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1.  Research: Treatment Effect of oral nutritional supplementation on wound healing in diabetic foot ulcers: a prospective randomized controlled trial 
Diabetic Medicine  2014;31(9):1069-1077.
Among people with diabetes, 10–25% will experience a foot ulcer. Research has shown that supplementation with arginine, glutamine and β-hydroxy-β-methylbutyrate may improve wound repair. This study tested whether such supplementation would improve healing of foot ulcers in persons with diabetes.
Along with standard of care, 270 subjects received, in a double-blinded fashion, (twice per day) either arginine, glutamine and β-hydroxy-β-methylbutyrate or a control drink for 16 weeks. The proportion of subjects with total wound closure and time to complete healing was assessed. In a post-hoc analysis, the interaction of serum albumin or limb perfusion, as measured by ankle–brachial index, and supplementation on healing was investigated.
Overall, there were no group differences in wound closure or time to wound healing at week 16. However, in subjects with an albumin level of ≤ 40 g/l and/or an ankle–brachial index of < 1.0, a significantly greater proportion of subjects in the arginine, glutamine and β-hydroxy-β-methylbutyrate group healed at week 16 compared with control subjects (P = 0.03 and 0.008, respectively). Those with low albumin or decreased limb perfusion in the supplementation group were 1.70 (95% CI 1.04–2.79) and 1.66 (95% CI 1.15–2.38) times more likely to heal.
While no differences in healing were identified with supplementation in non-ischaemic patients or those with normal albumin, addition of arginine, glutamine and β-hydroxy-β-methylbutyrate as an adjunct to standard of care may improve healing of diabetic foot ulcers in patients with risk of poor limb perfusion and/or low albumin levels. Further investigation involving arginine, glutamine and β-hydroxy-β-methylbutyrate in these high-risk subgroups might prove clinically valuable.
PMCID: PMC4232867  PMID: 24867069
2.  Differential efficacy of three cycles of CMF followed by tamoxifen in patients with ER-positive and ER-negative tumors: Long-term follow up on IBCSG Trial IX 
Annals of Oncology  2011;22(9):1981-1987.
Background: The benefit of adjuvant chemotherapy in postmenopausal patients with estrogen receptor (ER)-positive lymph node-negative breast cancer is being reassessed.
Patients and methods: After stratification by ER status, 1669 postmenopausal patients with operable lymph node-negative breast cancer were randomly assigned to three 28-day courses of ‘classical’ CMF (cyclophosphamide, methotrexate, 5-fluorouracil) chemotherapy followed by tamoxifen for 57 months (CMF→tamoxifen) or to tamoxifen alone for 5 years.
Results: ERs were positive in 81% of tumors. At a median follow-up of 13.1 years, patients with ER-positive breast cancers did not benefit from CMF [13-year disease-free survival (DFS) 64% CMF→tamoxifen, 66% tamoxifen; P = 0.99], whereas CMF substantially improved the prognosis of patients with ER-negative breast cancer (13-year DFS 73% versus 57%, P = 0.001). Similarly, breast cancer-free interval (BCFI) was identical in the ER-positive cohort but significantly improved by chemotherapy in the ER-negative cohort (13-year BCFI 80% versus 63%, P = 0.001). CMF had no influence on second nonbreast malignancies or deaths from other causes.
Conclusion: CMF is not beneficial in postmenopausal patients with node-negative ER-positive breast cancer but is highly effective within the ER-negative cohort. In the future, other markers of chemotherapy response may define a subset of patients with ER-positive tumors who may benefit from adjuvant chemotherapy.
