Guide oligonucleotide-dependent DNA linkage that facilitates controllable polymerization of microgene blocks

Faster and more efficient searches of a huge protein sequence space for the purpose of conducting experiments in protein evolution can be achieved through the development of a block shuffling-based evolution system. One of the key components of such a system is the accurate and efficient linkage of gene units. Here we introduce a new method that allows accurate and controllable linkage of microgene blocks. This method employs a thermostable DNA ligase that links two single-stranded microgene blocks when they hybridize a complementary guide oligonucleotide. At high temperature, the ligation of the microgene units is fully dependent on the guide oligonucleotide, which can exclude undesired polymer formation, including the incorporation of microgenes having illegitimate sizes and "head-to-head" and "tail-to-tail" ligation of blocks. We were also able to assemble three microgene units using two guide oligonucleotides. Using this method of controllable linkage should facilitate further development of a step-by-step system for the polymerization of gene blocks, leading to a versatile block shuffling-based protein evolution system.     

Molecular characterization of a novel beta1,3-galactosyltransferase for capsular polysaccharide synthesis by Streptococcus agalactiae type Ib

A group B streptococcus, Streptococcus agalactiae type Ib, produces a high-molecular-weight polysaccharide consisting of the following pentasaccharide repeating unit: -->4)-[alpha-D-NeupNAc-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. The type-specific capsular polysaccharide (CP) synthesis (cps) genes of this strain were cloned and analyzed. A cloned 10-kb DNA fragment contained cpsIbE to L and neu (neuraminic acid synthesis gene) B. Comparison of the gene products with those of S. agalactiae type Ia, which has a similar but distinct CP, showed that the translation products of cpsIa and cpsIb genes exhibited very high homology except for those of cpsJ and K. In the type Ia strain, cpsIaJ encodes beta1,4-galactosyltransferase, which catalyzes the transfer of galactose as the fourth monosaccharide of the sugar repeating unit. In the type Ib CP, this galactose forms a beta1,3-linkage to GlcNAc. The low homology between the type Ia and Ib CpsJs seems to reflect this difference. By enzymatic activity measurement, the cpsIbJ product was found to display beta1,3-galactosyltransferase activity. Furthermore, hydrophobic cluster analysis clarified the similarities and differences of the structures in N-terminal regions, including the DXD motif, between the galactosyltransferases.     

Identification and characterization of the UL56 gene product of herpes simplex virus type 2

The UL56 gene product of herpes simplex virus (HSV) has been shown to play an important role in viral pathogenicity. However, the properties and functions of the UL56 protein are little understood. We raised rabbit polyclonal antisera specific for the UL56 protein of HSV type 2 (HSV-2) and examined its expression and properties. The gene product was identified as three polypeptides with apparent molecular masses ranging from 32 to 35 kDa in HSV-2-infected cells, and at least one species was phosphorylated. Studies of their origins showed that the UL56 protein of HSV-2 is also translated from the upstream in-frame methionine codon that is not present in the HSV-1 genome. Synthesis was first detected at 6 h postinfection and was not abolished by the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies revealed that the UL56 protein localized to both the Golgi apparatus and cytoplasmic vesicles in HSV-2-infected and single UL56-expressing cells. Deletion mutant analysis showed that the C-terminal hydrophobic region of the protein was required for association with the cytoplasmic membrane and that the N-terminal proline-rich region was important for its translocation to the Golgi apparatus and cytoplasmic vesicles. Moreover, the results of protease digestion assays and sucrose gradient fractionation strongly suggested that UL56 is a tail-anchored type II membrane protein associated with lipid rafts. We thus hypothesized that the UL56 protein, as a tail-anchored type II membrane protein, may be involved in vesicular trafficking in HSV-2-infected cells.     

