Biomedical subjects

J K Ma

Publications and source records attributed to J K Ma.

At least 55 records · Page 3Linked to original sources

PubMed1993

Regulation of tight junction permeability by calcium mediators and cell cytoskeleton in rabbit tracheal epithelium.

The present study investigates the mechanisms controlling tight junction permeability of the tracheal epithelium, with an emphasis on the regulatory role of intra- and extracellular calcium as well as the cell cytoskeleton. The tracheas were isolated from rabbits and their junctional permeability barrier was investigated in vitro by means of transepithelial electrical resistance measurements and flux measurements of the radiolabeled paracellular tracer, 14C-mannitol. The effects of intra- and extracellular calcium were studied using the calcium ionophore A 23187 and EGTA, and that of the cytoskeleton was investigated using cytochalasin B. Intracellular calcium of the tracheal epithelium was monitored microfluorometrically using the specific calcium indicator, Fura-2 AM (acetoxymethyl ester). The results indicate that the tight junction permeability of the trachea was significantly increased upon treatment with all three of the test compounds, as evidenced by a substantial decrease in transepithelial electrical resistance and an increase in transepithelial flux of 14C-mannitol. The effects of EGTA and cytochalasin B on the tight junction permeability are fully reversible upon removal of the compounds from the bathing media. On the other hand, tissues treated with the calcium ionophore demonstrate a partial or no recovery in membrane permeability, depending on the intracellular calcium levels. Moderate and transient increases in intracellular calcium caused a partial reversibility of the membrane resistance, while high and sustained intracellular calcium levels induce a complete irreversibility of the membrane resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Transport and hydrolysis of enkephalins in cultured alveolar epithelial monolayers.

An in vitro cultured monolayer system of alveolar epithelial cells was used as a model to investigate transport and hydrolysis of two enkephalin peptides, Met-enkephalin (TGGPM) and [D-Ala2]Met-enkephalinamide (TAGPM), in pulmonary epithelium. Isolated alveolar type II cells formed continuous monolayers when grown on microporous tissue culture-treated polycarbonate filters in serum-free, hormonally defined medium. Transport and hydrolysis studies of enkephalins in the monolayer system obtained after 6 days in culture, using fluorescence reversed-phase HPLC, indicate a reduced but significant degradation of enkephalins in the alveolar epithelium compared to most other epithelia previously reported. Aminopeptidases and dipeptidyl carboxypeptidase represent two major hydrolytic enzymes for TGGPM, as indicated by the formation of the degradative products Tyr and Tyr-Gly-Gly, while dipeptidyl peptidase, which is responsible for the formation of Tyr-Gly, contributes much less. The enkephalinase inhibitor thiorphan failed to prevent the hydrolysis of TGGPM whereas the enkephalin analog TAGPM was relatively resistant to enzymatic cleavage. The rate of enkephalin transport across the alveolar epithelium was directly proportional to drug concentration and occurred irrespective of transport direction, suggesting passive diffusion as the major mechanism for transepithelial transport. Agents that affect paracellular transport pathways, e.g., EGTA and the calcium ionophore A-23187, greatly promoted the transport rate. The ionophore at high doses, in addition to promoting tight junction permeability, also caused cellular damage associated with a sustained rise in intracellular calcium levels, as indicated by nuclear propidium iodide fluorescence. The cultured monolayer of alveolar epithelium may be used to study pulmonary drug absorption, degradation, and toxicity.

Animals↗

PubMed1993

Mechanisms of hydroxyl free radical-induced cellular injury and calcium overloading in alveolar macrophages.

