Biomedical subjects

T H Ho

Publications and source records attributed to T H Ho.

At least 55 records · Page 3Linked to original sources

PubMed1991

Heat Shock Causes Selective Destabilization of Secretory Protein mRNAs in Barley Aleurone Cells.

The aleurone layer of GA(3)-stimulated barley (Hordeum vulgare L., cv Himalaya) grains is normally devoted to the synthesis and secretion of hydrolytic enzymes. Heat shock, however, suppresses the synthesis of the main hydrolytic enzyme, alpha-amylase, by destabilizing its otherwise highly stable mRNA (FC Belanger, MR Brodl, T-hD Ho [1986] Proc Natl Acad Sci USA 83: 1354-1358). In this paper we document that heat shock causes the suppression of the synthesis of some normal cellular proteins, while the synthesis of other normal cellular proteins is unaffected by heat shock. There are two major isozymic forms of alpha-amylase encoded by distinct mRNAs. The mRNA levels for both isozymic forms and the mRNA levels of two other secretory proteins, a protease and an endochitinase, were markedly reduced during heat shock. However, the levels of actin and beta-tubulin mRNAs, both nonsecretory proteins, were not diminished during heat shock. In addition, the levels of three other mRNA species detected by a set of unidentified cDNA clones (the sequence of one shows that it lacks a signal sequence) remained unchanged during heat shock. These data indicate that there are two classes of normal cellular protein mRNAs with regard to the effect of heat shock upon their persistence in the cell, and suggest that the distinction between them is whether or not they encode secretory proteins.

Journal Article↗

PubMed1990

Establishment and characterization of a new human cell line derived from ovarian clear cell carcinoma.

A new cell line, designated OCC1, was established from the ascitic fluid of a patient with a clear cell carcinoma of the ovary. The cell line grew well without interruption for over 12 months and over 80 passages. The doubling times of OCC1 were 36 and 38 hr at the 10th and 40th passages, respectively. Chromosomal analysis of the cell line showed hypertriploidy with modal number around 70-77. Several structural chromosomal abnormalities were consistently found. Electron microscopy revealed that OCC1 produced a basement membrane-like structure in vitro. Histological evaluation of xenografts from OCC1 in the 33th passage implanted and grown in nude (athymic) mice revealed a morphology identical to that of the original tumor.

Adenocarcinoma↗

PubMed1990

Heat shock proteins are not required for the degradation of alpha-amylase mRNA and the delamellation of endoplasmic reticulum in heat-stressed barley aleurone cells.

When barley (Hordeum vulgare) aleurone layers are heat shocked, the synthesis and secretion of alpha-amylase and other secretory proteins is arrested and the synthesis of heat shock proteins (hsps) is induced. alpha-Amylase mRNA, normally a very stable mRNA, is actively degraded during heat shock. In addition, endoplasmic reticulum (ER) is delamellated during heat shock, possibly causing the destabilization of the mRNA for the secreted alpha-amylase. To ascertain whether or not hsps play any role in the destabilization of alpha-amylase mRNA or in the delamellation process of ER, heat shocked cells were treated with the transcription inhibitor cordycepin, which effectively inhibits the synthesis of hsps yet does not affect alpha-amylase synthesis after this enzyme has been fully induced by gibberellic acid (12 hours). In the absence of hsp expression, heat shock still causes the destabilization of alpha-amylase mRNA and the delamellation of ER. Alternatively, the synthesis of hsps may be induced in the absence of temperature increase by incubating cells in the presence of arsenite. Arsenite-induced expression of some hsps in the absence of increased temperature does not result in the destabilization of alpha-amylase mRNA or in the delamellation of ER. If cordycepin or cycloheximide are used to inhibit hsp synthesis during heat shock, the tissue recovers from heat shock with normal recovery kinetics. Although hsps have been implicated in the establishment of thermotolerance, our observations indicate that hsps do not play a role in the other heat shock-induced changes observable in aleurone cells. Furthermore, if the synthesis of hsp mRNA is inhibited during heat shock (by cordycepin) hsp mRNAs are synthesized later, during recovery, indicating that there is a stable inducer of hsp synthesis in aleurone tissues.

