The tragedy of smoking, alcohol, and multiple substance use during pregnancy.

BACKGROUND: Antenatal substance use is a significant public health concern in South Africa (SA). Information on smoking, drinking and drug use during pregnancy was collected prospectively for the Safe Passage Study of the PASS (Prenatal Alcohol in Sudden infant death syndrome and Stillbirth) Network. OBJECTIVES: Data from 4 926 pregnant women in a community near Tygerberg Academic Hospital, Cape Town, were examined to determine whether associations between different substance use groups and postnatal infant outcomes at birth and 1 year were significant. METHODS: Gestational age (GA) was determined by earliest ultrasound. Maternal data were collected at enrolment or first antenatal visit. Substance use data were obtained at up to four occasions. Birthweight data were derived from medical records, and birthweight z-scores (BWZs) were specifically calculated using INTERGROWTH-21st study data. Statistical analyses were done with Statistica version 13.  Results. Women who used more substances enrolled later, were younger, and had smaller mid-upper arm circumferences (MUACs), less education and lower monthly income than women who used no substances (control group). Infants born to women who used more substances had lower GA at delivery, birthweight and BWZ than infants from the control group. At 1 year, infants born to women who used more substances had a lower weight, shorter length and smaller head circumference. Education was positively associated with all infant outcomes at birth and 1 year. MUAC was positively associated with infant BWZ, and weight and length at 1 year. Income was negatively associated with BWZ, but positively associated with all 1-year outcomes. CONCLUSION: Substance use during pregnancy affects infant outcomes at birth and 1 year of age. The addictive properties of substance use make cessation difficult, so prevention strategies should be implemented long before pregnancy. Higher maternal education, associated with better infant outcomes at birth and 1 year and acting as a countermeasure to substance use, is of paramount importance.

Triggering necroptosis in cisplatin and IAP antagonist-resistant ovarian carcinoma.

Ovarian cancer patients are typically treated with carboplatin and paclitaxel, but suffer a high rate of relapse with recalcitrant disease. This challenge has fostered the development of novel approaches to treatment, including antagonists of the 'inhibitor of apoptosis proteins' (IAPs), also called SMAC mimetics, as apoptosis-inducing agents whose action is opposed by caspase inhibitors. Surprisingly, IAP antagonist plus caspase inhibitor (IZ) treatment selectively induced a tumor necrosis factor-α (TNFα)-dependent death among several apoptosis-resistant cell lines and patient xenografts. The induction of necroptosis was common in ovarian cancer, with expression of catalytically active receptor-interacting protein kinase-3 (RIPK3) necessary for death, and in fact sufficient to compromise survival of RIPK3-negative, necroptosis-resistant ovarian cancer cells. The formation of a necrosome-like complex with a second critical effector, receptor-interacting serine-threonine kinase-1 (RIPK1), was observed. RIPK1, RIPK3 and TNFα were required for the induction of death, as agents that inhibit the function of any of these targets prevented cell death. Abundant RIPK3 transcript is common in serous ovarian cancers, suggesting that further evaluation and targeting of this RIPK3-dependent pathway may be of clinical benefit.

Hidden stressors in the clonogenic assay used in radiobiology experiments.

While clonogenic assays are extensively used in radiobiology, there is no widely accepted procedure for choosing the composition of the cell culture media. Cell line suppliers recommend a specific culture medium for each cell line, however a researcher will frequently customize this aspect of the protocol by supplementing the recommended support medium with additives. For example, many researchers add antibiotics, in order to avoid contamination of cells and the consequent loss of data, with little discussion of the influence of the antibiotics on the clonogenic survival of the cells. It is assumed that the effect of any variables in the growth medium on cell survival is taken into consideration by comparing the survival fraction relative to that of controls grown under the same conditions. In the search for better cancer treatment, the effect of various stressors on clonogenic cell survival is under investigation. This study seeks to identify and test potential stressors commonly introduced into the cell culture medium, which may confound the response to radiation.

Lift-off compensation for improved accuracy in ultrasonic lamb wave velocity measurements using electromagnetic acoustic transducers (EMATs).

