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Biomedical subjects

M R Brown

Publications and source records attributed to M R Brown.

At least 19 recordsLinked to original sources
PubMed1998

Identification of a steroidogenic neurohormone in female mosquitoes.

In the female mosquito, Aedes aegypti, neurohormones are released from the brain in response to a blood meal and stimulate the ovaries to secrete ecdysteroid hormones, which modulate yolk protein synthesis in the fat body. Neuropeptides with this bioactivity were isolated from head extracts, and partial sequences from these peptides when aligned gave a 31-residue sequence at the amino terminus. Oligonucleotide primers for this sequence were used to amplify with the polymerase chain reaction a genomic DNA product that hybridized to a clone from a head cDNA library. The cDNA encodes a 149-residue preprohormone that is processed into an 86-residue peptide, as indicated by the mass value obtained from the native peptide, with the expected amino-terminal sequence. After modification, the cDNA for the putative neurohormone was expressed in a bacterial system, and the purified peptide had high specific activity in bioassays, thus confirming that it is a steroidogenic gonadotropin, the first to be identified for invertebrates.

Aedes↗
PubMed1998

Mutations in PROP1 cause familial combined pituitary hormone deficiency.

Combined pituitary hormone deficiency (CPHD) in man denotes impaired production of growth hormone (GH) and one or more of the other five anterior pituitary hormones. Mutations of the pituitary transcription factor gene POU1F1 (the human homologue of mouse Pit1) are responsible for deficiencies of GH, prolactin and thyroid stimulating hormone (TSH) in Snell and Jackson dwarf mice and in man, while the production of adrenocorticotrophic hormone (ACTH), luteinizing hormone (LH) and follicle stimulating hormone (FSH) is preserved. The Ames dwarf (df) mouse displays a similar phenotype, and appears to be epistatic to Snell and Jackson dwarfism. We have recently positionally cloned the putative Ames dwarf gene Prop1, which encodes a paired-like homeodomain protein that is expressed specifically in embryonic pituitary and is necessary for Pit1 expression. In this report, we have identified four CPHD families with homozygosity or compound heterozygosity for inactivating mutations of PROP1. These mutations in the human PROP1 gene result in a gene product with reduced DNA-binding and transcriptional activation ability in comparison to the product of the murine df mutation. In contrast to individuals with POU1F1 mutations, those with PROP1 mutations cannot produce LH and FSH at a sufficient level and do not enter puberty spontaneously. Our results identify a major cause of CPHD in humans and suggest a direct or indirect role for PROP1 in the ontogenesis of pituitary gonadotropes, as well as somatotropes, lactotropes and caudomedial thyrotropes.

Adolescent↗
PubMed1998

Generation of a reproducible nutrient-depleted biofilm of Escherichia coli and Burkholderia cepacia.

An in vitro method of growing bacteria as a defined nutrient-depleted biofilm is proposed. The medium was defined nutritionally in terms of the quantitative composition and by the total amount of nutrient required to achieve a defined population size. Escherichia coli and Burkholderia cepacia were incubated on a filter support placed on a defined volume of solid medium. The change of biomass of the biofilm population was compared with the change in a planktonic culture. The size of the population in stationary phase was proportional to the concentration of limiting substrate up to 40 mumol cm-1 glucose for E. coli and up to 2.7 x 10(-9) mol cm-2 iron for B. cepacia. Escherichia coli growing exponentially had a growth rate of mu = 0.30 h-1 in a biofilm and mu = 0.96 h-1 in planktonic culture. The growth rate, mu, for exponentially growing B. cepacia in a biofilm was 1.12 h-1 and in planktonic culture 0.78 h-1. This method allows the limitation of the size of a biofilm population to a chosen value.

Biofilms↗
PubMed1998

Localization of a gene for otosclerosis to chromosome 15q25-q26.

Among white adults otosclerosis is the single most common cause of hearing impairment. Although the genetics of this disease are controversial, the majority of studies indicate autosomal dominant inheritance with reduced penetrance. We studied a large multi-generational family in which otosclerosis has been inherited in an autosomal dominant pattern. Five of16 affected persons have surgically confirmed otosclerosis; the remaining nine have a conductive hearing loss but have not undergone corrective surgery. To locate the disease-causing gene we completed genetic linkage analysis using short tandem repeat polymorphisms (STRPs) distributed over the entire genome. Multipoint linkage analysis showed that only one genomic region, on chromosome 15q, generated a lod score >2.0. Additional STRPs were typed in this area, resulting in a lod score of 3.4. STRPs FES (centromeric) and D15S657 (telomeric) flank the 14. 5 cM region that contains an otosclerosis gene.

