Validation of highly polymorphic fluorescent multiplex short tandem repeat systems using two generations of DNA sequencers

Validation studies are a crucial requirement before implementation of new genetic typing systems for clinical diagnostics or forensic identity. Two different fluorescence-based multiplex DNA profiling systems composed of amelogenin, HumD21S11 and HumFGA (referred to as multiplex 1A), and HumD3S1358, HumD21S11 and HumFGA (multiplex 1B) have been evaluated for use in forensic identification using the Applied Biosystems Model 373A and Prism 377 DNA Sequencers, respectively. Experiments were aimed at defining the limit of target DNA required for reliable profiling, the level of degradation that would still permit amplification of the short tandem repeat (STR) loci examined, and the robustness of each locus in the multiplexes after samples were exposed to environmental insults. In addition, the specificity of the multiplexes was demonstrated using nonhuman DNAs. Forensically relevant samples such as cigarette butts, chewing gum, fingernails and envelope flaps were processed using both an organic extraction procedure and a QIAamp protocol. DNAs and resultant multiplex STR profiles were compared. The validation of the triplex STR systems was extended to include over 140 nonprobative casework specimens and was followed with a close monitoring of initial casework (over 300 exhibits). Our results document the robustness of these multiplex STR profiling systems which, when combined with other multiplex systems, could provide a power of discrimination of approximately 0.9999.     

Sizzled: a secreted Xwnt8 antagonist expressed in the ventral marginal zone of Xenopus embryos

An expression cloning screen was used to isolate a novel gene homologous to the extracellular cysteine-rich domain of frizzled receptors. The gene (which we called sizzled for secreted frizzled) was shown to encode a soluble secreted protein, containing a functional signal sequence but no transmembrane domains. Sizzled (szl) is capable of inhibiting Xwnt8 as assayed by (1) dose-dependent inhibition of siamois induction by Xwnt8 in animal caps, (2) rescue of embryos ventralized by Xwnt8 DNA and (3) inhibition of XmyoD expression in the marginal zone. Szl can dorsalize Xenopus embryos if expressed after the midblastula transition, strengthening the idea that zygotic expression of wnts and in particular of Xwnt8 plays a role in antagonizing dorsal signals. It also suggests that inhibiting ventralizing wnts parallels the opposition of BMPs by noggin and chordin. szl expression is restricted to a narrow domain in the ventral marginal zone of gastrulating embryos. szl thus encodes a secreted antagonist of wnt signaling likely involved in inhibiting Xwnt8 and XmyoD ventrally and whose restricted expression represents a new element in the molecular pattern of the ventral marginal zone.     

Effect of Depo-Provera or Microgynon on the painful crises of sickle cell anemia patients

Forty-three homozygous (SS) female sickle cell anemic patients with a history of at least one painful crisis per month and desiring a reversible contraceptive were administered DMPA/3 months or Microgynon monthly. A third group of 16 surgically sterilized patients served as control. Patients were followed for 1 year to assess possible effects of the contraceptives on the patients' painful crises. No changes were observed in any of the groups in the hematological parameters. At the end of the study, 70% of the patients receiving DMPA were pain-free and only 16% of those still reporting painful crises rated them as intense. Patients receiving Microgynon also had an amelioration of the painful crises, although at a lower rate; after 12 months, 45.5% still experienced some crises. Although less marked than in the other groups, 50.5% of the control patients also reported an improvement of their painful crisis, which may be a result of closer medical care.     

Regioselectivity of nitroglycerin denitration by flavoprotein nitroester reductases purified from two Pseudomonas species

Two species of Pseudomonas capable of utilizing nitroglycerin (NG) as a sole nitrogen source were isolated from NG-contaminated soil and identified as Pseudomonas putida II-B and P. fluorescens I-C. While 9 of 13 laboratory bacterial strains that presumably had no previous exposure to NG could degrade low concentrations of NG (0.44 mM), the natural isolates tolerated concentrations of NG that were toxic to the lab strains (1.76 mM and higher). Whole-cell studies revealed that the two natural isolates produced different mixtures of the isomers of dinitroglycerol (DNG) and mononitroglycerol (MNG). A monomeric, flavin mononucleotide-containing NG reductase was purified from each natural isolate. These enzymes catalyzed the NADPH-dependent denitration of NG, yielding nitrite. Apparent kinetic constants were determined for both reductases. The P. putida enzyme had a Km for NG of 52 +/- 4 microM, a Km for NADPH of 28 +/- 2 microM, and a Vmax of 124 +/- 6 microM x min(-1), while the P. fluorescens enzyme had a Km for NG of 110 +/- 10 microM, a Km for NADPH of 5 +/- 1 microM, and a Vmax of 110 +/- 11 microM x min(-1). Anaerobic titration experiments confirmed the stoichiometry of NADPH consumption, changes in flavin oxidation state, and multiple steps of nitrite removal from NG. The products formed during time-dependent denitration reactions were consistent with a single enzyme being responsible for the in vivo product distributions. Simulation of the product formation kinetics by numerical integration showed that the P. putida enzyme produced an approximately 2-fold molar excess of 1,2-DNG relative to 1,3-DNG. This result could be fortuitous or could possibly be consistent with a random removal of the first nitro group from either the terminal (C-1 and C-3) positions or middle (C-2) position. However, during the denitration of 1,2-DNG, a 1.3-fold selectivity for the C-1 nitro group was determined. Comparable simulations of the product distributions from the P. fluorescens enzyme showed that NG was denitrated with a 4.6-fold selectivity for the C-2 position. Furthermore, a 2.4-fold selectivity for removal of the nitro group from the C-2 position of 1,2-DNG was also determined. The MNG isomers were not effectively denitrated by either purified enzyme, which suggests a reason why NG could not be used as a sole carbon source by the isolated organisms.     

Omental transposition flap for gross locally recurrent breast cancer

A randomized double-blind controlled trial was undertaken to study the efficacy of a cow udder ointment versus petroalatum alone. A total of 30 patients participated in the study. Sixteen patients completed the trial with 8 of 9 patients improving in the active group and 6 of 7 patients improving in the placebo group. This difference was not statistically significant. The use of a cow udder ointment for psoriasis cannot be supported, particularly with the potential for side effects.     

Apolipoprotein B and AI distributions in the United States, 1988-1991: results of the National Health and Nutrition Examination Survey III (NHANES III)

Serum apolipoproteins (apo) B and AI were measured in a probability sample of the noninstitutionalized US civilian population, ages > or = 4 years, which included non-Hispanic whites, non-Hispanic blacks, and Mexican-Americans. Apo B concentrations were the same in males and females, lower in black males than in other males, low in childhood (approximately 0.80 g/L) and increasing to approximately 1.2 g/L in adults, and higher in younger women on hormones. Apo AI was higher in females than males, higher in blacks than in others, remained constant from childhood to adulthood (approximately 1.35 g/L) in males, but increased with age (approximately 1.30 g/L to approximately 1.55 g/L) in females, and was higher in women taking hormones. These are the first national probability estimates of apo B and apo AI in the US and are referable to the WHO-IFCC First International Reference Materials for apo AI and B.