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Biomedical subjects

M H Hu

Publications and source records attributed to M H Hu.

At least 19 recordsLinked to original sources

Dendritic cells pulsed with gp96-peptide complexes derived from human hepatocellular carcinoma (HCC) induce specific cytotoxic T lymphocytes.

Dendritic cells (DCs) are one of the most potent antigen-presenting cells (APCs) capable of activating immune responses. Different forms of tumor antigens have been used to load DCs to initiate tumor-specific immune responses. Heat shock proteins (HSPs) are considered natural adjuvants which have the ability to chaperone peptides associated with them presented efficiently by interaction with professional APCs through specific receptors. In the present study, we used HSP, gp96-peptide complexes, derived from human hepatocellular carcinoma (HCC) cells as antigens for pulsing DCs. We found that gp96-peptide complexes derived from HCC cells induced the maturation of DCs by enhancing expression of human leukocyte antigen class II, CD80, CD86, CD40, and CD83. The matured DCs stimulated a high level of autologous T cell proliferation and induced HCC specific cytotoxic T lymphocytes, which specifically killed HCC cells by a major histocompatability complex (MHC) class I restricted mechanism. These findings demonstrate that DCs pulsed with gp96-peptide complexes derived from HCC cells are effective in activating specific T cell responses against HCC cells.

Antigen Presentation↗

Human papillomavirus type 16 E7 peptide(38-61) linked with an immunoglobulin G fragment provides protective immunity in mice.

OBJECTIVE: To explore whether the recombinant protein (Human papillomavirus (HPV) type16 E7 peptide(38-61) linked with an immunoglobulin G fragment) will generate protective immunity in mouse model. METHODS: In our study, we combined the HPV16 E7 peptide(38-61) with a murine IgG heavy chain constant region to construct a chimeric protein compound, which was highly expressed as inclusion bodies in a bacterial expression system with Escherichia coli. The purified chimeric protein was injected into C57BL/6 mice and the efficiency of the chimeric vaccine candidate was evaluated by antibody response assay, T cell proliferation assay, CTL assay, tumor challenge assay and therapeutic experiment. RESULTS: The chimeric vaccine candidate was able to induce anti-HPV antibodies as well as to elicit HPV16 E7-specific CTLs and T cell proliferation in a pre-clinical mouse model. It was also able to effectively protect mice against the challenge of HPV16-positive tumor cells, and to eradicate HPV16-expressing tumors in mice. CONCLUSIONS: The chimeric protein vaccine can induce E7-specific immune responses and protect mice against challenge of HPV16-positive tumor, even eradicate developed tumor. The results indicated a possibility to use the chimeric protein vaccine to protect human against HPV infection.

Animals↗

[The study of P68 RNA helicase on cell transformation].

The P68 nuclear protein is an ATP-dependent RNA helicase and RNA-dependent ATPase, and has been proved to have functions in the regulation of cell growth and division. Different phenotypes of P68 between tumouric cell lines (HeLa, TC3H10) and non-tumouric cell lines (NIH3T3 and NC3H10) were observed, and we reasoned that overexpression of P68 might profoundly affect cellular growth properties. We report here that overexpression human P68 by transfection of recombinant P68 expression vector in NIH3T3 and NC3H10 fibroblasts causes their tumorigenic transformation as determined by four criteria: (1) the morphology of transfectants from long fibric to spindle shaped; (2) formation of transformed foci on the monolayer of cells; (3) the transfectants acquired the ability to grow on soft agar forming cell microsphere cube; (4) tumor formation in nude mice after injection of transfected cells for 3-4 weeks.

3T3 Cells↗

Pleckstrin 2, a widely expressed paralog of pleckstrin involved in actin rearrangement.

