Mutations in myosin light chain kinase cause familial aortic dissections.

Mutations in smooth muscle cell (SMC)-specific isoforms of α-actin and β-myosin heavy chain, two major components of the SMC contractile unit, cause familial thoracic aortic aneurysms leading to acute aortic dissections (FTAAD). To investigate whether mutations in the kinase that controls SMC contractile function (myosin light chain kinase [MYLK]) cause FTAAD, we sequenced MYLK by using DNA from 193 affected probands from unrelated FTAAD families. One nonsense and four missense variants were identified in MYLK and were not present in matched controls. Two variants, p.R1480X (c.4438C>T) and p.S1759P (c.5275T>C), segregated with aortic dissections in two families with a maximum LOD score of 2.1, providing evidence of linkage of these rare variants to the disease (p = 0.0009). Both families demonstrated a similar phenotype characterized by presentation with an acute aortic dissection with little to no enlargement of the aorta. The p.R1480X mutation leads to a truncated protein lacking the kinase and calmodulin binding domains, and p.S1759P alters amino acids in the α-helix of the calmodulin binding sequence, which disrupts kinase binding to calmodulin and reduces kinase activity in vitro. Furthermore, mice with SMC-specific knockdown of Mylk demonstrate altered gene expression and pathology consistent with medial degeneration of the aorta. Thus, genetic and functional studies support the conclusion that heterozygous loss-of-function mutations in MYLK are associated with aortic dissections.

Neural reprogramming in retinal degeneration.

PURPOSE: Early visual defects in degenerative diseases such as retinitis pigmentosa (RP) may arise from phased remodeling of the neural retina. The authors sought to explore the functional expression of ionotropic (iGluR) and group 3, type 6 metabotropic (mGluR6) glutamate receptors in late-stage photoreceptor degeneration. METHODS: Excitation mapping with organic cations and computational molecular phenotyping were used to determine whether retinal neurons displayed functional glutamate receptor signaling in rodent models of retinal degeneration and a sample of human RP. RESULTS: After photoreceptor loss in rodent models of RP, bipolar cells lose mGluR6 and iGluR glutamate-activated currents, whereas amacrine and ganglion cells retain iGluR-mediated responsivity. Paradoxically, amacrine and ganglion cells show spontaneous iGluR signals in vivo even though bipolar cells lack glutamate-coupled depolarization mechanisms. Cone survival can rescue iGluR expression by OFF bipolar cells. In a case of human RP with cone sparing, iGluR signaling appeared intact, but the number of bipolar cells expressing functional iGluRs was double that of normal retina. CONCLUSIONS: RP triggers permanent loss of bipolar cell glutamate receptor expression, though spontaneous iGluR-mediated signaling by amacrine and ganglion cells implies that such truncated bipolar cells still release glutamate in response to some nonglutamatergic depolarization. Focal cone-sparing can preserve iGluR display by nearby bipolar cells, which may facilitate late RP photoreceptor transplantation attempts. An instance of human RP provides evidence that rod bipolar cell dendrite switching likely triggers new gene expression patterns and may impair cone pathway function.

Scheduling of continuous and discontinuous material flows with intermediate storage restrictions

This paper deals with scheduling batch (i.e., discontinuous), continuous, and semicontinuous production in process industries (e.g., chemical, pharmaceutical, or metal casting industries) where intermediate storage facilities and renewable resources (processing units and manpower) of limited capacity have to be observed. First, different storage configurations typical of process industries are discussed. Second, a basic scheduling problem covering the three above production modes is presented. Third, (exact and truncated) branch-and-bound methods for the basic scheduling problem and the special case of batch scheduling are proposed and subjected to an experimental performance analysis. The solution approach presented is flexible and in principle simple, and it can (approximately) solve relatively large problem instances with sufficient accuracy.

