Assessment of bacterial diversity in agricultural by-product compost by sequencing of cultivated isolates and amplified rDNA restriction analysis

An investigation of bacterial diversity in compost was performed using molecular chronometer in order to reveal its phylogeny. Thirty-three bacterial isolates isolated from compost were analyzed by 16S rRNA gene sequencing which revealed phylogenetic lineage of class Bacilli, γ, β-Proteobacteria, and Actinobacteria. Among these lineages, isolates belonging to class Bacilli consisted of species from genera Staphylococcus, Bacillus, Terribacillus, and Lysinibacillus. From phylum Actinobacteria: Microbacterium barkeri and Kocuria sp. were identified. Other bacterial groups had phylogenetic linkage with genera Comamonas and Acidovorax (class β-Proteobacteria); Serratia, Klebsiella, and Enterobacter (class γ-Proteobacteria). Similar isolates were analyzed through ARDRA. Amplified product of 16S rRNA gene from each isolates was subjected to cleavage by enzymes HpaII, HinfI, and MspI in separate reaction tubes. HpaII generated 2–6 bands ranging from 90–688 bp, HinfI generated 2–5 bands of 71–1,038 bp, and MspI 2–7 bands of 69–793 bp. The restriction patterns from HpaII, HinfI, and MspI were normalized separately and combined by means of pattern recognition software “Diversity Database.” HpaII had highest discrimination index (0.72) than HinfI (0.68) and MspI (0.65), and the combination of all three showed discrimination index (0.69). Numerical analysis of ARDRA patterns demonstrated sufficient phylogenetic information for characterizing bacterial diversity. Phylogenetic relationship obtained among isolates through ARDRA was compared with 16S rRNA gene sequence and ARDRA results showed sufficiently similar 16S rRNA gene sequence analysis, but not an overlapping. It has been observed that ARDRA technique facilitates the identification of bacteria in less than 36 h as compared to traditional 16S rRNA gene sequencing.     

Expression patterns of loricrin in various species and tissues

Abstract. In this study we analyzed the expression patterns of loricrin in various species and tissues using immunohistochemistry, immunoblotting and Northern blots. Loricrin is a glycine-, serine- and cysteine-rich protein expressed very late in epidermal differentiation in the granular layers of normal mouse and human epidermis. Later on in differentiation, loricrin becomes cross-linked as a major component into the cornified cell envelope by the formation of N^ɛ -(γ-glutamyl)lysine isopeptide bonds. This process either occurs directly or by the intermediate accumulation in L-keratohyaline granules of mouse epidermis and human acrosyringia. Loricrin was identified in all mammalian species analyzed by virtue of its highly conserved carboxy-terminal sequences revealing an electric mobility of ∼60 kDa in rodents, rabbit and cow and of ∼35 kDa in lamb and human on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Loricrin is expressed in the granular layer of all mammalian orthokeratinizing epithelia tested including oral, esophageal and fore-stomach mucosa of rodents, tracheal squamous metaplasia of vitamin A deficient hamster and estrogen induced squamous vaginal epithelium of ovary ectomized rats. Loricrin is also expressed in a few parakeratinizing epithelia such as BBN [N-butyl-N-(4–hydroxybutyl)nitrosamine]-induced murine bladder carcinoma and a restricted subset of oral and single vaginal epithelial cells in higher mammals. Our results provide further evidence that the program of squamous differentiation in internal epithelia of the upper alimentary tract in rodents and higher mammals differ remarkably. In addition, we also have noted the distinct distribution patterns of human loricrin and involucrin, another major precursor protein of the cornified cell envelope.     

The expression and modulation of IL-1 alpha in murine keratinocytes

Murine and human keratinocytes produce an IL-1-like factor that appears to be similar if not identical to monocyte-derived IL-1. IL-1 may be an important mediator in cutaneous inflammatory responses, however, little is currently known concerning factors that may modulate IL-1 expression in keratinocytes. To address this issue we examined the effect of LPS, UV, and the cell differentiation state on murine keratinocyte IL-1 mRNA expression. Our results indicated that as with the murine P388D1 monocyte cell line, PAM 212 keratinocytes constitutively express abundant amounts of IL-1 alpha mRNA. On exposure to LPS (100 micrograms/ml) for 8 h there was more than 10 times the increase in PAM 212 IL-1 alpha mRNA which was accompanied by a sixfold increase in supernatant IL-1 activity. Similarly UV irradiation had a significant effect on keratinocyte IL-1 alpha expression. High dose UV (300 mJ/cm2) inhibited PAM 212 IL-1 alpha expression at 4, 8, 24, 48 h post-UV whereas a lower dose of UV (100 mJ/cm2) inhibited UV at 4 and 8 h post-UV, but induced IL-1 expression at 24 and 48 h post-UV. The expression of IL-1 alpha varied with the differentiation state of the keratinocytes. Freshly removed newborn murine keratinocytes were found to constitutively express IL-1 alpha mRNA. Keratinocytes grown in low [Ca2+] tissue culture media (0.05 mM) for 6 days, functionally and phenotypically become undifferentiated and express increased quantities of IL-1 alpha mRNA, whereas cells grown in high [Ca2+] media (1.2 mM) for 6 days become terminally differentiated and IL-1 expression ceased. Keratinocytes cultured for 3 days in low [Ca2+] conditions expressed an intermediate level of IL-1 alpha. In contrast, little or no IL-1 beta mRNA was detected in either the PAM 212 cells or newborn murine keratinocytes. Thus LPS, UV, and cell differentiation state have a significant effect on expression of IL-1 alpha in murine keratinocytes.     

Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro

Epidermal differentiation is characterized by a series of coordinated morphological and biochemical changes which result in a highly specialized, highly organized, stratified squamous epithelium. Among the specific markers expressed in differentiating epidermis are (a) two early spinous cell proteins, keratins 1 and 10 (K1 and K10); and (b) two later granular cell proteins, filaggrin and a cornified envelope precursor (CE). In vitro, epidermal basal cells are selectively cultured in 0.05 mM Ca2+ medium, and terminal differentiation is induced when the Ca2+ concentration is increased to 1 mM. However, only a small fraction of the cells express the markers K1, K10, CE, or filaggrin in the higher Ca2+ medium. To explore the factors required for marker expression, cultured epidermal cells were exposed to intermediate Ca2+ concentrations and extracts were analyzed using specific antibody and nucleic acid probes for the four markers of interest. These studies revealed that marker expression was enhanced at a restricted concentration of Ca2+ in the medium of 0.10-0.16 mM. At this Ca2+ concentration, both protein and mRNA levels for each marker were substantially increased, whereas at higher or lower Ca2+ concentrations they were diminished or undetected. The percentage of cells expressing each marker was increased two- to threefold in the permissive Ca2+ medium as determined by immunofluorescence analysis. This optimal level of Ca2+ was required both to initiate and sustain marker expression. At the permissive Ca2+ concentration, expression of the markers was sequential and similar to the order of appearance in vivo. K1 was expressed within 8-12 h and K10 was expressed in the ensuing 12-24-h period. CE and filaggrin were expressed in the subsequent 24 h. Inhibition of K1 expression by cycloheximide suggested that an inducible protein was involved. Other investigators have determined that a shallow Ca2+ gradient exists in epidermis, where the basal cells and spinous cells are in a Ca2+ environment substantially below serum Ca2+ levels. These in vitro results suggest that the Ca2+ environment is a fundamental regulator of expression of epidermal differentiation markers and provide an explanation for the existence of the Ca2+ gradient in vivo.     

Shoot regeneration after fire or freezing temperatures and its relation to plant life-form for some heathland species

Shoot regeneration after prescribed burning or following the freezing temperatures of winter was monitored for nineteen heathland species present in an Arctostaphyleto-Callunetum community in northeast Scotland. Species whose renewal buds were near the surface of the ground started to grow earlier in the spring than species with renewal buds above the surface, but grouping species according to the position of their renewal bud (i.e. their life-form) did not account for all of the interspecific variation apparent. In the case of shoot regeneration after fire, species whose renewal buds were destroyed by fire because they were above-ground started to regenerate about the same time as species with belowground buds, protected from fire, but reached their maximum frequency of occurrence later. Grouping species by life-form was of limited value as a means of interpreting this interspecific variation in the timing of shoot regeneration after fire. It would be unwise to use plant life-form as the sole basis for interpreting or predicting a species' response to temperature stress when extreme temperatures occur regularly, as they do in heathland. The possible use of other plant traits to interpret and predict interspecific variation in the regeneration rate of heathland plants is discussed.Nomenclature follows Tutin et al. (1964–1980) for vascular plants. Acknowledgements. The Nature Conservancy Council and Mr J. J. Humphries kindly allowed Dinnet Moor to be used for the work presented here. One of us (RJR) received financial support for field work from the Natural Sciences and Engineering Research Council of Canada.     

Isopongaglabol and 6-methoxyisopongaglabol,two new hydroxyfuranoflavones from Pongamia glabra

Isopongaglabol and 6-methoxyisopongaglabol, two new hydroxyfuranoflavones, together with two furanoflavones 5-methoxyfurano(8,7-4″,5″)flavone and 5-methoxy-3′,4′-methylenedioxyfurano(8,7-4″,5″)flavone, two simple flavones, desmethoxykanugin and fisetin tetramethyl ether, a chromenoflavanone, ovalichromene B, two triterpenes, cycloart-23-ene-3β,25-diol and friedelin, and β-sitosterol-β-d-glucoside were isolated from the petrol and CHCl₃ extracts of the flowers of Pongamia glabra. The structures of isopongaglabol and 6-methoxyisopongaglabol have been established as 4′-hydroxyfurano(8,7-4″,5″)flavone and 4′-hydroxy-6-methoxyfurano(8,7-4″,5″)flavone, respectively, on the basis of the spectral evidence and they have been confirmed by synthesis.     

Charge-transfer complexes of some linear conjugated polyenes.

