Composition modulation by twinning in InAsSb nanowires.

We observe a composition modulated axial heterostructure in zincblende (ZB) InAs0.90Sb0.10 nanowires initiated by pseudo-periodic twin boundaries using scanning tunneling microscopy. The twin boundaries exhibit four planes with reduced Sb concentration due to a lower Sb incorporation during lateral overgrowth of a 4H wurtzite as compared to a ZB stacking sequence. We anticipate that this leads to compositional band offsets in addition to known structural band offsets present between 4H and ZB polytypes, changing the band alignment from type II to type I.

Modulation of behavioral networks by selective interneuronal inactivation.

Gamma-aminobutyric acid (GABA)-ergic disturbances are hallmark features of schizophrenia and other neuropsychiatric disorders and encompass multiple interneuronal cell types. Using bacterial artificial chromosome-driven, miRNA silencing technology we generated transgenic mouse lines that suppress glutamic acid decarboxylase 1 (GAD1) in either cholecystokinin (CCK)- or neuropeptide Y (NPY)-expressing interneurons. In situ lipidomic and proteomic analyses on brain tissue sections revealed distinct, brain region-specific profiles in each transgenic line. Behavioral analyses revealed that suppression of GAD1 in CCK+ interneurons resulted in locomotor and olfactory sensory changes, whereas suppression in NPY+ interneurons affected anxiety-related behaviors and social interaction. Both transgenic mouse lines had altered sensitivity to amphetamine albeit in opposite directions. Together, these data argue that reduced GAD1 expression leads to altered molecular and behavioral profiles in a cell type-dependent manner, and that these subpopulations of interneurons are strong and opposing modulators of dopamine system function. Furthermore, our findings also support the hypothesis that neuronal networks are differentially controlled by diverse inhibitory subnetworks.

Phosphine resistance in India is characterised by a dihydrolipoamide dehydrogenase variant that is otherwise unobserved in eukaryotes.

Phosphine (PH3) fumigation is the primary method worldwide for controlling insect pests of stored commodities. Over-reliance on phosphine, however, has led to the emergence of strong resistance. Detailed genetic studies previously identified two loci, rph1 and rph2, that interact synergistically to create a strong resistance phenotype. We compared the genetics of phosphine resistance in strains of Rhyzopertha dominica and Tribolium castaneum from India and Australia, countries having similar pest species but widely differing in pest management practices. Sequencing analysis of the rph2 locus, dihydrolipoamide dehydrogenase (dld), identified two structurally equivalent variants, Proline49>Serine (P49S) in one R. dominica strain and P45S in three strains of T. castaneum from India. These variants of the DLD protein likely affect FAD cofactor interaction with the enzyme. A survey of insects from storage facilities across southern India revealed that the P45/49S variant is distributed throughout the region at very high frequencies, in up to 94% of R. dominica and 97% of T. castaneum in the state of Tamil Nadu. The abundance of the P45/49S variant in insect populations contrasted sharply with the evolutionary record in which the variant was absent from eukaryotic DLD sequences. This suggests that the variant is unlikely to provide a strong selective advantage in the absence of phosphine fumigation.

Novel animal models for studying complex brain disorders: BAC-driven miRNA-mediated in vivo silencing of gene expression.

In schizophrenia, glutamic acid decarboxylase 1 (GAD1) disturbances are robust, consistently observed, cell-type specific and represent a core feature of the disease. In addition, neuropeptide Y (NPY), which is a phenotypic marker of a sub-population of GAD1-containing interneurons, has shown reduced expression in the prefrontal cortex in subjects with schizophrenia, suggesting that dysfunction of the NPY+ cortical interneuronal sub-population might be a core feature of this devastating disorder. However, modeling gene expression disturbances in schizophrenia in a cell type-specific manner has been extremely challenging. To more closely mimic these molecular and cellular human post-mortem findings, we generated a transgenic mouse in which we downregulated GAD1 mRNA expression specifically in NPY+ neurons. This novel, cell type-specific in vivo system for reducing gene expression uses a bacterial artificial chromosome (BAC) containing the NPY promoter-enhancer elements, the reporter molecule (eGFP) and a modified intron containing a synthetic microRNA (miRNA) targeted to GAD1. The animals of isogenic strains are generated rapidly, providing a new tool for better understanding the molecular disturbances in the GABAergic system observed in complex neuropsychiatric disorders such as schizophrenia. In the future, because of the small size of the silencing miRNAs combined with our BAC strategy, this method may be modified to allow generation of mice with simultaneous silencing of multiple genes in the same cells with a single construct, and production of splice-variant-specific knockdown animals.

