Relationship between c-Kit expression and proliferation in acute myeloblastic leukemia cell lines

We have proposed that the transmembrane receptor encoded by the c-Kit protooncogene and its ligand play an important role in regulating the proliferation of blasts cells in acute myeloblastic leukemia (AML). To test this hypothesis, immunobeads were used to separate blasts from three Kit-expression positive cell lines into strongly Kit-protein positive and weakly Kit-protein positive fractions. The strongly positive fraction had greater proliferative potential than the weakly positive fraction as assessed both by colony-formation in methylcellulose and growth of clonogenic cells in suspension. The reproducibility of the percentage of each blast population found in the strongly and weakly positive fractions provided evidence that Kit-protein expression is regulated. Kinetic experiments provided evidence for reversible transitions between strong and weak Kit protein expression. Thus regulated expression of the Kit receptor may be a mechanism for controlling blast cell growth in culture. © 1993 Wiley-Liss, Inc.     

A kinetic approach to defining the role of chemically modifiable residues at the active sites of enzymes.

Initial velocity studies can be used to define the function of chemically modifiable residues in the active site of a multisubstrate enzyme. Since the method relies on measuring biological activity, it has the advantage that it can be used with small amounts of relatively impure enzyme, but requires that modified enzyme molecules have some residual catalytic activity. The kinetic analysis of the modified enzyme can be carried out in the presence of some unmodified enzyme molecules. Consideration of examples of single- and multisubstrate enzymic reactions shows that interpretation of changes in apparent Km and apparent V values following chemical modification, in terms of effects on substrate binding or catalysis or both, requires a detailed knowledge of the kinetic reaction sequence. In the case of multisubstrate enzymes, it is necessary to ensure that, during the kinetic investigation, the concentrations of the non-varied substrates remain at near-saturating levels. For this reason, and because modification may induce changes in the rate-limiting step, a full kinetic analysis of the modified enzyme is advisable.     

A role for the transmembrane domain in the trimerization of the MHC class II-associated invariant chain.

MHC class II and invariant chain (Ii) associate early in biosynthesis to form a nonameric complex. Ii first assembles into a trimer and then associates with three class II alphabeta heterodimers. Although the membrane-proximal region of the Ii luminal domain is structurally disordered, the C-terminal segment of the luminal domain is largely alpha-helical and contains a major interaction site for the Ii trimer. In this study, we show that the Ii transmembrane domain plays an important role in the formation of Ii trimers. The Ii transmembrane domain contains an unusual patch of hydrophilic residues near the luminal interface. Substitution of these polar residues with nonpolar amino acids resulted in a decrease in the efficiency of Ii trimerization and subsequent class II association. Moreover, N-terminal fragments of Ii were found to trimerize independently of the luminal alpha-helical domain. Progressive C-terminal truncations mapped a homotypic association site to the first 80 aa of Ii. Together, these results implicate the Ii transmembrane domain as a site of trimer interaction that can play an important role in the initiation of trimer formation.     

Hypoxia-Induced Changes in the Bioactivity of Cytotrophoblast-Derived Exosomes

Migration of extravillous trophoblasts (EVT) into decidua and myometrium is a critical process in the conversion of maternal spiral arterioles and establishing placenta perfusion. EVT migration is affected by cell-to-cell communication and oxygen tension. While the release of exosomes from placental cells has been identified as a significant pathway in materno-fetal communication, the role of placental-derived exosomes in placentation has yet to be established. The aim of this study was to establish the effect of oxygen tension on the release and bioactivity of cytotrophoblast (CT)-derived exosomes on EVT invasion and proliferation. CT were isolated from first trimester fetal tissue (n = 12) using a trypsin-deoxyribonuclease-dispase/Percoll method. CT were cultured under 8%, 3% or 1% O2 for 48 h. Exosomes from CT-conditioned media were isolated by differential and buoyant density centrifugation. The effect of oxygen tension on exosome release (µg exosomal protein/10⁶cells/48 h) and bioactivity were established. HTR-8/SVneo (EVT) were used as target cells to establish the effect (bioactivity) of exosomes on invasion and proliferation as assessed by real-time, live-cell imaging (Incucyte™). The release and bioactivity of CT-derived exosomes were inversely correlated with oxygen tension (p<0.001). Under low oxygen tensions (i.e. 1% O2), CT-derived exosomes promoted EVT invasion and proliferation. Proteomic analysis of exosomes identified oxygen-dependent changes in protein content. We propose that in response to changes in oxygen tension, CTs modify the bioactivity of exosomes, thereby, regulating EVT phenotype. Exosomal induction of EVT migration may represent a normal process of placentation and/or an adaptive response to placental hypoxia.     

