Relationship between the administration of nicardipine hydrochloride and the development of delirium in patients on mechanical ventilation.

A history of hypertension is a known risk factor for delirium in patients in intensive care units, but the effect of antihypertensive agents on delirium development is unclear. Nicardipine, a calcium channel blocker, is widely used in ICU as a treatment agent for hypertensive emergency. This study investigated the relationship between the administration of nicardipine hydrochloride and delirium development in patients under mechanical ventilation. We conducted a medical chart review of 103 patients, who were divided into two groups according to the use of nicardipine hydrochloride. The prevalence of delirium was compared with respect to factors such as age, sex, laboratory data, and medical history, by multivariate analysis. 21 patients (20.4 %) were treated with nicardipine hydrochloride in 103 patients. The treatment and non-treatment groups differed significantly in age (72 vs. 65 years) and history of high blood pressure (57% vs. 11%). Multivariate analysis revealed that patients in the treatment group developed delirium significantly less often than those in the non-treatment group (19% vs. 48%). These results suggested that treatment of high blood pressure with nicardipine hydrochloride is a possible method for preventing the development of delirium.

The views of doctors on registration trials in a Japanese rural area: a survey of medical institutions registered to the Tokushima Network for Clinical Trials.

Tokushima University Hospital has established the Tokushima Network for Clinical Trials (TNCT) to promote clinical trials in the area in collaboration with the Tokushima Medical Association. The present study investigated the views of doctors towards registration trials in the TNCT. A questionnaire was provided to 49 clinics/hospitals registered to the TNCT in 2006 and 38 (78%) responded. It revealed that 48% of doctors were aware of registration trials and 87% were favourable towards participating as investigators in them. They considered close contact with developmental drugs, advancement of therapy and the opportunity to learn about state-of-the-art treatment as benefits of participation. The main areas of difficulty included management of adverse reactions and patients' refusal to take part. Many doctors wanted more opportunity to learn about trial-related issues such as regulations. The survey indicates that the TNCT needs to develop the infrastructure and enlighten participants to promote registration trials in this rural regional area.

Immediate effects of a rucksack type orthosis on the elderly with decreased lumbar lordosis during standing and walking.

The spinal orthosis, the so-called rucksack type orthosis (RO), has been used to relieve low back pain and fatigue during prolonged standing and walking for the elderly with spinal deformities. However, little is known about the RO's kinematical effects. Twenty-three elderly (78.9 +/- 6.9 years old) participated in experiment 1, and 13 elderly (78.4 +/- 7.9 years old) in experiment 2. They had decreased lumbar lordosis or lumbar kyphosis. In experiment 1, using the "Spinal Mouse", which can measure spinal curvature, the effects of the RO on posture during standing were investigated. In experiment 2, using electromyography, the effects of the RO on muscle activity during standing and walking were clarified. Lumbar curvature and the trunk angle of inclination during standing improved significantly when the RO was used. Back extensor muscle activities (T9, L3, and L5) during standing and walking decreased significantly when the RO was used. There were no significant differences in the activities of the upper trapezius and vastus lateralis during standing and walking. The present study suggests that the elderly with lumbar deformities might be able to stand and walk more efficiently with the RO. The RO could prove to be valuable in preservation therapy for the elderly with decreased lumbar lordosis or lumbar kyphosis.

Identification of a novel first exon in the human dystrophin gene and of a new promoter located more than 500 kb upstream of the nearest known promoter.

