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Biomedical subjects

D Kelly

Publications and source records attributed to D Kelly.

At least 127 records · Page 7Linked to original sources
PubMed1992

Induced reciprocal translocation in transgenic mice near sites of transgene integration.

Transgenic mice (JCP0 #18), heterozygous for an insertion of approximately 50 copies of the rat peripheral myelin (P0) protein cDNA, displayed a pattern of reduced litter size that suggested a chromosome rearrangement. Chromosome banding studies of fetal cells disclosed the presence of an apparently balanced translocation between a Chromosome (Chr) 1 and 14 with breakpoints at bands 1H3 and 14C3. In situ hybridization of biotin-labeled P0 rat cDNA probe to chromosome spreads and detection of specific signal with fluorescein isothiocyanate-conjugated avidin revealed a strong signal on the 1(14) translocation chromosome at the site of the breakpoint. A weaker signal was present near the breakpoint on the 14(1) derivative chromosome. These results suggest an etiologic relationship between the insertion of the transgene and the origin of the translocation. To further elucidate possible mechanisms, we first mapped the endogenous P0 gene (gene symbol Mpp). As previously reported (You et al., Genomics 9: 751, 1991), we found that Mpp is located on Chr 1 in the region of the translocation breakpoint in JCP0 mice. Subsequently, we have carried out pulsed-field gel and standard Southern analyses with P0 gene probes, but found no evidence for a direct involvement of the endogenous P0 gene in the process that generated the balanced reciprocal translocation. Thus, we favor the hypothesis that, during repair of DNA strand breakage--possibly induced by the microinjection procedure--the transgene copies were ligated to broken ends of Chrs 1 and 14. According to convention, this translocation is designated T(1;14)1Po. Homozygotes are phenotypically normal and breed well; they will be useful for genetic and physical mapping of Chrs 1 and 14.

Animals↗
PubMed1992

Transcriptional regulation of the murine k-FGF gene in embryonic cell lines.

Previous studies have shown that embryonal carcinoma (EC) cells express the fibroblast growth factor k-FGF; however, there is a large decrease in the expression of this gene when EC cells differentiate. In addition, it has been shown that differentiation of mouse F9 EC cells reduces the expression of a reporter gene under the control of both the putative human k-FGF promoter and an enhancer-like element that is located in the third exon of the k-FGF gene. Given the low degree of sequence similarity between the human k-FGF gene and the murine k-FGF gene upstream of the transcription start site, it was unclear whether human sequences mimic fully the regulation of the k-FGF gene in mouse cells. To address this question, we have examined the expression of gene constructs containing various regions of the murine k-FGF gene in two mouse EC cell lines and one mouse embryonic stem (ES) cell line. Our results demonstrate that the mouse 5' flanking region, like the human 5' flanking region, cannot support expression of the reporter gene. In both EC cell lines and the ES cell line, expression of the reporter gene is elevated 10- to 100-fold by the addition of a 316-bp region taken from the third exon of the murine k-FGF gene. In addition, we provide evidence that octamer binding proteins are involved in the regulation of the k-FGF gene. Last, this study has identified regions upstream of the transcription start site that appear to regulate the expression of the murine k-FGF gene in EC cells and in ES cells.

Animals↗
PubMed1992

Differential regulation of the transforming growth factor type-beta 2 gene promoter in embryonal carcinoma cells and their differentiated cells.

Previous studies have shown that EC cells do not express detectable levels of TGF-beta 2 or its mRNA until they differentiate. This suggested that differentiation influences the transcription of the TGF-beta 2 gene in this model system. To address this possibility, we have examined the activity of the TGF-beta 2 promoter in EC cells and their differentiated cells using gene constructs containing various portions of the TGF-beta 2 promoter inserted upstream of the reporter gene, chloramphenicol acetyltransferase (CAT). We determined that the level of CAT increases approximately ninefold when EC cells were induced to differentiate. Our studies also indicate that the TGF-beta 2 promoter contains at least two positive regulatory elements that are separated by a negative regulatory element. Finally, we have identified a CRE/ATF-like site that appears to be responsible for a positive regulatory element located between -77 and -40.

Base Sequence↗
PubMed1992

Thyroid-specific and hormone-dependent expression of rat thyroglobulin promoter fused with bacterial chloramphenicol acetyltransferase gene in transgenic mice.

The minimal promoter of rat thyroglobulin (TG) gene (168 bp) was fused with bacterial chloramphenicol acetyltransferase (CAT) gene, and transgenic mice carrying the TGCAT gene were produced. The minimal promoter is sufficient for thyroid-specific and hormone-dependent expression of TGCAT in transgenic mice. Deletion of a region between -128 and -92 bp (TGII), which is not required for the expression of TGCAT in transient expression assays but whose sequence is most extensively conserved among different species, appears to decrease frequency of the expression of TGCAT in transgenic mice. However, the same deletion apparently has no significant effect on TG promoter activity in stably transformed rat FRTL-5 cells.