Insights into phylogeny,sex function and age of Fragaria based on whole chloroplast genome sequencing

The cultivated strawberry is one of the youngest domesticated plants, developed in France in the 1700s from chance hybridization between two western hemisphere octoploid species. However, little is known about the evolution of the species that gave rise to this important fruit crop. Phylogenetic analysis of chloroplast genome sequences of 21 Fragaria species and subspecies resolves the western North American diploid F. vesca subsp. bracteata as sister to the clade of octoploid/decaploid species. No extant tetraploids or hexaploids are directly involved in the maternal ancestry of the octoploids.There is strong geographic segregation of chloroplast haplotypes in subsp. bracteata, and the gynodioecious Pacific Coast populations are implicated as both the maternal lineage and the source of male-sterility in the octoploid strawberries. Analysis of sexual system evolution in Fragaria provides evidence that the loss of male and female function can follow polyploidization, but does not seem to be associated with loss of self-incompatibility following genome doubling. Character-state mapping provided insight into sexual system evolution and its association with loss of self-incompatibility and genome doubling/merger. Fragaria attained its circumboreal and amphitropical distribution within the past one to four million years and the rise of the octoploid clade is dated at 0.372–2.05 million years ago.     

Patterns of pollen quantity and quality limitation of pre‐zygotic reproduction in Mimulus guttatus vary with co‐flowering community context

Pollen quantity limitation has been widely recognized as one of the main causes of plant reproductive failure in nature. However, its negative effects on fruit and seed production have been often confounded with those of low pollen quality (i.e. low conspecific pollen viability and/or slow pollen tube growth rate). The lack of differentiation between these two aspects of pollen limitation has resulted not only in a potential overestimation of the incidence of pollen quantity limitation but has led to a poor understanding of the factors and mechanisms that affect pollen quality limitation. Knowledge of the relative importance and underlying causes of both aspects of pollen limitation (quantity and quality) is required to fully understand the ecological and evolutionary consequences of pollen limitation in natural populations. Co‐flowering community context (e.g. species richness and conspecific density), in particular, can be an important driver of overall pollen limitation. However, how pollen quantity and quality limitation vary separately with the community context has not previously been examined, even though they may vary differently as they arise from different mechanisms, e.g. insect visitation rate versus inbreeding depression. Here we evaluate the effect of co‐flowering diversity and conspecific density on the relative importance of pollen quantity and quality limitation for Mimulus guttatus pre‐zygotic reproduction (i.e. pollen tube success) at serpentine seeps in California over two years. We found overall pollen limitation of pre‐zygotic reproductive success at all seeps regardless of the co‐flowering context. However, plants in high‐diversity/low‐ conspecific density communities were mostly limited by pollen quantity, whereas plants in low‐diversity/high‐conspecific density ones experienced stronger quality limitation – a pattern that was consistent across years. These results not only highlight the importance of conducting comprehensive studies on pollen limitation that evaluate both, its quantity and quality aspects, but are the first to show how their relative contribution to overall pollen limitation can vary with extrinsic factors such as the co‐flowering community context.     

Anatomical variation of orthotropic elastic moduli of the proximal human tibia

The anatomical variation of orthotropic elastic moduli of the cancellous bone from three human proximal tibiae was investigated using an ultrasonic technique. With this technique, it was possible to measure three orthogonal elastic moduli and three shear moduli from cubic specimens of cancellous bone as small as 8 mm per side. Correlation with mechanical tensile testing has shown this technique to offer a precise measure of cancellous modulus (Eten = 0.94Eult + 144.6 MPa, r2 = 0.96, n = 34). The cancellous bone of the proximal tibia was found to be very inhomogeneous, with the axial modulus ranging between 340 and 3350 MPa. A course map is presented, showing measured Young's moduli as a function of anatomical position. The anisotropy of the cancellous bone, determined by the relative differences between the three orthogonal moduli, was shown to be relatively constant over the entire range of cancellous densities tested. The relationship between the axial elastic modulus and the apparent density was found to be approximately linear, as reported by others for proximal tibial cancellous bone.     

The use of synthetic gonadotropin releasing hormone treatment in the collection of sheep embryos

Gonadotropin releasing hormone (GnRH) treatment was examined as a means of improving the efficacy of embryo collection in the sheep following intrauterine insemination of frozen-thawed semen. In summary, treatment consistently improved fertilization rates and the number of fertilized ova collected per ewe was enhanced compared with untreated ewes. The yield of fertilized ova in ewes treated with follicle stimulating hormone (FSH) was maximized by administering GnRH 36 h after progestagen treatment; 24 h was the preferred time in ewes treated with pregnant mare serum gonadotropin (PMSG). There was a significant (P < 0.001) increase in the percentage of unfertilized ova in the former treatment when GnRH was given at 24 h. An examination of the time of insemination (0, 6, 12 and 18 h before the median time of ovulation) indicated that fertilization rates were highest when insemination occurred at 6 h in both GnRH-treated ewes and in untreated ewes. In GnRH-treated ewes, the recovery of ova was lowest when insemination occurred at the time of ovulation. The number of motile frozen-thawed spermatozoa required for fertilization following treatment was estimated to be approximately 20 x 10(6) per uterine horn. GnRH-treatment also improved the yield of fertilized ova in sheep that were naturally mated, although this yield was lower than that obtained with intrauterine insemination of frozen-thawed semen. It is concluded that fertilization failure, a major problem in sheep embryo collection, can be eliminated through judicious use of GnRH treatment and properly timed intrauterine insemination.