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Biomedical subjects

S Z Huang

Publications and source records attributed to S Z Huang.

At least 19 recordsLinked to original sources
PubMed2001

Selection of in vitro produced, transgenic embryos by nested PCR for efficient production of transgenic goats.

The production of valuable pharmaceutical proteins using transgenic animals as bioreactors has become one of the goals of biotechnology. However, the efficiency of producing transgenic animals by means of pronuclear microinjection is low. This may be attributed in part to the low integration rate of foreign DNA. Therefore, a large number of recipients are required to produce transgenic animals. We recently developed a transgenic procedure that combined the techniques of goat oocyte in vitro maturation (IVM), in vitro fertilization (IVF), microinjection, preimplantation selection of the transgenic embryos with nested PCR and transferring the transgenic embryos into the recipient goat uterus to produce transgenic goats. Thirty-seven transgenic embryos determined by nested PCR were transferred to thirty-two recipient goats. In the end, four live-born kids were produced. As predicted, all the live kids were transgenic as identified by PCR as well as Southern blot hybridization, The integration rate was 100% (4/4) which was completely in accordance with the results of embryo preimplantation detection. The results showed a significant decrease in the number of recipients required as only 8 recipients (32/4) were needed to obtain one live transgenic goat. We suggest that the transgenic system described herein may provide an improved way to efficiently produce transgenic goats on a large scale.

Animals↗
PubMed2000

Reversal of aberrant splicing of beta-thalassaemia allele (IVS-2-654 C-->T) by antisense RNA expression vector in cultured human erythroid cells.

The antisense fragment targeting the aberrant splice sites of the beta-thalassaemia allele, IVS-2-654 C-->T (beta654), pretranscript was cloned into the mammalian expression vector, pcDNA3. The recombinant construct, pCMVA, was then used to repair the defective splicing of the beta654 mutant pretranscript in cultured beta654 erythroid cells by the lipofectin-mediated DNA transfection method. The total RNA was extracted at given time points after transfection and the effect of antisense RNA was studied by reverse transcription polymerase chain reaction (RT-PCR)-mediated mRNA quantitative assay, as well as globin chain microbiosynthesis. The antisense fragment transcribed from pCMVA effectively improved the beta654 splicing pattern in cultured erythroid cells. The level of correctly spliced transcript increased from 0.19 (day 0 after transfection) to 0.58 (day 8) in beta654/beta654 homozygous erythroid cells, and from 0.45 (day 0) to 0.83 (day 8) in beta654/betaA heterozygous erythroid cells, as determined by the ratio of normally spliced beta-globin transcript over total beta-globin transcript. Correspondingly, the ratios of globin chain biosynthesis (beta/alpha) increased from 0.16 (day 0) to 0.52 (day 8) in beta654/beta654 erythroid cells, and from 0.39 (day 0) to 0.84 (day 8) in beta654/betaA erythroid cells. Antisense RNA had no significant effect on the splicing pattern in betaA/betaA erythroid cells. The splicing pattern in transfected cells with pCMVA showed significant changes compared with that in untransfected cells and that in transfected cells with the control antisense fragment (human SRY gene sequence). In addition, we did not observe side-effects on cytological features after the introduction of pCMVA. All these results indicated that the antisense RNA transcribed from the mammalian expression vector pCMVA could efficiently and specifically suppress the aberrant splicing pattern of beta654 mutant pretranscript and restore the correct splicing pathway in vivo, leading to the improvement of globin chain biosynthesis in thalassaemic cells.

Adult↗
PubMed2000

[A study of transgenic IFV cattle integrated with human serum albumin gene].

The mammary gland expression vector (pcDNA 3.1-GCALBm) containing the full-length sequence of human serum albumin (hALB) cDNA and intron 1 as well as the goat beta-casien gene promoter and 5' up-stream regulatory sequence was constructed. The vector was micro-injected into bovine IVF eggs. The embryos were in vitro cultured to the late stage of morulae, and then few embryo cells were aspirated for the implantation detection of target gene integration and SRY DNA sequence using nested-PCR. Afterwards, ten integrated embryos were selected to transfer into eight recipients and three were pregnant. The pregnant rate was 37.5%(3/8). However, two were miscarried in mid-trimester but one was pregnant at term to deliver a male transgenic cattle integrated with hALB mini-gene. The transgenic efficiency was 12.5% (1/8).

