An analysis of the meaning of the concepts of "commitment" and "determination" in the study of patterns of development

The use of the term "commitment" by different authors was compared and the term itself was compared with the term "determination". Different authors understand the term "commitment" in different ways. It is proposed to preserve the terms "competence", "determination" (labile and stable) and "differentiation" in studies of normal development at stages preceding the appearance of organ rudiments in order to facilitate the use of the knowledge acquired by experimental embryology and to decipher these concepts at the molecular level. The meaning of the term "commitment" should be made more precise when describing the experimental results and also when assessing the results obtained by various authors and published in numerous papers and reviews.     

Effects of applied auxin,gibberellin and cytokinin on the activity of peroxidases in the peduncles of rose flowers

The activity of peroxidases in the proximal part of the flower peduncle of rose cv. Nubia was promoted by exogenous application of auxin but not by gibberellin or cytokinin. In cv. Mercedes the activity was promoted also by gibberellin and cytokinin. In the distal parts of the peduncles of both cultivars, peroxidase activity was not affected by any of the applied growth regulators. In young flowers of cv. Nubia the protein content of the penduncles was affected only by cytokinin, and in aged flowers only by auxin, while in Mercedes peduncles the content of protein was not affected by any of the applied growth regulators. The specific activity of peroxidases was promoted by auxin in peduncles of Nubia and by both auxin and cytokinin in peduncles of Mercedes flowers.     

The response of the skin of swine to increasing single exposures of 250-KVP X-rays

When the skin of the shoulder ("A" field) and lower back ("C" field) is irradiated through 10-cm-diameter fields with 250-kVp x-rays, having a HVL of 0.87 mm copper, a dose range is reached between approximately 1600 rads and 3000 rads in which a moist reaction is or is not formed. If a moist reaction is formed, it either heals completely, partially, or not at all. The evolution, time course, and dose dependence of the moist reaction that occurs following irradiation has been determined. The moist reaction is found at 17.5 +/- 0.6 days in the "A" field, and 20.8 +/-0.8 days in the "C" field. The reaction evolves to involve from 5 % to 100% of the field by 24.9 +/- 0.5 days in the "A" field and by 28.5 +/- 1.0 days in the "C" field. Healing is complete by 36.0 +/-1.0 days in the "A" field and by 38.0 +/- 1.3 days in the "C" field. The area of the field involved with a moist reaction at the time of maximal involvement is dose-dependent. The area of the field involved with a moist reaction at the time of complete healing is also dose-dependent. The dose at which 50 % of the fields were not healed was 2273 +/-103 rads in the "A" field, 2578 +/-141 rads in the "C" fields, and 2437 +/- 89 rads in the "A" and "C" fields. The values in the "C" field are significantly different from those in the "A" field except for the dose at which 50 % of the fields were not healed and the time at which the field was maximally healed.     

Functional states of neutrophils as suggested by whole blood chemiluminescence

Luminol-enhanced chemiluminescence (CL) of whole blood was examined in order to distinguish between activation states of phagocytic cells. The CL response of these cells was provoked by a phagocytic stimulus--polystyrene particles. Four functional states of phagocytes were proposed: "resting", "stand by", "activated" and "exhausted". The distinction was done on the basis of extent of the CL response to the particles, time pattern of the process, inhibition of CL by plasma and appearance of spontaneous light emission. Freshly drawn blood of healthy individuals exhibits the "resting" profile of CL, but that of patients with bacterial infection reveals CL patterns ascribed in this paper to the "stand by", "activated" or "exhausted" states of phagocytes. The "stand by", "activated" and "exhausted" behaviour of phagocytes in extravasated blood may be induced by preincubation of blood, stimulation with saline extract of Escherichia coli or N-formyl-Met-Leu-Phe, and by some manipulations involved in preparation of the purified neutrophils.     