PMCID: PMC3202167  PMID: 21282282
adjuvant chemotherapy; breast cancer; estrogen receptor; postmenopause; tamoxifen
3.  Survival Benefit of Adjuvant Radiation Therapy for Gastric Cancer following Gastrectomy and Extended Lymphadenectomy 
Purpose. Although randomized trials suggest a survival benefit of adjuvant chemotherapy and radiation therapy (XRT) for gastric adenocarcinoma, its use in patients who undergo an extended lymphadenectomy is less clear. The purpose of this study was to determine if a survival benefit exists in gastric cancer patients who receive adjuvant XRT following resection with extended lymphadenectomy. Methods. The SEER registry was queried for records of patients with resected gastric adenocarcinoma from 1988 to 2007. Multivariable Cox regression models were used to assess independent prognostic factors affecting overall survival (OS) and disease-specific survival (DSS). Results. Of 15,060 patients identified, 3,208 (21%) received adjuvant XRT. Adjuvant XRT was independently associated with improved OS (HR 0.67, CI 0.64–0.71) and DSS (HR 0.69, CI 0.65–0.73) in stages IB through IV (M0). This OS and DSS benefit persisted regardless of the extent of lymphadenectomy. Furthermore, lymphadenectomy with >25 LN resected was associated with improved OS and DSS compared with <15 LN or 15–25 LN. Conclusion. This population-based study shows a survival benefit of adjuvant XRT following gastrectomy that persists in patients who have an extended lymphadenectomy. Furthermore, removal of >25 LNs results in improved OS and DSS compared with patients who have fewer LNs resected.
PMCID: PMC3388431  PMID: 22778937
4.  The effects of a glucose load and sympathetic challenge on autonomic function in obese women with and without type 2 diabetes 
This study examined the effect of glucose ingestion on cardiac autonomic function in non-obese women and obese women with and without type 2 diabetes. Heart rate variability (HRV) was measured via continuous ECG and beat-by-beat blood pressure was recorded with finger photoplethysmography (Portapress) in a fasted state and in response to a 75 g glucose load in 42 middle-aged women (40–60 yr). Upright tilt was also utilized as an orthostatic stress to provide a clinically relevant challenge to the cardiovascular system. Significant main effects for log transformed (Ln) total power (TP, msec2) were observed with upright tilt (P<0.01) and glucose challenge (P<0.05). LnTP decreased in all groups in both the fasted and fed state with upright tilt (P<0.01), but glucose ingestion resulted in higher LnTP in the supine position only (P=0.008). Tilt resulted in a significant main effect for low frequency (LFnu, normalized units) and high frequency (Hfnu) power (P<0.000), while the glucose challenge had no effect on LFnu or HFnu power. LFnu approached significance for group differences (P=0.07), such that the non-obese had lower LF power than either of the obese groups. Sympathovagal balance (LnLF/HF ratio) was affected by position (P<0.000) and group (P<0.05), with a lower LnLF/HF in the non-obese than in the obese women. Baroreceptor sensitivity decreased (P<0.01) during upright tilt but was not changed by the glucose challenge. In conclusion, basal sympathovagal balance is higher in obese individuals with and without type 2 diabetes. Women with type 2 diabetes showed no differences in autonomic function with an orthostatic challenge or glucose load than non-diabetic, obese women. The glucose load did alter total spectral power in all of these middle-aged women, but had no impact on baroreceptor sensitivity.
PMCID: PMC1978097  PMID: 17512310
5.  Quantifying trade-offs: quality of life and quality-adjusted survival in a randomised trial of chemotherapy in postmenopausal patients with lymph node-negative breast cancer 
British Journal of Cancer  2004;91(11):1893-1901.
PMCID: PMC2409769  PMID: 15545973
breast cancer; adjuvant therapy; quality of life; quality-adjusted survival; Q-TWiST; utility
6.  Frequency of Isolation of Staphylococcus lugdunensis in Consecutive Urine Cultures and Relationship to Urinary Tract Infection 
Journal of Clinical Microbiology  2002;40(2):654-656.
Recent reports associate Staphylococcus lugdunensis with severe infection in humans. The frequency of this microorganism in urine cultures is unknown. Five hundred isolates of coagulase-negative staphylococci (CoNS) were recovered from 4,652 consecutive urine specimens submitted for culture to the Mayo Clinic Microbiology Laboratory. Thirty-one (6%) of 500 isolates of CoNS were identified as S. lugdunensis. In no case was S. lugdunensis isolated in pure culture; 29 (94%) of 31 S. lugdunensis isolates were part of mixed nonpathogenic flora. Medical records were reviewed for 30 of the 31 patients from whom these 31 isolates were isolated. Twenty-one (70%) of the 30 evaluable patients were not treated with antibiotics; the remaining 9 (30%) of 30 patients were treated with antibiotics that may be effective against S. lugdunensis. S. lugdunensis may be an unrecognized yet infrequent cause of urinary tract infection.
