Optimizing the expression of chimeric genes in plant cells

Summary The Serratia marcescens chiA gene encodes a secreted chitinase activity which contributes to the fungal growth inhibition exhibited by this bacterium. The coding region from the chiA gene was fused to the promoter and 3 polyadenylation region of the Agrobacterium nopaline synthase gene. Site-directed mutagenesis of specific nucleotides surrounding the initiating AUG of the coding sequence of this chimeric gene resulted in up to an eight-fold increase in the amount of chitinase protein detected in transformed plant tissue. Analysis of the chiA mRNA indicated that these nucleotides also affected mRNA levels. At least 50% of the chitinase protein produced in transformed tobacco cells was the same molecular weight as the S. marcescen secreted protein.     

The impact of augmentation mammaplasty: a follow-up study

Seventy-five women who had undergone augmentation mammaplasty responded to a questionnaire that obtained their perceptions of five areas of their personal and relationship functioning: (1) body and self-image, (2) attractiveness, (3) sensual sensitivity of breasts, (4) sexual life, and (5) relationship with partner. The women's perceptions of these areas before and after surgery were obtained retrospectively (between 3 months and 3 years after surgery). The 54 women in consistent relationships reported positive effects of the surgery on their relationship, although not to the extent that they had anticipated. However, regardless of this finding, these women perceived surgery to have had significant positive effects on their attractiveness, as well as on their body and self-image. Postoperatively, the partners of these women were viewed as having a significantly greater interest in sexual activity, as perceiving the women to be significantly more attractive, and as believing that the sexual relationship was significantly enhanced. The 21 women who were not in a consistent relationship also reported positive postoperative changes, although these were not statistically significant. Neither the women's age, length of time since surgery, nor the duration of the woman's relationship had any effect on the positive changes reported. The quantitative and qualitative data underscored the highly positive benefits of breast augmentation for the respondents.     

Radiosensitization in multifraction schedules. I. Evidence for an extremely low oxygen enhancement ratio

Stable monolayers of contact-inhibited C3H 10T1/2 cells were used in multifraction radiation experiments to measure the oxygen enhancement ratio (OER) at low doses/fraction under conditions where cell cycle effects (repopulation, redistribution) were minimal. Consistent with there being a dose-dependent reduction in the OER at low doses, an extremely low OER of 1.34 was measured after 20 fractions of 1.7 Gy every 12 h. The sparing effects of fractionating radiation doses were not apparent for cells irradiated under hypoxic conditions (i.e., multifraction survivals were lower than acute single-dose values) until doses exceeding 15 Gy were reached. This result suggested a deficiency in the recovery from sublethal and/or potentially lethal damage might exist after hypoxic irradiations, thereby reducing the OER. The capacity to repair potentially lethal damage was found to be nearly the same after hypoxic as compared to aerobic irradiations. However, there was an apparent absence of sublethal damage repair by 10T1/2 cells between two hypoxic irradiations which could be a major contributing factor to the extremely low OER value measured in this multifraction schedule.     

Fluoroaluminates mimic guanosine 5''-[gamma-thio]triphosphate in activating the polyphosphoinositide phosphodiesterase of hepatocyte membranes. Role for the guanine nucleotide regulatory protein Gp in signal transduction.

Fluoride and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) both activate the hepatocyte membrane polyphosphoinositide phosphodiesterase (PPI-pde) in a concentration-dependent manner. AlCl3 enhances the fluoride effect, supporting the concept that [A1F4]- is the active species. Analysis of the products of inositol lipid hydrolysis demonstrate that phosphatidylinositol bisphosphate is the major lipid to be hydrolysed. Guanosine 5'-[beta-thio]diphosphate (GDP beta S) is an inhibitor of activation of PPI-pde by both fluoride and GTP gamma S. These observations suggest that the guanine nucleotide regulatory protein (termed Gp) bears a structural resemblance to the well-characterized G-proteins of the adenylate cyclase system and the cyclic GMP phosphodiesterase system in phototransduction.     

