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Publications and source records attributed to Z Zheng.
The application of response surface method to optimal medium-screeing for inulinase production by Kluyveromyces sp. Y-85 has been investigated. The experiments of condition control for the enzyme production was carried out in a 15 L fermenter. A large scale fermentation (1000 L fermenter) was performed five batches, and average inulinase activity of 68.9 u/ml achieved.
The untransparent substrates on the epitaxial layer need not be etched out from chemistry etching to prepare the "window" through light. The optical absorption, optical nonlinearities and optical bistabilities of the semiconductor thin films with the nanostructure grown on the untransparent substrates can be directly measured by the end face coupling method of optical waveguide. Meanwhile, the several experiment examples of II - VI single crystal thin films and superlattices grown on GaAs substrate are demonstrated in this paper.
The entire reaction process in the fabrication of lead styphnate in situ was analyzed by using laser Raman spectrometer and FTIR spectrometer, including reaction time determination, testing of the reactants proportions, and identification of the resultants in terms of molecular structure.
We have measured survival after successive 2-Gy doses of X rays in HeLa cells and 1-Gy doses in cells of the nonimmortalized human fibroblast cell line AG15-22 under conditions where any effect of cell proliferation during multifraction X irradiation has been factored out. When HeLa cells in parallel series of (pseudo)hybrid spheroids (i.e. in agglomerates consisting of a mixture of supralethally irradiated HeLa feeder and viable HeLa cells) were exposed to n daily radiation doses and then trypsinized and exposed to the last dose, the surviving fraction at 2 Gy (SF2) declined exponentially from 0.55 +/- 0.01 to 0.31 +/- 0.01 after the fifth fraction. In monolayer HeLa cell cultures, the decline in SF2 was smaller but significant and was not influenced by the presence of feeder cells. Pure spheroids, composed entirely of viable HeLa cells, showed the same decline in SF2 as did monolayer cells. The cumulative-effect curve (i.e. the product of SF2 values) was linear-quadratic with the quadratic term increasing in the order monolayer, pure spheroids, pseudohybrid spheroids. SF2n and D0Eff (deduced from the initial SF2) severely underestimated cumulative radiosensitivity. This cumulative effect is clearly associated with the proximity of lethally irradiated cells and might be explained by differential population shifts in the course of the multifraction regimen. Similarly, AG15-22 cells irradiated with daily 1-Gy doses of X rays showed a larger increase in radiosensitivity when in hybrid spheroids than when in pure spheroids. However, for the AG15-22 cells, SF1 was twofold lower for the former than for the latter condition and remained constant for both conditions rather than decreasing with increasing fraction number. This large radiosensitizing effect remains unexplained.
This article is a report on a symposium held at Experimental Biology '98 in San Francisco, California. Recent developments in site-directed mutagenesis, computer-modeling, and mechanistic analysis of cytochromes P450 and flavin-containing monooxygenases are described. A unifying theme is the elaboration of general approaches for understanding and predicting the function of individual forms of these enzymes. A related goal is the production of soluble forms of mammalian cytochromes P450 for X-ray crystallography.