PMCID: PMC153380  PMID: 11825988
8.  Effects of intranasal cocaine on sympathetic nerve discharge in humans. 
Journal of Clinical Investigation  1997;99(4):628-634.
Cocaine-induced cardiovascular emergencies are mediated by excessive adrenergic stimulation. Animal studies suggest that cocaine not only blocks norepinephrine reuptake peripherally but also inhibits the baroreceptors, thereby reflexively increasing sympathetic nerve discharge. However, the effect of cocaine on sympathetic nerve discharge in humans is unknown. In 12 healthy volunteers, we recorded blood pressure and sympathetic nerve discharge to the skeletal muscle vasculature using intraneural microelectrodes (peroneal nerve) during intranasal cocaine (2 mg/kg, n = 8) or lidocaine (2%, n = 4), an internal local anesthetic control, or intravenous phenylephrine (0.5-2.0 microg/kg, n = 4), an internal sympathomimetic control. Experiments were repeated while minimizing the cocaine-induced rise in blood pressure with intravenous nitroprusside to negate sinoaortic baroreceptor stimulation. After lidocaine, blood pressure and sympathetic nerve discharge were unchanged. After cocaine, blood pressure increased abruptly and remained elevated for 60 min while sympathetic nerve discharge initially was unchanged and then decreased progressively over 60 min to a nadir that was only 2+/-1% of baseline (P < 0.05); however, plasma venous norepinephrine concentrations (n = 5) were unchanged up to 60 min after cocaine. Sympathetic nerve discharge fell more rapidly but to the same nadir when blood pressure was increased similarly with phenylephrine. When the cocaine-induced increase in blood pressure was minimized (nitroprusside), sympathetic nerve discharge did not decrease but rather increased by 2.9 times over baseline (P < 0.05). Baroreflex gain was comparable before and after cocaine. We conclude that in conscious humans the primary effect of intranasal cocaine is to increase sympathetic nerve discharge to the skeletal muscle bed. Furthermore, sinoaortic baroreflexes play a pivotal role in modulating the cocaine-induced sympathetic excitation. The interplay between these excitatory and inhibitory neural influences determines the net effect of cocaine on sympathetic discharge targeted to the human skeletal muscle circulation.
PMCID: PMC507844  PMID: 9045864
9.  Phase II metabolism of benzene. 
Environmental Health Perspectives  1996;104(Suppl 6):1183-1188.
The hepatic metabolism of benzene is thought to be a prerequisite for its bony marrow toxicity. However, the complete pattern of benzene metabolites formed in the liver and their role in bone marrow toxicity are not fully understood. Therefore, benzene metabolism was studied in isolated rodent hepatocytes. Rat hepatocytes released benzene-1,2-dihydrodiol, hydroquinone (HQ), catechol (CT), phenol (PH), trans-trans-muconic acid, and a number of phase II metabolites such as PH sulfate and PH glucuronide. Pretreatment of animals with 3-methylcholantrene (3-MC) markedly increased PH glucuronide formation while PH sulfate formation was decreased. Likewise, V79 cells transfected with the 3-MC-inducible rat UGT1.6 cDNA showed a considerable rate of PH and HQ glucuronidation. In addition to inducing glucuronidation of phenols, 3-MC treatment (reported to protect rats from the myelotoxicity of benzene) resulted in a decrease of hepatic CYP2E1. In contrast, pretreatment of rats with the CYP2E1-inducer isopropanol strongly enhanced benzene metabolism and the formation of phenolic metabolites. Mouse hepatocytes formed much higher amounts of HQ than rat hepatocytes and considerable amounts of 1,2,4-trihydroxybenzene (THB) sulfate and HQ sulfate. In conclusion, the protective effect of 3-MC in rats is probably due to a shift from the labile PH sulfate to the more stable PH glucuronide, and to a decrease in hepatic CYP2E1. The higher susceptibility of mice toward benzene may be related to the high rate of formation of the myelotoxic metabolite HQ and the semistable phase II metabolites HQ sulfate and THB sulfate.
PMCID: PMC1469749  PMID: 9118891
10.  An overview of benzene metabolism. 
Environmental Health Perspectives  1996;104(Suppl 6):1165-1171.