New estimation method for fatty acid composition in oil using near infrared spectroscopy

The absorption bands of cis-unsaturation and the carbon chain length of the fatty acid moieties in oil appear in the near infrared (NIR) wavelength region, especially around 1600-1800 nm. Using this region, a new estimation method for fatty acid composition analysis is proposed. Because the differences of the original NIR spectra are miniscule even in this region, the second derivative NIR spectra were examined in order to estimate the fatty acid composition in oil exclusively from the spectral patterns obtained. The parameters for calculating the second derivative NIR spectra were examined to make the spectral difference clearer. In any parameter, the absorption band was shifted to the shorter wavelength region when the unsaturation in fatty acid moieties increased, and it was shifted to the longer wavelength region when the carbon chain length increased. When the parameters were correct, this NIR method can estimate the fatty acid composition roughly, but simply, easily, and sometimes nondestructively.     

Molecular dissection of Salmonella FliH,a regulator of the ATPase FliI and the type III flagellar protein export pathway

FliH is a soluble component of the flagellar export apparatus that binds to the ATPase FliI, and negatively regulates its activity. The 235-amino-acid FliH dimerizes and interacts with FliI to form a hetero-trimeric (FliH)2FliI complex. In the present work, the importance of different regions of FliH was examined. A set of 24 scanning deletions of 10 amino acids was constructed over the entire FliH sequence, along with several combined deletions of 40 amino acids and truncations of both N- and C-termini. The mutant proteins were examined with respect to (i) complementation; (ii) dominance and multicopy effects; (iii) interaction with wild-type FliH; (iv) interaction with FliI; (v) inhibition of the ATPase activity of FliI; and (vi) interaction with the putative general chaperone FliJ. Analysis of the deletion mutants revealed a clear functional demarcation between the FliH N- and C-terminal regions. The 10-amino-acid deletions throughout most of the N-terminal half of the sequence complemented and were not dominant, whereas those throughout most of the C-terminal half did not complement and were dominant. FliI binding was disrupted by C-terminal deletions from residue 101 onwards, indicating that the C-terminal domain of FliH is essential for interaction with FliI. FliH dimerization was abolished by deletion of residues 101-140 in the centre of the sequence, as were complementation, dominance and interaction with FliI and FliJ. The importance of this region was confirmed by the fact that fragment FliHC2 (residues 99-235) interacted with FliH and FliI, whereas fragment FliHC1 (residues 119-235) did not. FliHC2 formed a relatively unstable complex with FliI and showed biphasic regulation of ATPase activity, suggesting that the FliH N-terminus stabilizes the (FliH)2FliI complex. Several of the N-terminal deletions tested permitted close to normal ATPase activity of FliI. Deletion of the last five residues of FliH caused a fivefold activation of ATPase activity, suggesting that this region of FliH governs a switch between repression and activation of FliI. Deletion of the first 10 residues of FliH abolished complementation, severely reduced its interaction with FliJ and uncoupled its role as a FliI repressor from its other export functions. Based on these data, a model is presented for the domain construction and function of FliH in complex with FliI and FliJ.     

Variability of ground reaction forces during treadmill walking.

The purpose of this study was to investigate whether or not the neuromuscular locomotor system is optimized at a unique speed by examining the variability of the ground reaction force (GRF) pattern during walking in relation to different constant speeds. Ten healthy male subjects were required to walk on a treadmill at 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 km/h. Three components [vertical (F(z)), anteroposterior (F(y)), and mediolateral (F(x)) force] of the GRF were independently measured for approximately 35 steps consecutively for each leg. To quantify the GRF pattern, five indexes (first and second peaks of F(z), first and second peaks of F(y), and F(x) peak) were defined. Coefficients of variation were calculated for these five indexes to evaluate the GRF variability for each walking speed. It became clear for first and second peaks of F(z) and F(x) peak that index variabilities increased in relation to increments in walking speed, whereas there was a speed (5.5-5.8 km/h) at which variability was minimum for first and second peaks of F(y), which were related to forward propulsion of the body. These results suggest that there is "an optimum speed" for the neuromuscular locomotor system but only for the propulsion control mechanism.     