Advanced production scheduling for batch plants in process industries

An Advanced Planning System (APS) offers support at all planning levels along the supply chain while observing limited resources. We consider an APS for process industries (e.g. chemical and pharmaceutical industries) consisting of the modules network design (for long–term decisions), supply network planning (for medium–term decisions), and detailed production scheduling (for short–term decisions). For each module, we outline the decision problem, discuss the specifi cs of process industries, and review state–of–the–art solution approaches. For the module detailed production scheduling, a new solution approach is proposed in the case of batch production, which can solve much larger practical problems than the methods known thus far. The new approach decomposes detailed production scheduling for batch production into batching and batch scheduling. The batching problem converts the primary requirements for products into individual batches, where the work load is to be minimized. We formulate the batching problem as a nonlinear mixed–integer program and transform it into a linear mixed–binary program of moderate size, which can be solved by standard software. The batch scheduling problem allocates the batches to scarce resources such as processing units, workers, and intermediate storage facilities, where some regular objective function like the makespan is to be minimized. The batch scheduling problem is modelled as a resource–constrained project scheduling problem, which can be solved by an efficient truncated branch–and–bound algorithm developed recently. The performance of the new solution procedures for batching and batch scheduling is demonstrated by solving several instances of a case study from process industries.

BMP-SIGNALING REGULATES A COMMON TRANSCRIPTIONAL PROGRAM TO CONTROL FACIAL FORM AND SKELETAL MORPHOGENESIS

Much of the craniofacial skeleton, such as the skull vault, mandible and midface, develops through direct, intramembranous ossification of the cranial neural crest (CNC) derived progenitor cells. Bmp-signaling plays critical roles in normal craniofacial development, and Bmp4 deficiency results in craniofacial abnormalities, such as cleft lip and palate. We performed an in depth analysis of Bmp4, a critical regulator of development, disease, and evolution, in the CNC. Conditional Bmp4 overexpression, using a tetracycline regulated Bmp4 gain of function allele, resulted in facial form changes that were most dramatic after an E10.5 Bmp4 induction. Expression profiling uncovered a signature of Bmp4 induced genes (BIG) composed predominantly of transcriptional regulators controlling self-renewal, osteoblast differentiation, and negative Bmp autoregulation. The complimentary experiment, CNC inactivation of Bmp2, Bmp4, and Bmp7, resulted in complete or partial loss of multiple CNC derived skeletal elements revealing a critical requirement for Bmp-signaling in membranous bone and cartilage development. Importantly, the BIG signature was reduced in Bmp loss of function mutants indicating similar Bmp-regulated target genes underlying facial form modulation and normal skeletal morphogenesis. Chromatin immunoprecipitation (ChIP) revealed a subset of the BIG signature, including Satb2, Smad6, Hand1, Gadd45g and Gata3 that was bound by Smad1/5 in the developing mandible revealing direct, Smad-mediated regulation. These data indicate that Bmp-signaling regulates craniofacial skeletal development and facial form by balancing self-renewal and differentiation pathways in CNC progenitors.

Toward the Development of Ethical Guidelines for Family Preservation

Family preservation workers need a standard set of ethical guidelines to assist them in providing their service in a proper manner. This paper describes how ethical codes have been developed for the "traditional" mental health care disciplines and why such codes are not sufficient for the type of work done in family preservation. The paper further provides examples of the types of ethical dilemmas family preservation workers encounter as well as suggestions for workers, supervisors, and agencies in dealing with such dilemmas.

Aboral ectoderm-specific expression and regulation of the LpS1 genes of {\it Lytechinus pictus\/}