We have identified a cDNA for pleckstrin 2 that is 39% identical and 65% homologous to the original pleckstrin. Like the original pleckstrin 1, this protein contains a pleckstrin homology (PH) domain at each end of the molecule as well as a DEP (Dishevelled, Egl-10, and pleckstrin) domain in the intervening sequence. A Northern blot probed with the full-length cDNA reveals that this homolog is ubiquitously expressed and is most abundant in the thymus, large bowel, small bowel, stomach, and prostate. Unlike pleckstrin 1, this newly discovered protein does not contain obvious sites of PKC phosphorylation, and in transfected Cos-7 cells, it is a poor substrate for phosphorylation, even after PMA stimulation. Cells expressing pleckstrin 2 undergo a dramatic shape change associated with actin rearrangement, including a loss of central F-actin and a redistribution of actin toward the cell cortex. Overexpression of pleckstrin 2 causes large lamellipodia and peripheral ruffle formation. A variant of pleckstrin 2 lacking both PH domains still had some membrane binding but did not efficiently induce lamellipodia, suggesting that the PH domains of pleckstrin 2 contribute to lamellipodia formation. This work describes a novel, widely expressed, membrane-associating protein and suggests that pleckstrin 2 may help orchestrate cytoskeletal arrangement.

Actins↗

Expression of a novel chimeric protein containing the A chain of tissue-type plasminogen activator and the B chain of pro-urokinase in insect cells using the baculovirus system.

A hybrid cDNA tu-pa, which contains Ser1-Thr263 of tissue-type plasminogen activator (t-PA) and Ser138-Leu411 of pro-urokinase (pro-UK) was constructed and expressed in the Sf9-AcNPV system. The expression level was approximate 2.5 mg/L. Tu-PA was purified via one-step affinity column conjugated with monoclonal antibody against the B chain of pro-UK, which showed a single band of approximate 60 kDa in SDS-PAGE. The specific activity of the chimeric protein on fibrin plate was 200,000 IU/mg protein. Tu-PA had a higher selectivity for fibrin than UK and pro-UK. Its activity can be promoted by CNBr degraded fibrin fragments as t-PA.

Animals↗

High level expression of recombinant plasminogen activator inhibitor-1 in Escherichia coli and generation of its mutants involving Asp125, Glu128 and Glu130.

Plasminogen activator inhibitor-1 (PAI-1) cDNA was expressed in Escherichia coli (E. coli) with high efficiency using a heat-inducible vector. About 100 mg of recombinant PAI-1 (rPAI-1) could be obtained from 1 liter of bacteria culture. rPAI-1 in inclusion bodies was purified by pI precipitation and Sephadex G-75 chromatography. After treatment with 4 mol/L guanidinium chloride and dialysis, the largely inactive PAI-1 gained considerably in activity as judged by its reaction with low molecular weight u-PA (LMW-u-PA). Degenerated oligonucleotides containing ApaI site and mutations at Asp125, Glu128, Glu130 in PAI-1 cDNA were synthesized. To facilitate the introduction of mutations, an ApaI site was first generated in PAI-1 cDNA using one of the oligonucleotides. Taking advantage of the APaI site, thirteen PAI-1 mutants involving Asp125, Glu128 and Glu130 were produced with these oligonucleotides using PCR. Most of the PAI-1 mutants had a similar activity as compared to wild type PAI-1, while some of the triple-site mutants had completely lost their activity.

DNA, Complementary↗

The relation between standing balance and walking function in children with spastic diplegic cerebral palsy.

To establish and compare the relationship between standing balance and walking performance, eight children with spastic diplegic cerebral palsy (CP) and 16 non-disabled, age- and sex-matched children were studied. The results showed that the children with CP had worse static balance stability in various sensory environments and dynamic balance (rhythmic shifting ability) than the non-disabled children. Moreover, the children with CP walked at a slower speed but at a greater physiological cost than the non-disabled children. In the children with CP, dynamic balance significantly correlated with walking function. It is suggested that rhythmic weight-shift training should be encouraged to improve the walking performance of children with CP.

Adolescent↗

Management for patients with advanced T4 epidermoid carcinoma of the esophagus.