Journal Article↗

PubMed1990

Cloning, sequence analysis, and expression of a cDNA encoding a plastid-localized heat shock protein in maize.

We have cloned and characterized a cDNA encoding a maize (Zea mays L.) heat shock protein (HSP), HSP26. The mRNA of HSP26 is present as a single mRNA species of 1.1 kilobase pairs in size and is detectable when maize seedlings are treated at 40 degrees C but not at 28 degrees C. Accumulation of HSP26 mRNA was detected after 10 minutes of incubation at 40 degrees C, reaching the maximum level after 1 hour. Comparison of the deduced amino acid sequence of maize HSP26 to other HSPs indicated a strong homology to the sequences of two nuclear encoded HSPs that are transported into the chloroplasts during heat shock: pea HSP21 and soybean HSP22. Maize HSP26 was also found to cross-react with anti-pea chloroplast HSP21 antibodies. Because of the sequence homology between maize HSP26, soybean HSP22, and pea HSP21, in vitro chloroplast protein import experiments were conducted. The in vitro synthesized maize HSP26 is specifically imported to the soluble fraction of the chloroplast and processed to a smaller polypeptide. The sequence homology and antibody cross-reactivity between maize HSP26 and pea HSP21 have allowed us to conclude that maize HSP26 is a nuclear-encoded, plastid-localized protein in maize.

Journal Article↗

PubMed1990

A major gibberellic Acid-induced barley aleurone cysteine proteinase which digests hordein : purification and characterization.

We previously described the purification and characterization of a 37,000 M(r) cysteine proteinase, designated EP-A, from gibberellic acid (GA(3))-induced barley (Hordeum vulgare L.) aleurone layers (S Koehler, T-HD Ho [1988] Plant Physiol 87: 95-103). A second, more abundant protease has now been purified from this tissue. This protease, designated EP-B, has an apparent M(r) of 30,000 on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It resolves into two bands during native isoelectric focusing with pl of 4.6 to 4.7. The analysis of hemoglobin digestion products by both gradient SDS-PAGE and Bio-Gel P2 chromatography, the inhibition of protease activity by E-64, leupeptin, iodoacetate, and p-hydroxymercuribenzoate, and N-terminal amino acid sequence analysis all indicate that EP-B is a cysteine proteinase. The first 22 amino acids at the N terminus of EP-B have been determined, and their sequence is 90% similar to that of EP-A. EP-B has properties similar to EP-A; however, EP-B is much more sensitive to high pH during gel electrophoresis and therefore is not detectable on native activity gels used to detect EP-A. Its pH optimum against azocasein and hemoglobin is 4.5 to 4.6. Both of these proteinases digest hordeins enriched for the B and D fractions into similar peptides of 25,000 to 2,000 M(r) as determined by gradient SDS-PAGE.

Journal Article↗

PubMed1990

Hormonal regulation, processing, and secretion of cysteine proteinases in barley aleurone layers.

Barley aleurone layers synthesize and secrete several proteases in response to gibberellic acid (GA3). Two major cysteine proteinases designated EP-A (37,000 M(r)) and EP-B (30,000 M(r)) have been described [Koehler and Ho (1988). Plant Physiol. 87, 95-103]. We now report the cDNA cloning of EP-B and describe the post-translational processing and hormonal regulation of both cysteine proteinases. Three cDNAs for cysteine proteinases were cloned from GA3-induced barley aleurone layers. Genomic DNA gel blot analysis indicated that these are members of a small gene family with no more than four to five different genes. The proteins encoded by two of these clones, pHVEP1 and 4, are 98% similar to each other and are isozymes of EP-B. The proteins contain large preprosequences followed by the amino acid sequence described as the mature N terminus of purified EP-B, and are antigenic to EP-B antiserum. The results of pulse-chase experiments indicated that the post-translational processing of large prosequences proceeds in a multistep fashion to produce the mature enzymes. Processing intermediates for EP-B are observed both in the aleurone layers and surrounding incubation medium, but only mature EP-A is secreted. The regulation of synthesis of EP-A, EP-B, and other aleurone cysteine proteinases was compared at the protein and mRNA levels. We conclude that barley aleurone cysteine proteinases are differentially regulated with respect to their temporal and hormonally induced expression.