Refined crystal structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin

"Aged" organophosphoryl conjugates of serine hydrolases differ from the corresponding "non-aged" conjugates in their striking resistance to nucleophilic reactivation. The refined X-ray structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin were compared in order to understand the molecular basis for this resistance of "aged" conjugates. "Aged" and "non-aged" crystalline organophosphoryl-gamma-chymotrypsin conjugates were obtained by prolonged soaking of native gamma-chymotrypsin crystals with appropriate organophosphates. Thus, a representative "non-aged" conjugate, diethylphosphoryl-gamma-chymotrypsin, was obtained by soaking native crystals with paraoxon (diethyl-p-nitrophenyl phosphate), and a closely related "aged" conjugate, monoisopropyl-gamma-chymotrypsin, was obtained by soaking with diisopropylphosphorofluoridate. In both crystalline conjugates, the refined structures clearly reveal a high occupancy of the active site by the appropriate organophosphoryl moiety within covalent bonding distance of Ser195 O gamma. Whereas in the "non-aged" conjugate both ethyl groups can be visualized clearly, in the putative "aged" conjugate, as expected, only one isopropyl group is present. There is virtually no difference between the "aged" and "non-aged" conjugates either with respect to the conformation of the polypeptide backbone as a whole or with respect to the positioning of the side-chains within the active site. In the "aged" conjugate, however, close proximity (2.6 A) of the negatively charged phosphate oxygen atom of the dealkylated organophosphoryl group to His57 N epsilon 2 indicates the presence of a salt bridge between these two moieties. In contrast, in the "non-aged" conjugate the DEP moiety retains its two alkyl groups; thus, lacking a negative oxygen atom, it does not enter into such a charge-charge interaction and its nearest oxygen atom is 3.6 A away from His57 N epsilon 2. It is suggested that steric constraints imposed by the salt bridge in the "aged" conjugate lie at the basis of its resistance to reactivation.     

Growth of rose flower peduncles and effects of applied plant growth regulators

Removal of flower buds results in abscission of peduncles of the rose cv. Nubia and cessation of peduncle growth in cv. Mercedes. Peduncle growth was inhibited when pistils and stamens were removed, but was not affected by removal only of sepals and petals. Growth of the decapitated peduncles of Mercedes was partially restored by the application of auxin in lanolin paste on the base of the peduncle and was completely restored by the application of gibberellin, while the combined application of auxin and gibberellin was the most effective for growth restoration. Growth of non-decapitated Nubia peduncles was promoted by application of gibberellin or gibberellin and auxin but not auxin alone.Peduncle elongation of both cultivars was not affected by application of cytokinin and the effect of gibberellin was antagonized by combined application with cytokinin. The peduncles strength (resistance to bending) was affected more strongly by auxin than by gibberellin, and most strongly when auxin and gibberellin were combined. The effect of auxin on the strength of peduncles, but not of gibberellin, was antagonized by application of cytokinin. Excised, non-decapitated flowering stems of Nubia treated with gibberellin and auxin in situ, showed, recovery of the peduncles from wilting after exposure to heat conditions.     

Characterization of oligosaccharides from an antigenic mannan of Saccharomyces cerevisiae

Mannans of the yeast Saccharomyces cerevisiae have been implicated as containing the allergens to which bakers and brewers are sensitive and also the antigen recognized by patients with Crohn's disease. A fraction of S. cerevisiae mannan, Sc500, having high affinity for antibodies in Crohn's patients has been characterized by NMR spectroscopy followed by fragmentation using alkaline elimination, partial acid hydrolysis and acetolysis. The released oligosaccharides were separated by gel filtration on a Biogel P4 column and analyzed by fluorescence labeling, HPLC and methylation analysis. The relationship between structure and antigen activity was measured by competitive ELISA. The antigenic activity of the original high molecular weight mannan could be ascribed to terminal Man13Man12 sequences which are rarely found in human glycoproteins but were over-represented in Sc500 compared to other yeast mannans.     

