Neural and smooth muscle development in the chicken gizzard

The development of the autonomic ganglia of Auerbach's plexus and gizzard smooth muscle was studied in chicken embryos. Nervous system and smooth-muscle-specific antibodies were employed in immunofluorescence stainings on tissue sections to investigate the temporal and spatial frame of neural and muscular differentiation in relation to each other. Subserosal clusters of neural cells were clearly demonstrable at embryonic day 5 (ED5), the earliest stage analysed, with the monoclonal antibody El (SGIII-1). Fine nerve fibres (ED6) and, later, large axon bundles projecting from subserosal neuron clusters towards the lumen were followed and found to reach the luminal border by ED11. Already in early development the area of the future laminar tendons on the ventral and dorsal surface of the gizzard was devoid of neuroblasts, and nerve fibres were not extending to the muscle-tendon borderline until ED16. Double stainings with antibodies to smooth muscle myosin (SMM) and El revealed that SMM expression, taken as an indicator for muscle differentiation, followed neural growth. It was first detectable in close apposition to the differentiating neuroblasts in the caudal and cranial portion of the gizzard at ED6. With further development, myosin expression proceeded inward towards the lumen in a wave which followed the ingrowth of E1-positive nerve fibres from the prospective Auerbach plexus. Neuromuscular differentiation deviated from this pattern in the lateral tendon area where nerve growth was delayed and myosin expression preceeded the arrival of E1-positive nerve fibres. The findings suggest that the gizzard could serve as a model system for the analysis of potential early nervous system imprints on smooth premuscle mesenchyme differentiation.     

Developmental regulation of presence of binding sites for neoglycoproteins and endogenous lectins in various embryonic stages of human lung,liver and heart

Protein-carbohydrate interactions are supposed to play key roles in the mechanisms of cell adhesion, biosignalling and intracellular routing, warranting the analysis of the developmental course of expression of epitopes of this system. Thus, a panel of carrier-immobilized carbohydrate ligands was used as probes, namely lactose,N-acetylgalactosamine,N-acetylglucosamine, mannose, fucose and maltose. Additionally, an antibody to an endogenous -galactoside-binding lectin (anti-galectin-1), the biotinylated lectin and two further human lectins, namely the macrophage migration inhibitory factor-binding sarcolectin and serum amyloid P component (SAP) that displays selectivity for sulphated sugars and mannose-6-phosphate, were included. They enabled us to assess the extent of the presence of respective binding sites in fixed sections from human lungs (pulmonary epithelial cells), livers (hepatocytes) and hearts (myocard cells) of 10–50 weeks gestation. Invariably, specific binding was detected in the three organ types, at least in certain stages. In most of the cases, the intensity of staining exhibited developmental regulation. The apparent patterns reveal similarities between the different cell types, as seen with immobilizedN-acetylglucosamine as well as with labelled galectin-1 and sarcolectin. However, drastic differences among such patterns with nearly opposite developmental courses do also occur, as detected for carrier-attached mannose and maltose residues. These results point to a potential importance for the detected glycohistochemical features in human development and substantiate the possibility of differential regulation of the presence of binding sites for distinct sugars within a certain organ and between the individual cell types of the monitored organs.     

Adsorption equilibrium and dynamics of lactase/CM-Sephadex system

Summary Partitioning behaviour and adsorption isotherms of lactase/CM-Sephadex system at equilibrium were investigated together with the adsorption kinetics in this study. Maximum adsorption was obtained at the pH values between 5.5–6.0. Adsorption isotherm was a close fit to the Langmuir model.Nomenclature a specific mass transfer area - D_m molecular diffusion coefficient (m²/sec) - e₁, e₂ charge of the protein and the gel - k apparent mass transfer coefficient (s^-1) - ka global mass transfer coefficient - f partition coefficient - K_p dissociation constant for adsorbent-adsorbate complex, (mg/mL solvent) - p equilibrium concentration of free enzyme, (mg free enzyme/mL solution) - q equilibrium concentration of adsorbed enzyme, (mg ads./mL gel) - q_m maximum adsorption capacity, (mg ads./ml gel) - Re particle Reynolds number - Sh Sherwood number - V_g/V gel volume (mL)/bulk solvent volume (mL) - Z dimensionless extent of adsorption - K_p/P_o , model parameter - (/) +1 , model parameter - V_g q_m / V P_o , model parameter     