Abscisic Acid↗

PubMed1990

Prevalence of human papillomavirus types 16 and 18 in cervical carcinomas: a study by dot and Southern blot hybridization and the polymerase chain reaction.

Histologically classified biopsies from 83 women with invasive cervical carcinoma were analyzed by dot blot hybridization for human papillomavirus (HPV) types 16 and 18 infection. Sixty of the 83 (72.3%) were found to contain HPV DNA, of which 43 (51.8%) contained HPV 16 DNA, 12 (14.5%) contained HPV 18 DNA and 5 (6.0%) contained both HPV 16 and 18 DNAs. Southern blot analysis on 65 specimens gave similar results. Of 23 specimens negative by dot blot, 21 were tested by the polymerase chain reaction. Seventeen of the 21 were positive for HPV DNA, of which 13 contained HPV 16 DNA and 4 contained both HPV 16 and 18 DNAs. In all, 95.1% (77/81) were positive for HPV 16 and/or 18 DNA sequences.

Adenocarcinoma↗

PubMed1990

Is genital human papillomavirus infection always sexually transmitted?

The occurrence of genital human papillomavirus (HPV) infection was studied prospectively by colposcopy and histology in 43 virginal and 162 sexually active women attending a colposcopy clinic. The study also included 111 husbands of the latter group. By colposcopic criteria, the prevalence of HPV infection was 51.1% in the virginal and 69.1% in the sexually active women. The prevalence was 77.1% among men whose wives had HPV infection compared to 13.3% among men whose wives did not have the infection (p less than 0.01). There was a strong association in genital HPV infection between husbands and wives as expected from a sexually transmissible disease. However, the high prevalence of the infection among the virginal women indicated that transmission of HPV by nonsexual modes was common. Genital HPV infection is ubiquitous and in women is not exclusively a venereal disease.

Adult↗

PubMed1989

A case-control study of cervix cancer in Singapore.

Cervix cancer is about twice as common in Asia as in the Western world and its incidence varies among different Asian ethnic groups. A study based in Singapore, the population of which comprises Chinese, Indians and Malaysians, offers the opportunity to evaluate whether the same risk factors are important in this part of the world as in the West. A total of 135 cases and an equal number of controls were interviewed and details concerning reproductive and sexual history, smoking, hygiene, socio-economic status and education were collected. Seventy-three cases had invasive cancer while 62 had micro-invasive disease or CIN III. The most important risk factors were parity and number of sexual partners. Smoking was rare in cases and controls and did not appear to be an important determinant of risk. Of the socio-economic factors, education appeared most predictive and lowered the risk. Age at first intercourse was strongly correlated with education (positively) and parity (negatively), but not with number of sexual partners. Biopsies were available for HPV DNA analysis in 38 cases and 37% were positive, mostly for HPV type 16. All these factors gave similar risks in invasive and preinvasive disease.

Adult↗

PubMed1988

Purification and characterization of gibberellic Acid-induced cysteine endoproteases in barley aleurone layers.

