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D Shire

Publications and source records attributed to D Shire.

59 records · Page 4Linked to original sources
PubMed1986

Scanning electron microscopy of Leishmania major in Phlebotomus papatasi.

The morphology of Leishmania major parasites and their interactions with various regions of the alimentary canal of Phlebotomus papatasi were studied by scanning electron microscopy. Parasites were observed to undergo development initiated with the ingestion of amastigotes and culminating in a characteristic distribution of four distinct morphological forms in various parts of the alimentary canal: namely, large numbers of elongate nectomonads in the abdominal mid-gut, haptomonad forms attached to the cuticle of the stomodeal valve, small spherical forms attached to the esophagus and masses of short promastigotes, believed to be the infective forms, lying free in the anterior thoracic mid-gut and the esophagus.

Animals↗
PubMedDate not supplied

The maximum rate of gene expression is dependent on the downstream context of unfavourable codons.

Presented here is an experimental demonstration of our theoretical predictions on the role of the downstream context of unfavourable codons in a gene on its expression level. Six non clustered AGG codons were inserted in the chloramphenicol acetyltransferase (cat) gene of E. coli and the expression of this modified gene (cat4) was compared with that of a cat gene in which four clustered AGG codons were inserted (cat2 gene). As predicted, the rate of production of the corresponding CAT4 and CAT2 proteins is equal as long as the rate of transcription of the gene does not exceed a given limit. When this limit is exceeded, production of CAT4 continues to increase, whereas CAT2 production decreases dramatically. Various consequences and possible applications of this downstream context effect are discussed.

Base Sequence↗
PubMedDate not supplied

Simultaneous quantitation of cytokine mRNAs in interleukin-1 beta stimulated U373 human astrocytoma cells by a polymerisation chain reaction method involving co-amplification with an internal multi-specific control.

The measurement of cytokine mRNA levels is of fundamental importance in the understanding of diverse pathological states. We present a simplification of a polymerase chain reaction-based technique which permits the simultaneous measurement of up to 20 cytokine mRNAs, together with those of several other cellular products, including beta 2-microglobulin and beta-actin. The technique makes use of internal standards bearing multiple PCR primer sites which are identical to those on the mRNAs to be assayed. Known quantities of the standards are added to the cellular RNA and the mixture is co-reverse transcribed and co-amplified. The simplifications described here are based on the fact that each pair of amplicons accumulates in a constant ratio even in the plateau phase of amplification. As a result, no preliminary experiments to determine the limits of the exponential phase of amplification are necessary; the same number of cycles may be chosen for all the mRNAs to be measured, whatever their level in the mixture might be; pipetting errors are avoided since all calculations are based upon the relative quantities of co-amplified material. Here we illustrate the method through a quantitative study of the expression of cytokine mRNAs in U373 human astrocytoma cells before and after stimulation with IL-1 beta. Quantitation was carried out either by incorporating radioactivity in the amplicons or by fluorescence measurements after propidium iodide staining. Only very low numbers of transcripts for IL-6, IL-8, CSF-1, MCP-1 and either Gro alpha or Gro beta were detectable in unstimulated cells. The levels of these cytokine mRNAs increased dramatically following IL-1 beta stimulation and, in addition, transcription of IL-1 beta, TNF alpha, GM-CSF, G-CSF, Gro gamma and MCP-1, some of which have not previously been detected in U373, was initiated in the stimulated cells. At the same time we found that transcripts for IL-2, IL-3, IL-4, IL-5, IFN gamma, huMlP1 alpha and huMlP1 beta were totally absent in this cell line. These results suggest a potentially important role for astrocytes in the local amplification of inflammatory responses in the brain.

Astrocytes↗
PubMedDate not supplied

Acridine-linked oligonucleotide probes for the short dAT-rich motifs in the 3'-untranslated region of cytokine genes.

We have investigated the use of oligonucleotide probes for identifying cDNA clones containing the short dAT-rich motifs found in the 3'-untranslated region of cytokine genes. To obtain sufficiently stable duplexes between the octameric probes used to identify genes containing the sequence dTATTTATT and its complement, it was necessary to couple an intercalating agent, an acridine derivative (acr), to the 5'-positions of the probes. The resulting octamers 5'-acr-dAATAAATA and, particularly, 5'-acr-dTATTTATT were successfully used to distinguish the complementary sequences in cDNA from internal, single point mismatched sequences. Southern blot analyses of plasmids containing IL-1 beta and IL-8 gave positive results with the 3' degenerate probe, 5'-acr-dTATTTATTN, clearly showing that the very short probe approach can be used in this type of analysis. Subsequently, in slot blot analyses we found that, even without the degenerate nucleotide, N, plasmids bearing cytokine sequences with at least 7 contiguous matched nucleotides could be unambiguously identified with 5'-acr-dTATTTATT. Unfortunately, because of the ubiquity of these dAT-rich sequences in bacterial DNA, it was not possible to use these probes for direct colony screening. In contrast to the results obtained with DNA, at the RNA level, with IL-1 beta mRNA bound to nitrocellulose, the hybrid formed with 5'-acr-dAATAAATA was very unstable, even in 1M LiCl solution at 2 degrees C; however, in the same salt solution the slightly longer acridine-coupled probes 5'-acr-dAATAAATAGGG and 5'-acr-dAAAGAACAA remained hybridized to their complementary sequences up to about 18 degrees C.

Acridines↗