Molecular computation by DNA hairpin formation.

Hairpin formation by single-stranded DNA molecules was exploited in a DNA-based computation in order to explore the feasibility of autonomous molecular computing. An instance of the satisfiability problem, a famous hard combinatorial problem, was solved by using molecular biology techniques. The satisfiability of a given Boolean formula was examined autonomously, on the basis of hairpin formation by the molecules that represent the formula. This computation algorithm can test several clauses in the given formula simultaneously, which could reduce the number of laboratory steps required for computation.

Gene rearrangements of T cell receptor beta and gamma chains in HTLV-I infected primary neoplastic T cells.

Rearrangements of T cell receptor beta and gamma chain (T beta and T gamma) genes were analyzed by Southern blot method in samples from 30 patients with adult T cell leukemia (ATL) and 17 patients with non-ATL T cell neoplasms. The DNA probes used were the constant and joining region of T beta gene and the joining region of T gamma gene. Rearranged bands of T beta gene on one or both allelic chromosomes were detected in all neoplastic T cells, even those of smoldering ATL, in which only a small percentage of peripheral blood T cells were detected as leukemic. T gamma gene was rearranged in the cells of all but one patient, the exception being one ATL patient. In order to test whether any given variable region (V) of T beta gene was expressed in ATL cells, two functionally rearranged V beta sequences of ATL were compared with a V beta sequence from T cells acute lymphoblastic leukemia cells. No significant homologies were noted among the three deduced gene product amino acid sequences, confirming that T beta molecules of ATL cells contained no specific structures in common. The observed heterogeneity of T beta and T gamma gene rearrangements in ATL cells further supported these findings.

Organization of the human hepatocyte growth factor-encoding gene.

Human genomic phage libraries were screened for the human hepatocyte growth factor (HGF)-encoding gene (HGF) using a cDNA encoding the human protein as a probe. Characterization of the clones revealed that this gene is composed of 18 exons interrupted by 17 introns spanning approx. 70 kb. The first exon contains the 5'-untranslated region and the signal peptide. The next ten exons encode the alpha-chain which contains four kringle structures. Each kringle domain is encoded by two exons as observed in other kringle-containing proteins. The twelfth exon contains the short spacer region between the alpha- and beta-chains and the remaining six exons comprise the beta-chain. The beta-chain is structurally similar to the catalytic domains of serine proteases; amino acid substitutions in the active site were found. The organization of the HGF gene is highly homologous to those of the serine proteases involved in blood coagulation and fibrinolysis, especially with that of plasminogen. This suggests that the human HGF gene is evolutionally related to these genes.

Augmenter of liver regeneration (ALR) may promote liver regeneration by reducing natural killer (NK) cell activity in human liver diseases.

Cytotoxicity of liver natural killer cells against regenerating hepatocytes has been reported as a possible mechanism of regeneration failure in fulminant hepatitis. An augmenter of liver regeneration (ALR) inhibits liver natural killer cell activity in rats. In this study, we measured hepatic expression of ALR mRNA, blood levels of ALR, and peripheral blood natural killer cell activity in patients with various types of acute liver disease to investigate the relationship between failure of liver regeneration and hepatic natural killer cells. Hepatic ALR mRNA expression was higher in liver disease patients than in non-liver disease controls, and a correlation was found between serum ALR values and hepatic levels of ALR mRNA. In acute liver injury, the serum ALR level also showed a negative correlation with NK activity. ALR was produced by and released from the liver at the time of hepatic injury. Our findings suggest that ALR may protect against failure of regeneration by inhibition of hepatic natural killer cell activity in acute liver injury.

Atypical presentation of vertebral bone metastasis from lung cancer.

The authors describe a case of lung cancer in a 55-year-old man who complained of back pain. Initial isotopic bone scanning showed no abnormality, however, magnetic resonance (MRI) imaging revealed bone metastasis in thoracic vertebral bone. Even when there is no typical findings of metastasis in bone scintigraphy, MRI imaging would be useful if vertebral bone metastasis is suspected. MRI imaging is an important modality to evaluate extraosseous extension and marrow invasion of metastatic tumors.

State transitions by molecules.

In our previous paper, we described a method by which a state machine is implemented by a single-stranded DNA molecule whose 3'-end sequence encodes the current state of the machine. Successive state transitions are performed in such a way that the current state is annealed onto an appropriate portion of DNA encoding the transition table of the state machine and the next state is copied to the 3'-end by extension with polymerase. In this paper, we first show that combined with parallel overlap assembly, a single series of successive transitions can solve NP-complete problems. This means that the number of necessary laboratory steps is independent from the problem size. We then report the results of two experiments concerning the implementation of our method. One is on isothermal reactions which greatly increase the efficiency of state transitions compared with reactions controlled by thermal cycles. The other is on the use of unnatural bases for avoiding out-of-frame annealing. The latter result can also be applied to many DNA-based computing paradigms.

Effect of respiratory rate on respiratory patterns in patients with chronic obstructive pulmonary disease.