A new mutation protocol for obtaining auxotrophic mutants of the yeastPhaffia rhodozyma

Summary A new mutation strategy, which involves -irradiation of cells followed by a selective enzymatic enrichment step, was worked out to obtain auxotrophic mutants from the astaxanthin-producing yeast P. rhodozyma. Under the optimized conditions described, different mutants suitable for strain improvement were isolated.     

Patterns of dispersal and seasonal migration in roe deer (Capreolus capreolus)

Roe deer ( Capreolus capreolus ) spacing behaviour, during the period between mother-fawn separation and home range establishment, was studied in southern Sweden during 1987–1992. Data were collected, using telemetry, in two non-hunted populations. Females dispersed either as yearlings, or as 2- or 3-year-olds after having migrated between non-overlapping summer and winter ranges for one and two seasons, respectively. Seasonal migration usually ended with permanent settlement in the new summer area. Males dispersed as one- or two-year-olds, or remained philopatric. Median distance moved was c. 2 km with no significant sex-bias. Both populations underwent one increase, and one stable phase. During the increase phase, the frequency of yearling dispersal was 70% in one of the populations, but dropped to 20% at high density. This drop paralleled a 14% decrease in yearling winter weights. In the other population, yearling dispersal frequency was 56% during the increase phase. This population stopped increasing at an intermediate density and dispersal frequency was kept at 75%. Weights remained high in this population. In neither of the populations, at intermediate and high densities, was a sex-bias in dispersal among yearlings evident. Dispersers were on average heavier than philopatrics. High juvenile winter weight did not predestinate yearlings to dispersal, whereas low weight seemed to prevent dispersal. On the basis of these relationships, we propose a hypothesis relating dispersal to body condition, in order to explain dispersal tendency at different population densities. Further, we suggest the reason for female migration is the advantage of residing in a group during winters when predation risk is high. Since it is difficult for females (but not for males) to join non-relatives, dispersers must go back to the natal area as long as they have too few offspring to form their own matriarchal group.     

Nod signal-induced plasma membrane potential changes in alfalfa root hairs are differentially sensitive to structural modifications of the lipochitooligosaccharide

Lipochitooligosaccharide Nod signals are important determinants of host specificity in the Rhizobium -legume symbiosis. The most rapid response of plant cells to the R. meliloti Nod signal NodRm-IV(C16:2,S) reported so far is the depolarization of the plasma membrane potential in alfalfa root hairs. In order to investigate whether this response may be part of a specific signal transduction cascade involved in the nodulation process, its specificity was studied with respect to host-specific modifications of the lipochitooligosaccharide. Five different Nod factors displaying different degrees of activity in inducing root hair deformation or cortical cell divisions on alfalfa were tested. The ability of the Nod factors to elicit plasma membrane depolarization correlated well with their activity in the bioassays. Removal of the sulfate group (NodRm-IV(C16:2)) led to inactivation of the Nod factor. An increase in the length of the chitooligosaccharide backbone (NodRm-V(C16:2,S)) or saturation of the acyl chain (NodRm-IV(C16:0,S)) resulted in severely reduced activity. In contrast, the O -acetyl group at the non-reducing terminus in NodRm-IV(Ac,C16:2,S), which confers substantially higher activity in long-term bioassays, did not enhance plasma membrane depolarization significantly in comparison with the non- O -acetylated factor. Thus, the rapid plasma membrane response is differentially sensitive to various structural motifs of the lipochitooligosaccharide. These data suggest that the different substituents modifying the basic Nod factor structure may have distinct functions, not all of them contributing to the interaction with a putative receptor in root hair cells. However, the overall specificity of the membrane depolarization for the cognate Nod factors raises the possibility that it is involved in a Nod signal transduction pathway.     

