Search PubMed⌕ Search

Biomedical subjects

G Zhu

Publications and source records attributed to G Zhu.

At least 217 records · Page 12Linked to original sources
PubMed1994

Characterization and chromosomal localization of the cornea-specific murine keratin gene Krt1.12.

Keratins are a group of water-insoluble proteins constituting paired acidic and basic keratin molecules that form 10-nm intermediate filaments in epithelial cells. Expression of the K3/K12 keratin pair characterizes the cornea-type differentiation. However, the mechanism that regulates this cornea-specific K12 expression remains unknown. To provide a better understanding of the cornea-specific expression, we have cloned the K12 cDNA (Liu, C.-Y., Zhu, G., Westerhausen-Larson, A., Converse, R., Kao, C. W.-C., Sun, T.-T., and Kao, W. W.-Y. (1993) Curr. Eye Res. 12, 963-974). In present studies, the murine K12 keratin gene (Krt1.12) was isolated and characterized. The murine Krt1.12 gene spans 6,567 base pairs of genomic DNA, and the mRNA encoding K12 keratin is distributed into eight exons. Chromosome mapping reveals that murine Krt1.12 is located within the Krt1 complex of mouse chromosome 11. In addition to the production of authentic K12 mRNA, the Krt1.12 gene gives rise to several alternate poly(A)+ RNAs by the use of alternative splicing in intron 2, an alternative promoter in intron 1, and/or both. Sequence analysis indicates that the transcripts derived from alternative splicing and/or the alternative promoter do not have a long open reading frame for keratin or keratin-like molecules. It is not known whether these alternate K12 poly(A)+ RNAs have any biological functions, e.g. regulation of K12 gene expression.

Amino Acid Sequence↗
PubMed1994

Complementing substitutions within loop regions 2 and 3 of the alpha/beta-barrel active site influence the CO2/O2 specificity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase.

An acetate-requiring mutant of the green alga Chlamydomonas reinhardtii, named 28-7J, has been recovered using chemical mutagenesis. It lacks ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) holoenzyme, and accumulates only a small amount of the chloroplast-encoded large subunit. Pulse/chase experiments revealed that large subunits and nuclear-encoded small subunits are synthesized at normal rates. Because the mutant strain displayed uniparental inheritance and failed to complement a known chloroplast rbcL gene mutant strain, the 28-7J rbcL gene was cloned and sequenced to identify the new mutation. A single base change was found that causes large-subunit arginine-217 to be replaced by serine. This substitution occurs within alpha-helix 2 of the alpha/beta-barrel active site. When photosynthesis-competent revertants were selected from mutant 28-7J, revertant R14-A was found to contain a second mutation within the rbcL gene. This intragenic suppressor mutation, named S14-A, causes alanine-242 to be replaced by valine within beta-strand 3. Holoenzyme from the R14-A double-mutant strain was found to have a 51% reduction in the CO2/O2 specificity factor, primarily due to a 91% decrease in the Vmax of carboxylation. The Km for ribulose 1,5-bisphosphate was increased 2-fold. Although the mutant substitutions are separated by 24 residues within the primary structure, they are close to each other in the tertiary structure. In fact, the substituted residues are also close to lysine-201, which must be carbamylated and coordinated with Mg2+ to activate the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Directed mutagenesis of chloroplast ribulosebisphosphate carboxylase/oxygenase. Substitutions at large subunit asparagine 123 and serine 379 decrease CO2/O2 specificity.

