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1.  Small intestine in lymphocytic and collagenous colitis: mucosal morphology, permeability, and secretory immunity to gliadin. 
Journal of Clinical Pathology  1997;50(6):527-529.
There is a recognised association between the "microscopic" forms of colitis and coeliac disease. There are a variety of subtle small intestinal changes in patients with "latent" gluten sensitivity, namely high intraepithelial lymphocyte (IEL) counts, abnormal mucosal permeability, and high levels of secretory IgA and IgM antibody to gliadin. These changes have hitherto not been investigated in microscopic colitis. Nine patients (four collagenous, five lymphocytic colitis) with normal villous architecture were studied. Small intestinal biopsies were obtained by Crosby capsule; small intestinal fluid was aspirated via the capsule. IEL counts were expressed per 100 epithelial cells, and intestinal IgA and IgM antigliadin antibody levels were measured by ELISA. Small intestinal permeability was measured by the lactulose:mannitol differential sugar permeability test. IEL counts were normal in all cases, median 17, range 7-30. Intestinal antigliadin antibodies were measured in six cases and were significantly elevated in two patients (both IgA and IgM). Intestinal permeability was measured in eight cases and was abnormal in two and borderline in one. These abnormalities did not overlap: four of nine patients had evidence of abnormal small intestinal function. Subclinical small intestinal disease is common in the two main forms of microscopic colitis.
PMCID: PMC500002  PMID: 9378824
2.  Role of epidermal growth factor and transforming growth factor α in the developing stomach 
AIMS—To determine whether epidermal growth factor (EGF) or the related transforming growth factor α (TGFα) may have a role in the developing human stomach; to substantiate the presence of EGF in human liquor in the non-stressed infant and whether EGF in amniotic fluid is maternally or fetally derived.
METHODS—The temporal expression and localisation of EGF, TGFα, and their receptors during fetal and neonatal life were examined in 20 fetal and five infant stomachs. Simultaneously, samples of amniotic fluid and fetal urine from 10 newborn infants were collected and assayed for EGF by radioimmunoassay.
RESULTS—EGF immunoreactivity was not noted in any of the specimens examined. In contrast, TGFα immunoreactivity was shown in mucous cells from 18 weeks of gestation onwards. EGF receptor immunoreactivity was seen on superficial mucous cells in gastric mucosa from 18 weeks of gestation onwards. The median concentration of EGF was 30 and 8.5 pg/ml in amniotic fluid and fetal urine, respectively, suggesting that EGF is not produced by the fetus.
CONCLUSIONS—This study adds weight to the hypothesis that swallowed EGF, probably produced by the amniotic membranes, and locally produced TGFα, may have a role in the growth and maturation of the human stomach.

 Keywords: epidermal growth factor; transforming growth factor α; EGF receptors; stomach
PMCID: PMC1720655  PMID: 9175944
3.  Use of methyl methacrylate resin for embedding bone marrow trephine biopsy specimens. 
Journal of Clinical Pathology  1997;50(1):45-49.
AIMS: To evaluate the use of methyl methacrylate resin as an embedding medium for undecalcified bone marrow trephine biopsy specimens. METHODS: About 2500 undecalcified bone marrow trephine biopsy specimens were processed, and embedded in methyl methacrylate resin. Semithin sections (2-3 microns) were stained by routine tinctorial and immunocytochemical staining methods with a wide range of antibodies using a standard streptavidin biotin horseradish peroxidase technique. Different antigen retrieval pretreatments were evaluated. RESULTS: Bone marrow trephine biopsy specimens are embedded routinely in methyl methacrylate at the Haematological Malignancy Diagnostic Service at The Leeds General Infirmary. Over 50 different primary antibodies are in current use; for the majority of these, microwave antigen retrieval or trypsin digestion, or both, is either essential or greatly enhances the results. CONCLUSIONS: Embedding bone marrow trephine biopsy specimens in methyl methacrylate resin retains morphology and permits reliable, high quality immunocytochemistry. This is particularly desirable for the demonstration of neoplastic cells in regenerative marrow after chemotherapy, and in the detection of residual disease after treatment. The use of methyl methacrylate for routine use on bone marrow trephine biopsy specimens is advocated.
PMCID: PMC499712  PMID: 9059356
5.  Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates. 
PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five polymorphisms differed by the presence of two to six copies of the 12-bp tandem repeat 5'-CAATATCAACAA-3'. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats are generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations.
PMCID: PMC168435  PMID: 9097438
6.  Molecular and biochemical characterization of xrs mutants defective in Ku80. 
Molecular and Cellular Biology  1997;17(3):1264-1273.