The dystrophin gene, which is mutated in patients with Duchenne and Becker muscular dystrophies, is the largest known human gene. Five alternative promoters have been characterized until now. Here we show that a novel dystrophin isoform with a different first exon can be produced through transcription initiation at a previously unidentified alternative promoter. The case study presented is that of a patient with Duchenne muscular dystrophy who had a deletion extending from the 5' end of the dystrophin gene to exon 2, including all promoters previously mapped in the 5' part of the gene. Transcripts from lymphoblastoid cells were found to contain sequences corresponding to exon 3, indicating the presence of new promoter upstream of this exon. The nucleotide sequence of amplified cDNA corresponding to the 5' end of the new transcript indicated that the 5' end of exon 3 was extended by 9 codons, only the last (most 3') of which codes for methionine. The genomic nucleotide sequence upstream from the new exon, as determined using inverse polymerase chain reaction, revealed the presence of sequences similar to a TATA box, an octamer motif and an MEF-2 element. The identified promoter/exon did not map to intron 2, as might have been expected, but to a position more than 500 kb upstream of the most 5' of the previously identified promoters, thereby adding 500 kb to the dystrophin gene. The sequence of part of the new promoter region is very similar to that of certain medium reiteration frequency repetitive sequences. These findings may help us understand the molecular evolution of the dystrophin gene.

Random multi-recombinant PCR for the construction of combinatorial protein libraries.

Development of a new methodology to create protein libraries, which enable the exploration of global protein space, is an exciting challenge. In this study we have developed random multi-recombinant PCR (RM-PCR), which permits the shuffling of several DNA fragments without homologous sequences. In order to evaluate this methodology, we applied it to create two different combinatorial DNA libraries. For the construction of a 'random shuffling library', RM-PCR was used to shuffle six DNA fragments each encoding 25 amino acids; this affords many different fragment sequences whose every position has an equal probability to encode any of the six blocks. For the construction of the 'alternative splicing library', RM-PCR was used to perform different alternative splicings at the DNA level, which also yields different block sequences. DNA sequencing of the RM-PCR products in both libraries revealed that most of the sequences were quite different, and had a long open reading frame without a frame shift or stop codon. Furthermore, no distinct bias among blocks was observed. Here we describe how to use RM-PCR for the construction of combinatorial DNA libraries, which encode protein libraries that would be suitable for selection experiments in the global protein space.

Specific bonding of puromycin to full-length protein at the C-terminus.

Puromycin, an analog of the 3' end of aminoacyl-tRNA, causes premature termination of translation by being linked non-specifically to growing polypeptide chains. Here we report the interesting phenomenon that puromycin acting as a non-inhibitor at very low concentration (e.g. 0.04 microM) can bond only to full-length protein at the C-terminus. This was proved by using a carboxypeptidase digestion assay of the products obtained by Escherichia coli cell-free translation of human tau 4 repeat (tau4R) mRNA in the presence of low concentrations of puromycin or its derivatives. The tau4R mRNA was modified to code for three C-terminal methionines, which were radioactively labeled, followed by a stop codon. The translation products could not be digested by carboxy-peptidase if puromycin or a derivative was present at the C-terminus of full-length tau4R. Puromycin and its derivatives at 0. 04-1.0 microM bonded to 7-21% of full-length tau4R, depending on the ability to act as acceptor substrates. Furthermore, the bonding efficiency of a puromycin derivative to tau4R was decreased by addition of release factors. These results suggest that puromycin and its derivatives at concentrations lower than those able to compete effectively with aminoacyl-tRNA can bond specifically to full-length protein at a stop codon. This specific bonding of puromycin to full-length protein should be useful for in vitro selection of proteins and for in vitro and in vivo C-terminal end protein labeling.

Natural killer cell-dependent suppression of systemic spread of human lung adenocarcinoma cells by monocyte chemoattractant protein-1 gene transfection in severe combined immunodeficient mice.

