Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume,hydraulic conductivity and permeability of CD34(+) cells to dimethyl sulphoxide

Umbilical cord blood (UCB) is an accepted treatment for the reconstitution of bone marrow function following myeloablative treatment predominantly in children and juveniles. Current cryopreservation protocols use methods established for bone marrow and peripheral blood progenitors cells that have largely been developed empirically. Such protocols can result in losses of up to 50% of the nucleated cell population: losses unacceptable for cord blood. The design of optimal cryopreservation regimes requires the development of addition and elution protocols for the chosen cryoprotectant; protocols that minimise damaging osmotic transients. The biophysical parameters necessary to model the addition and elution of dimethyl sulphoxide to and from cord blood CD34(+) cells have been established. An electronic particle counting method was used to establish the volumetric response of CD34(+) cells to changes in osmolality of the suspending medium. The non-osmotic volume of the cell was 0.27 of the cells isotonic volume. The permeation kinetics of CD34(+) cells to water and dimethyl sulphoxide were investigated at two temperatures, +1.5 and +20 degrees C. Values for the hydraulic conductivity were 3.2 x 10(-8) and 2.8 x 10(-7)cm/atm/s, respectively. Values for the permeability of dimethyl sulphoxide at these temperatures were 4.2 x 10(-7) and 7.4 x 10(-6)cm/s, respectively. Clonogenic assays indicated that the ability of CD34(+) cells to grow and differentiate was significantly impaired outside the limits 0.6-4x isotonic. Based on the Boyle van't Hoff plot, the tolerable limits for cell volume excursion were therefore 45-140% of isotonic volume. The addition and elution of cryoprotectant was modelled using a two-parameter model. Current protocols for the addition of cryoprotectant based on exposure at +4 degrees C would require additional time for complete equilibration of the cryoprotectant. During the elution phase current protocols are likely to cause CD34(+) cells to exceed tolerable limits. The addition of a short holding period during elution reduces the likelihood of this occurring.     

The single species hypothesis: truly dead and pushing up bushes, or still twitching and ripe for resuscitation?

Frank Livingstone proclaims himself to be the last living proponent of the single species hypothesis. In sharp contrast, a species-rich, bushy phylogeny is favored by most human paleontologists. Is Livingstone's proclamation merely contrarian posturing, or does closer inspection warrant reconsideration of just how speciose the hominin lineage is? The high-speciation perspective draws on evidence of speciosity in the Cercopithecoidea and punctuated equilibria theory for support. If blue monkeys and redtail monkeys are indistinguishable skeletally, this reasoning goes, or if red colobus and black and white colobus are likewise indistinguishable, should we not expect that there are more species of hominin than is apparent from skeletal evidence alone? A contrarian perspective notes that not all monkey taxa are speciose. Importantly, two broadly distributed, partly terrestrial monkeys have not speciated at all: vervets and baboons. Nor are monkeys the first choice as a hominin speciation model. If expectations of species numbers are based on the Hominoidea, a taxon more closely related to hominins, more similar in body size, and found in more hominin-like habitats than monkeys, a single-species perspective is more appealing. No great ape genus has even two sympatric species. Moreover, despite a separation of 1.6 Ma, West African chimpanzees have not speciated from Pt. troglodytes nor Pt. schweinfurthii. It is notable that no two contemporaneous species of hominin were separated by significantly more than this interval. A biological--as opposed to an ecological or geographical--species definition would place all hominins in a single, phenotypically diverse species. Since divergence from the chimpanzee, "species" distinctness in hominins may have been maintained by temporary allopatry and centripetal niche separation. The hominin lineage may have evolved as a single, phenotypically diverse, reticulately evolving species.     

