Morphological appearance of epidermal cells cultured on fibroblast-reorganized collagen gels

Summary Human foreskin fibroblasts were used to reorganize hydrated collagen gels into a dermal-like matrix, after which freshly isolated keratinocytes isolated from rabbit ear skin were placed on the surfaces of the matrices and cultured for up to 12 days. Transmission electron microscopy revealed 8–12 cell layers of epidermal cells organized in three distinct strata. The basal stratum consisted of cuboidal to columnar cells with typical complement of organelles, oval nuclei, and prominent tonofilaments inserting into desmosomes. Mitotic cells often were found at this level. There was no well-defined basement membrane region; rather, many of the cells appeared to be in close contact with collagen fibrils. The intermediate stratum of suprabasal cells consisted of elongated cells that had reduced organelles, but still were connected to each other by desmosomes. Finally, the superficial stratum of suprabasal cells contained cells that were completely flattened and often appeared to be sloughing off the apical surfaces of the cultures. Indirect immunofluorescence studies carried out on frozen sections revealed bullous pemphigoid antigen associated with basal epidermal cells; pemphigus vulgaris antigen around the epidermal cells of all strata, and keratin present in the epidermal cells of all strata. Filaggrin was observed in punctate and fibrillar arrangements in suprabasal cells. Fibronectin was found in a linear deposit at the dermal-epidermal junction and around the fibroblasts in the reorganized collagen gels. Type-IV collagen and laminin, however, were not detected.     

Is the Fatty Acid Composition of Phospholipids Important for the Function of the Circadian Clock in Neurospora crassa?

Effects of pheriylmethanol (PM), 2-phenylethanol (PE) and 3-phenyl-1-propanol(PP) on the conidiation rhythm and on the fatty acid compositionof phospholipids were examined in Neurospora crassa. The periodfor the conidiation rhythm was shortened in proportion to theconcentration used of PE of more than 1 mM. PM at 10 mM andPP at 3 mM, however, did not cause a decrease in the lengthof the period. PE did not affect the temperature compensationof the length of the period or the amplitude of the phase shiftby light. The ratio of linolenic acid to Hnoleic acid decreased in proportionto the concentrations of PE, PM, and PP in all the phospholipidsexamined. The proportion of phosphatidic acid+diphosphatidylglycerolto the total phospholipids also was decreased by these compounds.Period shortening by PE can not be explained by the change inthe phospholipid fatty acid composition. (Received April 18, 1983; Accepted June 22, 1983)     

Dielectric constant of sickle cell hemoglobin. Dielectric properties of sickle cell hemoglobin in solution and gel

The dielectric constants of sickle cell hemoglobin were determined before and after gelation. The dielectric properties of oxy and deoxy sickle cell hemoglobin in solution are nearly identical to those of oxy and deoxy hemoglobin A. Only in the gel state did deoxy sickle cell hemoglobin display dielectric behavior different from that in solution. Upon gelation of deoxy sickle cell hemoglobin, the dielectric constant showed a marked decrease, and the relaxation frequency shifted towards higher frequencies. This result suggests that dielectric constant measurement can be used for the investigation of the kinetics of polymerization of sickle cell hemoglobin molecules. Despite the marked decrease in the dielectric constant, deoxy sickle cell hemoglobin still showed a well-defined dielectric dispersion even in the gel state. This indicates that individual molecules have considerable freedom of rotation in gels. It was observed that the dielectric properties of gelled deoxy sickle cell hemoglobin were affected by electrical fields at the level of 10 to 20 V/cm. This observation suggests that electrical fields of moderate strengths are able to perturb the gel structure if the system is near the transition region. The non-linear electrical behavior of gelled sickle cell hemoglobin will be discussed further in subsequent papers.     

Study of dielectric behavior of DNA in shear gradient

The dielectric behavior of the DNA molecule is investigated in the presence of mechanical force as well as the electrical field. In the present experiment, the direction of the electrical field is perpendicular to that of the mechanical force. The dipole moment of polar molecules manifest itself as dielectric increment at low frequencies or as the conductance increment at high frequencies. These two quantities are closely related to each other by Eq. (1) in the text. Because of the difficulty due to electrode polarization at low frequencies, no useful information was obtained by investigating the dielectric increment in the present system. Therefore, the effect of shear gradient was studied by measuring the conductance increment at high frequencies with various velocity gradients. The conductance increment decreased when the shear was applied perpendicular to the electrical field. The conductance change is converted into the unit of dielectric constant; it was found that the dielectric increment of DNA solution decreases by as much as 85 percent. From these observations, it is concluded that the direction of the dipole moment in DNA is longitudinal rather than transverse. The same experiment was repeated with the random coil DNA and no anisotropy in the dielectric increment was observed.     