Animals↗
PubMed1999

The delta-globin RNA transcript level in beta-thalassemia carriers.

Increased levels of hemoglobin A(2) (HbA(2)) are present in most beta-thalassemia carriers. The mechanism of this effect is not understood, although the increase may result from transcriptional and posttranscriptional changes. In the present study, we quantitate delta-globin mRNA levels in peripheral-blood-enriched reticulocytes and characterize the variation of delta-mRNA levels in 30 beta-thalassemia heterozygotes who individually carry one of the four common Chinese beta-thalassemia alleles [codons 41/42 (-TTCT); codon 17 (A-->T); IVS-II-654 (C-->T); -28 (A-->G)]. A sensitive and quantitative competitive reverse-transcriptase polymerase chain reaction method was developed and used to assess the absolute amounts of delta-mRNA transcripts in these peripheral erythroid cells. The results showed a large increase in delta-mRNA amounts in all the carriers examined (72.3 +/- 9.0 amol/microg RNA) as compared with those in 12 controls (1.2 +/- 0.2 amol/ microg RNA). There was a direct correlation between the delta-mRNA levels and types of beta-thalassemia alleles; generally, the delta-mRNA levels are higher in heterozygotes for beta(0)-thalassemia mutations than beta(+)-thalassemia mutations. The delta-mRNA levels correlated inversely with hemoglobin and red cell indices but directly with HbA(2) levels in heterozygotes of each of the group of beta-thalassemia mutations. These results suggest that a greater impairment in beta-globin gene expression results in increased transcription of delta-globin gene and in a higher level of HbA(2).

Asian People↗
PubMed1999

[The gene expression of some cytokines and collagen proteins in rat bone tissue is related to estradiol (E2) and age].

30 female SD rats (3 months old) are equally divided into three groups: ovariectomy (OVX) rats, sham-operated (SHO) rats and 17 beta estradiol (E2) treated OVX rats. For each group, mRNA was isolated from long bone at one month and three months after surgery, respectively. mRNA was reverse transcribed into single strand cDNA and then used as a probe hybridizing to the DNA fragments of col I alpha(1), col I alpha(2), col III, col V, fibronectin, IL-1, IL-6, TGF-beta, LIF, TNF-alpha, TNF-beta by reverse northern and dot blot hybrization. The housekeeping gene, gapdh, was used as an internal control. The results show that in bone of rat, the stable expression of col I alpha (1), col I alpha(2) and col III are related to age not ovariectomy, while supplement with E2 can inhibit the expression of col III and col I alpha(2) completely. The expression of col V, IL-1, IL-6 can be inhibited by estrogen and recovered by removal of estrogen by OVX, then addition of E2 decreased it to the normal level. The expression of TGF-beta is also inhibited by estrogen. It increased during one month after overiectomy, and partially decreased in E2 complemented rat. Three months after surgery, the level of increasing and decreasing is less evident as two months ago. It seems that in young SD rat, the expression of TGF-beta is related to both estrogen and age.

Age Factors↗
PubMed1995

Hydroxyurea therapy in beta-thalassaemia intermedia: improvement in haematological parameters due to enhanced beta-globin synthesis.

The beta-thalassaemias represent a heterogenous group of diseases resulting from decreased erythroid beta-globin mRNA expression and imbalanced alpha/beta-globin chain synthesis which are manifest clinically by ineffective erythropoiesis and excessive haemolysis. Increasing levels of haemoglobin F (HbF) by pharmacological agents has been proposed to ameliorate the severity of the disease by improving the balance in globin chain synthesis. Hydroxyurea (HU), as an effective agent with low toxicity for activating gamma-globin gene, has been shown to enhance HbF synthesis in experimental animals and in patients with sickle cell anaemia. However, previous trials of HU in beta-thalassaemia patients are ambiguous, with a small number having increased HbF synthesis. In a recent study of HU effects in Chinese beta-thalassaemia patients we unexpectedly found that two unrelated patients with beta-thalassaemia intermedia demonstrated an improvement in the effectiveness of erythropoiesis reflected by an increase in haemoglobin concentration (from 4.1 to 6.3 g/dl, patient 1; from 6.5 to 9.7 g/dl, patient 2) and in red cell volume (from 68 to 104 fl, patient 1; from 68 to 85 fl, patient 2) after a period of excess of 300d of low-dosage HU treatment. These effects, however, appear to be due to increased beta-globin biosynthesis, because the percentage of HbF decreased in each patient as total Hb increased. This was reflected by changes in the beta/alpha ratio (from 0.301 to 0.581, patient 1; from 0.348 to 0.487, patient 2) with minimal changes in gamma-globin biosynthesis. We conclude that in addition to its known effects in stimulating gamma-globin production, hydroxyurea may have a more general role in augmenting globin synthesis, including beta-globin in some thalassaemia intermedia patients who maintain the capacity to express normal beta-globin chains.

