Respiratory responses to long-term temperature exposure in the box turtle,Terrapene ornata

Summary In late February, seven box turtles were collected with body temperatures between 7 and 9°C. Ventilation, gas exchange and end-tidal and were recorded at 5, 10, 15 and 25°C, the animals being at each temperature for 2 to 3 weeks. There was a pronounced diurnal rhythm of breathing frequency at all temperatures. At 5°C the mean 24-h frequency was only 3.7 breaths h^–1. At 15°C the frequency was 16 times higher with a 17-fold increase of ventilation. Oxygen uptake only changed from 3.4 to 12.7 ml·kg^–1·h^–1. Consequently, the ratio (ventilation, ml BTPS/O₂ uptake, ml STPD) increased from 12.5 at 5°C to 48 at 15°C, but decreased to 24 at 25°C. The decrease of this ratio during cold exposure contrasts with an increase of the ratio during cooling earlier reported for fresh water turtles,Pseudemys. Cutaneous CO₂ elimination was important at low temperature. This caused a decrease of the pulmonary gas exchange ratio so that end-tidal remained low at 5°C in spite of an end-tidal of only 54 Torr.     

Dynein at the nuclear envelope

Most cellular organelles are positioned through active transport by motor proteins. The authors discuss the evidence that dynein has important cell cycle-regulated functions in this context at the nuclear envelope.Most cellular organelles are positioned through active transport by motor proteins. This is especially important during cell division, a time when the organelles and genetic content need to be divided equally between the two daughter cells. Although individual proteins can attain their correct location by diffusion, larger structures are usually positioned through active transport by motor proteins. The main motor that transports cargoes to the minus ends of the microtubules is dynein. In nondividing cells, dynein probably transports or positions the nucleus inside the cells by binding to the nuclear envelope (NE; Burke & Roux, 2009). However, it appears that dynein also has important cell-cycle-regulated functions at the NE, as it is recruited to the NE every cell cycle just before cells enter mitosis (Salina et al, 2002; Splinter et al, 2010). Here, we discuss why dynein might be recruited to the NE for a brief period before mitosis.During late G2 or prophase the centrosomes separate to opposite sides of the nucleus, but remain closely associated with the NE during separation. This close association is probably mediated through NE-bound dynein, which ‘walks'' towards the minus ends of centrosomal microtubules, thereby pulling centrosomes towards the NE (Splinter et al, 2010; Gonczy et al, 1999; Robinson et al, 1999). We speculate that close association of centrosomes to the NE might have several functions. First, if centrosomes are not mechanically coupled to the NE, centrosome movement during separation will occur in random directions and chromosomes will not end up between the two separated centrosomes. In this scenario, individual kinetochores might attach more frequently to microtubules coming from both centrosomes (merotelic attachments), a defect that can result in aneuploidy, a characteristic of cancer. Second, centrosome-nuclear attachment also keeps centrosomes in close proximity to chromosomes, which might facilitate rapid capture of chromosomes by microtubules nucleated by the centrosomes after NE breakdown. This might not be absolutely essential, as chromosome alignment can occur in the absence of centrosomes. However, the spatial proximity of centrosomes and chromosomes at NE breakdown might improve the fidelity of kinetochore capture and chromosome alignment.In addition, dynein has also been suggested to promote centrosome separation in prophase in some systems (Gonczy et al, 1999; Robinson et al, 1999; Vaisberg et al, 1993), although not in others (Tanenbaum et al, 2008). Perhaps dynein, anchored at the NE just before mitosis, could exert force on microtubules emanating from both centrosomes, thereby pulling centrosomes apart. However, this force could also be produced by cortical dynein and specific inhibition of NE-associated or cortical dynein will be required to test which pool is responsible.Dynein has also been implicated in the process of NE breakdown itself, by promoting mechanical shearing of the NE. Two elegant studies showed that microtubules can tear the NE as cells enter mitosis (Salina et al, 2002; Beaudouin et al, 2002). One possibility is that microtubules growing into the NE mechanically disrupt it. Alternatively, NE-associated dynein might ‘walk'' along centrosomal microtubules and thereby pull on the NE, tearing it apart. However, testing the exact role of dynein in NE breakdown is complicated by the fact that centrosomes detach from the NE on inactivation of dynein and centrosomal microtubules stop growing efficiently into the NE. Thus, selective inhibition of dynein function will also be required to test this idea.Specific recruitment of dynein to the NE just before mitosis clearly suggests a role for dynein at the NE in preparing cells for mitosis. A major role of NE-associated dynein is to maintain close association of centrosomes with the NE during centrosome separation, which might be needed for efficient capture and alignment of chromosomes after NE breakdown, but additionally, NE-associated dynein could facilitate breakdown and contribute to centrosome separation in some systems.     

