New branched Porolithon species (Corallinales,Rhodophyta) from the Great Barrier Reef,Coral Sea,and Lord Howe Island

Porolithon is one of the most ecologically important genera of tropical and subtropical crustose (non-geniculate) coralline algae growing abundantly along the shallow margins of coral reefs and functioning to cement reef frameworks. Thalli of branched, fruticose Porolithon specimens from the Indo-Pacific Ocean traditionally have been called P. gardineri, while massive, columnar forms have been called P. craspedium. Sequence comparisons of the rbcL gene both from type specimens of P. gardineri and P. craspedium and from field-collected specimens demonstrate that neither species is present in east Australia and instead resolve into four unique genetic lineages. Porolithon howensis sp. nov. forms columnar protuberances and loosely attached margins and occurs predominantly at Lord Howe Island; P. lobulatum sp. nov. has fruticose to clavate forms and free margins that are lobed and occurs in the Coral Sea and on the Great Barrier Reef (GBR); P. parvulum sp. nov. has short (<2 cm), unbranched protuberances and attached margins and is restricted to the central and southern GBR; and P. pinnaculum sp. nov. has a mountain-like, columnar morphology and occurs on oceanic Coral Sea reefs. A rbcL gene sequence of the isotype of P. castellum demonstrates it is a different species from other columnar species. In addition to the diagnostic rbcL and psbA marker sequences, the four new species may be distinguished by a combination of features including thallus growth form, margin shape (attached or unattached), and medullary system (coaxial or plumose). Porolithon species, because of their ecological importance and sensitivity to ocean acidification, need urgent documentation of their taxonomic diversity.     

Changing synaptic specificities in the nervous system of Caenorhabditis elegans: Differentiation of the DD motoneurons

During postembryonic development, the DD motoneurons in the nematode Caenorhabditis elegans completely reorganize their pattern of synapses. Ablation of a pair of embryonic precursors results in the absence of this entire class of motoneurons. In their absence animals exhibit two developmentally distinct locomotory defects. The transition period from one defect to the other is correlated with the synaptic reorganization of the DD mns. Mutations in a gene (unc-123) have been isolated that exhibit locomotory defects similar to those of the ablated adult animals. Genetic and cellular analyses of one of these alleles suggest that the unc-123 gene product may be involved in the reestablishment of functional synapses in these neurons. © 1993 John Wiley & Sons, Inc.     

Isolation of DNA from yeasts

Methods are described that allow DNA to be prepared from widely different yeasts (Candida utilis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe). The methods are reliably reproducible, and the DNA obtained is of appropriate quality for the construction of gene libraries (upper limit of size range consistently 50-150 kbp). In method A, yeast cells are converted into spheroplasts by treatment with a highly purified mixture of enzymes from Trichoderma harzianum, the spheroplasts are lysed in a lauroylsarcosinate/EDTA buffer, and the lysate is incubated with proteinase K and then directly centrifuged through a cesium trifluoroacetate gradient. DNA is recovered from the appropriate fractions by ethanol precipitation, and the redissolved precipitate is incubated with ribonuclease. For the rest of the isolation, two protocols are given, one avoiding and one including phenol/chloroform extraction. In this way, DNA up to about 150 kbp in size can be obtained. In method B, spheroplasts are not made. Yeast cells are broken by grinding under liquid nitrogen and are then worked up in a manner similar to method A, protocol 2. Subsequent steps depend on the purpose for which the DNA is required. Traditional methods of sucrose or salt density gradient centrifugation or agarose gel electrophoresis are applicable for size selection. A sodium iodide/silica matrix technique allows fast and effective DNA recovery from agarose gels.     