Differences of the electrical and nicotinic receptor stimulation-evoked liberation of norepinephrine from chicken sympathetic neurons in culture: Possible involvement of different pools of the transmitter

We studied the release of [³H]norepinephrine from chicken sympathetic neurons in culture evoked by nicotinic and electrical stimulation with an intention to establish functional identity or nonidentity of the two stimuli in investigations of neurotransmitter release. Nicotinic stimulation evoked extracellular calcium dependent release of [³H]norepinephrine and the rise of intracellular calcium concentration. The release was completely blocked by nicotinic antagonists hexamethonium (100 mol/l) and mecamylamine (10 mol/l), and decreased by tetrodotoxin (0.3 mol/l) and -conotoxin (0.1 mol/l) to 17% and 27%, resp. The intracellular calcium response was decreased by nicotinic antagonists and tetrodotoxin, but not changed by -conotoxin. The electrical stimulation-evoked release was blocked by both tetrodotoxin and -conotoxin, and decreased by previous electrical, but not nicotinic, stimulation. The differential sensitivity to -conotoxin and tetrodotoxin, and the inability of nicotinic stimulation to decrease the liberation by following electrical stimulation may suggest the mobilization of different pools of the transmitter.     

Again on language of biology]

Some time ago I proposed in an Editorial in this journal some considerations on the language of biology. I concluded that, to realize an autonomy of such a language (and therefore of biology), we have to develop a valid language for biology. In such a context, it seemed to me that the term "metaphors" referred to the concepts concerning the information carried by genetic code, was a reasonable one. However, Barbieri's article in this issue of Rivista di Biologia / Biology Forum calls for a reply. Of course, we do not know very much in this field, even if we have some evidence that a sequence of bases on a DNA is not determined only by chance. In any case we can exclude that nature in this occasion has "invented" a code. Nature doesn't "invent" anything: it only follows its rules, that we name "laws of nature". Barbieri quotes the Morse code, but forgets to say that such a code is "conventional" in the sense that it is valid only because it is the result of an "agreement" between Morse and the users of that code. There is nothing more unnatural than a "code": with whom nature should actually have to "reach an agreement"? As a matter of fact, we interpret as "information" what happens by law of nature. Also Barbieri's thesis that genes and proteins are molecular artifacts, assembled by external agents, whereas generally molecules are determined by their bonds, i.e. by internal factors, is a disputable one. It is examined how much an external structure plays a role in ordinary chemical reactions. The "information" of physics is not a semantic information. For such information we can refer to history of literature, telegraphic offices, genetics or biochemistry.     

Purification of proteins of the Na/Cl cotransporter from membranes of Ehrlich ascites cells using a bumetanide-sepharose affinity column

Summary Bumetanide-binding proteins were isolated from membranes of Ehrlich ascites tumor cells by affinity chromatography. An affinity column was constructed with the active moiety of bumetanide as a ligand using 4-azidobumetanide, a photoactive analogue which inhibits Na/Cl cotransport in Ehrlich cells with high specificity. Covalent binding of the 4-azidobumetanide with Sepharose was promoted by photolysis. Membranes isolated from Ehrlich cells were solubilized withn-octylglucoside. Solubilized proteins retarded by the affinity column were readily eluted by bumetanide. In reducing gels the major proteins eluted by bumetanide were 76 kDa and 38–39 kDa. There were also two proteins of 32 to 35 kDa eluted in lesser amounts. No proteins retarded by the affinity column were eluted with extensive washing without bumetanide. Furthermore, bumetanide eluted no proteins from a control column lacking the specific ligand. Upon rechromatography with bumetanide in solution, bumetanide-eluted proteins were not retarded, but their purity was increased by the retardation of contaminating proteins. Bumetanide-binding protein purified in this manner were characterized further by electrophoresis in nonreducing, nondenaturing gels.     

