Biomedical subjects

J Fukushima

Publications and source records attributed to J Fukushima.

At least 73 records · Page 4Linked to original sources

PubMed1996

Purification and characterization of LasR as a DNA-binding protein.

In Pseudomonas aeruginosa, the activator protein LasR and a cognate autoinducer (AI) are required for expression of the elastase gene (lasB). In the present study, we investigated the binding properties of the P. aeruginosa lasR gene product. The LasR protein was overexpressed and purified as a glutathione S-transferase (GST) fusion protein. Using gel retardation and UV cross-linking analysis, we demonstrated that the GST-LasR could bind to a separate site in the lasB upstream operator regions 1 and 3 in the presence of the autoinducer. Regions 1 and 3 are located at 105 and 42 base pairs upstream, respectively, from the lasB transcriptional start site. Our present results clearly demonstrate that LasR is a specific DNA-binding protein that regulates the transcription of the lasB gene in the presence of an autoinducer.

4-Butyrolactone↗

PubMed1996

ErbB-2 expression is correlated with poor prognosis for patients with osteosarcoma.

BACKGROUND: It has been reported that the c-erbB-2 protooncogene is frequently amplified and overexpressed in many types of cancers, except sarcomas and hematological malignancies. METHODS: Expression of ErbB-2 in the tumors of 26 patients with conventional osteosarcoma was evaluated by immunoblotting. DNA from osteosarcoma tissues that expressed ErbB-2 were analyzed by Southern blot hybridization to examine gross rearrangement of the gene. The DNA was also surveyed for the presence of genetic mutation in the transmembrane domain of ErbB-2 by polymerase chain reaction-single-stranded DNA conformation polymorphism analysis. In addition, possible correlation of ErbB-2 expression with gender, age, histopathologic subtype, and response to chemotherapy was analyzed. Survival analysis was performed by the Kaplan-Meier test using the approximate chi-square statistic for the log-rank test. RESULTS: The ErbB-2 protein was detected in 11 of 26 osteosarcoma tissues (42%) by immunoblot analysis. Expression of ErbB-2 was confirmed by immunohistochemical studies using specific anti-ErbB-2 monoclonal antibody. However, neither amplification of the c-erbB-2 gene nor evidence of significant genetic mutation was found in these osteosarcomas. Expression of ErbB-2 examined by immunoblotting was most strongly correlated with early pulmonary metastases (P < 0.05). Among the entire group of 26 patients in this study, Kaplan-Meier life table survival of the patients with apparent ErbB-2 expression was significantly worse than that of the patients with little ErbB-2 expression (P < 0.01). CONCLUSIONS: In 42% of the osteosarcomas, the tumor cells expressed ErbB-2. Expression of ErbB-2 was strongly correlated with early pulmonary metastasis and poor survival rate for the patient. These data suggest that ErbB-2 plays a significant role in aggressive tumor growth and in the promotion of metastatic potential in osteosarcomas. ErbB-2 in the osteosarcoma tissues would be a useful prognostic marker for patients.

Adolescent↗

PubMed1996

Induction of HIV-1 Nef-specific cytotoxic T lymphocytes by Nef-expressing DNA vaccine.

Recently, some individuals who have remained seronegative despite definite exposure to HIV-1 have been reported. Among these individuals, an unusually high frequency of HIV-1 Nef-specific cytotoxic T lymphocytes was observed. Direct injection of plasmid DNA encoding foreign antigen can elicit both cell-mediated immunity and antibody responses (DNA vaccine). We constructed an HIV-1 Nef-expressing plasmid, and we induced HIV-1 Nef-specific cytotoxic T lymphocytes. This is the first report of inducing HIV-1 Nef-specific cytotoxic T lymphocytes by DNA vaccine.

Amino Acid Sequence↗

PubMed1996

Genetic control of in vivo tumor necrosis factor production in mice.