1H, 15N and 13C resonance assignments and secondary structure determination of the RNA-binding domain of E. coli rho protein

Summary Protein fragments containing the RNA-binding domain of Escherichia coli rho protein have been over-expressed in E. coli. NMR spectra of the fragment containing residues 1–116 of rho protein (Rho116) show that a region of this protein is unfolded in solution. Addition of (dC)₁₀ to this fragment stabilizes the folded form of the protein. The fragment comprising residues 1–130 of rho protein (Rho130) is found to be stably folded, both in the absence and presence of nucleic acid. NMR studies of the complex of Rho 130 with RNA and DNA oligonucleotides indicate that the binding-site size, affinity, and specificity of Rho 130 are similar to those of intact rho protein; therefore, Rho 130 is a suitable model of the RNA-binding domain of rho protein. NMR line widths as well as titration experiments of Rho130 complexed with oligonucleotides of various lengths suggest that Rho130 forms oligomers in the presence of longer oligonucleotides. ¹H, ¹⁵N and ¹³C resonance assignments were facilitated by the utilization of two pulse sequences, CN-NOESY and CCH-TOCSY. The secondary structure of unliganded Rho130 has been determined by NMR techniques, and it is clear that the RNA-binding domain of rho is more structurally similar to the cold shock domain than to the RNA recognition motif.Abbreviations Rho116, Rho130 protein containing the first 116 (130) residues of rho - CSD cold shock domain - RRM RNA recognition motif - RBD RNA-binding domain - IPTG isopropyl -D-thiogalactopyranoside - EDTA ethylenediaminetetraacetic acid - NOE nuclear Overhauser enhancement     

Disruption of microfilaments alters laminin synthesis but not laminin trafficking in NHEKin vitro

Summary Laminin synthesis and deposition are concomitant with the development of a basal lamina between the human epidermis and the underlying dermis. One of the challenges in tissue engineering of human epidermal models is to develop substrates and conditions that encourage the development of a basement membrane. The purpose of this study was to determine if actin filaments and/or microtubules are involved in the synthesis/secretion of laminin by normal human epidermal keratinocytes (NHEK)in vitro. NHEK synthesize and secrete laminin subunits B1, B2, and M but little, if any, of laminin subunit A. Data indicate that disruption of microfilaments by the destabilizing agent, cytochalasin D, had no apparent effect on the relative synthesis rates of most cytosolic proteins as, revealed by one-dimensional sodium dodecyl sulfate (SDS) gel electrophoresis. This drug, however, increased laminin B2 synthesis several fold over untreated controls. This enhanced synthetic rate was independent of the type of collagen, matrix on which the NHEK were grown. Similar increases in synthesis of the M and B1 laminin chains were not observed. To determine if this increase in synthesis lead to increases in laminin B2 secretion, laminin B2 was immunoprecipitated from both the apical and basal domains of NHEK cells grown on microporous membranes. While more laminin B1, B2, and M were secreted basally than apically, an observation consistent with laminin’s role in basal lamina formation, cytochalasin D had no apparent effect on either basal or apical laminin B2 secretion. Experiments with the microtubule destabilizer, nocodazole, showed no similar effects on laminin synthesis and/or secretion. We conclude that (a) disruption of the actin network in NHEK selectively increases the synthesis of laminin B2, (b) the secretion of laminin B2 from NHEK cells is not governed by either the microfilamentous cytoskeleton or the amount of laminin synthesized by NHEK, and (c) disruption of the microtubular network does not alter laminin synthesis or secretion.     

Enhanced sunflower oil utilization and gamma-linolenic acid production by Mucor circinelloides f.circinelloides CBS 108.16 in the presence of acetate

World Journal of Microbiology and Biotechnology -     

Extensive variations and basic features in the alcohol dehydrogenase-sorbitol dehydrogenase family