Single millimeter wave treatment does not impair gastrointestinal transit in mice

Millimeter wave treatment (MWT) is based on those biological effects that develop following skin exposure to low power electromagnetic waves. This method of treatment is in wide clinical use in several Eastern European countries for treatment of a variety of conditions, including pain syndromes. However, most treatment modes of MWT were developed empirically, and certain indications and contraindications for the use of MWT remain to be established. In our previous blind experiments we have shown that the hypoalgesic effect of MWT may be quantitatively evaluated, and most probably mediated by the neural system in general, and the system of endogenous opioids in particular. Taking in consideration a well-known ability of opioids to cause gastrointestinal disturbances, which could limit clinical application of MWT, the main aim of the present study was to investigate whether a single MWT, that can produce opioid-related hypoalgesia, may also retard gut transit and colorectal passage in mice. The charcoal meal test was used to quantitatively evaluate upper gastrointestinal transit, and the glass bead test was employed to examine colonic propulsion in mice. MWT was applied to the nose area of mice. The MWT characteristics were: frequency = 61.22 GHz; incident power density = 15 mW/cm(2); and duration = 15 min. The results obtained have shown that MWT does not significantly change small intestinal or colonic transit in mice, and thus suppression of gastrointestinal motility should not be a setback in the clinical use of MWT.     

Analyzing mRNA-protein complexes using a yeast three-hybrid system

RNA-protein interactions are essential for the proper execution and regulation of every step in the life of a eukaryotic mRNA. Here we describe a three-hybrid system in which RNA-protein interactions can be analyzed using simple phenotypic or enzymatic assays in Saccharomyces cerevisiae. The system can be used to detect or confirm an RNA-protein interaction, to analyze RNA-protein interactions genetically, and to discover new protein or RNA partners when only one is known. Multicomponent complexes containing more than one protein can be detected, identified, and analyzed. We describe the method and how to use it, and discuss applications that bear particularly on eukaryotic mRNAs.     

The use and abuse of commercial kits used to detect autoantibodies

The detection of autoantibodies in human sera is an important approach to the diagnosis and management of patients with autoimmune conditions. To meet market demands, manufacturers have developed a wide variety of easy to use and cost-effective diagnostic kits that are designed to detect a variety of human serum autoantibodies. A number of studies over the past two decades have suggested that there are limitations and concerns in the use and clinical application of test results derived from commercial kits. It is important to appreciate that there is a complex chain of users and circumstances that contributes to variations in the apparent reliability of commercial kits. The goal of this review is to identify the principal links in this chain, to identify the factors that weaken the chain and to propose a plan of resolution. It is suggested that a higher level of commitment and partnership between all of the participants is required to achieve the goal of improving the quality of patient care through the use of autoantibody testing and analysis.     

Low power millimeter wave irradiation exerts no harmful effect on human keratinocytes in vitro

Low power millimeter wave (LP-MW) irradiation has been successfully used in clinical practice as an independent and/or supplemental therapy in patients with various diseases. It is still not clear, however, whether exposed skin is directly affected by repeated LP-MW irradiation and whether cells of the epidermis can be activated by the absorbed energy. Keratinocytes, the most numerous component of the epidermis are believed to manifest functional responses to physical stimuli. In this study we analyzed whether LP-MW irradiation modulated the production of chemokines, including RANTES and IP-10 of keratinocytes in vitro. We also investigated whether LP-MW irradiation induces a heat stress reaction in keratinocytes, and stimulates heat shock protein 70 (Hsp70) production. Vital staining of keratinocytes with carboxyfluorescein succinimidyl ester and ethidium bromide was used to analyze the MW effect on the viability of adherent cells. In addition, we studied the effect of LP-MW irradiation on intercellular gap junctional communication in keratinocyte monolayers by Lucifer yellow dye transfer. We found no significant changes in constitutive RANTES and inducible IP-10 production following LP-MW irradiation. LP-MW exposure of keratinocyte monolayers did not alter Hsp70 production, unlike exposure to higher power MWs (HP-MW) or hyperthermia (43 degrees C; 1 h). LP-MW irradiation and hyperthermia did not alter the viability of adherent keratinocytes, while HP-MW irradiation induced cellular damage within the beam area. Finally, we found no alteration in the gap junctional intercellular communication of keratinocytes following LP-MW irradiation, which on the other hand, was significantly increased by hyperthermia. In summary, we detected no harmful effect of LP-MW irradiation on both keratinocyte function and structure in vitro, although these cells were sensitive to higher MW power that developed heat stress reaction and cellular damage. Our results provide further evidence that LP-MW irradiation does not induce evidence of skin inflammation or keratinocyte damage and that its clinical application appears to be safe.     

