A sharp peak of the zero-temperature penetration depth at optimal composition in BaFe2(As(1-x)P(x))2.

In a superconductor, the ratio of the carrier density, n, to its effective mass, m*, is a fundamental property directly reflecting the length scale of the superfluid flow, the London penetration depth, λ(L). In two-dimensional systems, this ratio n/m* (~1/λ(L)(2)) determines the effective Fermi temperature, T(F). We report a sharp peak in the x-dependence of λ(L) at zero temperature in clean samples of BaFe(2)(As(1)(-x)P(x))(2) at the optimum composition x = 0.30, where the superconducting transition temperature T(c) reaches a maximum of 30 kelvin. This structure may arise from quantum fluctuations associated with a quantum critical point. The ratio of T(c)/T(F) at x = 0.30 is enhanced, implying a possible crossover toward the Bose-Einstein condensate limit driven by quantum criticality.

Nodal versus nodeless behaviors of the order parameters of LiFeP and LiFeAs superconductors from magnetic penetration-depth measurements.

High-precision measurements of magnetic penetration depth λ in clean single crystals of LiFeAs and LiFeP superconductors reveal contrasting behaviors. In LiFeAs the low-temperature λ(T) shows a flat dependence indicative of a fully gapped state, which is consistent with previous studies. In contrast, LiFeP exhibits a T-linear dependence of superfluid density infinity λ(-2), indicating a nodal superconducting order parameter. A systematic comparison of quasiparticle excitations in the 1111, 122, and 111 families of iron-pnictide superconductors implies that the nodal state is induced when the pnictogen height from the iron plane decreases below a threshold value of ~1.33 Å.

[Estimating probabilities and dealing with mutations in paternity testing--verification of DNA testing with commercially available STR kits].

Since Jeffreys devised a DNA fingerprint in 1985, DNA analysis has been applied to paternity testing. The progress of the techniques, especially the development of polymerase chain reaction (PCR), makes it possible to type 10-15 short tandem repeat (STR) loci in paternity testing by a single test tube. When using a well qualified database, we can now obtain a paternity index (PI) as high as 10(6) in usual trio cases. Furthermore, the DNA testings are now applied to unusual cases, such as personal identification of Japanese war orphans left in China. Here, we reviewed how to calculate the PI likelihood ratio and exclusion probability in a trio case, a motherless case, parent identification without reliable evidence of mother and child relationship, and a sibling case. We also reviewed how to handle single exclusion cases usually derived from a single mutation that is no longer rare when many STR loci are used. Finally, we emphasized the importance of ethical, legal and social counseling for clients in paternity testing. From that point of view, paternity tests by mail should not be allowed because of lack of such counseling.

Cloning and sequencing of a novel human gene which encodes a putative hydroxylase.

Using a conventional two-hybrid technique with MAWD as bait protein, a novel full-length cDNA was isolated and sequenced from a human liver cDNA library. This cDNA consists of 2,575 base pairs and has a predicted open reading frame encoding 255 amino acids. Overall, it is similar to the catalytic enzyme PHZF, catalyzing the hydroxylation of phenazine-1-carboxylic acid to 2-hydroxyphenazine-1-carboxylic acid. Polymerase chain reaction-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 10q21.1 near the marker D10S210.

A carboxy-terminally truncated form of the human immunodeficiency virus type 1 Vpr protein induces apoptosis via G(1) cell cycle arrest.