We report on the genetic effect on in vivo production of tumor necrosis factor (TNF)-alpha induced by lipopolysaccharides (LPS) using various congenic mouse strains. B10.A, Bl0.A(3R), B10.AQR, B10.A(5R), and B10.S(7R) produced significantly high TNF-alpha compared with B10.BR, B10.S, C57BL/10, B10.A(2R), B10.A(4R), B10.G, B10.DA(80NS), and B10.RIII(71NS). This suggests that LPS-induced TNF-alpha production is genetically controlled by H-2. Mice with the same alleles on K, A, E, or S loci produced various (high or low) levels of TNF-alpha, thus indicating that regulatory genes are located outside these loci. All strains with H-2Dd produced significantly high levels of TNF-alpha, but strains with other alleles in the H-2D locus produced low levels. Thus, TNF-alpha production appears to be genetically linked to H-2D itself or H-2D linked genes and the allele d is linked to a high responder gene. This was the case with the A background. C3H/HeN (H-2k), however, showed a high TNF-alpha production, suggesting the presence of another controlling gene outside H-2. In addition, high TNF-alpha productivity was transmitted into F1 mice (B10.A X B10.BR) in a dominant fashion. Both LPS-stimulated and unstimulated TNF-alpha mRNA expression in splenic macrophages were enhanced in high responder strains. Thus, we conclude that TNF-alpha production is closely related to genes within or linked to the H-2D locus as well as others outside H-2.

Animals↗

PubMed1996

Simple-spike activity of floccular Purkinje cells responding to sinusoidal vertical rotation and optokinetic stimuli in alert cats.

To understand how the cerebellar flocculus is involved in the processing of semicircular canal signals in the vertical vestibulo-ocular reflex (VOR), we analyzed the simple-spike activity of floccular Purkinje (P) cells that was modulated by sinusoidal pitch rotation, and then analyzed their activity during presentation of sinusoidal vertical optokinetic stimuli in alert, head-fixed cats. The great majority of P cells also responded to optokinetic stimuli with peak discharge near peak stimulus velocity. Eighty percent of P cells that responded to both pitch and optokinetic stimuli showed increased activity when the directions of the resultant eye movements were the same. During rapid modification of the VOR induced by visual pattern movement, modulation amplitudes of the cells tested increased together with the eye velocity increase. Maximal activation directions of these cells studied during vertical rotation in many planes were near the vertical canal planes, similar to those in our previous studies. The remaining 20% of P cells showed increased discharge for the same direction of stimulus movement. These results suggest that the activity of the majority of pitch-responding P cells contains, at least partly, a vertical eye velocity component during presentation of vestibular or optokinetic stimuli in addition to canal inputs during pitch rotation.

Animals↗

PubMed1996

Cross axis vestibulo-ocular reflex induced by pursuit training in alert monkeys.

We examined the effect of conflicting vestibular and smooth pursuit information on the vestibulo-ocular reflex (VOR) in alert monkeys. Sinusoidal whole body rotation was applied either in the pitch or yaw plane while presenting a target spot that moved orthogonally to the rotation plane. The monkeys were rewarded for tracking the spot and eye movements induced by rotation alone were examined every 15-30 min in complete darkness. Orthogonal eye movement responses to rotation were not observed before training but appeared after about 30 min of training. The gains (eye/chair) of the orthogonal component increased up to 0.2 after 1-2 h and were largest at the training frequency and approximately in phase with the stimulus; phase advanced at lower frequencies and lagged at higher frequencies. Amplitude tuning was also demonstrated when examined using different amplitudes at a constant frequency after training. Two hours of training to fixate an earth-stationary spot projected onto a patterned visual background that moved orthogonally to the rotation plane during rotation, did not induce a cross axis response. These results indicate that pursuit training during VOR is effective in inducing cross-axis eye movement responses that are tuned to the metrics of the training stimulus.

Adaptation, Physiological↗

PubMed1996

Adaptive changes in human smooth pursuit eye movement.

Adaptive changes in initial eye velocity of pursuit eye movement were examined in nine normal subjects using a target that moved in a multiple ramp fashion. Significant changes in initial eye velocity occurred rapidly after training in six of the subjects. The magnitude and direction of the induced changes were a function of the training conditions. Adaptive changes started 100-200 ms after onset of pursuit eye movement (usually 140 ms), suggesting that the late (but not early) component of initial eye velocity was under adaptive control by our training paradigms.

Adaptation, Physiological↗

PubMed1996

Chromophore reorientations in the early photolysis intermediates of bacteriorhodopsin.