The long form of CDK2 arises via alternative splicing and forms an active protein kinase with cyclins A and E

We have reinvestigated the long form of cyclin-dependent kinase (CDK)2 that is expressed in many rodent cells. We show that the mRNA encoding CDK2L arises by alternative splicing and that the encoded protein can bind to, and be activated by, cyclins A and E. The complex of CDK2L with cyclin A has about half the specific activity of the equivalent CDK2-cyclin A complex. Also, CDK2L--cyclin A is inhibited to the same extent and by the same concentrations of p21(CIP1) as CDK2--cyclin A. The nucleotide sequences of intron V in the human and murine CDK2 genes, where the sequences encoding the 48-residue insert in CDK2L are located, show very high conservation in the position of the alternatively spliced exon and its surroundings. Despite this, we were not able to detect significant expression of CDK2L in human cell lines, although a low level is expressed in COS-1 cells from monkeys.     

Protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein

Protein misfolding is the basis of a number of human diseases and presents an obstacle to the production of soluble recombinant proteins. We present a general method to assess the solubility and folding of proteins in vivo. The basis of this assay is structural complementation between the alpha- and omega- fragments of beta-galactosidase (beta-gal). Fusions of the alpha-fragment to the C terminus of target proteins with widely varying in vivo folding yield and/or solubility levels, including the Alzheimer's amyloid beta (A beta) peptide and a non-amyloidogenic mutant thereof, reveal an unambiguous correlation between beta-gal activity and the solubility/folding of the target. Thus, structural complementation provides a means of monitoring protein solubility/misfolding in vivo, and should find utility in the screening for compounds that influence the pathological consequences of these processes.     

The molecular dynamics of pain control

Pain is necessary for survival, but persistent pain can result in anxiety, depression and a reduction in the quality of life. The discriminative and affective qualities of pain are both thought to be regulated in an activity-dependent fashion. Recent studies have identified cells and molecules that regulate pain sensitivity and the parallel pathways that distribute nociceptive information to limbic or sensory areas of the forebrain. Here, we emphasize the cellular and neurobiological consequences of pain, especially those that are involved in the generation and maintenance of chronic pain. These new insights into pain processing will significantly alter our approach to pain control and the development of new analgesics.     

Central nervous system in cerebral malaria: 'Innocent bystander' or active participant in the induction of immunopathology?

Cerebral malaria (CM) is a major life-threatening complication of Plasmodium falciparum infection in humans, responsible for up to 2 million deaths annually. The mechanisms underlying the fatal cerebral complications are still not fully understood. Many theories exist on the aetiology of human CM. The sequestration hypo-thesis suggests that adherence of parasitized erythrocytes to the cerebral vasculature leads to obstruction of the microcirculation, anoxia or metabolic disturbances affecting brain function, resulting in coma. This mechanism alone seems insufficient to explain all the known features of CM. In this review we focus on another major school of thought, that CM is the result of an over-vigorous immune response originally evolved for the protection of the host. Evidence in support of this second hypothesis comes from studies in murine malaria models in which T cells, monocytes, adhesion molecules and cytokines, have been implicated in the development of the cerebral complications. Recent studies of human CM also indicate a role for the immune system in the neurological complications. However, it is likely that multiple mechanisms are involved in the induction of cerebral complications and both the presence of parasitized erythrocytes in the central nervous system (CNS) and immunopathological processes contribute to the pathogenesis of CM. Most studies examining immunopathological responses in CM have focused on reactions occurring primarily in the systemic circulation. However, these also do not fully account for the development of cerebral complications in CM. In this review we summarize results from human and mouse studies that demonstrate morphological and functional changes in the resident glial cells of the CNS. The degree of immune activation and degeneration of glial cells was shown to reflect the extent of neurological complications in murine cerebral malaria. From these results we highlight the need to consider the potentially important contribution within the CNS of glia and their secreted products, such as cytokines, in the development of human CM.     