Adult↗
PubMed1998

Increasing resistance of planktonic and biofilm cultures of Burkholderia cepacia to ciprofloxacin and ceftazidime during exponential growth.

The change in resistance of Burkholderia cepacia to ceftazidime and to ciprofloxacin during the exponential phase and up to the onset of stationary phase was assessed along the growth curve in batch culture. B. cepacia was grown in planktonic culture and in a biofilm on a membrane support. Resistance increased progressively during the exponential phase, being increased by ten-fold about every four generations. Bacteria grown in a biofilm were about 15 times more resistant than equivalent planktonic-grown bacteria. The growth rate was not the key factor for the development of resistance. The growth phase and the mode of growth have a fundamental impact on the susceptibility of B. cepacia towards antimicrobial agents. Bacteria growing at the same rate may differ greatly in their resistance to antimicrobial agents.

Animals↗
PubMed1998

Central hypothyroidism reveals compound heterozygous mutations in the Pit-1 gene.

Mutations in the gene encoding the Pit-1 transcriptional activator interfere with the embryologic determination and ultimate functions of anterior pituitary cells that produce growth hormone (GH), prolactin (Prl) and thyroid-stimulating hormone (TSH). Central hypothyroidism is often the presenting feature of combined pituitary hormone deficiency (CPHD), but it is not detected in screening programs that rely upon elevation of TSH. We report a child whose hypothyroidism was recognized clinically at age 6 weeks, and subsequently found to have GH and Prl as well as TSH deficiency. With thyroxine and GH replacement he has reached the 70th percentile for height and has normal intelligence. Molecular analysis of genomic DNA for Pit-1 revealed the presence of compound heterozygous recessive mutations: a nonsense mutation in codon 172 and a novel missense mutation substituting glycine for glutamate at codon 174. This case is the first demonstration of CPHD due to compound heterozygous Pit-1 point mutations, as most reported cases of the CPHD phenotype involve either the dominant negative R271W allele or homozygosity for recessive Pit-1 mutations. Therefore, in cases of CPHD, the possibilities of compound heterozygosity for two different Pit-1 mutations, or homozygosity for mutations in the epigenetic gene, Prop-1, should be considered.

Alleles↗
PubMed1998

Exogenous nitric oxide enhances neutrophil cell death and DNA fragmentation.

Inhaled nitric oxide (NO) is an important new therapeutic agent used to treat pulmonary arterial hypertension in a variety of disease states. However, the effects of NO on cells in the lung are uncertain. Previously, we have shown that NO gas depresses neutrophil oxidative cell function and increases neutrophil cell death. The purpose of this in vitro study was to determine the mechanism of neutrophil death. We hypothesized that NO hastened cell death by inducing apoptosis. To mimic the clinical environment of patients with respiratory failure, we also studied the effects of hyperoxia on neutrophil cell viability and apoptosis. Isolated human neutrophils were exposed to 80% O2 (O2), NO at 20 ppm in room air (NO/RA), 20 ppm NO blended with 80% O2 (NO/O2), or RA alone (control) for 2 to 24 h. Experiments were repeated with NO concentrations of 5 and 50 ppm and with 20 ppm in the presence of superoxide dismutase (SOD). Neutrophils were also incubated in the absence or presence of neutrophil stimulant fMLP (10 nM). Neutrophil cell viability was measured by fluorescence viability/cytotoxicity assay. Neutrophil apoptosis was assessed by cell death detection ELISA for histone-associated DNA fragments, TdT transferase-mediated fluorescence-labeled dUTP nick end labeling (TUNEL) assay, and DNA fragmentation gel electrophoresis. NO/O2-exposed neutrophils showed decreased viability at 2 h (31.7 +/- 3.7%, mean % viability +/- SD) compared with control (94.7 +/- 4.7%), O2 (75.6 +/- 9.3%), and NO/RA (62.8 +/- 14.9%; P < 0.05 by ANOVA; n = 9). Although control neutrophils demonstrated marked apoptosis at 24 h, there was no significant apoptosis at 2, 4, or 6 h (P < 0.001 by Kruskal-Wallis, n = 20) as assessed by ELISA and TUNEL assays. When compared with RA controls at 2 h, neutrophils exposed to NO/O2 showed significantly more apoptosis (292% of control, range: 106 to 2,488%, P < 0.001 by ANOVA and Kruskal-Wallis) but not with exposure to NO/RA or O2 alone. These findings were confirmed by TUNEL assay (n = 4, P < 0.05). NO/ RA and NO/O2-exposed neutrophils demonstrated both evidence of necrosis and enhanced DNA fragmentation at 2 h by gel electrophoresis (n = 2). Fifty parts per million NO produced similar findings, but exposure to 5 ppm NO did not induce significant DNA fragmentation. Coincubation with SOD inhibited NO/ O2-associated apoptosis, suggesting peroxynitrite contributed to cell death. Stimulation with fMLP did not alter apoptosis induced in neutrophils exposed to NO/RA or NO/O2. We conclude that exogenous NO gas, at clinically relevant concentrations under hyperoxic conditions, induces cell death in neutrophils in part by enhancing DNA fragmentation.