The photoselection-induced time-resolved linear dichroism of a bacteriorhodopsin suspension of purple membrane from 350 to 750 nm is measured by a new pseudo-null measurement technique. In combination with time-resolved absorption measurements, these linear dichroism measurements are used to determine the reorientation of the retinal chromophore of bacteriorhodopsin from 50 ns to 50 microseconds after photolysis. This time range covers the times when the K photointermediate decays to form L, as well as the early times during the formation of the M intermediate in the photocycle. An analysis of the photoselection-induced linear dichroism measured directly, along with the absorbance changes polarized parallel to the linearly polarized excitation, shows that the anisotropy is invariant over this time period, implying that the photolyzed chromophore rotates less than 8 degrees C with respect to unphotolyzed chromophores during this part of the photocycle.

Anisotropy↗

PubMed1996

Further evidence for the specific involvement of the flocculus in the vertical vestibulo-ocular reflex (VOR).

We examined the simple-spike activity of floccular Purkinje (P) cells during sinusoidal pitch rotation and vertical optokinetic stimuli in alert, head-fixed cats. The great majority of pitch-responding P cells also responded to optokinetic stimuli with increased activity when the directions of the resultant eye movements were the same. During rapid modification of the VOR induced by visual pattern movement, modulation amplitudes of the cells tested increased together with the eye velocity increase. Maximal activation directions of these cells studied during vertical rotation in many planes were near the vertical canal planes. These results suggest that the activity of the majority of pitch-responding P cells contains a vertical eye velocity component during vestibular or optokinetic stimuli in addition to canal inputs during pitch rotation.

Animals↗

PubMed1996

The human foamy virus Bel-1 transcription factor is a sequence-specific DNA binding protein.

The Bel-1 transcriptional transactivator encoded by human foamy virus (HFV) can efficiently activate gene expression directed by both the HFV long terminal repeat (LTR) and internal (Int) promoter elements. By DNA footprinting and gel retardation analysis, we demonstrate that Bel-1 can specifically bind to discrete sites in both the LTR and Int promoter elements in vitro. However, transactivation of the HFV LTR by Bel-1 was observed to require not only the promoter-proximal Bel-1 binding site identified in vitro but also additional promoter-distal sequences. These data suggest that Bel-1 binding is necessary but not sufficient for efficient transactivation of Bel-1-responsive promoters in mammalian cells and therefore raise the possibility that Bel-1 function may require the action of a cellular DNA binding protein(s). Importantly, these data demonstrate that Bel-1 is unique among retroviral regulatory proteins in being a sequence-specific DNA binding protein.

Base Sequence↗

PubMed1996

[Coronary risk factors in children of young coronary artery disease patients].

PURPOSE: To determine whether children and adolescents, whose fathers have established coronary artery disease (CAD), have increased prevalence of coronary risk factors (RF). METHODS: The frequencies of abnormal values of lipid variables, glucose, blood pressure, obesity index (calculated through Newen-Goldstein index), smoking and electrocardiographic alterations (ECG), were assessed in 280 descendents of young revascularized patients (< 55 years). The study population was divided in two groups according to age, respectively GA (2 to 12 years) and GB (12 to 19 years). Eventual influences of age, gender, obesity and smoking on lipid variable were evaluated through variance analysis. RESULTS: Of the study population, 48.2% and 44.6% had total cholesterol (TC) and LDL-C respectively above the desirable values; 21.7% and 26.1% had values similar to adults under increased risk. Triglyceridemia (TG) > 200mg/dl was found in 1.4% of the sample and lower values of HDL-C in 16.8%. Overweight and obesity were observed in 13.1% and 20.0% of the patients and influenced TG levels in GB. Smoking occurred in 10.4%; hypertension in 3 cases and none had abnormal glucose levels or ECG. CONCLUSION: Healthy children of fathers with established CAD, exhibit a high frequency of altered lipid profile and increased body weight. The results suggest the need for early identification of RF in offspring of young CAD patients, thus emphasizing changes in risk profile and improving lifestyle.

Adolescent↗

PubMed1995

A macromolecular multicomponent peptide vaccine prepared using the glutaraldehyde conjugation method with strong immunogenicity for HIV-1.

