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The Drosophila melanogaster developmental gene g1 encodes a variant zinc-finger-motif protein.

In Drosophila melanogaster, the mechanisms involved in the pattern formation of complex internal organs are still largely unknown. However, the identity of the molecular determinants that control the development of these specific tissues is emerging from the combined use of genetic and molecular approaches. We have cloned a gene that is expressed in the mesoderm, one of the fundamental embryonic germ layers which gives rise to internal structures, such as the musculature. Here, we describe the molecular characterization of this gene, designated as g1. The nucleotide (nt) sequence of its cDNA shows an open reading frame of 852 nt, which encodes a 32-kDa protein with two putative zinc fingers, and a serine/glutamine/proline-rich region. These features indicate a functional role for g1, which remains to be elucidated, in regulating gene expression during mesoderm formation.

Urea substitutes toxic formamide as destabilizing agent in nucleic acid hybridizations with RNA probes

Since their introduction some three decades ago, methods for hybridization analysis of nucleic acids immobilized on solid supports have evolved to improve the sensitivity, speed, and convenience of their application. However, in many cases these methods still require the use of solutions containing formamide, a recognized hazardous solvent with potential toxicity. Here, we have compared the efficiency of urea to that of formamide as denaturing agent in nucleic acid hybridization with RNA probes. We show that urea at concentrations of 2–4 molar in solution performs as good as 50% formamide to reduce heterologous background hybridization in Northern blotting experiments realized at 68°C. Presence of urea at higher concentrations resulted in reduced hybridization sensitivity, possibly due to increased viscosity. When tested in Southern blot analysis of genomic DNA, our results revealed that the use of urea in hybridization solution is also suitable to carry out single‐copy gene detection. Together, these findings show that urea can efficiently and safely replace formamide in solutions.

Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signal-regulated kinases 1 and 2.

In the treatment of autoimmune diseases, intravenous Igs (IVIg) are assumed to modulate immune cells through the binding of surface receptors. IVIg act upon definite human B cell populations to modulate Ig repertoire, and such modulation might proceed through intracellular signaling. However, the heterogeneity of human B cell populations complicates investigations of the intracellular pathways involved in IVIg-induced B cell modulation. The aim of this study was to establish a model allowing the screening of IVIg signal transduction in human B cell lines and to attempt transposing observations made in cell lines to normal human B lymphocytes. Nine human B cell lines were treated with IVIg with the goal of selecting the most suitable model for human B lymphocytes. The IgG(+) DB cell line, whose response was similar to that of human B lymphocytes, showed reduced IVIg modulation following addition of PD98059, an inhibitor of extracellular signal-regulated protein kinase 1/2 (ERK1/2). The IVIg-induced ERK1/2 phosphorylation was indeed proportional to the dosage of monomeric IVIg used when tested on DB cells as well as Pfeiffer cells, another IgG(+) cell line. In addition, two other intermediates, Grb2-associated binder 1 (Gab1) and Akt, showed increased phosphorylation in IVIg-treated DB cells. IVIg induction of ERK1/2 phosphorylation was finally observed in peripheral human B lymphocytes, specifically within the IgG(+) B cell population. In conclusion, IVIg immunomodulation of human B cells can thus be linked to intracellular transduction pathways involving the phosphorylation of ERK1/2, which in combination with Gab1 and Akt, may be related to B cell antigen receptor signaling.

CD40-activated B cells from patients with systemic lupus erythematosus can be modulated by therapeutic immunoglobulins in vitro

IntroductionAberrant signaling within and between B and T cells, considered to be central in systemic lupus erythematosus (SLE), could depend on enhanced CD40-CD154 activation. As a result, autoreactive B cells, normally anergic, differentiate and secrete antibodies attacking several normal tissues. Thus restorating B cell homeostasis might help control this disease. In this study, two facets of SLE B cells were investigated, namely their in vitro response to CD40-CD154 and the effect of treatment with human immunoglobulins for intravenous use (IVIg).Materials and MethodsBlood samples from SLE patients and healthy volunteers were obtained and used to isolate B cells, which were activated through CD40 in the presence or absence of IVIg. The phenotype, proliferation, and differentiation of the SLE B cells were determined and compared with those of control B cells using flow cytometry and standard ELISA.ResultsIn this model, CD40-activated SLE B cells, as control B cells, proliferated and differentiated and were characterized by the emergence of CD19loCD38++CD138+CD27++ cells. IVIg treatment of the CD40-activated SLE B cells resulted in higher differentiation, characterized by increased secretion rates of IgG and IgM, as reported previously for control B cells.ConclusionsTaken as a whole, such accelerated differentiation of CD40-activated B cells suggests that IVIg may participate in re-equilibration of the antibody repertoire by replacing pathological antibodies by de novo harmless antibodies.

