Effects of pulsatile flow on cultured vascular endothelial cell morphology

Endothelial cells (EC) appear to adapt their morphology and function to the in vivo hemodynamic environment in which they reside. In vitro experiments indicate that similar alterations occur for cultured EC exposed to a laminar steady-state flow-induced shear stress. However, in vivo EC are exposed to a pulsatile flow environment; thus, in this investigation, the influence of pulsatile flow on cell shape and orientation and on actin microfilament localization in confluent bovine aortic endothelial cell (BAEC) monolayers was studied using a 1-Hz nonreversing sinusoidal shear stress of 40 +/- 20 dynes/cm2 (type I), 1-Hz reversing sinusoidal shear stresses of 20 +/- 40 and 10 +/- 15 dynes/cm2 (type II), and 1-Hz oscillatory shear stresses of 0 +/- 20 and 0 +/- 40 dynes/cm2 (type III). The results show that in a type I nonreversing flow, cell shape changed less rapidly, but cells took on a more elongated shape than their steady flow controls long-term. For low-amplitude type II reversing flow, BAECs changed less rapidly in shape and were always less elongated than their steady controls; however, for high amplitude reversal, BAECs did not stay attached for more than 24 hours. For type III oscillatory flows, BAEC cell shape remained polygonal as in static culture and did not exhibit actin stress fibers, such as occurred in all other flows. These results demonstrate that EC can discriminate between different types of pulsatile flow environments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epistasis in maize (Zea mays L.)

Summary Three flint and three dent maize (Zea mays L.) inbred lines, their possible F₁ crosses, F₂ and backcross progenies, and all possible three-way crosses were evaluated in a three-year experiment for yield, ear moisture, and plant height. The purpose was to estimate genetic parameters in European breeding materials from (i) generation means analysis, (ii) diallel analysis of generation means, and (iii) analysis of F₁ and three-way cross hybrids. Method (i) was based on the F-metric model and methods (ii) and (iii) on the Eberhart-Gardner (1966) genetic model; both models extended for heterotic maternal effects.Differences among generation means for yield and plant height were mainly attributable to dominance effects. Epistatic effects were significantly different from zero in a few crosses and considerably reduced heterosis in both traits. Additive x additive and domiance x dominance effects for yield were consistently positive and negative, respectively. Significant maternal effects were established to the advantage of generations with a heterozygous seed parent. In the diallel analysis, mean squares for dominance effects were greater than for additive effects for yield and plant height but smaller for ear moisture. Though significant for yield and plant height, epistatic variation was small compared to additive and dominance variation. Estimates of additive x additive epistasis for yield were significantly negative in 11 of 15 crosses, suggesting that advantageous gene combinations in the lines had been disrupted by recombination in the segregating generations. The analysis of hybrids supported the above findings regarding the analysis of variance. However, the estimates of additive x additive epistasis for yield were considerably smaller and only minimally correlated with those from the diallel analysis. Use of noninbred materials as opposed to materials with different levels of inbreeding is considered the main reason for the discrepancies in the results.     

Biological activity in vivo of insulin analogues modified in the N-terminal region of the B-chain

[AlaB5]Insulin as well as a hybrid analogue of insulin and "insulin-like growth factor" (IGF-I), in which the N-terminal amino-acid sequence H-Phe-Val-Asn-Gln- of the B-chain has been replaced by the tripeptide H-Gly-Pro-Glu-of IGF-I, have been prepared by the partial-synthetic route. Their biological activity in vivo has been compared with that of other analogues in rabbits, mice and rats as far as data are available. These rodents respond differently, rats being less sensitive to modifications than rabbits and mice. The results explain unexpected discrepancies discussed in previous papers.     