Apoptosis↗
PubMed1998

Compound heterozygous deletion of the PROP-1 gene in children with combined pituitary hormone deficiency.

Mutations in the prophet of Pit-1 gene (PROP1) have been shown to be responsible for combined pituitary hormone deficiency (CPHD) with deficiencies of growth hormone (GH), Prolactin (Prl), thyroid-stimulating hormone (TSH) and gonadotropins. We previously reported that homozygosity for a 2bp deletion in exon 2 (296delGA) accounted for CPHD in three patients from two Russian families. Here we report a second mutational hot spot in exon 2. This 2bp 149delGA deletion results in a frame shift that leads to the same serine to stop codon change at codon 109 (S109X). The predicted proteins are each truncated at residue 108 but diverge from the wild type sequence at different points in the homeodomain. Compound heterozygosity for the two mutations (149delGA/296delGA) was detected in 5 of 14 CPHD children from 4 families (36%). This provides the first evidence of heterozygosity for two common deletions as a cause of CPHD in Russian children.

Adolescent↗
PubMed1997

Distinctions between CD8+ and CD4+ T-cell regenerative pathways result in prolonged T-cell subset imbalance after intensive chemotherapy.

Rapid recovery of CD4+ T cells after intensive chemotherapy is limited by an age-dependent decline in thymopoiesis. Here we sought to determine whether similar limitations exist for CD8+ T-cell regeneration. After intensive chemotherapy, CD8+ T cells had a faster effective doubling time than CD4+ T cells (median, 12.6 v 28.2 days, P < .05). Accordingly, at 3 months posttherapy, mean CD8+ T-cell number had returned to baseline, whereas mean CD4+ T-cell number was only 35% of pretherapy values (P < .05). These differences were primarily due to very rapid expansion of CD8+CD57+ and CD8+CD28- subsets. At 3 months posttherapy, there was no relationship between age and CD8+ T-cell number (R = -.02), whereas CD4+ T-cell number was inversely related to age (R = -.66) and there were no discernible differences in CD8+ recovery among patients with or without thymic enlargement, whereas CD4+ recovery was enhanced in patients with thymic enlargement after chemotherapy (P < .01). Therefore thymic-independent pathways of T-cell regeneration appear to rapidly regenerate substantial numbers of CD8+, but not CD4+ T cells, resulting in prolonged T-cell subset imbalance after T-cell depletion. These inherent distinctions between CD4+ v CD8+ T-cell regeneration may have significant implications for immunotherapeutic strategies undertaken to eradicate minimal residual neoplastic disease after cytoreductive chemotherapy.

Adolescent↗
PubMed1997

The spectrum of growth-hormone insensitivity.

