Transformation of a psychrotrophic bacterium with a plasmid from a mesophile

The psychrotrophic bacteriumBacillus psychrophilus was transformed with the broadhost-range plasmid pC194. The ability of the transformant to express chloramphenicol (CAM) resistance and the possible effects of such expression on the physiology of the psychrotroph were examined. The transformant exhibited growth rates, filament formation at elevated temperatures, synthesis of cold shock proteins and cold acclimation proteins, similar to the parentalB. psychrophilus.     

Characterization of a new strain of a purple nonsulfur bacterium from a thermal spring

A new budding purple nonsulfur bacterium of the genus Rhodobacter (strain Ku-2) was isolated from a mat of a moderately thermal spring (Baikal rift zone, Buryatia, Russia). The bacterium had lamellar photosynthetic membranes, which are typical of only one Rhodobacter species, Rba. blasticus. The cells contained spheroidene carotenoids and bacteriochlorophyll a (Bchl a). In vivo absorption spectrum of the cells, with the major maximum at 863 nm and an additional peak at 887 nm, is characteristic of the pigment-protein complexes of Bchl a-containing membranes. The previously described Rba. blasticus strains do not exhibit the 887-nm maximum. The new isolate was photoheterotrophic, with optimal growth occurring at 35°C, 3 g/L NaCl, and pH 7–8. The DNA G+C content was 64.4 mol %. The similarity between the 16S rRNA gene sequences of strain Ku-2 and the Rba. blasticus type strain was 98.7%. The PufM amino acid sequences of strain Ku-2 and the earlier studied Rba. blasticus type strain were 89.5 % identical. Thus, strain Ku-2 belongs to the genus Rhodobacter and is phylogenetically close to Rba. blasticus.     

Irreversible inhibition of a monoclonal antibody by a nitrophenyl ester

TEPC-15 is a phosphorylcholine-binding mouse myeloma protein which reacts with an ester-containing phosphorylcholine, thep-nitrophenyl ester of 6-(phosphorylcholine)hexanoic acid (PEPCH). The rate of nitrophenolate release mediated by the antibody ispH-dependent and increases with increasingpH. The antibody becomes inactive during the reaction with the ester. The inactive antibody is not reactivated even after treatment with hydroxylamine. Antibody activity is associated with the Fab' fragment. These observations together with thepH profile of the reaction suggest that the ester acylates a lysine side chain near the antibody-binding site.A preliminary discussion of this work was presented at the 78th Annual Meeting of the American Society of Biological Chemists, Philadelphia, Pennsylvania, 1987.     

Perioperative Management of a Patient with a Nonresectable Pheochromocytoma

ObjectiveTo describe the clinical course of a patient with a nonresectable pheochromocytoma during urgent hip surgery.MethodsTo describe the clinical management and postoperative outcome of the patient and review the relevant literature.ResultsAn 85-year-old male with a nonresectable pheochromocytoma required urgent hip surgery following a traumatic hip fracture. He was perioperatively managed with phenoxybenzamine, metyrosine, and metoprolol to avoid potential pheochromocytoma-related complications. He remained hemodynamically stable and recovered from the surgery without complications.ConclusionsThis case illustrates the successful management of a patient requiring urgent surgery in the setting of a nonresectable pheochromocytoma, which is rarely described in the literature. (Endocr Pract. 2013;19: e74-e76     

Recognition of a genus-wide antigen ofLegionella by a monoclonal antibody

A monoclonal antibody was produced against a cytoplasmic membrane protein that appears to be common to all species of the genusLegionella. The antibody was positive in polyacrylamide gel electrophoresis and Western blotting with extracts of all of 22 species type strains ofLegionella that were tested. The apparent molecular mass of the protein varied from 57.2 to 62.1 kilodaltons for the 23 species type strains ofLegionella. An enzyme-linked immunosorbent assay (ELISA) was developed with the monoclonal antibody to enable rapid screening of clinical and environmental isolates forLegionella. All of 23 species type strains ofLegionella that were tested were strongly positive with the monoclonal antibody in the ELISA. Among 27 other bacterial species and 84 strains that were tested, onlyBordetella ssp. andAcinetobacter lwoffii were cross-reactive in the ELISA. These two cross-reactive species are readily distinguishable fromLegionella by culture characteristics. The monoclonal antibody may also be useful in tests to detect the genus-wide antigen in body fluids of patients with legionellosis.     