We enrolled 22 patients with chronic obstructive pulmonary disease (COPD) and 20 normal subjects as controls. Using a hot-wire flow meter, we obtained tidal volume (VT), duty ratio (Ti/Ttot), and mean inspiratory flow (VT/Ti) as measures of respiratory pattern at two different respiratory rates; 0.5 Hz and 1.5 Hz. At 0.5 Hz, there were significant differences in Ti/Ttot and VT/Ti. At 1.5 Hz, patients with COPD had significantly lower values of VT and VT/Ti. Furthermore, we calculated the change ratios from 0.5 Hz to 1.5 Hz in these parameters as new parameters of respiratory pattern change by respiratory rate. VT0.5/1.5 and VT/Ti0.5/1.5 significantly correlated with FEV1.0/FVC. The findings suggest that the parameters of the respiratory pattern may change depending on respiratory rates, and that the ratios of those parameters obtained at two different rates could be helpful in diagnosing airflow obstruction.

[Ventilatory effects of halothane and enflurane in dogs].

The ventilatory effects of halothane and enflurane were studied in permanently tracheostomized dogs at the same anesthetic depth of 1 MAC. Inspiratory and expiratory durations were longer and tidal volume greater during enflurane than during halothane anesthesia. Mean inspiratory flow rate and minute ventilation during enflurane anesthesia were less than those during halothane anesthesia. As a result, end-expiratory carbon-dioxide concentration was higher during enflurane than during halothane anesthesia. When end-expiratory carbon-dioxide concentration was held at 7.5%, tidal volume was not different between the two anesthetics, while the difference of other parameters still remained. In addition, the magnitude of Hering-Breuer reflex determined by end-expiratory airway occlusion was essentially identical between halothane and enflurane anesthesia. The present results indicate that 1) depressant effect of enflurane on respiratory drive is greater than that of halothane and 2) the two anesthetics act on the respiratory timing mechanism differently.

[Factors contributing to an increase in DLco (steady state) after exercise in healthy men].

We studied factors contributing to an increase of DLco (steady state) from rest to exercise in 10 healthy men. DLco(ss), Dm, and Vc were measured under three different conditions, rest, constant load exercise (50 watts), and hyperventilation (equal to the tidal volume and respiratory rate of exercise). DLco(ss) increased significantly during exercise and hyperventilation compared with at rest. DLco(ss) also increased significantly during exercise, compared with hyperventilation. During constant load exercise (50 watts) increased Dm and Vc, caused by increased ventilation, together with increased of Vc caused by increased of pulmonary blood flow resulted in an increase in DLco(ss).

[One year follow-up of pulmonary rehabilitation in patients with pulmonary emphysema--physiological outcome].

To evaluate the long-term effects of pulmonary rehabilitation on physiological outcome, 12 patients with pulmonary emphysema were enrolled in an inpatient pulmonary rehabilitation program for 6 weeks. After discharge from the hospital, they were followed up for 1 year. The pulmonary rehabilitation program consisted of breathing retraining, thoracic mobilization, exercise training, and patient education. Although the subjects did not participate in outpatient maintenance group sessions after their discharge, they continued breathing retraining and exercise training at home. Their vital capacity improved significantly, and was sustained over the course of the year; other pulmonary functions, however, did not change significantly. Maximum exercise load increased 31% after the rehabilitation program; although it was 18% higher than baseline at follow-up one year later, that was not a significant change. The follow-up data on exercise traming had generally deteriorated 1 year after the rehabilitation program. The change in maximum exercise load from baseline before and after the inpatient pulmonary rehabilitation program correlated closely with the change in maximum exercise load thereafter to follow-up one year later (R = 0.62). We conclude that it is pessible to estimate long-term change in exercise capacity on the basis of short-term changes achieved during inpatient pulmonary rehabilitation. It may be necessary to develop maintenance programs of some kind to help pulmonary emphysema patients retain the benefits of pulmonary rehabilitation over the longer term.

[Alpha 1-antitrypsin deficiency (Siiyama) with pulmonary emphysema].

A 44-year-old man was admitted to the hospital with dyspnea on exertion. Chest radiographs and pulmonary function tests showed evidence of pulmonary emphysema. Serum alpha 1-antitrypsin (alpha 1-AT) could not be detected by nephelometry, immuno-electrophoresis, or iso-electric focusing. However, allele-specific PCR revealed a genotype homozygotic for an alpha 1-AT deficient variant of the Siiyama allele. An elder sister of the proband was also a homozygous carrier of the Siiyama allele. The amino acid sequence for normal alpha 1-AT variants had been substituted by Arg101-Val213-Glu376 in the proband, demonstrating that the alpha 1-antitrypsin-deficient Siiyama variant in this pedigree was derived from M 1 (Val213).

Effects of enflurane on contractile response of canine trachealis muscle.

