An ultrastructural examination of the role of cell membrane surface coat material during neurulation

Data from neural crest cultures indicate that cell surface coat material (CSM) is directly involved in cellular migration and events surrounding differentiation. To investigate whether the CSM also has a morphogenetic role, embryos of the amphibian Ambystoma maculatum were examined ultrastructurally throughout the stages of neurulation. Segments of the neural axis were fixed in glutaraldehyde-containing Alcian blue 8GX, which reportedly enhances preservation of CSM, and were postfixed in OsO4 containing 1 percent lanthanum nitrate, which stains the CSM. The medial groove formed by the appearance of the neural ridges contains a large amount of CSM and numerous vesicles coated with lanthanum-positive material. In contrast, the lateral ridge surfaces are covered by a small amount of uniformly distributed CSM and a paucity of vesicles. As the ridges begin to fold there is a progressive increase in the amount of CSM within the presumptive neural tube region. Further convergence of the neural folds is accompanied by an increase of CSM at their leading edges. As the folds approximate each other, lanthanum-positive material physically bridges the gap. However, as the apposing tissue actually abuts to form the neural tube, no CSM is observed in the remaining interspace. The specific distribution and sequential accumulation of cell CSM during the events of neurulation strongly suggest its direct participation in the morphogenetic process.     

Morphogenesis in the slime mold Dictyostelium discoideum. 1. The accumulation and erasure of "morphogenetic information".

When developing cultures of the cellular slime mold Dictyostelium discoideum are disaggregated and morphogenesis immediately reinitiated, they recapitulate the morphogenetic scheme, but at an increased rate. Employing this feature of the system, we have identified time periods when “information” accumulates for specific morphogenetic events. In this case, we have defined “morphogenetic information” as the reduction in time for the appearance of a particular morphology during morphogenetic recapitulation. Accumulated information can be erased by disaggregating developing cultures and reinoculating them into liquid growth medium. Erasure occurs as a discrete event and can be blocked by inhibiting protein synthesis.By comparing morphogenesis in log-phase and stationary-phase cultures, at least two parallel timers have been distinguished, one specific for aggregation and one or more specific for morphogenetic events following aggregation. Approaches to the molecular identity of morphogenetic information and morphogenetic timers are discussed.     

HIF-1α Polymorphism in the Susceptibility of Cervical Spondylotic Myelopathy and Its Outcome after Anterior Cervical Corpectomy and Fusion Treatment

Background

To investigate the association between the single nucleotide polymorphism (SNP) of hypoxia-inducible factor1 α (HIF-1α) and the susceptibility to cervical spondylotic myelopathy (CSM) and its outcome after surgical treatment.

Method

A total of 230 CSM patients and 284 healthy controls were recruited. All patients received anterior cervical corpectomy and fusion (ACF) and were followed for 12 months. The genotypes for two HIF-1α variants (1772C>T and 1790G>A) were determined.

Results

In the present study, we found that the HIF-1α polymorphism at 1790G>A significantly affects the susceptibility to CSM and its clinical features, including severity and onset age. In addition, the 1790A>G polymorphism also determines the prognosis of CSM patients after ACF treatment. The GG genotype of 1790G>A polymorphism is associated with a higher risk to develop CSM, higher severity and earlier onset age. More importantly, we found that the 1790G>A polymorphism determines the clinical outcome in CSM patients who underwent ACF treatment.

Conclusion

Our findings suggest that the HIF-1α 1790G>A polymorphism is associated with the susceptibility to CSM and can be used as predictor for the clinical outcome in CSM patients receiving ACF treatment.     

