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Biomedical subjects

J Kim

Publications and source records attributed to J Kim.

At least 937 records · Page 52Linked to original sources
PubMed1994

Single amino acid substitutions of alpha 1-antitrypsin that confer enhancement in thermal stability.

A recombinant alpha 1-antitrypsin variant which increased thermal stability was obtained from random mutagenesis followed by screening. The clone was identified as having a single mutation of Phe51-->Cys. Heat deactivation of purified recombinant alpha 1-antitrypsin produced in Escherichia coli revealed that the mutation slowed down the deactivation rate 10-fold at 57 degrees C, increasing thermal stability of recombinant protein to almost that of natural glycosylated plasma form. The mutant protein also exhibited increased stability against denaturant. The urea-induced unfolding monitored by the changes in fluorescence intensity at 360 nm showed that the mutation shifted midpoint of the transition from 1.9 M to 2.8 M. The mutation site is particularly interesting in that some genetic variants mapped at adjacent positions were shown previously to cause aggregation of the polypeptides, while the Phe51-->Cys mutation decreased aggregation rate significantly during heat deactivation. The association rate constant with porcine pancreatic elastase revealed that the mutation did not affect inhibitory activity significantly. The site identified may be critical for regulating stability of alpha 1-antitrypsin. Characterization of various single amino acid substitutions at position 51 suggests that volume and flexibility of hydrophobic side chain at the site are critical factors for enhancing the stability of alpha 1-antitrypsin.

Amino Acid Sequence↗
PubMed1994

The chromaffin granule and synaptic vesicle amine transporters differ in substrate recognition and sensitivity to inhibitors.

Classical studies using bovine chromaffin granules have defined the physiologic and pharmacologic properties of the vesicular amine transporter that packages monoamine transmitters into intracellular vesicles for subsequent regulated release. The recent isolation of two distinct but closely related cDNA clones encoding vesicular amine transport suggests that the activity expressed in the brain (synaptic vesicle amine transporter or SVAT) may differ significantly from the previously described adrenal gland activity (chromaffin granule amine transporter or CGAT). A direct comparison of the two transporters now shows that SVAT has a higher affinity than CGAT for monoamine substrates, in particular for histamine. In addition, SVAT shows approximately 10-fold greater sensitivity to tetrabenazine than CGAT. [3H]Dihydrotetrabenazine shows no detectable binding to CGAT but does bind to SVAT, accounting for the differential sensitivity. Furthermore, methamphetamine preferentially inhibits transport by SVAT relative to CGAT, apparently by competing at the site of amine recognition rather than by disrupting the vesicular pH gradient. These previously unsuspected differences in the storage of monoamine transmitter in the central nervous system and the adrenal gland may help to account for several classic pharmacological observations.

1-Methyl-4-phenylpyridinium↗
PubMed1994

Nitrogenase and biological nitrogen fixation.

Biological nitrogen fixation is catalyzed by the nitrogenase enzyme system which consists of two metalloproteins, the iron (Fe-) protein and the molybdenum-iron (MoFe-) protein. Together, these proteins mediate the ATP-dependent reduction of dinitrogen to ammonia. Recent crystallographic analyses of Fe-protein and MoFe-protein have revealed the polypeptide fold and the structure and organization of the unusual metal centers in nitrogenase. These structure provide a molecular framework for addressing the mechanism of the nitrogenase-catalyzed reaction. General features of the nitrogenase system, including conformational coupling of nucleotide hydrolysis, aspects of the cluster structures, and the general spatial organization of redox centers within the protein subunits, are relevant to a wide range of biochemical systems.

Adenosine Triphosphate↗
PubMed1994

Activation of p53 gene expression in premalignant lesions during head and neck tumorigenesis.

