cpDNA intergene sequences corroborate restriction site data for reconstructingRubiaceae phylogeny

Representatives of seven genera from five tribes ofRubiaceae have been compared in respect to a non-coding intergene cpDNA region of about 1000 bp, situated between the atpB and the rbcL genes. The resulting most parsimonious PAUP cladogram corresponds very well with one based on total cpDNA restriction site data obtained byBremer & Jansen (1991). The two different molecular analyses thus corroborate each other and contribute to an improved systematic arrangement of the large family, e.g., in respect to placing the tribeHedyotideae clearly into the subfamilyRubioideae, closer toRubieae than toPsychotrieae.     

Chaetomella acutiseta produces chaetomellic acids A and B which are reversible inhibitors of farnesyl-protien transferase

Chaetomellic acids A and B, isolated from Chaetomella acutiseta, are specific inhibitors of farnesyl-protein transferase that do not inhibit geranylgeranyl transferase type 1 or squalene synthase. Chaetomellic acids A and B are reversible inhibitors, resemble farnesyl diphosphate and probably inhibit FPTase by substituting for farnesyl diphosphate. Chaetomellic acid production appears to be widespread within the genus Chaetomella. Correspondence to: R. B. Lingham     

Genetic and biochemical properties of an extracellular neutral metalloprotease from Staphylococcus hyicus subsp. hyicus

The gene encoding the extracellular neutral metalloprotease ShpI from Staphylococcus hyicus subsp. hyicus was cloned. DNA sequencing revealed an ORF of 1317 nucleotides encoding a 438 amino acid protein with Mr of 49698. When the cloned gene was expressed in Staphylococcus carnosus, a 42 kDa protease was found in the culture medium. The protease was purified from both S. carnosus (pCAshp1) and S. hyicus subsp. hyicus. The N-terminal amino acid sequences of the two proteases revealed that ShpI is organized as a pre-pro-enzyme with a proposed 26 amino acid signal peptide, a 75 amino acid hydrophilic pro-region, and a 337 amino acid extracellular mature form with a calculated Mr of 38394. The N-termini showed microheterogeneity in both host strains. ShpI had a maximum proteolytic activity at 55°C and pH 7.4–8.5. The protease, which had a low substrate specificity, could be inhibited by metal- and zinc-specific inhibitors, such as EDTA and 1,10-phenanthroline. Insensitivity to phosphoramidon separates ShpI from the thermolysin-like family. The conserved Zn²⁺ binding motif, the only homology to other proteases, and the reactivation of the apoenzyme by Zn²⁺, indicated that Zn²⁺ is the catalytic ion. Ca²⁺ very probably acts as a stabilizer. We also demonstrated the presence of a second extracellular protease in S. hyicus subsp. hyicus.     

Phagocytosis of Intravenously Administered Particles by Leukocytes Adhered to the Aortic Endothelium of the Rat

Phagocytosis has been used to characterize on a functional basis leukocytes adhered to the aortic endothelium of the rat. After intravenous administration of particles, phagocytosis was observed microscopically in esterase-positive leukocytes adhered to the endothelium in whole mounts of aorta. Polybead^R blue and red, 0.5 and 1 μm particle size, were inadequate because they were insufficiently colored to be identified individually at 400. Fluoresbrite^tm YG 0.25 and 0.50 μm at doses of 0.2 and 2 ± 0.3 m1/100 g, respectively, produced endothelial lesions. The same occurred with Monastral blue B^R (MbB) at 0.3 ml/100 g, red iron at 2 ± 16 mg/100 g and India ink at different concentrations depending on the supplier. At lower particle doses, lesions were not found. Deferoxamine mesylate 1.5 mg/100 g intravenous and allopurinol 5 mg/100 g intraperitoneal administered before the particles diminished the number and intensity of lesions. In none of the cases studied was the percentage of phagocytic cells greater than 50%. Clearance curves of MbB and Fluoresbrite^tm indicated rapid disappearance of particles from the blood. Results indicate that administration of particulate suspensions is not a good method for characterizing the phagocytic leukocytes adhering to the aortic endothelium because low doses produce rapid clearance of particles, thus impeding sufficient leukocyte loading, and higher doses produce endothelial lesions that often impair reliable counting of the adhering leukocytes.     

