Basic fibroblast growth factor and insulin-like growth factor I are strong mitogens for cultured mouse keratinocytes

Basic fibroblast growth factor (bFGF), but not acidic fibroblast growth factor (aFGF), was found to be mitogenic for cultured mouse keratinocytes. A six-to-nine fold increase in 3H-thymidine (3H-dT) incorporation into the acid insoluble pool and a similar increase of the labeling index can be measured when bFGF, at a concentration between 1 and 10 ng/ml, is added to keratinocytes arrested in serum-free and growth factor-free medium with a Ca++-concentration below 0.1 mM. The half-maximal response is observed between 0.2 and 0.7 ng/ml. In the same culture system, insulin-like growth factor I/somatomedin C (IGF-I) and insulin act as mitogens. IGF-I shows half-maximal stimulation with 2-3 ng/ml, insulin with 100-500 ng/ml. Basic FGF, IGF-I and insulin can be classified as strong stimulators of DNA synthesis in mouse keratinocytes. In this regard they are comparable to epidermal growth factor, which shows a half-maximal stimulation at a concentration between 1.5-2 ng/ml. These results show that in addition to mesenchymal cells, FGF is a growth factor not only for neuroectodermal cells, but ectodermal cells in general. They further support the idea that the growth promoting effect of insulin on keratinocytes may be mediated by the IGF-I receptor.     

Genetic diversity among progenitors and elite lines from the Iowa Stiff Stalk Synthetic (BSSS) maize population: comparison of allozyme and RFLP data

Summary Data for restriction fragment length polymorphisms (RFLPs) of 144 clone-enzyme combinations and for 22 allozyme loci from 21 U.S. Corn Belt maize (Zea mays L.) inbreds were analyzed. The genetic materials included 14 progenitors of the Iowa Stiff Stalk Synthetic (BSSS) maize population, both parents of one missing BSSS progenitor, four elite inbreds derived from BSSS, and inbred Mo17. Objectives were to characterize the genetic variation among these 21 inbreds for both allozymes and RFLPs, to compare the results from both types of molecular markers, and to estimate the proportion of unique alleles in the BSSS progenitors. Genetic diversity among the 21 inbreds was substantially greater for RFLPs than for allozymes, but the percentages of unique RFLP variants (27%) and unique allozyme alleles (25%) in the BSSS progenitors were similar. Genetic distances between inbreds, estimated as Rogers' distance (RD), were, on average, twice as large for RFLP (0.51) as for allozyme data (0.24). RDs obtained from allozyme and RFLP data for individual line combinations were only poorly correlated (r = 0.23); possible reasons for discrepancies are discussed. Principal component analysis of RFLP data, in contrast to allozyme data, resulted in separate groupings of the ten BSSS progenitors derived from the Reid Yellow Dent population, the four BSSS elite lines, and Mo17. The remaining six BSSS progenitors were genetically rather diverse and contributed a large number of rare alleles to BSSS. The results of this study corroborate the fact that RFLPs are superior to allozymes for characterizing the genetic diversity of maize breeding materials, because of (1) the almost unlimited number of markers available and (2) the greater amount of polymorphisms found. In particular, RFLPs allow related lines and inbreds with common genetic background to be identified, but a large number of probe-enzyme combinations is needed to estimate genetic distances with the precision required.Joint contribution from Cereal and Soybean Research Unit, USDA, Agricultural Research Service, and Journal Paper No. J-14236 of the Iowa Agricultural and Home Economics Experiment Station, Projects 2818 and 2778     

Tryptophan residues of creatine kinase: a fluorescence study

Spectroscopic studies of rabbit skeletal muscle creatine kinase (CPK) and its complexes with adenosine phosphates have long suggested the occurrence of a tryptophan residue at or near the coenzyme binding sites [K?gi, J. H. R., Li, T.-K., & Vallee, B. L. (1971) Biochemistry 10, 1007-1015; Price, N. C. (1972) FEBS Lett. 24, 21-23]. This conjecture was further supported by nuclear Overhauser effect (NOE) 1H NMR studies indicating through-space interactions between protons of the adenine ring of bound ADP and one or more aromatic side chains of the proteins [Vasák, M., Nagayama, K., Wüthrich, K., Mertens, M. L., & K?gi, J. H. R. (1979) Biochemistry 18, 5050-5055]. Further evidence for a tryptophan residue in the environment of the active site has now been obtained by fluorescence-quenching studies using iodide and acrylamide as external quenchers. Thus, while by the addition of iodide the tryptophan fluorescence of unliganded CPK is reduced to about 75% of the unquenched control, no such effect is manifested upon addition of this quencher to the CPK.ADP and CPK.ATP complexes. Similarly, the relative effectiveness of quenching of the CPK-coenzyme complexes by acrylamide is only about 60% of that measured in the unliganded enzyme. Both these data and the spectral characteristics of the quenched fluorescence suggest that coenzyme binding perturbs a tryptophan residue that is close to the active site and that is partially exposed to the solvent. The differential effectiveness of external quenchers on unliganded and liganded CPK allows the determination of the ligand binding equilibria by fluorescence-quenchability titration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of molecular markers linked to the Agropyron elongatum-derived leaf rust resistance gene Lr24 in wheat