Excessive production of reactive oxygen radicals by alveolar macrophages is proposed to play an important role in oxidative lung injury. A major product oxygen radical formation is the highly reactive hydroxyl radical (.OH) generated via a biologic Fenton reaction. In addition to its known ability to induce lipid peroxidation, recent studies have suggested that the .OH may exert its cytotoxic effect through the alteration of [Ca2+]i homeostasis. To test this potential mechanism as well as to investigate the relationship between .OH and Ca2+ overloading in cytotoxic injury, isolated rat alveolar macrophages were exposed to externally generated radical system, H2O2 (0.01 to 1 mM) and Fe2+ (1 mM) and their [Ca2+]i levels and cell injury were monitored using quantitative fluorescence microscopy with the aid of the specific Ca2+ indicator, Fura-2, and membrane integrity indicator, propidium iodide. Electron spin resonance measurements using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) confirmed the production of the .OH radical by this system. Upon the addition of the radicals, the macrophages displayed a rapid initial rise in [Ca2+]i which was followed by a slower but more pronounced [Ca2+]i elevation that reached a level 3 to 5 times higher than the basal level. This process preceded cell death as evident by nuclear propidium iodide fluorescence. Depletion of extracellular Ca2+ inhibited both the [Ca2+]i response and cell injury. Preincubation of the cells with the Ca2+ channel blocker verapamil or .OH radical scavenger mannitol similarly inhibited the [Ca2+]i rise and loss of viability. Firefly luciferase assay of cellular ATP content demonstrated that the alterations in [Ca2+]i following .OH treatment preceded the depletion of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Characterization and applications of antibodies produced in plants.

Of the variety of compounds expressed in transgenic plants, antibodies offer probably the widest range of applications. The antibodies appear to possess all of the functional characteristics of antibody derived from hybridoma cells, although further study will be required to determine the effect of the difference in heavy chain glycosylation. More work will also be directed toward the assembly, accumulation, stability and secretion of plant antibodies. The effect of the signal sequence on the expression and assembly of antibodies has been shown and further methods of optimizing transgenic protein accumulation in plants almost certainly exist. Plant antibody technology is still in its infancy. However, it offers enormous potential in "mix-and-match" antibody engineering, and the construction of multimeric immunoglobulin complexes may be feasible relatively easily, for the first time. Furthermore, as there is an enduring interest in using antibodies for therapeutic purposes, agricultural production and distribution offers a means of obtaining large quantities of antibodies at a relatively low cost.

Amino Acid Sequence↗

PubMed1992

Monoclonal antibody engineering in plants.

Techniques for plant transformation have been developed to such an extent that a number of foreign genes are currently being introduced into transgenic plants. Tobacco plants that produce monoclonal antibodies are of interest, because in addition to synthesis of two gene products (i.e. the heavy and light chains), the two polypeptides need to be assembled correctly, in order to result in a functional antibody. The studies on a catalytic antibody suggest that this is the case, and that the antibody functions identically to the native murine-derived antibody. The only difference observed was in the glycosylation of the heavy chain. Further transgenic plants are being generated to produce monoclonal antibodies that may be used therapeutically (and are therefore required in large quantities), or to provide disease resistance in plants. In addition, the ability of plants to assemble antibody complexes is being investigated further, to study the possibility of generating secretory IgA, which consists of heavy and light chains as well as two additional polypeptide units.

Antibodies, Monoclonal↗

PubMed1992

A mechanism of passive immunization with monoclonal antibodies to a 185,000 M(r) streptococcal antigen.

The cell surface streptococcal antigen (SA) I/II of 185,000 M(r) is an immunodominant molecule that expresses one or more adhesion determinants. A series of 14 monoclonal antibodies (MAb) to defined parts of SA I/II were generated and some of these were used in passive immunization of macaques. Topical administration of selected MAb to the teeth of macaques prevented colonization of endogenous or implanted exogenous Streptococcus mutans for a period of 1 year. Significant reduction of both smooth surface and fissure caries was found in macaques who had MAb (Guy's 1) applied to their teeth, as compared with saline-treated animals. A series of in vivo passive immunization experiments was then carried out in 57 human subjects. Topical application of MAb to SA I/II prevented colonization of both artificially implanted exogenous strains of S. mutans, as well as natural recolonization by indigenous S. mutans. The properties of the protective MAb were then investigated and the epitope specificity within the SA I/II molecule was found to be essential but not the isotype specificity of the immunoglobulin (Ig). The requirement for complement activating and the phagocyte binding sites of the Fc fragment of MAb was not essential, as the F(ab')2 fragment of the MAb was as protective as the intact IgG, but the Fab fragment failed to prevent recolonization of S. mutans. Prevention of recolonization was specifically restricted to S. mutans, as the proportion of other organisms, such as S. sanguis, failed to show a significant change. The surprising feature of these experiments was that protection of re-colonization of S. mutans lasted up to 2 years, although MAb was applied for only 3 weeks and functional MAb was detected on the teeth only 3 days following application of the MAb. The long-term protection could therefore not be accounted for by a persistence of MAb on the teeth, but may be due to a shift in the microbial balance in which other bacteria occupy the ecological niche vacated by S. mutans, resulting in colonization resistance to S. mutans. Gene cloning and sequencing the SA from S. mutans, S. sobrinus and S. sanguis identified a conserved region (residues 955-1213) which on Southern hybridization and partial DNA sequence analysis was also found in 19 alpha-haemolytic oral streptococci. The results suggest that the SA molecule may constitute a family of adhesins in oral alpha haemolytic streptococci.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

