Purification and characterization of a 60-kDa protein from oat, formerly known as a TCP1-related chaperone

Recently, Mummertet al. [Nature 363, 644–648 (1993)] isolated a proposed TCP1-related chaperone. Here we report several findings concerning the protein which they sequenced. Two similar N-terminal sequences were obtained from this abundant 60-kDa protein. Internal sequences were also acquired by protease digestion. Initially it was believed the protein was able to completely inhibit citrate synthase aggregation, but later purifications demonstrated that the 60-kDa polypeptide lacked both chaperone activity and the previously reported kinase activity [Grimmet al., Planta 178, 199–206 (1989)]. It is now our belief that this protein is neither a chaperone nor a kinase.     

Materials for the fungus flora of Japan (48)

A coelomycetePolymorphum quercinum, newly found on the branches ofFagus crenata in Japan, is described.(47): Kobayashi, T. et al., Mycoscience 35: 399–401, 1994.     

Transinhibition and Voltage-gating in a Fungal Nitrate Transporter

We have applied enzyme kinetic analysis to electrophysiological steady-state data of Zhou et al. (Zhou, J.J., Trueman, L.J., Boorer, K.J., Theodoulou, F.L., Forde, B.G., Miller, A.J. 2000. A high-affinity fungal nitrate carrier with two transport mechanisms. J. Biol. Chem. 275:39894–9) and to new current-voltage-time records from Xenopus oocytes with functionally expressed NrtA (crnA) 2H⁺-NO ₃ ^– symporter from Emericella (Aspergillus) nidulans. Zhou et al. stressed two Michaelis-Menten (MM) mechanisms to mediate the observed nitrate-induced currents, I _{NO 3} ^– . We show that a single straightforward reaction cycle describes the data well, pointing out that during exposure to external substrate, S = (2H⁺+NO ₃ ^– )_o, the product concentration inside, [P] = [H⁺] _i ² · [NO ₃ ^– _i, may rise substantially near the plasma membrane, violating the condition [P] [S] for MM kinetics. Here, [P] and its changes during experimentation are treated explicitly. K _1/2 20 µM for I _{NO 3} ^– at pH_o from Zhou et al. is confirmed. According to our analysis, NrtA operates between about 0.2 and 0.6 of the electrical distance in the membrane (outside 0, inside 1). In absence of thermodynamic gradients, the predominant orientation of the binding site(s) is probably inwards. The activity of the enzyme is sensitive to the transmembrane voltage, V, with an apparent gating charge of +1.0 ± 0.5 for inactivation, and transition probabilities of 0.3–1.3 s^–1 at V = 0. This gating mode impedes loss of cellular NO ₃ ^– during depolarization.     

Materials for the fungus flora of Japan (47)

Albophoma yamanashiensis isolated from a forest soil in Yamanashi Prefecture is described as a new genus belonging to Nectrioidaceae, Coelomycetes.(46): Hosoya, T. et al., Trans. Mycol. Soc. Japan 34: 429–432, 1993.     

Heterologous gene expression in <Emphasis Type="Italic">Thermus thermophilus</Emphasis>: β-galactosidase,dibenzothiophene monooxygenase,PNB carboxy esterase, 2-aminobiphenyl-2,3-diol dioxygenase,and chloramphenicol acetyl transferase

Enzymes from thermophiles are preferred for industrial applications because they generally show improved tolerance to temperature, pressure, solvents, and pH as compared with enzymes from mesophiles. However, nearly all thermostable enzymes used in industrial applications or available commercially are produced as recombinant enzymes in mesophiles, typically Escherichia coli. The development of high-temperature bioprocesses, particularly those involving cofactor-requiring enzymes and/or multi-step enzymatic pathways, requires a thermophilic host. The extreme thermophile most amenable to genetic manipulation is Thermus thermophilus, but the study of expression of heterologous genes in T. thermophilus is in its infancy. While several heterologous genes have previously been expressed in T. thermophilus (Fridjonsson et al. in J Bacteriol 184:3385–3391, 2002, Koyama et al. in Appl Environ Microbiol 56:2251–225, 1990, Lasa et al. in J Bacteriol 174:6424–6431, 1992, Mathew et al. in Appl Environ Microbiol 58:421–425, 1992, Takagi et al. in J Ind Microbiol Biotechnol 23:214–217, 1999, Tamakoshi et al. in Extremophiles 5:17–22 2001), the data reported here include the first examples of the functional expression of a gene from an archaeal hyperthermophile (bglA from Pyrococcus woesei), a cofactor-requiring enzyme (dszC from Rhodococcus erythropolis IGTS8), and a two-component enzyme (carBa and carBb from Sphingomonas sp. GTIN11). A thermostable derivative of pnbA from Bacillus subtilis was also expressed, further expanding the list of genes from heterologous hosts that have been expressed in T. thermophilus.     