Female↗
PubMed1994

Experimental studies on immune-complex in situ type glomerulonephritis in rabbits treated with mai-luo-tong and natural indigo.

By injection of C-BSA, immune-complex in situ type glomerulonephritis was duplicated in rabbits and treated with Mai-Luo-Tong and natural Indigo. The results showed that proteinuria in the treated groups M and Q was decreased. The difference between group M and control group is statistically significant (P < 0.05). Under light and electron microscope, although glomerular basement membrane was irregularly thickened and subepithelial dense electron deposited in the treated group, but microthrombus, erythrocytes and platelets aggregation and leukocytes impaction were not seen within glomerular capillaries. Also in groups C, Q, M, mesenteric cell count was 99.40 +/- 18.53, 92.87 +/- 17.89, 66.55 +/- 7.75 respectively, the M. Q groups are compared with group C, the result is of statistical significance (P < 0.05) and there is no apparent glomerular fibrosis in the treated groups.

Animals↗
PubMed1994

RNA transcripts of the beta-thalassaemia allele IVS-2-654 C-->T: a small amount of normally processed beta-globin mRNA is still produced from the mutant gene.

IVS-2-654 C-->T is a common Chinese beta-thalassaemia mutation. Previous studies report that this mutation resulted in the formation of an abnormally spliced mRNA and the absence of detectable normal beta-globin mRNA, hence the mutation was considered to cause beta o-thalassaemia. We recently used the method of PCR amplified cDNA copies of circulating erythroid cell mRNA to analyse the mutant gene transcripts and found that this IVS-2-654 mutation does not abolish normal RNA processing entirely, but that a significant amount (over 15%) of normally processed beta-globin mRNA is produced. Microglobin chain biosynthetic analysis using the HPLC method showed that beta-globin chain was also present in the blood of patients with IVS-2-654 C-->T mutation. Accordingly, this mutant allele leads to a beta (+)-thalassaemia. Further, the methodology described in this paper provides a new approach towards the detection of RNA transcripts of beta-thalassaemia alleles as well as the study of gene expression in beta-thalassaemia and other genetic diseases.

Alleles↗
PubMed1994

Amplification of the C-erbB-2(HER-2/neu) proto-oncogene in ovarian carcinomas.

C-erbB-2(HER-2/neu) proto-oncogene is mainly expressed in epithelial tissue and activated due to its amplification. Amplification of the C-erbB-2 proto-oncogene has been associated with poor prognosis in human ovarian cancer. Our study was to examine whether amplification is more frequently observed in ovarian cancer, or it is associated with poor prognosis of human ovarian cancer in China. The DNA of ovarian cancers was extracted and consequently digested with restriction endonuclease EcoRI, electrophoresed in 0.8% agarose gels and blotted onto nitrocellulose filter with Southern transferring method. It was then hybridized with a 32P-labelled C-erbB-2 probe and subsequently underwent autoradiography. The result has shown that the C-erbB-2(HER-2/neu) gene was amplified in 8 of 26 human ovarian cancers (30.8%). The clinical data showed that all of the 8 cases with the amplified C-erbB-2 were in their advanced stage (III-IV). Five of the patients died from 2 to 4 months after operation. These data suggest that amplification of the C-erbB-2 gene may play a role in the pathogenesis of ovarian carcinoma; it is frequently observed in advanced ovarian cancer and is associated with poor prognosis for these patients.