The transport barrier of epithelia: a comparative study on membrane permeability and charge selectivity in the rabbit.

The transport barrier of the epithelial presents one of the major problems limiting the effective use of these tissues as alternate delivery routes for macromolecules such as peptides and proteins. In the present study, two membrane transport properties, namely, the permeability and permselectivity of the shunt pathway, were investigated and compared in various tissues including the nasal, tracheal, bronchial, buccal, rectal, vaginal, corneal, epidermal, duodenal, jejunal, ileal, and colonic epithelia. Membrane permeability was evaluated using a combined method based on electrical conductance and flux measurements of a hydrophilic fluorescent probe, 6-carboxy fluorescein (CF). Membrane permselectivity or the charge discriminating ability of the membrane was evaluated by KCl diffusion potential measurements. The results indicate that all epithelia under investigation possess a relatively high degree of permeation barrier and are highly selective for the absorption of positively charged solutes. Shunt path permeability was found to vary greatly among tissues from different epithelia, whereas membrane charge selectivity was relatively constant in these tissues. A good correlation was observed between membrane electrical conductance and steady-state flux of CF, indicating a paracellular transport of the compound. The rank order of the intrinsic membrane permeability was as follows: intestinal approximately nasal greater than or equal to bronchial greater than or equal to tracheal greater than vaginal greater than or equal to rectal greater than corneal greater than buccal greater than skin. Membrane permselectivity, expressed as the ratio of transport number (positive over negative), ranges from 1.78 for the buccal to 1.33 for the rectal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Effects of bisbenzylisoquinoline alkaloids on alveolar macrophages: correlation between binding affinity, inhibitory potency, and antifibrotic potential.

The Chinese have conducted extensive studies concerning the medicinal properties of plant products. In this investigation the ability of three bisbenzylisoquinoline alkaloids to inhibit particle-induced activation of alveolar macrophages was evaluated and this inhibitory potential was correlated with the ability of those drugs to bind to membrane components. Tetrandrine, i.e., an herbal medicine used as an antifibrotic agent in China, was a potent inhibitor of particle-stimulated oxygen consumption, superoxide release, and hydrogen peroxide secretion by alveolar macrophages. Tetrandrine also exhibited substantial binding affinity for membrane lipids and alveolar macrophages. In contrast, tubocurine, an analogue with little antifibrotic potential, exhibited low binding affinity and had little effect on macrophage activation. Methoxyadiantifoline, an alkaloid of unknown antifibrotic potential, exhibited inhibitory and binding properties similar to those of tetrandrine. The data indicate that a strong relationship exists between the antifibrotic potential of these alkaloids and their ability to bind to alveolar macrophages and inhibit particle-induced activation of these phagocytes. These drugs should serve as useful probes to evaluate the role of alveolar macrophages in pulmonary fibrosis.

Administration, Oral↗

PubMed1991

Cosolvency of dimethyl isosorbide for steroid solubility.