Benzene toxicity involves both bone marrow depression and leukemogenesis caused by damage to multiple classes of hematopoietic cells and a variety of hematopoietic cell functions. Study of the relationship between the metabolism and toxicity of benzene indicates that several metabolites of benzene play significant roles in generating benzene toxicity. Benzene is metabolized, primarily in the liver, to a variety of hydroxylated and ring-opened products that are transported to the bone marrow where subsequent secondary metabolism occurs. Two potential mechanisms by which benzene metabolites may damage cellular macromolecules to induce toxicity include the covalent binding of reactive metabolites of benzene and the capacity of benzene metabolites to induce oxidative damage. Although the relative contributions of each of these mechanisms to toxicity remains unestablished, it is clear that different mechanisms contribute to the toxicities associated with different metabolites. As a corollary, it is unlikely that benzene toxicity can be described as the result of the interaction of a single metabolite with a single biological target. Continued investigation of the metabolism of benzene and its metabolites will allow us to determine the specific combination of metabolites as well as the biological target(s) involved in toxicity and will ultimately lead to our understanding of the relationship between the production of benzene metabolites and bone marrow toxicity.
PMCID: PMC1469747  PMID: 9118888
11.  Prevention of tumor formation in a mouse model of Burkitt's lymphoma by 6 weeks of treatment with anti-c-myc DNA phosphorothioate. 
Molecular Medicine  1995;1(6):647-658.
BACKGROUND: Transgenic mice bearing a murine immunoglobulin enhancer/c-myc fusion transgene (Emu-myc) provide a useful model for Burkitt's lymphoma. MATERIALS AND METHODS: Groups of 12 Emu-myc mice were treated prophylactically for 6 weeks after weaning with anti-c-myc DNA phosphorothioate (20 mg/kg/day), scrambled control DNA, or saline, delivered by micro-osmotic pumps. RESULTS: Half of the mice treated with saline or scrambled control DNA displayed palpable tumors by 8-9 weeks after birth, and 95% of them did so by 16 weeks, but 75% of the mice treated with antisense DNA were still free of tumors at the age of 26 weeks. Antisense therapy ablated MYC antigen in the spleens of tumor-bearing mice. Plasma physiological parameters indicated no acute toxicity. CONCLUSIONS: Long-term tumor resistance after anti-c-myc DNA therapy implies induction of a host response. Prophylactic anti-c-myc DNA therapy might prevent lymphoma in asymptomatic individuals displaying c-myc translocations.
PMCID: PMC2229988  PMID: 8529131
12.  Physician awareness of fetal alcohol syndrome: a survey of pediatricians and general practitioners. 
OBJECTIVE: To determine the knowledge, clinical experience and perceived needs for resource materials of Saskatchewan physicians in regard to fetal alcohol syndrome (FAS) and alcohol-related birth defects. DESIGN: Mailed survey. SETTING: Saskatchewan. PARTICIPANTS: All 48 pediatricians and half (394) of the family physicians (FPs) and general practitioners (GPs) practising in Saskatchewan received a questionnaire. The numbers of physicians who completed it were 24 and 249 respectively. RESULTS: The pediatricians were more likely than the other physicians to be aware of FAS and to have diagnosed at least one case of FAS. Among the FPs and GPs, the year of graduation from medical school was a significant factor in their knowledge of FAS and their diagnostic practices. Those who graduated before 1974, the year FAS was first described in the medical literature, were less likely than the more recent graduates to be aware of FAS and to ask their patients about alcohol use during pregnancy but were more likely to feel comfortable discussing alcohol-related issues in families. All of the groups reported a need for more information about FAS and for resources on alcohol-related issues in general. CONCLUSIONS: Saskatchewan physicians are aware of FAS but have expressed a need for more information about FAS, particularly for parents, as well as physician training materials and information about where to refer patients with FAS and parents with alcohol-related problems.