Optical anisotropy of synthetic polynucleotides. II. Calculation of pi-electron polarizability of uracil and adenine

The interchain packing energy of two-, three-, and four-stranded α-helical ropes has been investigated. Poly-L -alanine was chosen as a model, and the interchain energies were calculated for various combinations of chain sense as a function of interchain distance, axial orientation and displacement. For comparison, the packing energies of the analogous assemblies of straight α-helices were also computed. Only those systems in which the chains were equivalent, i.e., related by line symmetry, were considered. The results indicate that for structures in which all the chains are equivalent the interchain packing energy favors the coiled coil arrangements.     

Effect of ions on the dielectric relaxation of DNA

The dielectric relaxation of DNA solutions has been investigated with and without extraneous ions covering a wide frequency range. The effect of monovalent ions such as Na, K, and Li as well as divalent ions such as Mg, Ca, and Hg have been included in the study. These ions are found to have a profound effect on the dielectric increment and the relaxation time without affecting the molecular dimension drastically. This dielectric effect is interpreted as indicating the importance of counterion fluctuation on the low frequency dielectric constant of DNA in solution. The effect of an organic ion, tetra-methylammoniun bromide, has also been studied. This ion has no noticeable effect. A simple theory is derived on the basis of a microscopic model to account for the effect of external ions on the dielectric behavior of solutions of DNA.     

The Large Isoform of Myelin-Associated Glycoprotein Is Scarcely Expressed in the Quaking Mouse Brain

Two polypeptide isoforms of myelin-associated glycoprotein (MAG) with molecular masses of 72 and 67 kDa are produced by alternative splicing of the exon 12 portion. Our previous work has demonstrated that in the quaking mouse brain this alternative splicing is lacking and that the mRNA coding the large MAG isoform (L-MAG) is scarcely expressed, whereas that of small MAG isoform (S-MAG) is overexpressed. In the present study, we prepared antisera specific to the S-MAG and L-MAG amino acid residues, respectively. Immunoblots showed that the L-MAG band was scarcely detectable in the quaking mouse brain, whereas the S-MAG band had an apparently higher molecular mass than in the normal control. Our immunohistochemical study also showed that L-MAG was scarcely stained in the quaking mouse brain. These results seemed to reflect a reduction in content of L-MAG mRNA and abnormal glycosylation in the quaking mouse brain.     

Production of Dihomo-γ-Linolenic Acid by a Δ5-Desaturase-Defective Mutant of Mortierella alpina 1S-4

A mutant, which has low Δ5-desaturase activity, of an arachidonic acid-producing fungus, Mortierella alpina 1S-4, was shown to be a novel potent producer of dihomo-γ-linolenic acid (DHGA). On submerged culture under optimal conditions for 6 days at 28°C in a 10-liter fermentor, the mutant produced 3.2 g of DHGA per liter of culture broth (123 mg/g of dry mycelia), which accounted for 23.4% of the total mycelial fatty acids. Mycelial arachidonic acid amounted to only 19 mg/g of dry mycelia (0.5 g/liter of culture broth), which accounted for 3.7% of the total mycelial fatty acids. The other major mycelial fatty acids were palmitic acid (11.0%), stearic acid (12.8%), oleic acid (22.7%), linoleic acid (8.9%), γ-linolenic acid (6.5%), and lignoceric acid (7.8%). More than 97 mol% of the DHGA produced was found in the triglyceride fraction irrespective of the growth temperature employed (12 to 28°C).     

Typing of Verotoxins by DNA Colony Hybridization with Poly- and Oligonucleotide Probes,a Bead-Enzyme-Linked Immunosorbent Assay,and Polymerase Chain Reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.     

Expression of a moderately anionic peroxidase is induced by aluminum treatment in tobacco cells: Possible involvement of peroxidase isozymes in aluminum ion stress

To clarify the mechanism of aluminum (Al) toxicity and Al tolerance, we isolated a new clone (pAL201) from a tobacco cDNA library. Northern blot hybridization analysis indicated that the expression of pAL201 is induced by Al treatment and phosphate (P₁) starvation. The complete cDNA sequence suggested that this clone encodes a moderately anionic peroxidase (EC 1.11.1.7). Analysis by isoelectric focussing indicated that a moderately anionic peroxidase (approximately pI 6.7) and two cationic peroxidases (pI 9.2 and 9.7) in the soluble fraction are activated by Al treatment and P₁ starvation, while two moderately anionic isozymes are repressed by these stresses. We suppose that Al ion stress can control the activity of some peroxidase isozymes, one of which is probably induced by enhanced gene expression of pAL201. There is a possibility that some of these isozymes have some functions in Al ion stress.