The immunogenicity of a newly constructed macromolecular multicomponent peptide vaccine candidate against human immunodeficiency virus type 1 (HIV-1) was compared with that of previously reported vaccine candidates. This vaccine candidate is composed of a macromolecular multicomponent peptide complex consisting of three V3 region peptides, one Gag region peptide, and CD4-binding site peptide and was constructed using the multiple-antigen peptide and glutaraldehyde methods. Sera from rabbits immunized with this newly constructed vaccine showed strong antibody titers against each constituent peptide antigen. Furthermore, these antibodies exhibited strong neutralizing and antifusion activity toward HIV-1IIB, HIV-1MN, and fresh isolates from Japanese HIV-seropositive individuals. These results show that this new vaccine candidate has the capacity to induce strong, polyvalent immunogenicity and therefore may prove to be a powerful peptide vaccine against HIV-1 infection.

AIDS Vaccines↗

PubMed1995

Neutralization of HIV-1 by secretory IgA induced by oral immunization with a new macromolecular multicomponent peptide vaccine candidate.

Control of pandemic infection of human immunodeficiency virus type 1 (HIV-1) requires some means of developing mucosal immunity against HIV-1 because sexual transmission of the virus occurs mainly through the mucosal tissues. However, there is no evidence as yet that the secretory immunoglobulin A (IgA) antibody induced by immunization with antigens in experimental animals can neutralize HIV-1. We demonstrate here that oral immunization with a new macromolecular peptide antigen and cholera toxin (CT) induces a high titre (1:2") of gut-associated and secretory IgA antibody to HIV-1. Using three different neutralizing assays, we clearly demonstrate that this secretory IgA antibody is able to neutralize HIV-1IIIB, HIV-1SF2 and HIV-1MN. Our new approach may prove to be important in the development of a mucosal vaccine that will provide protection of mucosal surfaces against HIV-1.

AIDS Vaccines↗

PubMed1995

Antibody responses raised against a conformational V3 loop peptide of HIV-1.

The amino acid sequence of the principal neutralizing determinant (PND) of 224 cases of human immunodeficiency virus type 1 (HIV-1) was determined and the most frequently occurring sequence was used as a peptide antigen for studying virus-specific antibody responses. In our present study, a linear peptide of the most frequent PND was first synthesized and then oxidized to create a disulfide-bridged loop conformation. Then, in order to construct a macromolecular structure for the purpose of increasing antigenicity, the synthetic peptide was conjugated to a core peptide. We compared the immunogenicity of the disulfide-bridged loop PND peptide antigen (AG4) and the linear PND peptide antigen (AG5). After immunizing rabbits 5 and 6 times with both peptides, the results obtained using ELISA revealed that AG4 (conformational-loop type) was more capable of inducing a high titer of antigen-specific antibodies than was AG5 (linear type). Despite an amino acid sequence homology of 72%, a 1:8 dilution of serum raised against AG4 inhibited 81.9% of HIV-1IIIB-mediated cell fusion, suggesting that conformational V3 loop peptide is able to elicit an antibody response which is strongly HIV-1-specific.

Amino Acid Sequence↗

PubMed1995

Discharge characteristics of vestibular and saccade neurons in the rostral midbrain of alert cats.

1. The interstitial nucleus of Cajal (INC) is involved in generation of vertical eye movements. To further explore the neural substrates of INC involvement in eye movements, we examined the discharge characteristics of neurons in the rostral midbrain that discharge for vestibular stimulation and saccades in alert cats. We analyzed 75 vestibular and saccade neurons (VSNs) that showed characteristic discharge during pitch rotation. Of these, 50 exhibited gradually increasing activity during upward slow phases and burst of action potentials during downward fast phases induced by downward pitch. The remaining 25 cells showed the opposite response pattern: gradually increasing activity during downward slow phases and a burst during upward fast phases induced by upward pitch. We classified these cells as downward VSNs and upward VSNs, respectively. The effects of electrical stimulation of the contralateral vestibular nerve were tested for 13 downward VSNs and 2 upward VSNs; all of them were activated at short latencies. 2. All vertical VSNs also showed burst activity associated with spontaneous saccades. The preferred direction for the burst was always the same for saccades and vestibular fast phases. The onset of burst activity preceded the onset of fast eye movement by an average of 35 +/- 14 ms (mean +/- SD). During fast eye movement in other directions, many of these cells also showed an inconsistent burst and the burst often began after the onset of saccades. Half of the upward VSNs examined showed a pause in activity during downward fast eye movement. 3. Burst parameters in the preferred directions were correlated with saccade parameters in half of the vertical VSNs examined, although the correlation coefficients were typically 0.5-0.6. 4. All vertical VSNs had irregular resting activity. Half of the downward VSNs examined showed eye-position sensitivity toward the direction opposite to the preferred direction for the burst activity but only if the period of analysis was restricted to discharge shortly before and after saccades. 5. All downward VSNs examined during sinusoidal rotation in several vertical planes received strong excitatory input from the contralateral anterior canal. Three of four upward VSNs examined received contralateral posterior canal excitation; the exception received ipsilateral posterior canal excitation.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1994

Intracellular receptor-type transcription factor, LasR, contains a highly conserved amphipathic region which precedes the putative helix-turn-helix DNA binding motif.