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine with various biological activities, including augmentation of cytotoxic activity of monocytes and natural killer (NK) cells. The present study was undertaken to determine whether transfection of the MCP-1 gene into lung cancer cells affected their tumorigenicity and metastatic potential by the NK cell-mediated mechanism. The human MCP-1 gene inserted into an expression vector (BCMGSNeo) was transfected into human lung adenocarcinoma (PC-14) cells. There was no difference in in vitro proliferation between MCP-1 gene-transfected PC-14 cells and the parent cells or mock-transfected cells. The tumorigenicity and in vivo tumor growth of MCP-1 gene-transfected PC-14 cells were similar to those of the parent cells or mock-transfected cells when tumor cells were injected into the s.c. space of NK cell-intact severe combined immunodeficient (SCID) mice. Although parent cells and mock-transfected cells inoculated i.v. formed lung metastatic colonies and pleural effusion, MCP-1 gene transfectants reduced the systemic spread in NK cell-intact SCID mice. Interestingly, these modulations in a systemic spread by MCP-1 gene transfection were not observed in NK cell-depleted SCID mice. Decreased survival of MCP-1 gene transfectants in the lung was observed in NK cell-intact SCID mice but not in NK cell-depleted SCID mice. Recombinant MCP-1 or the supernatant of MCP-1 gene transfectants enhanced the cytotoxicity of human CD56+ NK cells and spleen cells of SCID mice against PC-14 cells. These findings suggest that locally produced MCP-1 suppresses tumor progression by a NK cell-mediated mechanism, depending on organ microenvironment.

Asparagusate dehydrogenases and lipoyl dehydrogenase from asparagus mitochondria.

1. Lipoyl dehydrogenase (NADH: lipoamide oxidoreductase, ED 1.6.4.3) and two asparagusate dehydrogenases from asparagus mitochondria were purified by a series of steps, freezing and thawing, sodium dodecylsulfate extraction, and chromatography on Sephadex G-200 and DEAE-cellulose. 2. Lipoyl dehydrogenase was highly specific for alpha-lipoic acid, which could not be replaced at all by asparagusic acid. Each of the asparagusate dehydrogenases was capable of reducing both asparagusic and alpha-lipoic acids by using NADH as hydrogen donor. 3. Reduction of alpha-lipoic cid with NADH by lipoyl dehydrogenase was activated by NAD, but that of asparagusic acid by asparagusate dehydrogenase was inactivated by NAD. 4. Lipoyl dehydrogenase and two asparagusate dehydrogenases differed in electrophoretic mobility on polyacrylamide gels.

Foldability of barnase mutants obtained by permutation of modules or secondary structure units.

Modules, defined as stable, compact structure units in a globular protein, are good candidates for the construction of novel foldable proteins by permutation. Here we decomposed barnase into six modules (M1-M6) and constructed 23 barnase mutants containing permutations of the internal four (M2-M5) out of six modules. Globular proteins can also be subdivided into secondary structure units based on the extended structures that control the mutual relationships of the modules. We also decomposed barnase into six secondary structure units (S1-S6) and constructed 21 barnase mutants containing permutations of the internal four (S2-S5) out of six secondary structure units. Foldability of these two types of mutants was assessed by means of circular dichroism, fluorescence, and 1H-NMR measurements. A total of 15 of 23 module mutants and 15 of 21 secondary structure unit mutants formed definite secondary structures, such as alpha-helix and beta-sheet, at 20 microM owing to intermolecular interactions, but most of them converted to random coil structures at a lower concentration (1 microM). Of the 44 mutants, only two, M3245 and S2543, gave distinct near-UV CD spectra. S2543 especially showed definite signal dispersion in the amide and methyl regions of the 1H-NMR spectrum, though M3245 did not. Furthermore, urea-induced unfolding of S2543 monitored by far-UV CD and fluorescence measurements showed a distinct cooperative transition. These results strongly suggest that S2543 takes partially folded conformations in aqueous solution. Our results also suggest that building blocks such as secondary structure units capable of taking different stable conformations by adapting themselves to the surrounding environment, rather than building blocks such as modules having a specified stable conformation, are required for the formation of foldable proteins. Therefore, the use of secondary structure units for the construction of novel globular proteins is likely to be an effective approach.

Cytolysis of human dendritic cells by autologous lymphokine-activated killer cells: participation of both T cells and NK cells in the killing.