Creation of a non-mycorrhizal control for a bioassay of AM effectiveness

and application times 2 weeks before sowing, at sowing and 1 week after sowing were investigated. Various Finnish field soils with their indigenous AMF communities were used. The main test plant species was oil-seed flax (Linum usitatissimum). In a comparison of sampling time, barley (Hordeum vulgare) was also used and phytotoxicity was studied additionally on red clover (Trifolium pratense), barley and pea (Pisum sativum) mutants. Sampling in the spring after the thaw resulted in the highest infectivity and AM response and the clearest differences between soils with varying AM potential. No evidence of temporal variation in benomyl effectiveness on mycorrhiza was found. The dose of benomyl sufficient to create a control with suppressed mycorrhization was 20 mg per kg soil at target moisture incorporated in the soil. Plant growth reduction in irradiated soil was observed with benomyl application 1 week after sowing only with flax and red clover. The most effective application time for benomyl was immediately before sowing.     

Intersession reliability of a posturo-stabilometric test,using a force platform

Aim of the studyTo evaluate the intersession reliability of a posturo-stabilometric examination.MethodsSingle blind clinical trial conducted in two sessions over two weeks.44 healthy volunteers free from postural and temporomandibular disorders. All the subjects complied with the criteria for completing the study.All the subjects underwent two sessions of posturo-stabilometric examinations in different visual and mandibular conditions.Sway area, sway length and the coordinates of the center of pressure were evaluated and statistically analyzed using the Intraclass correlation coefficient (ICC).ResultsAll the posturo-stabilometric parameters seemed to have an excellent reproducibility with overall ICCs higher than 70% and good confidence intervals except for the sway area (ICC 0.422 with CI 0.283–0.560 with open eyes and ICC 0.554 with CI 0.424–0.683 with closed eyes).ConclusionsThe posturo-stabilometric examination carried out using a force platform has a good intrasession and intersession reliability, especially considering sway velocity, COP X and COP Y parameters. The force platform usefulness in analyzing static posture is confirmed in any medical field.     

Performance of a membrane-dialysis bioreactor with a radial-flow fixed bed for the cultivation of a hybridoma cell line

A bioreactor system for the continuous cultivation of animal cells with a high potential for scale-up is presented. This reactor system consists of radial-flow fixed-bed units coupled with a dialysis module. The dialysis membrane enables the supply of low-molecular-weight nutrients and removal of toxic metabolites, while high-molecular-weight nutrients and products (e.g. monoclonal antibodies) are retained and accumulated. This concept was investigated on the laboratory scale in a bioreactor with an integrated dialysis membrane. The efficiency of the reactor system and the reproducibility of the cell activity (hybridoma cells) under certain process conditions could be demonstrated in fermentations up to 77 days. Based on model calculations, an optimized fermentation strategy was formulated and experimentally confirmed. Compared to chemostat cultures with suspended cells, a ten-times higher mAb concentration (383 mgl⁻¹) could be obtained. The highest volumetric specific mAb production rate determined was 6.1 mg mAb (1 fixed bed)⁻¹ h⁻¹.     