Rumen fermentation patterns and nutrient digestion in lambs fed cottonseed meal supplemental diets

Twenty crossbred 3–4-month-old male lambs of uniform body weight were used to assess the effect of raw and processed cottonseed (Gossypium) meals (CSM) on nutrient utilisation and rumen fermentation patterns. Lambs were assigned to five dietary treatments in a completely randomised design and fed isonitrogenous and isocaloric concentrate mixtures containing 30% deoiled groundnut cake (DGNC) (control), 40% of either raw, 45 min cooked, 1% calcium hydroxide (Ca(OH)₂) or iron (1 part free gossypol (FG):0.3 parts iron) treated CSM. The concentrate mixtures were fed to meet 80% of crude protein requirements (NRC, 1985) with ad libitum access to chopped maize hay (Zea mays). A metabolic trial of 6 days duration was conducted after 135 days of feeding, and rumen liquor was collected for 2 days at its end. Incorporation of 40% raw CSM resulted in reductions (P<0.01) in the digestibility of dry matter and organic matter in comparison to control fed lambs. Cooking and Ca(OH)₂ treatment of CSM improved digestibilities of these nutrients, to levels comparable to those in control fed lambs. The ether extract (P<0.05) and energy (P<0.01) digestibilities in lambs fed Ca(OH)₂ treated CSM were higher compared to those lambs fed raw CSM diets. Acid detergent fibre and cellulose digestibilities were lower (P<0.05) on cooked and iron treated CSM fed lambs. Retention of nitrogen and energy did not differ among diets. The digestible and metabolisable energy content of the Ca(OH)₂ treated CSM incorporated diet was higher (P<0.01) than the other CSM containing diets, but similar to DGNC containing ration. Rumen pH and total volatile fatty acid concentration was similar among diets, but NH₃–N was lower (P<0.05) on the cooked CSM diets versus the control. Feeding of lambs with raw CSM amounting to 17.9% of total dry matter intake (i.e. 303 mg FG intake per day) adversely affected utilisation of nutrients and rumen fermentation. This depression was alleviated to the greatest extent by 45 min cooking and 1% calcium hydroxide treatment. Iron treatment of CSM had little overall benefit.     

Distribution and characterization of plasmid-related sequences in the chromosomal DNA of different thermophilic Methanobacterium strains

The genomes of several thermophilic members of the genus Methanobacterium were analyzed for homology to the related restriction-modification plasmids pFVI and pFZ1 from M. thermoformicicum strains THF and Z-245, respectively. Two plasmid regions, designated FR-I and FR-II, could be identified with chromosomal counterparts in six Methanobacterium strains. Multiple copies of the pFVI-specific element FR-I were detected in the M. thermoformicicum strains CSM3, FF1, FF3 and M. thermoautotrophicum ΔH. Sequence analysis showed that one FR-I element had been integrated in almost identical sequence contexts into the chromosomes of the strains CSM3 and AH. Comparison of the FR-I elements from these strains with that from pFVI revealed that they consisted of two subfragments, boxI (1118 bp) and boxII (383 bp), the order of which is variable. Each subfragment was identical on the sequence level with the corresponding plasmid-borne element and was flanked by terminal direct repeats with the consensus sequence A(A/T)ATTT. These results suggest that FR-I represents a mobile element. FR-II was located on both plasmids pFVI and pFZI, and on the chromosome of M. thermoformicicum strains THF, CSM3 and HN4. Comparison of the nucleotide sequences of the two plasmid FR-II copies and that from the chromosome of strain CSM3 showed that the FR-II segments were approximately 2.5–3.0 kb in size and contained large open reading frames (ORFs) that may encode highly related proteins with an as yet unknown function.     

Alterations in the cell envelope of Escherichia coli late in bacteriophage T4 infection

The cell envelope of Escherichia coli was examined for changes during late stages of bacteriophage T4 infection. Late events in T4 infection are shown to result in (i) a reduction in the effectiveness of membrane separation procedures employing either isopycnic sucrose gradient centrifugation or selective solubilization of inner membrane by detergent (Sarkosyl or Triton X-100), (ii) the appearance of a 54 000 dalton host protein in membrane preparations, (iii) the adventitious presence of detergent-resistant phage morphogenetic structures in membrane preparations, and (iv) a decrease in the activity of NADH oxidase and an apparent alteration in its association with inner membrane. These modifications occur regardless of the state of the $\text{e}$ and $\text{t}$ genes of T4.     