DNA polymerase activities of human milk.

DNA polymerases have been partially purified from human milk. A DNA polymerase detected by phosphocellulose chromatography is similar to the enzymes of RNA tumor viruses in that a hybrid of polyriboadenylate and oligodeoxythymidylate is a better template than is DNA. However, this polymerase differed from that of the RNA tumor viruses in its chromatographic behavior. Three different methods of detecting "reverse transcriptase" activity failed to correlate with the donor's family history of cancer.

Crossinhibitory activities of Ngn1 and Math1 allow specification of distinct dorsal interneurons.

Distinct classes of neurons are generated from progenitor cells distributed in characteristic dorsoventral patterns in the developing spinal neural tube. We define restricted neural progenitor populations by the discrete, nonoverlapping expression of Ngn1, Math1, and Mash1. Crossinhibition between these bHLH factors is demonstrated and provides a mechanism for the generation of discrete bHLH expression domains. This precise control of bHLH factor expression is essential for proper neural development since as demonstrated in both loss- and gain-of-function experiments, expression of Math1 or Ngn1 in dorsal progenitor cells determines whether LH2A/B- or dorsal Lim1/2-expressing interneurons will develop. Together, the data suggest that although Math1 and Ngn1 appear to be redundant with respect to neurogenesis, they have distinct functions in specifying neuronal subtype in the dorsal neural tube.

Gene interactions constrain the course of evolution of phosphine resistance in the lesser grain borer, Rhyzopertha dominica.

Phosphine, a widely used fumigant for the protection of stored grain from insect pests, kills organisms indirectly by inducing oxidative stress. High levels of heritable resistance to phosphine in the insect pest of stored grain, Rhyzopertha dominica have been detected in Asia, Australia and South America. In order to understand the evolution of phosphine resistance and to isolate the responsible genes, we have undertaken genetic linkage analysis of fully sensitive (QRD14), moderately resistant (QRD369) and highly resistant (QRD569) strains of R. dominica collected in Australia. We previously determined that two loci, rph1 and rph2, confer high-level resistance on strain QRD569, which was collected in 1997. We have now confirmed that rph1 is responsible for the moderate resistance of strain QRD369, which was collected in 1990, and is shared with a highly resistant strain from the same geographical region, QRD569. In contrast, rph2 by itself confers only very weak resistance, either as a heterozygote or as a homozygote and was not discovered in the field until weak resistance (probably due to rph1) had become ubiquitous. Thus, high-level resistance against phosphine has evolved via stepwise acquisition of resistance alleles, first at rph1 and thereafter at rph2. The semi-dominance of rph2 together with the synergistic interaction between rph1 and rph2 would have led to rapid selection for homozygosity. A lack of visible fitness cost associated with alleles at either locus suggests that the resistance phenotype will persist in the field.

Heme synthetase deficiency in human protoporphyria. Demonstration of the defect in liver and cultured skin fibroblasts.

The final step in heme biosynthesis is chelation of porphyrin with Fe++ catalyzed by the mitochondrial enzyme heme synthetase. We have employed a sensitive radiochemical assay for this enzyme, using 59Fe and deuteroporphyrin or protoporphyrin as substrates. In this method iron is maintained in the ferrous state, oxygen is excluded from the incubation system, and labeled heme product is extracted into ethyl acetate. This assay has been used to measure the activity of heme synthetase in homogenates of liver, obtained by needle biopsy, and in sonicates of human skin fibroblasts, cultured in vitro. In addition, activity of the first enzyme of the heme synthetic pathway, delta-aminolevulinic acid synthetase, has been measured in fibroblast lysates. Lysates of fibroblasts from eight patients with protoporphyria had activities of delta-aminolevulinic acid synthetase which did not differ significantly from those of eight normal fibroblast lines, whereas activity of heme synthetase, with either deuteroporphyrin or protoporphyrin as substrate, was markedly decreased in sonicates of skin fibroblasts from these patients, the mean being 8% of control with deuteroporphyrin and 14% with protoporphyrin as substrate. In homogenates of liver from five patients with protoporphyria, activity of heme synthetase was also significantly less than that found in six patients without prophyria, the mean being 13% of control with protoporphyrin as substrate. These results provide evidence that decreased activity of heme synthetase is the basic defect in the heme synthetic pathway in protoporphyria. This deficiency is probably responsible for protoporphyrin accumulation and hence the biochemical and clinical features observed in protoporphyria.