The Spec genes serve as molecular markers for examining the ontogeny of the aboral ectoderm lineage of sea urchin embryos. These genes are activated at late-cleavage stage only in cells contributing to the aboral ectoderm of Strongylocentrotus purpuratus and encode 14,000-17,000 Da calcium-binding proteins. A comparative analysis was undertaken to better understand the mechanisms underlying the activation and function of the Spec genes by investigating Spec homologues from Lytechinus pictus, a distantly related sea urchin. Spec antibodies cross-reacted with 34,000 Da proteins in L. pictus embryos that displayed a similar ontogenetic pattern to that of Spec proteins. One cDNA clone, LpS1, was isolated by hybridization to a synthetic oligonucleotide corresponding to a calcium-binding domain or EF-hand. The LpS1 mRNA has developmental properties similar to those of the Spec mRNAs. LpS1 encodes a 34,000 Da protein containing eight EF-hand domains, which share structural homology with the Spec EF-hands; however, little else in the protein sequence is conserved, implying that calcium-binding is important for Spec protein function. Genomic DNA blot analysis showed two LpS1 genes, LpS1$\alpha$ and LpS1$\beta$, in L. pictus. Partial gene structures for both LpS1$\alpha$ and $\beta$ were constructed based on genomic clones isolated from an L. pictus genomic library. These revealed internal duplications of the LpS1 genes that accounted for the eight EF-hand domains in the LpS1 proteins. Sequencing analysis showed there was little in common among the 5$\sp\prime$-flanking regions of the LpS1 and Spec genes except for the presence of a binding site for the transcription factor USF.^ A sea urchin gene-transfer expression system showed that 762 base pairs (bp) of 5$\sp\prime$-flanking DNA from the LpS1$\beta$ gene were sufficient for correct temporal and spatial expression of reporter genes in sea urchin embryos. Deletions at the 5$\sp\prime$ end to 511, 368, or 108bp resulted in a 3-4 fold decrease in chloramphenicol acetyltransferase (CAT) activity and disrupted the restricted activation of the lac Z gene in aboral ectoderm cells.^ A full-length Spec1 protein and a truncated LpS1 protein were induced and partially purified from an in vitro expression system. (Abstract shortened with permission of author.) ^

The interaction of P160 BCR with BCR-ABL gene products unique to Philadelphia chromosome leukemias

The BCR gene is involved in the pathogenesis of Philadelphia chromosome-positive (Ph$\sp1$) leukemias. Typically, the 5$\sp\prime$ portion of BCR on chromosome 22 becomes fused to a 5$\sp\prime$ truncated ABL gene from chromosome 9 resulting in a chimeric BCR-ABL gene. To investigate the role of the BCR gene product, a number of BCR peptide sequences were used to generate anti-BCR antibodies for detection of BCR and BCR-ABL proteins. Since both BCR and ABL proteins have kinase activity, the anti-BCR antibodies were tested for their ability to immunoprecipitate BCR and BCR-ABL proteins from cellular lysates by use of an immunokinase assay. Antisera directed towards the C-terminal portions of P160 BCR, sequences not present in BCR-ABL proteins, were capable of co-immunoprecipitating P210 BCR-ABL from the Ph$\sp1$- positive cell line K562. Re-immunoprecipitation studies following complete denaturation showed that C-terminal BCR antisera specifically recognized P160 BCR but not P210 BCR-ABL. These and other results indicated the presence of a P160 BCR/P210 BCR-ABL protein complex in K562 cells. Experiments performed with Ph$\sp1$-positive ALL cells and uncultured Ph$\sp1$-positive patient white blood cells established the general presence of BCR/BCR-ABL protein complexes in BCR-ABL expressing cells. However, two cell lines derived from Ph$\sp1$-positive patients lacked P160 BCR/P210 BCR-ABL complexes. Lysates from one of these cell lines mixed with lysates from a cell line that expresses only P160 BCR failed to generate BCR/BCR-ABL protein complexes in vitro indicating that P160 BCR and P210 BCR-ABL do not simply oligomerize.^ Two-dimensional tryptic maps were performed on both BCR and BCR-ABL proteins labeled in vitro with $\sp{32}$P. These maps indicate that the autophosphorylation sites in BCR-ABL proteins are primarily located within BCR exon 1 sequences in both P210 and P185 BCR-ABL, and that P160 BCR is phosphorylated in trans in similar sites by the activated ABL kinase of both BCR-ABL proteins. These results provide strong evidence that P160 BCR serves as a target for the BCR-ABL oncoprotein.^ K562 cells, induced to terminally differentiate with the tumor promoter TPA, show a loss of P210 BCR-ABL kinase activity 12-18 hours after addition of TPA. This loss coincides with the loss of activity in P160 BCR/P210 BCR-ABL complexes but not with the loss of the P210 BCR-ABL, suggesting the existence of an inactive form of P210 BCR-ABL. However, a degraded BCR-ABL protein served as the kinase active form preferentially sequestered within the remaining BCR/BCR-ABL protein complex.^ The results described in this thesis form the basis for a model for BCR-ABL induced leukemias which is presented and discussed. ^

