No abstract

Complex regional pain syndrome type I (CRPS I, formerly known as reflex sympathetic dystrophy) is a painful neuropathic disorder that develops after trauma affecting the limbs without overt nerve injury. Clinical features are spontaneous pain, hyperalgesia, impairment of motor function, swelling, changes in sweating, and vascular abnormalities. In this study, the pathophysiological mechanisms of vascular abnormalities were investigated. Furthermore, the incidence, sensitivity and specificity of side differences in skin temperature were defined in order to distinguish patients with definite CRPS I from patients with extremity pain of other origin. In 25 CRPS I patients and two control groups (20 healthy subjects and 15 patients with other types of extremity pain), cutaneous sympathetic vasoconstrictor activity was altered tonically by the use of controlled thermoregulation. Whole-body temperature changes were induced with a thermal suit in which cold or hot water circulated. The vascular reflex response (skin blood flow, laser Doppler flowmetry, skin temperature, infrared thermometry) was analysed to quantify sympathetic outflow. Measurements were performed during a complete thermoregulatory cycle, i.e. during the entire spectrum of sympathetic vasoconstrictor activity from high (whole-body cooling) to low sympathetic activity (whole-body warming). Venous noradrenalin levels were determined bilaterally in five CRPS patients. (i) Three distinct vascular regulation patterns were identified related to the duration of the disorder. In the "warm" (acute) type of regulation, the affected limb was warmer and perfusion values were higher than in the contralateral limb during the entire spectrum of sympathetic activity. In the "intermediate" type of regulation the limb was either warmer or colder. In the "cold" (chronic) type of regulation, skin temperature and perfusion values were lower on the affected side during the entire spectrum of sympathetic vasoconstrictor activity. (ii) Noradrenalin levels were lower on the affected side, even in chronic patients with considerable cutaneous vasoconstriction. (iii) Temperature and blood flow differences between the two sides were dynamic and most prominent at a high to medium level of vasoconstrictor activity. (iv) In both control groups, there were only minor side differences in flow and temperature. In conclusion, it is suggested that, in CRPS I, unilateral inhibition of sympathetic vasoconstrictor neurones leads to a warmer affected limb in the acute stage. Secondary changes in neurovascular transmission may lead to vasoconstriction and cold skin in chronic CRPS I, whereas sympathetic activity is still depressed. Vascular abnormalities are dynamic. The maximal skin temperature difference that occurs during the thermoregulatory cycle distinguishes CRPS I from other extremity pain syndromes with high sensitivity and specificity.

Obligately endosymbiotic bacteria living in the cytoplasm of ciliates of the genus Euplotes constitute the new genus Polynucleobacter gen. nov. These endosymbionts are commonly known as omicron and omicronlike particles. The best-studied form, as well as the type species, is omicron from stock 15 of Euplotes aediculatus. We propose the name Polynucleobacter necessarius gen. nov., sp. nov., for this bacterium.Endosymbiotic bacteria often are seen in protozoa. However, only a few of them have been studied extensively, and several of these are still referred to by Greek letters, as is customary for cytoplasmic elements. Among the endosymbionts which are well characterized but do not have binary names are omicron and the omicronlike endosymbionts. The purpose of the present paper is to name this group of symbionts in accordance with the rules of the Bacteriological Code. The name Polynucleobacter was selected to indicate a characteristic feature of this genus: all members show multiple nucleoids easily visible in these endosymbionts after staining with acetocarmin, acetoorcein, or deoxyribonucleic acid (DNA)-fluorescent dyes. The epithet necessarius was chosen to indicate the relationship of omicron and its host Euplotes aediculatus, which depend upon each other (3).

