Murine Brca1: sequence and significance for human missense mutations

We have cloned and sequenced a mouse homologue of the humanbreast and ovarian cancer susceptibility gene, BRCA1. The predictedmouse Brca1 protein is composed of 1812 amino acids. The murineprotein is 60% identical and 72% similar to the human BRCA1protein. Two regions of high homology have been identified betweenthe two proteins. First is the Cys3—His—Cys4 typezinc-finger domain that is identical between the two proteins.The second region is defined by 115 amino acids near the carboxyend of the Brca 1 protein that is 83% identical to human BRCA1sequence. Seven of eight amino acids involved in human missensemutations that are associated with the disease were found tobe conserved between the two species. In contrast, most of theamino acids that are involved in polymorphic variations werenot conserved. We therefore propose that the interspecies conservationof predicted amino acid sequences can be used as an additionalcriterion to determine the significance of human missense mutations.     

Calmodulin permanently associates with rat olfactory CNG channels under native conditions

An important mechanism by which vertebrate olfactory sensory neurons rapidly adapt to odorants is feedback modulation of the Ca(2+)-permeable cyclic nucleotide-gated (CNG) transduction channels. Extensive heterologous studies of homomeric CNGA2 channels have led to a molecular model of channel modulation based on the binding of calcium-calmodulin to a site on the cytoplasmic amino terminus of CNGA2. Native rat olfactory CNG channels, however, are heteromeric complexes of three homologous but distinct subunits. Notably, in heteromeric channels, we found no role for CNGA2 in feedback modulation. Instead, an IQ-type calmodulin-binding site on CNGB1b and a similar but previously unidentified site on CNGA4 are necessary and sufficient. These sites seem to confer binding of Ca(2+)-free calmodulin (apocalmodulin), which is then poised to trigger inhibition of native channels in the presence of Ca(2+).     

Conservation of receptor antagonist anti-tumor activity by epidermal growth factor receptor antibody expressed in transgenic corn seed

Recombinant protein production in plants such as corn is a promising means to generate high product yields at low comparable production cost. The anti-EGFR monoclonal antibody C225, cetuximab, is a well-characterized receptor antagonist antibody recently approved for the treatment of refractory colorectal cancer. We initiated a study to test and compare the functional activity of glycosylated and aglycosylated C225 produced in stable transgenic corn seed. Both corn antibodies were shown to be functionally indistinguishable from mammalian-derived C225 in demonstrating high-affinity binding to the EGF receptor, blocking of ligand-dependent signaling, and inhibiting cell proliferation. In addition, consistent with cetuximab, both corn antibodies possessed strong anti-tumor activity in vivo. Acute dose primate pharmacokinetic studies, however, revealed a marked increase in clearance for the glycosylated corn antibody, while the aglycosylated antibody possessed in vivo kinetics similar to cetuximab. This experimentation established that corn-derived receptor blocking monoclonal antibodies possess comparable efficacy to mammalian cell culture-derived antibody, and offer a cost effective alternative to large-scale mammalian cell culture production.     

Enhanced Toxicity for Mice of Combinations of Antibiotics with Escherichia coli Cells or Salmonella typhosa Endotoxin

Enhanced lethality for BALB/c mice has been observed after the administration of Salmonella typhosa endotoxin with either actinomycin D, cycloheximide, or nogalamycin. The dose of actinomycin D required to kill half of the mice (LD₅₀) was 0.8 mg/kg in normal animals, 0.35 mg/kg in mice administered 0.08 mg of endotoxin per kg, and 0.28 mg/kg in mice administered 0.2 mg of endotoxin per kg. The LD₅₀ of endotoxin in normal mice was 12 mg/kg and in mice given 0.4 mg of actinomycin D per kg was 0.067 mg/kg. The LD₅₀ of actinomycin D in mice administered 1.8 × 10⁸ live Escherichia coli cells per kg or 1.8 × 10⁹ heat-killed E. coli cells per kg was reduced to 0.4 mg/kg. The LD₅₀ of cycloheximide was 181 mg/kg in normal animals and 28 mg/kg in mice administered 4 mg of endotoxin per kg. The LD₅₀ of endotoxin in mice given 120 mg of cycloheximide per kg was 0.02 mg/kg. Enhanced lethality due to various combinations of cycloheximide and endotoxin was abolished by pretreatment of mice with endotoxin. The LD₅₀ of nogalamycin was 21 mg/kg in normal mice and 13 mg/kg in mice receiving 1 mg of endotoxin per kg.     