Available data concerning the treatment of patients with advanced T4 esophageal carcinoma are limited. A consecutive series of 42 patients with advanced T4M0 epidermoid carcinoma of the esophagus were studied from June 1987 to July 1992. The aim of this study was to evaluate the efficacy of various therapeutic modalities, and further evaluate the therapeutic options. The various therapeutic modalities included the following: Group I, feeding jejunostomy or endoesophageal intubation, 6 patients; Group II, palliative subtotal esophagectomy only, 8 patients; Group III, bypass procedures without tumor resection, 9 patients; Group IV, nutritional support and then treatment with irradiation (n=8) or concurrent radio-chemotherapy (n=4), 12 patients; Group V, subtotal esophagectomy, followed by aggressive concurrent radiochemotherapy, 7 patients. The total prescribed irradiation dose was 60 Gy (10 Gy/5 fractions/week). A combination regimen of chemotherapy consisted of cisplatin, 5-fluorouracil, and leucovorin (PFL regimen). For the patients undergoing esophagectomy or bypass procedures (n=24), the rates of operative complication and mortality were 45.8% and 25%, respectively. Side effects of adjuvant therapy (n=24) consisted of main airway irritation (100%), mucositis or gastrointestinal symptoms (83.3%), hematologic toxicity (79.2%), esophagitis or gastric ulcer (62.5%), alopecia (37.5%), and pneumonia (20.8%). The mortality due to toxicity of adjuvant therapy was 21.1% (4/19 patients). The mean survival times for each of the different groups was 1.9+/-0.5 months for Group I, 4.8+/-1.6 months for Group II, 5.2+/-1.2 months for Group III, 7.3+/-2.0 months for Group IV, and 20.3+/-2.5 months for Group V, respectively. Compared with patients of Groups I--IV, the Group V patients had a significantly superior one-year survival rate (P<0.01). Our results demonstrated that esophagectomy followed by concurrent irradiation and PFL combination chemotherapy may provide a significant improvement in the quality of life and survival for appropriate patients with advanced T4M0 epidermoid carcinoma of the esophagus. Furthermore, more than one cycle of PFL regimen chemotherapy may result in a better prognosis. During the performance of such an aggressive treatment, the utmost care must be taken with the patient's nutrition and to prevent pulmonary complications.

Aged↗

Validity of force platform measures for stance stability under varying sensory conditions.

Maintaining stance stability under varying sensory environment is an essential function in the elderly and among patients. Testing sensory organization ability of standing balance, the Sensory Organization Test (SOT), has become a standard procedure in many clinical and laboratory settings. The stance stability can be quantified by two forceplate measures in the SOT: the equilibrium score (ES) and the sway area (SA). This study compares the validity of the ES and the SA in detecting gender, trial, and sensory effects on stability in twenty (ten male, ten female) healthy young adults. Subjects were tested under six sensory conditions: eyes open (EO), eyes closed (EC), sway-referenced vision (Vs), sway-referenced support surface (Ss), eyes closed sway-referenced support surface (ECSs), and sway-referenced visual surround and support surface (VsSs). A visual surround and/or the support surface were tilted proportionately to the subject's spontaneous sway in the sway-referenced conditions. Three trials, 20-second for each trial, were repeated for each sensory condition. Above results demonstrated that the Pearson correlation coefficients between the ES and the SA were all highly significant (p < .0001) except for the first trial of the EO condition. The consistencies in which the two measures discriminated among sensory conditions were tested by the Kendall's coefficient of concordance. The Kendall's coefficient for the ES (W = .843) and the SA (W = .866) were high and similar. Separate ANOVA procedure for the ES and SA revealed that both measures satisfactorily detected a significant sensory condition and trial effects and insignificant gender effect. We can conclude that ES and SA are valid measures of stance stability during the SOT. Our results confirm that healthy young adults have a poorer postural stability when the visual and somatosensory inputs are simultaneously altered. Moreover, the learning effect is observed during repeated trials within test conditions.