Antigenic interrelationship between the 40-kilodalton cytokeratin polypeptide and desmoplakins

We describe here antigenic cross-reactivity between the human 40-kilodalton cytokeratin polypeptide [Moll et al] and components of bovine desmosomal plaque, namely desmoplakins I and II. This relationship was revealed by an antibody (KM 4.62), raised against cytoskeletal preparation of cultured human breast adenocarcinoma cells (MCF-7) and selected by immunoblotting and immunofluorescent labeling. In cultured human cells that contain the 40-kD cytokeratin, antibody KM 4.62 labeled arrays of filaments throughout the cytoplasm. This antibody labeled the basal layer of nonkeratinizing squamous epithelia as well as various simple (normal and malignant) epithelia and epithelial elements of the thymus. In liver tissue, labeling was obtained only in bile ducts and canaliculi but not in the hepatocytes. In bovine cells and tissues, on the other hand, immunofluorescent labeling with antibody KM 4.62 was strictly confined to desmosomes. This was verified by double immunolabeling with both antibody KM 4.62 and specific cytokeratin or desmosomal antibodies. Immunoblotting analysis indicated that the former antibody reacts specifically with the high molecular weight components of the bovine desmosomal plaque, namely desmoplakins I and II. These immunochemical results suggest that bovine desmoplakins share same structural relationship with the human acidic, 40-kD cytokeratin.     

Human tissue kallikrein. I. Isolation and characterization of human pancreatic kallikrein from duodenal juice

Human pancreatic kallikrein was purified from duodenal juice by ion exchange chromatography on DEAE-Sepharose and immunoaffinity chromatography. Thus, an enzyme preparation with a specific activity (using Ac-Phe-Arg-OEt as substrate) of 1 000 U/mg protein was obtained. A specific biological activity of 1310 KE/mg protein was measured in the dog blood pressure assay and of 0.361 HMW kininogen-U/mg, corresponding to the liberation of 383 micrograms bradykinin-equivalents per mg enzyme per min from HMW kininogen in the rat uterus assay. In dodecyl sulfate gel electrophoresis one protein band corresponding to a molecular mass of 27 kDa was obtained. Using gel filtration on Ultrogel AcA-44 a molecular mass of 40 kDa was measured. The amino-acid composition was determined and isoleucine and alanine were identified as the only N-terminal amino-acid residues. On isoelectric focusing four protein bands with isoelectric points of 5.60, 5.65, 5.70 and 5.85 were separated. The bimolecular velocity constant for the inhibition by diisopropyl fluoro phosphate was determined as 10.5 l x mol-1 x min-1. The dissociation constant Ki of the human pancreatic kallikrein-aprotinin complex was calculated to be 1.5 x 10(-10)M. The kinetic constants for the kallikrein-catalysed hydrolysis of Ac-Phe-Arg-OEt and D Val-Leu-Arg-Nan were determined. Immunological studies showed a close relationship between the human pancreatic kallikrein and other human tissue kallikreins, especially with human urinary kallikrein. Detergents such as Triton X-100, Tween 20 and lysolecithin, as well as human serum albumin, activated the human pancreatic kallikrein preparation.     

Sources of sucrose translocated from illuminated sugar beet source leaves

A search for source leaf sucrose pools that differed in their relation to export was carried out in photosynthesizing leaves of Beta vulgaris L. The time course of depletion of [¹⁴C]sucrose in a leaf in unlabeled CO₂ following steady state labeling provided evidence for two distinct sucrose pools. After the start of the light period, leaf blade sucrose remained constant although it exchanged between the two pools. Newly synthesized sucrose destined for export passed through one pool more rapidly than through the other. All of the leaf blade sucrose appeared to exchange with export sucrose. Modeling and regression analysis of [¹⁴C]sucrose data provided a means for estimating the size of the two pools. From 20 to 40% of the sucrose was calculated to be present in the pool that provided the less direct path to export; this was likely vacuolar sucrose. The remainder of the sucrose in the blade was probably in the cytoplasm and veins. Added amounts of leaf blade sucrose, produced in response to elevated CO₂, appeared to be stored mainly in the vacuolar compartment.     

De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin

Poly(A)+ RNA isolated from bovine muzzle epidermis was microinjected into nonepithelial cells containing only intermediate-sized filaments of the vimentin type. In recipient cells keratin polypeptides are synthesized and assemble into intermediate-sized filaments at multiple dispersed sites. We describe the time course and the pattern of de novo assembly of keratin filaments within living cells. These filaments were indistinguishable, by immunofluorescence and immunoelectron microscopic criteria, from keratin filament arrays present in true epithelial cells. The presence of extended keratin fibril meshworks in these injected cells is compatible with cell growth and mitosis. Double immunolabeling revealed that newly assembled keratin was not codistributed with microfilament bundles, microtubules or vimentin filaments. We suggest that assembly mechanisms exist which in vivo sort out newly synthesized cytokeratin polypeptides from vimentin.