Comparison of 15N- and 13C-determined parameters of mobility in melittin

Backbone and tryptophan side-chain mobilities in the 26-residue, cytolytic peptide melittin (MLT) were investigated by ¹⁵N and ¹³C NMR. Specifically, inverse-detected ¹⁵N T₁ and steady-state NOE measurements were made at 30 and 51 MHz on MLT at 22 °C enriched with ¹⁵N at six amide positions and in the Trp¹⁹ side chain. Both the disordered MLT monomer (1.2 mM peptide at pH 3.6 in neat water) and -helical MLT tetramer (4.0 mM peptide at pH 5.2 in 150 mM phosphate buffer) were examined. The relaxation data were analyzed in terms of the Lipari and Szabo model-free formalism with three parameters: _m, the correlation time for the overall rotation; S², a site-specific order parameter which is a measure of the amplitude of the internal motion; and _e, a local, effective correlation time of the internal motion. A comparison was made of motional parameters from the ¹⁵N measurements and from ¹³C measurements on MLT, the latter having been made here and previously [Kemple et al. (1997) Biochemistry, 36, 1678–1688]. _m and _e values were consistent from data on the two nuclei. In the MLT monomer, S² values for the backbone N-H and C-H vectors in the same residue were similar in value but in the tetramer the N-H order parameters were about 0.2 units larger than the C-H order parameters. The Trp side-chain N-H and C-H order parameters, and _e values were generally similar in both the monomer and tetramer. Implications of these results regarding the dynamics of MLT are examined.     

Stress‐responsive accumulation of plastid chaperonin 60 during seedling development

Plastid chaperonin 60 (cpn60) is a chloroplast protein, presumed to assist in assembly and folding of plastid proteins. Although molecular chaperones often accumulate significantly in response to stress, this has never been demonstrated for cpn60. In this study, the accumulation of cpn60 in Nicotiana seedlings during their development was followed under different stress conditions. It was found that cpn60 accumulates markedly in developing seedlings in response to tentoxin and several other (but not all) stresses. Cpn60 accumulates only during a narrow period of seedling development. It is proposed that cpn60 accumulation under stress is developmentally regulated.     

Autoantibodies to components of the mitotic apparatus

Autoantibodies directed to a variety of cellular antigens and organelles are a feature of autoimmune diseases. They have proven useful in a clinical setting to establish diagnosis, estimate prognosis, follow disease progression, alter therapy, and initiate new investigations. Cellular and molecular biologists have used autoantibodies as probes to identify molecules involved in key cellular processes. One of the most interesting sets of autoantibodies are those that target antigens within the mitotic apparatus (MA). The MA includes chromosomes, spindle microtubules and centrosomes. The identification, localization, function, and clinical relevance of MA autoantigens is the focus of this review. Abbreviations: ATP – adenosine triphosphate; CENP – centromere protein; CREST – calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia; HMG – high mobility group; IB – intercellular bridge; IIF – indirect immunofluorescence; MAPs – microtubule associated proteins; NuMA – nuclear mitotic apparatus; NOR – nucleolar organizer; PBC – primary biliary cirrhosis; PM – polymyositis; Pol I, II, III – RNA polymerases; RA-rheumatoid arthritis; SLE – systemic lupus erythematosus; SS – Sjögren's syndrome; SSc – systemic sclerosis; topo – topoisomerase.     

A three-hybrid screen identifies mRNAs controlled by a regulatory protein

RNA-protein interactions are important in many biological contexts. Identification of the networks that connect regulatory proteins to one another and to the mRNAs they control is a critical need. Here, we use a yeast three-hybrid screening approach to identify RNAs that bind a known RNA regulatory protein, the Saccharomyces cerevisiae PUF protein, Mpt5p. The assay selects RNAs that bind in vivo using simple phenotypes and reporter genes. It enables rapid analyses of the affinity and specificity of the interaction. We show that the method identifies mRNAs that are genuinely regulated by the protein in vivo, and that it complements biochemical strategies, yielding a set of mRNAs that overlap with, but are distinct from, those obtained by biochemical means. The approach we describe facilitates construction of protein-RNA linkage maps.