Chloroplast-encoded large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) are insoluble when separated from the holoenzyme or expressed in Escherichia coli, limiting directed mutagenesis to prokaryotic enzymes. In the present study, we performed directed mutagenesis and chloroplast transformation with the large subunit gene of Chlamydomonas reinhardtii. Two separate mutations were created that are known to influence the CO2/O2 specificity of prokaryotic enzymes. The asparagine 123 to glycine and serine 379 to alanine substitutions gave rise to photosynthesis-deficient mutants that synthesize normal levels of holoenzyme. The Vmax for carboxylation was reduced more than 95% and the Km(CO2) was increased more than 3-fold for both mutant enzymes. Km (O2) was slightly reduced for the glycine 123 enzyme, but increased more than 5-fold for the alanine 379 enzyme. CO2/O2 specificity factors for both enzymes are decreased by more than 70%. Km values for ribulose 1,5-bisphosphate are not significantly affected, but binding affinities for the transition-state analog 2-carboxy-D-arabinitol 1,5-bisphosphate are reduced. The changes brought about by these substitutions in the eukaryotic large subunit are different from the changes observed in prokaryotic enzymes.

Animals↗
PubMed1994

Dynamics of the expression of cytoskeleton components and adherens molecules by fibroblastic cells in alkali-burned and lacerated corneas.

To provide a better understanding of the role of fibroblastic cells during corneal wound-healing, we examined the expression of cytoskeleton components (i.e. smooth muscle alpha-actin (alpha-SMA), vimentin, desmin), adherens molecules (vinculin and talin) and cellular fibronectin in alkali-burned and lacerated rabbit corneas. Alkali-burned and lacerated corneas, which had healed for various periods of time (1 day to 45 days), were excised and subjected to immunohistochemical studies with monoclonal antibodies against alpha-SMA, vimentin, desmin, vinculin, talin and cellular fibronectin. Monoclonal antibody against alpha-SMA reacted with fibroblastic cells in injured corneas but did not react with keratocytes in normal corneas. Anti-desmin antibody did not react with any corneal cells in normal or injured corneas except the muscle cells in the newly-formed capillary of injured corneas. The results indicate that the fibroblastic cells in injured corneas have the characteristics of myofibroblasts. The number of myofibroblasts in granulation tissues increased and peaked within 3 weeks of injury and then declined. Electron microscopy revealed that some fibroblastic cells in the lacerated cornea which had healed for 4 weeks contained dense chromatins. In situ 3'-end labeling with terminal nucleotide transferase indicating that some of the fibroblastic cells contained nicked genomic DNA. These observations imply that apoptosis plays a role in regulating the number of myofibroblasts in the injured corneas. Antibodies against cellular fibronectin, vinculin and talin react with the fibroblastic cells in the injured corneas, but not with the keratocytes of normal corneas. Examination with transmission electron microscopy demonstrated the presence of microtendon and fibronexis associated with fibroblastic cells and the presence of stress fiber within fibroblastic cells. The results indicate that the fibroblastic cells may cause corneal wound contraction, which in turn contributes to the formation of opaque scar tissues.

Animals↗
PubMed1994

Amplification of ionophore resistance in a field strain of Eimeria tenella by treating free sporozoites with monensin in vitro.

Monensin resistance in a field strain of Eimeria tenella (FS139) was successfully amplified by an in vitro method. Sporozoites of parent FS139, designated as FS139(0), were treated with monensin at 5 micrograms ml-1 before inoculation into chickens. Oocysts developing from these drug-treated sporozoites were designated FS139(5). Sporozoites of FS139(5) were then treated with a higher level of monensin (25 micrograms ml-1), which produced another line called FS139(25). Sporozoites of FS139(25) received a monensin treatment of 100 micrograms ml-1 which yielded line FS139(100). After one passage in chickens, sporozoites of FS139(0), FS139(25) and FS139(100) were treated with either 0, 1, 25, or 100 micrograms ml-1 of monensin and inoculated into chicken primary kidney cell cultures to observe sporozoite invasion rates and development of first generation schizonts. Data on invasion rates and development of schizonts showed that FS139(100) was the most drug-resistant line while FS139(25) was the second most resistant line compared with the parent line FS139(0). These in vitro treatment techniques could be used to develop resistant coccidia for laboratory study of the physiological mechanisms of resistance.