Viral protein R (Vpr) of human immunodeficiency virus type 1 inhibits cell proliferation by arresting the cell cycle at the G(2) phase and inducing to apoptosis after G(2) arrest. We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation via a novel pathway that is distinct from G(2) arrest. However, the mechanism of this effect of C81 is unknown. We demonstrate here that C81 can induce apoptosis via G(1) arrest of the cell cycle. Immunostaining for various markers of stages of the cell cycle and flow cytometry analysis of DNA content showed that most HeLa cells that had been transiently transfected with a C81 expression vector were arrested at the G(1) phase and not at the G(2) or S phase of the cell cycle. Staining for annexin V, which binds phosphatidylserine on the plasma membrane, as an early indicator of apoptosis and measurement of the activity of caspase-3, a signaling molecule in apoptotic pathways, indicated that C81 is a strong inducer of apoptosis. Expression of C81 induced the condensation, fragmentation, and clumping of chromatin that are typical of apoptosis. Furthermore, the kinetics of the C81-induced G(1) arrest were closely correlated with changes in the number of annexin V-positive cells and the activity of caspase-3. Replacement of Ile or Leu residues by Pro at positions 60, 67, 74, and 81 within the leucine zipper-like domain of C81 revealed that Ile60, Leu67, and Ile74 play important roles both in the C81-induced G(1) arrest and in apoptosis. Thus, it appears that C81 induces apoptosis through pathways that are identical to those utilized for G(1) arrest of the cell cycle. It has been reported that Ile60, Leu67, and Ile74 also play an important role in the C81-induced suppression of growth. These results suggest that the suppression of growth induced by C81 result in apoptosis that is independent of G(2) arrest of the cell cycle.

Mechanism of the incidental production of a melanin-like pigment during 6-demethylchlortetracycline production in Streptomyces aureofaciens.

The secondary metabolite 6-demethylchlortetracycline (6-DCT), which is produced by Streptomyces aureofaciens, is used as a precursor of semisynthetic tetracyclines. Strains that produce 6-DCT also produce a melanin-like pigment (MP). The correlation between MP production and 6-DCT production was investigated by using S. aureofaciens NRRL 3203. Production of both MP and 6-DCT was repressed by phosphate or ammonium ions, suggesting that syntheses of these compounds are controlled by the same regulators. Ten chlortetracycline-producing recombinants were derived from 6-DCT-producing mutant NRRL 3203 by gene replacement. All of the recombinants produced chlortetracycline but not MP, indicating that MP production is the results of a defect in the 6-methylation step and suggesting that the polyketide nonaketideamide is a common intermediate leading to MP as well as 6-DCT. To further examine the possibility that MP might be synthesized via the 6-DCT-biosynthetic pathway, mutants defective in 6-DCT biosynthesis were derived from a 6-DCT producer. Some of these mutants were able to produce MP, while others, including mutants with mutations in the gene encoding anhydrotetracycline oxygenase, an enzyme catalyzing the penultimate step in the pathway, produced neither 6-DCT nor MP. Production of 6-DCT and production of MP were restored simultaneously by integrative transformation with the corresponding 6-DCT-biosynthetic genes, indicating that some of 6-DCT-biosynthetic enzymes are indispensable for MP production. These findings suggest that a defect in the 6-methylation step results in redirection of carbon flux from a certain intermediate in the 6-DCT-biosynthetic pathway to a shunt pathway and results in MP production.

Molecular cloning and characterization of human MAWD, a novel protein containing WD-40 repeats frequently overexpressed in breast cancer.

A full-length cDNA clone encoding a novel protein containing WD-40 repeats, which were frequently involved in protein-protein interactions, was isolated and sequenced. This clone had a predicted open reading frame (ORF) encoding 350 amino acids possessing six repeats of WD-40 motif. It was most closely homologous to TRIP-1, a phosphorylation substrate of the transforming growth factor-beta type II receptor. In the process of characterizing the function of the new gene product, we found that overexpression of the gene seemed to activate mitogen-activated protein kinase and to promote anchorage-independent growth of the cells. Moreover, the gene product was frequently overexpressed in human tumor breast tissues compared with their normal breast tissues, suggesting that the gene might be involved in the tumor progression. Radiation hybrid mapping placed the gene into human chromosome 12q11-12 near the marker D12S1593.

[Complications in home treatment of patients with severe disturbances of consciousness].