Molecular analysis of $\beta$1,4-galactosyl-transferase localization to the cell surface and participation in cell adhesion

$\beta$1,4-Galactosyltransferase (GalTase) is unusual among the glycosyltransferases in that it is found in two subcellular compartments where it performs different functions. In the trans-Golgi complex, GalTase participates in oligosaccharide biosynthesis as do other glycosyltransferases. GalTase is also found on the cell surface, where it associates with the cytoskeleton and functions as a receptor for extracellular oligosaccharide ligands. Although we know much regarding GalTase function on the cell surface, little is known about the mechanisms underlying its transport to the plasma membrane. Cloning of the GalTase gene revealed that there are two GalTase proteins (i.e., long and short) with different size cytoplasmic tails. This raises the possibility that differences in the cytoplasmic domain of GalTase may influence its subcellular distribution. The object of this study was to examine this hypothesis directly through the use of molecular, immunological, and biochemical approaches.^ To examine whether the two GalTase proteins are targeted to different subcellular compartments, F9 embryonal carcinoma cells were transfected with either long or short GalTase cDNAs and intracellular and cell surface enzyme levels measured. Cell surface GalTase activity was enriched in cells overexpressing the long, but not the form of short GalTase. Furthermore, a dominant negative mutation in cell surface GalTase was created by transfecting cells with GalTase cDNAs encoding a truncated version of long GalTase devoid of the extracellular catalytic domain. Overexpressing the complete cytoplasmic and transmembrane domains of long GalTase led to a loss of GalTase-dependent cellular adhesion by specifically displacing surface GalTase from its cytoskeletal associations. In contrast, overexpressing the analogous truncated protein of short GalTase had no effect on cell adhesion. Finally, chloramphenicol acetyltransferase (CAT) reporter proteins were used to determine directly whether the cytoplasmic domains of long and short GalTase were responsible for differential subcellular distribution. The cytoplasmic and transmembrane domains of long GalTase led to CAT expression on the ceil surface and its association with the detergent-insoluble cytoskeleton; the analogous fusion protein containing short GalTase was restricted to the Golgi compartment. These results suggest that the cytoplasmic domain unique to long GalTase is responsible for targeting a portion of this protein to the cell surface and associating it with the cytoskeleton, enabling it to function as a cell adhesion molecule. ^

Functional studies of homeobox gene {\it Cart1\/} during mouse development

Cart1 is a paired-class homeobox-containing gene that is expressed in head mesenchyme, branchial arches, limb buds, and various cartilages during embryogenesis. To understand the role of Cart1 during mammalian development, I generated Cart1-mutant mice by gene targeting in mouse embryonic stem cells. Cart1-homozygous mutants were born alive but all died soon after birth. Most had acrania (absence of the cranial vault) and meroanencephaly (absence of part of the brain). In situ hybridization studies showed that Cart1 is expressed specifically in forebrain mesenchyme but not in midbrain or hindbrain mesenchyme nor in the neural tube. Developmental studies revealed a transient deficiency of forebrain mesenchyme cells due to apoptosis associated with a delay in neural tube closure in that region. Subsequently, the forebrain region became filled with mesenchyme and closed, however, the midbrain neural tube region never initiated closure and remained open. These results suggest that Cart1 is required for the survival of forebrain mesenchyme and that its absence disrupts cranial neural tube morphogenesis by blocking the initiation of closure in the midbrain region, and this ultimately leads to the generation of lethal craniofacial defects. Prenatal treatment of Cart1 homozygous mutants with folic acid suppressed the development of the acrania/meroanencephaly phenotype. Thus, Cart1 mutant mice provide a novel animal model for understanding the cellular, molecular, and genetic etiology of neural tube defects and for the development of prenatal therapeutic protocols using folic acid. ^

The role of conserved region 1 of Escherichia coli sigma-70 in transcription initiation