Application of a two-phenotype color-shift model with heterogeneous growth to a rat hepatocarcinogenesis experiment

The color-shift model (CSM) was introduced by Kopp-Schneider et al. [1] to describe formation and progression of foci of altered hepatocytes (FAH). It incorporates the field-effect hypothesis which postulates that entire colonies of altered hepatocytes simultaneously alter their phenotype. In the original CSM, FAH grow with deterministic growth rate and change their phenotype after an exponentially distributed waiting time. A modification of the original color-shift model (CSMbeta) is presented here in which the growth rate varies from focus to focus according to a beta distribution. The concept of an exponentially distributed waiting time to phenotype change is modified to the concept of a random radius at which phenotype changes and this radius is modelled as beta distributed. The original and the modified CSM are applied to data from an initiation-promotion rat hepatocarcinogenesis experiment with diethylnitrosomorpholine (DEN) and N-nitrosomorpholine (NNM), in which two phenotypes of FAH were observed in hematoxilin/eosin (H&E) stained liver sections. The Cramer-von-Mises Distance is used as a measure for the discrepancy between empirical and theoretical size distributions. Comparisons of model fit show that considerable improvement is obtained for CSMbeta compared to the original CSM.     

Effects of non-enzymatic browning reaction intensity on in vitro ruminal protein degradation and intestinal protein digestion of soybean and cottonseed meals

The effects of non-enzymatic browning reactions on in vitro ruminal gas production and in vitro ruminal and intestinal crude protein (CP) digestibilities of soybean (SBM) and cottonseed (CSM) meals were investigated. Non-enzymatically browned SBM and CSM samples were prepared using two xylose levels (10 or 30 g/kg dry matter), two heating lengths (30 or 60 min) and two heating temperatures (120 or 150 °C) for a total of one untreated (commercially solvent-extracted, Control) and eight treated samples for each protein source. The control SBM had higher (P<0.001) in vitro ruminal CP degradability values than the treated samples. Intestinal protein digestibility and total-tract CP digestibility of CSM and SBM were affected by the treatment (P<0.01). The results of the study indicate that not only ruminal CP degradability is reduced but also intestinal and total-tract CP digestibilities may be lowered depending on protein source and intensity of the non-enzymatic browning reaction.     

Crossveinless-2 is required for the relocalization of Chordin protein within the vertebral field in mouse embryos

Bone morphogenetic proteins (BMPs), as well as the BMP-binding molecules Chordin (Chd), Crossveinless-2 (CV2) and Twisted Gastrulation (Tsg), are essential for axial skeletal development in the mouse embryo. We previously reported a strong genetic interaction between CV2 and Tsg and proposed a role for this interaction in the shaping of the BMP morphogenetic field during vertebral development. In the present study we investigated the roles of CV2 and Chd in the formation of the vertebral morphogenetic field. We performed immunostainings for CV2 and Chd protein on wild-type, CV2^−/− or Chd^−/− mouse embryo sections at the stage of onset of the vertebral phenotypes. By comparing mRNA and protein localizations we found that CV2 does not diffuse away from its place of synthesis, the vertebral body. The most interesting finding of this study was that Chd synthesized in the intervertebral disc accumulates in the vertebral body. This relocalization does not take place in CV2^−/− mutants. Instead, Chd was found to accumulate at its site of synthesis in CV2^−/− embryos. These results indicate a CV2-dependent flow of Chd protein from the intervertebral disc to the vertebral body. Smad1/5/8 phosphorylation was decreased in CV2^−/−vertebral bodies. This impaired BMP signaling may result from the decreased levels of Chd/BMP complexes diffusing from the intervertebral region. The data indicate a role for CV2 and Chd in the establishment of the vertebral morphogenetic field through the long-range relocalization of Chd/BMP complexes. The results may have general implications for the formation of embryonic organ-forming morphogenetic fields.     