We have been providing home treatment for patients with severe disturbance of consciousness requiring various medical treatments for about 10 years. In the 65 cases we have encountered, we studied complications during the course of the home treatment. The proportions of infections, decubiti and convulsions were found to be very high. With infections in particular, conditions worsened rapidly in many cases, so we made it a principle to have the patients hospitalized early. We also investigated changes in serum albumin levels, peripheral blood lymphocyte count and prognostic nutritional index (PNI). Patients who develop infections repeatedly and succumb to early death often show low values for PNI and lymphocyte count. The low values are considered useful as one indicator of the general conditions of patients under home treatment. For the management of patients with severe disturbances of consciousness, it is important to keep a close liaison with other departments or hospitals and strengthen home nursing sections.

The potential contribution of MVR-PCR to paternity probabilities in a case lacking a mother.

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) was applied to a paternity case lacking a mother to evaluate the paternity probability. After three flanking polymorphic sites at each of MS31A and MS32 loci were investigated from the child and alleged father, allele-specific MVR-PCR was performed using genomic DNA. It was confirmed that one allele in the child was identical to that in the alleged father at both loci. Mapped allele codes were compared with allele structures established from population surveys. No perfect matches were found although some motifs were shared with other Japanese alleles. The paternity index and probability of paternity exclusion at these two MVR loci were then estimated, establishing the power of MVR-PCR even in paternity cases lacking a mother.

Hyperproduction of tryptophan by Corynebacterium glutamicum with the modified pentose phosphate pathway.

A classically derived tryptophan-producing Corynebacterium glutamicum strain was recently significantly improved both by plasmid-mediated amplification of the genes for the rate-limiting enzymes in the terminal pathways and by construction of a plasmid stabilization system so that it produced more tryptophan. This engineered strain, KY9218 carrying pKW9901, produced 50 g of tryptophan per liter from sucrose after 80 h in fed-batch cultivation without antibiotic pressure. Analysis of carbon balances showed that at the late stage of the fermentation, tryptophan yield decreased with a concomitant increase in CO2 yield, suggesting a transition in the distribution of carbon flow from aromatic biosynthesis toward the tricarboxylic acid cycle via glycolysis. To circumvent this transition by increasing the supply of erythrose 4-phosphate, a direct precursor of aromatic biosynthesis, the transketolase gene of C. glutamicum was coamplified in the engineered strain by using low- and high-copy-number plasmids which were compatible with the resident plasmid pKW9901. The presence of the gene in low copy numbers contributed to improvement of tryptophan yield, especially at the late stage, and led to accumulation of more tryptophan (57 g/liter) than did its absence, while high-copy-number amplification of the gene resulted in a tryptophan production level even lower than that resulting from the absence of the gene due to reduced growth and sugar consumption. In order to assemble all the cloned genes onto a low-copy-number plasmid, the high-copy-number origin of pKW9901 was replaced with the low-copy-number one, generating low-copy-number plasmid pSW9911, and the transketolase gene was inserted to yield pIK9960. The pSW9911-carrying producer showed almost the same fermentation profiles as the pKW9901 carrier in fed-batch cultivation without antibiotic pressure. Under the same culture conditions, however, the pIK9960 carrier achieved a final tryptophan titer of 58 g/liter, which represented a 15% enhancement over the titers achieved by the pKW9901 and pSW9911 carriers.

Mechanism of alanine hyperproduction by arthrobacter oxydans HAP-1: metabolic shift to fermentation under nongrowth aerobic conditions

Arthrobacter oxydans HAP-1 hyperproduces DL-alanine in a non-growth-associated manner. We found that decreased activities of pyruvate dehydrogenase and of the enzyme catalyzing NADH oxidation in the stationary phase are paralleled by a shift of pyruvate metabolism to alanine synthesis by L-alanine dehydrogenase. We propose that this enzyme functions as an electron sink even under aerobic conditions.

