Oxygen: a master regulator of pancreatic development?

Beyond its role as an electron acceptor in aerobic respiration, oxygen is also a key effector of many developmental events. The oxygen‐sensing machinery and the very fabric of cell identity and function have been shown to be deeply intertwined. Here we take a first look at how oxygen might lie at the crossroads of at least two of the major molecular pathways that shape pancreatic development. Based on recent evidence and a thorough review of the literature, we present a theoretical model whereby evolving oxygen tensions might choreograph to a large extent the sequence of molecular events resulting in the development of the organ. In particular, we propose that lower oxygenation prior to the expansion of the vasculature may favour HIF (hypoxia inducible factor)‐mediated activation of Notch and repression of Wnt/β‐catenin signalling, limiting endocrine cell differentiation. With the development of vasculature and improved oxygen delivery to the developing organ, HIF‐mediated support for Notch signalling may decline while the β‐catenin‐directed Wnt signalling is favoured, which would support endocrine cell differentiation and perhaps exocrine cell proliferation/differentiation.     

Immunochemistry of the HLA class II molecules isolated from a mouse cell transfected with DQ alpha and beta genes from a DR4 haplotype

Cells from a mouse B lymphoma were transfected by DQ alpha and DQ beta genes derived from a DR4 haplotype. Quantitatively, the resulting expression of human class II molecules was similar to that of human B lymphoblastoid cell lines. Qualitatively, the transformant class II molecules differed from normal class II molecules in their carbohydrate moiety. As for their antigenic specificity, they were shown to carry two determinants previously identified on DQ molecules controlled by DR4 haplotypes, i. e., DQw3 and DCHON. The transformant molecules did not carry a third DR4-associated specificity, DC5 (equivalent to TA10), and must possess a structure allelic to DC5. However, no corresponding alloantigenic specificity was detected by a screening of relevant alloantisera.     

Electro-insertion of xeno-glycophorin into the red blood cell membrane

The electroporation technique, with field strengths slightly below the critical value Ec for electroporation of red blood cells (RBC), enables the insertion of xeno-proteins into the RBC membrane without damaging the cells. The electro-insertion has been used to insert biotinylated human glycophorin into human RBC membrane and human glycophorin into murine RBC membrane. Binding anti-human glycophorin antibody (10F7) to the murine RBC bearing human glycophorin indicates extracellular orientation of inserted glycophorin. Insertion of about 10(5) glycophorin molecule per cell has been estimated by whole cell ELISA.     

Serological recognition of HLA-DR allodeterminant corresponding to DNA sequence involved in gene conversion

HLA class 11 molecules were isolated from mouse L cells transfected with a DR gene and an allele, 52a, of locus DR III from an HLA-homozygous cell line, AVL, of the DR3 haplotype. The isolated molecules were found to possess a new allospecificity, named TR81. This specificity behaved allelic to the previously described DR III locus. The TR81 specificity was also present on the DR I gene product of the DR3 haplotype. The nucleotide sequence of the gene encoding TR81 differs from TR81-negative DR genes of the DRw52 family in only two codons, both located in the regions known to be involved in a gene conversion event. Consequently, the following conclusions can be formulated. (a) TR81 is a bi-locus specificity and allelic to TR22 only in its DR III locus localization. (b) The TR81 specificity is the phenotypic counterpart of the gene conversion event which led to the generation of the DR I gene of the DR3 haplotype. (c) One or both individual amino acid substitutions in the first domain of the DR chain are responsible for the TR81 allospecificity. (d) Since TR81 is expressed on the DR I chain of the DR3 haplotype, it is possible that TR81 and DR3 represent the same serological specificity.     

Detection of complex alleles by direct analysis of DNA heteroduplexes

DNA molecules derived from three alleles of the HLA-DRB3 locus and differing from each other at several nucleotide sites were denatured and cross-hybridized. Each allelic combination was found to generate a pair of heteroduplexes of different mobility. Their retardation as compared to homoduplexes was proportional to the number of mismatches. In each heteroduplexes pair the component possessing the highest number of Pyr-Pyr oppositions was the most retarded. The results are those predicted by a theoretical model implying a correlation between base-pair opening and bending of the DNA double helix. These observations introduce a new HLA typing method at the genomic level and indicate an experimental approach to the analysis of the superhelical DNA conformation as related to different types of base oppositions.     

Studies on Analogues of Succinic Semialdehyde as Substrates for Succinate Semialdehyde Dehydrogenase from Rat Brain

The methyl ester of succinic semialdehyde (SSA) was examined as a substrate for succinate semialdehyde dehydrogenase (SSADH) from rat brain. It was found that the ester can be oxidized by the enzyme. Values of Km for SSA-Me were higher than for those for SSA, and for this substrate the enzyme showed a substrate-dependent inhibition. This finding suggests that the carboxylate group of SSA is not essential in the process of inhibition of SSADH by the substrate. Cyclopropyl analogues of SSA, cis- and trans-1-formyl-cyclopropan-2-carboxylic acids, were also individually tested as substrates of SSADH. Only the trans isomer was found to be oxidized to the corresponding dicarboxylic acid; it inhibited the enzyme in the same range of concentrations as SSA. The above data suggest that, as for gamma-aminobutyric acid, SSA is present in an unfolded, transoid conformation at the active site of SSADH.     

