Global gene expression analysis of bovine somatic cell nuclear transfer blastocysts and cotyledons.

Low developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos is a universal problem. Abnormal placentation has been commonly reported in SCNT pregnancies from a number of species. The present study employed Affymetrix bovine expression microarrays to examine global gene expression patterns of SCNT and in vivo produced (AI) blastocysts as well as cotyledons from day-70 SCNT and AI pregnancies. SCNT and AI embryos and cotyledons were analyzed for differential expression. Also in an attempt to establish a link between abnormal gene expression patterns in early embryos and cotyledons, differentially expressed genes were compared between the two studies. Microarray analysis yielded a list of 28 genes differentially expressed between SCNT and AI blastocysts and 19 differentially expressed cotyledon genes. None of the differentially expressed genes were common to both groups, although major histocompatibility complex I (MHCI) was significant in the embryo data and approached significance in the cotyledon data. This is the first study to report global gene expression patterns in bovine AI and SCNT cotyledons. The embryonic gene expression data reported here adds to a growing body of data that indicates the common occurrence of aberrant gene expression in early SCNT embryos.

Structural requirements for the modulatory effect of 6-substituted pterins on interleukin 2 receptor binding.

(6R)-5,6,7,8-Tetrahydrobiopterin is produced by stimulated human T lymphocytes, and is known to affect various aspects of interleukin-2-directed T cell proliferation. Using an increased apparent affinity of interleukin 2 receptor to interleukin 2 as a measure of activity, this study explores whether other 6-substituted pterins might have the same effect, and what structural features are necessary for activity. Of the compounds tested, only the T-lymphocyte-derived (6R)-5,6,7,8-tetrahydrobiopterin was active. The diastereomeric (6S)-5,6,7,8-tetrahydrobiopterin was inactive, as were 7,8-dihydrobiopterin, sepiapterin, 5,6,7,8-tetrahydroneopterin, 6,7-dimethyl-5,6,7,8-tetrahydropterin and 6-hydroxymethylpterin. 7,8-Dihydroneopterin and neopterin were also found to be inactive. It follows that neither of these compounds participates in the feedback modulation of IL-2 receptor affinity, although both of them can be detected upon IFN-gamma stimulation of human monocytes/macrophages. A computer-based molecular modelling study of (6R)-5,6,7,8-tetrahydrobiopterin and (6R)-5,6,7,8-tetrahydroneopterin revealed substantial differences in overall shape between the two molecules, with certain features figuring prominently in the low-energy conformers of (6R)-5,6,7,8-tetrahydrobiopterin.

The yeast recombinational repair protein Rad59 interacts with Rad52 and stimulates single-strand annealing.

The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.

A novel allele of RAD52 that causes severe DNA repair and recombination deficiencies only in the absence of RAD51 or RAD59.

With the use of an intrachromosomal inverted repeat as a recombination reporter, we have shown that mitotic recombination is dependent on the RAD52 gene, but reduced only fivefold by mutation of RAD51. RAD59, a component of the RAD51-independent pathway, was identified previously by screening for mutations that reduced inverted-repeat recombination in a rad51 strain. Here we describe a rad52 mutation, rad52R70K, that also reduced recombination synergistically in a rad51 background. The phenotype of the rad52R70K strain, which includes weak gamma-ray sensitivity, a fourfold reduction in the rate of inverted-repeat recombination, elevated allelic recombination, sporulation proficiency, and a reduction in the efficiency of mating-type switching and single-strand annealing, was similar to that observed for deletion of the RAD59 gene. However, rad52R70K rad59 double mutants showed synergistic defects in ionizing radiation resistance, sporulation, and mating-type switching. These results suggest that Rad52 and Rad59 have partially overlapping functions and that Rad59 can substitute for this function of Rad52 in a RAD51 rad52R70K strain.

Multiple-event study of bioretention for treatment of urban storm water runoff.