Using in series ammonium sulfate precipitation, gel filtration, and DEAE anion exchange high performance liquid chromatography, we have purified to homogeneity a protease of M(r) 37,000 secreted from barley (Hordeum vulgare L. cv Himalaya) embryoless half-seeds. This protease exists in three isozymic forms whose synthesis and secretion from barley aleurone layers was shown to be a gibberellic acid (GA(3))-dependent process (R Hammerton, T-HD Ho 1986 Plant Physiol 80: 692-697). This protease constitutes a major portion of the protease activity secreted from half-seeds between 72 to 96 hours of incubation in the presence of GA(3) as detected on activity gels containing hemoglobin as the substrate. Analysis of digestion products by urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that this protease is an endoprotease, therefore it is designated as barley endoprotease-A (EP-A). Inhibitor studies demonstrated that EP-A belongs to the cysteine class of endoproteases. The optimum pH for EP-A activity was 5.0, and the temperature optimum was 45 degrees C. Comparison of cyanogen bromide generated peptide fragments and NH(2)-terminal sequence analyses of the three individual EP-A isozymes demonstrates that they are very similar to each other. The NH(2)-terminal sequence shows extensive sequence homology to the NH(2)-terminal sequence of papain and several other cysteine proteinases. We also provide evidence that EP-A is not ;aleurain,' a putative cysteine proteinase encoded by a GA(3)-induced barley cDNA clone (JC Rogers, D Dean, GR Heck 1985 Proc Natl Acad Sci USA 82:6512-6516).

Journal Article↗

PubMed1988

Hormonal regulation of alpha-amylase expression in barley aleurone layers : the effects of gibberellic Acid removal and abscisic Acid and phaseic Acid treatments.

The expression of alpha-amylase isozymes in barley (Hordeum vulgare L.) aleurone layers is known to be maximally induced between 12 and 20 hours after addition of the phytohormone, gibberellic acid (GA(3)). Addition of another hormone, abscisic acid (ABA), or its metabolite, phaseic acid, during this time period resulted in reduced alpha-amylase expression. Expression of the high isoelectric point alpha-amylase isozyme group was affected much more by both of these treatments than was expression of the low isoelectric point alpha-amylase isozyme group. Addition of either the translation inhibitor cycloheximide or the transcription inhibitor cordycepin prevented the decrease in alpha-amylase mRNA levels after ABA treatment. Cordycepin also prevented the decreases in alpha-amylase expression that result from phaseic acid treatment. Midcourse GA(3) removal experiments were performed to determine whether ABA treatment and the removal of GA(3) have analogous effects on alpha-amylase expression. It was found that cordycepin treatment also prevented decreases in alpha-amylase mRNA levels resulting from GA(3) removal. We conclude that the suppression of alpha-amylase expression caused by ABA or midcourse GA(3) removal is dependent on continuous RNA and protein synthesis.

Journal Article↗

PubMed1988

Human papillomavirus in dysplasia and carcinoma of the cervix in Singapore.

A prospective study was conducted in Singapore in 1985 where 107 women with abnormal cervical smears were studied for cervical neoplasia and its association with the human papillomaviruses (HPV), using HPV 11, 16 and 18 DNA as probes. Cervical biopsies were performed for histology as well as for DNA Southern Blot hybridization studies to detect the presence of HPV 11, 16 or 18 genome. The prevalence of the various types of papillomavirus DNA in cervical tissue samples from cervical carcinoma and dysplasias is presented. HPV types 16 and 18 were found in 5 of 8 (63%) cases of invasive squamous cell carcinoma (SCC), in 12 of 41 (29%) cases of squamous carcinoma-in-situ (CIN 3) and in 3 of 48 (6%) cases of lower grade dysplasia. None of our cases of SCC were associated with unknown HPV types detected using a mixture of HPV 16 or 18 DNA under conditions of low stringency. These unknown HPV types were present in approximately one quarter of our cases of dysplasia of all grades.

Adult↗

PubMed1987

Biochemical properties and hormonal regulation of barley nuclease.

