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Biomedical subjects

M Chee

Publications and source records attributed to M Chee.

16 recordsLinked to original sources
PubMed1991

Enzymatic multiplex DNA sequencing.

The problem of reading DNA sequence films has been reformulated using an easily implemented, multiplex version of enzymatic DNA sequencing. By utilizing a uniquely tagged primer for each base-specific sequencing reaction, the four reactions can be pooled and electrophoresed in a single lane. This approach has been previously proposed for use with fluorescently labelled probes (1), and is analogous to the principle used in four-dye fluorescence sequencing except that the signals are resolved following electrophoresis (2). After transfer to a nylon membrane, images are obtained separately for each of the four reactions by hybridization using oligonucleotide probes. The images can then be superimposed to reconstitute a complete sequence pattern. In this way the correction of gel distortion effects and accurate band registration are considerably simplified, as each of the four base-specific ladders require very similar corrections. The methods therefore provide the basis for a second generation of more accurate and reliable film reading programs, as well as being useful for conventional multiplex sequencing. Unlike the original multiplex protocol (3), the approach described is suitable for small projects, as multiple cloning vectors are not used. Although more than one vector can be utilized, only a library of fragments cloned into any single phage, phagemid or plasmid vector is actually required, together with a set of tagged oligonucleotide primers.

Base Sequence↗
PubMed1990

Herpesviruses: a study of parts.

Dormancy, an adaptation to survival in a hostile environment, is a trait common to herpesviruses. Two other features are a large (0.1-0.25 Mb) and mutile (inherently easily mutable) genome. The complete nucleotide sequences of four herpesviruses have recently been determined. This database is unparalleled in allowing the comparative evolutionary study of a complex group of viruses in eukaryotes. In this article, we examine aspects of herpesvirus diversity in the light of recent studies which have revealed characteristics that unify the family at the genetic level.

DNA Replication↗
PubMed1990

Human herpesvirus 6 is closely related to human cytomegalovirus.

A sequence of 21,858 base pairs from the genome of human herpesvirus 6 (HHV-6) strain U1102 is presented. The sequence has a mean composition of 41% G + C, and the observed frequency of CpG dinucleotides is close to that predicted from this mononucleotide composition. The sequence contains 17 complete open reading frames (ORFs) and part of another at the 5' end of the sequence. The predicted protein products of two of these ORFs have no recognizable homologs in the genomes of other sequenced human herpesviruses (i.e., Epstein-Barr virus [EBV], human cytomegalovirus [HCMV], herpes simplex virus [HSV], and varicella-zoster virus [VZV]). However, the products of nine other ORFs are clearly homologous to a set of genes that is conserved in all other sequenced herpesviruses, including homologs of the alkaline exonuclease, the phosphotransferase, the spliced ORF, and the major capsid protein genes. Measurements of similarity between these homologous sequences showed that HHV-6 is clearly most closely related to HCMV. The degree of relatedness between HHV-6 and HCMV was commensurate with that observed in comparisons between HSV and VZV or EBV and herpesvirus saimiri and significantly greater than its relatedness to EBV, HSV, or VZV. In addition, the gene for the major capsid protein and its 5' neighbor are reoriented with respect to the spliced ORFs in the genomes of both HHV-6 and HCMV relative to the organization observed in EBV, HSV, and VZV. Three ORFs in HHV-6 have recognizable homologs only in the genome of HCMV. Despite differences in gross composition and size, we conclude that the genomes of HHV-6 and HCMV are closely related.

Amino Acid Sequence↗
PubMed1990

Focal seizure in non-ketotic hyperglycaemia.

Structural brain lesions are the common causes of focal seizure. We report two patients who presented with focal fits with non-ketotic hyperglycaemia. There were no focal brain lesions to account for the fits. The fit of one patient was aborted by intravenous phenytoin while the other by controlling the blood sugar.

Aged↗
PubMed1989

Identification of the major capsid protein gene of human cytomegalovirus.