Dendritic cells (DC) play a key role in the initiation of immune response by stimulating the naive T cells. The fate of DC after the initiation of immune response is not clearly understood. Although there are few reports implicating natural killer (NK) cells in the elimination of DC, killing of DC by LAK cells, and specifically by T cells, has not been studied. In this study, we observed that DC, generated from monocytes, in vitro in the presence of granulocyte-macrophage colony-stimulating factor, interleukin-4 (IL-4), and tumor necrosis factor alpha were susceptible to cytolysis by lymphokine-activated killer (LAK) cells induced in the presence of IL-2 and IL-15 but not IL-12 alone. However, LAK cells induced by a combination of IL-12 and suboptimal dose of IL-2 were cytotoxic to DC. When purified lymphocytes were activated with IL-2, the CD8+/CD57- fraction (T-LAK), but not the CD8-/CD57+ fraction (NK-LAK) was cytotoxic to autologous DC. However, when unseparated peripheral blood mononuclear cells were used to generate LAK cells, both T-LAK and NK-LAK fractions showed equal cytotoxicity against autologous DC. Monoclonal antibodies against CD54, CD11a, and CD18 significantly inhibited the cytolysis, indicating that the killing involves the engagement of CD54 with its ligands.

Origins of globular structure in proteins.

Since natural proteins are the products of a long evolutionary process, the structural properties of present-day proteins should depend not only on physico-chemical constraints, but also on evolutionary constraints. Here we propose a model for protein evolution, in which membranes play a key role as a scaffold for supporting the gradual evolution from flexible polypeptides to well-folded proteins. We suggest that the folding process of present-day globular proteins is a relic of this putative evolutionary process. To test the hypothesis that membranes once acted as a cradle for the folding of globular proteins, extensive research on membrane proteins and the interactions of globular proteins with membranes will be required.

STABLE: protein-DNA fusion system for screening of combinatorial protein libraries in vitro.

We have developed a new method that permits the complete in vitro construction and selection of peptide or protein libraries. This method relies on an in vitro transcription/translation reaction compartmentalized in water in oil emulsions. In each emulsion compartment, streptavidin (STA)-fused polypeptides are synthesized and attached to the encoding DNA via its biotin label. The resulting protein-DNA fusion molecules recovered from the emulsion can be subjected to affinity selection based on the properties of the peptide portion, whose sequence can be determined from that of its DNA-tag. This method, named 'STABLE' (STA-biotin linkage in emulsions), should be useful for rapid in vitro evolution of proteins and for ligand-based selection of cDNA libraries.

Insertional gene fusion technology.

The classical 'end to end' gene fusion technique has widely been used for monitoring gene expression, biological screening and purification of recombinant proteins. Recent progress with the 'insertional' gene fusion approach, on the other hand, has demonstrated that this technique can be utilized for membrane protein topology analysis, display of randomized protein libraries and design of biosensor proteins. In this review, we describe examples of insertional gene fusion and compare the old and new gene fusion techniques.

Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution.

Protein-engineering techniques have been adapted for the molecular design of biosensors that combine a molecular-recognition site with a signal-transduction function. The optical signal-transduction mechanism of green fluorescent protein (GFP) is most attractive, but hard to combine with a ligand-binding site. Here we describe a general method of creating entirely new molecular-recognition sites on GFPs. At the first step, a protein domain containing a desired molecular-binding site is inserted into a surface loop of GFP. Next, the insertional fusion protein is randomly mutated, and new allosteric proteins that undergo changes in fluorescence upon binding of target molecules are selected from the random library. We have tested this methodology by using TEM1 beta-lactamase and its inhibitory protein as our model protein-ligand system. 'Allosteric GFP biosensors' constructed by this method may be used in a wide range of applications including biochemistry and cell biology.

Permutation of modules or secondary structure units creates proteins with basal enzymatic properties.

The RNase activity of barnase mutants obtained by the permutation of modules or secondary structure units was investigated. Four of the 45 mutants had weak but distinct RNase activity, and they had unique optimum pHs and temperatures like natural enzymes. One of the active mutants had an ordered conformation, but the others did not. An active mutant having disordered conformation formed an ordered conformation in the presence of GMP, which is an inhibitor of this mutant. These results indicate that the amino acid sequences derived from barnase have sufficient plasticity to be rearranged into different proteins with basal enzymatic properties.

