Transient transfection of 293T cells was utilized to produce high-titer murine recombinant retroviral vectors for clinical studies. This system was initially optimized by gene transfer using different retroviral envelope proteins into activated human CD4+ T lymphocytes in vitro. Higher titer and infectivity were obtained than with stable murine producer lines; titers of 0.3-1 X 10 7 infectious units per milliliter for vectors encoding the green fluorescent protein (GFP) were achieved. Virions pseudotyped with envelope proteins from gibbon ape leukemia virus or amphotropic murine leukemia virus resulted in gene transfer of 50% in CD4+ human T lymphocytes with this marker. Gene transfer of Rev M10 with this vector conferred resistance to HIV infec- tion compared with negative controls in the absence of drug selection. Thus, the efficiency of transduction achieved under these conditions obviated the need to include selection to detect biologic effects in T cells. Fi- nally, a protocol for the production of large-scale supernatants using transient transfection was optimized up to titers of 1.9 X 10 7 IU/ml. These packaging cells can be used to generate high-titer virus in sufficient quan- tities for clinical studies and will facilitate the rapid, cost-effective generation of improved retroviral, lentivi- ral, or other viral vectors for human gene therapy.
A quantitative approach to assessing the image quality of mammograms at screening centres has been developed. In this study an expert radiologist made a subjective judgement of the overall film blackening for each mammogram taken on a sequential series of 100 women attending screening over a period of three days on a single screening system. To relate these subjective judgements to quantitative measurements each mammogram was digitised and the distribution of film densities analysed. Average film densities in the main breast area were found to vary from 1.2 to 2.4, with an average of 1.8 for oblique views and 1.7 for CC views. The densities measured on quality control films of 40 mm thick PMMA exposed at 28 kV and 30 kV, and processed at the same time as the clinical films, were 1.52 and 1.67 respectively. Mammograms were judged satisfactory when film densities in the main breast region were in the range 0.8 to 2.8 with average densities of about 1.6 to 2.0. The method described allows the ideal film blackening to be quantitatively assessed.
Recombinant lambda phages were isolated that resulted from recombination between the lambda genome and plasmid pBR322 in Escherichia coli, even though these deoxyribonucleic acids (DNAs) did not share extensive regions of homology. The characterization of these recombinant DNAs by heteroduplex analysis and restriction endonucleases is described. All but one of the recombinants appeared to have resulted from reciprocal recombination between a site on lambda DNA and a site on the plasmid. In general, there were two classes of recombinants. One class appeared to have resulted from recombination at the phage attachment site that probably resulted from lambda integration into secondary attachment sites on the plasmid. Seven different secondary attachment sites on pBR322 were found. The other class resulted from plasmid integration at other sites that were widely scattered on the lambda genome. For this second class of recombinants, more than one site on the plasmid could recombine with lambda DNA. Thus, the recombination did not appear to be site specific with respect to lambda or the plasmid. Possible mechanisms for generating these recombinants are discussed.
A study was conducted to evaluate the effects of various precombustion chamber design, operating, and control parameters on the exhaust emissions of a natural gas engine. Analysis of the results showed that engine-out total hydrocarbons and oxides of nitrogen (NOx) can be reduced, relative to conventional methods, through prechamber design. More specifically, a novel staged prechamber yielded significant reductions in NOx and total hydrocarbon emissions by promoting stable prechamber and main chamber ignition under fuel-lean conditions. Precise fuel control was also critical when balancing low emissions and engine efficiency (i.e., fuel economy). The purpose of this paper is to identify and explain positive and deleterious effects of natural gas prechamber design on exhaust emissions.
Shiga toxin (STX) is a ribosome-inactivating cytotoxin produced by Shigella dysenteriae serotype 1. The enzymatic domain of the STX A polypeptide has been defined by introducing amino- and carboxy-terminal deletions in the polypeptide and assessing activity in a cell-free translation system. Three recombinant forms of StxA which possess enzymatic activity were genetically fused to a 165-amino-acid polypeptide derived from CD4, the cellular receptor for human immunodeficiency virus type 1 (HIV-1). This strategy eliminated the STX receptor-binding subunit and directed the hybrid toxins to cells expressing the HIV-1 surface glycoprotein gp120. A bacterial lysate containing these toxin chimeras killed the HIV-1-infected T-cell line 8E5 but was not cytotoxic toward the uninfected parental cell line A3.01. This cytotoxic activity was specifically inhibited by monoclonal antibodies which block the interaction between CD4 and gp120. These StxA-CD4 hybrids add to the repertoire of recombinant fusion proteins which possess the capacity to selectively kill HIV-1-infected T cells.