The amino acid composition and NH2-terminal amino acid sequence of barley nuclease (EC 3.1.30.2) were determined. The amino acid composition is similar to that of mung bean nuclease, and therefore the biochemical properties of barley nuclease were characterized and compared with those of mung bean and other plant nucleases. The 3'-nucleotidase activity of barley nuclease is greater for purine than for pyrimidine ribonucleotides. The enzyme has little activity towards ribonucleoside 2' and 5'-monophosphates, and deoxyribonucleoside 3' and 5'-monophosphates, and is also inactive towards the 3'-phosphoester linkage of nucleoside cyclic 2',3' and 3',5'-monophosphates. The enzyme hydrolyzes dinucleoside monophosphates, showing strong preference for purine nucleosides as the 5' residues. Barley nuclease shows significant base preference for homoribonucleic acids, catalyzing the hydrolysis of polycytidylic acid greater than polyuridylic acid greater than polyadenylic acid much greater than polyguanylic acid. The enzyme also has preference for single-stranded nucleic acids. Hydrolysis of nucleic acids is primarily endonucleolytic, whereas the products of digestion possess 5'-phosphomonoester groups. Nuclease activity is inhibited by ethylenediaminetetraacetic acid and zinc is required for reactivation. Secretion of nuclease from barley aleurone layers is dependent on the hormone gibberellic acid [Brown, P.H. and Ho, T.-h. D. (1986) Plant Physiol. 82, 801-806]. Consistent with these results, gibberellic acid induces up to an eight-fold increase in the de novo synthesis of nuclease in aleurone layers. The secreted enzyme is a glycoprotein having an apparent molecular mass of 35 kDa. It consists of a single polypeptide having an asparagine-linked, high-mannose oligosaccharide. The protein portion of the molecule has a molecular mass of 33 kDa.

Amino Acid Sequence↗

PubMed1987

Absence of heat shock protein synthesis in isolated mitochondria and plastids from maize.

Examination of the proteins synthesized by isolated mitochondria, chloroplasts, or proplastids from maize tissues showed that a heat treatment at 40 degrees C does not induce or enhance the synthesis of any protein when compared to preparations treated at the control temperature of 28 degrees C. These observations are consistent with the results obtained by labeling proteins in vivo under sterile conditions. In vivo labeling in the presence of cycloheximide during heat shock showed no heat shock protein synthesis. Labeling in the presence of chloramphenicol during heat shock showed a similar heat shock protein pattern as in the absence of the inhibitor. It is concluded that maize organelles do not synthesize heat shock proteins and that, if present, they may be due to bacterial contamination.

Chloramphenicol↗

PubMed1987

Phytic Acid Metabolism in Lily (Lilium longiflorum Thunb.) Pollen.

The accumulation of phytic acid during development of lily (Lilium longiflorum Thunb.) pollen and its degradation during germination have been studied. A substantial amount of phytic acid accumulates in lily pollen by 5 days before anthesis, and little change occurs during subsequent maturation. Mature lily pollen contains 7 to 8 micrograms phytic acid per milligram pollen. Considerable degradation of phytic acid occurs by 15 minutes of incubation in glucose culture medium, and very little is left by 3 hours. No partially phosphorylated myo-inositol accumulates during germination. The breakdown of phytic acid proceeds at a constant rate during this time period. The rate is calculated to be 0.037 microgram phytic acid/milligram pollen/minute. Two phytases are detected in germinated lily pollen extract using high performance liquid chromatography with an anion exchange column (diethylaminoethyl-5PW). The results suggest that one of the phytases is already present in mature ungerminated lily pollen and the other one is newly synthesized during germination from a long-lived, pre-existing mRNA.

Journal Article↗

PubMed1987

Intracellular localization of heat shock proteins in maize.

