Experimental studies on a lethal gene (1) in the Mexican axolotl, Ambystoma mexicanum.

1. Gene ? is a recessive lethal factor found in the white strain of axolotls. Animals heterozygous for the gene are phenotypically normal. When mated with each other they give offspring 25% of which exhibit the lethal effects of the gene. 2. The ?/? homozygotes develop normally to an advanced embryonic stage (Harrison stage 40) before the effects of the gene are first manifested. They then come to display a characteristic combination of abnormalities, including a disproportionately small head, small and poorly developed eyes, abnormal poorly developed gills, undifferentiated limb buds, and reduced overall growth rate. They may feed briefly, but soon stop and invariably die within a few weeks of the time of hatching. 3. The action of gene ? has been analyzed by parabiosing mutant and normal embryos, and by grafting various organ primordia reciprocally between mutant and normal embryos. Parabiosis to normal embryos fails to correct the abnormalities of the mutants, although their survival may be somewhat prolonged. Grafts of mutant organ primordia (eye, limb, gill, pronephros, gonad, head) also invariably fail to show improved development or to survive on normal hosts; normal organ primordia develop normally on mutant hosts so long as the mutant survives. These experiments indicate that gene ? is a recessive autonomous cell lethal affecting all of the organ systems during late embryonic and early larval development.     

Primary structure of the nucleotide binding domain of the Ca,Mg-ATPase from cardiac sarcoplasmic reticulum

The nucleotide binding domain of the active site of the Ca,Mg-ATPase of cardiac sarcoplasmic reticulum (SR) has been isolated using fluorescein isothiocyanate (FITC) as an active site label and sequenced. After removal of non-specifically incorporated FITC with hydroxylamine, the amount of label incorporated was stoichiometric with residual ATPase activity, demonstrating that the label was incorporated uniquely at the active site. The SR was succinylated before digestion by trypsin in order to obtain a peptide of sufficient length to determine if the cardiac SR ATPase is a candidate for the unidentified cDNA clone recently sequenced by MacLennan et al. (Nature 316: 696-700, 1985). The sequence of the labeled SR peptide, obtained by affinity chromatography on a FITC antibody column, was T S M S K M F K G P E V I D R. This sequence was identical with that predicted by the unidentified clone and is significantly different from the sequence reported by Kirley et al. (Biochem. Biophys. Res. Commun. 130: 732-738, 1985) for a FITC labeled peptide isolated from cardiac SR.     

Comparison of multiple molecular dynamics trajectories calculated for the drug-resistant HIV-1 integrase T66I/M154I catalytic domain

HIV-1 integrase (IN) is an essential enzyme for the viral replication and an interesting target for the design of new pharmaceuticals for multidrug therapy of AIDS. Single and multiple mutations of IN at residues T66, S153, or M154 confer degrees of resistance to several inhibitors that prevent the enzyme from performing its normal strand transfer activity. Four different conformations of IN were chosen from a prior molecular dynamics (MD) simulation on the modeled IN T66I/M154I catalytic core domain as starting points for additional MD studies. The aim of this article is to understand the dynamic features that may play roles in the catalytic activity of the double mutant enzyme in the absence of any inhibitor. Moreover, we want to verify the influence of using different starting points on the MD trajectories and associated dynamical properties. By comparison of the trajectories obtained from these MD simulations we have demonstrated that the starting point does not affect the conformational space explored by this protein and that the time of the simulation is long enough to achieve convergence for this system.     

Peptides inhibit selectin-mediated cell adhesion in vitro, and neutrophil influx into inflammatory sites in vivo