Dimethyl isosorbide (DMI), which is currently under investigation for its potential use as a pharmaceutical vehicle and drug permeation enhancer, is a water-miscible liquid with relatively low viscosity. The solubilization behavior of DMI as a cosolvent for nonpolar drugs was characterized via dielectric constant measurements of binary solvent systems containing DMI and either water, propylene glycol (PG), or polyethylene glycol (PEG). Evidence from the dielectric constant profiles and NMR studies suggest that DMI undergoes complexation with water and PG, but not with PEG, through hydrogen bonding interactions. The solvent complexation exhibited a major effect on the solubilities of prednisone, dexamethasone, and prednisolone in the mixed solvent systems. Maximum solubility of each drug was found to occur near a DMI/water or DMI/PG concentration ratio of 1:2. In the DMI-PEG mixed system, while there is no apparent interaction between DMI and PEG molecules, the solubility of prednisone was found to increase with decreasing dielectric constant.

Dexamethasone↗

PubMed1991

Restriction fragment length polymorphisms and sequence variation within the spaP gene of Streptococcus mutans serotype c isolates.

A restriction fragment length polymorphism study was undertaken to determine the extent and location of heterogeneity within spaP encoding the Mr 185,000 cell surface protein P1 (antigen I/II) of Streptococcus mutans serotype c isolates. The gene was found to be highly conserved except for a central variable (V) region predicted to encode less than 150 amino acids. Sequence analysis identified two V-region variants. These differences were independent of the geographic source of the isolates. Southern analysis using synthetic oligonucleotide probes indicated that nonretention of P1 (I/II) by some isolates is not due to a deletion of the 3'-terminal DNA necessary to encode an intact carboxy terminus.

Antigens, Bacterial↗

PubMed1991

Sequence and structural analysis of surface protein antigen I/II (SpaA) of Streptococcus sobrinus.

Streptococcal antigen I/II or the surface protein antigen A (SpaA) of Streptococcus sobrinus is an adhesin which mediates binding of the organism to tooth surfaces. The complete sequence of the gene which encodes SpaA has been determined. The gene consists of 4,584 bp and encodes a protein of 1,528 amino acid residues. The deduced amino acid sequence shows extensive homology with those of the cell surface adhesins from Streptococcus mutans serotypes c and f and from Streptococcus sanguis. Structural analysis of the N-terminal region (residues 50 to 550), which is rich in alanine and includes four tandem repeats of an 82-residue sequence, suggests that it adopts an alpha-helical coiled-coil conformation. Cell surface hydrophobicity may be associated with this region. The C-terminal region is more conserved and includes two tandem repeats of a 39-residue proline-rich sequence. A further proline-rich sequence in this region is predicted to span the cell wall. Although a hydrophobic sequence is present in the C-terminal region, it appears to be too short to span the cell membrane. Anchoring of SpaA in the cell membrane may therefore require some form of posttranslational modification or association with another membrane protein.

Adhesins, Bacterial↗

PubMed1991

Conservation of the gene encoding streptococcal antigen I/II in oral streptococci.

The spaP gene of Streptococcus mutans serotype c encodes a major cell surface protein, streptococcal antigen (SA) I/II, with an Mr of 185,000, that is thought to be involved in bacterial adhesion to teeth. Proteins with significant amino acid sequence homology to SA I/II have also been found in S. sobrinus and S. sanguis. The objectives of this study were to investigate the conservation of the spaP gene in the mutans groups of streptococci and to determine whether homologous genes were present in other species of alpha-hemolytic streptococci. DNA extracted from representative strains of 19 streptococcal species was examined by Southern hybridization and partial DNA sequence analysis. A series of five overlapping DNA probes from the spaP gene were amplified by the polymerase chain reaction and used in the Southern hybridizations. The entire gene was found to be well conserved in all strains of S. mutans serotypes c, e, and f investigated. A probe from the 3' region of the gene, which encodes residues 857 to 1207 of the SA I/II protein, hybridized with DNA from a number of mutans streptococci, as well as with DNA from nonmutans alpha-hemolytic streptococci. Conservation within this region was further demonstrated by sequencing gene fragments of two strains of S. intermedius and S. oralis. The results show that some regions of the spaP gene are highly conserved not only in the mutans group of streptococci but also in other nonmutans alpha-hemolytic streptococci. This suggests that a family of cell surface proteins which, by analogy with the 185,000-Mr SA I/II of S. mutans, could be involved in bacterial adhesion might exist.