With the goal of identifying a potential intermediate biomarker in the multistep process of head and neck cancer development, we conducted immunohistochemical analyses for p53 expression in 33 patients with head and neck squamous cell carcinomas whose tissue sections contained adjacent normal epithelium, hyperplastic, and/or dysplastic lesions. Fifteen of 33 (45%) squamous cell carcinomas of the head and neck expressed p53, but none of four normal control patients (cancer-free nonsmokers) expressed detectable p53 in oral mucosa specimens. To determine when p53 expression is initiated during head and neck tumorigenesis, we examined the normal and premalignant lesions adjacent to the tumors. Five of 24 (21%) samples of normal epithelium adjacent to tumors, 7 of 24 (29%) samples of hyperplasia, and 9 of 20 (45%) samples of dysplasia expressed p53. Quantitative image analysis demonstrated not only a gradual increase in the amount of p53 expression as tissue abnormalities progressed but also a topological change in expression. Whereas p53 expression, when present, was limited to the basal layer in normal epithelium adjacent to tumor, the expression of p53 expanded into the parabasal and superficial layers in hyperplasia and dysplasia. We conclude that p53 expression can be altered in very early phases of head and neck tumorigenesis. Thus, it may be an excellent candidate for risk assessment and may serve as an intermediate biomarker in chemoprevention trials.

Base Sequence↗
PubMed1994

Dual-energy x-ray absorptiometry of raloxifene effects on the lumbar vertebrae and femora of ovariectomized rats.

A new potential therapeutic agent for postmenopausal osteoporosis, raloxifene, previously known as keoxifene, was evaluated by x-ray densitometry and more traditional techniques in quantitating the short-term (4-5 weeks) effects of ovariectomy on bones from 6-month-old rats. A Hologic QDR 1000/W and, to a limited extent, a Lunar DPXL, was used to quantitate ovariectomy, estrogen replacement, and raloxifene effects on vertebrae, femora, and tibiae. Both instruments performed well with precisions of 1.6% (Hologic) and 0.9% (Lunar) for anesthetized rats, which improved to 0.4% (Hologic) and 0.5% (Lunar) when the same rats were frozen. The lumbar vertebrae L1-4 showed a 12% decrease in bone mineral density 4 weeks after ovariectomy, compared with a 9% decrease for femora. Tibiae were also examined, but edge-detection problems prevented reproducible analysis of this site in vivo. The decrease in bone mineral density postovariectomy, especially for femora, was found to include both an increase in the projected area and a slight but not significant decrease in the bone mineral content of L1-4 and femora. These changes in density parameters of femora were supported by a decrease in dry weight and volume and a marginal increase in the second moment of inertia I for the identical femora examined ex vivo. Examination of individual lumbar vertebrae L1-5 suggested that the bone mineral density of L3 changes most dramatically in response to ovariectomy, but present techniques lack the spatial resolution and precision to quantitate bone changes reliably in individual vertebrae. 17 beta-Estradiol administered at 100 micrograms/kg/day subcutaneously inhibited ovariectomy effects on L1-4 bone mineral density, femoral moment of inertia, dry weight, and volume and to a lesser extent, femoral bone mineral density. A nonsteroidal compound, raloxifene HCl, at 1 mg/kg/day per os, had bone effects and effects on body weight that were largely indistinguishable from those of 17 beta-estradiol; however, raloxifene did not produce the uterotrophic effects observed with estrogen. The half-maximal efficacious dose of raloxifene on L1-4 bone mineral density was between 0.1 and 1.0 mg/kg/day per os. These data show that dual-energy x-ray absorptiometry compares favorably with traditional methods in quantitating bone changes caused by ovariectomy in small rodents, that L1-4 is a more sensitive region than whole femora in evaluating the effect of estrogen deficiency on bone loss, and the raloxifene may have promise as a treatment for conditions characterized by excessive bone loss after ovariectomy.

Absorptiometry, Photon↗
PubMed1994

Preparation and characterization of a monoclonal antibody that inhibits myoblast fusion of avian skeletal myoblasts.

To investigate the mechanism of myoblast fusion using quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells), we prepared monoclonal antibodies against a cell surface antigen involved in myogenic differentiation. For this, a Balb/c mouse was immunized with the membrane fraction of QM-RSV cells, and hybridomas producing monoclonal antibodies were raised by fusion of spleen cells from the immunized mouse with myeloma cells. By analysis of the hybridoma supernatants, we obtained a monoclonal antibody, termed H-145, that strongly inhibited myoblast fusion. H-145 inhibited myoblast fusion dose-dependently, and its effect was readily reversed by its removal. H-145 promoted biochemical differentiation of the cells until 48 h. It did not affect a fusion-commitment step to differentiation, but inhibited a later step. Indirect immunofluorescence and immunoblot analyses showed that the antigen reacting with H-145 was a glycoprotein with a molecular weight of approximately 116 kDa. This antigen is present throughout differentiation, but as differentiation progresses, its expression increases and its distribution on the cell surface changes. The antigen purified by H-145 affinity chromatography failed to react with beta 1-integrin, alpha 5-integrin, NCAM, or N-cadherin on immunoblotting. Thus, H-145 antigen differs from these components that are known to be associated with myogenic differentiation. Consequently, the results suggest that H-145 antigen may be a new cell surface antigen associated with cell differentiation.