The crystalline texture of a sheet metal strongly affects its formability, so having knowledge of this texture is of great industrial relevance. The texture of rolled sheet metals, such as aluminium and steel, may be determined by ultrasonic measurement of the velocity of the zero order symmetric (S(0)) Lamb wave as a function of angle to the rolling direction. Electromagnetic acoustic transducers (EMATs) may perform this measurement without contacting the sample, therefore reducing perturbation to the plate wave system, as they are electromagnetically coupled to the sheet. The EMAT system measurements are non-destructive and may be made in real time, therefore offering advantages over the conventional techniques such as X-ray and neutron diffraction. It has been noticed that in the two EMAT pitch-catch system, the apparent arrival times of the ultrasonic waves change with variation in lift-off (distance between sample and transducer) due to impedance and aperture effects. For precise and accurate texture parameters to be obtained, accurate absolute ultrasonic velocity measurement is required and hence lift-off must be compensated for. This is of particular importance to online inspection systems where constant lift-off may be difficult to maintain. The impedance behaviour of various coil geometries has been investigated as a function of lift-off and frequency and compared to the received ultrasonic signal and the drive current pulse profile. Theoretical models have been used to explain the observed behaviour, and hence a scheme has been proposed for the compensation of lift-off effects in real time.

Development of an advanced multimode automatic ultrasonic texture measurement system for laboratory and production line application.

We present work on the development of an ultrasonic texture measurement system for sheet metals using non-contact transducers, suitable for use both in the laboratory and on the production line. Variation of the velocity of the zero-order symmetric (S0) Lamb wave is used to determine the crystallographic texture of polycrystalline metal sheets ranging in thickness from 0.1 to 3 mm. This system features improvements on previous state-of-the-art ultrasonic technology in that it probes velocity over a continuous range of angles using only two electromagnetic acoustic transducers (EMATs). This is demonstrated to offer a significant improvement in accuracy and allows the detection and investigation of asymmetric anisotropies in the sheets. Another advantage of the system is its potential for combining several different measurements using a single pair of transducers. The capability is demonstrated for through-thickness shear wave measurements as well as the zero-order symmetric Lamb wave measurements which are the primary means of determining the texture. The change between generating Lamb and through-thickness bulk waves can be made entirely by changing the electrical circuit connected to the EMATs without modifying the transducer assembly in any way. Measurement of all of the above waves can provide information on the sheet thickness and other physical properties of the sheet in addition to texture. Certain texture parameters can be calculated from both Lamb and shear wave velocities, allowing self-calibration of the system.

Ribosome exchange revisited: a mechanism for translation-coupled ribosome detachment from the ER membrane.

In current models, ribosome release from the endoplasmic reticulum (ER) is coupled to the termination of protein translation. Thus, coincident with termination, membrane-bound ribosomes dissociate into their component subunits and are released into the cytosol. Here, we review past and current data and propose that the affinity of the ribosome for the ER membrane is decreased during translation, with ribosome release occurring when a membrane-bound ribosome is engaged in the synthesis of a protein lacking a signal sequence. Our model emphasizes a role for the conformation of the large ribosomal subunit in the regulation of membrane affinity and provides a mechanism for translation-coupled ribosome release.

The MN1-TEL fusion protein, encoded by the translocation (12;22)(p13;q11) in myeloid leukemia, is a transcription factor with transforming activity.

The Tel gene (or ETV6) is the target of the translocation (12;22)(p13;q11) in myeloid leukemia. TEL is a member of the ETS family of transcription factors and contains the pointed protein interaction (PNT) domain and an ETS DNA binding domain (DBD). By contrast to other chimeric proteins that contain TEL's PNT domain, such as TEL-platelet-derived growth factor beta receptor in t(5;12)(q33;p13), MN1-TEL contains the DBD of TEL. The N-terminal MN1 moiety is rich in proline residues and contains two polyglutamine stretches, suggesting that MN1-TEL may act as a deregulated transcription factor. We now show that MN1-TEL type I, unlike TEL and MN1, transforms NIH 3T3 cells. The transforming potential depends on both N-terminal MN1 sequences and a functional TEL DBD. Furthermore, we demonstrate that MN1 has transcription activity and that MN1-TEL acts as a chimeric transcription factor on the Moloney sarcoma virus long terminal repeat and a synthetic promoter containing TEL binding sites. The transactivating capacity of MN1-TEL depended on both the DBD of TEL and sequences in MN1. MN1-TEL contributes to leukemogenesis by a mechanism distinct from that of other chimeric proteins containing TEL.

Regulation of ribosome detachment from the mammalian endoplasmic reticulum membrane.

In current models, protein translocation in the endoplasmic reticulum (ER) occurs in the context of two cycles, the signal recognition particle (SRP) cycle and the ribosome cycle. Both SRP and ribosomes bind to the ER membrane as a consequence of the targeting process of translocation. Whereas SRP release from the ER membrane is regulated by the GTPase activities of SRP and the SRP receptor, ribosome release from the ER membrane is thought to occur in response to the termination of protein synthesis. We report that ER-bound ribosomes remain membrane-bound following the termination of protein synthesis and in the bound state can initiate the translation of secretory and cytoplasmic proteins. Two principal observations are reported. 1) Membrane-bound ribosomes engaged in the synthesis of proteins lacking a signal sequence are released from the ER membrane as ribosome-nascent polypeptide complexes. 2) Membrane-bound ribosomes translating secretory proteins can access the translocon in an SRP receptor-independent manner. We propose that ribosome release from the ER membrane occurs in the context of protein translation, with release occurring by default in the absence of productive nascent polypeptide-membrane interactions.