Virilization of a female infant by a maternal adrenocortical carcinoma

ObjectiveTo describe a possible mechanism underlying the partial virilization of a 46, XX infant by a functional maternal adrenocortical carcinoma (ACC).MethodsWe performed immunocytochemical staining of tumor sections for luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors. In addition, related reports in the literature are discussed.ResultsA previously healthy mother developed a large cortisoland androgen-producing stage III adrenal tumor that did not interfere with conception or early morphogenesis. The tumor eluded detection until after delivery of a partially virilized 46, XX female infant with ambiguous genitalia. Immunohistochemical staining of tumor sections revealed overexpression of the LH/hCG receptor. Virilization of the genetically female fetus may have resulted from hCG-stimulated steroid secretion by the ACC.ConclusionBecause hypercortisolism and hyperandrogenism are associated with menstrual disturbances and spontaneous abortion, pregnancy in patients with functional adrenal tumors is uncommon. Rarely, maternal steroid excess from a functional adrenal tumor has caused 46, XX disordered sex differentiation. This unusual case demonstrates the influence of hCG on the functionality of an ACC and demonstrates the rare phenomenon of virilization of a female infant by a functional maternal adrenal tumor. (Endocr Pract. 2011;17:e26-e31)     

Dosage effect of a gene with a regulating effect on anthocyanin synthesis in a trisomic Petunia hybrida

A Petunia hybrida inbred line (W 28) has white flowers with red spots on the corolla. These spots are the result of back mutations of an unstable allele of the gene Anl for anthocyanin synthesis. Among the progeny of a population of selfed plants a primary trisomic with red-spotted white flowers was found. The reversion frequency was more than twice as high as compared with disomic plants of the same family.It was found that the chromosome in triplicate was not the chromosome on which the gene Anl is localized. It can be concluded that there is an independently segregating factor which influences the frequency of back mutations of the Anl locus. Twin spots were found among the flowers of the trisomic. They consisted of two adjacent sectors, one with a spot frequency equal to that of the flowers of disomic plants, and the other with a spot frequency more than twice as high as that of the trisomic. Probably an irregular distribution of the extra chromosome resulted in one sector with the normal diploid number of chromosomes, and an adjacent sector with two extra chromosomes. The reversion frequencies in the sector suggest that the factor which affects the reversion frequency of the unstable alleles of Anl exhibits a dosage effect.     

Diversity of a soybean rhizobial population adapted to a Cerrados soil

One hundred isolates were trapped by soybean (Glycine max) plants inoculated with a soil from the Cerrados, the main producing area in Brazil. The soil was originally void of rhizobia able to nodulate soybean, and 15 years before received inoculant containing Bradyrhizobium elkanii strains SEMIA 587 and SEMIA 5019; the area has been annually cropped with soybean since then, but with no further inoculation for the past 7 years. Enormous diversity was observed among the isolates, with thirteen serologically distinct groups, twelve protein and seven lipopolysaccharide profiles; no more than five isolates shared similar characteristics. An unexpected feature was that 48% of the isolates showed multiple reactions with the antisera to the serogroups established in the soils. Also 40% of the isolates reacted with the antiserum to B. japonicum strain SEMIA 566, that has never been introduced into the soil, probably due to dispersion from other cropping areas, associated with its high saprophytic competence; 13% of the isolates did not react with any of the antisera. Nodulation and N₂ fixation capacity also varied considerably among the isolates. Although one third of the isolates were fast growers with an acid reaction in vitro, and many formed pseudo-nodules on common bean (Phaseolus vulgaris), they shared several properties with the Bradyrhizobium inoculant strains. A high level of genetic diversity was confirmed when the DNAs were amplified with BOX and RPO1 primers, and several isolates were positioned in far different clusters in the analysis of interspersed repetitive or nif-directed sequences. Moreover, serological properties showed higher correlation with BOX than with RPO1 products. The high diversity could be attributed both to lateral transfer of genetic material between inoculant and indigenous strains and to genomic rearrangements during the adaptation to the Cerrados, and may play an important role as a biological buffer, avoiding the dominance of a particular strain.     