PMCID: PMC1337654  PMID: 7712419
13.  Tracking the Response of Burkholderia cepacia G4 5223-PR1 in Aquifer Microcosms 
The introduction of bacteria into the environment for bioremediation purposes (bioaugmentation) requires analysis and monitoring of microbial population dynamics to define persistence and activity from both efficacy and risk assessment perspectives. Burkholderia cepacia G4 5223-PR1 is a Tn5 insertion mutant which constitutively expresses a toluene ortho-monooxygenase that degrades trichloroethylene (TCE). This ability of G4 5223-PR1 to degrade TCE without aromatic induction may be useful for bioremediation of TCE-containing aquifers and groundwater. Thus, a simulated aquifer sediment system and groundwater microcosms were used to monitor the survival of G4 5223-PR1. The fate of G4 5223-PR1 in sediment was monitored by indirect immunofluorescence microscopy, a colony blot assay, and growth on selective medium. G4 5223-PR1 was detected immunologically by using a highly specific monoclonal antibody which reacted against the O-specific polysaccharide chain of the lipopolysaccharides of this organism. G4 5223-PR1 survived well in sterilized groundwater, although in nonsterile groundwater microcosms rapid decreases in the G4 5223-PR1 cell population were observed. Ten days after inoculation no G4 5223-PR1 cells could be detected by selective plating or immunofluorescence. G4 5223-PR1 survival was greater in a nonsterile aquifer sediment microcosm, although after 22 days of elution the number of G4 5223-PR1 cells was low. Our results demonstrate the utility of monoclonal antibody tracking methods and the importance of biotic interactions in determining the persistence of introduced microorganisms.
PMCID: PMC1388346  PMID: 16534928
14.  Antibiotic therapy for ocular infection. 
Western Journal of Medicine  1994;161(6):579-584.
Infections of the eye can rapidly damage important functional structures and lead to permanent vision loss or blindness. Broad-spectrum antibiotics should be administered to the appropriate site of infection as soon as a diagnosis is made. Topical drops are preferred for corneal and conjunctival infections. Intravitreal antibiotics, and possibly subconjunctival and parenteral antibiotics, are preferred for endophthalmitis. Parenteral antibiotics are recommended for infection in deep adnexal structures. We review specific aspects of antibiotic therapy for ocular and periocular infection.
PMCID: PMC1022739  PMID: 7856158
15.  Effects of metal treatment on DNA repair in polyamine-depleted HeLa cells with special reference to nickel. 
Environmental Health Perspectives  1994;102(Suppl 3):51-55.
Human cells depleted of the naturally occurring polyamines putrescine, spermidine, and spermine exhibit altered chromatin structure and marked deficiencies in DNA replicative and repair processes. Similar effects have been observed following treatment of normal mammalian cells with various heavy metal salts. In an attempt to better understand how metals interfere with normal DNA metabolic processes, a series of studies was carried out in which the toxicity and repair-inhibitory properties of various metals were evaluated in polyamine-depleted HeLa cells. Cytotoxicity of copper, zinc, magnesium, and cadmium was not altered in cells carrying lower polyamine pools. However, the sensitivity to nickel was markedly increased upon polyamine depletion, a condition that was readily reversed by polyamine supplementation. Nucleoid sedimentation analysis indicated that a greater amount of nickel-induced DNA damage occurred in polyamine-depleted cells than in normal cells, possibly serving as the basis for the increased sensitivity. Both polyamine depletion and nickel treatment result in decreased repair of DNA strand breaks and decreased cloning efficiency following X-ray and ultraviolet irradiation. Nickel treatment of polyamine-depleted cells resulted in synergistic sensitivity to both radiation treatments. None of the other metals tested enhanced X-ray or ultraviolet sensitivity of polyamine-depleted cells. Analysis of retarded repair sites following ultraviolet irradiation indicated those sites to be nonligatable in polyamine-depleted and nickel-treated cells, suggesting a block in the normal gap-sealing process.
PMCID: PMC1567407  PMID: 7843137
16.  Mortality patterns among the youth of a northeastern American Indian cohort. 
Public Health Reports  1993;108(3):403-407.
Mortality patterns prevalent among American Indian youth have not been well documented. This investigation reports on mortality patterns among the Seneca Nation of Indians from January 1, 1955, through December 31, 1989. The study cohort consisted of 3,033 Seneca tribal members born during the study period. Deaths occurring among cohort members younger than age 25 were identified through a computer match against New York State vital record files. Sex-specific standardized mortality ratios were calculated on the basis of mortality patterns exhibited by the general population of New York State, exclusive of New York City. Males exhibited significantly elevated mortality for all causes combined, for deaths due to all accidents combined, for motor vehicle accidents, and for suicide. Females demonstrated significantly elevated mortality from all accidents combined, for motor vehicle accidents, and for all other types of accidents. Age-specific mortality patterns also varied both by sex and by calendar time. These findings are important to consider in the design of programs aimed at reducing premature mortality among American Indian populations from preventable causes of death.