We have previously demonstrated that steroidogenic acute regulatory protein (StAR) is essential for the rate-limiting step in the acute regulation of steroidogenesis, which is the transport of cholesterol from the outer to the inner mitochondrial membrane. We have hypothesized that this transport occurs as the 37-kilodalton (kDa) precursor form of StAR is imported into the mitochondria and processed to its 30-kDa mature forms. Using an in vitro transcription and translation system in the presence of mitochondria isolated from unstimulated mouse MA-10 Leydig tumor cells, we now directly show that the 37-kDa form is indeed the cytosolic precursor of StAR and can be processed by mitochondria to all four 30-kDa mature forms. To determine the subcellular location of StAR in steroidogenic cells, ultrastructural immunocytochemistry was performed in adrenal zona fasciculata cells using the protein A-gold technique. We show that StAR is associated exclusively with the mitochondria. There, StAR is primarily localized in the intermembrane space and the intermembrane space side of the cristae membrane. StAR was shown to induce steroid production in isolated mitochondria. StAR protein was expressed in COS1 cells and the cell lysate, which was shown to contain abundant levels of StAR by Western blot analysis, was incubated with mitochondria isolated from unstimulated MA-10 cells. In these experiments, StAR increased steroid production by at least 4-fold over control mock-transfected lysate, and this increase was time and dose dependent. Furthermore, the increase in steroid production induced by StAR-containing lysate was not observed when COS1 lysate containing high levels of another mitochondrially imported protein, adrenodoxin, was used. We conclude from these results that in response to tropic hormone stimulation of steroidogenic cells, StAR is synthesized as a 37-kDa precursor, imported into the mitochondria, processed to its 30-kDa mature forms, and localized to the intermembrane space. During import and processing in vitro, StAR induces steroid production in isolated mitochondria in a specific manner.
The steroidogenic acute regulatory (StAR) protein promotes intramitochondrial delivery of cholesterol to the cholesterol side-chain cleavage system, which catalyzes the first enzymatic step in all steroid synthesis. Intriguingly, substrate cholesterol derived from lipoprotein can upregulate StAR gene expression. Moreover, substrate oxysterols have been suggested to also play a role. To investigate whether oxysterols can regulate StAR expression, two steroidogenic cell lines, mouse Y1 adrenocortical and MA-10 Leydig tumor cells, were treated with various oxysterols and steroids, including 25-hydroxycholesterol (25 OHC), 22(R)OHC and 20alphaOHC. The majority of these compounds rapidly increased StAR protein levels within as little as 1 h. The most potent oxysterols were 20alphaOHC for Y1 and 25 OHC for MA-10 cells. After 8 h, StAR mRNA abundance also increased whereas there were no detected changes in promoter activity. Thus, in contrast to lipoprotein, oxysterols acutely increase StAR protein levels independently of mRNA abundance, and later increase mRNA levels independently of new gene transcription. Therefore, we propose that oxysterols modulate steroidogenesis at two levels. First, oxysterols may be important in post-transcriptional regulation of StAR activity and production of steroids for paracrine action. Secondly, through direct conversion to steroid, oxysterols may account in part for StAR-independent steroid production in the body.
In the mountains of Peru, globular colonies of Nostoc commune (Nostocales) are collected in the highland lakes by the indigenous people, who call them llullucha. They are consumed locally, traded for maize, or sold, eventually entering the folk markets of Cusco and other neighboring cities. Throughout highland Peru, Nostoc commune is highly salient as a seasonal dietary item, being eaten alone, or in picante-a local stew-and is said to be highly nutritious. Nostoc commune has been known to produce unusual amino acids, including those of the mycosporine group, which possibly function to prevent UV damage [1]. We analyzed 21 different Nostoc commune spherical colonies from 7 different market collections in the Cusco area for the presence of b-N-methylamino-l-alanine (BMAA), a neurotoxic amino acid [2] produced by diverse taxa of cyanobacteria [3], using four different analytical techniques (HPLC-FD, UPLC-UV, UPLC-MS, LC-MS-MS). We found using all four techniques that BMAA was present in the samples purchased in the Peruvian markets at levels ranging from 2–22µg/g. Since BMAA has been putatively linked to neurodegenerative disease, it would be of interest to know if the occurrence of neurodegenerative illness is greater among individuals who consume llullucha in Peru.