Data from a Study of a Neuroallergic Family

Migraine and other types of headaches, serous meningites, encephalomyelites related to the employment of serums and vaccines, including Alazhuanin's hemorrhagic encephalitis, acute hemorrhagic encephalitis, parainfectious encephalites, nervous system damage of arthritic origin (primarily chorea minor), vascular diseases of the nervous system in periarteritis nodosa, cerebral thromboangiitis obliterans in Winiwarter-Buerger's disease, serous and toxic neurites and polyneurites, the Guillain-Barre syndrome, Landry's paralysis, polyneurites resulting from treatment with salvarsan and sulfonamides, certain cases of Meniere's syndrome, demyelinating diseases of the nervous system (multiple sclerosis, related acute and chronic disseminated encephalites, encephalitis periaxialis diffusa, and other forms of sclerous leuco encephalites and, finally, epilepsy (1-24) are diseases of the nervous system associated with immune pathology reactions to various antigens (foods, inhalational, medicamental, viral, bacterial and other foreign agents, and autoantigens).     

Recognition of a Flipped Base in a Hairpinloop DNA by a Small Peptide

Two tiny hairpin DNAs, CORE (dAGGCTTCGGCCT) and AP2 (dAGGCTXCGGCCT; X: abasic nucleotide), fold into almost the same tetraloop hairpin structure with one exception, that is, the sixth thymine (T6) of CORE is exposed to the solvent water (Kawakami, J. et al., Chem. Lett. 2001, 258–259). In the present study, we selected small peptides that bind to CORE or AP2 from a combinatorial pentapeptide library with 2.5 × 10⁶ variants. On the basis of the structural information, the selected peptide sequences should indicate the essential qualifications for recognition of the hairpin loop DNA with and without a flipped base. In the selected DNA binding peptides, aromatic amino acids such as histidine for CORE and glutamine/aspartic acid for AP2 were found to be abundant amino acids. This amino acid preference suggests that CORE-binding peptides use π–π stacking to recognize the target while hydrogen bonding is dominant for AP2-binding peptides. To investigate the binding properties of the selected peptide to the target, surface plasmon resonance was used. The binding constant of the interaction between CORE and a CORE-binding peptide (HWHHE) was about 1.1 × 10⁶ M^?1 at 25°C and the resulting binding free energy change at 25°C (ΔG°₂₅) was ?8.2 kcal mol^?1. The binding of the peptide to AP2 was also analyzed and the resulting binding constant and ΔG°₂₅ were about 4.2 × 10⁴ M^?1 and ?6.3 kcal mol^?1, respectively. The difference in the binding free energy changes (ΔΔG°₂₅) of 1.9 kcal mol^?1 was comparable to the values reported in other systems and was considered a consequence of the loss of π–π stacking. Moreover, the stabilization effect by stacking affected the dissociation step as well as the association step. Our results suggest that the existence of an aromatic ring (T6 base) produces new dominant interactions between peptides and nucleic acids, although hydrogen bonding is the preferable mode of interaction in the absence of the flipping base. These findings regarding CORE and AP2 recognition are expected to give useful information in the design of novel artificial DNA binding peptides.     

Continuous flow cultures of a HeLa cell line as a basis for a steady supply of Rubella virus

A HeLa cell line was propagated in semicontinuous suspension culture, 85 liters final volume, and in continuous flow culture with a volume of 300 ml. or 5 liters in an autoclavable medium to which 8% calf serum had been added. A medium containing 0.1% Methocel and 2% calf serum was also tested. Maximum productivity was obtained at a dilution rate of 0.33 day^?1 with a cell density of about 1.0 × 10⁶ cells/ml. The same cell line was also infected with Rubella virus and the production of virus was followed at the 5-liter cultivation level.     

Study of a madurella grisea strain isolated from a foot mycetoma

Madurella grisea has been isolated from a madura foot with black grains. The fungi classification was made based on the macro and micro-morphology characteristics of the culture. Difficulties with the interpretation of biochemistry tests were analized. The study is completed with trials of in vitro sensibility for different antifungic agents.     