Cloning of the transketolase gene and the effect of its dosage on aromatic amino acid production in Corynebacterium glutamicum.

Transketolase is a key enzyme of the non-oxidative pentose phosphate pathway. The effect of its overexpression on aromatic amino acid production was investigated in Corynebacterium glutamicum, a typical amino-acid-producing organism. For this purpose, the transketolase gene of the organism was cloned on the basis of its ability to complement a C. glutamicum transketolase mutant with pleiotropically shikimic-acid-requiring, ribose- and gluconic-acid-negative phenotype. The gene was shown by deletion mapping and complementation analysis to be located in a 3.2-kb XhoI-SalI fragment of the genome. Amplification of the gene by use of low-, middle-, and high-copy-number vectors in a C. glutamicum strain resulted in overexpression of transketolase activities as well as a protein of approximately 83kDa in proportion to the copy numbers. Introduction of the plasmids into a tryptophan and lysine co-producer resulted in copy-dependent increases in tryptophan production along with concomitant decreases in lysine production. Furthermore, the presence of the gene in high copy numbers enabled tyrosine, phenylalanine and tryptophan producers to accumulate 5%-20% more aromatic amino acids. These results indicate that overexpressed transketolase activity operates to redirect the glycolytic intermediates toward the nonoxidative pentose phosphate pathway in vivo, thereby increasing the intracellular level of erythrose 4-phosphate, a precursor of aromatic biosynthesis, in the aromatic-amino-acid-producing C. glutamicum strains.

Large-scale preparation of high-molecular weight DNA from buccal mucosa.

Four non-invasive methods of sampling DNA from buccal mucosa, simple rinses, scrubbing with cotton balls, scrubbing with toothbrushes and rinses after scrubbing with toothbrushes, were investigated. Scrubbing with toothbrushes yielded 5.79 +/- 5.56 microg of DNA rich in high-molecules, while less than one eighth the amount was recovered by scrubbing with cotton balls. Rinses after scrubbing with toothbrushes gave 50.0 +/- 46.0 microg of DNA and simple rinses 34.4 +/- 35.7 microg, although the DNA was considerably degraded. DNA specimens obtained from buccal cells were shown to be more or less in the process of degradation including apoptosis. For minisatellite analysis, only DNA prepared by scrubbing with toothbrushes could be used, while all specimens could be applied to PCR analyses. Since scrubbing with toothbrushes is painless and harmless, we recommend this method. Subsequent rinsing will yield a large amount of DNA suitable for many PCR analyses.

Change in chemoattractant responsiveness of developing axons at an intermediate target.

Developing axons reach their final targets as a result of a series of axonal projections to successive intermediate targets. Long-range chemoattraction by intermediate targets plays a key role in this process. Growing axons, however, do not stall at the intermediate targets, where the chemoattractant concentration is expected to be maximal. Commissural axons in the metencephalon, initially attracted by a chemoattractant released from the floor plate, were shown to lose responsiveness to the chemoattractant when they crossed the floor plate in vitro. Such changes in axon responsiveness to chemoattractants may enable developing axons to continue to navigate toward their final destinations.

A self-defense gene homologous to tetracycline effluxing gene essential for antibiotic production in Streptomyces aureofaciens.

By Northern blot analyses with DNA probes carrying 6-demethylchlortetracycline (6-DCT) biosynthetic genes from Streptomyces aureofaciens NRRL3203, a highly expressed gene (tcrC) was detected in a high titer producing mutant derived from the parental strain NRRL3203 by NTG mutagenesis. The analysis of the nucleotide sequence of the 2.8-kb BamHI fragment containing tcrC gene showed that the predicted tcrC gene product is a protein consisting of 512 amino acids. The deduced amino acid sequence had a high level identity with that of the self-defense gene (tet347) of Streptomyces rimosus, known to mediate oxytetracycline efflux. The tcrC gene-inactivated strains generated from strain NRRL3203 by gene replacement had a 90% decrease in the level of resistance to tetracycline and the antibiotic productivity when compared with the parental strain.