Identification and characterization of a new human ETS-family transcription factor, TEL2, that is expressed in hematopoietic tissues and can associate with TEL1/ETV6.

The ETS family of proteins is a large group of transcription factors implicated in many aspects of normal hematopoietic development, as well as oncogenesis. For example, the TEL1/ETV6 (TEL1) gene is required for normal yolk sac angiogenesis, adult bone marrow hematopoiesis, and is rearranged or deleted in numerous leukemias. This report describes the cloning and characterization of a novel ETS gene that is highly related to TEL1 and is therefore called TEL2. The TEL2 gene consists of 8 exons spanning approximately 21 kilobases (kb) in human chromosome 6p21. Unlike the ubiquitously expressed TEL1 gene, however, TEL2 appears to be expressed predominantly in hematopoietic tissues. Antibodies raised against the C-terminus of the TEL2 protein were used to show that TEL2 localizes to the nucleus. All ETS proteins can bind DNA via the highly conserved ETS domain, which recognizes a purine-rich DNA sequence with a GGAA core motif. DNA binding assays show that TEL2 can bind the same consensus DNA binding sequence recognized by TEL1/ETV6. Additionally, the TEL2 protein is capable of associating with itself and with TEL1 in doubly transfected Hela cells, and this interaction is mediated through the pointed (PNT) domain of TEL1. The striking similarities of TEL2 to the oncogenic TEL1, its expression in hematopoietic tissues, and its ability to associate with TEL1 suggest that TEL2 may be an important hematopoietic regulatory protein.

Ribosome-independent regulation of translocon composition and Sec61alpha conformation.

In this study, the contributions of membrane-bound ribosomes to the regulation of endoplasmic reticulum translocon composition and Sec61alpha conformation were examined. Following solubilization of rough microsomes (RM) with digitonin, ribosomes co-sedimented in complexes containing the translocon proteins Sec61alpha, ribophorin I, and TRAPalpha, and endoplasmic reticulum phospholipids. Complexes of similar composition were identified in digitonin extracts of ribosome-free membranes, indicating that the ribosome does not define the composition of the digitonin-soluble translocon. Whereas in digitonin solution a highly electrostatic ribosome-translocon junction is observed, no stable interactions between ribosomes and Sec61alpha, ribophorin I, or TRAPalpha were observed following solubilization of RM with lipid-derived detergents at physiological salt concentrations. Sec61alpha was found to exist in at least two conformational states, as defined by mild proteolysis. A protease-resistant form was observed in RM and detergent-solubilized RM. Removal of peripheral proteins and ribosomes markedly enhanced the sensitivity of Sec61alpha to proteolysis, yet the readdition of inactive ribosomes to salt-washed membranes yielded only modest reductions in protease sensitivity. Addition of sublytic concentrations of detergents to salt-washed RM markedly decreased the protease sensitivity of Sec61alpha, indicating that a protease-resistant conformation of Sec61alpha can be conferred in a ribosome-independent manner.

Mutations in the murine fitness 1 gene result in defective hematopoiesis.

Identification and characterization of mutations that disrupt normal hematopoiesis are essential for understanding the genetic pathways that control the development and regulation of the mammalian hematopoietic system. Previously, the fitness 1 gene was identified by five, independent mutations in N-ethyl-N-nitrosourea (ENU) saturation mutagenesis experiments within the albino (c) region of mouse chromosome 7 (MMU7). We report here that fit1 mutants are anemic, display numerous peripheral blood defects, and are deficient in early hematopoietic progenitor cell populations. The number of both erythroid and myeloid progenitors, as well as B cells, are reduced. These results implicate fit1 involvement in normal hematopoiesis and suggest that further characterization of the fit1 gene, and the five presumed point mutations of the gene, will lead to an improved understanding of normal hematopoiesis in the mouse.

Cloning and characterization of the galE locus of Pasteurella haemolytica A1.