PMCID: PMC1403396  PMID: 8497581
17.  Features of the adeno-associated virus origin involved in substrate recognition by the viral Rep protein. 
Journal of Virology  1993;67(10):6096-6104.
We previously demonstrated that the adeno-associated virus (AAV) Rep68 and Rep78 proteins are able to nick the AAV origin of DNA replication at the terminal resolution site (trs) in an ATP-dependent manner. Using four types of modified or mutant substrates, we now have investigated the substrate requirements of Rep68 in the trs endonuclease reaction. In the first kind of substrate, portions of the hairpinned AAV terminal repeat were deleted. Only deletions that retained virtually all of the small internal palindromes of the AAV terminal repeat were active in the endonuclease reaction. This result confirmed previous genetic and biochemical evidence that the secondary structure of the terminal repeat was an important feature for substrate recognition. In the second type of substrate, the trs was moved eight bases further away from the end of the genome. The mutant was nicked at a 50-fold-lower frequency relative to a wild-type origin, and the nick occurred at the correct trs sequence despite its new position. This finding indicated that the endonuclease reaction required a specific sequence at the trs in addition to the correct secondary structure. It also suggested that the minimum trs recognition sequence extended three bases from the cut site in the 3' direction. The third type of substrate harbored mismatched base pairs at the trs. The mismatch substrates contained a wild-type sequence on the strand normally cut but an incorrect sequence on the complementary strand. All of the mismatch mutants were capable of being nicked in the presence of ATP. However, there was substantial variation in the level of activity, suggesting that the sequence on the opposite strand may also be recognized during nicking. Analysis of the mismatch mutants also suggested that a single-stranded trs was a viable substrate for the enzyme. This interpretation was confirmed by analysis of the fourth type of substrate tested, which contained a single-stranded trs. This substrate was also cleaved efficiently by the enzyme provided that the correct strand was present in the substrate. In addition, the single-stranded substrate no longer required ATP as a cofactor for nicking. Finally, all of the substrates with mutant trss bound the Rep protein as efficiently as the wild-type did. This finding indicated that the sequence at the cut site was not involved in recognition of the terminal repeat for specific binding by the enzyme. We concluded that substrate recognition by the AAV Rep protein involves at least two and possibly as many as four features of the AAV terminal repeat.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC238031  PMID: 8396670
18.  The toxicology of benzene. 
Benzene is metabolized, primarily in the liver, to a series of phenolic and ring-opened products and their conjugates. The mechanism of benzene-induced aplastic anemia appears to involve the concerted action of several metabolites acting together on early stem and progenitor cells, as well as on early blast cells, such as pronormoblasts and normoblasts to inhibit maturation and amplification. Benzene metabolites also inhibit the function of microenvironmental stromal cells necessary to support the growth of differentiating and maturing marrow cells. The mechanism of benzene-induced leukemogenesis is less well understood. Benzene and its metabolites do not function well as mutagens but are highly clastogenic, producing chromosome aberrations, sister chromatid exchange, and micronuclei. Benzene has been shown to be a multi-organ carcinogen in animals. Epidemiological studies demonstrate that benzene is a human leukemogen. There is need to better define the lower end of the dose-response curve for benzene as a human leukemogen. The application of emerging methods in biologically based risk assessment employing pharmacokinetic and mechanistic data may help to clarify the uncertainties in low-dose risk assessment.
PMCID: PMC1519582  PMID: 8354177
19.  Binding properties of replication protein A from human and yeast cells. 
Molecular and Cellular Biology  1992;12(7):3050-3059.
Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70, 32, and 14 kDa). We have analyzed the DNA-binding properties of both human and yeast RP-A in order to gain a better understanding of their role(s) in DNA replication. Human RP-A has high affinity for single-stranded DNA and low affinity for RNA and double-stranded DNA. The apparent affinity constant of RP-A for single-stranded DNA is in the range of 10(9) M-1. RP-A has a binding site size of approximately 30 nucleotides and does not bind cooperatively. The binding of RP-A to single-stranded DNA is partially sequence dependent. The affinity of human RP-A for pyrimidines is approximately 50-fold higher than its affinity for purines. The binding properties of yeast RP-A are similar to those of the human protein. Both yeast and human RP-A bind preferentially to the pyrimidine-rich strand of a homologous origin of replication: the ARS307 or the simian virus 40 origin of replication, respectively. This asymmetric binding suggests that RP-A could play a direct role in the process of initiation of DNA replication.