Animals↗
PubMed1992

Morphological changes and alterations in regional intrarenal blood flow induced by graded renal ischemia.

A model of renal ischemia was used to study morphological changes and alterations in intrarenal blood flow. Renal artery blood flow was reduced from 120 to 20 ml./minute (normal 172 +/- 14) for 3 weeks. Morphological changes were assessed histologically, and by electronmicroscopy. Intrarenal blood flow was determined using microspheres. Flow rates less than 80 ml./minute resulted in a progressive loss of renal volume with arterial thrombosis and renal infarction at 20 ml./minute. Histological changes included loss of glomerular volume, tubular dilatation (60 ml./minute), tubular cast formation (50 ml./minute) tubular atrophy, interstitial fibrosis, arteriolar thickening (40 ml./minute) and glomerular hyalinization (30 ml./minute). Electronmicroscopy changes at 60 ml./minute (loss of glomerular microvasculature, unfolding of glomerular vascular tuft, appearance of blind ending vessels) progressed to disruption of glomerular architecture noted at 30 ml./minute. Narrowing of medullary blood vessels (60 ml./minute) and neovascularisation (40 ml./minute) was observed. Progressive ischemia decreased medullary, inner cortical and outer cortical blood flow (5.9 to 2.1 ml./minute/gm.) p less than 0.01, with a compensatory increase to the opposite kidney.

Animals↗
PubMed1992

Characterization and autoradiographic localization of the epidermal growth factor receptor in the jejunum of neonatal and weaned pigs.

Receptors for epidermal growth factor (EGF) were characterized on the intestinal membranes of newborn, sucking and weaned pigs. 125I-labelled EGF (125I-EGF) binding to membrane homogenates was time-dependent, saturable, linearly correlated to membrane protein and reversible. Analysis of saturation curve data revealed a single class of 125I-EGF binding sites in both newborn and weaned pigs. Receptor levels tended to be higher in weaned than in newborn pigs; the converse was true for the receptor affinity. In contrast, virtually no binding sites were found on the intestinal membranes of sucking pigs. Autoradiography in vitro of jejunal sections of newborn and weaned pigs demonstrated 125I-EGF receptors on both microvillar and basolateral surfaces of enterocytes, suggesting that luminal EGF could influence developmental processes in the intestine either directly or indirectly following transcytosis of the ligand.

Animals↗
PubMed1992

Molecular genetic study of the frequency of monosomy 22q11 in DiGeorge syndrome.

It is well established that DiGeorge syndrome (DGS) may be associated with monosomy of 22q11-pter. More recently, DNA probes have been used to detect hemizygosity for this region in patients with no visible karyotypic abnormality. However, DGS has also been described in cases where the cytogenetic abnormality does not involve 22q11; for instance, four cases of 10p- have been reported. In this study we have prospectively analyzed patients, by using DNA markers from 22q11, to assess the frequency of 22q11 rearrangements in DGS. Twenty-one of 22 cases had demonstrable hemizygosity for 22q11. Cytogenetic analysis had identified interstitial deletion in 6 of 16 cases tested; in 6 other cases no karyotype was available. When these results are combined with those from our previous studies, 33 of 35 DGS patients had chromosome 22q11 deletions detectable by DNA probes.

Chromosome Deletion↗
PubMed1992

High-grade squamous intraepithelial lesions and invasive carcinoma following the report of three negative Papanicolaou smears: screening failures or rapid progression?

The cytologic smears and histopathologic specimens of 18 patients developing cervical intraepithelial neoplasia 3 (CIN3) and two developing invasive squamous carcinoma following the report of at least three negative Papanicolaou tests were studied. A median number of 9.5 smears per patient procured over a median interval of 93.5 mo were reviewed. Twenty-eight (22.7%) of 123 reportedly negative smears revealed a squamous intraepithelial lesion (SIL), 17 (13.8%) were unsatisfactory, 14 (11.4%) lacked an endocervical component, and 37 (30.1%) were classified as atypical squamous cells of undetermined significance (ASQUS) on reexamination. Fourteen (50%) of 28 smears originally misclassified as negative contained fewer than 100 SIL cells and five (17.8%) were severely inflamed. One patient whose smears were misclassified as negative had an atrophic cervix, one had SIL cells primarily in thick sheets, and two had small CIN3 cells resembling squamous metaplasia. Six patients (30%) had a single false negative smear, seven (35%) had multiple false negative smears, seven (35%) had two or more unsatisfactory smears reported as negative, seven had at least two smears lacking an endocervical component, and six had at least two smears taken during pregnancy. Thirteen patients had abnormal smears classified as ASQUS or high-grade SIL (HSIL) but never had a specimen showing only a low-grade SIL (LSIL). This study demonstrates that early signs of SIL may be difficult to recognize cytologically and that poor quality specimens and inadequate sampling may contribute to false negative diagnoses.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Thyrotoxic periodic paralysis in a Jamaican male patient.

