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Biomedical subjects

L L Dunn

Publications and source records attributed to L L Dunn.

15 recordsLinked to original sources
PubMed2006

The function of melanotransferrin: a role in melanoma cell proliferation and tumorigenesis.

Melanotransferrin (MTf) or melanoma tumor antigen p97 is an iron (Fe) binding transferrin homolog expressed highly on melanomas and at lower levels on normal tissues. It has been suggested that MTf is involved in a variety of processes such as Fe metabolism and cellular differentiation. Considering the crucial role of Fe in many metabolic pathways, for example, DNA synthesis, it is important to understand the function of MTf. To define the roles of MTf, two models were developed: (i) an MTf knockout (MTf-/-) mouse and (ii) downregulation of MTf expression in melanoma cells by post-transcriptional gene silencing (PTGS). Examination of the MTf-/- mice demonstrated no differences compared with wild-type littermates. However, microarray analysis showed differential expression of molecules involved in proliferation such as Mef2a, Tcf4, Gls and Apod in MTf-/- mice compared with MTf+/+ littermates. Considering the role of MTf in melanoma cells, PTGS was used to downregulate MTf mRNA and protein levels by >90 and >80%, respectively. This resulted in inhibition of proliferation and migration. As found in MTf-/- mice, in melanoma cells with suppressed MTf expression, hMEF2A and hTCF4 were upregulated compared with parental cells. Furthermore, when melanoma cells with decreased MTf expression were injected into nude mice, tumor growth was markedly reduced, suggesting a role for MTf in proliferation and tumorigenesis.

Animals↗
PubMed2005

Examination of the distribution of the transferrin homologue, melanotransferrin (tumour antigen p97), in mouse and human.

Melanotransferrin (MTf) is a transferrin homologue initially identified in melanoma cells. Serum transferrin (Tf) contains two iron (Fe)-binding sites and plays a vital role in Fe transport. However, human MTf has only a single, high affinity, Fe-binding site. Furthermore, while isolated MTf can bind Fe, it plays little role in Fe uptake by cells and its function remains elusive. To further understand the biological role of this molecule, we examined the expression profile of mouse MTf (mMTf) and human MTf (hMTf) and the splice variant of the latter. Analysis of mMTf in 18 normal mouse tissues and 4 embryonic stages (7-17 days) using an RNA dot blot demonstrated it was expressed at high levels in the pancreas, salivary gland and epididymis of the adult, while embryonic tissues showed low expression. The expression pattern was very different from that of mouse transferrin receptor 1 (TfR1) mRNA, which was found at high levels in the spleen and embryo. Using the more sensitive RT-PCR technique, mMTf expression was demonstrated across all 24 normal mouse tissues assessed. Analysis of the mMTf genomic sequence predicted only one mMTf transcript, although two putative transcripts were found in the testis using Northern blotting. An alternate hMTf transcript, h delta MTf, has been identified by others, although its tissue distribution was not previously examined. In human heart and skeletal muscle, three putative hMTf transcripts were identified at approximately 2, 3 and 4 kb, the smallest transcript being consistent with h delta MTf. The two larger transcripts were also found in 10 other human tissues. The h delta MTf transcript was detected using RT-PCR and Southern blotting in tumour-derived cell lines, with the highest expression being identified in melanoma cells. Immunohistochemistry showed that hMTf was expressed primarily within epithelia. In fact, the most pronounced expression was within the epidermis of the skin, tubules of the kidney and the ducts of sweat and salivary glands. The distribution of MTf and its splice variants may provide clues to their possible biological roles.

Alternative Splicing↗
PubMed2001

A prospective study of psychosocial job strain and birth outcomes.

We conducted a prospective study in Alabama to examine the relation between job strain during pregnancy and birth outcomes. We hypothesized that maternal jobs high in demands and low in control would lead to lower birth weight in offspring. The sample consisted of 480 black and white women, 20 to 34 years of age receiving early prenatal care. Adjusting for standard covariates, women with high strain jobs had babies with birth weights 190 gm lower than those born to mothers in low strain jobs or unemployed (95% CI = 48 gm, 333 gm). Black women experienced a greater effect from job strain than white women.

Adult↗
PubMed1998

The N-terminal domain of IkappaB alpha masks the nuclear localization signal(s) of p50 and c-Rel homodimers.

Members of the Rel/NF-kappaB family of transcription factors are related to each other over a region of about 300 amino acids called the Rel Homology Domain (RHD), which governs DNA binding, dimerization, and binding to inhibitor. At the C-terminal end of the RHD, each protein has a nuclear localization signal (NLS). The crystal structures of the p50 and RelA family members show that the RHD consists of two regions: an N-terminal section which contains some of the DNA contacts and a C-terminal section which contains the remaining DNA contacts and controls dimerization. In unstimulated cells, the homo- or heterodimeric Rel/NF-kappaB proteins are cytoplasmic by virtue of binding to an inhibitor protein (IkappaB) which somehow masks the NLS of each member of the dimer. The IkappaB proteins consist of an ankyrin-repeat-containing domain that is required for binding to dimers and N- and C-terminal domains that are dispensable for binding to most dimers. In this study, we examined the interaction between IkappaB alpha and Rel family homodimers by mutational analysis. We show that (i) the dimerization regions of p50, RelA, and c-Rel are sufficient for binding to IkappaB alpha, (ii) the NLSs of RelA and c-Rel are not required for binding to IkappaB alpha but do stabilize the interaction, (iii) the NLS of p50 is required for binding to IkappaB alpha, (iv) only certain residues within the p50 NLS are required for binding, and (v) in a p50-IkappaB alpha complex or a c-Rel-IkappaB alpha complex, the N terminus of IkappaB alpha either directly or indirectly masks one or both of the dimer NLSs.

