Nonequilibrium freezing of one-cell mouse embryos. Membrane integrity and developmental potential.

A thermodynamic model was used to evaluate and optimize a rapid three-step nonequilibrium freezing protocol for one-cell mouse embryos in the absence of cryoprotectants (CPAs) that avoided lethal intracellular ice formation (IIF). Biophysical parameters of one-cell mouse embryos were determined at subzero temperatures using cryomicroscopic investigations (i.e., the water permeability of the plasma membrane, its temperature dependence, and the parameters for heterogeneous IIF). The parameters were then incorporated into the thermodynamic model, which predicted the likelihood of IIF. Model predictions showed that IIF could be prevented at a cooling rate of 120 degrees C/min when a 5-min holding period was inserted at -10 degrees C to assure cellular dehydration. This predicted freezing protocol, which avoided IIF in the absence of CPAs, was two orders of magnitude faster than conventional embryo cryopreservation cooling rates of between 0.5 and 1 degree C/min. At slow cooling rates, embryos predominantly follow the equilibrium phase diagram and do not undergo IIF, but mechanisms other than IIF (e.g., high electrolyte concentrations, mechanical effects, and others) cause cellular damage. We tested the predictions of our thermodynamic model using a programmable freezer and confirmed the theoretical predictions. The membrane integrity of one-cell mouse embryos, as assessed by fluorescein diacetate retention, was approximately 80% after freezing down to -45 degrees C by the rapid nonequilibrium protocol derived from our model. The fact that embryos could be rapidly frozen in the absence of CPAs without damage to the plasma membrane as assessed by fluorescein diacetate retention is a new and exciting finding. Further refinements of this protocol is necessary to retain the developmental competence of the embryos.     

Meiotic recombination in an Irish family with beta-thalassaemia

Using the technique of allele-specific priming of the polymerase chain reaction (PCR), the C-T substitution in codon 39 was identified as the cause of -thalassaemia in an Irish family. Analysis of the restriction fragment length polymorphisms (RFLPs) in the -globin gene cluster established linkage of the -thalassaemia mutation to a particular -haplotype but indicated that a recombinational event had occurred in the paternal chromosome in the younger of two affected children. Non-paternity was excluded by DNA fingerprinting analysis with hypervariable minisatellite probes. This is the fourth case of recombination in the -globin gene cluster to be reported. The event has occurred 5 of the polymorphic RsaI site at position-550 bp upstream of the -globin gene mRNA Cap site, within the 9.1-kb region that has been shown to be a hot spot for recombination in the -globin gene cluster.     

Characterization and classification of virulent lactococcal bacteriophages isolated from a Cheddar cheese plant

C.N. CASEY, E. MORGAN, C. DALY AND G.F. FITZGERALD, 1993. Twenty-two bacteriophages, isolated from cheese-vat whey samples over a period of 4 years, were found to be active against one or more of four different strains of Lactococcus lactis subsp. cremoris used in a defined strain starter system in an Irish Cheddar cheese factory. All the phages were small isometric-headed with non-contractile tails, a baseplate and a collar; they had genome sizes of approximately 30 kb, and belonged to a single DNA hybridization group. All 22 phages could be classified into four distinct groups based on restriction analysis which overlapped perfectly with those based on host range. Each group of phage examined showed cross-reactive host ranges. None of the phage DNAs hybridized to the chromosomes of any of the seven cultures used in the factory during the four cheesemaking seasons, and neither could phages be induced from any of the strains by mitomycin-C or ultraviolet light treatment.     

Activation-induced T-cell death is cell cycle dependent and regulated by cyclin B.

Developing thymocytes and some T-cell hybridomas undergo activation-dependent programmed cell death. Although recent studies have identified some critical regulators in programmed cell death, the role of cell cycle regulation in activation-induced cell death in T cells has not been addressed. We demonstrate that synchronized T-cell hybridomas, irrespective of the point in the cell cycle at which they are activated, stop cycling shortly after they reach G2/M. These cells exhibit the diagnostic characteristics of apoptotic cell death. Although p34cdc2 levels are not perturbed after activation of synchronously cycling T cells, cyclin B- and p34cdc2-associated histone H1 kinase activity is persistently elevated. This activation-dependent induction of H1 kinase activity in T cells is associated with a decrease in the phosphotyrosine content of p34cdc2. We also demonstrate that transient inappropriate coexpression of cyclin B with p34cdc2 induces DNA fragmentation in a heterologous cell type. Finally, in T cells, cyclin B-specific antisense oligonucleotides suppress activation-induced cell death but not cell death induced by exposure to dexamethasone. We therefore conclude that a persistent elevation of the level of cyclin B kinase is required for activation-induced programmed T-cell death.     