Expression of a Self-Incompatibility Glycoprotein (S2-Ribonuclease) from Nicotiana alata in Transgenic Nicotiana tabacum.

In Nicotiana alata, self-incompatibility is controlled by a single locus, designated the S-locus, with multiple alleles. Stylar products of these alleles are ribonucleases that are secreted mainly in the transmitting tract tissues. N. tabacum plants were transformed with constructs containing the S2-cDNA and genomic S2-sequences from N. alata that were linked to the cauliflower mosaic virus 35S promoter. Unlike other genes controlled by this promoter, the genes were expressed most highly in mature floral organs. This pattern of expression was observed at both the protein and RNA levels. The S2-glycoprotein was detected in the stylar transmitting tract tissues of the transgenic plants. The transgene product was secreted, had ribonuclease activity, and was glycosylated with the correct number of glycan chains. However, the maximum level of S2-glycoprotein in styles of the transgenic plants was approximately 100-fold lower than that found in N. alata styles carrying the S2-allele. Perhaps because of this lower protein level, the plants showed no changes in the incompatibility phenotype.

Effect of condensed tannin extract supplementation on growth performance, nitrogen balance, gas emissions, and energetic losses of beef steers.

Condensed tannins (CT) may decrease greenhouse gas emissions and alter the site of N excreted by ruminants. We evaluated the effect of top-dressing a steam-flaked corn-based finishing diet (14.4% CP and NEg 1.47 Mcal/kg) for beef cattle with a commercially available CT extract at 3 levels (0, 0.5, and 1.0% of diet, DM basis). Angus-crossbred steers ( = 27; 350 ± 32 kg initial BW) were individually fed via Calan gates for 126 d. Diet digestibility and N balance were estimated after 34 and 95 d on feed (Phase 1 and Phase 2, respectively) using titanium dioxide as a marker of fecal output and the creatinine:BW ratio as a marker for urine output. Ruminal CH and metabolic CO fluxes were measured using a GreenFeed system (C-Lock Inc., Rapid City, SD) for 2 sampling periods that coincided with fecal and urine sampling. Urine energy loss was estimated from urine N excretion, assuming all excreted N was urea. Oxygen consumption was estimated from CO production assuming a respiratory quotient of 1.05. Average daily gain (2.08, 2.14, and 2.08 kg/d for 0, 0.5, and 1.0% CT, respectively) and G:F did not differ ( = 0.88) among treatments. Starch intake and OM intake did not differ ( ≥ 0.42) among treatments during each phase. Apparent total tract starch digestibility during Phase 1 linearly decreased ( = 0.04) with inclusion of CT. Apparent total tract digestibility of OM and starch were not different among treatments ( ≥ 0.13) during Phase 2. Nitrogen intake did not differ ( ≥ 0.16) among treatments during each phase, but fecal N excretion linearly increased ( = 0.05) with inclusion of CT during Phase 1. Urinary N excretion was not different ( ≥ 0.39) among treatments during both phases, but urinary N as a proportion of total N excretion linearly decreased ( = 0.01) when CT was included in the diet during Phase 1. Retained N was not different ( ≥ 0.27) among treatments during each phase. Fluxes of CO were similar ( ≥ 0.37) among treatments during both phases. No differences ( ≥ 0.23) were observed for percentage of GE intake lost as CH (2.99, 3.12, and 3.09% in Phase 1 and 3.54, 3.55, and 4.35% in Phase 2) for 0, 0.5, and 1.0% CT, respectively. No difference ( ≥ 0.42) was observed for heat production lost as a percent of GE intake during both phases. Growth performance, gas emissions, and energetic losses were not affected by the inclusion CT in a steam-flaked corn-based finishing diet.

