Low-level constitutional mosaicism of a de novoBRCA1 gene mutation.

BACKGROUND: Pathogenic BRCA1 mutations are usually inherited. Constitutional low-level BRCA1 mosaicism has never been reported. METHODS: Next-generation sequencing (NGS) of cancer gene panel of germline and tumour DNA in a patient with early onset, triple-negative breast cancer. RESULTS: Constitutional de novo mosaicism (5%) for a pathogenic (c.1953dupG; p.Lys652Glufs*21) BRCA1mutation was detected in leukocytes, buccal tissue and normal breast tissue DNA, with ∼50% mutation in tumorous breast tissue. CONCLUSION: This is the first reported case of low-level, multiple tissue, constitutional mosaicism in BRCA1, and highlights the need to consider deep sequencing in affected individuals clinically suspected of having cancer predisposition whose tumours display a BRCA mutation.

Antitumor effect of a copper coordination compound with superoxide dismutase-like activity.

Growth of Ehrlich carcinomas in inbred CBA mice was retarded by im administration of Cu(II)(3,5-diisopropylsalicylate)2 (CuDIPS). CuDIPS is a low molecular weight (mol wt = 503) copper coordination compound that exhibits superoxide dismutase (SOD)-like activity. It has been used as an anti-inflammatory agent and is lipid-soluble. This property enables the compound to penetrate membranes, thus becoming an intracellular O2- scavenger. In the tumor system studied, the amounts of both copper- and zinc-containing SOD (CuZnSOD) and manganese-containing SOD are reduced. Injection of Orgotein (CuZnSOD from bovine liver) had no significant effect on tumor growth and host survival. When CuDIPS was administered at various doses, reduction in tumor size, delay of metastasis, and a significant increase in survival of the hosts were observed.

Possible role of glutathione in the antitumor effect of a copper-containing synthetic superoxide dismutase in mice.

The effect of glutathione and a glutathione reductase inhibitor on the antitumor effect of Cu(II)(3,5-diisopropylsalicylate)2 (CuDIPS) was studied. CuDIPS is a low-molecular-weight copper coordination compound that exhibits superoxide dismutase-like activity. CuDIPS had antitumor activity against intraperitoneal Ehrlich ascites carcinoma in Swiss mice. A single ip injection of glutathione partially eliminated the antitumor effect of CuDIPS, whereas a single ip injection of 1,3-bis(2-chloroethyl)-1-nitrosourea enhanced the antitumor effect of CuDIPS. These results are consistent with the hypothesis that CuDIPS exerts part of its antitumor effect by producing H2O2.

Postirradiation treatment with copper(II)2(3,5-diisopropylsalicylate)4 enhances radiation recovery and hemopoietic regeneration.

We have previously reported that copper(II)2(3,5-diisopropylsalicylate)4 (Cu-DIPS), administered 3 h before exposure to lethal irradiation, significantly increased the survival rate of mice. Agents that can improve recovery from irradiation are of particular importance for accidental radiation exposure if they are effective when given after exposure. In the present study, we showed that Cu-DIPS had radiation recovery activity when administered subsequent to radiation exposure. Mice were exposed to 800 cGy irradiation and 3 h later injected with vehicle or 20, 40, or 60 mumol/kg Cu-DIPS. The 30-day survival rate was significantly increased at all doses of Cu-DIPS tested. Survival increased from 47% for vehicle-treated mice to 78% (p less than 0.001) for mice treated with 40 mumol/kg. The recovery of hemopoietic activity was assessed in similarly treated mice 14 and 24 days after irradiation. The postirradiation Cu-DIPS treatment significantly increased spleen weights, bone marrow cellularity, and hemopoietic activity in the spleen and bone marrow compared to vehicle-treated controls. Enhanced recovery of hemopoietic activity included both committed progenitor granulocyte-macrophage colony-forming units (GM-CFU) and more primitive stem cells (endogenous spleen colony-forming units, CFU-Se). The number of CFU-Se at 14 days, the number of bone marrow GM-CFU at 24 days, and bone marrow cellularity at 24 days appear to be better predictors of survival rates than other parameters.

Copper(II)2(3,5-diisopropylsalicylate)4 stimulates hemopoiesis in normal and irradiated mice.