Mechanical Properties of Chondrocytes Estimated from Different Models of Micropipette Aspiration

In this study, two viscoelastic creep expressions for the aspirated length of individual solid-like cells undergoing micropipette aspiration (MPA) were derived based on our previous studies wherein the cell size relative to the micropipette and the cell compressibility were taken into account. Next, three mechanical models of MPA, the half-space model (HSM), incompressible sphere model (ICSM), and compressible sphere model (CSM), were employed to fit the MPA data of chondrocytes. The results indicated that the elastic moduli and viscoelastic parameters of chondrocytes for the ICSM and CSM exhibited significantly higher values than those from the HSM (p < 0.001) because of the considerations of the geometric parameter (ξ) and the compressibility of the cell (ν). For the normal chondrocytes, the elastic moduli obtained from the ICSM and CSM (ν = 0.3) were 47.4 and 78.9% higher than those from the HSM. In the viscoelasticity, the parameters k₁, k₂, and μ for the ICSM were respectively increased by 37.8, 37.9, and 39.0% compared to those from the HSM, whereas for the CSM (ν = 0.3), the above parameters were 135, 314, and 257% higher compared to those from the HSM. And with the increase of ξ and ν, the above mechanical parameters decreased. Furthermore, the thresholds of ξ varying with ν were obtained for the given values of relative errors caused by the HSM in the elastic and viscoelastic parameters. The above findings obviously indicated that the geometric parameter of MPA and the Poisson’s ratio of a cell have marked influences on the determination of cellular mechanical parameters by MPA and thus should be considered in the pursuit of more accurate investigations of the mechanical properties of cells.     

Rapid accumulation and disappearance of plasma membrane proteins during development of wild-type and mutant strains of Dictyostelium discoideum

Plasma membranes were purified from the cellular slime mold Dictyostelium discoideum at different developmental stages and the protein compositions compared. The protein components of the plasma membrane of vegetative cells are largely conserved during development. Specific morphogenetic events are accompanied by synthesis and accumulation of several new proteins which are subsequently lost as development progresses. Proteins with apparent molecular weights of 38,000, 36,500 and 10,000 to 12,000 rapidly accumulate during the first six hours of development and then disappear from the plasma membrane after 12 hours. Later in development, several new high molecular weight proteins are synthesized and appear in the membrane. The pattern of accumulation of membrane proteins in wild-type and mutant strains suggests that appearance of membrane proteins is linked to a dependent sequence of events.     

The influence of some elicitors on growth and morphogenesis of Hypericum perforatum L. callus cultures

We studied the effects of elicitors, such as mannan, gβ-1,3-glucan, ancymidol, and cork crumbs, on morphogenetic and biosynthetic potencies of shoot cultures of Hypericum perforatum L. In the presence of these elicitors, different morphogenetic structures of H. perforatum callus cultures were formed. A correlation was found between the morphogenetic processes and induction of hypericin and pseudohypericin biosynthesis in the callus cultures.     

Effect of lipids on the structure and rheology of gels formed by canine submaxillary mucin

Rheological experiments have shown that canine submaxillary mucin (CSM) forms gels in aqueous solution at low ionic strength and in 6M GdnHCl. Examination of specimens of intact CSM and also its subunits prepared by reduction and carboxymethylation showed that the presence of lipid increases the gel-forming capability, probably as a result of enhancement of the intermolecular hydrophobic interactions. The rheological evidence for gelation is that substantially larger values of the oscillatory storage modulus, G'(ω), and the dynamic complex viscosity, η^*(ω), are observed for lipid-containing CSM. TMs is backed up by electron micrographs of freeze fractured specimens, where we observe a network morphology in which the cross-links are formed as a result of non-bonded interactions between a number of CSM chains. The intermolecular interactions responsible for gelation probably involve hydrophobic association between the interdigitated oligosaccharides, and/or between the non-glycosylated regions of the protein core, and can occur even in a highly chaotropic medium (6M GdnHCl). In contrast to previous experiments with porcine submaxillary mucin and human tracheobronchial mucin, which form microphase-separated gels in aqueous solution, CSM solutions undergo macroscopic phase separation into polymerrich (gel) and polymer-poor (sol) phases. These data point to stronger hydrophobic interactions in lipid-containing CSM.     