Animals↗
PubMed1994

Lineage-specific and differentiation-dependent expression of K12 keratin in rabbit corneal/limbal epithelial cells: cDNA cloning and northern blot analysis.

Corneal epithelial cells synthesize an acidic (55 kDa) K12 and a basic (64 kDa) K3 keratin as their major differentiation products during an advanced stage of differentiation. In this paper, we describe the cDNA cloning of rabbit K12 keratin. We used a 36 base pairs (bp) oligonucleotide corresponding to a consensus sequence of many known acidic keratins as a probe to screen a cDNA library of normal rabbit corneal epithelium. Several partial cDNA clones were isolated. Hybrid-selection showed that the 3'keratin chain-specific portion of the cDNA hybridizes with K12 mRNA. A rabbit antiserum raised against the C-terminus of the cDNA-deduced amino acid sequence recognizes, in immunoblotting, the K12 keratin. In situ hybridization showed that K12 mRNA is present in all cell layers of central corneal epithelium, but in only the suprabasal cells of limbal epithelium indicating a parallel expression pattern between K12 and K3. Cultured rabbit corneal epithelial cells initially synthesize K14/K5 keratins, but later when the cells become heavily stratified they synthesize large quantities of K12 and K3 mRNAs, as detected by Northern blotting. Cultured esophageal epithelial cells do not make K12 mRNA confirming the tissue-specificity of K12 expression. Although it has been suggested that conjunctival epithelial cells can trans-differentiate into a bona fide corneal epithelium, we showed here that cultured conjunctival cells do not synthesize significant amounts of K12/K3 mRNAs. These results strongly suggest that conjunctival epithelial cells, whose differentiation can be modulated significantly by the extracellular matrix, form a lineage intrinsically distinct from the corneal/limbal epithelial lineage.

Amino Acid Sequence↗
PubMed1994

[Surgical treatment of orbital fracture].

In last six years, 51 cases (34 males, 17 females) of orbital deformities caused by fracture were treated. According to careful examinations, x-ray and CT scan, the operation plan was made. The coronal incision or plus lower eyelid incision were employed. Subperiosteal dissection began from the upper margin of the orbit to release input soft tissue and reposition the eyeball. Rib bone was grafted for orbital defects. Canthoplasty was used if necessary. The surgical results were satisfactory.

Accidents, Traffic↗
PubMed1994

[The efficacy and side reactions in humans immunized with Danish strain of dried BCG of Shanghai].

A procedure of keeping Danish BCG strain 2 is reported. The strain is cultivated in Bile potato Medium and Suton Potato Medium alternatively with incubation periods of two weeks. Lyophilized vaccine produced by using seeds derived from this strain gives better immunogenicity and lower side reaction to new-born babies and has better thermostability in compare with vaccines derived from strains of Beijing, Brazil, CCCP, France of Hungary.

Animals↗
PubMed1994

Sequence specificity in the recognition of the epidermal growth factor receptor by the abl Src homology 2 domain.

The transforming activity of the abl gene product requires a functional src homology 2 (SH2) domain. An assay was developed to evaluate this function by examining binding of a bacterially-expressed abl SH2 domain to the activated EGF receptor, used as a surrogate tyrosine phosphorylated protein. The sequence specificity of this interaction has been explored with a series of point mutants of EGF receptor. Analysis of equilibrium binding reveals that substitution of Tyr1086 for Phe in the EGF receptor produced a 10-fold reduced affinity for abl SH2 domain binding as compared to the wildtype receptor. Moreover, a phosphorylated peptide modeled on the sequences surrounding Tyr1086 specifically inhibits abl SH2 binding, with an IC50 of approximately 10 microM. Evaluation of a series of additional peptides, modeled on the Tyr1086 sequence, revealed that the carboxy terminal residues directly next to the phosphotyrosine were particularly critical to this binding. Molecular modeling studies of the pTyr1086 peptide revealed the potential hydrophobic, ionic and hydrogen bonding interactions involved in the functions of the abl SH2 domain.

3T3 Cells↗