Animals↗
PubMed1994

Ribosome-binding sites on chloroplast rbcL and psbA mRNAs and light-induced initiation of D1 translation.

Chloroplast ribosome-binding sites were identified on the plastid rbcL and psbA mRNAs using toeprint analysis. The rbcL translation initiation domain is highly conserved and contains a prokaryotic Shine-Dalgarno (SD) sequence (GGAGG) located 4 to 12 nucleotides upstream of the initiator AUG. Toeprint analysis of rbcL mRNA associated with plastid polysomes revealed strong toeprint signals 15 nucleotides downstream from the AUG indicating ribosome binding at the translation initiation site. Escherichia coli 30S ribosomes generated similar toeprint signals when mixed with rbcL mRNA in the presence of initiator tRNA. These results indicate that plastid SD sequences are functional in chloroplast translation initiation. The psbA initiator region lacks a SD sequence within 12 nucleotides of the initiator AUG. However, toeprint analysis of soluble and membrane polysome-associated psbA mRNA revealed ribosomes bound to the initiator region. E. coli 30S ribosomes did not associate with the psbA translation initiation region. E. coli and chloroplast ribosomes bind to an upstream region which contains a conserved SD-like sequence. Therefore, translation initiation on psbA mRNA may involve the transient binding of chloroplast ribosomes to this upstream SD-like sequence followed by scanning to localize the initiator AUG. Illumination 8-day-old dark-grown barley seedlings caused an increase in polysome-associated psbA mRNA and the abundance of initiation complexes bound to psbA mRNA. These results demonstrate that light modulates D1 translation initiation in plastids of older dark-grown barley seedlings.

Bacterial Proteins↗
PubMed1994

Vir-115 gene product is required to stabilize D1 translation intermediates in chloroplasts.

The nuclear gene mutant of barley, vir-115, shows a developmentally induced loss of D1 synthesis that results in inactivation of Photosystem II. Translation in plastids isolated from 1 h illuminated vir-115 seedlings is similar to wild type. In wild-type barley, illumination of plants for 16 to 72 h results in increased radiolabel incorporation into the D1 translation intermediates of 15-24 kDa. In contrast, these D1 translation intermediates were not observed in vir-115 plastids isolated from plants illuminated for 16-72 h. In addition, after 72 h of illumination, radiolabel incorporation into D1 was undetectable in vir-115 plastids. The level and distribution of psbA mRNA in membrane-associated polysomes was similar in wild-type and vir-115 mutant plastids isolated from plants illuminated for 16-72 h. Toeprint analysis showed similar levels of translation initiation complexes on psbA mRNA in vir-115 and wild-type plastids. These results indicate that translation initiation and elongation of D1 is not significantly altered in the mutant plastids. Ribosome pausing on psbA mRNA was observed in wild-type and vir-115 mutant plastids. Therefore, the absence of D1 translation intermediates in mutant plastids is not due to a lack of ribosome pausing on psbA mRNA. Based on these results, it is proposed that vir-115 lacks or contains a modified nuclear-encoded gene product which normally stabilizes the D1 translation intermediates. In wild-type plastids, ribosome pausing and stabilization of D1 translation intermediates is proposed to facilitate assembly of cofactors such as chlorophyll with D1 allowing continued D1 synthesis and accumulation in mature chloroplasts.

Bacterial Proteins↗
PubMed1994

Fluid dynamic study of the moving actuator type total artificial heart by image-processing technique.

Flow velocity inside the artificial ventricle of a moving actuator type total artificial heart is investigated by the image-processing technique. Optical flow is estimated from two sequential images of flow patterns to find flow-velocity distribution. The region with high velocity inside the artificial ventricle is found from the estimated flow-velocity distribution. Fluid velocity in a simple glass conduit is analysed and can be estimated by calculating optical flow.