Amino Acid Sequence↗

PubMed1990

Sequencing and characterization of the 185 kDa cell surface antigen of Streptococcus mutans.

The gene spa P (formerly designated as spa P1) encoding the Mr 185,000 surface antigen I/II of Streptococcus mutans, serotype c (strain NG5) has been sequenced. The deduced amino acid sequence of antigen I/II (1561 residues) includes a putative signal peptide (residues 1-38), as well as a transmembrane region (residues 1537-1556). The N-terminal part of the protein (residues 39-550) is particularly rich in alanine and includes three tandem repeats of a sequence of 82 residues. This region is predicted to be alpha-helical, adopting a coiled-coil formation, and may account for the cell surface hydrophobicity associated with expression of antigen I/II. In contrast the C-terminal region (residues 800-1549) is proline-rich, favouring an extended conformation. Comparison with the sequence determined from Strep. mutans strain MT8148 showed that antigen I/II is highly conserved with the exception of a short central region (residues 750-805). N-terminal sequencing of purified antigens I and II components indicated that antigen I extends from the amino-terminus of the intact Mr 185,000 surface antigen while antigen II extends from residue 996.

Amino Acid Sequence↗

PubMed1990

Prevention of colonization of Streptococcus mutans by topical application of monoclonal antibodies in human subjects.

Topical application of Streptococcus mutans-specific MAbs was examined as a means of preventing colonization in the mouth. The MAbs recognize the cell surface antigen (SA I/II) of Strep. mutans and also bind to the intact bacterial cell. In experiments with implantation of an exogenous Strep. mutans strain, specific MAb significantly reduced the levels of implantation when compared with that in controls given a non-specific MAb. In a second investigation, the effect of MAb on recolonization by indigenous Strep. mutans was studied in subjects after treatment with a topical antibacterial mouthwash (chlorhexidine), which had decreased Strep. mutans to undetectable levels. In control subjects, the indigenous Strep. mutans started to recolonize within days, but those receiving MAb remained free of Strep. mutans for up to 2 yr. By using different Strep. mutans-specific MAbs, it was found that the serotype and epitope specificities of the IgG MAb were essential, but that the IgG sub-class may not be important. No clinical side-effects were detected in any of the subjects receiving MAb and there was no evidence of serum, salivary or gingival fluid antibody responses against the antibody. Local passive immunization with MAbs is a safe method for preventing colonization of Strep. mutans in the mouth.

Administration, Topical↗

PubMed1990

An investigation into the mechanism of protection by local passive immunization with monoclonal antibodies against Streptococcus mutans.

Local oral passive immunization with Streptococcus mutans-specific monoclonal antibody (MAb) (Guy's 13) prevented recolonization by indigenous S. mutans in human volunteers who had first been treated with a conventional antibacterial agent (chlorhexidine). The F(ab')2 fragment of the MAb was as protective as the intact immunoglobulin G, but the Fab fragment of the molecule failed to prevent recolonization of S. mutans. In subjects receiving the MAb Fab fragment, S. mutans levels in dental plaque and saliva reappeared at a similar rate to that found in sham-immunized subjects who received either saline or a nonprotective MAb. In vitro, MAb had no bacteriostatic or bacteriocidal effect on S. mutans. However, S. mutans grown in the presence of either intact immunoglobulin G MAb or the F(ab')2 fragment formed very long chains, which resulted in clumping of the cells. S. mutans grown with either saline or the MAb Fab fragment formed significantly shorter chains, more characteristic of streptococcal growth in liquid media. The results suggest that the two binding sites of the MAb molecule may be an essential feature for preventing streptococcal colonization but that the ability to bind to phagocytes and activate complement which resides in the Fc fragment is not essential. Protection against colonization by S. mutans lasting up to 2 years was observed in immunized subjects, although MAb was applied over a period of only 3 weeks. Furthermore, functional MAb was detected up to 3 days following application of MAb to the teeth. The long-term protection could not be accounted for by a persistence of MAb on the tooth surface, and we have suggested that it may be due to a shift in the balance of the oral flora which discouraged recolonization by S. mutans. However, examination of the proportions of Streptococcus sanguis and veillonella species in the recolonization experiments failed to reveal a significant change in the proportions of either organism, which returned to approximately the preexperimental levels in both the immunized and control groups. These findings confirm the in vivo functional specificity of the MAb to S. mutans but are not consistent with the suggestion that S. sanguis or veillonella take over the niche vacated by S. mutans, unless the shift in the proportion of these organisms cannot be detected by the method used.