Adult↗

Production of human insulin in an E. coli system with Met-Lys-human proinsulin as the expressed precursor.

The construction of a gene encoding Lys-human proinsulin, its direct expression in E. coli, and the simple purification procedure are described here. The temperature inducible promotor was employed for induction in a very short time. The expression level could reach 20-30%. After simple downstream processing and only one step of Sephadex G50 purification, 150 mg recombinant Lys-human proinsulin with a purity of up to 90% could be obtained easily from 1 L of high density fermentation medium. The obtained product is in the form of Met-Lys-human proinsulin because of the failure of the bacterial host to remove the initiator methionine residue. The Lys-human proinsulin could be changed into human insulin by trypsin and carboxypeptidase B treatment in later steps. After separation with DEAE-Sephadex A25, human insulin with expected amino acid composition and full native biological activity could be obtained with a yield of 50 mg/L of fermentation medium.

Base Sequence↗

Double-C-peptide human proinsulin.

A fusion gene encoding double-C-peptide human proinsulin was constructed by insertion of a DNA fragment encoding human C-peptide into the 5'-terminal C-peptide coding sequence of a synthetic human proinsulin gene with correct reading frame and over-expressed in E. coli. The purified double-C-peptide human proinsulin shows decreased activity in receptor binding and insulin immune assays as compared with human proinsulin. Disulphide bond reconstitution studies demonstrate that there is not much more influence of the protein concentration on the yield of refolded double-C-peptide human proinsulin. The double-C-peptide human proinsulin shows a 1.86-fold human C-peptide immune activity as compared with that of human proinsulin and gives a good yield of the molecule with correct disulphide bonds in reconstitution studies strongly suggesting the existence of very flexible conformation of the C-peptide.

Amino Acid Sequence↗

Multisensory training of standing balance in older adults: I. Postural stability and one-leg stance balance.

BACKGROUND: The effects of standing balance training on the ability to maintain stability in both static two-leg and one-leg stance were tested in healthy older adults. METHODS: Subjects (age range 65-90 years) were randomly assigned to a training (n = 12) or control group (n = 12). Training subjects received a 10-hour balance training program which selectively manipulated sensory inputs from the visual, vestibular, and somatosensory systems. RESULTS: Training subjects showed significantly improved stability (root-mean-square values of anteroposterior platform torque) after training in five of the eight training conditions (when somatosensory inputs were changed or when two or more sensory systems were simultaneously manipulated) (p < .006). When tested 4 weeks after completion of training, subjects (a) fell less frequently when the ankle/foot somatosensory inputs were minimized and (b) stood longer on one leg than the control group (p < .001). CONCLUSIONS: Balance training designed to improve intersensory interaction could effectively improve balance performance in healthy older adults.

Accidental Falls↗

Multisensory training of standing balance in older adults: II. Kinematic and electromyographic postural responses.

BACKGROUND: The purpose of this study was to analyze the electromyographic and kinematic characteristics of postural responses of 24 healthy older adults both prior to and immediately after a multisensory balance training period. It was hypothesized that the muscle and movement characteristics of postural responses would be optimized in the group after training. METHODS: Balance was tested by determining muscle and kinematic response characteristics used in compensating for support surface displacements. Twenty-four subjects were randomly assigned to a training group or a control group. The training group received a multisensory balance training program during a 15-day training period (described in the companion article). RESULTS: The results showed that the training (a) significantly shortened the onset latency of the neck flexor muscle (p < .05); (b) showed a trend toward decreasing the response frequency of antagonist muscles; (c) showed a trend toward increasing the response frequency of the trunk flexor muscles; and (d) showed a trend toward decreasing the maximal excursion of the first-trial of the ankle joint rotation. CONCLUSIONS: From these results and those of the companion article, it was concluded that the multisensory balance training program had an effect in optimizing the muscle and movement characteristics of postural response in the training group older adults.

Accidental Falls↗

Biochemical properties of recombinant mutants of nonglycosylated single chain urokinase-type plasminogen activator.