Pheromone 3 mRNA of the ciliate Euplotes octocarihatus contains three in-frame UGA codons that are translated as cysteines. This was revealed from cDNA sequencing and from plasma desorption mass spectrometry of cleaved pheromone 3 in connection with pyridylethylation of the fragments. N-terminal sequence analysis of carboxymethylated protein confirmed this conclusion for the first of the three UGA codons. Besides UGA the common cysteine codons UGU and UGC are also used to encode cysteine. UAA functions as a termination codon. Preparation of RNA. Total RNA was prepared by disruption of 1-3 x 107 cells in 8 M urea/4 M LiCl in a PotterElvehjem homogenizer, followed by precipitation on ice overnight. RNA was collected by centrifugation, dissolved in 10 mM Mops, pH 7.5/0.5% SDS, and extracted three times with phenol/chloroform/isoamyl alcohol (25:24:1) and once with chloroform/isoamyl alcohol (24:1). Total RNA was then precipitated by addition of 0.1 volume of 4 M LiCl and 2.5 volumes of absolute ethanol.Poly(A)+ RNA was prepared by affinity chromatography on oligo(dT)-cellulose (Bethesda Research Laboratories) as recommended by the supplier with the exception that Mops was used as the buffer instead of Tris. Poly(A)+ RNA was precipitated by the addition of LiCI and ethanol as described above and redissolved in water. Quantity and quality were determined spectrophotometrically by measuring absorption at 260 and 280 nm (14).cDNA Synthesis and Cloning. The cDNA library was constructed (15) in the vector AgtlO. The cDNA was treated with S1 nuclease and ligated with EcoRI linkers prior to its insertion into the EcoRI site of the vector. The pheromone 3 gene was identified by plaque hybridization with the synthetic oligodeoxynucleotide 5'-GTRTANGGYTCYTCCCA-3', corresponding to the N terminus of the secreted pheromone, and was isolated by standard techniques (14).DNA Sequencing. Eight positively hybridizing plaques were obtained from 105 transformants. Five of them were further subcloned for sequencing by the dideoxy chain-termination method. Their nucleotide sequences were determined from double-stranded and single-stranded templates (pUC12, pT7T3, M13mpl8, and M13mp19 as sequencing vectors) according to the sequencing strategy outlined in Fig. 1 1To whom reprint requests should be addressed.

The ciliate Euplotes octocarinatus and some close relatives of it are triggered by predator-released substances to undergo morphogenetic changes that inhibit their engulfment. The changes occur within a few hours and do not require cell division. They are perpetuated during reproduction so long as the concentration of the morphogen is maintained. The ability of Euplotes to respond to predator-produced signals by a defensive change in cell architecture probably provides an effective mechanism for damping population oscillations ofboth prey and predators andfosters coexistence. The signal-induced cell transformation merits study for its own sake because of its developmental implications.

The hypothesis is advanced that all freshwater Euplotes species with a 9 type 1 fronto‐ventral cirrus pattern (E. patella type) depend upon bacteria‐like endosymbionts. Aposymbiotic cells of these species are unable to divide. The hypothesis is based on the investigation of 40 different freshwater Euplotes stocks collected in Germany, France, the USA, and Japan. No symbionts were found in E. crenosus and E. palustris, freshwater species with 10 fronto‐ventral cirri, nor in E. muscicola, a representative of the freshwater Euplotes group with a 9 type 2 fronto‐ventral cirrus pattern (E. affinis type). Characteristic for the essential endosymbionts are multiple nucleoids, a feature described earlier for omikron, an indispensable symbiont of E. aediculatus. Although the symbionts differ from omikron and among each other in size, shape, and their average number per host, they are believed to be related to omikron. In two stocks a different type of bacterium was found in which no defined nucleoids can be detected. Transfer of this symbiont into aposymbiotic cells, originally carrying omikron, revealed that it can restore the ability to multiply. Similarly, omikron was also able to restore the ability to divide in cells freed of this symbiont. It is assumed that this different type of symbiont is a secondary invader of Euplotes which displaced the original omikron‐like endosymbiont. Some of the stocks were found to carry, in addition to omikron‐like symbionts, other symbiotic bacteria; E. daidaleos carries in addition an alga. The findings suggest that the freshwater Euplotes species with a 9 type 1 cirrus pattern are closely related to each other and evolved from an ancestor (probably of cirrotype 10) which already was dependent upon endosymbionts of the omikron type. It supports the view that the two subgroups of freshwater Euplotes forms with a cirrotype of 9 have evolved independently from each other from species with 10 fronto‐ventral cirri by losing a cirrus at different positions.

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We propose that the amino acid residues 57/58 and 60/61 of eukaryotic release factors (eRF1s) (counted from the N-terminal Met of human eRF1) are responsible for stop codon recognition in protein synthesis. The proposal is based on amino acid exchanges in these positions in the eRF1s of two ciliates that reassigned one or two stop codons to sense codons in evolution and on the crystal structure of human eRF1. The proposed mechanism of stop codon recognition assumes that the amino acid residues 57/58 interact with the second and the residues 60/61 with the third position of a stop codon. The fact that conventional eRF1s recognize all three stop codons but not the codon for tryptophan is attributed to the flexibility of the helix containing these residues. We suggest that the helix is able to assume a partly relaxed or tight conformation depending on the stop codon recognized. The restricted codon recognition observed in organisms with unconventional eRF1s is attributed mainly to the loss of flexibility of the helix due to exchanged amino acids. ß