Efficient repetitive alteration of the mouse Huntington's disease gene by management of background in the tag and exchange gene targeting strategy

The introduction of subtle mutations to predetermined locations in the mouse genome has aided in the assessment of gene function and the precise modeling of inherited disorders. Subtle mutations can be engineered into the mouse genome by the tag and exchange gene targeting strategy (Askew et al., 1993; Stacey et al., 1994; Wu et al., 1994). This two-step method involves both a positive and a negative selection. The negative selection step typically generates a large amount of undesired background that may prevent the practical recovery of gene targeted clones (Vazquez et al., 1998). In this work we describe a strategy to effectively manage this background by calculation of a tolerable level of background for a specific targeting event, pre-screening for clones with low background, subcloning and growth of cell lines under selection. This strategy was used to repeatedly and efficiently alter the mouse Huntington's disease homologue (Hdh) resulting in an average of 15 percent of the clones having the desired modification. Analysis of the remaining background clones showed they arose de novo by a mechanism that involved physical loss of the marker rather than mutation or inactivation. We calculated the rate of loss of this marker as 8.3×10^–6 events/cell/generation. We further show that the exchanged clones retained the capacity to contribute to the mouse germline demonstrating the utility of this strategy in the production of mouse lines with Hdh variants.     

Statistics of canonical RNA pseudoknot structures

In this paper we study canonical RNA pseudoknot structures. We prove central limit theorems for the distributions of the arc-numbers of k-noncrossing RNA structures with given minimum stack-size τ over n nucleotides. Furthermore we compare the space of all canonical structures with canonical minimum free energy pseudoknot structures. Our results generalize the analysis of Schuster et al. obtained for RNA secondary structures [Hofacker, I.L., Schuster, P., Stadler, P.F., 1998. Combinatorics of RNA secondary structures. Discrete Appl. Math. 88, 207–237; Jin, E.Y., Reidys, C.M., 2007b. Central and local limit theorems for RNA structures. J. Theor. Biol. 250 (2008), 547–559; 2007a. Asymptotic enumeration of RNA structures with pseudoknots. Bull. Math. Biol., 70 (4), 951–970] to k-noncrossing RNA structures. Here k2 and τ are arbitrary natural numbers. We compare canonical pseudoknot structures to arbitrary structures and show that canonical pseudoknot structures exhibit significantly smaller exponential growth rates. We then compute the asymptotic distribution of their arc-numbers. Finally, we analyze how the minimum stack-size and crossing number factor into the distributions.     

A joint coordinate system proposal for the study of the trapeziometacarpal joint kinematics

The International Society of Biomechanics (ISB) has recommended a standardisation for the motion reporting of almost all human joints. This study proposes an adaptation for the trapeziometacarpal joint.

The definition of the segment coordinate system of both trapezium and first metacarpal is based on functional anatomy. The definition of the joint coordinate system (JCS) is guided by the two degrees of freedom of the joint, i.e. flexion–extension about a trapezium axis and abduction–adduction about a first metacarpal axis. The rotations obtained using three methods are compared on the same data: the fixed axes sequence proposed by Cooney et al., the mobile axes sequence proposed by the ISB and our alternative mobile axes sequence. The rotation amplitudes show a difference of 9° in flexion–extension, 2° in abduction–adduction and 13° in internal–external rotation.

This study emphasizes the importance of adapting the JCS to the functional anatomy of each particular joint.     