Bioretention is a novel best management practice for urban storm water, employed to minimize the impact of urban runoff during storm events. Bioretention consists of porous media layers that can remove pollutants from infiltrating runoff via mechanisms that include adsorption, precipitation, and filtration. However, the effectiveness of bioretention in treating repetitive inputs of runoff has not been investigated. In this study, a bioretention test column was set up and experiments proceeded once every week for a total of 12 tests. Through all 12 repetitions, the infiltration rate remained constant (0.35 cm/min). All 12 tests demonstrated excellent removal efficiency for TSS, oil/grease, and lead (99%). For total phosphorus, the removal efficiency was about 47% the system removal efficiency ranged from 2.3% to 23%. Effluent nitrate concentration became higher than the influent concentration during the first 28 days and removal efficiency ranged from 9% to 20% afterward. Some degree of denitrification was apparently proceeding in the bioretention system. Overall, the top mulch layer filtered most of TSS in the runoff and prevented the bioretention media from clogging during 12 repetitions. Runoff quality was improved by the bioretention column.

Modulation of interleukin 2 high affinity binding to human T cells by a pyrimidodiazepine insect metabolite.

An insect metabolite containing the little known pyrimido[4,5-b][1,4]diazepine ring system has been found to act as an effective mimic of tetrahydrobiopterin in its ability to modulate the affinity of interleukin 2 (IL-2) for its receptors on human T cells. Semi-empirical molecular orbital calculations reveal that while tetrahydrobiopterin has considerable flexibility, the pyrimidodiazepine has rather few conformational options and offers a useful model for exploring the nature of the pterin binding site.

Mutations in noncoding regions of the proteolipid protein gene in Pelizaeus-Merzbacher disease.

BACKGROUND: Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive dysmyelinating disorder of the CNS. Duplications or point mutations in exons of the proteolipid protein (PLP) gene are found in most patients. OBJECTIVE: To describe five patients with PMD who have mutations in noncoding regions of the PLP gene. METHODS: Quantitative multiplex PCR and Southern blot analyses were used to detect duplication of the PLP gene, and DNA sequence analysis, including exon-intron borders, was used to detect mutation of the PLP gene. RESULTS: Duplication of the PLP gene was ruled out, and mutations were identified in noncoding regions of five patients in four families with PMD. In two brothers with a severe form of PMD, a G to T transversion at IVS6+3 was detected. This mutation resulted in skipping of exon 6 in the PLP mRNA of cultured fibroblasts. A patient who developed nystagmus at 16 months and progressive spastic ataxia at 18 months was found to have a 19-base pair (bp) deletion of a G-rich region near the 5' end of intron 3 of the PLP gene. A patient with a T to C transition at IVS3+2 and a patient with an A to G transition at IVS3+4 have the classic form of PMD. These, like the 19-bp deletion, are in intron 3, which is involved in PLP/DM20 alternative splice site selection. CONCLUSIONS: Mutations in introns of the PLP gene, even at positions that are not 100% conserved at splice sites, are an important cause of PMD.

Removal of Cu(II) and Cd(II) from aqueous solution by seafood processing waste sludge.

Dried waste slurry generated in seafood processing factories has been shown to be an effective adsorbent for the removal of heavy metals from dilute solutions. Characterization of the sludge surface with scanning electron microscope and X-ray microanalyzer were carried out to evaluate the components on the sludge surface that are related to the adsorption of metal ions. Aluminum and calcium, as well as organic carbon are distributed on the surface of sludge. Alkalimetric titration was used to characterize the surface acidity of the sludge sample. The surface acidity constants, pKa1s and pKa2s, were 5.80 and 9.55, respectively. Batch as well as dynamic adsorption studies were conducted with 10(-5) to 5 x 10(-3) M Cu(II) and Cd(II). A surface complexation model with the diffuse layer model successfully predicted Cu(II) and Cd(II) removals in single metal solutions. Predictions of sorption in binary-adsorbate systems based on single-adsorbate data fits represented competitive sorption data reasonably well over a wide range of conditions. The breakthrough capacity found from column studies was different for each metal ion and the data reflect the order of metal affinity for the adsorbent material very well.

Loading estimates of lead, copper, cadmium, and zinc in urban runoff from specific sources.