Effects of energy supplementation on energy losses and nitrogen balance of steers fed green-chopped wheat pasture I: Calorimetry.

Cattle grazing wheat pasture in the southern Great Plains are sometimes fed an energy supplement; however, the benefits of supplementation on nutrient balance, energy metabolism, and greenhouse gas emissions have not been elucidated. Therefore, we used 10 British crossbred steers (206 ± 10.7 kg initial BW) in a respiration calorimetry study to evaluate the effects of energy supplementation on energy losses, N balance, and nutrient digestibility of steers fed green-chopped wheat forage. The study design was an incomplete replicated 4 × 4 Latin square with treatments in a 2 × 2 factorial arrangement. Steers ( = 8) were assigned to 1 of 2 BW blocks (4 steers per block) with dietary factors consisting of 1) no supplementation (CON) or supplemented with a steam-flaked corn-based energy supplement (that also contained monensin sodium) at 0.5% of BW daily (SUP) and 2) NEm intakes of 1 times (1x) or 1.5 times (1.5x) maintenance. Wheat forage was harvested daily and continuously fed as green-chop to steers during the 56-d study. There were no differences ( ≥ 0.32) between CON and SUP for OM (78.3 vs. 80.7%, respectively) or NDF (68.3 vs. 64.8%, respectively) digestibility. At the 1.5x level of intake, there was no difference ( ≥ 0.16) in energy lost in feces (4.27 vs. 3.92 Mcal/d) or urine (0.58 vs. 0.55 Mcal/d), heat production (8.69 vs. 8.44 Mcal/d), or retained energy (3.10 vs. 3.46 Mcal/d) between supplementation treatments. Oxygen consumption (1,777 vs. 1,731 L/d; = 0.67) and CO production (1,704 vs. 1,627 L/d; = 0.56) of CON and SUP steers, respectively, were not different; however, SUP steers tended to have ( = 0.06) lower CH production (115 vs 130 L/d) than CON steers. Methane, as a proportion of GE intake, was similar for CON (6.87%) and SUP (6.07%; = 0.18), as was the ME:DE ratio ( = 0.24; 86.3% for CON and 87.9% for SUP). Fractional N excretion in urine and feces, as a proportion of total N excreted ( ≥ 0.84) or N intake ( ≥ 0.63), was not different between treatments. Calculated NEm and NEg values for CON were 1.76 and 1.37 Mcal/kg DM, respectively, whereas the NEm and NEg values for the SUP treatment were 2.32 and 1.61 Mcal/kg DM, respectively. Calculated NE values for steers fed additional energy were approximately 17.5% greater than the expected difference in energy content. This was probably the result of the inconsistent response at the 1x DMI level. Under these circumstances, energy supplementation did appear to enhance NEm and NEg value of the supplemented wheat forage diet.

Bacterial artificial chromosome transgenic analysis of dynamic expression patterns of regulator of G-protein signaling 4 during development. II. Subcortical regions.

A large family of regulator of G protein signaling (RGS) proteins modulates signaling through G-protein-coupled receptors. Previous studies have implicated RGS4 as a vulnerability gene in schizophrenia. To begin to understand structure-function relationships, we have utilized bacterial artificial chromosome (BAC) methods to create transgenic mice that express green fluorescent protein (GFP) under the control of endogenous RGS4 enhancer elements, circumventing the lack of suitable antibodies for analysis of dynamic patterns of expression. This report follows from the accompanying mapping paper in cerebral cortex, with a focus on developmental and mature expression patterns in subcortical telencephalic, diencephalic and brainstem areas. Based on reporter distribution, the data suggest that alterations in RGS4 function will engender a complex phenotype of increased and decreased neuronal output, with developmental, regional, and cellular specificity.

Bacterial artificial chromosome transgenic analysis of dynamic expression patterns of regulator of G-protein signaling 4 during development. I. Cerebral cortex.