The role of glycosylation on the enzymatic properties of single chain urokinase-type plasminogen activator (scu-PA) was investigated by site-specific mutagenesis of the glycosylated Asn-302 residu to Gln. In addition, the role of the NH2-terminal polypeptide chain and of the Cys-148 to Cys-279 interchain disulphide bond on the activity of non-glycosylated scu-PA was investigated. Therefore, variants of recombinant scu-PA (rscu-PA) were produced by transfecting Chinese hamster ovary cells with cDNA encoding rscu-PA N302Q (rscu-PA with Asn-302 to Gln mutation), rscu-PA C279A,N302Q (rscu-PA with Cys-279 to Ala and Asn-302 to Gln mutations) or rscu-PA del(N2-F157)C279A,N302Q (rscu-PA C279A,N302Q with deletion of Asn-2 through Phe-157). These mutants were purified to homogeneity from conditioned cell culture medium and were obtained essentially as single chain molecules with specific activities on fibrin plates of (mean +/- S.E.; n = 6) 45,000 +/- 5000. IU/mg, 19,000 +/- 800 IU/mg and < or = 100 IU/mg for rscu-PA N302Q, rscu-PA C279A,N302Q and rscu-PA del(N2-F157)C279A,N302Q, respectively, as compared to 64,000 +/- 2600 IU/mg for wild-type rscu-PA obtained in the same expression system. Plasmin quantitatively converts rscu-PA N302Q and rscu-PA C279A,N302Q to amidolytically active two-chain derivatives with a specific activity of 56,000 IU/mg and 32,000 IU/mg, respectively, as compared to 75,000 IU/mg for wild-type rscu-PA. Plasminogen activation as a function of time was comparable for rscu-PA N302Q and wild-type rscu-PA, and somewhat slower for rscu-PA C279A,N302Q. In a human plasma milieu in vitro, consisting of a 125I-fibrin labeled plasma clot submerged in plasma, 50 percent clot lysis in 2 h required 2.2 micrograms/ml rscu-PA N302Q and 6.0 micrograms/ml rscu-PA C279A,N302Q, as compared to 3.2 micrograms/ml wild-type rscu-PA. In contrast, rscu-PA del(N2-F157)C279A,N302Q was not converted to an amidolytically active two chain derivative by plasmin, and did not induce significant plasminogen activation in purified systems or clot lysis in a human plasma milieu. Following bolus injections in hamsters, the initial half-lives (1.8-2.6 min) and the plasma clearances (0.6-1.5 ml min-1) were comparable for wild-type rscu-PA and for the three rscu-PA mutants. These results suggest that the fibrinolytic activity in a plasma milieu in vitro and the in vivo turnover of rscu-PA are not markedly affected by the absence of carbohydrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Biochemical properties of recombinant single-chain urokinase-type plasminogen activator mutants with deletion of Asn2 through Phe157 and/or substitution of Cys279 with Ala.