We have previously reported that copper(II)2(3,5-diisopropylsalicylate)4 (Cu-DIPS) significantly increased the survival rate of mice exposed to lethal irradiation. To examine whether Cu-DIPS affected hemopoietic activity, groups of mice were treated with Cu-DIPS or vehicle and assayed for in vitro interleukin 3 (IL-3)-dependent colony-forming units (CFU-C) and for committed progenitor granulocyte-macrophage CFU (GM-CFU). Cu-DIPS increased the number of splenic IL-3 CFU-C by five- to sixfold 7 days after treatment and splenic GM-CFU by 12-fold on day 24. These increases were accompanied by a 50% increase in spleen weight. Bone marrow IL-3 CFU-C and GM-CFU were not affected at 7 or 14 days after treatment, but were somewhat depressed at 24 days. In irradiated (8.0 Gy) mice treated with Cu-DIPS or vehicle, splenic IL-3 CFU-C and GM-CFU were undetectable 7 days after irradiation, but recovered more rapidly in Cu-DIPS-treated mice. By 24 days splenic IL-3 CFU-C in Cu-DIPS-treated mice recovered to 150% of normal (unirradiated) values and GM-CFU recovered to 270% of normal, whereas irradiated control values remained at 25% and 7%, respectively. The recovery of bone marrow hemopoiesis was slower than spleen, but 42 days after irradiation Cu-DIPS-treated mice had higher levels of bone marrow IL-3 CFU-C (eightfold) and GM-CFU (4.6-fold) than vehicle-treated mice. Cu-DIPS stimulated sixfold increases in renewable, pluripotent stem cells as measured by the in vivo assay of endogenous colony-forming units (CFU-Se).

Mucopolysaccharidosis VII in a Cat Caused by 2 Adjacent Missense Mutations in the GUSB Gene.

BACKGROUND: Mucopolysaccharidoses (MPS) are common lysosomal storage disorders causing typically progressive skeletal and ocular abnormalities. OBJECTIVES: To describe the clinic features, metabolic profile and a unique mutation in a domestic shorthair (DSH) kitten with MPS VII. ANIMALS: Affected kitten and 80 healthy cats. METHODS: Serum lysosomal enzyme activities and urinary glycosaminoglycan (GAG) accumulation were assessed. Exons of the ß-glucuronidase gene (GUSB) were sequenced from genomic DNA and genotyping was conducted. RESULTS: A 3-month-old DSH cat was presented for stunted growth, paresis, facial dysmorphia, multiple skeletal deformities, and corneal opacities. Evaluation of blood smears disclosed metachromatic granules in leukocytes and a urinary mucopolysaccharide spot test was positive. The proband had no GUSB activity but normal or increased activities for other lysosomal enzymes. Sequencing of the GUSB gene from the proband and comparison to the sequence of 2 healthy cats and the published feline genome sequence demonstrated 2 unique single base transitions (c.1421T>G and c.1424C>T) in exon 9, altering 2 adjacent codons (p.Ser475Ala and p.Arg476Trp). These amino acid changes are in a highly conserved domain of the GUSB protein and nontolerable to maintain function. Moreover, the p.Arg476Trp mutation previously has been identified in human patients. None of the other clinically healthy cats had these mutations. CONCLUSIONS AND CLINIC IMPORTANCE: The diagnostic approach to MPS disorders is delineated. This is only the second mutation known to cause MPS VII in cats. Similarly, 2 different mutations have been described in MPS VII dogs, thereby showing the molecular heterogeneity of MPS VII in companion animals.

Lack of effects of copper gluconate supplementation.

A double-blind study was done giving 10 mg of copper/day as copper gluconate or placebo capsules for 12 wk. The seven subjects receiving copper gluconate had no change in the level of copper in the serum, urine, or hair. There was also no change in the levels of zinc or magnesium. There was also no significant change in levels of hematocrit, triglyceride, SGOT, GGT, LDH, cholesterol, or alkaline phosphatase. The side effects of nausea, diarrhea, and heartburn were the same in the subjects receiving copper gluconate and subjects receiving placebo capsules.

Matching simulation and experiment: a new simplified model for simulating protein folding.

Simulations of simplified protein folding models have provided much insight into solving the protein folding problem. We propose here a new off-lattice bead model, capable of simulating several different fold classes of small proteins. We present the sequence for an alpha/beta protein resembling the IgG-binding proteins L and G. The thermodynamics of the folding process for this model are characterized using the multiple multihistogram method combined with constant-temperature Langevin simulations. The folding is shown to be highly cooperative, with chain collapse nearly accompanying folding. Two parallel folding pathways are shown to exist on the folding free energy landscape. One pathway contains an intermediate--similar to experiments on protein G, and one pathway contains no intermediates-similar to experiments on protein L. The folding kinetics are characterized by tabulating mean-first passage times, and we show that the onset of glasslike kinetics occurs at much lower temperatures than the folding temperature. This model is expected to be useful in many future contexts: investigating questions of the role of local versus nonlocal interactions in various fold classes, addressing the effect of sequence mutations affecting secondary structure propensities, and providing a computationally feasible model for studying the role of solvation forces in protein folding.