Binding Sites↗
PubMed1995

The PEST-like sequence of I kappa B alpha is responsible for inhibition of DNA binding but not for cytoplasmic retention of c-Rel or RelA homodimers.

In most cells, proteins belonging to the Rel/NF-kappa B family of transcription factors are held in inactive form in the cytoplasm by an inhibitor protein, I kappa B alpha. Stimulation of the cells leads to degradation of the inhibitor and transit of active DNA-binding Rel/NF-kappa B dimers to the nucleus. I kappa B alpha is also able to inhibit DNA binding by Rel/NF-kappa B dimers in vitro, suggesting that it may perform the same function in cells when the activating signal is no longer present. Structurally, the human I kappa B alpha molecule can be divided into three sections: a 70-amino-acid N terminus with no known function, a 205-residue midsection composed of six ankyrin-like repeats, and a very acidic 42-amino-acid C terminus that resembles a PEST sequence. In this study we examined how the structural elements of the I kappa B alpha protein correlate with its functional capabilities both in vitro and in vivo. Using a battery of I kappa B alpha mutants, we show that (i) a dimer binds a single I kappa B alpha molecule, (ii) the acidic C-terminal region of I kappa B alpha is not required for protein-protein binding and does not mask the nuclear localization signal of the dimer, (iii) the same C-terminal region is required for inhibition of DNA binding, and (iv) this inhibition may be accomplished by direct interaction between the PEST-like region and the DNA-binding region of one of the subunits of the dimer.

Amino Acid Sequence↗
PubMed1993

The role of O6-alkylguanine-DNA alkyltransferase in protecting Rat4 cells against the mutagenic effects of O6-substituted guanine residues incorporated in codon 12 of the H-ras gene.

The role of rat O6-alkylguanine-DNA alkyltransferase (AGT) in modulating mutagenesis by O6-substituted guanines in the rat H-ras gene was examined. Rat4 cells were transfected with vectors carrying O6-methyl-, O6-ethyl- or O6-benzylguanine residues in place of the normal guanines at either the first, second, or both the first and second positions in codon 12 (GGA) of the H-ras coding sequence. The percentage of transformed colonies was determined for cells grown in normal medium or in medium containing O6-benzylguanine to completely deplete AGT. In parallel experiments with O6-methylguanine-containing vectors, the percentage of cellular DNA harboring codon 12 mutations was determined for normal cells and cells lacking AGT. A reasonable correspondence was observed between the percentage of mutated DNA and the percentage of transformed colonies produced in both types of cells. The results indicate that the contribution of AGT to the repair of O6-substituted guanine damage decreases as the O6 substituent is changed from methyl > ethyl > benzyl. Additionally, cellular AGT appears to repair an O6-methylguanine more readily at the first position of codon 12 than the second position. However, other repair mechanisms in these mammalian cells appear to play a major role in correcting low levels of O6-substituted guanine damage including O6-methylguanine damage.

Base Sequence↗
PubMed1993

Analysis of multiple mRNAs from pathogenic equine infectious anemia virus (EIAV) in an acutely infected horse reveals a novel protein, Ttm, derived from the carboxy terminus of the EIAV transmembrane protein.

Transcription of pathogenic equine infectious anemia virus (EIAV) in an acutely infected horse was examined by using the polymerase chain reaction and nucleotide sequencing. Four spliced transcripts were identified in liver tissue, in contrast to the multiplicity of alternatively spliced messages reported for in vitro-propagated human immunodeficiency virus, simian immunodeficiency virus, and, to a lesser extent, EIAV. Nucleotide sequence analysis demonstrated that three of these mRNAs encode known viral proteins: the envelope precursor, the product of the S2 open reading frame, and the regulatory proteins Tat and Rev. The fourth transcript encodes a novel Tat-TM fusion protein, Ttm. Ttm is a 27-kDa protein translated from the putative tat CTG initiation codon and containing the carboxy-terminal portion of TM immediately downstream from the membrane-spanning domain. p27ttm is expressed in EIAV-infected canine cells and was recognized by peptide antisera against both Tat and TM. Cells transfected with ttm cDNA also expressed p27ttm, which appeared to be localized to the endoplasmic reticulum or Golgi apparatus by indirect immunofluorescence. The carboxy terminus of lentiviral TM proteins has previously been shown to influence viral infectivity, growth kinetics, and cytopathology, suggesting that Ttm plays an important role in the EIAV life cycle.