The intracellular oxidation of 2',7'-dichlorofluorescin in murine T lymphocytes

Intracellular reactive oxygen species (ROS) production by activated murine T lymphocytes was investigated by analyzing intracellular dichlorofluorescin (DCFH(2)) oxidation in lymph node cells (LNC). An increase in DCFH(2) oxidation in LNC induced by phorbol myristate acetate (PMA) was detected by flow cytometry. It was confirmed that this increase was present in Thy1(+) LNC. We examined the contribution to intracellular DCFH(2) oxidation of ROS released by leukocytes other than T cells present in the LNC suspension. Superoxide dismutase, catalase, and glutathione/glutathione peroxidase inhibited the PMA-induced increase in intracellular DCFH(2) oxidation. Furthermore, PMA failed to elicit DCFH(2) oxidation in LNC isolated from mice lacking a functional NADPH oxidase (gp91(phox) gene knockout mice), but this response could be restored in these cells by the addition of T cell-depleted LNC from wild-type litter mates. This study highlights the necessity for caution in using the DCFH(2) assay to demonstrate specific intracellular ROS production in heterogeneous cell populations. It also suggests that cells other than T cells in lymph node populations may, through production of ROS, influence the intracellular redox state of T lymphocytes.     

Peroxisome proliferators compete and ameliorate Hcy-mediated endocardial endothelial cell activation

To determine whether homocysteine(Hcy)-mediated activation of endocardial endothelial (EE) cells isameliorated by peroxisome proliferator-activated receptor (PPAR), weisolated EE cells from mouse endocardium. Matrix metalloproteinase(MMP) activity and intercellular adhesion molecule (ICAM)-1 in EE cellswere measured in the presence and absence of Hcy, and ciprofibrate (CF;PPAR- agonist) or 15-deoxy-^12,14-prostaglandinJ₂ (PGJ₂; PPAR- agonist) by zymography andWestern blot analyses, respectively. Results suggest that Hcy-mediated MMP activation and ICAM-1 expression are ameliorated by CF and PGJ₂. To test the hypothesis that Hcy competes with otherligands for binding to PPAR and -, we prepared cardiac nuclearextracts. Extracts were loaded onto an Hcy-cellulose affinity column.Bound proteins were eluted with CF and PGJ₂. To determineconformational changes in PPAR upon binding to Hcy, we measured PPARfluorescence at 334 nm. Dose-dependent increase in PPAR fluorescencedemonstrated a primary binding affinity of 0.32 ± 0.06 µM. There wasdose-dependent quenching of PPAR fluorescence byfluorescamine-homocysteine (F-Hcy). PPAR- fluorescence quenching wasabrogated by the addition of CF but not by PGJ₂. PPAR-fluorescence quenching was abrogated by the addition ofPGJ₂ but not by CF. These results suggest that Hcy competeswith CF and PGJ₂ for binding to PPAR- and -,respectively, indicating a role of PPAR in amelioration of Hcy-mediatedEE dysfunction.

     

Simultaneous analysis of chromosomes and chromosome-associated proteins in mammalian oocytes and embryos

Cytogenetic analyses of mammalian eggs and preimplantation embryos have been limited by the difficult and tedious task of preparing chromosomes from single cells or small numbers of cells. In this report we describe a new technique that is both reliable and comparatively simple. Further, since the technique does not use the conventional 3:1 methanol:acetic acid fixative, it has the advantage of producing high-resolution chromosome preparations without destroying chromosome-associated proteins. Thus, this method provides a sensitive means of conducting studies of a heretofore inaccessible period of mammalian development, and of studying proteins thought to mediate both meiotic chromosome segregation and chromatin modifications in the preimplantation embryo.     

Dose and litter allocations in the design of teratological studies for detecting hormesis

BACKGROUND: Hormesis is being recognized in the field of toxicology due to the stimulating effects of some toxic compounds at low exposure levels. Therefore, it is desirable that experimental designs for toxicological studies be flexible enough to aid in the detection of hormetic effects. Current designs may still not have enough power to do this. METHODS: A simulation study was conducted to determine teratological study designs that would yield more power over standard designs in detecting hormesis. Developmental toxicity endpoints of interest are the number of dead/resorbed or malformed fetuses in a litter. The simulation designs mimic teratological experiments in terms of sample size and number of dose levels. Modified designs with even dose spacing at low levels and reallocated litters are investigated to determine the power of hormetic detection. RESULTS: Designs with reallocated litters (with more litters at low exposure levels than at high levels) and even dose spacing have more power than those with equal litters per group and uneven dose spacing. CONCLUSIONS: Through appropriate modifications of current experimental designs, such as reallocation of litters and even dose spacing, we can better detect hormetic effects.