An evaluation of anatomical and functional knee axis definition in the context of side-cutting

Side-cutting is commonly used to evaluate knee joint kinematics and kinetics in the context of anterior cruciate ligament injury risk. Many existing side-cutting studies fail to clearly define the orientation of the femoral frame and the knee axis, making comparisons between studies difficult. A femoral frame constructed using the ISB or existing functional methods does not necessarily have a medial-lateral axis that is aligned with the axis of the knee. A functional frame that directly aligns with the medial-lateral knee axis was compared to the ISB anatomical frame and the Besier functional frame (Besier et al., 2003) to determine whether the chosen frame would affect the interpretation of side-cutting data. Kinematic and kinetic variables were calculated during three side-cutting manoeuvres of 28 subjects. Differences in mean frame orientation were correlated with the differences in mean knee angle during side-cutting. The differences between the ISB anatomical frame and the functional frames were significantly correlated with the differences in superior-inferior and medial-lateral axis orientations. Coefficients of multiple correlation showed a good to high (CMCs≥0.74) similarity between frames for knee angles and moments. Using a femoral anatomical frame rather than a functional frame most significantly affected offset rather than cross talk in knee angles and moments measured during side-cutting. There were no significant differences in offset or cross talk between the two functional methods. Maximum differences of <4° for frontal plane knee angle requires cautious interpretation but differences <8Nm for knee joint moment were not thought to affect the interpretation of side-cutting data when comparing between studies.     

A joint coordinate system proposal for the study of the trapeziometacarpal joint kinematics

The International Society of Biomechanics (ISB) has recommended a standardisation for the motion reporting of almost all human joints. This study proposes an adaptation for the trapeziometacarpal joint. The definition of the segment coordinate system of both trapezium and first metacarpal is based on functional anatomy. The definition of the joint coordinate system (JCS) is guided by the two degrees of freedom of the joint, i.e. flexion-extension about a trapezium axis and abduction-adduction about a first metacarpal axis. The rotations obtained using three methods are compared on the same data: the fixed axes sequence proposed by Cooney et al., the mobile axes sequence proposed by the ISB and our alternative mobile axes sequence. The rotation amplitudes show a difference of 9 degrees in flexion-extension, 2 degrees in abduction-adduction and 13 degrees in internal-external rotation. This study emphasizes the importance of adapting the JCS to the functional anatomy of each particular joint.     

Morpho-histological study of somatic embryo-like structures in hypocotyl cultures of<Emphasis Type="Italic"> Pelargonium</Emphasis> ×<Emphasis Type="Italic"> hortorum</Emphasis> Bailey

Somatic embryo-like structures were produced from the hypocotyls of ten cultivars of Pelargonium × hortorum using the protocols of Marsolais et al. (1991; Can J Bot 69:1188–1193) and Slimmon et al. (1991; Plant Cell Rep 10:587–589) and their embryonic natures evaluated. Nine cultivars responded, and 937 structures were formed. Regeneration corresponded well with published data. The somatic embryo-like structures were globular- to leaf-shaped or similar to shoots. A root pole was never visible. Histological examinations confirmed the lack of bipolarity and revealed vascular connections to the explant in the more developed structures. Therefore, these structures cannot be classified as somatic embryos. The importance of these results is discussed in terms of evaluating published protocols for the propagation of these pelargoniums by somatic embryogenesis from hypocotyls.Communicated by H. Lörz     

Pregnancy-associated changes in oligomannose oligosaccharides of human and bovine uromodulin (Tamm-Horsfall glycoprotein)

The urinary glycoprotein uromodulin (Tamm-Horsfall glycoprotein) exhibits a pregnancy-associated ability to inhibit antigen-specific T cell proliferation, and the activity is associated with a carbohydrate moiety [Muchmore and Decker (1985) Science 229:479–81; Hessionet al., (1987) Science 237:1479–84; Muchmore, Shifrin and Decker (1987) J Immunol 138:2547–53]. We report here that the Man₆₍₇₎GlcNAc₂-R glycopeptides derived from uromodulin inhibit antigen-specific T cell proliferation by 50% at 0.2–2 M, and further studies, reported elsewhere, confirm that oligomannose glycopeptides from other sources are also inhibitory, with Man₉GlcNAc₂-R the most inhibitory of those tested [Muchmoreet al., J Leukocyte Biol (in press)]. In this work, we have extended the observation of pregnancy-associated inhibitory activity to a second species, and have compared the oligomannose profile of Tamm-Horsfall glycoprotein (nonpregnant) with that of uromodulin (pregnant) derived from both human and bovine sources. Surprisingly, there was a pregnancy-associated decrease in the total content of oligomannose chains due predominantly to a reduction in Man₅GlcNAc₂-R and Man₆GlcNAc₂-R. Man₇GlcNAc₂-R, which did not decrease with pregnancy, comprised a significantly greater proportion of the total oligomannose chains in pregnant vs. nonpregnant samples from both species (human; 34.6% vs. 25.9%: bovine; 14.4% vs. 7.2%).     