The enzyme UDP-galactose 4-epimerase (GalE) is involved in one of the major steps of galactose metabolism in bacteria. In many cases, GalE is required for the biosynthesis of extracellular polysaccharide materials such as lipopolysaccharide (LPS) and capsule. Mutants defective in galE have been shown to exhibit reduced virulence. Here we describe the cloning and characterization of the galE gene from the bovine pathogen Pasteurella haemolytica A1. This was achieved by the complementation of a Salmonella typhimurium galE mutant with a P. haemolytica A1 plasmid bank. Analysis of six clones recovered on minimal media with galactose as the carbon source showed that they all contained the same recombinant plasmid with a 5-kbp DNA insert. The galE-complementing activity was localized to a 2.2-kbp DNA region by subcloning. Biochemical, immunological, and phage sensitivity analyses of the recombinant LPS in S. typhimurium showed that it is essentially identical to that of the wild type. In vivo expression studies showed that a 37-kDa protein is expressed from the complementing plasmids, and the presence of GalE activity was confirmed by an assay for epimerase activity. Nucleotide sequence analysis of the cloned DNA identified the galE gene. Comparison of the deduced amino acid sequence analysis of P. haemolytica A1 GalE with published data showed high-level homology, 81.6%, with the GalE of Haemophilus influenzae type b. However, the sequences flanking galE do not show similarity with any other gal gene, suggesting that P. haemolytica A1 galE is not linked to the other genes of the gal operon, as is the case for Neisseria meningitidis, Neisseria gonorrhoeae, and H. influenzae. The separation of galE from the classical gal operon genes was confirmed by Southern blot hybridization studies, and a physical map showing the relative positions of galE, galT, and galK was constructed.

Physical localization of eed: a region of mouse chromosome 7 required for gastrulation.

In the mouse, the embryonic ectoderm development (eed) region is defined by deletions encompassing the albino (c) locus of chromosome 7. The region is located 1-2 cM distal to the c locus and was of undetermined size. Embryos homozygous for deletions removing eed display defects in axial organization during gastrulation. Two loci, identified by chemical mutagenesis, are known to map within the eed interval. One, l7Rn5, probably represents the gene required for gastrulation. The second, l7Rn6, is required for survival after birth. fit1, a third locus identified by chemical mutagenesis, maps distal to the eed interval and is also required for survival after birth. A 900-kb YAC contig has been constructed, and deletion breakpoints defining the limits of the regions containing these loci have been localized. Their positions place the eed region within a maximum 150-kb interval at the proximal end of the contig, while fit1 maps to a 360-kb interval within the middle of the contig. Several clusters of rare-cutting restriction sites map within these regions and represent potential locations of candidate genes.

Cloning and characterization of a gene from Pasteurella haemolytica A1 involved in lipopolysaccharide biosynthesis.

A Pasteurella haemolytica A1 gene involved in the biosynthesis of a moiety on the core of the lipopolysaccharide molecule has been cloned and characterized. Escherichia coli clones which carry this gene showed an alteration of its lipopolysaccharide migration profile on tricine SDS-PAGE and exhibited resistance to the core-specific phage U3. In addition, lipopolysaccharide extracted from the E. coli clones was recognized by an anti-corespecific antiserum, but not by antiserum specific for the O antigen of P. haemolytica A1 lipopolysaccharide. Nucleotide sequence analysis of the cloned DNA identified an open reading frame (lpsA) coding for a protein of 263 amino acids which showed significant homology with a Haemophilus influenzae type b lipooligosaccharide biosynthesis gene. PCR amplification of genomic DNA, using primers based on the P. haemolytica A1 lpsA sequence, yielded products from only the A biotypes of P. haemolytica.

Genetic and physical mapping of the fitness 1 (fit1) locus within the Fes-Hbb region of mouse chromosome 7.

Mutations at the fit1 locus affect normal pre- and post-natal development by retarding growth and reducing viability. We report mapping of the fit1 locus, by trans-complementation crosses to mice carrying deletions of the albino (c) locus in Chromosome (Chr) 7, to a subregion of the c-deletion complex within the Mod2-sh1 interval. The fit1 locus, which is currently defined by five N-ethyl-N-nitrosourea (ENU)-induced mutations, was found to map in a subregion between the eed and exed loci. A restriction fragment containing a deletion breakpoint that genetically defines the proximal border of fit1 was cloned, providing a DNA probe (RN302) that maps proximal to fit1. Long-range mapping with this probe, and with a DNA probe that maps distal to the fit1 interval, established that the region containing at least part of the fit1 gene is 530 kb or less. Positioning of fit1 between deletion breakpoints, and the isolation and mapping of a DNA probe proximal to it, should facilitate the cloning and molecular characterization of fit1, as well as of the eed locus and the tightly linked l(7)5Rn and l(7)6Rn loci.

Molecular analysis of radiation-induced albino (c)-locus mutations that cause death at preimplantation stages of development.