The objective of this study was to identify molecular markers linked to the wheat leaf rust resistance gene Lr24 derived from Agropyron elongatum (3DL/3Ag translocation). Two near isogenic lines (NILs), ‘Arina’ and Lr24/7 ^* “Arina”, were screened for polymorphism at the DNA level with 115 RFLP probes. Twenty-one of these probes map to the homoeologous group 3. In addition, 360 RAPD primers were tested on the NILs. Six RFLP probes showed polymorphism between the NILs, and 11 RAPD primers detected one additional band in the resistant NIL. The genetic linkage of the polymorphic markers with Lr24 was tested on a segregating F₂ population (150 plants) derived from a cross between the leaf rust resistant Lr24/7 ^* “Arina” and the susceptible spelt (Triticum spelta) variety ‘Oberkulmer’. All 6 RFLP markers were completely linked to Lr24: one was inherited as a codominant marker (PSR1205), one was in coupling phase (PSR1203) and 4 were in repulsion phase (PSR388, PSR904, PSR931, PSR1067) with Lr24. The localization of these probes on chromosome 3D was confirmed by nulli-tetrasomic analysis. Distorted genotypic segregation was found for the Codominant RFLP marker PSR1205. This distortion can be explained by the occurrence of hemizygous plants. One of the 11 RAPD markers (OPJ-09) also showed complete linkage to theLr24 resistance gene. The polymorphic RAPD fragment was cloned and sequenced. Specific primers were synthesized, and they produced an amplification product only in the resistant plants. This specific marker allows a reliable and rapid screening of a large number of genotypes in practical breeding. Analysis of 6 additional lines containing Lr24 revealed that 3 lines have a smaller chromosomal segment of A. elongatum than lines derived from ‘Agent’, a commonly used gene donor for the Lr24 resistance gene.     

Capillary blood perfusion during postischemic reperfusion in striated muscle.

Monitoring of nutritive blood flow in muscle is of particular importance to reconstructive surgeons, since ischemia/reperfusion in striated muscle is known to result in postischemic microvascular perfusion failure. Laser Doppler flowmetry has recently been introduced as an easy-to-use, noninvasive technique for continuous monitoring of microvascular tissue perfusion. Despite its popularity, there exists a great deal of controversy as to what actually generates the laser Doppler signal recorded from a given tissue. Intravital microscopy is a technique for direct visualization of the nutritional circulation in tissue. By using intravital microscopy, direct measurements of blood perfusion in individual segments of the nutritional microcirculation can be made. In 22 Syrian golden hamsters we performed laser Doppler flowmetry and intravital microscopy measurements in muscle tissue prior to and during reperfusion after 4 hours of tourniquet ischemia using the dorsal skinfold chamber model. Intravital microscopy (n = 10) revealed a heterogeneous capillary perfusion during the early reperfusion phase with a decrease (p less than 0.01) in functional capillary density to 49.4 +/- 17.0 percent of control. No recovery was observed after 24 hours of reperfusion. Laser Doppler flowmetry (n = 12) showed a parallel reduction of capillary red blood cell flux during the early perfusion phase to 43.9 +/- 22.6 percent of control values (p less than 0.01), and no recovery was observed after 24 hours of reperfusion. However, the laser Doppler flowmetry technique was not able to detect the capillary perfusion inhomogeneities shown by intravital microscopy. Postischemic reperfusion in striated muscle is characterized by a decrease in functional capillary density and a heterogeneous capillary perfusion. Laser Doppler flowmetry is a useful tool for monitoring microvascular tissue perfusion, although in striated muscle of the hamster it must be considered that accurate nutritional "capillary" flow readings can be grossly overestimated if larger vessels, such as arterioles and collecting venules, are contained in the measuring field of the laser Doppler probe.     

Stimulation of DNA synthesis and myo-inositol incorporation in mammalian cells

Phosphatidylinositol (PI) synthesis and its role in controlling the cell cycle has been investigated using fibroblasts and liver cells in culture. PI synthesis as measured by incorporation of [³H]-myo-inositol into trichloroacetic acid precipitable material during 0–60 min after serum or growth factor stimulation of serum-starved cells is increased in primary fetal rat liver cells, rat embryo fibroblasts, and 3T3 mouse cells. In contrast, growth stimulation of 3T3 cells and hepatocytes rendered quiescent in G₁ by amino acid starvation is not accompanied by increased incorporation of [³H]-myo-inositol into trichloroacetic acid precipitable material. This suggests that those cells might be arrested at a different point in G₁ than cells arrested by serum depletion. Inhibition of PI synthesis by δ-hexachlorocyclohexane (HCH), a steric analog of myo-inositol, during early times (e.g., 0–4 hr) after growth stimulation, reversibly blocks initiation of DNA synthesis in 3T3 cells. The results support the idea that increased PI synthesis in response to growth stimulation in the cell types studied here is a prerequisite for progression through G₁ and subsequent entry into S phase.     

Diet influences the intake target and mitochondrial functions of Drosophila melanogaster males

In this study, we examine the dietary protein to carbohydrate ratio (P:C) on the mitochondrial functions of two Drosophila melanogaster mtDNA haplotypes. We investigated multiple physiological parameters on flies fed with either 1:12 P:C or 1:3 P:C diets. Our results provide experimental evidence that a specific haplotype has a reduction of complex I activity when the flies are fed with the 1:12 P:C diet. This study is of particular importance to understand the influence of diet on mitochondrial evolution in invasive and broadly distributed species including humans.