We have cloned and sequenced the lasR gene, which is involved in the transcriptional activation of several pathogenic factors, from Pseudomonas aeruginosa IFO3455 and PA103. These clones were predicted to be an open reading frame of 239 amino acids as reported for the PAO1 strain. There is only a single base change resulting in an amino acid exchange from M145 (PAO1) to I (IFO3455). PA103 DNA differs with PAO1 DNA in two bases resulting in only a single amino acid substitution from R179 to W. When the IFO3455 LasR was expressed in a PA103 strain which is known to be LasR defective, proteinase gene activation was detected, however, when PA103 LasR was expressed, no enhancement was measurable. From these results, it appears that the amino acid substitution of R179 to W inactivated LasR activity. This substitution is located in the highly conserved sequence found in many transcription factors, including sigma factors, and may disrupt amphipathic alpha-helix, predicted for the 176 to 189 region, which precedes the putative helix-turn-helix DNA binding motif. We presumed that these three helices may contribute to specific DNA binding.

Amino Acid Sequence↗

PubMed1994

NF-kappa B and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene.

Expression of the human monocyte chemoattractant protein-1 (hMCP-1) is ubiquitous in various cell types and is increased by a wide variety of stimuli. We initially found that the effects of various stimuli, including IL-1 beta, TNF-alpha, and 2-O-tetradecanoylphorbol 13-acetate, on the expression of hMCP-1 mRNA were quite different among A172 glioblastoma cells, HT1080 fibrosarcoma cells, and SKLMS1 leiomyosarcoma cells. These findings suggested that hMCP-1 expression is regulated both in a stimulus-specific and a tissue-specific manner. To elucidate the mechanism underlying this stimulus-specific and tissue-specific regulation, we isolated a hMCP-1 5'-flanking genomic DNA fragment and sequenced it extensively up to bp 3011 upstream from the transcriptional start site. Among many putative cis-elements, we identified two cis-elements critical for the transcription of the hMCP-1 gene. The first element is a remote kappa B binding site located far upstream between bp -2612 and -2603 that was important for IL-1 beta-, TNF-alpha-, and 2-O-tetradecanoylphorbol 13-acetate-induced enhancer activity. Mutation at the kappa B consensus site resulted in a complete loss of these stimulus-induced enhancer activities. The second element is a GC box located between bp -64 and -59 that was important for the maintenance of basal transcriptional activity. Overexpression of rSp1 resulted in increased hMCP-1 transcriptional activity, possibly suggesting the role of Sp1 in controlling basal hMCP-1 transcription via this GC box. These results together indicate that hMCP-1 expression is controlled by at least two distinct regulatory elements: a kappa B site and a GC box that seem to be associated with stimulus-specific and tissue-specific regulation, respectively.

Base Sequence↗

PubMed1994

Voluntary control of saccadic eye movement in patients with frontal cortical lesions and parkinsonian patients in comparison with that in schizophrenics.

To investigate whether the abnormalities of antisaccades in schizophrenics could be explained by a dysfunction of the frontal cortex, we examined 10 patients with frontal cortical lesions and 22 patients with idiopathic Parkinson's disease with mild symptoms (Yahr I-II) using the same tasks, and compared the results with those obtained in schizophrenics. The frontal patients with lesions covering the frontal eye field and prefrontal cortex showed more errors, longer latencies, and lower peak velocities in the antisaccade task, despite giving normal results in the visually guided saccade task. This was similar to the results observed in schizophrenics. Parkinsonian patients did not consistently show a significant difference in the antisaccade task. These results indicate specific abnormalities of antisaccades in schizophrenics and patients with frontal cortical lesions but not consistently in Parkinsonian patients. This suggests that the abnormalities of antisaccades in schizophrenics might be explained by a frontal cortical dysfunction.

Adult↗