Cell competence forAgrobacterium-mediated DNA transfer inPisum sativum L.

Distribution and properties of pea (Pisum sativum L.) cells, competent forAgrobacterium-mediated transformation were analysed byin situ histochemical detection of GUS (-glucuronidase) activity, 4 d after inoculation with engineeredAgrobacterium tumefaciens. The vector system consisted of the hypervirulent disarmed strain EHA101 and the binary plasmid pIBGUS, carrying an intron-containing, 35S-promotor drivengusA (oruidA) gene and two selectable marker genes. Cells competent for transformation were mainly restricted to the dedifferentiating cells neighbouring the vascular system of cotyledon and epicotyl explants. A standardized assay was developed, allowing determination and quantification of factors influencing number and distribution of competent cells. In etiolated seedlings, competence for transformation decreased with the distance of the epicotyl explant from the shoot apex and was specifically induced by the exogenous application of auxins. Transient expression ofgusA afterAgrobacterium-mediated DNA transfer was dramatically reduced upon application of cell-cycle and DNA replication inhibitors aphidicolin, colchicine and nalidixic acid. GUS expression after direct DNA transfer of double-stranded plasmid DNA (via PEG into protoplasts or via particle bombardment of epicotyl segments) was independent of cell-division/DNA replication.A GUS-positive mutant of EHA101 was constructed to allowin situ analysis of attaching bacteria within the plant tissue. Attachment and invasion was inhibited by well-developed cuticula but was restored after chloroform treatment of the tissue surface. Moreover, no correlation was found between distribution of attaching bacteria and the pattern of transformation-competent cells.     

Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes

Fragments of the proximal half of mouse chromosome 17 including the t-complex region were microdissected from metaphase spreads. DNA was isolated from a pool of such fragments, and was cloned on microscale. Individual clones were used to probe genomic digests of DNA from a pair of Chinese hamster cell lines with or without mouse chromosome 17, and livers of congenic inbred lines of mice carrying wild-type and/or t-haplotype forms of chromosome 17. The data obtained indicate that 95% of the low copy number microclone inserts recognize DNA sequences present on mouse chromosome 17. It has been possible to use one-third of these clones to identify restriction-fragment-length polymorphisms between wild-type and t-haplotype DNA on a congenic background. These results demonstrate that these clones have been derived from the t-complex or regions closely linked to it. Clones of this type should provide starting points for a molecular analysis of this region of the mouse genome.     

The calcium hypothesis of cystic fibrosis

Data have been presented which suggests that various CF cell types show evidence of alterations in calcium homeostasis. The significance of these observations and the exact nature of the putative calcium defect in CF remains to be elucidated. It must also be determined whether this possible defect is primary, or is secondary or tertiary to some more basic lesion. The data reviewed suggests that altered calcium homeostasis may play some focal role in the aetiology or the pathogenesis of CF.     

Defective regulation of the cytosolic Ca2+ activity in parathyroid cells from patients with hyperparathyroidism

The parathyroid hormone (PTH) release and cytosolic Ca²⁺ activity were determined in normal bovine parathyroid cells and parathyroid cells obtained from patients with hyperparathyroidism (HPT). There was a sigmoid relation between the cytosolic Ca²⁺ activity and the extracellular calcium concentration between 0.5 and 6.0 mmol/l. The PTH release was inhibited in parallel with the rise in the cytosolic Ca²⁺ activity. Both the hormone release and the cytosolic Ca²⁺ activity were lower in cells from human adenomas and hyperplastic glands~ and in comparison with the bovine preparations these ceils had higher set points for the cytosolic Ca²⁺ activity and PTH release. There was a close correlation between the individual set points for the cytosolic Ca²⁺ activity and PTH release in a material containing both normal and pathological cells. The results indicate that the abnormal PTH release characteristic of HPT is due to a defective regulation of the cytosolic Ca²⁺ activity.