A case of thyrotoxic periodic paralysis occurring in a Black Jamaican male patient is described. Diagnosis is based on history and confirmed by evaluation of serum electrolyte during attacks and thyroid function studies. The pathophysiology, associations, therapy and prognosis are discussed. It is important that clinicians recognise the condition as all forms of periodic paralysis are amenable to treatment, and progressive weakness can be prevented or even reversed.

Adult↗
PubMed1991

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene, and expression of inactive mutant enzyme protein in E. coli.

A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G985. The same assay consistently revealed A985 in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G985 mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.

Acyl-CoA Dehydrogenases↗
PubMed1991

Investigation of paediatric liver disease.

The investigation of children with liver disease falls into two categories: the investigation of the cholestatic baby and the investigation of the older child (over 2 years) with hepatomegaly. The approach to investigation is directed by the clinical features and employs many different investigational methods including biochemistry, haematology, radiology, electrophysiology and histology. As the clinical presentation of many diseases is similar, it is appropriate to perform a variety of first-line tests, proceeding to more complex investigations only as indicated.

Child↗
PubMed1991

Ontogenic expression of histo-blood group antigens in the intestines of suckling pigs: lectin histochemical and immunohistochemical analysis.

The expression of intestinal histo-blood group AO and related antigens was investigated in piglets during an 8 week suckling period. Lectin histochemical and immunohistochemical analyses were undertaken on sections of resin-embedded intestinal tissue and semi-quantitative scoring systems were adopted for categories of lectins and monoclonal antibodies reactive with carbohydrate moieties present in core, backbone and terminal oligosaccharide sequences of histo-blood group antigens. Distinct age-related changes were observed in the terminal glycosylation of both secretory and membrane glycoconjugates. Histo-blood group A antigen was identified in intestinal mucin 5 weeks after birth and the precursor H antigen was found in goblet cells at week 1. H antigen was undetectable on intestinal membranes during the first 3 weeks of suckling but a conspicuous and sustained level of this form of fucosylation was apparent during the latter half of the suckling period. More complex membrane glycosylation involving further fucosylation and/or the expression of A antigen, was evident in the latter part of the suckling period. These temporal changes in membrane and secretory glycosylation may be physiologically important during intestinal adaptation and development in young pigs.

Age Factors↗
PubMed1991

The influence of lactation products on the temporal expression of histo-blood group antigens in the intestines of suckling pigs: lectin histochemical and immunohistochemical analysis.

Lectins and carbohydrate-specific monoclonal antibodies were used as cytochemical probes to investigate the possible influence of lactation products on the expression of intestinal membrane and secretory glycoconjugates in suckling piglets. Two different lactational regimes were compared; the first involved normal rearing of piglets for 8 weeks on a single dam and the second involved repeated cross-fostering of littermates onto recently farrowed sows, thereby restricting them to early milk. Five histo-blood group phenotypes were recognized within the piglet population: two immature phenotypes, 'O immature' or 'Oi' and 'A immature' or 'Ai', and three mature forms, 'O', 'A' and '-'. Under the normal suckling regime the transition from immature to mature OA phenotypes was evident at the fifth week post partum. However, in the repeatedly cross-fostered piglets this transition was evident much earlier at 3 weeks post partum. It is suggested that qualitative or quantitative variations in milk composition during the sow's lactation may significantly influence the expression of intestinal histo-blood group antigens in her suckling young.

Age Factors↗
PubMed1991

Polyamide profiles of porcine milk and of intestinal tissue of pigs during suckling.

Previous studies have suggested that luminal polyamines can directly influence intestinal differentiation of neonatal rats. The present investigation has demonstrated the presence of high levels of polyamines in porcine milk and in the intestinal tissues of suckling pigs. The quantities of polyamines in sow's milk sampled between wk 1 and 8 of lactation were determined using high performance liquid chromatography (HPLC). The concentration of milk spermidine (SPD) remained constant over the first 3 to 4 wk of lactation but increased 4-fold between wk 4 and 7. Neither putrescine nor spermine (SPN) were detected in any of the milk samples. During intestinal development the mucosal SPD/SPN ratio was elevated between wk 1 and 3 and wk 5 and 7. The latter period of increase corresponded with the surge in milk SPD concentration. It is suggested that milk SPD is taken up from the intestinal lumen and is involved in potentiating intestinal differentiation during the latter part of the suckling period.