Signaling through G-protein-coupled receptors is modulated by a family of regulator of G protein signaling (RGS) proteins that have been implicated in several neurological and psychiatric disorders. Defining the detailed expression patterns and developmental regulation of RGS proteins has been hampered by an absence of antibodies useful for mapping. We have utilized bacterial artificial chromosome (BAC) methods to create transgenic mice that express GFP under the control of endogenous regulator of G-protein signaling 4 (RGS4) enhancer elements. This report focuses on expression patterns in the developing and mature cerebral cortex. Based on reporter distribution, RGS4 is expressed by birth in neurons across all cortical domains, but in different patterns that suggest region- and layer-specific regulation. Peak expression typically occurs before puberty, with complex down-regulation by adulthood. Deep and superficial neurons, in particular, vary in their patterns across developmental age and region and, in primary sensory cortices, layer IV neurons exhibit low or no expression of the GFP reporter. These data suggest that altering RGS4 function will produce a complex neuronal phenotype with cell- and subdomain-specificity in the cerebral cortex.

Viral reverse transcriptase suppression associated with erythroid differentiation of Friend leukemia cells.

The Friend erythroleukemia cell line T3-C12, which produces Friend murine leukemia virus (F-MuLV) and can be induced to synthesize hemoglobin by dimethyl sulfoxide (DMSO), was monitored for viral RNA-dependent DNA polymerase reverse transcriptase (RT) activity. The amount of viral 60-70S RNA released from DMSO-treated cells was unaffected or increased compared to that from control cells, while RT activity from treated cells was decreased. Accordingly, the specific activity in F-MuLV from DMSO-treated cells expressed as RT/70S RNA was decreased to 8% of the control activity. The 5-bromo-2'-deoxyuridine added to cultures containing DMSO reversed the differentiation process, and the F-MuLV thus treated did not exhibit the reduced RT activity normally observed in DMSO-treated virus. Cell-free F-MuLV incubated with and without DMSO showed the same RT activity, indicating that DMSO itself did not inhibit RT activity. However, when F-MuLV-containing pellets from control and DMSO-treated culture fluids were mixed, there was marked inhibition of the control RT activity, suggesting that RNase hybrid activity was stimulated or that an inhibitor was produced. Assays of F-MuLV-RNase hybrid released from control and DMSO-treated cells showed no difference in activity, indicating that a specific inhibitor of RT was produced or activated. Additions of certain nucleotide triphosphates to RT incubation mixtures did not result in any stimulation of RT activity in DMSO-treated F-MuLV, suggesting that phosphatase was not responsible for the observed inhibition. The results suggested that DMSO treatment of T3-C12 cells caused a reduction in viral RT activity by stimulating the production of an inhibitor, the nature of which is unknown.

Evidence for multiple sites of regulation of heme synthesis in murine erythroleukemia cells.

Friend murine erythroleukemia (MEL) cells can be induced to differentiate along the erythroid pathway by such dissimilar agents as dimethyl sulfoxide (DMSO), butyrate, inhibitors of DNA synthesis, and certain highly polar agents. This study showed differential biochemical effects of the potent inducers DMSO and butyrate on the heme biosynthetic pathway in three clones of Friend MEL cells. When the cells were incubated with combinations of DMSO and butyrate, hemoglobin production was less than that amount produced when each inducer was incubated singly with the cells. Procaine, a local anesthetic that competes with Ca2+ and thus affects membrane permeability, slightly inhibited DMSO-mediated hemoglobin production but almost tripled the level stimulated by butyrate alone. Similarly, EDTA, which also can bind Ca2+ and which can modify the activity of heme biosynthetic enzymes, also inhibited hemoglobin production mediated by DMSO, whereas it stimulated hemoglobin production in cells exposed to butyrate. Total porphyrin accumulation was greater in DMSO-treated cells than in butyrate-treated cells, which suggests that butyrate induces the enzymes of the heme pathway more efficiently than does DMSO. DMSO and butyrate may affect the heme biosynthetic pathway by multiple mechanisms or, alternatively, they may affect the multistep pathway at various points, producing partial blocks or incomplete enzyme induction.

Augmentation of hematoporphyrin uptake and in vitro-growth inhibition of L1210 leukemia cells by succinylacetone.