Deletion mutations at the albino (c) locus have been useful for continuing the development of fine-structure physical and functional maps of the Fes-Hbb region of mouse chromosome 7. This report describes the molecular analysis of a number of radiation-induced c deletions that, when homozygous, cause death of the embryo during preimplantation stages. The distal extent of these deletions defines a locus, pid, (preimplantation development) genetically associated with this phenotype. The proximal breakpoints of eight of these deletions were mapped with respect to the Tyr (tyrosinase; albino) gene as well as to anonymous loci within the Fah-Tyr region that are defined by the Pmv-31 viral integration site and by chromosome-microdissection clones. Rearrangements corresponding to the proximal breakpoints of two of these deletions were detected by Southern blot analysis, and a size-altered restriction fragment carrying the breakpoint of one of them was cloned. A probe derived from this deletion fusion fragment defines a locus, D7Rn6, which maps within (or distal to) the pid region, and which discriminates among the distal extents of deletions eliciting the pid phenotype. Extension of physical maps from D7Rn6 should provide access both to the pid region and to loci mapping distal to pid that are defined by N-ethyl-N-nitrosourea-induced lethal mutations.

Direct photoaffinity labeling of rat liver carbamoyl phosphate synthetase I with ATP.

The adenine subsites of the ATP sites of rat liver carbamoyl phosphate synthetase I have been localized by direct photoaffinity labeling with ATP. The synthetase is known to utilize two molecules of ATP, apparently in mechanistically discrete steps and at separate ATP sites. UV irradiation of the synthetase in the presence of [alpha-32P]ATP resulted in the incorporation of label. Peptide analysis of the ATP-photolabeled synthetase demonstrated that the labeling was extremely selective. To localize the sites of ATP photoincorporation to discrete regions of the synthetase which appear to be structural domains, the enzyme was photolabeled with [alpha-32P]ATP and subjected to limited proteolytic digestion. Consideration of these data indicated that the internal domains B and C were preferentially labeled and that there was lesser, but significant, labeling of the N-terminal domain A. Omission of the required allosteric activator N-acetylglutamate from the photolabeling mixture resulted in an approximately 60% decrease in label incorporation and an accompanying decrease in the extent of label incorporation in domain B. Consideration of these N-acetylglutamate effects, together with previous findings on the effects of the allosteric activator, confirmed the following functional identification of the ATP sites: domain B participates in binding the molecule of ATP involved in bicarbonate activation, whereas domain C participates in binding the molecule of ATP involved in carbamate phosphorylation.

Location of the ATP gamma-phosphate-binding sites on rat liver carbamoyl-phosphate synthetase I. Studies with the ATP analog 5'-p-fluorosulfonylbenzoyladenosine.

The gamma-phosphate subsites of the MgATP sites of rat liver carbamoyl-phosphate synthetase I have been defined by use of the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The synthetase utilizes two molecules of MgATP, apparently in mechanistically discrete steps and at separate MgATP sites. Sequence analysis has revealed internal duplication within the synthetase molecule (Nyunoya, H., Broglie, K.E., Widgren, E.E., and Lusty, C.J. (1985) J. Biol. Chem. 260, 9346-9356) and, based on sequence similarity with other nucleotide-binding proteins, potential ATP sites have been predicted for each of the duplicated sequences. The present FSBA studies have identified four peptides within carbamoyl-phosphate synthetase I that are involved in binding MgATP. Differential effects of N-acetylglutamate, a required allosteric activator, on the interaction of FSBA with the peptides were utilized to develop the following model for two distinct MgATP sites. Peptides 631-638 and 1327-1348 (with Cys1327 and/or Cys1337 modified by FSBA) apparently form part of the binding site for the MgATP involved in bicarbonate activation. Peptides 1310-1317 and 1445-1454 (with Tyr1450 modified by FSBA) apparently form part of the binding site for the MgATP involved in phosphorylation of enzyme-bound carbamate. Each of these MgATP sites contains a peptide from one of the internal duplicated regions of the enzyme molecule, which have previously been suggested as containing MgATP sites (Nyunoya, H., Broglie, K. E., Widgren, E. E., and Lusty, C. J. (1985) J. Biol. Chem. 260, 9346-9356; Powers-Lee, S. G., and Corina, K. (1987) J. Biol. Chem. 262, 9052-9056), as well as a peptide from the flexible C-terminal region.

Affinity labeling of spinach phosphoribulokinase subsequent to S-methylation at Cys16.

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme, S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparent Kd of 3-4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [14C]reagent per mole of enzyme subunit. Amino acid analysis of the [14C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.