OBJECTIVES: To summarize the steps that led to the recognition of the Laron syndrome of growth-hormone (GH) insensitivity as a recessive disorder that is caused by mutations in the growth hormone receptor (GHR) gene, to discuss the different types of mutations that have been found in the GHR gene, and to examine whether the degree of growth impairment in affected homozygotes depends on the specific type of GHR mutation. RESULTS: A broad spectrum of abnormalities in the GHR gene have been reported. These abnormalities range from deletions of multiple exons, through deletions of a small number of base pairs, nonsense mutations, missense mutations that alter GH-binding affinity or impair receptor processing, to mutations that exert their deleterious effects by altering messenger RNA splicing and result in the loss of portions of the GHR. CONCLUSIONS: Different abnormalities in the GHR gene have different effects on the concentrations of circulating GH-binding protein that represents the extracellular portion of the GHR. Homozygosity or compound heterozygosity for the different mutations produces a fairly uniform phenotype of severe postnatal growth retardation. The differences in height standard deviation scores between persons appear to depend more on age, sex, and nutritional status than on the specific type of GHR mutation.

Age Factors↗
PubMed1997

Common office problems in pediatric urology and gynecology.

The number of genital problems that pediatricians encounter is substantial. The most common ones have been reviewed in this article. Perhaps the most important point to reinforce is the appropriateness of nonintervention in uncircumcised boys whose foreskins have not become retractile during early school years. Without infections or pathologic phimosis, these boys do well, and most foreskins become retractile as they approach puberty. Abnormalities beyond those discussed or those not fitting the anticipated pattern probably warrant specialty referral.

Child↗
PubMed1997

Effect of reduction of nitric oxide on plasma and kidney tissue angiotensin II levels.

Nitric oxide synthase (NOS) blockade increases blood pressure (BP) and modifies glomerular and tubular function. Angiotensin II (AII) blockade restores glomerular and tubular function but does not lower BP. We measured plasma renin activity (PRA), plasma (AIIp), and kidney tissue (AIIk) AII with radioimmunoassay to investigate the dissociation between renal and systemic effects of NOS blockade. Two period clearance studies followed by plasma and renal tissue harvesting were performed in seven groups of rats. Groups 1 and 1A served as controls. Groups 2 and 2A received NaCl-NaHCO3 during the first period and N(G)-monomethyl-L-arginine (L-NMMA, 0.5 mg/kg/min) during the second period. Group 3 was similar to group 2 but renal perfusion pressure (RPP) was maintained constant by using an aortic snare. Groups 4 and 4A received N(G)-nitro-L-arginine-methyl ester (L-NAME, 5 mg/100 mL of drinking water) for 2 weeks. NOS blockers decreased AIIp (group 1, 74 +/- 7 pg/mL; group 2, 22 +/- 1 pg/mL; group 3, 26 +/- 1 pg/mL; group 4, 19 +/- 3 pg/mL). The decrease in AIIp was a direct effect of L-NMMA independent of changes in perfusion pressure, as AIIp was similar in group 3 (normal RPP) and groups 2 and 4 (increased RPP). Measurements of PRA and AIIp demonstrated a similar reduction in PRA and AIIp in rats treated with NOS blocker. Although NOS blockers decreased AIIp, acute or chronic administration of NOS blockers did not modify AIIk (group 1, 1,192 +/- 51; group 2, 1,354 +/- 85; group 3, 1,348 +/- 180; group 4, 1,276 +/- 172 pg/kidney). Our findings demonstrate that NO blockers produce a dissociation between plasma and kidney AII levels. This dissociation can explain the beneficial effects of AII blockers on renal function and their lack of antihypertensive effects in anesthetized rats treated with NOS blockers.

Angiotensin II↗
PubMed1997

A novel locus for autosomal dominant nonsyndromic hearing loss, DFNA13, maps to chromosome 6p.

Nonsyndromic hearing loss (NSHL) is the most common type of hearing impairment in the elderly. Environmental and hereditary factors play an etiologic role, although the relative contribution of each is unknown. To date, 39 NSHL genes have been localized. Twelve produce autosomal dominant hearing loss, most frequently postlingual in onset and progressive in nature. We have ascertained a large, multigenerational family in which a gene for autosomal dominant NSHL is segregating. Affected individuals experience progressive hearing loss beginning in the 2d-4th decades, eventually making the use of amplification mandatory. A novel locus, DFNA13, was identified on chromosome 6p; the disease gene maps to a 4-cM interval flanked by D6S1663 and D6S1691, with a maximum two-point LOD score of 6.409 at D6S299.

Adolescent↗
PubMed1997

Mechanisms of the bactericidal activity of low amperage electric current (DC).