Increased expression of a hemimethylated oriC binding protein, SeqA, in an aphA mutant

In Escherichia coli, the origin of DNA replication, oriC, becomes transiently hemimethylated at the GATC sequences immediately after initiation of replication and this hemimethylated state is prolonged because of its sequestration by a fraction of outer membrane. This sequestration is dependent on a hemimethylated oriC binding protein such as SeqA. We previously isolated a clone of phage λgtll called hobH, producing a LacZ fusion protein which recognizes hemimethylated oriC DNA. Very recently, Thaller et al. (FEMS Microbiol. Lett. 146 (1997)191–198)found that the same DNA segment encodes a non-specific acid phosphatase, and named the gene aphA. We show here that the interruption of the aphA reading frame by kanamycin resistance gene insertion, abolishes acid phosphatase (NAP) activity. Interestingly, in the membrane of the null mutant, the amount of SeqA protein is about six times higher than that in the parental strain, suggesting the existence of a regulatory mechanism between SeqA and NAP expression.     

An Investigation of the Immediate Effect of Static Stretching on the Morphology and Stiffness of Achilles Tendon in Dominant and Non-Dominant Legs

AimsThis study was undertaken to investigate the immediate effect of static stretching on normal Achilles tendon morphology and stiffness, and the different effect on dominant and non-dominant legs; and to evaluate inter-operator and intra-operator reliability of using shear-wave elastography in measuring Achilles tendon stiffness.Methods20 healthy subjects (13 males, 7 females) were included in the study. Thickness, cross-sectional area and stiffness of Achilles tendons in both legs were measured before and after 5-min static stretching using grey-scale ultrasound and shear-wave elastography. Inter-operator and intra-operator reliability of tendon stiffness measurements of six operators were evaluated.ResultsResult showed that there was no significant change in the thickness and cross-sectional area of Achilles tendon after static stretching in both dominant and non-dominant legs (p > 0.05). Tendon stiffness showed a significant increase in non-dominant leg (p < 0.05) but not in dominant leg (p > 0.05). The inter-operator reliability of shear-wave elastography measurements was 0.749 and the intra-operator reliability ranged from 0.751 to 0.941.ConclusionShear-wave elastography is a useful and non-invasive imaging tool to assess the immediate stiffness change of Achilles tendon in response to static stretching with high intra-operator and inter-operator reliability.     

Immunogold localization of acyl carrier protein in plants and Escherichia coli: Evidence for membrane association in plants

Immunogold labelling was used to study the distribution of acyl carrier protein (ACP) in Escherichia coli and a variety of plant tissues. In E. coli, ACP is distributed throughout the cytoplasm, confirming the observation of S. Jackowski et al. (1985, J. Bacteriol., 162, 5–8_. In the mesocarp of Avocado (Persea americana) and maturing seeds of oil-seed rape (Brassica napus cv. Jet Neuf), over 95% of the ACP is localised to plastids. The protein is almost exclusively located in the chloroplasts of leaf material from oil-seed rape. Approximately 80% of the gold particles associated with the ACP were further localized to the thylakoid membrane of the chloroplast. Since acetyl-CoA carboxylase has been reported to be localized to the thylakoid membrane (C.G. Kannangara and C.J. Jensen, 1975, Eur. J. Biochem., 54, 25–30), these results are consistent with the view that the two sequential enzymes in fatty-acid synthesis are in close spacial proximity.Abbreviations ACC acetyl CoA carboxylase - ACP acyl carrier protein - FAS fatty-acid synthetase     

Net nitrogen mineralization in soils under six indigenous tree species,an abandoned pasture and a secondary forest in the atlantic lowlands of costa rica