Adenocarcinoma, Clear Cell↗
PubMed1994

Treatment of beta-thalassemia with hydroxyurea (HU)--effects of HU on globin gene expression.

A newly developed method of RT-PCR/competitive PCR for measuring the relative and absolute content of globin mRNAs as well as micro-globin chain biosynthetic assay have been used to study the alterations of globin gene expressions in the patients with beta-thalassemia pre- and post-hydroxyurea (HU) treatment. It was found for the first time that HU had the effect of enhancing beta-globin gene expression in some patients. Two cases with beta-thalassemia who were subjected to HU treatment for over two years showed a marked increase in beta-globin mRNA level and beta-globin chain synthesis, resulting in more effective erythropoiesis and the alleviation of clinical symptoms.

Adult↗
PubMed1994

[The relation between prognosis and amplification of the c-erB-2 (HER-2/neu) proto-oncogene in ovarian carcinomas].

C-erbB-2 (HER-2/neu) proto-oncogene is mainly expressed in epithelial tissue and activated due to its amplification. Amplification of the C-erbB-2 proto-oncogene is associated with poor prognosis in human ovarian cancer. We examined whether amplification of C-erbB-2 is common in ovarian carcinoma or is associated with poor prognosis. The DNA of ovarian carcinoma was extracted and consequently digested with restriction endonuclease EcoRI, electrophoresed in 0.8% agarose gels and blotted onto nitrocellulose filter with Southern transfering method. It was hybridized with a 32p-labelled C-erbB-2 probe and subsequently underwent autoradiography. It was shown that the C-erbB-2 (HER-2/neu) gene was amplified in 8 of 26 human ovarian carcinomas (30.8%). Clinically the 8 patients with the amplified C-erbB-2 were in their advanced stage (III-IV). Five of the patients died from 2 to 4 months after operation. These findings suggest that amplification of the C-erbB-2 gene may play a role in the pathogenesis of ovarian carcinoma, it is frequently observed in advanced ovarian carcinoma and associated with poor prognosis for these patients.

DNA, Neoplasm↗
PubMed1994

Sexing bovine embryos using PCR amplification of bovine SRY sequence.

This study analyses the bovine SRY DNA sequence by direct sequencing procedure, followed by the designation of the PCR primers specific for bovine SRY. Using PCR amplification of bovine SRY gene, the embryo sex was determined. The results of the embryo sex identification were confirmed after the embryo transfer and pregnancies.

Animals↗
PubMed1993

A new de novo mutation (A113T) in HMG box of the SRY gene leads to XY gonadal dysgenesis.

We describe a new point mutation in the SRY gene of a Chinese XY female with gonadal dysgenesis (Swyer syndrome). Using the double stranded DNA cycle sequencing method, a single nucleotide substitution of G-->A was identified at codon 113 of the patient's SRY gene, resulting in a conservative amino acid change from alanine (A) to threonine (T) at a residue that lies within the putative DNA binding motif. With this mutation, one MnlI recognition site is abolished and a new BsmAI site is present in the DNA sequence of the SRY gene; therefore, it is easily detected by analysis of the digestion of the amplified SRY DNA fragment on an electrophoretic agarose gel. In situ hybridisation to the XY female's chromosomes showed that her mutant SRY gene was indeed located on the short arm of her Y chromosome. The SRY mutation in the XY female reported here occurred de novo, as sequence analysis showed that it was not present in her father or other family members.

Adult↗
PubMed1993

[Research on immune complex in situ type glomerulonephritis treated with mai-luo-tong in rabbits].

Applying c-BSA to duplicate immune complex in situ type glomerulonephritis in rabbits and treating it with Blood Circulation Promoting and Stasis-Removing Drugs Mai-Luo-Tong, the results showed that proteinuria in the treated group was decreased significantly, as compared to the control group (P < 0.01). Under light and electron microscope, although glomerular basement membrane was irregularly thickened and subepithelial dense electron deposits were found in both groups, but histopathologic damage in the treated group less than that of control one. In the treated group micro-thrombus, erythrocytes and platelets aggregation, leukocytes impaction were not seen within glomerular capillary. Also in the treated group mesangial cell proliferation and granulocyte infiltration were decreased significantly (P < 0.01) and there was no apparent glomerular fibrosis in former group.

Animals↗