Induction of maturation in small Xenopus laevis oocytes

The competence of Xenopus laevis oocytes in various stages of growth to respond to progesterone treatment was investigated. Full-grown (stage 6) oocytes undergo nuclear membrane dissolution and resume meiosis in response to progesterone exposure, while smaller oocytes (stages 3-5; less than 1100 micron in diameter) do not. The defect which prevents 750- to 1050-micron oocytes from responding to progesterone can be overcome by microinjecting cytoplasm withdrawn from a stage 6 oocyte. Germinal vesicle breakdown in these small oocytes occurs on a timetable similar to that of stage 6 oocytes exposed to progesterone and is accompanied by a twofold increase in protein synthesis as well as the activation of MPF. The results argue that a cytoplasmic factor(s) which probably first appears at late stage 5 is required for progesterone responsiveness. The identity and role of the factor(s) in the development of maturation competence and the regulation of maternal mRNA translation are discussed.     

A nonendocytotic mechanism for the selective uptake of high density lipoprotein-associated cholesterol esters

We have previously described in rats the selective uptake of HDL-associated cholesterol esters (traced by [3H]cholesteryl oleyl ether) in excess of the uptake of HDL-associated apoA-I. In the present studies we show that the mechanism also exists in cultured cells of human and mouse origin as well. This selective uptake represents a net uptake of cholesterol esters and not an isotope exchange, as shown by mass flux studies in adrenal cells. Inhibitors of receptor recycling, chloroquine, monensin, and colchicine, inhibited uptake of apoA-I from HDL by Hep G-2 human hepatoma cells to about the same extent as a reference protein, asialofetuin, but inhibited uptake of the cholesteryl ether tracer much less. Levels of NaN3 which effectively inhibited sucrose pinocytosis inhibited uptake of apoA-I to about the same extent but did not inhibit uptake of the cholesteryl ether at all. Thus, not only receptor recycling, but endocytosis as well, appears not to be involved in selective uptake. This conclusion was supported by studies in which synthetic HDL particles were made to contain two neutral lipid core tracers; one of them, the [3H]cholesteryl ether previously used, was selectively taken up, whereas the other, [14C]sucrose octaoleate, was excluded from selective uptake. Thus, selective uptake cannot involve endocytosis of the entire lipid core, but may involve other specific transfer mechanisms.     

Behavioural and physiological adaptations to a burrowing lifestyle in the snake blenny, Lumpenus lampretaeformis, and the red band-fish, Cepola rubescens

Observations have been made on the mode of burrow construction in the snake blenny, Lumpenus lampretaeformis , under laboratory conditions. It appears that head probing and lateral oscillations of the body are principally responsible for the excavation of the burrow which is completed within 24 h. The burrow structure has been analysed in detail, showing a mean depth of 7.2 cm with a maximum observed length of 73 cm, with most systems between 20 and 35 cm in length. Initially linear burrows with two openings are usually provided with a small side tunnel, giving the system a characteristic Y-shape.
Burrow irrigation was investigated for the first time in L. lampretaeformis. The mean duration of burrow irrigation, by flexions of the tail of the fish, was 21 s with over 13 min h⁻¹ spent in irrigating the burrow. The mean water displacement per irrigation period was 3.1 ml. The PO ₂ and PCO ₂ were measured in both surface water and within the burrow system of L. lampretaeformis. Surface water values for PO ₂ were high (> 150 Torr) and PCO ₂ low (<0.4 Torr). Hypoxic and hypercapnic conditions were measured in the burrow system itself, with PO ₂ values ranging between 57 and 129 Torr and PCO ₂ rising to > 1.3 Torr in some burrows.
A comparative study of Cepola rubescens burrows indicated similar surface water PO ₂ and PCO ₂ values as in L. lampretaeformis. Burrow water PO ₂ values ranged between 60 and 94 Torr, with PCO ₂ values as high as 1.5 Torr being recorded. These results are discussed in relation to the adaptation of both species to a burrowing lifestyle.     

On the tertiary structure of the extracellular domains of the epidermal growth factor and insulin receptors

Alignment of the sequences, the identification of conserved residue patterns and secondary structure predictions indicate that the extra-cellular regions of the human and Drosophila epidermal growth factor (EGF), c-erb-B2 and human insulin receptors each contain two large, homologous domains (L) which are probably comprised of at least four short alpha-helices followed by turns of conserved length and beta-strands. In the human and Drosophila EGF and c-erb-B2 receptors these homologous domains are each followed by a series of smaller cystine-rich domains (S) to give a gene-duplicated structure of L1S11S12S13L2S21S22S23. In the human insulin receptor, the second series of cystine domains is replaced by a different sequence. These duplicated structures are probably organised as a pseudo-symmetrical dimer. There are two 'hyper-variable' regions, one at the end of the large domains and one in the cystine-rich sequences, which are candidates for hormone or growth-factor binding.