Mark A. Pickett

Identification of a specific pattern of vascular endothelial growth factor mRNA expression in human placenta and cultured placental fibroblasts

Reverse transcription and the polymerase chain reaction (PCR) were used to detect vascular endothelial growth factor (VEGF) mRNA in human placental tissue and cultured placental fibroblasts obtained during the first trimester of pregnancy. The primers for VEGF corresponded to areas in exon 4 and exon 8 of the VEGF gene. After one round of PCR three products, equivalent to VEGF121, VEGF165 and VEGF189, were detected within placental tissue and cultured placental fibroblasts. A further round of PCR revealed the presence of two more products equivalent to VEGF206 and VEGF145. Thus, in addition to the production of readily secreted forms of VEGF (VEGF121 and VEGF165), the placenta produces several transcripts expected to increase the growth factor pool of the extracellular matrix.

The major outer-membrane proteins of Chlamydia trachomatis serovars A and B: intra-serovar amino acid changes do not alter specificities of serovar- and C subspecies-reactive antibody-binding domains

The major outer-membrane protein (MOMP) of Chlamydia trachomatis is a promising candidate antigen for chlamydial vaccine development. We have sequenced the MOMP genes for a serovar A and a serovar B isolate and have compared these new sequences with those already reported. Intra-serovar changes in the inferred amino acid sequences of the surface-exposed variable segments known to be responsible for binding of neutralizing antibody were observed. Nevertheless, epitope mapping with solid-phase peptides showed that these intra-serovar changes did not affect the binding of serovar- and subspecies-specific, potentially protective antibodies. Variable segment 1 of C. trachomatis serovar A contained two adjacent antibody-binding sites, one of which was C-subspecies specific while the other was serovar A specific. Therefore the subspecies binding site for C-complex organisms is in variable segment 1, whilst that for B-complex organisms is in variable segment 4. This work shows that MOMP sequences are relatively stable within the serovar categorization for isolates taken decades apart from different continents. Within a given serovar, however, limited interchange of functionally related amino acids may occur without impairing the binding of serovar-specific antibody.

Reverse transcription with nested polymerase chain reaction shows expression of basic fibroblast growth factor transcripts in human granulosa and cumulus cells from in vitro fertilisation patients

Basic fibroblast growth factor (bFGF) is a potent angiogenic factor that has also been implicated in granulosa cell and oocyte maturation. We now report the expression of messenger ribonucleic acid (mRNA) encoding bFGF in human granulosa and cumulus cells obtained at oocyte recovery in in vitro fertilisation patients. It was necessary to use the sensitive technique of a nested polymerase chain reaction (PCR) after reverse transcription (RT) to detect transcripts. This finding in conjunction with a recent report showing the presence of transcripts for transforming growth factor beta (TGF beta) in the same type of cells by PCR indicates that mechanisms are in place for controlling extracellular proteolysis and cell differentiation.

High-level expression and epitope localization of the major outer membrane protein of Chlamydia trachomatis serovar L1

Fragments of the gene encoding the major outer membrane porin protein (MOMP) of Chlamydia trachomatis serovar L1 were ligated into the pUC plasmid vectors to give a series of overlapping recombinant sex pressing MOMP from the lac promoter. Induction of this promoter with IPTG leads to high‐level expression of the recombinant porin protein. Electron microscopy shows the presence of insoluble inclusions within the Escherichia coli host cells. Probing the expressed MOMP fragments with a set of monoclonal antibodies permitted localization of the four binding sites (epitopes)of primary‐sequence‐dependent monoclonal antibodies that exhibit genus‐, species‐, subspecies and type (serovar)‐specific reactivities.

Chlamydiaphage Chp2, a skeleton in the ?X174 closet: scaffolding protein and procapsid identification

Chlamydiaphage Chp2 is a member of the family Microviridae, of which bacteriophage phiX174 is the type species. Although grouped in the same family, the relationship between the Microviridae coliphages and the Chp2-like viruses, which infect obligate intracellular parasitic bacteria, is quite distant, with major differences in structural protein content and scaffolding protein dependence. To investigate the morphogenesis of Chp2, large particles were isolated from infected Chlamydophila abortus by equilibrium and rate zonal sedimentation. A monoclonal antibody that recognizes only assembled viral coat proteins was used in these detection assays. Thus, the detected particles represent virions and/or postcapsid formation assembly intermediates. Two distinct particle types were detected, differing in both protein and DNA content. Filled particles lacked VP3, the putative internal scaffolding protein, whereas empty particles contained this protein. These results indicate that VP3 is a scaffolding protein and that the isolated VP3-containing particles most likely represent Chp2 procapsids.

Intracellular Parasitism of Chlamydiae: Specific Infectivity of Chlamydiaphage Chp2 in Chlamydophila abortus

The obligate intracellular nature of chlamydiae presents challenges to the characterization of its phages, which are potential tools for a genetic transfer system. An assay for phage infectivity is described, and the infectious properties of phage Chp2 were determined.

In vivo and in vitro effects of mutagenesis of active site tyrosine residues of mercuric reductase

X‐ray crystal structure analysis of mercuric reductase suggested that the binding site for Hg2+ consisted of two tyrosine residues, Tyr264 and Tyr605, as well as two cysteine residues, Cys207 and Cys628. We have previously shown that mutagenesis of Tyr605 to Phe lowered the k cat of the enzyme 6‐fold, whereas the same mutation of Tyr264 resulted in a reduction of 160‐fold [(1993) Biochemistry 32, 7475–7478]. Tyr605 occupies the same position in mercuric reductase as the active site His residue in the related enzyme glutathione reductase. The mutation of Tyr605 of mercuric reductase to a His residue produced a 24‐fold decrease in k cat and a 15‐fold decrease in k cat and a 15‐fold decrease in K m. The in vivo resistance to Hg2+ of E. coli strains carrying wild type or mutant mer A genes correlated with the in vitro measurements of k cat/K m for mercuric reductase activity.

Comparison of 3′ and 5′ biotin labelled oligonucleotides for in situ hybridisation

Oligonucleotide probes enzymatically labelled at the 3′-end with biotin have been used successfully to detect target RNA and DNA in combination with in situ hybridisation. Addition of multiple biotin residues to the 3′-end increases the hybridisation signals, but it is not known whether the same principle is applicable to the 5′-end. We have labelled a 35-base oligonucleotide during synthesis with 1, 5 and 12 biotin molecules at the 5′-end and compared it to conventional 3′-labelling. In additional experiments the probes were labelled at both ends. Probes were applied to histological sections obtained from paraffin-embedded cell-clot-complexes that contain uninfected and Rhinoviral-infected cells, using a standard in situ hybridisation protocol with appropriate controls. Hybridisation signals were compared for intensity of cytoplasmic signal and sensitivity as number of positive cells. Both parameters increased in parallel with higher numbers of biotin residues attached to the 5′-end and 12 biotin residues were almost as effective as 3′-enzymatic tailing. The sensitivity could be increased above that of either 3′- or 5′-labelling by the addition of residues at both ends of the probe. The 5′-attachment of biotin residues can extend the value of oligonucleotide probes employed for in situ hybridisation and yield increased sensitivity when combined with 3′-enzymatic labelling.