In order to elucidate the possibility of costimulatory molecules-mediated immuno or immuno-gene therapy for human hematological malignancies, we analyzed 30 hematopoietic cell lines and cells obtained from 48 patients with hematological malignancies for the expression of costimulatory molecules such as CD80 and CD86. The 30 hematopoietic cell lines were composed of 4 cell lines derived from the patients with T-cell acute lymphoblastic leukemia (T-ALL), 3 from Philadelphia chromosome positive ALL (Ph1+ALL), 8 from acute myeloblastic leukemia (AML), 3 from acute promyelocytic leukemia (APL), 8 from chronic myeloid leukemia at blast crisis (CML-BC), 3 from Burkitt's lymphoma and one from follicular cell lymphoma. The expression of CD80 or CD86 was frequent on cell lines derived from the patients with CML-BC or Burkitt's lymphoma, while it was rare on cell lines from T-ALL. Subsequently we analyzed the cells obtained from 48 patients with hematological malignancies, which consisted of 6 samples from patients with ALL, 30 from AML, 2 from CML-BC, 3 from B-cell lymphoma and one from each acute mixed leukemia (AMixL), adult T cell leukemia (ATL), T-cell large granular lymphocytic leukemia (T-LGL leukemia), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome (MDS)-RAEB in T, multiple myeloma (MM) or T-cell lymphoma. Among all the 48 cases, all cases except one case with CLL and two with B cell lymphoma were demonstrated to be negative for CD80 on the neoplastic cells. CD86 and HLA-DR were shown to be expressed in 50% and 88% of total 48 cases respectively. In 30 AML samples, CD86 was positive in 15 cases (50%), which was sharply in contrast with the finding that CD80 was not detected in any AML samples. HLA-DR was expressed in 25 AML samples (83%). We also treated seven human hematopoietic cell lines with IFN-gamma, IL-12 or IL-15 and observed whether these cytokines could induce or enhance the expression of CD40, CD54, CD58 and HLA-DR as well as CD80 and CD86. The present study demonstrated that the expression of CD86 could be upregulated not only by IFN-gamma, but also by IL-12 or IL-15 in some cell lines. These findings suggested the possibility that the absence of CD80 on neoplastic cells may be associated with the lack of efficient anti-tumor immunity in most patients with hematological malignancies and that the immuno or immuno-gene therapy manipulating the expression of costimulatory molecules such as CD80 may be a useful treatment modality for hematological malignancies.
Previous studies have shown that the guinea pig inferior mesenteric artery receives spinal sensory vasodilatory innervation, which can be activated by colon distention and electrical nerve stimulation. In the present study, we investigated the hypotheses that nitric oxide synthase (NOS) is present in guinea pig primary sensory neurons in the dorsal root ganglion (DRG) and in nerve fibers surrounding the mesenteric arteries, and that nitric oxide (NO) is a sensory neurotransmitter in the inferior mesenteric artery in vitro. Double-labeling immunohistochemistry showed that neuronal NOS-IR was found in 12% of cells of guinea pig thoracic and lumbar DRGs; in 95.1% of these cells it was colocalized with substance P (SP), and SP immunoreactivity (SP-IR) was present in 23% of cells of the same DRGs. Neuronal NOS-like immunoreactivity was localized in nerve fibers surrounding guinea pig mesenteric artery and 25% of them were double stained with SP-IR. Endothelium-denuded inferior mesenteric artery preparations in vitro were incubated with guanethidine (30 microns, 30 min) and pre-contracted with methoxamine (30 microns). The NO donors, sodium nitroprusside (1 micron) and L-nitrosocysteine (300 microns), produced 91.0 +/- 5.5 and 90.4 +/- 9.6% vasodilatation of total vasodilatation in the vessel segments, respectively, which was capsaicin- or tetrodotoxin-insensitive. Repetitive electrical field stimulation of the preparations produced a frequency-dependent vasodilatation which was reduced by pretreatment with capsaicin or by tetrodotoxin (10 microns). The NOS inhibitor N omega-nitro-L-arginine (L-NNA) (100 microns, 30 min) diminished the nerve-evoked vasodilatation from 41.8 +/- 8.4 to 21.4 +/- 9.7% at 2 Hz and from 50.8 +/- 5.6 to 19.0 +/- 7.3% at 15 Hz (P < 0.05), whereas NG-nitro-L-arginine methyl ester (L-NAME, 100 microns-1 mM) did not significantly inhibit the relaxation. The stereo isomer nitro-D-arginine (D-NNA, 100 microns, 30 min) was ineffective. These findings suggest that NO is a neurotransmitter released from primary sensory nerves which mediates vasodilation in vitro.