Urban stormwater runoff is being recognized as a substantial source of pollutants to receiving waters. A number of investigators have found significant levels of metals in runoff from urban areas, especially in highway runoff. As an initiatory study, this work estimates lead, copper, cadmium, and zinc loadings from various sources in a developed area utilizing information available in the literature, in conjunction with controlled experimental and sampling investigations. Specific sources examined include building siding and roofs; automobile brakes, tires, and oil leakage; and wet and dry atmospheric deposition. Important sources identified are building siding for all four metals, vehicle brake emissions for copper and tire wear for zinc. Atmospheric deposition is an important source for cadmium, copper, and lead. Loadings and source distributions depend on building and automobile density assumptions and the type of materials present in the area examined. Identified important sources are targeted for future comprehensive mechanistic studies. Improved information on the metal release and distributions from the specific sources, along with detailed characterization of watershed areas will allow refinements in the predictions.

Experience with a planned exposure program for the control of enzootic transmissible gastroenteritis in swine.

Oral inoculation of pregnant sows and gilts with a homogenate of pig intestines containing live, virulent transmissible gastroenteritis (TGE) virus was associated with significant (P < 0.01) reduction of mortality in nursery pigs in a herd affected with enzootic TGE. The mortality of weaned pigs from April through June 1981, when sows were not vaccinated or inoculated, was 9.3%. Mortality of weanling pigs from July through December 1981 was 5.7% (P < 0.01), and stayed consistently between 2.6 and 4.8% from 1982 through 1990. After beginning the exposure program, there were no clinical signs attributable to TGE in the farrowing house or nursery until 1986, when recrudescence of TGE was evident in the nursery. Although the source of the virus could not be substantiated, recrudescence suggested that TGE virus would not be eradicated as long as exposure was continued. The most plausible explanation for the decrease in the incidence of diarrhea and mortality in the nursery pigs after initiation of the planned exposure program is that a higher, longer lasting amount of lactogenic immunity was provided to suckling pigs. Presumably over time, this resulted in less TGE virus carryover into the nursery by weanling pigs, thereby reducing and confining the environmental challenge to the nursery. Strict all-in, all-out pig movement in the nursery with thorough cleaning and disinfecting presumably further reduced the environmental challenge.

Laboratory study of biological retention for urban stormwater management.

Urban stormwater runoff contains a broad range of pollutants that are transported to natural water systems. A practice known as biological retention (bioretention) has been suggested to manage stormwater runoff from small, developed areas. Bioretention facilities consist of porous soil, a topping layer of hardwood mulch, and a variety of different plant species. A detailed study of the characteristics and performance of bioretention systems for the removal of several heavy metals (copper, lead, and zinc) and nutrients (phosphorus, total Kjeldahl nitrogen [TKN], ammonium, and nitrate) from a synthetic urban stormwater runoff was completed using batch and column adsorption studies along with pilot-scale laboratory systems. The roles of the soil, mulch, and plants in the removal of heavy metals and nutrients were evaluated to estimate the treatment capacity of laboratory bioretention systems. Reductions in concentrations of all metals were excellent (> 90%) with specific metal removals of 15 to 145 mg/m2 per event. Moderate reductions of TKN, ammonium, and phosphorus levels were found (60 to 80%). Little nitrate was removed, and nitrate production was noted in several cases. The importance of the mulch layer in metal removal was identified. Overall results support the use of bioretention as a stormwater best management practice and indicate the need for further research and development.

Project HITE (health individualization and teacher education): a health curriculum for 3-, 4- and 5-year olds.

South Carolina has been a national leader in such health problems as cardiovascular disease, syphillis and gonorrhea. Since many of the problems could be attributed to poor health habits, education of the state's residents seemed in order. The education/re-education of inaccessible adults with firmly established health habits--poor or otherwise--was not feasible, but the education of impressionable, readily accessible three-, four- and five-year olds was possible. Through a grant from the federal government, an individualized health curriculum that could accommodate the differences of the learners and include vital health content was developed. This project of developing and distributing the health curriculum to teachers of young children was called Project HITE (Health Individualization and Teacher Education).

Effects of amino acid substitutions in and around the arginine-glycine-aspartic acid (RGD) sequence on fertilization and parthenogenetic development in mature bovine oocytes.