Mechanomyogram and electromyogram responses of upper limb during sustained isometric fatigue with varying shoulder and elbow postures

To investigate the behavior of mechanomyogram (MMG) and electromyogram (EMG) signals in the time and frequency domains during sustained isometric contraction, MMG and surface EMG were obtained simultaneously from four muscles: upper trapezius (TP), anterior deltoid (DL), biceps brachii (BB), and brachioradialis (BR) of 10 healthy male subjects. Experimental conditions consisted of 27 combinations of 9 postures [3 shoulder angles (SA): 0 degree, 30 degrees, 60 degrees and 3 elbow angles (EA): 120 degrees, 90 degrees, 60 degrees] and 3 contraction levels: 20%, 40%, and 60% of maximum voluntary contraction (MVC). Subjective evaluations of fatigue were also assessed using the Borg scale at intervals of 60, 30, and 10 sec at 20%, 40%, and 60% MVC tests, respectively. The mean power frequency (MPF) and root mean square (RMS) of both signals were calculated. The current study found clear and significant relationships among physiological and psychological parameters on the one hand and SA and EA on the other. EA's effect on MVC was found to be significant. SA had a highly significant effect on both endurance time and Borg scale. In all experimental conditions, significant correlations were found between the changes in MPF and RMS of EMG in BB with SA and EA (or muscle length). In all four muscles, MMG frequency content was two or three times lower than EMG frequency content. During sustained isometric contraction, the EMG signal showed the well-known shift to lower frequencies (a continuous decrease from onset to completion of the contraction). In contrast, the MMG spectra did not show any shift, although its form changed (generally remaining about constant). Throughout the contraction, increased RMS of EMG was found for all tests, whereas in the MMG signal, a significant progressive increase in RMS was observed only at 20% MVC in all four muscles. This supports the hypothesis that the RMS amplitude of the MMG signal produced during contraction is highly correlated with force production. Possible explanations for this behavioral difference between the MMG and EMG signals are discussed.     

Detection of underlying characteristics of nuclear chromatin patterns of thyroid tumor cells using texture and factor analyses

BACKGROUND: Aspiration biopsy cytology of thyroid tumors has been used more frequently in recent times to differentiate between malignant and benign lesions. Chromatin patterns of the tumor cell nuclei are one of most important factors for cytologic diagnosis. The interpretation of nuclear chromatin patterns is subjective and more difficult than that of nuclear size or shape. In the present report, we investigated how to detect underlying chromatin characteristics of benign and malignant thyroid tumor cells by means of texture and factor analyses. METHODS: We employed a computer-aided system in which light microscopy was combined with an image processor and monochrome camera. Using this system, 100 randomly selected cells in a Papanicolaou stained, aspiration biopsy cytologic smear in each case of 39 benign and malignant thyroid tumor cases were digitized. We applied two-dimensional and higher texture analyses with the use of co-occurrence and run-length matrices to analyze the chromatin patterns. Factor analysis was used to determine whether a large number of independent variables actually measured one or more underlying common variables. RESULTS: According to parameters with high factor-loading values, the morphologic chromatin characters were classified into three categories according to heterogeneity, contrast, and homogeneity of chromatin patterns. On the basis of analyses with these morphologic categories, nuclei of papillary carcinoma showed higher contrast of chromatin patterns than did those of the benign group. Moreover, there was a variety of contrasting chromatin patterns among cells in each papillary carcinoma case in comparison with the benign group. In contrast, follicular carcinomas showed a significant difference in the standard deviation of factor 3, which indicated more monotonous chromatin patterns among cells in each follicular carcinoma case than in each benign case. CONCLUSION: We believe that this technique, using texture and factor analyses, is useful in the detection of underlying characteristics of nuclear chromatin patterns in aspiration biopsy cytology.     

Feedback regulation of pancreatic secretion by peptide YY

The present status of our understanding of the feedback regulation of pancreatic secretion by peptide YY (PYY) released from the distal intestine is reviewed. Exocrine pancreatic secretion is primarily controlled by the cephalic (the vagus nerve), gastric (acid and pepsin secretion, and nutrients delivered into the duodenum by gastric emptying), and intestinal (secretin and CCK) mechanisms. PYY acts on the multiple sites in the brain and gut, and inhibits pancreatic secretion by regulating these primary control mechanisms. The involvement of Y(1) and Y(2) receptors has been suggested in the regulation of pancreatic secretion. However, it remains to be studied which site of action or receptor subtype is physiologically most important for this regulation.