The sigma (σ) subunit of eubacterial RNA polymerase is essential for initiation of transcription at promoter sites. σ factor directs the RNA polymerase core subunits ( a2bb′ ) to the promoter consensus elements and thereby confers selectivity for transcription initiation. The N-terminal domain (region 1.1) of Escherichia coli σ70 has been shown to inhibit DNA binding by the C-terminal DNA recognition domains when σ is separated from the core subunits. Since DNA recognition by RNA polymerase is the first step in transcription, it seemed plausible that region 1 might also influence initiation processes subsesquent to DNA binding. This study explores the functional roles of regions 1.1 and 1.2 of σ70 in transcription initiation. Analysis in vitro of the transcriptional properties of a series of N-terminally truncated σ70 derivates revealed a critical role for region 1.1 at several key stages of initiation. Deletion of the first 75 to 100 amino acids of σ70 (region 1.1) resulted in both a slow rate of transition from a closed promoter complex to a DNA-strand-separated open complex, as well as a reduced efficiency of transition from the open complex to a transcriptionally active open complex. These effects were partially reversed by addition of a polypeptide containing region 1.1 in trans. Therefore, region 1.1 not only modulates DNA binding but is important for efficient transcription initiation, once a closed complex has formed. A deletion of the first 133 amino acids which removes both regions 1.1 and 1.2 resulted in arrest of initiation at the earliest closed complex, suggesting that region 1.2 is required for open complex formation. Mutagenesis of region 1.1 uncovered a mechanistically important role for isoleucine at position 53 (I53). Substitution of I53 with alanine created a σ factor that associated with the core subunits to form holoenzyme, but the holoenzyme was severely deficient for promoter binding. The I53A phenotype was suppressed in vivo by truncation of five amino acids from the C-terminus of σ 70. These observations are consistent with a model in which σ 70I53A fails to undergo a critical conformational change upon association with the core subunits, which is needed to expose the DNA-binding domains and confer promoter recognition capability upon holoenzyme. To understand the basis of the autoinhibitory properties of the σ70 N-terminal domain, in the absence of core RNA polymerase, a preliminary physical assessment of the interdomain interactions within the σ70 subunit was launched. Results support a model in which N-terminal amino acids are in close proximity to residues in the C-terminus of the σ 70 polypeptide. ^

Neuropathology and pathogenesis of HIV encephalopathies

The nervous system is frequently affected in patients with the acquired immune deficiency syndrome (AIDS). In addition to opportunistic CNS infections and cerebral lymphomas, approx. 20% of the patients develop HIV-associated encephalopathies. Two major histopathological manifestations are observed. HIV leukoencephalopathy (progressive diffuse leukoencephalopathy) is characterized by a diffuse loss of myelin in the deep white matter of the cerebral and cerebellar hemispheres, with scattered multinucleated giant cells and microglia but scarce or absent inflammatory reaction. HIV encephalitis (multinucleated giant cell encephalitis) is associated with accumulations of multinucleated giant cells, inflammatory reaction and often focal necroses. In some patients, both patterns may overlap. In order to identify the HIV genome in the CNS, brain tissue from 27 patients was analyzed for the presence of HIV gag sequences using the polymerase chain reaction (PCR) and primers encoding a 109 base pair segment of the gag gene. Amplification of HIV gag succeeded in all 5 patients with clinical and histopathological evidence for HIV encephalopathy but was negative in the 20 AIDS patients with opportunistic bacterial, parasitic and/or viral infections or with cerebral lymphomas. These results strongly suggest that the evolution of histopathologically recognizable HIV-encephalopathies closely correlates with the presence and/or tissue concentration of HIV. Since there were no cases with amplified HIV DNA in the absence of HIV-associated tissue lesions, we conclude that harboring and replication of HIV in the CNS rapidly causes corresponding clinical and morphological changes of HIV-associated encephalopathies. In two children with severe HIV encephalomyelitis, large amounts of HIV gag and env transcripts were detected in affected areas of the brain and spinal cord by in situ hybridization.(ABSTRACT TRUNCATED AT 250 WORDS)

Male Dominance in the New Zealand Longfin Eel Population of a New Zealand River: Probable Causes and Implications for Management