Analysis of a case of somatic instability in a strain of maize from India

A case of somatic instability affecting aleurone colour in a strain of maize from India with flint background was analysed. The somatic instability is localized to theC ¹ (Inhibitor) allele ofC locus on the short arm of chromosome 9. Molecular tests indicated thatAc is not present in the Indian stock and the evidence is consistent with the involvement of theEn (Spm) transposable element in the instability. The presence of theEn (Spm)-like element in the stock would suggest that these elements have been present in the maize genome for a long time. A new allele ofshrunken (sh1) gene with a somewhat unorthodox breeding behaviour is also described.     

Living inside a sponge skeleton: the association of a sponge,a macroalga and a diatom

The relationships between sponges and macroalgae have been poorly investigated, especially in temperate waters. This work describes the consortium between the dictyoceratid sponge Dysidea pallescens and the red alga Acrochaetium spongicola permeating spongin fibres in the Mediterranean Sea; moreover, this is the first report of a diatom, Amphora pediculus, living also inside the spongin skeleton. The annual trend of the total algal biomass did not vary over time in relation to the temperature, irradiance and incorporation of foreign bodies. Our analyses, conducted by light and electron microscopy, suggest that both the macroalga and the diatom invade the skeleton of the sponge from the external environment, and that the benthic diatom, epiphyte of the macroalga, is passively carried inside the fibres through the growth of Acrochaetium spongicola. All the examined samples of D. pallescens showed that the macroalga permeated at least some fibres, while the presence of the diatom was occasional. The superficial layer of the sponge, thin and reticulate, likely allows the passage of the light and ensures the algal survival inside the sponge tissue. The high occurrence of the association with A. spongicola, together with the morphological adaptations of the sponge favouring the algal life, suggest that the relationship is mutualistic. The possible benefits for the involved partners are hypothesized. The taxonomy and ecology of endozoic Acrochaetiales are controversial due to the reduced size of thalli, the absence of peculiar diagnostic characters and unknown reproductive structures. Therefore, detailed studies on the relationships between the algae and their hosts will provide helpful information for the algal identification.     

The molecular structure of a complex of a 2,6-diimino-pyridine as a bidentate ligand with molybdenum carbonyl

Reaction of the pyridyl-diimine ligand, 2,6-[1- (2,2-dimethylpropanimino)ethyl]pyridine (1) with Mo(CO)₆ has unexpectedly yielded [Mo(CO)₄1], in which the normally tridentate ligand, even under forcing conditions, is bidentate. A single crystal X-ray structure determination reveals the distorted octahedral molecule with the MoC distances trans to the nitrogen donor atoms 0.1 Å shorter than the other two MoC distances. Other bond lengths suggest delocalization of charge in the chelate ring and this is reinforced by proton NMR data which suggests electron flow from the metal into the pyridine ring.     

Structural dynamics of a lytic peptide interacting with a supported lipid bilayer

The interaction of a melittin mutant with a 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)-supported lipid bilayer was studied with the use of time-resolved evanescent wave-induced fluorescence spectroscopy (TREWIFS) and evanescent wave-induced time-resolved fluorescence anisotropy measurements (EW-TRAMs). The mutant peptide was labeled at position K14 with AlexaFluor 430 and retained the lytic activity characteristic of native melittin. The fluorescence decay kinetics of the conjugate was found to be biexponential with a short-lived component, τ₁, due to photoinduced electron transfer between AlexaFluor 430 and proximal side chains within or between the peptides. The longer-lived component, τ₂, was sensitive to the polarity of the microenvironment at or near the K14 position of the peptide. Upon interaction with a DPPC-supported bilayer, the proportional contribution of τ₁ increased, indicating a conformational change of the peptide. The values of τ₁ and τ₂ indicate that the AlexaFluor 430 probe experienced an environment with an equivalent polarity no less than that of methanol. EW-TRAMs data from the melittin mutant revealed hindered rotational motions of the AlexaFluor 430 probe both in the plane and perpendicular to the plane of the supported lipid bilayer. The data indicate a highly ordered and polar environment near the center of the melittin helix consistent with the formation of a toroidal pore.