The contribution of the NH2-terminal polypeptide chain and of the Cys148-Cys279 interchain disulphide bond to the enzyme activity of urokinase-type plasminogen activator (u-PA) was studied using site-specific mutagenesis. Recombinant single-chain u-PA (rscu-PA) variants were produced by transfecting Chinese hamster ovary cells with cDNA encoding des(Asn2-Phe157)rscu-PA (rscu-PA with deletion of Asn2-Phe157), [Ala279]rscu-PA (rscu-PA with Cys279----Ala mutation) or des(Asn2-Phe157)[Ala279]rscu-PA [des(Asn2-Phe157)rscu-PA with Cys279----Ala mutation]. Des(Asn2-Phe157)rscu-PA, [Ala279]rscu-PA and des(Asn2-Phe157)[Ala279]rscu-PA, purified from conditioned cell culture medium, were obtained as nearly homogeneous single-chain molecules with Mr approximately 30,000, 54,000 and 30,000, and specific fibrinolytic activities on fibrin plates of (mean +/- SD; n = 3) 860 +/- 150 IU/mg, 43.0 +/- 2.5 IU/micrograms and 240 +/- 20 IU/mg, respectively, compared to 69.0 +/- 4.3 IU/micrograms for wild-type rscu-PA obtained in the same expression system. The plasminogen activating potential in a buffer milieu of [Ala279]rscu-PA was somewhat lower than that of rscu-PA, but that of both deletion mutants was virtually abolished. In a human plasma milieu in vitro, consisting of a radiolabelled human plasma clot submerged in plasma, 50% clot lysis in 2 h required 6.5 micrograms/ml [Ala279]rscu-PA or 3.4 micrograms/ml rscu-PA, whereas with both deletion mutants no significant clot lysis was observed with up to 16 micrograms/ml. Treatment of [Ala279]rscu-PA or rscu-PA with plasmin resulted in quantitative conversion to two-chain molecules and was associated with an increase in specific amidolytic activity from about 600 IU/mg to 62.5 IU/micrograms for [Ala279]rscu-PA as compared to an increase from about 0.3 IU/micrograms to 75.0 IU/micrograms for rscu-PA. In contrast, no significant amidolytic activity could be generated by treatment of des(Asn2-Phe157)rscu-PA or des(Asn2-Phe157)[Ala279]rscu-PA with plasmin. The u-PA B-chain, isolated from plasmin-treated [Ala279]rscu-PA, had enzymic properties which were comparable to those of rtcu-PA, with respect to specific fibrinolytic activity, amidolytic activity, kinetics of plasminogen activation and clot-lysis activity in a human plasma milieu in vitro. Following bolus injection into hamsters, the plasma clearances were comparable (0.7-1.1 ml/min) for wild-type rscu-PA and for the three truncated rscu-PA mutants.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

Vacuole sign and nodule sign in early peripheral bronchogenic carcinoma. Diagnostic value and pathologic correlation.

We analyzed the radiological and pathological characteristics of 30 peripheral lung masses 1.5-3 cm in diameter. We describe the vacuole and the nodule sign. Retrospective examination of 100 conventional tomograms of peripheral pulmonary masses and comparison with the histological diagnoses allowed us to determine the usefulness of these two signs in differentiating small peripheral bronchogenic carcinomas from tuberculomas.

Adenocarcinoma↗

Studies on the role of the phi X174 gene A protein in phi X viral strand synthesis. II. Effects of DNA replication of mutations in the 30-nucleotide icosahedral bacteriophage origin.

phi X174 viral strand circular DNA can be synthesized in vitro from phi X174 replicative form I (RFI) DNA in the presence of the phi X A protein, the Escherichia coli DNA polymerase III elongation system, the E. coli rep helicase, and the E. coli single-stranded DNA binding protein. M13mp9 or pBR322 RFI DNAs containing a 30-base pair sequence found at the phi X origin of replication supported phi X A protein synthesis as well as the phi X template, giving rise to a net molar excess of deoxynucleotide incorporation. In this paper, we show that mutations in positions 1-3 of the 30-nucleotide origin replicated at a lower efficiency than plasmids containing the wild-type origin, because of a deficiency in the reinitiation reaction. Mutations in positions 4-7, upstream of the phi X A protein cleavage site, failed to support replication because of their inability to support nicking. An origin containing a mutation at the residue to which the phi X A protein is covalently linked to the DNA was an active template that supported a net molar excess of incorporation. Mutations at the 3' end of the origin region, retaining only the first 21-25 nucleotides of the 30-base pair origin, failed to support replication because of impaired binding of the phi X A protein to the template and consequently poor nicking. A construct bearing the first 28 nucleotides of the origin supported wild-type replication, as did a plasmid containing a 28-mer origin with a point mutation at position 26, but this latter construct also appeared to be partially deficient in phi X A protein binding activity. These results are consistent with the presence of a phi X A protein binding domain at the 3' end of the origin.

Bacteriophage phi X 174↗