidylcholine (PC12). Cell membrane permeability barriers (dye exclusion), acrosomal status (pisum sativum (PSA) lectin binding), and intracellular Ca2+ (Fluo3 fluorescence) were evaluated utilizing flow cytometry and fluorescence microscopy. By these methods the AR is resolved into four kinetically distinct steps: (a) PC12 transfer to the sperm plasma membrane (PM); (b) increased permeability of the PM to extracellular Ca2+; (c) localized leakage of acrosomal contents at the anterior tip of the sperm; and (d) vesiculation of sperm membranes and complete exposure of acrosomal contents. Evidence for PC12 transfer to sperm includes transfer of a fluorescent PC12 analogue from vesicles to cells and the absence of detectable vesicle--cell fusion. The fusion inducing properties of PC12 appear to reside in the lipid head group as neither dilauroylphosphatidylethanolamine nor dilauroylphosphatidylglycerol stimulated the AR. The effect of PC chain length on AR induction closely parallels the aqueous phase solubility of the lipid tested. The rate and extent of the AR depend on the extracellular calcium concentration. Cells treated in the absence of calcium do not undergo the AR, but do so rapidly (less than 1 min) upon subsequent addition of calcium. This role of Ca2+ is partially filled by Sr2+, but not by Ba2+ or Mg2+. The rate of the AR decreases with decreasing temperature and the AR occurs very slowly below 27 degrees C. Simultaneous evaluation of intracellular calcium and acrosomal status reveals the kinetic relationship between Ca2+ influx and the exposure of acrosomal contents. N-Ethylmaleimide preincubation arrests PC12-treated sperm at an intermediate stage in the AR, characterized by punctate PSA binding over the tip of the sperm head. The AR, a developmentally regulated, receptor-mediated fusion event, synchronously induced here in vitro, provides a useful model for mechanistic studies of exocytosis.     

Stem deformity inPinus radiata plantations in south-eastern Australia

Plantations of radiata pine (P. radiata D.Don) on soils previously under legume based pastures have a high incidence of stem deformity compared with forest soils. A comparison of soil properties and tree nutrition of 5 to 7 year-old radiata pine on former pastures in the first part of the study showed that stem deformity was strongly correlated with mineralisation of soil N and in particular with nitrification. Other soil properties that have changed as a result of pasture improvement, e.g. pH, available P and Mn, were only partially correlated with stem deformity. In the second part of the study, the role of N availability and other soil properties in the expression of deformity was further investigated in a separate field experiment on soils formerly under native eucalypt forest, tobacco cropping, and improved pasture. Young radiata pine plantings were treated with lime, phosphorus, and nitrogen applied as urea and sodium nitrate. Liming increased soil pH by around 1.5 units, raised exchangeable Ca²⁺ and decreased available Mn. Soil mineral N content was only marginally affected by liming. Superphosphate increased soil available P and raised levels of P in foliage. Changes in soil pH, availability of P, Mn, and B did not affect growth or stem deformity at any of the sites. In contrast, application of N fertilisers at 200 and 600 kg N ha^-1 increased mineral N content and stimulated nitrification, particularly at the forest site. The high rate of N fertiliser increased basal area at the forest site by 45%, but also raised the level of stem deformity from 12% to 56%. At the tobacco and pasture sites, this treatment did not increase growth and did not significantly raise stem deformity above the already high basic level of deformity (63%). Implications of stem deformity in young plantations of radiata pine on potential utilisation later in the rotation are discussed.