Iron(III)-adriamycin and Iron(III)-daunorubicin complexes: physicochemical characteristics, interaction with DNA, and antitumor activity

Fe(III) complexes of two anthracyclines, adriamycin and daunorubicin, have been studied. Using potentiometric and spectroscopic measurements, we have shown that adriamycin and daunorubicin form two well-defined species with Fe(III), which can be formulated as respectively Fe(HAd)3 and Fe(HDr)3. In these formulas, HAd and HDr stand for adriamycin and daunorubicin in which the 1,4-dihydroxy-anthraquinone moiety is half-deprotonated. Both complexes are six-membered chelates. The stability constant is beta = (2.5 +/- 0.5) X 10(28) for both complexes. Interaction with DNA has been studied showing that, despite strong coordination to Fe(III), anthracyclines are able to intercalate between DNA bases pairs, releasing the metal. These complexes display antitumor activity against P 388 leukemia that compares with that of the free drug. Fe(HAd)3, unlike adriamycin, does not catalyze the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase. Moreover, it is shown that the triferric adriamycin compound so called "quelamycin" is in fact a mixture of Fe(HAd)3 and polymeric ferric hydroxide.     

Leukocyte LFA-1, OKM1, p150,95 deficiency syndrome: functional and biosynthetic studies of three kindreds

Three patients (2 female, 1 male) with recurrent infection, granulocytosis, impaired pus formation, and/or delayed umbilical cord separation were identified. Assessments of polymorphonuclear leukocytes (PMN)/monocyte function in each patient revealed profound abnormalities of adherence and adherence-dependent functions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of their PMN lysates demonstrated a deficient or absent protein(s) of 138 kilodaltons (gp 138). Na3HB4 labeling demonstrated the absence of a major cell surface glycoprotein complex in each patient. Among parental and sibling PMN suspensions, functional assessments revealed no consistent abnormalities, although variably diminished gp138 was identified by SDS-PAGE and Na3HB4 labeling. Analysis by fluorescence-activated cell sorting and monoclonal antibodies (MAb) to LFA-1 alpha, OKM1 alpha, and their common beta subunit demonstrated a severe or total deficiency of PMN/monocyte surface expression of each protein among all patients; intermediate values were observed for parental and affected sibling suspensions, findings consistent with an autosomal recessive mode of inheritance for this disorder. Cell surface labeling (125I) and immunoprecipitation with the same MAb demonstrated the absence of these glycoproteins in addition to a 150-kilodalton protein (p150,95). Identical abnormalities of surface expression of patient lymphocytes blast-transformed with phytohemagglutinin (PHA) or Epstein-Barr virus were demonstrated. Further, significantly diminished natural killer cell cytotoxicity was observed for each patient tested. PHA blast-transformed patient lymphocytes labeled with [35S]methionine demonstrated a total absence of the beta molecule but indicated the presence of an LFA-1 alpha precursor. These findings indicate that LFA-1 alpha synthesis and surface expression require beta association. It is concluded that impaired inflammatory function in this disorder is casually related to a heritable deficiency of critical "adhesive" leukocyte glycoproteins.     

Communicating junctions and calmodulin: Inhibition of electrical uncoupling inXenopus embryo by calmidazolium

Summary This paper reports the inhibitory effects of calmidazolium (CDZ), a calmodulin inhibitor, on electrical uncoupling by CO₂. Membrane potential and coupling ratio (V ₂/V₁) are measured in two neighboring cells ofXenopus embryos (16 to 64 cell stage) for periods as long as 5.5 hr. Upon exposure to 100% CO₂, control cells consistently uncouple even if the CO₂ treatments are repeated every 15 min for 2.5 hr. CDZ (5×10^–8–1×10^–7 m) strongly inhibits uncoupling. The inhibition starts after 30, 50 and 60 min of treatment with 1×10^–7, 7×10^–8 and 5×10^–8 m CDZ, respectively, is concentration-dependent and partially reversible. In the absence of CO₂, CDZ also improves electrical coupling. CDZ has no significant effect on membrane potential and nonjunctional membrane resistance. These data suggest that calmodulin or a calmodulin-like protein participates in the uncoupling mechanism.     

Analysis of the coliphage T5 DNA ejection process with free and liposome-associated TonA protein.

Outer membrane protein TonA, the receptor for coliphage T5, has been partially purified and incorporated into the phospholipid bilayer of liposomes. Adsorption of the phage to its receptor in either a free or liposome-associated form is fast and sufficient to trigger the ejection of encapsidated DNA. In both in vitro systems the exit of DNA from the phage capsid is a very slow process. Ejected DNA can partially accumulate inside the liposome aqueous compartment, but the transfer from the phage head to the liposome internal space is never complete, perhaps because the liposome volume is too small. The presence of polyamines or divalent cations (magnesium) or both in the incubation medium diminished the extent of DNA ejection, possibly by stabilizing DNA inside the head. DNA movement was slowed as the temperature was decreased from 37 to 18 degrees C. Furthermore, incubation at 4 degrees C totally prevented this DNA movement, even if a large part of the DNA had already exited the capsid.