The gene product defective in radiosensitive CHO mutants belonging to ionizing radiation complementation group 5, which includes the extensively studied xrs mutants, has recently been identified as Ku80, a subunit of the Ku protein and a component of DNA-dependent protein kinase (DNA-PK). Several group 5 mutants, including xrs-5 and -6, lack double-stranded DNA end-binding and DNA-PK activities. In this study, we examined additional xrs mutants at the molecular and biochemical levels. All mutants examined have low or undetectable levels of Ku70 and Ku80 protein, end-binding, and DNA-PK activities. Only one mutant, xrs-6, has Ku80 transcript levels detectable by Northern hybridization, but Ku80 mRNA was detectable by reverse transcription-PCR in most other mutants. Two mutants, xrs-4 and -6, have altered Ku80 transcripts resulting from mutational changes in the genomic Ku80 sequence affecting RNA splicing, indicating that the defects in these mutants lie in the Ku80 gene rather than a gene controlling its expression. Neither of these two mutants has detectable wild-type Ku80 transcript. Since the mutation in both xrs-4 and xrs-6 cells results in severely truncated Ku80 protein, both are likely candidates to be null mutants. Azacytidine-induced revertants of xrs-4 and -6 carried both wild-type and mutant transcripts. The results with these revertants strongly support our model proposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5) silenced by hypermethylation. Site-directed mutagenesis studies indicate that previously proposed ATP-binding and phosphorylation sites are not required for Ku80 activity, whereas N-terminal deletions of more than the first seven amino acids result in severe loss of activities.
PMCID: PMC231851  PMID: 9032253
7.  Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers. 
Journal of Bacteriology  1997;179(3):818-824.
Bacillus anthracis causes anthrax and represents one of the most molecularly monomorphic bacteria known. We have used AFLP (amplified fragment length polymorphism) DNA markers to analyze 78 B. anthracis isolates and six related Bacillus species for molecular variation. AFLP markers are extremely sensitive to even small sequence variation, using PCR and high-resolution electrophoresis to examine restriction fragments. Using this approach, we examined ca. 6.3% of the Bacillus genome for length mutations and ca. 0.36% for point mutations. Extensive variation was observed among taxa, and both cladistic and phenetic analyses were used to construct a phylogeny of B. anthracis and its closest relatives. This genome-wide analysis of 357 AFLP characters (polymorphic fragments) indicates that B. cereus and B. thuringiensis are the closest taxa to B. anthracis, with B. mycoides slightly more distant. B. subtilis, B. polymyxa, and B. stearothermophilus shared few AFLP markers with B. anthracis and were used as outgroups to root the analysis. In contrast to the variation among taxa, only rare AFLP marker variation was observed within B. anthracis, which may be the most genetically uniform bacterial species known. However, AFLP markers did establish the presence or absence of the pXO1 and pXO2 plasmids and detected 31 polymorphic chromosomal regions among the 79 B. anthracis isolates. Cluster analysis identified two very distinct genetic lineages among the B. anthracis isolates. The level of variation and its geographic distribution are consistent with a historically recent African origin for this pathogenic organism. Based on AFLP marker similarity, the ongoing anthrax epidemic in Canada and the northern United States is due to a single strain introduction that has remained stable over at least 30 years and a 1,000-mile distribution.
PMCID: PMC178765  PMID: 9006038
8.  Alpha-adaptin, a marker for endocytosis, is expressed in complex patterns during Drosophila development. 
Molecular Biology of the Cell  1997;8(8):1391-1403.
A Drosophila cDNA encoding a structural homologue of the mammalian coated vesicle component alpha-adaptin (AP2 adaptor complex) has been cloned and sequenced. The mammalian and invertebrate sequences are highly conserved, especially within the amino terminal region, a domain that mediates interactions with other components within the AP2 complex and with specific receptors tails. Mammalian alpha-adaptins are encoded by two genes; however, Drosophila alpha-adaptin has a single gene locus, within polytene bands 21C2-C3 on the left arm of the chromosome 2, closely adjacent to the paired homeobox gene aristaless. There seem to be at least two Drosophila alpha-adaptin transcripts expressed, plausibly by alternative splicing. One of the transcripts is more abundant during early embryogenesis and may be of maternal origin. We have studied the distribution of the alpha-adaptin protein throughout embryogenesis and at the neuromuscular junction of the third instar larva. During cellularization of the blastoderm embryo, the protein is seen between and ahead of the elongating nuclei, and then redistributes to the cell surface during gastrulation. These observations suggest a role for endocytosis in cellularization and are consistent with the finding that dynamin (the shibire gene product), another component of the endocytic mechanism, is required for cellularization. At later stages of embryogenesis, alpha-adaptin is expressed in complex and dynamic patterns. It is strongly induced in elements of the central and peripheral nervous system (e.g., in neuroblasts, the presumptive stomatogastric nervous system, and the lateral chordotonal sense organs), in the Garland cells, the adult midgut precursors, the antenno-maxillary complex, the endoderm, the fat bodies, and the visceral mesoderm. In the larva, alpha-adaptin is localized at the plasma membrane in the synaptic boutons of the neuromuscular junctions. The cells expressing high levels of alpha-adaptin are known or expected to support high levels of endocytosis; thus, this coated vesicle protein seems to be an excellent marker for endocytic activity. The expression patterns of dynamin, detected in the embryo by in situ hybridization methods, are very similar to those reported here for alpha-adaptin reflecting the likely coordinated expression of endocytic components. Taken together with previous evidence, our results suggest that endosomal vesicle trafficking, membrane recycling, and the regulation of endocytosis play critical roles in the wide range of developmental processes.
PMCID: PMC276164  PMID: 9285813

Results 1-8 (8)