The HYP2 gene of Saccharomyces cerevisiae is essential for aerobic growth: characterization of different isoforms of the hypusine-containing protein Hyp2p and analysis of gene disruption mutants

In Saccharomyces cerevisiae, hypusine-containing proteins are encoded by two closely related genes, HYP1 and HYP2, which are regulated reciprocally by oxygen and heme. We have purified the aerobically expressed hypusine-containing proteins from yeast. The three proteins detected (two isoforms, which differ in their pI values, and a degradation product thereof, lacking the N-terminal 10 amino acid residues) are all encoded by HYP2. The N-terminus of both isoforms is formed by acetylation of a serine residue after cleavage of the first methionine. Cells mutant for hyp2 are unable to grow aerobically. However, under anaerobic conditions these mutants display no obvious phenotype, presumably because the strictly anaerobically expressed HYPI gene product (Hyp1p) is present. This implies that Hyp1p and Hyp2p fulfill very similar functions. In fact, Hyp1p can substitute for Hyp2p under aerobic conditions, when expressed under the control of the GAL1 promoter in hyp2 mutant cells.Abbreviations HYP1 and HYP2 S. cerevisiae genes encoding hypusine-containing protein Hyplp and Hyp2p, respectively     

Update on proliferation-associated antibodies applicable to formalin-fixed paraffin-embedded tissue and their clinical applications

Summary This review provides an update on proliferation-associated antibodies applicable to immunohistochemical techniques in formalin-fixed paraffin-embedded tissue. New insights into proliferating cell nuclear antigen (PCNA) and antibodies to PCNA are presented. The characterization of the protein recognized by the Ki-67 antibody has enabled production of a new range of antibodies (monoclonal and polyclonal) which have immunostaining profiles similar to those of the original antibody. A new proliferation-associated antibody, KiS1, is described. The clinical applications of antibodies to PCNA in human material are summarized, and the limitations of these studies are discussed.     

A Rhizobium tropici DNA region carrying the amino-terminal half of a nodD gene and a nod-box-like sequence confers host-range extension

Rhizobium tropici CIAT899 is a broad-host-range strain that, in addition to Phaseolus, nodulates other plant legumes such as Leucaena and Macroptilium. The narrow-host-range of Rhizobium leguminosarum biovars phaseoli (strain CE3) and trifolii (strain RS1051) can be extended to Leucaena esculents and Phaseolus vulgaris plants, respectively, by the introduction of a DNA fragment 521 bp long, which carries 128 amino acids of the amino-terminal region of a nodD gene from R. tropici, as well as a putative nod-box-like sequence, divergently oriented. The 521 bp fragment, in the presence of L. esculenta or P. vulgaris root exudates, induced a R. leguminosarum bv. viciae nodA-lacZ fusion in either a CE3 or RS1051 background, respectively.     

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.     

AUXOSPORULATION IN CYCLOTELLA OCELLATA (BACILLARIOPHYCEAE) UNDER NATURAL AND EXPERIMENTAL CONDITIONS1

Growth and sexual reproduction in a population of Cyclotella ocellata Pantocseck were studied during one annual cycle in a reservoir and in short-term enclosure experiments performed in situ involving different nutrient conditions and concentrations of zooplankton species. Three phases of auxosporulation in this diatom were distinguishable morphologically: 1) preauxospore, from the beginning of zygote formation until the valves were longitudinally separated, 2) primary auxospore, when the zygote grew too large to fit inside the valves and before it reached its full size, and 3) mature auxospore, characterized by a well-developed, markedly scalloped edge. Under experimental and natural conditions, sexual reproduction was associated with changes in cell size. In the natural system, the auxospore appeared to act as a resting structure during conditions adverse for population growth. A threshold population of small cells appeared to be necessary for sexual reproduction in the natural system, whereas auxosporulation was associated with phosphorus fertilization in the enclosures. In both environments only cells smaller than 9.5 μm in diameter were capable of auxospore formation. Our results suggest that, once having reached the critical cell size, the factors that trigger sexual reproduction may depend on ambient environmental conditions.