Juvenile polyposis syndrome (JPS; MIM 174900) is an autosomal dominant condition with incomplete penetrance characterized by hamartomatous polyps of the gastrointestinal tract and a risk of gastrointestinal cancer. Gastrointestinal hamartomatous polyps are also present in Cowden syndrome (CS; MIM 158350) and Bannayan-Zonana syndrome (BZS; also called Ruvalcaba-Myhre-Smith syndrome; MIM 153480). The susceptibility locus for both CS and BZS has recently been identified as the novel tumor suppressor gene PTEN, encoding a dual specificity phosphatase, located at 10q23.3. A putative JPS locus, JP1, which most likely functions as a tumor suppressor, had previously been mapped to 10q22-24 in both familial and sporadic juvenile polyps. Given the shared clinical features of gastrointestinal hamartomatous polyps among the three syndromes and the coincident mapping of JP1 to the region of PTEN, we sought to determine whether JPS was allelic to CS and BZS by mutation analysis of PTEN and linkage approaches. Microsatellite markers spanning the CS/BZS locus (D10S219, D10S551, D10S579, and D10S541) were used to compute multipoint lod scores in eight informative families with JPS. Lod scores of < -2.0 were generated for the entire region, thus excluding PTEN and any genes within the flanking 20-cM interval as candidate loci for familial JPS under our statistical models. In addition, analysis of PTEN using a combination of denaturing gradient gel electrophoresis and direct sequencing was unable to identify a germline mutation in 14 families with JPS and 11 sporadic cases. Therefore, at least a proportion of JPS cases are not caused by germline PTEN alteration or by an alternative locus at 10q22-24.
The majority of familial medullary thyroid neoplasms are associated with germ-line mutations of the RET proto-oncogene, yet very little is known about the mechanisms involved in the pathogenesis of familial and sporadic nonmedullary thyroid tumors. A subset of thyroid tumors have loss of heterozygosity of chromosome 10q22-23, a region harboring the gene responsible for Cowden disease, an autosomal dominant hamartoma syndrome associated with thyroid and breast tumors. PTEN/MMAC1/TEP1 codes for a dual-specificity phosphatase and is likely a tumor suppressor gene. We sought to determine the PTEN status in a series of epithelial thyroid neoplasms. We studied 95 sporadic thyroid tumors, of which 39 were papillary thyroid carcinomas (PTCs), 12 were follicular carcinomas, 9 were anaplastic carcinomas, 5 were Hürthle cell carcinomas, 21 were nonfunctioning follicular adenomas, and 9 were Hürthle cell adenomas. Direct sequencing of PCR-amplified products was performed for all nine exons of PTEN. Two polymorphic markers, one located in intron 8 and another, a dinucleotide repeat marker, AFMa086wg9, located within intron 2, were analyzed in paired blood-tumor DNA samples to assess hemizygous deletions of PTEN. We found a somatic frameshift mutation in one PTC, which was expected to generate a premature stop codon 2 amino acids downstream. Twenty-six % of informative benign tumors (four follicular adenomas and three Hürthle cell adenomas) and only 3 of 49 (6.1%) informative malignant tumors (one PTC, one follicular carcinoma, and one anaplastic carcinoma) showed evidence of hemizygous deletion of PTEN (P = 0.046). We conclude that a subset of thyroid tumors have somatic deletions of the PTEN gene, predominantly the benign forms, and that small intragenic mutations of PTEN are infrequent in thyroid tumors. We speculate that other mechanisms of PTEN inactivation, rather than small intragenic mutations, might occur in the hemizygously deleted samples and act as the "Knudson second hit." Alternatively, other tumor suppressor genes mapping to chromosome 10q22-23 could be the actual targets for such deletions and thus represent the various hits in the pathway of multistep carcinogenesis.
The density, molecular isoform, and posttranslational modifications of CD44 can markedly influence growth and metastatic behavior of tumors. Many CD44 functions, including some involving tumors, have been attributed to its ability to recognize hyaluronan (HA). However, only certain CD44-bearing cells bind soluble or immobilized HA. We now show that CD44 made by wild-type Chinese hamster ovary (CHO-K1) cells and a ligand-binding subclone differ with respect to N-linked glycosylation. While both bear CD44 with highly branched, complex-type glycoforms, CD44 expressed by the wild type was more extensively sialylated. CHO-K1 cells which failed to recognize HA when grown in culture gained this ability when grown as a solid tumor and reverted to a non-HA-binding state when returned to culture. The ability of CHO-K1 cells to recognize HA was also reversibly induced when glucose concentrations in the medium were reduced. Glucose restriction influenced CD44-mediated HA binding by many but not all, of a series of murine tumors. Glucose concentrations and glycosylation inhibitors only partially influenced CD44 receptor function on resting murine B lymphocytes. These observations suggest that glucose levels or other local environmental conditions may markedly influence glycosylation pathways used by some tumor cells, resulting in dramatic alteration of CD44-mediated functions.