Conserved organization of genes for biosynthesis of chlortetracycline in Streptomyces strains.

By genomic Southern blot analysis, the DNA sequences homologous to the gene cluster responsible for biosynthesis of 6-demethyl-chlortetracycline in Streptomyces aureofaciens NRRL3203 were shown to be highly conserved in independent chlortetracycline- or tetracycline producing Streptomyces strains. By contrast, oxytetracycline-producing Streptomyces strains had no hybridization with the cluster DNA.

Cloning and nucleotide sequence of the gene responsible for chlorination of tetracycline.

Two cosmid clones containing distinct types of self-defense gene of a 6-demethylchlortetracycline producer, Streptomyces aureofaciens NRRL3203, were isolated. The gene responsible for chlorination of tetracycline (chl gene) was subcloned from one of the cosmid clones by complementation of a chlorination-deficient mutant, using a gene cloning system for strain NRRL3203 developed in this study. The nucleotide sequence analysis of a 4.4-kb SacI-BamHI fragment containing the chl gene showed that the predicted product of the chl gene is a polypeptide of 452 amino acids, and that the chl gene was preceded by two open reading frames, which could endode polypeptides of 50 kDa and 32 kDa, respectively. A search for sequences homologous to these ORFs found that the former product strongly resembles that of the 6-hydroxylation enzyme for oxytetracycline biosynthesis, and that the latter product has a weak but significant similarity to the hydroxyindole O-methyltransferase of bovine pineal gland. By Northern blot analysis, these three genes were suggested to be polycistronically transcribed.

Guidance of cerebellofugal axons in the rat embryo: directed growth toward the floor plate and subsequent elongation along the longitudinal axis.

To elucidate guidance mechanisms of brain commissural axons, we examined the navigation of cerebellofugal axons. Axons were labeled by implantation of the fluorescent tracer Dil into the cerebellar plate (CP) of fixed, flat whole-mount embryonic rat brain. Axons initially grew straight toward the ventral midline floor plate (FP) in the rostral hindbrain and then, after crossing it, made a right-angled turn to grow either caudally or rostrally along the longitudinal axis. In collagen gel culture, CP axons showed directed growth toward both FP explants and heterologous cells expressing netrin-1, a FP-derived chemoattractant for spinal commissural axons. These results suggest that CP axons are guided to the midline by FP-derived chemoattractant(s) and then reoriented, possibly by another guidance cue, for longitudinal extension. Considering that the basic structures of the neural tube, including the FP, extend up to the caudal diencephalon, these results suggest that common guidance mechanisms operate for ventrally decussating commissural axons in both the brain and spinal cord.

Cloning of the ATP phosphoribosyl transferase gene of Corynebacterium glutamicum and application of the gene to L-histidine production.

Corynebacterium glutamicum mutants lacking ATP phosphoribosyl transferase (PRT) were selected by complementation with the Escherichia coli PRT gene. The recombinant plasmid pCH13 carrying a wild type PRT gene from C. glutamicum T106 was obtained in one of the mutants, LH13. Transformants, LH13/pCH13 and T106/pCH13, had three times higher PRT specific activity than T106. The plasmid pCH99 specifying the PRT, which was desensitized to feedback inhibition by L-histidine fifty-fold higher than the wild type PRT, was derived from pCH13. L-Histidine productivity of C. glutamicum F81, was markedly decreased by pCH13, but increased twice by pCH99. In cultivation in jar fermentors, F81/pCH99 continued to accumulate L-histidine through fermentation and yielded to the titer of 22/5 g/liter, while F81 accumulated only 11.5 g/liter due to production retardation halfway through fermentation. Moreover, F81/pCH99 had a larger production rate than F81 even in its production phase. These results indicate that the yield improvement results from amplification of the highly desensitized PRT provided by pCH99.