The mechanisms whereby low amperage (10-100 microA) electric current (DC) is bactericidal were investigated with Staphylococcus epidermidis and Staphylococcus aureus. A zone of inhibition test involving the insertion of an anode and cathode into an agar plate inoculated with a lawn of bacteria was used to study the antimicrobial activity of electric current. A zone of inhibition was produced around the cathode when 10 microA (DC) was applied for 16 h. The diameter of the zone was greatly reduced in the presence of catalase. There was no zone around the cathode when the test was carried out under anaerobic conditions. H2O2 was produced at the cathode surface under aerobic conditions but not in the absence of oxygen. A salt-bridge apparatus was used to confirm that H2O2 was produced at the cathode and chlorine at the anode. The antimicrobial activity of low amperage electric current under anaerobic conditions and in the absence of chloride ions against bacteria attached to the surface of a current carrying electrode was also investigated. Antibacterial activity was reduced under anaerobic conditions, which is compatible with the role of H2O2 as a primary bactericidal agent of electricity associated with the cathode. A reduction in chloride ions did not significantly reduce the antibacterial activity suggesting that chlorine plays only a minor role in the bactericidal activity towards organisms attached to anodal electrode surfaces. The localized production of H2O2 and chlorine and the intrinsic activity due to electric current may offer a useful method for eradicating bacteria from catheter surfaces.

Catalase↗
PubMed1997

Resource utilization and patient morbidity in head and neck reconstruction.

Many papers have addressed the technical aspects of free tissue transfer in head and neck cancer patients. However, there has not been a critical assessment of the impact of free tissue transfer on resource utilization and patient morbidity compared with pedicle flap reconstructions. Two cohorts of patients derived from 245 consecutive reconstructions were tightly matched by age, site, stage, and histology, yielding 44 patient pairs differing in method of reconstruction. Patients undergoing free flap reconstruction spent more time in the operating room than those reconstructed with pedicled flaps (993 min vs. 777 min, P < 0.0001). The group with free flap reconstruction spent fewer days in the surgical intensive care unit and hospital (2 days vs. 2.5 days; 18.5 days vs. 22.6 days). This difference is attributed to the paucity of postoperative complications in the group with free flap reconstruction (fistula formation 4.5% vs. 21%, P < 0.04). These data indicate that the continued use of sophisticated reconstructive techniques in head and neck cancer patients is economically sound as assessed by patient morbidity and resource utilization.

Critical Care↗
PubMed1997

Insulin-mediated secretion of ecdysteroids from mosquito ovaries and molecular cloning of the insulin receptor homologue from ovaries of bloodfed Aedes aegypti.

The reproductive cycle of female mosquitoes is activated by ingestion of blood from vertebrate hosts. Shortly after feeding, neurohormones are released from the brain neurosecretory system and stimulate the ovaries to secrete ecdysteroids, which are necessary for vitellogenesis by the fat body. Because bombyxins, which are insulin-like peptides, stimulate ecdysteriodgenesis in silkworm larvae, we tested porcine insulin and found that it activates ecdysteroidogenesis and protein synthesis in ovaries isolated from unfed mosquitoes. To further characterize the regulation of ecdysteroidogenesis in female mosquitoes, we cloned the mosquito insulin receptor (MIR) homologue from ovarian mRNA. The sequence of the extracellular domain shows moderate homologies with vertebrate and Drosophila insulin receptor homologues, as well as with the insulin receptor-related receptor, the latter being an "orphan" receptor with an unknown function. In the intracellular domain, high homologies are observed, particularly in those subdomains that are responsible for ATP binding and kinase activity. Northern blot analysis of MIR demonstrated a highly specific expression in ovaries, and cloning experiments indicated its presence in the brain. Recombinant MIR extracts from a baculovirus expression system contained high constitutive kinase activity in the presence of manganese or magnesium. Activation was independent of a ligand. SDS-gel analysis suggested that the recombinant receptor was not post-translationally processed into an alpha- and beta-subunit as was expected from a putative cleavage signal. Enzymatic properties of the proreceptor are presented: the Km for ATP was between 15 and 50 microM in the presence of a synthetic substrate: maximal kinase activity to 100-fold over basic activity was reached in the presence of 1 mM manganese. Stimulation of key oogenic processes by porcine insulin and identification of a MIR indicate that insulin-like neurohormones may have an important regulatory role in mosquito oogenesis.

Aedes↗