Nitrogen mineralization, nitrification potentials, pH, total N, C, extractable P and cations were measured in soils under 4-year-old, mono-specific stands of six fast-growing, native tree species, an abandoned pasture, and a 20-year-old secondary forest, as part of a study on the use of indigenous tree species for rehabilitation of soil fertility on degraded pastures at the La Selva Biological Station in the Atlantic humid lowlands of Costa Rica. Soil net nitrification potential rates were higher under two N-fixing, leguminous species,Stryphnodendron microstachyum Poepp. et Endl. (1.1–1.9 mg kg^–1 day^–1) andDalbergia tucurensis Donn. Smith (0.7–1.5 mg kg^–1 day^–1), than under the non-N-fixing trees in the plantation,Vochysia guatemalesis Don. Sm.,Vochysia ferruginea Mart,Dipteryx panamensis (Pittier) Record and Mell andHyeronima alchorneoides Fr. Allemao (0.2–0.8 mg kg^–1 day^–1). Values under the N-fixing trees were comparable to those found in secondary forest. There were no statistically significant differences in soil total N or in other nurtients between the species. Results of pH measurements done before and after incubation did not show any clear evidence of a pH drop attributable to nitrification.     

Activation of flavonoid biosynthesis in roots of Vicia sativa subsp. nigra plants by inoculation with Rhizobium leguminosarum biovar viciae

Infective (nodulating) Rhizobium leguminosarum biovar viciae (R.l. viciae) bacteria release Nod factors which stimulate the release of nodulation gene-inducing flavanones and chalcones from roots of the host plant Vicia sativa subsp. nigra (K. Recourt et al., Plant Mol Biol 16: 841–852; H.P. Spaink et al., Nature 354: 125–130). The hypothesis that this release results from increased synthesis of flavonoids was tested by studying the effect of inoculation of V. sativa with infective and uninfective R.l. viciae bacteria on (i) activity of L-phenylalanine ammonia-lyase, (ii) level of chalcone synthase mRNA, and (iii) activity of (eriodictyol) methyltransferase in roots. Consistent with the hypothesis, each of these parameters was found to increase 1.5 to 2-fold upon inoculation with infective R.l. viciae bacteria relative to the situation for uninoculated roots and for roots inoculated with uninfective rhizobia.     

Reproducible reference frame for in vitro testing of the human vertebrae

Definition of an anatomical reference frame is necessary for in vitro biomechanical testing. Nevertheless, there is neither a clear recommendation, nor consensus in the literature concerning an anatomical reference frame for in vitro testing of the human vertebrae. The scope of this work is to define a reference frame for the human vertebrae for in vitro applications. The proposed anatomical reference frame relies on alignment of well-defined points on the endplates, and on two landmarks on the posterior wall. The repeatability of the proposed alignment procedure has been tested in vitro by 5 operators, on 7 specimens. Furthermore, the feasibility and repeatability of the proposed procedure was assessed in silico, using CT-scans of the same specimens.     

Sensor-to-body calibration procedure for clinical motion analysis of lower limb using magnetic and inertial measurement units

Magnetic and Inertial measurement units (MIMUs) have become exceedingly popular for ambulatory human motion analysis during the past two decades. However, measuring anatomically meaningful segment and joint kinematics requires virtual alignment of the MIMU frame with the anatomical frame of its corresponding segment. Therefore, this paper presents a simple calibration procedure, based on MIMU readouts, to align the inertial frame of the MIMU with the anatomical frames, as recommended by ISB. The proposed calibration includes five seconds of quiet standing in a neutral posture followed by ten consecutive hip flexions/extensions. This procedure will independently calibrate MIMUs attached to the pelvis, thigh, shank, and foot. The accuracy and repeatability of the calibration procedure and the 3D joint angle estimation were validated against the gold standard motion capture system by an experimental study with ten able-bodied participants. The procedure showed high test-retest repeatability in aligning the MIMU frame with its corresponding anatomical frame, i.e., the helical angle between the MIMU and anatomical frames did not significantly differ between the test and retest sessions (except for thigh MIMU). Compared to previously introduced procedures, this procedure attained the highest inter-participant repeatability (inter-participant coefficient of variations of the helical angle: 20.5–42.2%). Further, the proposed calibration would reduce the offset errors of the 3D joint angle estimation (up to 12.8 degrees on average) compared to joint angle estimation without calibration (up to 26.3 degrees on average). The proposed calibration enables MIMU to measure clinically meaningful gait kinematics.