To investigate the potential use of E. coli cytosine deaminase (CD) gene instead of the commonly used HSV-TK gene in the gene therapy of brain tumors, we constructed a retrovirus vector carrying the CD gene. We then transduced a rat glioma cell line C6 with CD gene by the retrovirus vector. Transduction of the CD gene made C6 cells become highly sensitive to the anti-fungi drug 5-fluorocytosine (5FC). IC50 for 5FC was 6,000 microM in CD-negative cells, while it was 3 microM in CD-positive cells. Mixed cellular assay showed that CD-positive cells had a strong "bystander effect" on CD-negative cells when exposed to 5FC. Significant anti-tumor effects were observed in nude mice bearing s.c. tumors derived from CD-positive cells when these animals were given 250 mg/kg 5FC twice a day for 20 consecutive days. A marked decrease in tumor weight occurred when a mixture containing 50% CD-positive and 50% CD-negative C6 cells was injected s.c., followed by 5FC treatment, suggesting the bystander effect in vivo. Concerning the pharmacokinetics of 5FC, especially its high oral bio-availability and good penetration into cerebrospinal fluid, we suppose that the combination of CD-gene transfer and 5FC oral administration may have potential use in the gene therapy of brain tumors.
The relationships of the corticotropin-like intermediate lobe peptide (CLIP)/ACTH-immunoreactive axons with the serotoninergic and non-serotoninergic neurons in the dorsal raphe nucleus of the rat were examined by means of a double label immunocytochemical method. It is suggested that the rare contacts established by the CLIP/ACTH-immunoreactive fibers with serotoninergic neurons (cell bodies and dendrites) are not under a synaptic from. In contrast, the contacts with non-serotoninergic neurons were predominantly formed with dendrites and showed a substantial number of synapses.
The susceptibility gene for Cowden disease (CD), an autosomal dominant inherited cancer syndrome, has recently been mapped to an approximately 6-cM interval on chromosome subband 10q22-23 between the markers D10S541 and D10S564. CD is characterized by hamartomas of many organ systems, including the thyroid, breast, skin, and gastrointestinal tract, as well as carcinoma of the thyroid and breast. Follicular thyroid adenomas and carcinomas are significant component tumors in CD; thus, we sought to examine their sporadic counterpart tumors for loss of heterozygosity (LOH) of microsatellite markers in the 20-cM region within and flanking the Cowden critical interval. In all, 38 sporadic thyroid tumors were analyzed. LOH within the CD interval was observed in 5 of 19 (26%) follicular thyroid adenomas and 1 of 9 (11%) Hürthle cell adenomas. Furthermore, of these adenomas with LOH, 3 of 4 (75%) were atypical follicular adenomas, whereas 2 of 15 (13%) were typical follicular adenomas. Surprisingly, no LOH was detected in this region in 10 follicular carcinomas. The shortest region of overlap includes the markers D10S1735 and D10S1739. If the LOH observed in these sporadic tumors is related to the CD gene, then the Cowden critical interval can be revised to lie within the interval defined by D10S579 and D10S564. LOH in this narrow interval implicates the CD gene, or another gene in that interval, in follicular thyroid tumorigenesis. However, this does not explain the lack of LOH in follicular carcinomas. Taken together, it may instead be evidence against a stepwise progression from atypical adenomas to carcinomas. Alternatively, sporadic thyroid adenoma formation may be independent of that locus, but loss of this region could prevent carcinoma formation, thus implying that the CD gene may be an oncogene or growth promoter.
Much has been learned recently by experimental manipulation of the structure of CD44 and assessment of the resulting functions. However, even greater structural variation is naturally introduced by CD44-bearing cells. A structural model is now available for the portion of CD44 that recognizes hyaluronan, but it is clear that all domains of the molecule influence CD44 functions.