Bis(3,5-diisopropylsalicylato)copper(II), a potent radioprotectant with superoxide dismutase mimetic activity.

Superoxide disproportionation may partially account for the noteworthy radioprotectant activity of bis(3,5-diisopropylsalicylato)copper(II) [CuII(3,5-dips)2]. Groups of mice treated with CuII(3,5-dips)2 3 or 24 h before exposure to a lethal dose of gamma-radiation had survival rates of 33% and 58%, respectively. These results suggest that copper complexes might be developed for protection of normal tissues in association with cancer radiotherapy and protection against occupational exposures to hazardous radiation.

Commercial discharge packs and breast-feeding counseling: effects on infant-feeding practices in a randomized trial.

A randomized controlled trial was conducted to evaluate two interventions for prolonging the duration of breast-feeding in a multiethnic sample of 343 low-income urban women. One intervention compared research breast-feeding bedside counseling by a trained counselor, who also made eight telephone calls during the first 3 months of the infant's life, with the routine breast-feeding counseling provided in the hospital by nurses. The other intervention compared commercial discharge packs provided by formula companies with research discharge packs designed to be consistent with the WHO Code of Marketing of Breastmilk Substitutes. When infants were 4 months old, a telephone interviewer unaware of treatment status contacted 95% (324/343) of the women to determine the infants' feeding and health histories. Compared with routine counseling, research counseling delayed the first introduction of solid foods to the infant's diet (P = .03, one-tailed) but did not exert a statistically significant effect on breast-feeding by 4 months' postpartum. Women who received the research discharge pack, compared with those who received the commercial pack, were more likely to prolong exclusive breast-feeding (P = .004, one-tailed), to be partially breast-feeding at 4 months postpartum (P = .04, one-tailed), and to delay the daily use of solid foods in the infant's diet (P = .017, one-tailed). Among the women who received research counseling, the research discharge pack was associated with lower rates of rehospitalization of infants than was the commercial pack (1% v 14%; P = .014, two-tailed). We conclude that in high-risk maternity populations, commercial discharge materials for breast-feeding women should be replaced by materials consistent with the WHO Code.

Parental response to repeat testing of infants with 'false-positive' results in a newborn screening program.

Forty-five percent of the parents of 60 infants being retested in a newborn screening program for metabolic disorders understood that their infant was undergoing retesting because the first test result was abnormal. Fifty-five percent had incorrect or incomplete information, believing that retesting was routine, or that mistakes had been made in the original testing procedure, or they report being told nothing specific about the testing. Parents who were aware that the initial test was abnormal were no more anxious or depressed while waiting for the repeat test results than other parents. At a second interview after learning the normal results of the repeat test, both those parents informed of the initial abnormal result as well as those not informed were less anxious and depressed. However, 36% of the parents of these normal infants reported concern about the health of their infant because of the repeat testing. This concern was not related to a parent's knowledge that the initial test result was abnormal, but was greater in parents reporting that they had not received sufficient information about the screening/testing process and its significance for the health of their infant.

Pediatrician involvement in breast-feeding promotion: a national study of residents and practitioners.