On adsorption of some electron-acceptor molecules on the solid films of all-trans-beta-carotene, beta-apo-8'-carotenal, astacene and methylbixin a new absorption band appears on the longer-wavelength side of the spectrum in addition to the original bands. The position of this new band is dependent on the electron affinity (EA) of the acceptor molecules, and the intensity of this band increases with the amount of adsorbed acceptor molecules. A linear relationship between the vmax. of the new band and EA was observed. The value of the ionization potential of the polyenes estimated from such linear relationship agrees satisfactorily with the value obtained by other methods. It has been concluded that the polyenes behave as electron donor and first form molecular charge-transfer complexes (of type [polyene . I2] with iodine) with electron acceptors, these finally dissociating to yield ionic complexes (of type [polyene . I+] with iodine).     

Characterization of human loricrin. Structure and function of a new class of epidermal cell envelope proteins

We have isolated and characterized a full-length cDNA clone encoding human loricrin. Curiously, this protein displays major differences from the recently described mouse loricrin (Mehrel, T., Hohl, D., Nakazawa, H., Rothnagel, J.A., Longley, M.A., Bundman, D., Cheng, C.K., Lichti, U., Bisher, M.E., Steven, A. C., Steinert, P.M., Yuspa, S.H., and Roop, D.R. (1990) Cell 61, 1103-1112). Although both proteins are glycine-serine-cysteine-rich, the sequences have not been conserved. However, analysis of the sequences reveals a common motif of quasi-peptide repeats of an aliphatic or aromatic amino acid residue followed by several glycine and/or serine and cysteine residues. These sequences are interspersed and flanked by short glutamine- or glutamine/lysine-rich peptides. Thus loricrins consist of a family of cell envelope proteins of highly variable sequences that nevertheless retain common structural elements. We show that unlike all other putative protein components of the cell envelope, loricrins are highly insoluble, due at least in part to cross-linking by disulfide bonds. Furthermore, we have isolated four peptides from purified human cell envelopes that contain recognizable loricrin sequences and which are cross-linked by the N epsilon-(gamma-glutamyl)lysine isodipeptide bond. The presence of such bonds thus affords an explanation for the extraordinary insolubility of loricrin by cross-linking to the cell envelope and can also explain the low steady-state levels of monomeric loricrin in cytoskeletal extracts of epidermis. This study represents the first report of this isodipeptide cross-link in a protein component of the cornified cell envelope. We propose a model for the structure of loricrin in which (i) the unusual glycine-serine-rich sequences adopt a flexible loop conformation, indexed on the recurrent aliphatic residues; (ii) inter- or intramolecular isodipeptide and disulfide cross-links induce or stabilize folding of loricrin so as to form a more compact rosette-like structure; and (iii) the presence of the flexible glycine-rich loops necessarily will impact a flexible character to the cell envelope and entire epithelium.     

Physical locus of the DNA polymerase gene and genetic maps of bacteriophage T5 mutants.

Segments of DNA that contained the DNA polymerase gene of bacteriophage T5 were isolated. The physical locus of the gene was identified by transforming Escherichia coli with purified DNA fragments generated by restriction enzyme digestions, and the transformed cells were used to rescue amber mutants of T5 with mutations in the gene for DNA polymerase. The method is applicable to any other gene that has mutations with low reversion frequencies. We studied the following mutations of the T5 DNA polymerase gene, reading from left to right by the standard convention (D. J. McCorquodale, Crit. Rev. Microbiol. 4:101-159, 1975): D7, D8, aml, ts5E-ts53, am6, and D9. These loci were found to reside within three pieces of DNA with a total length of 3,600 base pairs. Because the structural gene for T5 DNA polymerase is estimated to be 2,600 base pairs long, the whole structural gene may reside in these segments. These are located 58.3 to 61.3% of the distance from the left end of the DNA. The left-end piece of the DNA (1,100 base pairs) containing the polymerase gene has loci D7 and D8, and the right-end piece (1,600 base pairs) has locus D9, according to the results of the transformation assay. These results are consistent with the genetic map.     

Interaction of a DNA-binding protein, the product of gene D5 of bacteriophage T5, with double-stranded DNA: effects on T5 DNA polymerase functions in vitro.

The gene D5 product (gpD5) of bacteriophage T5 is a DNA-binding protein that binds preferentially to double-stranded DNA and is essential for T5 DNA replication, yet it inhibits DNA synthesis in vitro. Mechanisms of inhibition were studied by using nicked DNA and primed single-stranded DNA as a primer-template. Inhibition of T5 DNA polymerase activity by gpD5 occurred when double-stranded regions of DNA were saturated with gpD5. The 3' leads to 5' exonuclease associated with T5 DNA polymerase was not very active with nicked DNA, but inhibition of hydrolysis of substituents at 3'-hydroxyl termini by gpD5 could be observed. T5 DNA polymerase appears to be capable of binding to the 3' termini even when double-stranded regions are saturated with gpD5. The interaction of gpD5 with the polymerases at the primer terminus is apparently the primary cause of inhibition of polymerization.