Cowden disease (CD) is an autosomal dominant cancer predisposition syndrome associated with an elevated risk for tumours of the breast, thyroid and skin. Lhermitte-Duclos disease (LDD) cosegregates with a subset of CD families and is associated with macrocephaly, ataxia and dysplastic cerebellar gangliocytomatosis. The common feature of these diseases is a predisposition to hamartomas, benign tumours containing differentiated but disorganized cells indigenous to the tissue of origin. Linkage analysis has determined that a single locus within chromosome 10q23 is likely to be responsible for both of these diseases. A candidate tumour suppressor gene (PTEN) within this region is mutated in sporadic brain, breast and prostate cancer. Another group has independently isolated the same gene, termed MMAC1, and also found somatic mutations throughout the gene in advanced sporadic cancers. Mutational analysis of PTEN in CD kindreds has identified germline mutations in four of five families. We found nonsense and missense mutations that are predicted to disrupt the protein tyrosine/dual-specificity phosphatase domain of this gene. Thus, PTEN appears to behave as a tumour suppressor gene in the germline. Our data also imply that PTEN may play a role in organizing the relationship of different cell types within an organ during development.
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Causative germline missense mutations in the RET proto-oncogene have been associated with over 92% of families with the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2). MEN 2A is characterized primarily by medullary thyroid carcinoma (MTC) and pheochromocytoma, both tumors of neural crest origin. Parathyroid hyperplasia or adenoma is also seen in MEN 2A, but rarely in MEN 2B, which has additional stigmata, including a marfanoid habitus, mucosal neuromas, and ganglioneuromatosis of the gastrointestinal tract. In familial MTC, MTC is the only lesion present. Somatic RET mutations have also been identified in a subset of sporadic MTCs, pheochromocytomas, and rarely, small cell lung cancer, but not in sporadic parathyroid hyperplasias/adenomas or other neuroendocrine tumors. Glial cell line-derived neurotrophic factor (GDNF) and its receptor molecule GDNFR-alpha, have recently been identified as members of the RET ligand binding complex. Therefore, the genes encoding both GDNF and GDNFR-alpha are excellent candidates for a role in the pathogenesis of those MEN 2 families and sporadic neuroendocrine tumors without RET mutations. No mutations were found in the coding region of GDNF in DNA samples from 9 RET mutation negative MEN 2 individuals (comprising 6 distinct families), 12 sporadic MTCs, 17 sporadic cases of parathyroid adenoma, and 10 small cell lung cancer cell lines. Therefore, we find no evidence that mutation within the coding regions of GDNF plays a role in the genesis of MEN 2 and sporadic neuroendocrine tumors.
Lysosomal proteases, although tightly regulated under physiological conditions, are known to contribute to cell injury after various forms of tissue ischemia have occurred. Because cathepsin B is a prominent lysosomal protease found in brain parenchyma, the authors hypothesized that it may contribute to neuronal cell death after focal cerebral ischemia. The authors measured the expression and spatial distribution of cathepsin B within the ischemic brain in 43 animals by means of immunohistochemical analysis in a rat model of transient middle cerebral artery (MCA) occlusion. Cathepsin B activity was also measured within specific ischemic brain regions by using an in vitro assay (22 animals). In addition, the authors tested the therapeutic effect of preischemic intraventricular administration of stefin A, a cysteine protease inhibitor, on the volume of cerebral infarction after transient MCA occlusion (15 animals). Increased cathepsin B immunoreactivity was detected exclusively within the ischemic neurons after 2 hours of reperfusion following a 2-hour MCA occlusion. Cathepsin B immunolocalization in the ischemic region decreased by 24 hours of reperfusion, but then increased by 48 hours of reperfusion because the infarct was infiltrated by inflammatory cells. Increased immunolocalization of cathepsin B in the inflammatory cells located in the necrotic infarct core continued through 7 days of reperfusion. Cathepsin B enzymatic activity was significantly increased in the ischemic tissue at 2, 8, and 48 hours, but not at 24 hours of reperfusion after 2 hours of MCA occlusion. Continuous intraventricular infusion of stefin A, before 2 hours of MCA occlusion (15 animals), significantly reduced infarct volume compared with control animals (12 animals): the percentage of hemispheric infarct volume was 20+/-3.9 compared with 33+/-3.5 (standard error of the mean; p = 0.025). These data indicate that neuronal cathepsin B undergoes increased expression and activation within 2 hours of reperfusion after a 2-hour MCA occlusion and may be a mechanism contributing to neuronal cell death. Intraventricular infusion of stefin A, an inhibitor of cathepsin B, significantly reduces cerebral infarct volume in rats.