OBJECTIVE: Physician support for breast-feeding mothers has been shown to improve breast-feeding rates, but no evaluation of the adequacy of physicians' breast-feeding-related training has been conducted. This study was designed to assess pediatricians' knowledge, attitudes, training, and activities related to breast-feeding promotion. METHODS: Surveys were mailed to a national random sample of pediatric residents (n = 999) and practitioners (n = 610) who were board certified within the previous 3 to 5 years. RESULTS: Response rates were 74% for residents and 69% for practitioners. Although more than 90% of respondents agreed that pediatricians should be involved in breast-feeding promotion, their clinical knowledge and experience did not suggest a high degree of competency. For example, practitioners were only slightly more aware of breast-feeding's protective effect against otitis media (71% vs 60%), and more than one quarter of both groups did not agree that exclusive breast-feeding is the most beneficial form of infant nutrition. Clinical advice often included inappropriate recommendations for breast-feeding termination or formula supplementation; only 64% of practitioners and 52% of residents knew that supplementing during the first few weeks of life may cause breast-feeding failure. For both groups, prior personal breast-feeding experience (ie, respondent or spouse had breast-fed an infant for 2 or more weeks) was a major determinant of improved clinical knowledge, more frequent activity, and greater self-confidence and perceived effectiveness in the area of breast-feeding promotion. Residents reported that the breast-feeding instruction provided during training was primarily in lecture format, with limited clinical opportunities to practice skills needed to assist breast-feeding mothers. Reflecting on their own training, more than 70% of practitioners recommended that more time be devoted to direct patient interaction and practice of counseling and problem-solving skills. CONCLUSIONS: These results indicate that residency training does not adequately prepare pediatricians for their role in breast-feeding promotion. Improvements in residency training and innovative continuing education programs should be implemented to help pediatricians meet the needs of their breast-feeding patients.

Absorption-edge transmission technique using Ce- 139 for measurement of stable iodine concentration.

We have investigated a technique for measuring stable iodine concentrations by absorption-edge transmission measurements using a Ce 139 radiation source. The lanthanum daughter emits characteristic x-rays whose energies just bracket the absorption edge of iodine at 33.2 keV. Relative transmission of these x-rays is sensitive to iodine concentration in the sample, but is relatively insensitive to other elements. By applying energy-selective beam filtration, it is possible to determine the relative transmission of these closely spaced x-ray energies with NaI(Tl) detectors. Optimizations of sample thickness, detector thickness, and Ce-139 source activity are discussed. Using sample volumes of about 10 ml, one can determine iodine concentration to an uncertainty (standard deviation) of +/- 5 microgram/ml with a 5-mCi source in a measurement time of 400 sec. Potential clinical applications of the in vitro technique are discussed, along with comparative aspects of the Ce-139 technique and other absorption and fluorescence techniques for measuring stable iodine.

Deadtime characteristics of Anger cameras.

The deadtime characterisitcs of Anger cameras are analyzed from theoretical and experimental points of view. An investigation of two Searle Radiographics cameras revealed that they had both paralyzable and nonparalyzable components, the deadtimes of which varied with analyzer window width, the energy of the gamma rays counted, and the position of the "time constant selector" switch in the A-scope module of these cameras. A mathematical model for two-component systems is presented.

Methods of correcting Anger camera deadtime losses.

Three different methods of correcting for Anger camera deadtime loss were investigated. These included analytic methods (mathematical modeling), the marker-source method, and a new method based on counting "pileup" events appearing in a pulse-height analyzer window positioned above the photopeak of interest. The studies were done with 99mTc on a Searle Radiographics camera with a measured deadtime of about 6 musec. Analytic methods were found to be unreliable because of unpredictable changes in deadtime with changes in radiation scattering conditions. Both the marker-source method and the pileup-counting method were found to be accurate to within a few percent for true counting rates of up to about 200 K cps, with the pileup-counting method giving better results. This finding applied to sources at depths ranging up to 10 cm of presdwood. The relative merits of the two methods are discussed.

Animal evaluation of technetium-99m triamide mercaptide complexes as potential renal imaging agents.

Technetium-99m mercaptoacetylglycylglycylglycine (MAG3), a [99mTc]triamide mercaptide (N3S) compound has been synthesized in an attempt to obviate the stereochemistry problems associated with the diamide dimercaptide (N2S2) ligands. Because initial studies have been promising, the terminal glycine on the MAG3 compound has been varied to create a new series of N3S compounds. Twelve new N3S complexes were initially screened in mice and the more promising complexes, 99mTc mercaptoacetylgylcylglycyl-glycine [( 99mTc]MAG3), 99mTc mercaptoacetylgylcylglycyl-L-alanine [( 99mTc]MAG2-Ala), and both complexes of 99mTc mercaptoeacetylglycylglycyl-L-asparagine [( 99mTc]MAG2-Asn) and 99mTc mercaptoacetylglycylglycyl-L-glutamine [( 99mTc]MAG2-Gln), were further evaluated in rats utilizing constant infusion blood clearances, extraction efficiencies and protein binding assays. The renal excretion of all these complexes compared favorably with simultaneously administered [131I]OIH and [125I]iothalamate. The triamide mercaptide complexes represent a new ligand class for 99mTc, which may provide a variety of complexes for the evaluation of renal tubular function.