Succinylacetone (SA; 4,6-dioxoheptanoic acid), a specific inhibitor of delta-aminolevulinic acid dehydrase (ALAD) (the second enzyme of the heme biosynthetic pathway), was tested for its effect in L1210 cells from inbred DBA/2 mice. ALAD from broken L1210 cells was completely inhibited by 1 microM SA, but in whole cells activity was decreased only 83% after incubation of the cells with 2.5 mM SA for 3 days. When incubated with hematoporphyrin (HP), L1210 cells rapidly took up porphyrin from the medium, and this uptake could be augmented by pretreatment of the cells with SA; but this enhancement of porphyrin uptake occurred gradually over a period of days. When SA-treated and untreated L1210 cells were incubated with increasing concentrations of HP in the medium, SA-treated cells reached the saturation concentration of cellular porphyrin at lower medium HP concentrations than did untreated cells. Growth of L1210 cells could be inhibited by 2 mM SA or more. Addition of increasing amounts of serum to cultures of cells containing SA did not reverse the growth inhibition due to SA. Porphyrin uptake from HP in the medium in nonmalignant fibroblast line 3T3 was much lower than in L1210 cells and could not be enhanced by incubation of the cells with SA.

Erythroid differentiation in cultured Friend leukemia cells treated with metabolic inhibitors.

The induction of erythroid differentiation in the T3-C12 clone of Friend leukemia cells by dimethyl sulfoxide is accompanied by reduction in viral RNA-dependent DNA polymerase activity with increased cellular delta-aminolevulinic acid synthetase activity and hemoglobin synthesis. These cells were treated with a variety of compounds to determine whether other durgs are capable on inducing erythroid differentiation. While several hormones, inhibitors of RNA synthesis, organic solvents, inhibitors of DNA polymerase, sulfhydryl inhibitors, and inducers of delta-aminolevulinic acid synthetase administered singly did not stimulate hemoglobin synthesis like dimethyl sulfoxide, inhibitors of DNA and RNA synthesis such as adriamycin, mitomycin C, and hydroxyurea:mithramycin were synergistic in stimulating erythroid differentiation.

Uptake of hematin and growth of malignant murine erythroleukemia cells depleted of endogenous heme by succinylacetone.

Heme levels and growth of malignant murine erythroleukemia cells in heme-free medium are drastically reduced by incubation of these cells in the presence of 4,6-dioxoheptanoic acid [succinylacetone (SA)]. When hematin was added to the culture medium of heme-depleted cells, the intracellular heme levels returned to normal, and growth inhibition produced by SA was also reversed. However, when cells depleted of heme by growth in heme-free medium containing SA were placed in heme-free medium without SA, heme levels were restored to normal, and growth was resumed. Hematin uptake in both untreated and heme-depleted malignant murine erythroleukemia cells exhibited biphasic kinetics, with a rapid phase of about 2 min followed by a slower uptake. The rate of uptake of exogenous hematin was slightly greater at 37 degrees than at 20 degrees. Although supplementation of heme-free medium with exogenous hematin increased total cellular heme in both untreated and heme-depleted malignant murine erythroleukemia cells, the fraction of heme in the 20,000 X g sediment was unaffected. A nonmalignant fibroblastic cell line, 3T3, exhibited little or no capacity to take up exogenous hematin.

Surgery for cyanotic heart disease in the first year of life.

Data are reviewed on 248 patients less than 1 year old who presented with a diagnosis of cyanotic heart disease between January 1976 and January 1982. No infant had had prior surgical treatment. The patients were classified according to diagnosis: tetralogy of Fallot, transposition of the great arteries, pulmonary atresia and anomalies of the tricuspid atresia or single ventricle type. Other remote forms of cyanotic heart disease were excluded from the analysis. Management of these patient groups is discussed in relation to their potential for corrective surgery early in infancy or later. The proper selection of palliative procedures that will permit bilateral growth and development of pulmonary arteries and equal distribution of pulmonary blood flow is emphasized. Morbidity and mortality in each patient group are discussed.

Two-dimensional echocardiographic recognition and repair of subvalvular mitral aneurysm of the left ventricle in an infant.

The clinical features, diagnostic studies and surgical treatment of a subvalvular mitral aneurysm of the left ventricle are described. The infant presented at 9 weeks of age with large apical ventricular septal defects and pulmonary hypertension. The subvalvular aneurysm was an incidental finding. Both lesions were treated surgically.