Blood Flow Velocity↗
PubMed1994

Perceived motion in the vector sum direction.

The visual system is known to extract both Fourier (e.g. contour) and non-Fourier (e.g. texture boundary) motion signals from moving patterns. Recent evidence suggests that both Fourier and non-Fourier motion signals are involved in the analysis of rigid two-dimensional image motion. To determine how these signals are combined, we have measured perceived directions for plaids composed of either two non-Fourier components or one Fourier plus one non-Fourier component. In all cases the visual system combines these components to produce perceived motion in the vector sum direction, even when this deviates by as much as 53 deg from the intersection of constraints direction. This result was predicted by a model for pattern motion that combines Fourier and non-Fourier component motion signals using a vector sum operation.

Humans↗
PubMed1994

Catalytic properties and potential of an extracellular protease from an extreme halophile.

An extracellular protease has been isolated and partially purified from the extreme halophile Halobacterium halobium (ATCC 43214). The major enzyme component has a M(r) of 66,000 and is highly dependent upon salt concentrations near saturation for catalytic activity and stability. In aqueous solutions, a decrease in the NaCl concentration from 4 to 1 M results in an increase of nearly three orders of magnitude in the first-order rate constant of inactivation at 30 degrees C. Salt effects the stability of the enzyme in a cooperative manner, with a Hill coefficient of 4.1, which is similar to that of other enzymes from extreme halophiles. The enzyme activity is dramatically affected by the salt concentration, with a loss of 2.5 orders of magnitude in kcat/Km in going from 4 to 0 M NaCl. This loss in catalytic efficiency is primarily due to a dramatic increase in the Km for the substrate in low-salt media. Thermodynamic analysis revealed that this Km increase was mainly the result of increased solubility of the synthetic peptide substrate in low-salt media, which dramatically increases the ground-state stability of the substrate. This results in an effectively reduced substrate partitioning from the bulk solution into the enzyme's active site and an increased value of Km. The halophilic protease is also active in DMF/water mixtures, albeit with novel catalytic properties. In 33% (v/v) DMF in aqueous buffer, the esterase activity of the enzyme is ca. 80-fold higher than the corresponding amidase activity. This contrasts to the situation in pure aqueous buffer, in which the esterase activity is only fourfold higher than the amidase activity. The increased esterase activity relative to amidase activity prompted us to investigate the use of the protease in kinetically controlled peptide synthesis. The enzyme has a broad acyl donor substrate specificity and can effectively use amino acid esters of Phe, Tyr, Trp, Ser, Gly, and Ala. The enzyme is significantly more selective for the amino acid amide, preferring Gly in the P'1 site. A series of glycine-containing oligopeptides have been prepared in yields up to 76% without degradation due to secondary hydrolysis.

Amidohydrolases↗
PubMed1994

A standard experimental 'chemical burn'.

To establish a standard method for producing experimental cutaneous injuries caused by contact with corrosive liquids, we modified an apparatus and method recommended by Walker and Mason in 1967 to produce experimental thermal burns. The resulting procedure proved to be safe, reproducible, humane and efficient and can be used with a wide variety of corrosive liquids.

Animals↗
PubMed1994

Immunohistochemical study on proliferating cell nuclear antigen expression in ameloblastomas.

The aim of this study is to evaluate the proliferating activity of ameloblastomas and its correlation to the biological behaviour according to each histological type. 38 cases of solid and unicystic ameloblastomas were reviewed including 1 case of ameloblastic carcinoma and 1 recurrent case. An anti-PCNA antibody, PC10(Dako), was applied for the detection of proliferating cell nuclear antigen (PCNA) in paraffin embedded tissue sections. Also 20 cases of dentigerous cysts were reviewed. In conclusion, we observed that there was no difference between the proliferating activities of the different histological types of solid ameloblastomas. Because 1 case of ameloblastic carcinoma and one recurrent case revealed remarkably high PCNA reactivity, we believe that PCNA would be useful in showing the differentiation between benign and malignant ameloblastomas. In unicystic ameloblastomas, plexiform intraluminal growth was considered to be an important feature in tumorous transformation of cystic epithelium.

Ameloblastoma↗