Morphologic and biochemical heterogeneity of lysosomes]

By means of isopycnic centrifugation in the continuous density gradient of sucrose two subfractions of lysosomes were isolated from rat liver homogenates: a "light" one (with the floating density p=1.13) and a "heavy" one (p=1.24). Electron microscopic, enzymatic and electron microscope enzymatic analysis of the isolated subfractions showed that the "light" subfraction consisted mainly of newly-formed primary lysosomes, while the "heavy" one was presented by secondary lysosomes. Parallel biochemical investigations demonstrated a considerable enzymatic heterogeneity of the two lysosomal subfractions: the "light" subfraction was characterized by a high specific activity of acid DNase, acid RNase and beta-galactosidase, and by almost total absence of beta-glucosidase activity, while the "heavy" one was characterized by a high specific activity of beta-glucosidase, beta-glucuronidase and beta-N-acetylglucosaminidase. Possible causes of enzyme heterogeneity of rat liver lysosomes are discussed.     

The role of neurochemical mechanisms of the dorsal raphe nucleus in various models of anxiety states in rats

Dopamine and serotonin microinjection in the dorsal raphe nucleus of rats does not influence the alarm state in the test of "threatening situation" avoidance, but increases or weakens the state of alarm in the rest of "illuminated site" avoidance. Local injection of GABA in this midbrain formation weakens the alarm state in the test of "threatening situation" avoidance but is not effective in the test of "illuminated site" avoidance. Chemical stimulation of the raphe nucleus by glutaminic acid does not influence the two different experimentally modelled states of alarm, but modulates the mechanisms controlling the instinct of darkness preference by rats.     

The endocrine cells of the rectum of adult ox.

In order to identify the endocrine cell types in various parts of the Ruminant gut, we have applied ultrastructural, both morphological and cytochemical, techniques, in parallel to the histochemical ones, to study the rectal mucosa of the adult Ox. In these studies we show that: "EC" cells, of the intestinal type, contain predominantly pleiomorphic granules, which are very electron dense and heavily reactive to "Masson" and "Grimelius" methods; "L" cells are recognizable by their numerous granules, which are fairly homogeneous in shape and osmiophilia. They do not react with "Masson" and are weak or negative to Grimelius s reaction. These granules occur near to others that are less dense, unreactive to "Masson", and that contain an argyrophilic matrix, with an eccentric electron dense core, which does not react with silver; "F-like" cells contain granules which are variable in shape, size and osmiophilia. They are unreactive to "Masson" and weak or unreactive to Grimelius silver; "H" cells contain few, small and uniformly osmiophilic granules. These are unreactive to "Masson" and uniformly reactive to "Grimelius". Our data suggest that the morphology, frequency and distribution of the cell types we have identified in the mucosa of the bovine rectum correspond with those reported in large intestine and rectum of Monogastrics, as by other authors described.     

Identification and mapping of cleistogamy genes in barley

Cleistogamy is a closed type of flowering with ensured self-pollination and an important trait to study evolutionary development in flower organs, reproduction systems, gene flow, and disease control. Still, very limited information is available about the genetic control and regulatory mechanism of this trait in barley. In this work, from the eight crosses between cleistogamous and chasmogamous accessions, five crosses generated chasmogamous F₁ plants and their F₂ plants segregated as 3 chasmogamous:1 cleistogamous, whereas three crosses generated cleistogamous F₁ plants, and their F₂ plants segregated as 1 chasmogamous:3 cleistogamous. Although a single gene was responsible for the control of cleistogamy in these two groups of crosses, the direction of dominance was opposite, suggesting two genes, cly1 and Cly2, for the genetic control of cleistogamy in barley. Epistatic type of gene interaction between the two loci was detected. In the analysis of 99 recombinant inbred lines of Azumamugi × Kanto Nakate Gold and doubled haploid lines of Harrington × Mikamo Golden, where in both crosses F₁ was chasmogamous, the cly1 locus has been mapped on chromosome 2HL. Using the analysis of the F₂ population of Misato Golden and Satsuki Nijo where F₁ was cleistogamous, the Cly2 locus was mapped in the same region of chromosome 2HL. Because the cly1 and Cly2 loci were mapped in the same region in these three different mapping populations, it was concluded that the expression of cleistogamy is under the control of two tightly linked genes or different alleles of the same gene.     