PMCID: PMC364519  PMID: 1320195
20.  Temporal expression of the human alcohol dehydrogenase gene family during liver development correlates with differential promoter activation by hepatocyte nuclear factor 1, CCAAT/enhancer-binding protein alpha, liver activator protein, and D-element-binding protein. 
Molecular and Cellular Biology  1992;12(7):3023-3031.
The human class I alcohol dehydrogenase (ADH) gene family consists of ADH1, ADH2, and ADH3, which are sequentially activated in early fetal, late fetal, and postnatal liver, respectively. Analysis of ADH promoters revealed differential activation by several factors previously shown to control liver transcription. In cotransfection assays, the ADH1 promoter, but not the ADH2 or ADH3 promoter, was shown to respond to hepatocyte nuclear factor 1 (HNF-1), which has previously been shown to regulate transcription in early liver development. The ADH2 promoter, but not the ADH1 or ADH3 promoter, was shown to respond to CCAAT/enhancer-binding protein alpha (C/EBP alpha), a transcription factor particularly active during late fetal liver and early postnatal liver development. The ADH1, ADH2, and ADH3 promoters all responded to the liver transcription factors liver activator protein (LAP) and D-element-binding protein (DBP), which are most active in postnatal liver. For all three promoters, the activation by LAP or DBP was higher than that seen by HNF-1 or C/EBP alpha, and a significant synergism between C/EBP alpha and LAP was noticed for the ADH2 and ADH3 promoters when both factors were simultaneously cotransfected. A hierarchy of ADH promoter responsiveness to C/EBP alpha and LAP homo- and heterodimers is suggested. In all three ADH genes, LAP bound to the same four sites previously reported for C/EBP alpha (i.e., -160, -120, -40, and -20 bp), but DBP bound strongly only to the site located at -40 bp relative to the transcriptional start. Mutational analysis of ADH2 indicated that the -40 bp element accounts for most of the promoter regulation by the bZIP factors analyzed. These studies suggest that HNF-1 and C/EBP alpha help establish ADH gene family transcription in fetal liver and that LAP and DBP help maintain high-level ADH gene family transcription in postnatal liver.
PMCID: PMC364516  PMID: 1620113
21.  The cost of breast cancer recurrences. 
British Journal of Cancer  1992;65(3):449-455.
Information about the costs of recurrent breast cancer is potentially important for targeting cost containment strategies and analysing the cost-effectiveness of breast cancer control programmes. We estimated these costs by abstracting health service and consumable usage data from the medical histories of 128 patients, and valuing each of the resources used. Resource usage and costs were summarised by regarding the recurrence as a series of episodes which were categorised into five anatomical site-based groups according to the following hierarchy: visceral, central nervous system (CNS), bone, local and other. Hospital visits and investigations comprised 78% of total costs for all episodes combined, and there were significant differences between the site-based groups in the frequency of hospital visits and most investigations. Total costs were most accurately described by separate linear regression models for each group, with the natural logarithm of the cost of the episode as the dependent variable, and predictor variables including the duration of the episode, duration squared, duration cubed and a variable indicating whether the episode was fatal. Visceral and CNS episodes were associated with higher costs than the other groups and were more likely to be shorter and fatal. A fatal recurrence of duration 15.7 months (the median for our sample) was predicted to cost $10,575 (Aus + 1988; or 4,877 pounds). Reduction of the substantial costs of recurrent breast cancer is likely to be a sizable economic benefit of adjuvant systemic therapy and mammographic screening. We did not identify any major opportunities for cost containment during the management of recurrences.
PMCID: PMC1977600  PMID: 1558803
22.  Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo. 
Journal of Clinical Investigation  1991;88(5):1613-1621.