Characterization of random-sequence proteins displayed on the surface of Escherichia coli RNase HI.

In a previous study, random-sequence proteins of 120-130 amino acid residues were inserted into the surface loop region of the enzyme, Escherichia coli RNase HI [Doi et al. (1997) FEBS Lett. 402, 177-1801. Here we established that the RNase H activity of the insertion mutants is correlated with their secondary structure contents evaluated by circular dichroism measurement at 222 nm. The random-sequence insert of a mutant enzyme possessing relatively high RNase H activity was detached from the RNase HI scaffold, and its characterization indicated that the random-sequence protein maintains its secondary structure after separation from the scaffold. Thus, the structural features of random-sequence proteins were suggested to be monitored by measuring the activity of the scaffold enzyme into which these proteins have been inserted.

Fluorescence labeling of the C-terminus of proteins with a puromycin analogue in cell-free translation systems.

We have developed a new method for the C-terminus-specific fluorescence labeling of proteins. This method is based on the experimental finding that a fluorescent puromycin analogue at lower concentrations bonds efficiently to the C-terminus of mature proteins in cell-free translation systems using mRNA without a stop codon. This labeling is performed under moderate conditions and its labeling efficiency is in the range of 50-95%. Here we demonstrate a protein-protein interaction assay using fluorescence polarization measurement. This labeling method should also be useful for other rapid molecular interaction assays without purification of the labeled proteins, such as fluorescence correlation spectroscopy.

Insertion of foreign random sequences of 120 amino acid residues into an active enzyme.

Random sequences of 120-130 amino acid residues were inserted into a surface loop region of Escherichia coli RNase HI. This library was screened and about 10% of the clones were found to retain RNase H activity. Subsequent random mutagenesis led to an increase in RNase H activity and solubility of the protein. The inserted regions were found not to contribute to the secondary structure of the mutant protein. The high frequency of insertion of flexible sequences and the increase in the protein's function by further mutagenesis simulate one of the events in protein evolution.

In vitro virus: bonding of mRNA bearing puromycin at the 3'-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro.

Adequate means for genotype assignment to phenotype is essential in evolutionary molecular engineering. In this study, construction of 'in vitro virus' was carried out in which a genotype molecule (mRNA) covalently binds to the phenotype molecule (protein) through puromycin on the ribosome in a cell-free translation system. Bonding efficiency was approximately 10%, thus indicating a population of the in vitro virus to have approximately 10(12) protein variants, this number being 10(4) that in the phage display. The in vitro virus is useful for examining protein evolution in a test tube and the results may possibly serve as basis for a general method for selecting proteins possessing the most desirable functions.

Immunological circumvention of multiple organ metastases of multidrug resistant human small cell lung cancer cells by mouse-human chimeric anti-ganglioside GM2 antibody KM966.

serum against SBC-3/DOX cells to a similar extent compared with parental SBC-3 cells. Pretreatment of human effector cells with various cytokines induced further enhancement of the KM966-dependent ADCC against SBC-3/DOX cells. Intravenous injection of SBC-3 or SBC-3/DOX cells into natural killer (NK) cell-depleted severe combined immunodeficient (SCID) mice developed metastases in multiple organs (liver, kidneys and lymph nodes). Interestingly, SBC-3/DOX cells produced metastases more rapidly than SBC-3 cells, suggesting more aggressive phenotype of SBC-3/DOX cells than their parental cells in vivo. Systemic treatment with KM966, given on days 2 and 7, drastically inhibited the formation of multiple-organ metastases produced by both SBC-3 and SBC-3/DOX cells, indicating that KM966 can eradicate metastasis by SCLC cells irrespective of MDR phenotype. These findings suggest that the mouse-human chimeric KM966 targets the GM2 antigen, and might be useful for the immunological circumvention of multiple-organ metastases of refractory SCLC.