Amino Acid Sequence↗
PubMed1989

Designed diagnostic restriction fragment length polymorphisms for the detection of point mutations in ras oncogenes.

The polymerase chain reaction (PCR) technique has greatly facilitated the identification of ras oncogenes by allele-specific hybridization of the PCR-amplified DNA to radioactively labelled oligonucleotide probes. In this study, we describe a different method which employs designed mismatch primers that create diagnostic restriction fragment length polymorphisms (RFLPs). This procedure allows the identification of point mutations in the amplified DNA without the use of any radioactive probes. We apply this method to the detection of specific point mutations in the rat H- and K-ras oncogenes in carcinogen-induced tumors. We also suggest strategies for the diagnostic RFLP analysis of most point mutations in the 12th and 61st codons of human ras oncogenes. This simple method is especially suitable for analyzing minuscule amounts of tissue samples where only a fraction of cells may carry activated oncogenes.

Alleles↗
PubMed1987

Pigmented corneal rings in neonates with liver disease.

We performed eye examinations in neonates to determine whether pigmented corneal rings were present in infants with hepatic disease, as in adults. None of 16 control infants had pigmented corneal rings, but 10 (71%) of 14 infants with cholestatic liver disease had such rings. Most rings could be seen without slit lamp by direct inspection of the cornea. Furthermore, the rings appeared to resolve over time. Liver dysfunction tended to be more severe in patients with corneal rings. We speculate that abnormal tissue accumulation of copper may be present in many infants with cholestatic liver disease. Ophthalmologic examination for pigmented corneal rings or determination of serum copper levels may need to be performed in patients at high risk with hepatic disease to monitor for excessive copper accumulation.

Copper↗
PubMed1987

Fatty acid enhancement of the quantum yield for the formation of lumirubin from bilirubin bound to human albumin.

During phototherapy for neonatal jaundice, bilirubin is converted into a number of different polar photoproducts. Because fatty acids are bound to human albumin in vivo and have been shown to affect the binding of bilirubin to albumin, we examined the effect of various fatty acids on the photochemistry of albumin-bound bilirubin. Fatty acids of carbon chain length of 10 or more were found to increase by as much as 3-fold the quantum yield for formation of lumirubin, an intramolecularly cyclized isomer of bilirubin. The binding of these same fatty acids was found to affect visible absorption and circular dichroic spectra of the bound bilirubin, a finding previously interpreted to be the result of a conformational change in the bilirubin. The increased quantum yield for the formation of lumirubin appears to be the result of an allosterically induced conformational change in bilirubin which is bound to albumin.

Bilirubin↗
PubMed1985

Selected opioids and responding for intracranial reinforcement.

Rats fixed with chronically indwelling electrodes for intracranial stimulation (ICS) of the lateral hypothalamus pressed a lever during daily sessions for a fixed intensity of ICS. Before some sessions, they were given placebo, or ethylketocyclazocine (EKC) in racemic or isomeric forms [either (+)EKC or (-)EKC]. Naloxone (NX) was also given with the agents. The racemate facilitated pressing across a narrow range of small doses (centered about 0.02 mg/kg). At no dose did (-)EKC, a potent analgesic, facilitate pressing and typically depressed it. (+)EKC, at doses of 0.04 and 0.08 mg/kg, facilited pressing. These data provide further confirmation that opioid analgesia and ability to enhance pressing are separable. When NX was given with a large dose of the racemate, paradoxically pressing for ICS was facilitated. Apparently, NX selectively blocked the effects of (-)EKC. SKF 10047 was also administered in racemic and isomeric forms. All three forms produced some facilitation of pressing at small doses (e.g., 0.75 mg/kg) and depressed pressing at large doses (e.g., 5.0 mg/kg).

Animals↗
PubMedDate not supplied

Opioids, benzodiazepines and intake of ethanol.

Deprived rats were given the opportunity to take water or a sweetened ethanol solution for one hour/day. Across days, intake of the ethanol solution increased. Doses of morphine prior to an opportunity to drink increased avidity for the ethanol solutions, while doses of chlordiazepoxide did not. The opioid antagonist naloxone decreased intake, whereas, the benzodiazepine antagonist Ro 15-1788 failed to do so. These findings confirm that certain opioids can increase ethanol intake, and further specify that, under the same testing regimen, benzodiazepines do not reliably modify rats' propensity to drink a solution containing ethanol

Alcohol Drinking↗
PubMedDate not supplied

Selected opioids, ethanol and intake of ethanol.

Rats were given an opportunity to drink tap water or a sweetened ethanol solution once a day. Across initial days of opportunity, rats increased their intake of the ethanol solution. Prior to some days' sessions with presented fluids, rats received either an injection of placebo (the carrier of drugs) or doses of ethylketocyclozocine, diprenorphine, or ethanol. Diprenorphine increased rats' intake of the ethanol solution compared to placebo. The other agents did not reliably modify intakes. These findings support a conclusion that selected activity in opioid systems of brain increase the propensity to drink alcoholic beverages.

Alcohol Drinking↗