SpecialEscherichia coli serotypes among enterotoxigenic strains from diarrhoea in adults and children

106 enterotoxigenicEscherichia coli strains from children and adults from many parts of the world were serotyped for O and H antigens. Some OH types,i.e. O6H16, O8H9, O15H11, O25H42, O78H11 and O78H12, were found repeatedly from different geographical locations. Some of these OH serotypes were only found rarely among more than 20000E. coli strains collected over many years from different locations and sources. It is suggested that these special OH serotypes represent clones which have been selected to the special conditions in the small intestine and selected to carry the plasmids necessary to provoke diarrhoea.     

The relationship of age and cardiovascular fitness to cognitive and motor processes

Older and younger aerobically trained and sedentary adults participated in an S1-S2-S3 paradigm designed to elicit event-related potential (ERP) and behavioral responses to determine the influence of cardiovascular fitness on cognitive and motor processes. The paradigm provided warning (S1) as to the difficulty level of an upcoming decision task (S2). Participants had to decide the taller of two bars on presentation of S2 but hold their response until S3, to which they indicated their choice motorically. Results revealed age-related differences for ERP measures as older participants showed increased amplitude of the stimulus preceding negativity (SPN) prior to S2, and longer latencies and equipotentiality of P3 in response to S2. Fitness effects were also observed for the contingent negative variation (CNV) with decreased amplitude for fit relative to sedentary individuals. Age interacted with fitness for P3 latency to S2 as older sedentary individuals showed the longest latency followed by older fit and both younger groups. No significant group differences were observed for reaction time (RT) to S3. Therefore, physical fitness is associated with attenuation of cognitive decline in older individuals and greater economy of motor preparation for both young and older participants.     

Genetic linkage analysis identifies new proximal and distal flanking markers for the X-linked agammaglobulinemia gene locus, refining its localization in Xq22

Genetic linkage analysis has been instrumental in mapping thegene for X-linked agammaglobulinemia (XLA) to the proximal longarm of the human X chromosome, to Xq22. Due to the relativerarity of this disease the localization of the gene within Xq22has remained imprecise. We have investigated twenty-nine familiesaffected by XLA and have found no recombinants with the DXS178locus in over 30 informative meioses. DXS178 is now the mostreliable and informative locus for use in pre-natal diagnosisand carrier detection of XLA. In addition, we have identifiednew closely linked proximal and distal flanking markers forXLA, DXS442 and DXS101, respectively. These loci are separatedby 2cM, considerably reducing the extent of DNA within whichthe XLA locus can be contained. This will open up the way formore directed positional cloning efforts for the isolation ofthe XLA gene.     

Instability in the faecal flora of a patient suffering from food-related irritable bowel syndrome

The faecal microbial flora of a patient with severe irritable bowel syndrome related to multiple food intolerances was very variable and contained a high proportion of facultative bacteria and an unusual incidence of Clostridium species.     

Vaccinia virus K1L protein mediates host-range function in RK-13 cells via ankyrin repeat and may interact with a cellular GTPase-activating protein

The K1L protein of vaccinia virus is required for its growth in certain cell lines (RK-13 and human). The cowpox host-range protein CP77 has been shown to complement K1L function in RK-13 cells, despite a lack of homology between the two proteins except for ankyrin repeats. We investigated the role of ankyrin repeats of K1L protein in RK-13 cells. The growth of a recombinant vaccinia virus, with K1L gene mutated in the most conserved ankyrin repeat, was severely impaired. Infection with the mutant virus caused shutdown of cellular and viral protein synthesis early in infection. We also investigated the interaction of K1L protein with cellular proteins and found that K1L interacts with the rabbit homologue of human ACAP2, a GTPase-activating protein with ankyrin repeats. Our result suggests the importance of ankyrin repeat for host-range function of K1L in RK-13 cells and identifies ACAP2 as a cellular protein, which may be interacting with K1L.