Structural comparisons of sorbitol dehydrogenase with zinc-containing 'long' alcohol dehydrogenases reveal distant but clear relationships. An alignment suggests 93 positional identities with horse liver alcohol dehydrogenase (25% of 374 positions) and 73 identities with yeast alcohol dehydrogenase (20%). Sorbitol dehydrogenase forms a link between these distantly related alcohol dehydrogenases and is in some regions more similar to one of them that they are to each other. 43 residues (11%) are common to all three enzymes and include a heavy over-representation of glycine (half of all glycine residues in sorbitol dehydrogenase), showing the importance of space restrictions in protein structures. Four regions are well conserved, two in each domain of horse liver alcohol dehydrogenase. They are two segments close to the active-site zinc atom of the catalytic domain, and two in the central beta-pleated sheet strands of the coenzyme-binding domain. These similarities demonstrate the general importance of internal and central building units in proteins. Large variations affect a region adjacent to the third protein ligand to the active-site zinc atom in horse liver alcohol dehydrogenase. Such changes at active sites of related enzymes are unusual. Other large differences concern the segment around the non-catalytic zinc atom of horse liver alcohol dehydrogenase; three of its four cysteine ligands are absent from sorbitol dehydrogenase. Three segments with several exchanges correspond to a continuous region with superficial areas, inter-domain contacts and inter-subunit interactions in the catalytic domain of alcohol dehydrogenase. They may correlate with the altered quaternary structure of sorbitol dehydrogenase. Regions corresponding to top and bottom beta-strands in the coenzyme-binding domain of the alcohol dehydrogenase are also little conserved. Within sorbitol dehydrogenase, a large segment shows an internal similarity. The two distantly related alcohol dehydrogenases and sorbitol dehydrogenase form a triplet of enzymes illustrating basic protein relationships. They are ancestrally close enough to establish similarities, yet sufficiently divergent to illustrate changes in all but fundamental properties.     

Sorbitol dehydrogenase. The primary structure of the sheep-liver enzyme

The first primary structure for a sorbitol dehydrogenase has been determined by analysis of the tetrameric enzyme from sheep liver. The [14C]carboxymethylated protein was cleaved with CNBr and proteolytic enzymes. Peptides were purified by several methods, often utilizing exclusion chromatography for pre-fractionation and reverse-phase high-performance liquid chromatography for final purification. Different methods of sequence analysis complemented each other, mainly the manual dimethylaminoazobenzene isothiocyanate method and and the use of liquid-phase sequencer degradations. All eight major CNBr fragments were purified and form the basis of the work. Three minor CNBr fragments derived from an acid cleavage and from a partly resistant Met-Thr bond were also obtained, as well as evidence for a contaminating homologous polypeptide. Most of the tryptic peptides were purified, including all with methionine residues, thus overlapping the CNBr fragments. Combined, all data permit the deduction of a 354-residue amino acid sequence for the polypeptide chain of sorbitol dehydrogenase. The N terminus is acyl-blocked, the C terminus is formed by a proline residue, tryptophan is the least common residue (two, at positions 50 and 301) and there are 10 cysteine residues, including the residue previously shown to be especially reactive (at position 43). Similarities to 'long' alcohol dehydrogenases have functional implications.     

Radioimmunoassay of carbonic anhydrase III in rat tissues.

A specific and sensitive radioimmunoassay for the rat carbonic anhydrase III isoenzyme was developed. High concentrations of carbonic anhydrase III were detected in soleus muscle and male liver. Female liver and other skeletal muscles contained significantly lower concentrations, and only trace amounts were found in heart, prostate, kidney, brain, plasma, urine and, possibly, erythrocytes.     

Role of glutathione in the regulation of hepatic cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme of bile acid biosynthesis

The effect of in vivo variation of hepatic glutathione (using diethyl maleate and L-cysteine) on in vitro cholesterol 7 alpha-hydroxylase activity was studied in male Sprague-Dawley rats. Cholesterol 7 alpha-hydroxylase activity in glutathione-depleted rats (ca. 10% of control glutathione) was significantly reduced compared to that in vehicle-injected controls. While L-cysteine treatment of glutathione-depleted animals increased glutathione levels somewhat (ca. 20% of control glutathione), they were still significantly less than control levels. Similarly, cholesterol 7 alpha-hydroxylase activity in the partially glutathione replete animals was approximately 50% greater than that in the glutathione-depleted animals, but still significantly less than that in the controls. The rate of 7 alpha-hydroxylation of cholesterol was found to be dependent on liver glutathione content. The calculated maximal rate was 34.4 picomoles/mg/min with a half maximal activity at 1.89 mumoles glutathione/gm liver. These results suggest that hepatic glutathione may be an important modulator of in vivo activity of cholesterol 7 alpha-hydroxylase.