Integrins have been shown to be involved in the process of fertilization and many integrin-ligand interactions are mediated through the recognition of an arginine-glycine-aspartic acid (RGD) sequence. Despite the fact the RGD domain is a principal player in determining the functional characteristics of an adhesive protein, increasing evidence has accumulated implicating the amino acids flanking the RGD sequence in determining the functional properties of the RGD-containing protein. A set of linear peptides in which the amino acid sequence in and around the RGD tri-peptide was modified was synthesized to better understand the specificity of the RGD-receptor interaction. Mature oocytes were fertilized in vitro in the presence of RGD-containing and RGD-modified peptides. Both the RGD-containing and RGD-modified peptides impaired the ability of sperm to fertilize bovine oocytes, illustrated by a reduction in cleavage. The linear modified RGD containing peptides were also examined for their ability to induce parthenogenetic development with the objective of providing a linear RGD peptide with greater biological activity than the one (GRGDSPK) used previously (Campbell et al., 2000). The data demonstrate the specificity of the receptor for the RGD sequence, further implicate the involvement of integrins in the process of bovine fertilization, and illustrate the importance of the amino acids surrounding the RGD sequence in determining the binding and functional properties of RGD-containing peptides. The data support the findings that a linear RGD peptide can block fertilization and that amino acids around the RGD sequence have an impact on the biological activity of the receptor.

Targeting the vulnerable in emergency situations: who is vulnerable?

BACKGROUND: Emergencies such as wars and natural disasters increase the vulnerability of affected populations and expose these populations to risks such as disease, violence, and hunger. Emergency public health interventions aim to mitigate these effects by providing basic minimum requirements, reducing vulnerability, and reducing exposure to risk. Targeted services are generally aimed at children under 5. Mortality rates among young children are higher than the crude mortality rate (CMR) among the whole population in emergency settings, so attention is focused on this age group. However, even under normal conditions mortality is higher in young children. This analysis compared the relative risk of death for young children with that for older children and adults under normal conditions and in emergency settings. METHODS: Mortality data from refugee camps set up in response to three different emergencies were examined. Baseline mortality rates in the refugees' countries of origin were calculated from published data. Relative risks between normal and emergency conditions were calculated and compared. FINDINGS: Mortality rates were higher among children under 5 than among older children and adults both under normal circumstances and in the emergency setting in camps in Tanzania, Uganda, and Zaire. However, the relative risk for under-5 versus over-5 mortality was smaller under emergency conditions than under normal circumstances. Thus, children over 5 and adults are disproportionately more affected by exposure to emergency risks than are younger children. INTERPRETATION: If the objective of intervention, to reduce mortality, is to be achieved, the population over the age of 5 cannot be ignored. Emergency public health needs to develop specific tools to investigate risk in other age groups (as well as children under 5), to identify causes, and to design programmes to address such needs.

A mutational analysis of the 5' HoxD genes: dissection of genetic interactions during limb development in the mouse.

Using gene targeting in mice, we have undertaken a systematic mutational analysis of the homeobox-containing 5' HoxD genes. In particular, we have characterized the limb defects observed in mice with independent targeted disruptions of hoxd-12 and hoxd-13. Animals defective for hoxd-12 are viable, fertile, and appear outwardly normal yet have minor autopodal defects in the forelimb which include a reduction in the bone length of metacarpals and phalanges, and a malformation of the distal carpal bone d4. The limb phenotypes observed in hoxd-13 mutant mice are more extensive, including strong reductions in length, complete absences, or improper segmentations of many metacarpal and phalangeal bones. Additionally, the d4 carpal bone is not properly formed and often produces an extra rudimentary digit. To examine the genetic interactions between the 5' HoxD genes, we bred these mutant strains with each other and with our previously characterized hoxd-11 mouse to produce a series of trans-heterozygotes. Skeletal analyses of these mice reveal that these genes interact in the formation of the vertebrate limb, since the trans-heterozygotes display phenotypes not present in the individual heterozygotes, including more severe carpal, metacarpal and phalangeal defects. Some of these phenotypes appear to be accounted for by a delay in the ossification events in the autopod, which lead to either the failure of fusion or the elimination of cartilaginous elements. Characteristically, these mutations lead to the overall truncation of digits II and V on the forelimb. Additionally, some trans-animals show the growth of an extra postaxial digit VI, which is composed of a bony element resembling a phalange. The results demonstrate that these genes interact in the formation of the limb. In addition to the previously characterized paralogous interactions, a multitude of interactions between Hox genes is used to finely sculpt the forelimb. The 5' Hox genes could therefore act as a major permissive genetic milieu that has been exploited by evolutionary adaptation to form the tetrapod limbs.