Expression of gooseberry-proximal in the Drosophila developing nervous system responds to cues provided by segment polarity genes

SummarySegment polarity genes define the cell states that are required for proper organization of each metameric unit of the Drosophila embryo. Among these, the gooseberry locus has been shown to be composed of two closely related genes which are expressed in an overlapping single-segment periodicity. We have used specific antibodies raised against the protein product of the gooseberry proximal (gsb-p) gene to determine the spatial distribution of this antigen in wild type embryos, and to monitor the effects of segment polarity mutants on the pattern of the gsb-p protein distribution. We find that the gsb-p protein accumulates beneath each posterior axonal commissure in the progeny of neuroblasts deriving from the epidermal compartments of wingless (wg) and engrailed (en) expression. The results of this analysis support the idea that gsb-p has a specific role in the control of cell fates during neurogenesis, and indicate that en and wg provide critical positional cues to define the domain in which gsbp will be activated. Furthermore, these data suggest that, in order to be expressed in the embryonic CNS, gsb-p may preliminarily require activity of the gooseberry-distal gene in the epidermis.

Anomalous cleavage of histone H1 from Physarum polycephalum

Abstract The H1 histone from amoebae of the slime mould Physarum polycephalum is non-enzymatically cleaved in mildly acidic pH conditions, while calf histones are unaffected. One of the degradation products, the CP1 polypeptide, migrates slightly faster than Physarum H1 on polyacrylamide gels. Since the CP1 polypeptide can be produced during histone isolation and purification, it could be mistaken for a histone H1 subtype. Whether the cleavage of H1 to CP1 occurs in vivo during the life cycle of P. polycephalum is unknown. The molecular weight of the CP1 polypeptide was estimated at 26000 on SDS-polyacrylamide gels. Since the apparent molecular weight of Physarum H1 was 28800, the CP1 polypeptide must have resulted from the cleavage of H1 near one or both termini. Partial chymotryptic digestions of Physarum H1 and the CP1 polypeptide suggested that the CP1 fragment is most likely produced by the cleavage of a short portion of the C-terminal region of Physarum H1. The CP1 polypeptide could be used in amino acid sequencing or in studies to determine the physical structure and function of different regions of the Physarum H1 histone.

An Overview of Apoptosis in Blood Transfusion

Introduction Control of cell death is essential for normal development, differentiation and tissue homeostasis. This tight control applies as well for components of blood cells, such as erythrocytes for instance. The average life span of an erythrocyte is about 120 days. Considering that the human body has 3 to 5 x 10” erythrocytes in circulation, it is estimated that about 1% of all erythrocytes must therefore die every day and, in order to sustain the levels of circulating erythrocytes, be replaced by an equal number of newly differentiated cells. What are the mechanisms involved in the destruction of aged erythrocytes in vivo and what happens of these cells in transfusion plastic bags during 5, 21 or 42 days? Do storage conditions modify the viability of the other blood components? What are the putative clinical impacts of the presence of dying or dead cell products in transfused patients? In the following, I present a brief overview of the apoptotic form of cell death and of its role in transfusion medicine. Apoptosis Cells die by two primary processes: necrosis or apoptosis (1,2). Necrosis refers to the accidental cell death that occurs in response to harmful insults such as physical damage, hypoxia, hyperthermia and chemical injury. Morphologically, necrosis is characterized by early mitochondria1 swelling and failure, dysfunction of the plasma membrane with loss of homeostasis, cell swelling, and rupture. The loss of cell membrane integrity with release of cell contents, including proteases and lysozymes, induces an inflammatory reaction that can affect the surrounding tissues, often causing substantial damage to neighboring cells. Apoptosis, or programmed cell death, describes the highly conserved gene-regulated processes by which cells are removed under normal conditions when they reach the end of their lifespan. are damaged, or superfluous (3). This major physiological mechanism of cell removal is now known to be involved in divergent phenomena such as hair loss, digit formation from limb buds, migration of dermal cells upward to the epidermis, and during lymphocyte development. The apoptotic processes are characterized by a sequence of morphological alterations, which are clearly distinguishable from necrosis. In cells undergoing apoptosis, there is ruffling, blebbing. and condensation of the plasma and nuclear membranes, and, subsequently, aggregation of nuclear chromatin. Mitochondria and ribosomes retain their gross structure and at least partial function. There is disruption of the cytoskeletal architecture; the cell shrinks and then fragments into a cluster of membrane-enclosed “apoptotic bodies” of variable size that contain a variety of intact cytoplasmic organelles and some nuclear fragments. Several of the biochemical events underlying these morphological changes have been characterized. The degradation of nuclear lamins and cytoskeletal proteins by an activated proteolytic cascade involving a family of proteases called caspases, is a central component of the apoptotic machinery resulting in the disassembly of the cell (4). Another main biochemical feature of apoptosis is DNA cleavage at the internucleosomal sites, by calcium-activated endonucleases (5 ) . This results in the typical DNA laddering on agarose gels, which is considered the biochemical hallmark of apoptosis. Tissue transglutaminase is also a specific protein whose induction and activation has been clearly demonstrated to occur during apoptosis (6). This