Animals↗
PubMed1991

Digestive development of the early-weaned pig. 1. Effect of continuous nutrient supply on the development of the digestive tract and on changes in digestive enzyme activity during the first week post-weaning.

Gastric intubation was adopted to examine the effect of continuous nutrient supply on digestive development of the pig during the immediate post-weaning period. The 14 d-weaned animals were slaughtered at 3, 5 and 7 d post-weaning (3W, 5W and 7W respectively) and the suckled animals were slaughtered at 14 and 22 d of age (14SR and 22SR respectively). The weight of the pancreas (g/kg bodyweight) was significantly greater (P less than 0.05) in the 5W and 7W groups, as was the weight of large intestine (g/kg) in all weaned groups (P less than 0.01) compared with sow-reared pigs. The stomach weight (g/kg) tended to be greater in the weaned groups. Weaning, in conjunction with a continuous nutrient supply, did not significantly alter the time-related changes in the weight of the small intestine (SI) or the SI mucosa, although both variables tended to be lowest in the 3W group. However, there was a 20% reduction in the protein content of the mucosa within the first 3 d post-weaning (P less than 0.01) which persisted during the 7 d experimental period. Lactase, (beta-galactosidase; EC 3.2.1.23) activity (mumol/g protein and mol/d) of the 7W group was reduced to approximately 40% of the 22SR value. Hence, continuous nutrient supply may have delayed, but did not prevent, the loss of lactase activity at weaning. The activity of sucrase (sucrose-alpha-glucosidase; EC 3.2.1.48) was significantly higher in 22SR compared with 14SR animals. Sucrase activity in weaned pigs was intermediate to the values for sow-reared pigs whereas maltase (alpha-glucosidase; EC 3.2.1.20) and glucoamylase (glucan 1,4-alpha-glucosidase; EC 3.2.1.3) were significantly increased in relation to their sow-reared counterparts. Continuous nutrient supply did not prevent the reduction in villous height and the crypt hypertrophy associated with weaning. The results of the present study suggest that there may be some degree of interaction between nutrient intake and gut development during the immediate post-weaning period but that there is also a component of the adaptive response which is independent of nutrient intake. They confirm the rapid substrate induction of the brush-border glucoamylases and indicate the importance of considering total as well as specific enzyme activity for satisfactory interpretation of changes in digestive function.

Animal Nutritional Physiological Phenomena↗
PubMed1991

Digestive development of the early-weaned pig. 2. Effect of level of food intake on digestive enzyme activity during the immediate post-weaning period.

Gastric intubation was adopted as a means of comparing the effect of two feeding levels, continuous nutrient supply (C) and restricted nutrient supply (R), on the digestive development of pigs weaned at 14 d of age, during the first 5 d post-weaning. The absolute weights of the stomach and the pancreas were significantly greater (P less than 0.001) in C compared with R pigs. The effect was not significant for pancreas weight when expressed per kg body-weight but was significant (P less than 0.05) for stomach weight. The weights of the small intestine (SI), SI mucosa and total mucosal protein were significantly higher (P less than 0.001) in C pigs but protein content per g mucosa was similar in the C and R groups. There was no significant effect of treatment on the activity of lactase (beta-glucosidase; EC 3.2.1.23) or sucrase (sucrose-alpha-glucosidase; EC 3.2.1.48) irrespective of the basis of comparison used. The specific activity (mumol/min per g protein) of maltase (alpha-glucosidase; EC 3.2.1.20) and of glucoamylase (glucan-1,4-alpha-glucosidase; EC 3.2.1.3) were similar in C and R groups but activities of maltase (mumol/g mucosa) (P less than 0.05), and maltase and glucoamylase (mol/d) (P less than 0.01) were significantly higher in C pigs. Villous height and crypt depth were significantly greater in C pigs (P less than 0.001 and P less than 0.05 respectively). Enteroglucagon was significantly (P less than 0.05) higher in C compared with R pigs. Xylose absorption and the digestibility of energy were not affected by treatment. Digestibility of dry matter, organic matter, crude protein (nitrogen x 6.25) and carbohydrate were significantly higher (P less than 0.001, P less than 0.01, P less than 0.05 and P less than 0.001 respectively) in R pigs compared with C pigs but the differences were small, ranging from 1.3 to 2.5%. These results demonstrate that (1) nutrient intake in the weaned pig affects the anatomy, morphology and function of the gut, (2) there is considerable 'spare capacity' for digestion of cereal-based diets even in pigs weaned at 14 d of age, (3) measurements in vitro of digestive function are of limited value unless supported by information in vivo on absorption/digestibility.

Animal Nutritional Physiological Phenomena↗