The selectins are cell adhesion molecules whose carbohydrate-bindingdomain (C-type lectin) is thought to be involved in leukocyteadhesion to activated vascular endothelium in the inflammatoryprocess. A series of peptides, based on a conserved region (⁴⁸YYWIGIRK⁵⁵-NH₂)of the lectin domain of E-, L- and P-selectins, were analysedfor their ability to block selectin-mediated cell adhesion invitro, and neutrophil infiltration into sites of inflammationin vivo. The peptides inhibited the adhesion of myeloid cellsto recombinant forms of E- and P-selectin. The adhesion of myeloidcells to human endothelial cells, stimulated to express E-selectin,was also inhibited by the peptides. Finally, the peptides blockedthe adhesion of lymphocytes, expressing L-selectin, to highendothelial venules in lymph nodes which contain the ligandfor L-selectin. A clear structure-activity relationship wasestablished when peptides of different amino acid chain lengthswere tested in these assays. Peptides lacking tyrosine residues(e.g. WIGIR-NH₂) at their amino terminus were poor inhibitorsof selectin-mediated cell adhesion in vitro. The peptides thatwere found to be inhibitors of cell adhesion in vitro were alsofound to inhibit (up to 70%) neutrophil infiltration into sitesof inflammation in a thioglycollate-induced peritonitis mousemodel system. They also significantly reduced (>50%) themigration of neutrophils into cytokine-treated skin. These resultsstrongly suggest that compounds based on these tyrosine-containing,selectin-derived peptides could be used as anti-inflammatorytherapeutic agents. inflammation neutrophils peptides selectins     

Use of lectins for detection of electrophoretically separated glycoproteins transferred onto nitrocellulose sheets

A method of rapidly identifying lectin-binding glycoproteins separated by polyacrylamide gel electrophoresis is described. The method is particularly useful for comparing the glycoprotein content of different cell types and fractions. Normal rat liver, Novikoff hepatoma, and rat mammary tumor cell line 13762 MAT-B were fractionated to give purified nuclei and other fractions defined by their sedimentation properties in low ionic strength buffer. The subcellular fractions were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, transferred to nitrocellulose sheets, and localized by an immunochemical method to identify lectin-binding activities. The localization pattern of concanavalin A and wheat germ agglutinin-binding activities in the fractions from the three cell types showed the greatest similarities between the glycoprotein contents of normal liver and Novikoff hepatoma fractions. On a per-cell basis the purified nuclei from each of the cell types contained less activity overall than did other particulate cell fractions. Washing the nuclei from normal liver and Novikoff hepatoma, but not MAT-B cells, in nonionic detergent removed or depressed most of the lectin-binding activities. However, two major bands were unaffected by the detergent. One of these localized with wheat germ agglutinin at an apparent molecular weight of 62,000 in the nuclei of all three cell types. The other localized with concanavalin A at an apparent molecular weight of 200,000 in normal liver and Novikoff hepatoma nuclei.     

Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics‐centric study investigates a possible link between protein paucimannosylation, an under‐studied class of human N‐glycosylation [Man_1‐3GlcNAc₂Fuc_0‐1], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non‐cancerous specimens are profiled from 467 published and unpublished PGC‐LC‐MS/MS N‐glycome datasets collected over a decade. PMGs, particularly Man_2‐3GlcNAc₂Fuc₁, are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0–50.2%). Analyses of paired (tumor/non‐tumor) and stage‐stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N‐acetyl‐β‐hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.     

Light-Regulated Gravitropism in Seedling Roots of Maize

Red light-induced changes in the gravitropism of roots of Zea mays variety Merit is a very low fluence response with a threshold of 10⁻⁹ moles per square meter and is not reversible by far red light. Blue light also affects root gravitropism but the sensitivity of roots to blue is 50 to 100 times less than to an equal fluence of red. In Z. mays Merit we conclude that phytochrome is the sole pigment associated with light-induced changes in root gravitropism.     

Assessing the skill of yes/no predictions

Summary Should healthy, middle‐aged women receive precautionary mammograms? Should trauma surgeons use the popular TRISS score to predict the likelihood of patient survival? These are examples of questions confronting us when we decide whether to use a yes/no prediction. In order to trust a prediction we must show that it is more valuable than would be our best guess of the future in the absence of the prediction. Calculating value means identifying our loss should the prediction err and examining the past performance of the prediction with respect to that loss. A statistical test to do this is developed. Predictions that pass this test are said to have skill. Only skillful predictions should be used. Graphical and numerical methods to identify skill will be demonstrated. The usefulness of mammograms is explored.