Structure of the parathyroid glands,as revealed by different methods of fixation

Summary Stereology and semi-automatic image analysis were used with the aim of comparing the structure of parathyroid glands from untreated adult Mongolian gerbils fixed by immersion with those fixed by perfusion. Subclassification of the chief cells based upon the staining affinity or electron density of the cytoplasm was readily performed only in glands fixed by immersion, and so-called atrophic cells were observed only in these glands. The atrophic cells were often surrounded by light chief cells. In glands fixed by perfusion, light chief cells were only rarely encountered. A significant difference between glands fixed by immersion and those fixed by perfusion was found only with regard to the form of cells and nuclei, those fixed by perfusion being more spherical. When comparing individual cells within glands fixed by immersion, light chief cells were more spherical and had a significantly larger nuclear and cellular size, and a lower mitochondrial volume density than the intermediate/dark chief cells. Otherwise there were no significant differences in any of the parameters investigated. These data indicate that occurrence of socalled light chief cells and atrophic cells is a result of improper fixation. The results of this study do not favour the concept of a functional cycle with a simultaneous occurrence of active and inactive cells within parathyroid glands.     

The effect of humoral factors on phagocytic activity of central macrophages in the erythroblast islet culture

A 24-hour cultivation of erythroblast islets (El) in presence of erythropoietin (EP) and macrophage colony stimulating factor (MCSF) led to a concentration-dependent enhancement of phagocytic activity (PA) of the El's "central" macrophages. The latter phenomenon was revealed in "mature" El classes only. The PA of "central" macrophages in "proliferating" El appeared to be affected neither by EP, nor by MCSF.     

Fractionation of wheat gliadin and glutenin subunits by two-dimensional electrophoresis and the role of group 6 and group 2 chromosomes in gliadin synthesis

Summary Subunits of wheat endosperm proteins have been fractionated by two-dimensional electrophoresis. To determine which subunits in the two-dimensional electrophoretic pattern belong to gliadin or glutenin the endosperm proteins have also been fractionated by a modified Osborne procedure and by gel filtration on Sephadex G-100 and Sepharose CL-4B prior to separation by two-dimensional electrophoresis.The control of production of five major grain protein subunits is shown to be determined by chromosomes 6A, 6B and 6D by comparing two-dimensional electrophoretic protein subunit patterns of aneuploid lines of the variety Chinese Spring. From these and previous studies it is concluded that some , and gliadins (molecular weights by SDS-PAGE 30,000 to 40,000) are specified by genes on the short arms of homoeologous Group 6 chromosomes, the gliadins (molecular weights by SDS-PAGE 50,000 to 70,000) are specified by genes on the short arms of homoeologous Group 1 chromosomes and the glutenin subunits (molecular weights by SDS-PAGE > 85,000) are specified by genes on the long arms of homoeologous Group 1 chromosomes.No major gliadins or glutenin subunits were absent when any of the chromosomes in homoeologous Groups 2, 3, 4, 5 or 7 were deleted. However two gliadins whose presumed structural genes are on chromosome 6D were absent in aneuploid stocks of Chinese Spring carrying two additional doses of chromosome 2A. Two out of thirty-three intervarietal or interspecific chromosome substitution lines examined, involving homoeologous Group 2 chromosomes, lacked the same two gliadins. All the subunits in the other thirty-one chromosome substitution lines were indistinguishable from those in Chinese Spring. It is therefore concluded that the major variation affecting gliadin and glutenins in wheat is concentrated on the chromosomes of homoeologous Groups 1 and 6 but Group 2 chromosomes are candidates for further study.An endosperm protein controlled by chromosome 4D in Chinese Spring is shown to be a high molecular weight globulin.     