The coding region for the major capsid protein (MCP) of human cytomegalovirus (HCMV) was identified by comparing the protein sequence with the respective sequences of herpes simplex virus (HSV), Epstein-Barr virus, and varicella-zoster virus. The predicted length of the HCMV MCP was 1,370 amino acids. Comparison of the MCP sequences of the different human herpesviruses showed a homology of 25% to the MCP of HSV type 1, a homology of 29% to the MCP of Epstein-Barr virus, and a homology of 23% to the MCP of varicella-zoster virus. A subfragment of the HSV type 1 KpnI i fragment encoding the MCP VP5 cross-hybridized with the HCMV HindIII U fragment containing part of the MCP gene. Northern (RNA) blot analyses with subclones out of the coding region for the HCMV MCP detected one large transcript of about 8 kilobases. A portion of the open reading frame was expressed in Escherichia coli plasmid pBD2 IC2OH as a beta-galactosidase fusion protein and was used to generate polyclonal antibodies in New Zealand White rabbits. The obtained antisera reacted in Western immunoblots with the MCP of purified HCMV virions. A monoclonal antibody against the human MCP and a monospecific rabbit antiserum against strain Colburn of simian cytomegalovirus detected the fusion protein as well as the MCP of purified virions in immunoblots.

Amino Acid Sequence↗
PubMed1979

Effects of pulsatile perfusion pressure and storage on hearts preserved for 24 hours under hypothermia, for transplantation.

Canine hearts preserved for 24 hours under hypothermic pulsatile perfusion at a systolic pressure of 25 mm Hg had better perfusion and transplantation survival results than hearts perfused at 50 or 80 mm Hg. Also, hearts perfused at a systolic pressure of 25 mm Hg did better than simple hypothermically stored hearts or fresh allografts. These findings indicate that hearts are adequately perfused for 24 hours under hypothermia for transplantation at a systolic pressure of 25 mm Hg.

Animals↗
PubMed1979

Concanavalin a: modification of kidney graft immunogenicity in a double transplant model.

This study determines the effectiveness of flush graft pretreatment with concanavalin A (Con A) in minimally immunosuppressed recipients receiving double kidney transplantation. Significant prolongation of survival was seen when both kidneys from the same unrelated donor were treated with Con A, when both kidneys from different unrelated donors were treated with Con A, or when the allogeneic kidney was treated with Con A, and the syngeneic kidney was untreated. There was no significant prolongation in kidney allograft survival when one of the two allogeneic kidneys was treated with Con A, whether they came from the same, or different donors. If only the syngeneic kidney was treated with Con A and no treatment was given to the allogeneic kidney, also no prolongation of survival was observed. Thus, this study fully agrees with previous data indicating that Con A is not acting by systemic immunosuppression, but by local changes that modify the kidney immunogenicity. The theoretical implications associated with the prolongation of graft survival after flush pretreatment with Con A are activation of suppressor T cells, enhancement, physico-chemical modifications of the cell membrane and/or others.

Animals↗
PubMed1979

Preservation of the pancreas for transplantation.

Twenty-four hour hypothermic bloodless pulsatile perfusion of the canine pancreas did not have any detrimental effect on the survival rate of minimally immunosuppressed recipients. On the contrary, there was a favorable trend toward improved long term survival of perfused and transplanted pancreas. Fresh grafts or those stored under hypothermia survived for shorter periods of time than did the perfused grafts. Apancreatic dogs survived only for a few days after pancreatectomy. Hypothermic pulsatile perfusion appeared to be a better method of preservation than hypothermic storage of pancreatic allografts.

Amylases↗
PubMed1977

Preservation of canine hearts after warm ischemia (zero to thirty minutes) and one to two days of hypothermic storage. A comparative analysis of crystalloid and colloid solutions with different osmolarity and ion composition.

The effect of extracellular crystalloid (Ringer's) and colloid (silica gel fraction [SGF]) solutions, and intracellular crystalloid (Sacks) and colloid (modified silica gel fraction [MSGF]) solutions for canine heart preservation in a 24 to 48 hour model of hypothermic storage and zero to 30 minutes of warm ischemia was compared. Canine hearts flushed with an intracellular colloid solution (MSGF) had better survival rates after transplantation than did the hearts flushed with intracellular crystalloid solutions (Sacks). Better survival results also were observed in the group of hearts flushed with extracellular colloid (SGF) solutions than extracellular crystalloid (Ringer's) solutions. The most important theoretical factor in heart preservation appears to be hyperosmolarity and elevated concentration of potassium, proteins, and glucose.

Animals↗