The intracellular distribution of the maize root heat shock proteins (hsp) was studied as a step toward understanding their physiological function. Linear sucrose density centrifugation was employed to separate organelles so the relative quantities of hsp in different subcellular compartments could be analyzed in a single preparation. Gradient fractions were assayed for the presence of hsp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and for marker enzyme activities. Analyses of 15 to 60% gradients showed five hsp to be organelle associated. Hsp 25 and 72 were in fractions containing closely equilibrating Golgi and endoplasmic reticulum marker activities, while hsp 18, 29, and 72 were in fractions containing overlapping plasma membrane, mitochondria, and glyoxysomal marker activities. Hsp larger than 72 kilodaltons were not present in gradient fractions. A second fractionation scheme achieved better separation of the two sets of closely equilibrating organelles. When a 13,000g centrifugation step to remove mitochondria was employed prior to gradient centrifugation, hsp 29 was absent from the gradient fractions. If the buoyant density of the endoplasmic reticulum was shifted by either maintaining the ribosomes on the membrane or removing them, a corresponding shift in the equilibrium positions of hsp 25 and 72 occurred. Hsp 18 and 70 remained in plasma membrane-containing fractions irrespective of these treatments.

Journal Article↗

PubMed1986

Heat shock causes destabilization of specific mRNAs and destruction of endoplasmic reticulum in barley aleurone cells.

In response to a phytohormone, gibberellic acid, the aleurone layers of barley seeds synthesize and secrete alpha-amylases, which are coded by a set of stable mRNAs. When aleurone layers are subjected to heat shock treatment, the synthesis of alpha-amylase is suppressed while heat shock proteins are induced. The suppression of alpha-amylase synthesis is not the result of translational control as reported in several other systems. Rather, the sequences of alpha-amylase mRNA are rapidly degraded during heat shock as shown by in vitro translation and dot blot hybridization with a cDNA probe. Upon recovery from heat shock, the tissue resumes the synthesis of alpha-amylase in 2-4 hr. However, in the presence of a transcription inhibitor, cordycepin, the resumption of synthesis of alpha-amylase does not take place, indicating that new transcription of alpha-amylase genes is necessary for this recovery process. The degradation of alpha-amylase mRNAs correlates with the rapid destruction of endoplasmic reticulum as observed by electron microscopy, a phenomenon that has not been reported previously as a heat shock response. Since alpha-amylase mRNA is associated with the endoplasmic reticulum via membrane-bound polyribosomes, we suggest that the destruction of the endoplasmic reticulum during heat shock causes the destabilization and the eventual degradation of alpha-amylase mRNA.

Deoxyadenosines↗

PubMed1986

Hormonal regulation of the development of protease and carboxypeptidase activities in barley aleurone layers.

Carboxypeptidase and protease activities of hormone-treated barley (Hordeum vulgare cv Himalaya) aleurone layers were investigated using the substrates N-carbobenzoxy-Ala-Phe and hemoglobin. A differential effect of gibberellic acid (GA(3)) on these activities was observed. The carboxypeptidase activity develops in the aleurone layers during imbibition without the addition of hormone, while the release of this enzyme to the incubation medium is enhanced by GA(3). In contrast, GA(3) is required for both the production of protease activity in the aleurone layer and its secretion. The time course for development of protease activity in response to GA(3) is similar to that observed for alpha-amylase. Treating aleurone layers with both GA(3) and abscisic acid prevents all the GA(3) effects described above. Carboxypeptidase activity is maximal between pH 5 and 6, and is inhibited by diisopropylfluorophosphate and p-hydroxymercuribenzoate. We have observed three protease activities against hemoglobin which differ in charge but are all 37 kilodaltons in size on sodium dodecyl sulfate polyacrylamide gels. The activity of the proteases can be inhibited by sulfhydryl protease inhibitors, such as bromate and leupeptin, yet is enhanced by 2-fold with 2-mercaptoethanol. In addition, these enzymes appear to be active against the wheat and barley storage proteins, gliadin and hordein, respectively. On the basis of these characteristics and the time course of GA(3) response, it is concluded that the proteases represent the GA(3)-induced, de novo synthesized proteases that are mainly responsible for the degradation of endosperm storage proteins.

Journal Article↗