Intracellular effects of phage phi W-14 DNA on transformation of Bacillus subtilis

Summary Uptake of transforming DNA by competent Bacillus subtilis cells in the presence of phage W-14 DNA (in which half the thymine residues are replaced by -putrescinyl-thymine) is accompanied by a decrease in the amount of trichloracetic acid-precipitable label of the former retained by recipient cells during subsequent incubation. Fractionation of lysates of cells incubated for 0.5 min at 37°C after DNA uptake at 30°C in the presence of low concentrations of W-14 DNA (0.1 g/ml) demonstrated the presence of single-stranded transforming DNA molecules, typical for DNA taken up by B. subtilis. The intracellular effect of W-14 DNA was enhanced by an increase in its concentration (to 0.5–1 g/ml), or by increasing the temperature of uptake (to 37°C). With either of these treatments transforming DNA taken up was found in the form of a broad asymmetric band, indicative of degradation, and partially located at the density characteristic for single-stranded molecules. Fractionation of lysates of cells treated (0.1 g/ml) or untreated with W-14 DNA, and incubated for 20 min at 37°C after DNA uptake, showed disappearance of the single-stranded band. Donor DNA label was then found exclusively in the recipient DNA band, its amount being lower in samples treated with W-14 DNA. The influence of a high concentration of W-14 DNA on retention of transforming DNA label was correlated with its effect on transformation. On exposure to low concentrations of phage DNA, such a correlation was observed only after longer periods of incubation, due to slower intracellular degradation of homologous DNA taken up. The results are consistent with the proposal that W-14 DNA-induced reduction in efficiency of transformation is due to intracellular stimulation of transforming DNA degradation, leading to a decrease in the number of donor molecules available for recombination with the recipient chromosome.     

Influence of TNFalpha on the sialylation of mucins produced by a transformed cell line MM-39 derived from human tracheal gland cells

In order to investigate the influence of inflammation on the peripheral glycosylation of airway mucins, a human respiratory glandular cell line (MM-39) was treated by TNF. The expression and the activity of sialyl- and fucosyl-transferases, involved in the biosynthesis of peripheral carbohydrate determinants like sialyl-Lewis x, were investigated by RT-PCR and by HPAEC respectively. The mRNA steady-state level of sialyl- (ST3Gal III) and of fucosyl- (FUT3) transferases was moderately up-regulated by TNF; a 52% increase of 2,3-sialyltransferase activity was also observed in TNF-stimulated MM-39 cells. After metabolic radio-labelling with [³H]glucosamine and [³H]fucose, the mucins released inthe culture supernatant were purified by Sepharose CL-4B, density-gradient centrifugation and treatment with glycosaminoglycans-degrading enzymes. The mucins, released in the culture supernatant from control MM-39 cells, were constituted by two populations of molecules having the same 1.39–1.44 mg/ml density but carrying either high or low amounts of sialic acid residues at their periphery. TNF was able to increase the sialylation of the weakly sialylated mucins. This effect and the enhancement of the 2,3-sialyltransferase activity by TNF argue in favour of a regulation of the mucin sialylation by this pro-inflammatory cytokine. Despite the moderate overexpression of FUT3, no fucosylation of mucins produced by MM-39 cells was induced by TNF. In conclusion, the influence of TNF on the sialylation of mucins could explain why the mucins from infected patients suffering either from cystic fibrosis or from chronic bronchitis are more sialylated.     

Characterization of cellulolytic enzyme complexes obtained from mutants of Trichoderma reesei with enhanced cellulase production

Summary The cellulolytic enzyme complexes secreted by the fungus Trichoderma reesei QM 9414 and its mutants M 5, M 6, MHC 15, and MHC 22 were characterized by determining their specific filter-paper (FP)-, carboxymethylcellulase (C_x)-and -glucosidase (G)-activities. They were characterised further by measuring their C_x and G profiles after separation on an isoelectrofocusing column over the pH range 3–10. While the overall FP-activity was roughly equal in all preparations, the specific -glucosidase activity was highest in mutants MHC 15 and MHC 22 which are distingiushed morphologically from the parent strain, QM 9414, by a higher degree of branching of their hyphae. Two peaks of -glucosidase activity were detected by isoelectric focusing in preparations from QM 9414 and M 6, none in the enzyme from the mutant M 5 while 3 and 4 peaks respectively were found in preparations from morphological mutants MHC 15 and MHC 22. The higher -glucosidase activity in these last two preparations was also reflected in the higher glucose to cellobiose ratio in the initial stages of cellulose hydrolysis by the individual enzyme preparations.