Modulation of Drosophila retinal epithelial integrity by the adhesion proteins capricious and tartan

Background

The development of the Drosophila eye imaginal disc requires complex epithelial rearrangements. Cells of the morphogenetic furrow are apically constricted and this leads to a physical indentation in the epithelium. Posterior to the furrow, cells start to rearrange into distinct clusters and eventually form a precisely patterned array of ommatidia. These morphogenetic processes include regulated changes of adhesion between cells.

Methodology/Principal Findings

Here, we show that two transmembrane adhesion proteins, Capricious and Tartan, have dynamic and complementary expression patterns in the eye imaginal disc. We also describe novel null mutations in capricious and double null mutations in capricious and tartan. We report that they have redundant functions in regulating the architecture of the morphogenetic furrow and ommatidial spacing.

Conclusions/Significance

We conclude that Capricious and Tartan contribute to the adhesive properties of the cells in the morphogenetic furrow and that this regulated adhesion participates in the control of spacing ommatidial clusters.     

Local surface differentiations at the vegetal pole of the eggs of Nassarius reticulatus,Buccinum undatum,and Crepidula fornicata (Gastropoda,Prosobranchia)

In the eggs of the gastropods Nassarius reticulatus, Buccinum undatum, and Crepidula fornicata, local surface differentiations are described, using transmission and scanning electron microscopy. The differentiations consist of special microvilli (Nassarius), ridges composed of microvilli (Buccinum), or extensive surface folds (Crepidula). They occur on the polar lobes of these eggs and it is argued that a relation may exist between the surface structures and the localization or expression of the morphogenetic factors present in the polar lobes. The presence of the surface differentiations before the appearance of the polar lobe and their persistence after resorption of the lobes are shown in Nassarius and Crepidula eggs. Evidence is presented that the differentiated area at the vegetal pole of the Nassarius eggs corresponds to the former contact area of the oocyte with the follicle cells in the ovary. This correspondence is considered as an argument in favor of the theory that the egg cortex may locally acquire special properties under the influence of the surrounding structures in the ovary.     

Nkx genes regulate heart tube extension and exert differential effects on ventricular and atrial cell number

Heart formation is a complex morphogenetic process, and perturbations in cardiac morphogenesis lead to congenital heart disease. NKX2-5 is a key causative gene associated with cardiac birth defects, presumably because of its essential roles during the early steps of cardiogenesis. Previous studies in model organisms implicate NKX2-5 homologs in numerous processes, including cardiac progenitor specification, progenitor proliferation, and chamber morphogenesis. By inhibiting function of the zebrafish NKX2-5 homologs, nkx2.5 and nkx2.7, we show that nkx genes are essential to establish the original dimensions of the linear heart tube. The nkx-deficient heart tube fails to elongate normally: its ventricular portion is atypically short and wide, and its atrial portion is disorganized and sprawling. This atrial phenotype is associated with a surplus of atrial cardiomyocytes, whereas ventricular cell number is normal at this stage. However, ventricular cell number is decreased in nkx-deficient embryos later in development, when cardiac chambers are emerging. Thus, we conclude that nkx genes regulate heart tube extension and exert differential effects on ventricular and atrial cell number. Our data suggest that morphogenetic errors could originate during early stages of heart tube assembly in patients with NKX2-5 mutations.     

A model of network formation by Physarum plasmodium: Interplay between cell mobility and morphogenesis

The plasmodium of Physarum polycephalum has attracted much attention due its intelligent adaptive behavior. In this study, we constructed a model of the organism and attempted to simulate its locomotion and morphogenetic behavior. By modifying our previous model, we were able to get closer to the actual behavior. We also compared the behavior of the model with that of the real organism, demonstrating remarkable similarity between the two.