Effects of meal size and correction technique on gastric emptying time: studies with two tracers and opposed detectors.

Geometric-mean correction of gastric radioactivity can be used to correct for the distribution, depth, and attenuation of the radionuclide. Anterior image counts were compared with the geometric mean of anterior and posterior counts, using a computer-assisted gamma camera. Phantom and human studies using Tc-99m and In-111 were used to evaluate the difference between anterior-only and geometric-mean data. The effect of meal size on gastric emptying was studied using Tc-99m and In-111 simultaneously as solid-phase and liquid-phase markers. Differences between the anterior data and AP-PA data corrected by geometic mean were compared in ten healthy male subjects. The average half-emptying times for solid food, with 1692-, 900-, and 300-g meals, were 277, 146, and 77 min, respectively. Average half-emptying times for liquid were 178, 81, and 38 min for the same meals.

Perception of radiation hazards.

The health risks of radiation have been carefully studied and are relatively well understood in comparison with other risks to the human environment. Public perception of these risks often is distorted, due in part to lack of familiarity with the actual risk levels involved. There is a need for dissemination to the public of accurate information on radiation risks as well as to patients and volunteer subjects for studies involving radiation exposures. Often such information can be presented meaningfully by comparing the risks of radiation exposure with other, more familiar risks. Natural background radiation is a universally present and generally accepted source of risk, and thus serves as one reference against which to compare the risks of other radiation exposures. Natural background radiation averages about 100 mrem/yr, but much higher levels are encountered in some parts of the US (400 mrem/yr) and worldwide (2,000 mrem/yr). These variations are due primarily to differences in cosmic ray intensity with altitude and in terrestrial radiation originating from soil and rocks. Radiation risks also may be compared with the risks of other human activities, both voluntary and involuntary. The former are useful for comparisons with the risks of voluntary radiation exposures such as occupational exposure and participation in medical or research procedures involving radiation. Involuntary radiation exposure, such as might result from the transportation and disposal of radioactive waste, poses a more complicated issue. Comparisons of such exposures to natural background radiation levels and their variations are helpful. Two other concepts that have been proposed for assessing the relative risk of low-level radiation exposure are "de minimus risk" and "probability of causation." The former suggests that there is some minimal level of involuntary risk that can be considered acceptable, provided it carries with it some benefit to society or the individual. The latter is a concept that has been introduced in legislation to decide compensation for alleged injuries from radiation exposures.

Stable superoxide dismutase (SOD)-mimetic ternary human serum albumin-Cu(II)(3,5-diisopropylsalicylate)2/Cu(II)2(3,5-diisopropylsalicylate)4 complexes in tissue distribution of the binary complex.

Copper(II)2(3,5-diisopropylsalicylate)4 [Cu(II)2(3,5-DIPS)4] has been found to have antiinflammatory, antiulcer, anticancer, anticonvulsant, antimutagenic, antidiabetic, analgesic, and radiation protection and recovery activities. It has also been found to reduce ischemia-reperfusion injury. Because of these activities it was of interest to understand how this compound is transported in the body to affected tissues. Evidence supporting the suggested formation of ternary human serum albumin (HSA)-Cu(II)(3,5-DIPS)2 or Cu(II)2(3,5-DIPS)4 complexes was obtained using ultraviolet spectrophotometry, dialysis, and atomic absorption spectrophotometry or atomic emission spectroscopy. Superoxide dismutase (SOD)-mimetic activity was also determined using the xanthine/xanthine oxidase/cytochrome c system. Ultraviolet spectra of aqueous solution mixtures of Cu(II)2(3,5-DIPS)4 in equilibrium with 2Cu(II)(3,5-DIPS)2 and HSA as well as aqueous solutions of solid Cu(II)2(3,5-DIPS)4 obtained by stirring the solid with an aqueous solution of HSA showed no obvious change in absorbance to indicate ternary complex formation. However, comparison of ultraviolet spectra taken before and after dialysis supports the suggested bonding of Cu(II)(3,5-DIPS)2 or Cu(II)2(3,5-DIPS)4 to HSA. Comparison of copper concentrations before and after dialysis also supports the suggested bonding of Cu(II)(3,5-DIPS)2 or Cu(II)2(3,5-DIPS)4 to HSA. Based upon these data it is plausible that Cu(II)(3,5-DIPS)2 or Cu(II)2(3,5-DIPS)4 form stable ternary complexes with HSA. These stable ternary complexes were also found to have SOD-mimetic activity.