The promoter of the human dihydrofolate reductase (DHFR) gene contains two consensus binding sites for the DNA binding protein Sp1. DNAse protection and gel mobility shift assays demonstrate binding of recombinant Sp1 to both decanucleotide Sp1 binding sequences which are located 49 and 14 base pairs upstream of the transcription start site. The more distal of the two binding sites exhibits a somewhat higher affinity for Sp1. The G-C specific DNA binding drug, mithramycin, binds to both consensus sequences and prevents subsequent Sp1 binding. Promoter-dependent in vitro transcription of a DHFR template is selectively inhibited by mithramycin when compared to the human H2b histone gene. A similar effect is also noted in vivo. Mithramycin treatment of MCF-7 human breast carcinoma cells containing an amplified DHFR gene induces selective inhibition of DHFR transcription initiation, resulting in a decline in DHFR mRNA level and enzyme activity. This selective inhibition of DHFR expression suggests that it is possible to modulate the overexpression of the DHFR gene in methotrexate resistant cells.
PMCID: PMC295684  PMID: 1834700
23.  Years of potential life lost among a Native American population. 
Public Health Reports  1989;104(3):279-285.
The determination of years of potential life lost (YPLL) can aid in monitoring changes in premature mortality among various population groups. While premature mortality has been shown to differ among blacks and whites, patterns of YPLL have not been well established among other racial groups. The Seneca Nation of Indians (SNI) is a Native American group residing primarily in western New York State (NYS). A review of SNI necrology records revealed that 55 percent (510 of 924) of the deaths between 1955 and 1984 occurred before 65 years of age. The proportion of premature deaths among males exceeded the proportion in females. SNI males demonstrated an increased risk of premature death (odds ratio = 1.43) relative to SNI females. Both the percentage of premature deaths and the number of YPLL per death were greater among SNI members compared with NYS residents. Almost one-half of all YPLL among the SNI were attributable to accidents and injuries. Heart disease, digestive diseases, and malignant neoplasms also represented important contributors to YPLL for both SNI males and females. This investigation identifies important causes of premature death among a Native American population and underscores the preventable nature of premature loss of life.
PMCID: PMC1579922  PMID: 2498978
24.  Evidence for covalent attachment of the adeno-associated virus (AAV) rep protein to the ends of the AAV genome. 
Journal of Virology  1990;64(12):6204-6213.
We have demonstrated that when the covalently joined ends of linear adeno-associated virus (AAV) DNA are resolved in vitro, the virus-encoded Rep protein becomes covalently attached to the 5' ends of the DNA. The covalent bond is between a tyrosine residue of the AAV Rep protein and a 5' phosphate of a thymidine residue in the AAV genome. Only the Rep protein encoded by the AAV p5 promoter, Rep68, was capable of becoming covalently attached to the ends of the AAV genome; the Rep proteins encoded by the p19 promoter were not. We also investigated some of the requirements for the complete in vitro resolution reaction. Inhibitor studies suggested that terminal resolution required DNA polymerase delta, ATP, and the deoxyribonucleoside triphosphates but did not require the remaining ribonucleoside triphosphates, DNA polymerase alpha, RNA polymerase II, or topoisomerases I and II. Finally, purified AAV Rep68, when added to the crude cytosol from uninfected HeLa cells, was sufficient for resolution. This suggested that terminal resolution relies on host enzymes and the virus-encoded p5 Rep proteins.
PMCID: PMC248795  PMID: 2173787
25.  Activation of bone marrow phagocytes following benzene treatment of mice. 
Techniques in flow cytometry/cell sorting were used to characterize the effects of benzene and its metabolites on subpopulations of bone marrow cells. Treatment of male Balb/c mice with benzene (880 mg/kg) or a combination of its metabolites, hydroquinone and phenol (50 mg/kg), resulted in a 30 to 40% decrease in bone marrow cellularity. Flow cytometric analysis revealed two subpopulations of bone marrow cells that could be distinguished by their size and density or granularity. The larger, more dense subpopulation was found to consist predominantly of macrophages and granulocytes as determined by monoclonal antibody binding and by cell sorting. Benzene treatment had no selective cytotoxic effects on subpopulations of bone marrow cells. To determine if benzene treatment activated bone marrow phagocytes, we quantified production of hydrogen peroxide by these cells using the fluorescent indicator dye, 2',7'-dichlorofluorescein diacetate. We found that macrophages and granulocytes from bone marrow of treated mice produced 50% more hydrogen peroxide in response to the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate than did cells from control animals. It is hypothesized that phagocyte activation and production of cytotoxic reactive oxygen intermediates may contribute to hematotoxicity induced by benzene.
PMCID: PMC1568140  PMID: 2676504

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