Molecular analysis of the feline immunodeficiency virus protease: generation of a novel form of the protease by autoproteolysis and construction of cleavage-resistant proteases.

The feline immunodeficiency virus (FIV) protease is essential for virion maturation and subsequent viral replication in that it cleaves the Gag and Gag/Pol polyproteins at eight sites to release the respective structural proteins and enzymes. During purification of a recombinant FIV protease (PR), we noted that it underwent autoproteolysis (autolysis) to give discrete cleavage products. These additional PR cleavage sites were defined using N-terminal amino acid sequence analysis and mass spectrometry. Protease breakdown products were also found in FIV virions and were of the same apparent molecular weights as the in vitro autolysis products. Four primary PR autolysis sites were blocked via substitution of either the P1 amino acid with a beta-branched amino acid or the P1' amino acid with lysine. Cleavage-resistant PRs which had Km and k(cat) values similar to those of FIV PR were constructed. An autolysis time course determined that blocking all four primary autolysis sites yielded a cleavage-resistant PR which was enzymatically stable. Concomitant with autolysis is the generation of an N-terminally truncated form of the PR (Thr6/PR) which has enhanced stability with respect to that of FIV PR. A structural basis for the Thr6/PR activity is presented, as are the possible roles of autolysis in the viral replication cycle.     

Further cholinergic aspects of carotid body chemotransduction of hypoxia in cats

Fitzgerald, Robert S., Machiko Shirahata, and Tohru Ide.Further cholinergic aspects of carotid body chemotransduction ofhypoxia in cats. J. Appl. Physiol.82(3): 819-827, 1997.From the 1930s into the 1970s, the role ofacetylcholine (ACh) in the carotid body's chemotransduction of hypoxiawas debated. Since the late 1970s, the issue has been pursued onlyintermittently or not at all. The purpose of this study was to testagain with a new preparation the hypothesis that ACh is an excitatoryneurotransmitter in the cat carotid body's chemotransduction ofhypoxia. We tested the effect of the specific nicotinic blockermecamylamine and the muscarinic blocker of all five muscarinicreceptors, atropine. We further tested the effects ofM₁ andM₂ muscarinic-receptor blockers.The carotid body region was selectively perfused with hypoxicKrebs-Ringer bicarbonate (KRB) solutions that were blocker free orcontained varying doses of the blockers. Both mecamylamine and atropinereduced the response to hypoxic KRB in a dose-related manner. TheM₂ muscarinic-receptor blockersgallamine and AFDX 116 increased the response to hypoxic KRB, whereasthe M₁ muscarinic-receptor blockerpirenzepine reduced the response to hypoxic KRB. These data areconsistent with an excitatory role for ACh in the carotid bodychemotransduction of hypoxia in the cat.

     

Isoforms of an endogenous lectin in rabbit bone marrow

A lectin which may mediate inter-erythroblast associations during red blood cell development in rabbit bone marrow has previously been purified and characterised. We have now detected different forms of this lectin in purified preparations and crude tissue extracts, by isoelectric focusing in agarose gels followed by rocket immunoelectrophoresis and by indirect antibody staining of focused proteins blotted onto nitrocellulose paper. These minor antigens are probably isoforms of the bone marrow lectin previously characterised.     

Scanning electron microscopy of Treponema pallidum (Nichols strain) attached to cultured mammalian cells.

This paper describes the attachment of Treponema pallidum (Nichols strain) to cultured mammalian cells as a visualized by scanning electron microscopy. Treponemes were incubated for 3 hr with cultured cells derived from normal rabbit testes or human skin epithelium, then fixed, processed with critical-point drying, and examined with a Cambridge Mark 2A scanning electron microscope. Large numbers of treponemes became attached to the cultured cells without altering the morphological integrity of the cultured cells. Attachment appeared to involve a very close physical proximity of treponemes to the cultured cells; at the site of attachment, no changes such as swelling or indentation of the cultured cell surface were observed. The addition of ruthenium red to the fixatives produced a treponemal-associated surface precipitate. This material, which is probably mucopolysaccharide and/or phospholipid, may be important in protecting the organisms against host defense mechanisms; in addition, it may be involved in the serological unresponsiveness of freshly prepared suspensions of T. pallidum.