Kinetic PCR analysis: real-time monitoring of DNA amplification reactions

We describe a simple, quantitative assay for any amplifiable DNA sequence that uses a video camera to monitor multiple polymerase chain reactions (PCRs) simultaneously over the course of thermocycling. The video camera detects the accumulation of double-stranded DNA (dsDNA) in each PCR using the increase in the fluorescence of ethidium bromide (EtBr) that results from its binding duplex DNA. The kinetics of fluorescence accumulation during thermocycling are directly related to the starting number of DNA copies. The fewer cycles necessary to produce a detectable fluorescence, the greater the number of target sequences. Results obtained with this approach indicate that a kinetic approach to PCR analysis can quantitate DNA sensitively, selectively and over a large dynamic range. This approach also provides a means of determining the effect of different reaction conditions on the efficacy of the amplification and so can provide insight into fundamental PCR processes.     

A Drosophila homolog of bovine smg p25a GDP dissociation inhibitor undergoes a shift in isoelectric point in the developmental mutant quartet

The Drosophila developmental mutation quartet causes late larval lethality and small imaginal discs and, when expressed in the adult female, has a lethal effect on early embryogenesis. These developmental defects are associated with mitotic defects, which include a low mitotic index in larval brains and incomplete separation of chromosomes in mitosis in the early embryo. quartet mutations also have a biochemical effect, i.e., a basic shift in isoelectric point in three proteins. We have purified one of these proteins, raised an antibody to it, and isolated and sequenced its cDNA. At the amino acid level, the sequence shows 68% identity and 81% similarity to bovine smg p25a GDP dissociation inhibitor (GDI), a regulator of ras-like small GTPases of the rab/SEC4/YPT1 subfamily. The correlation between a basic shift in isoelectric point in Drosophila GDI in quartet mutant tissue and the quartet developmental phenotype raises the possibility that a posttranslational modification of GDI is necessary for its function and that GDI function is essential for development.     

Molecular cloning of a novel melanocortin receptor

Using the technique of the polymerase chain reaction primed with oligonucleotides based on the homologous transmembrane regions of seven transmembrane G protein-linked receptors, we isolated three full-length human genes that encode a novel subgroup of this receptor family. Recently, two of these receptors were identified as specific for alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropic hormone. We report the molecular cloning and pharmacologic characterization of a third member of this subgroup. The gene for this receptor encodes a protein of 361 amino acids in length. Its pharmacology characterizes it as an MSH receptor specific to the heptapeptide core common to adrenocorticotropic hormone and alpha-, beta-, and gamma-MSH. By Northern blot hybridization and polymerase chain reaction, it is expressed in brain, placental, and gut tissues but not in melanoma cells or in the adrenal gland. These findings may yield insight into the physiology of peptides derived from pro-opiomelanocortin post-translational processing.     

The prevalence of campylobacters and arcobacters in broiler chickens

Chicken carcasses from a supermarket and from a poultry abattoir were examined using methods designed to isolate as many strains of campylobacters and related organisms as possible. Strains of arcobacter, but no campylobacters, were isolated from every carcass after enrichment. Campylobacter jejuni subsp. jejuni was isolated from all carcasses examined by direct plating and other Campylobacter-like strains were isolated from nine out of 15 abattoir carcasses by direct plating but not after enrichment. Only the Camp. jejuni subsp. jejuni strains could be identified to species level using a readily available identification scheme and/or a commercial identification kit. Examination of caecal contents from the 15 abattoir poultry yielded Camp. jejuni subsp. jejuni and Campylobacter-like strains from 15 and eight by direct plating, and from six and nine after enrichment, respectively. Four sites in the intestine of the abattoir birds (60 samples) were examined for arcobacters and only one strain was isolated. This indicates that arcobacters are probably not normal inhabitants of the poultry intestine. Poultry is a rich source of other campylobacteria besides the thermophilic Campylobacter spp.     

Abdominal wall hematoma]

We describe three case reports of hematomas in the abdominal wall muscles and discuss etiology, diagnostic findings and treatment. Abdominal wall hematomas are uncommon, and the condition is often misdiagnosed. One of our patients was laparotomised owing to suspected appendicitis, and one patient presented a tuberculous abscess that may have been an infected hematoma. Ultrasonographic examination or CT gives the correct diagnose. Conservative management is the treatment of choice unless bleeding is severe or the course is complicated by infection.