The endemic New Zealand longfin eel Anguilla dieffenbachi (hereafter, longfin eel), is overfished, and in southern South Island, New Zealand, rivers have recently become predominated by males. This study examined length and age at sexual differentiation in male eels in the Aparima River catchment (area, 1,375 km(2); mean flow, 20 m(3.)s(-1)) and the sex ratio and distribution of eels throughout the catchment. Longfin eels differentiated into males mostly at lengths from 300 to 460 mm and ages from 10 to 25+ years. Females were rare: Of 738 eels examined for sexual differentiation, 466 were males and 5 were females, and a few others, not examined, were large enough to be female. These counts suggest a male : female ratio among differentiated longfin eels of 68:1. Of 31 differentiated shortfin eels A. australis, less common in the Aparima River, 26 were females. Male longfin eels were distributed throughout the main stern and tributaries; undifferentiated eels were more prevalent in lower and middle reaches and in the main stem than in upper reaches and tributaries. In other studies, male longfin eels predominated commercial catches in the Aparima and four other southernmost rivers, by 2.4:1 to 13.6:1 males to females. The Aparima River had the most skewed sex ratio. Longfin eel catches from the Aparima River will become more male predominated because few sublegal-size females were present. The length-frequency distributions of eels in the present samples and in the commercial catches were truncated just above minimum legal size (about 460 mm), showing that few females escape the fishery. Historically, females predominated these rivers. The recent change in sex ratio is attributable partly to selective harvest of females, and partly to changes in the structure of the population from fishing, such that differentiation into males has been favored. Longevity, delayed sexual maturity, semel-parity, and endemism with restricted range make the longfin eel particularly vulnerable to overfishing.

Looking the cow in the eye: deletion in the NID1 gene is associated with recessive inherited cataract in Romagnola cattle

Cataract is a known condition leading to opacification of the eye lens causing partial or total blindness. Mutations are known to cause autosomal dominant or recessive inherited forms of cataracts in humans, mice, rats, guinea pigs and dogs. The use of large-sized animal models instead of those using mice for the study of this condition has been discussed due to the small size of rodent lenses. Four juvenile-onset cases of bilateral incomplete immature nuclear cataract were recently observed in Romagnola cattle. Pedigree analysis suggested a monogenic autosomal recessive inheritance. In addition to the cataract, one of the cases displayed abnormal head movements. Genome-wide association and homozygosity mapping and subsequent whole genome sequencing of a single case identified two perfectly associated sequence variants in a critical interval of 7.2 Mb on cattle chromosome 28: a missense point mutation located in an uncharacterized locus and an 855 bp deletion across the exon 19/intron 19 border of the bovine nidogen 1 (NID1) gene (c.3579_3604+829del). RT-PCR showed that NID1 is expressed in bovine lenses while the transcript of the second locus was absent. The NID1 deletion leads to the skipping of exon 19 during transcription and is therefore predicted to cause a frameshift and premature stop codon (p.1164fs27X). The truncated protein lacks a C-terminal domain essential for binding with matrix assembly complexes. Nidogen 1 deficient mice show neurological abnormalities and highly irregular crystal lens alterations. This study adds NID1 to the list of candidate genes for inherited cataract in humans and is the first report of a naturally occurring mutation leading to non-syndromic catarct in cattle provides a potential large animal model for human cataract.

Evidence of trepanation in Late Iron Age Switzerland (420–240BC)

In Europe the procedure of trepanation is known since the Neolithic and is still practiced today in East African native tribes. Trepanation is the oldest known surgical intervention and is defined as the intentional penetration of the cranial vault with removal of piece of a skull bone. Therefore, it is believed that neurosurgery is one of the world’s oldest professions. In this study two skulls with lesions from the Late Iron Age cemetery of Münsingen (420–240 BC), Switzerland, are presented. Aim of the study was to analyse the lesions and to assess whether they were caused by surgical interventions. Sex and age of the individuals were determined by current morphologic-anthropological methods. Radiological examinations were performed with a multislice CT-scanner. Different trepanation methods and signs of healing are discussed. In Switzerland about 33 skulls with possible trepanations dating from Neolithic to medieval times are known and are presented in a short review. Studies of ancient surgical interventions provide important information of pre- and early historic populations. Trepanations of the skull such as the presented cases prove a profound understanding of the human body in ancient times.