The effect of enflurane anesthesia on bronchomotor tone was studied in 15 dogs, using isolated tracheal segments in situ. In six animals, the changes of tracheal tension in response to changing enflurane concentration were continuously measured while keeping tidal volume, respiratory frequency, and end-tidal PCO2 constant. In nine other dogs, the contractile response to a brief period of apnea was assessed in three planes of anesthesia, namely 0% (baseline anesthesia with pentobarbital), 1%, and 2% of inspired enflurane concentration. In addition, effects of hypercapnia, hypoxia, and changing tidal volume were also examined in three of these nine animals. Furthermore, the magnitude of contraction elicited by electrical stimulation of the recurrent laryngeal nerve (RLN) was determined at each depth of anesthesia in six animals. Increasing enflurane concentrations invariably decreased resting tracheal tension as well as the magnitude of contraction in response to apnea and RLN stimulation in a dose-dependent manner. These data suggest that enflurane not only decreases unstimulated canine bronchomotor tone but also inhibits contraction produced by various stimuli used in the present experiments.

Changes in breathing patterns after lung operations.

To assess the effects of thoracic surgery on breathing patterns without conscious intervention, we analyzed and compared the breathing patterns observed immediately before and after surgical procedure while anesthesia was maintained with enflurane and nitrous oxide at a constant level in 15 patients undergoing thoracic surgery. Our results showed that immediately after the surgical procedure, respiratory frequency (f) was significantly higher and tidal volume (V(T)) was significantly smaller than those observed immediately before the surgical procedure. The increased f during the period immediately after the surgical procedure was due to shortening of both inspiratory time (T(I)) and expiratory time (T(E)) without changing the ratio of T(I) to the total breath time (T(tot)). Despite the significant decrease in V(T) observed immediately after the surgical procedure, the value of negative pressure during the occluded inspiration (P(max)) was significantly higher than that observed immediately before the surgical procedure. These results indicate that a rapid, shallow type of breathing pattern can occur after thoracic surgery without conscious intervention.

Analysis of Agrobacterium tumefaciens plasmid pTiC58 replication region with a novel high-copy-number derivative.

The origin of replication, ori, of the nopaline tumor-inducing plasmid, pTiC58, mapped in a region that shares sequence homology with octopine plasmids pTiAch5 and pTiB6. Within this region, the minimum amount of DNA necessary for maintaining autonomous replication was a 2.6-kilobase region, which also comprised the incompatibility function inc. pTiC58 derivatives containing inc were incompatible with Agrobacterium tumefaciens plasmids pTiC58, pTiD1439, pTiAch5, pTi15955, and pTiA5 and were compatible with A. rhizogenes plasmid pRi12. Situated adjacent to the origin region was a 1.5-kilobase par segment involved in stable inheritance of pTiC58 under nonselective growth conditions. When par was present, plasmid maintenance approached that of the wild-type pTiC58. Rapid loss from the cell population was observed for plasmids not containing this locus. Another 1.5-kilobase region, cop, positively regulated pTiC58 copy number, enabling certain pTiC58 derivatives to exist at a copy number up to 80 times higher than that of wild-type pTiC58. Deletions within the cop locus resulted in reduced copy number. The ori/inc regions were flanked on either side by the par and cop loci.

Two types of immunoglobulin-negative Abelson murine leukemia virus-transformed cells: implications for B-lymphocyte differentiation.

Both alleles of immunoglobulin (Ig) heavy-chain joining region (JH) genes in three Ig-negative Abelson murine leukemia virus (Ab-MuLV)-transformed cell lines were characterized by DNA cloning and nucleotide sequence determination. These studies unambiguously identified two distinct types of Ig-negative B-lineage cells. The first type of cell (e.g., R8) is an "immature pre-B cell," and it contains at least one intermediate recombinant structure containing heavy-chain diversity (DH) and JH sequences but no variable region (VH) sequence. This type of cell, which has also been characterized by other investigators, generates mu-positive sublines during subsequent culturing of cells and represents a precursor stage to pre-B cells. The second type of cell (e.g., RAW253) is an "abortive pre-B cell," in that both JH alleles contain nonfunctional VH-DH-JH structures. The nucleotide sequence determinations in this study demonstrated that these nonfunctional V-D-J structures were generated by nonproductive somatic recombinations, involving either out-of-phase joining events, or the formation of termination codons in the DH coding sequences. The identification of abortive pre-B cells suggests that the recombinational joining of Ig VH, DH, and JH segments is not actively regulated by a putative recombinase to preserve the translational reading frame. This in turn implies that a large portion of precursor cells at the early stage of B-cell differentiation are abortive and possibly blocked to further differentiation.

Identification of pTiC58 plasmid-encoded proteins for virulence in Agrobacterium tumefaciens.

Analyses were made of the host-dependent-variation (hdv) locus of the virulence (vir) region of the pTiC58 plasmid of Agrobacterium tumefaciens. The hdv locus is comprised of at least four genes that encode polypeptides of 13, 15, 29, and 28 kDa. Insertion of transposon Tn5 in the first gene abolishes the expression of all four genes in vitro and in vivo. Nucleotide sequence analysis of the hdv locus revealed four open reading frames tandemly arranged with spacer sequences having no promoter-like sequences and lacking the ability to bind A. tumefaciens RNA polymerase. These studies suggest that the hdv locus is comprised of at least four genes arranged in an operon in the vir region. The protein products of these genes are likely to function in some aspect of the host-range determination of A. tumefaciens.