Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Molding of the regenerate in mandibular distraction: clinical experience

Initial clinical experience with distraction osteogenesis has demonstrated the risk of developing postdistraction malocclusion that requires secondary orthodontic correction. In addition, optimal mandibular form is not always achieved. Both animal studies and preliminary clinical investigations have suggested that the regenerate can be successfully "molded" during active mandibular distraction. The authors have applied this concept clinically to obtain a more desirable occlusal relationship in a group of mandibular distraction patients. Eleven patients are described in whom angulation of the distraction device or intermaxillary/interdental elastics were employed to mold the regenerate. Two representative case studies are provided to illustrate the principles. When using elastic traction to close an anterior open bite, care must be taken that extrusion of individual teeth is minimized by distributing the force over the entire dental arch, especially the basilar portions of the jaws. The authors demonstrate that molding of the regenerate can be successfully accomplished not only during device activation but also early in the consolidation period. The outer limit of the time window in which molding is effective remains to be defined.     

Endoscopic approach for the resection of forehead masses

Over the past several years, surgery aided by the endoscope has come into favor for a number of reasons. Because it is minimally invasive surgery, it has less morbidity, thus, reduced postoperative pain and complications. It results in earlier mobilization and shorter hospitalization, and most importantly, it contributes to an improved cosmetic appearance as a result of a shortened incision line concealed within the hairline in most cases. We have proposed an alternative approach to the surgical resection of forehead masses by means of the endoscope, which has proven to be useful not only for diagnosis but also as a therapeutic tool for the removal of forehead lesions. This report described the clinical experience with the removal of forehead masses in four patients. The cases illustrated the feasibility and ease of resecting a variety of forehead masses with excellent cosmetic results. We hope that more plastic surgeons will use the proposed technique and will continue to explore the safe limits of endoscopic plastic surgery.     

Xanthomonas campestris pv. campestris gum Mutants: Effects on Xanthan Biosynthesis and Plant Virulence

Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. It is composed of polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490–2500, 1993). A cluster of 12 genes in a region designated xpsI or gum has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of X. campestris gum mutants, we report experimental data for assigning functions to the products of the gum genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain gum and non-gum mutations. In addition, we provide evidence that the C-terminal domain of the gumD gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of X. campestris against the plant.     

The past, present and future of cell-free protein synthesis

Recent technical advances have revitalized cell-free expression systems to meet the increasing demands for protein synthesis. Cell-free systems offer several advantages over traditional cell-based expression methods, including the easy modification of reaction conditions to favor protein folding, decreased sensitivity to product toxicity and suitability for high-throughput strategies because of reduced reaction volumes and process time. Moreover, improvements in translation efficiency have resulted in yields that exceed a milligram of protein per milliliter of reaction mix. We review the advances on this expanding technology and highlight the growing list of associated applications.     

Efficient generation of insect-based cell-free translation extracts active in glycosylation and signal sequence processing

A novel method for generation of insect-based cell-free translation extracts is presented. The protocol can be completed in less than an hour, and the resulting extracts are extremely proficient in N-linked glycosylation and signal sequence processing. No specialized equipment other than that usually present in an ordinary biochemistry laboratory is required. The novel approach dramatically reduces cost and time while rendering enhanced lysates compared to previously published strategies.