Adult↗

PubMed1989

Specificity of monoclonal antibodies in local passive immunization against Streptococcus mutans.

Local oral passive immunization in human subjects with a monoclonal antibody (MoAb) raised against the 185-kD antigen I/II from S. mutans significantly reduced or prevented oral colonization of an exogenous strain of the organism. In subjects sham-immunized with either saline or an unrelated MoAb, however, significantly greater proportions of S. mutans persisted for a longer duration than in those immunized with the specific anti-streptococcal MoAb. Recolonization of indigenous S. mutans after this organism was reduced to undetectable levels by an antimicrobial agent has also been completely prevented with specific MoAb. Indeed, S. mutans was not detected for a period of over 1 year, as compared with recolonization within 10-82 days in the control subjects. The specificity of MoAb in preventing colonization of the streptococci was studied with four MoAb. This revealed that: (1) the sub-class of antibody is not an essential factor, as both MoAb Guy's 1 and 13 prevented colonization, although Guy's 1 is an IgG2a and Guy's 13 is an IgG1 class of antibody; (2) serotype specificity is important, as MoAb Guy's 9, which only recognizes S. sobrinus (serotypes d and g), does not prevent colonisation by S. mutans (serotype c); (3) neither protein nor carbohydrate nature of the putative adhesin was a determining factor, because MoAb Guy's 1 recognizes a carbohydrate and Guy's 13 a protein determinant and both MoAb prevented adherence of S. mutans; and (4) epitope specificity appears to be the most important factor in preventing adherence of S. mutans, as MoAb Guy's 11 and 13 share the same serotype specificity and both recognize a protein determinant, yet only Guy's 13 prevents colonisation. The long duration of protection from re-colonization by indigenous S. mutans, lasting about 1 year after application of the specific MoAb was stopped, cannot be accounted for by functional MoAb remaining on the teeth. We suggest that initially the MoAb prevents colonization by S. mutans and that the ecological niche vacated by this streptococcus is filled by other organisms from the oral flora, thereby discouraging re-colonization by S. mutans.

Adult↗

PubMed1989

[Double-cup replacement in the treatment of tuberculosis of the hip. Long-term follow-up studies].

24 tuberculous hip joints were treated by radical debridement and double-cup replacement in one single stage. They healed up primarily and remained well up to the time of follow-up, a period of 5 years and 7 months on average, except one which recurred 1 month after operation and persisted until removal of the implants. Slipping of head cup occurred in 4 cases in 10 to 12 months post-operatively. The slipped cups were removed or received, showing fibrous drinking of the head stump. Factors influencing and the time taking place of slipping of head cup along with it management were discussed and described. It is shown that radical debridement and double-cup replacement could serve to cure the tuberculosis and preserve the function of the hip joint.

Adolescent↗

PubMed1989

[Observations on the effect of omental transplantation to the traumatized spinal cord].

Experiments with 2 groups of different animals were carried out to observe the effect of wrapping the traumatized spinal cord with transplanted omentum. In the first group, the spinal cords of 20 rabbits were uniformly impacted with a force of 11.2g x 10cm = 112gcf, and the cord specimens prepared in different times after injury were examined under light and electron microscopes. In the second group, the spinal cords of 14 cats were equally struck with a force of 25g x 20cm = 500 gcf, and the animals were checked for motor function and somatosensory evoked potential (SEP) of their hind limbs. The results found in the rabbits in the first group showed that the transplanted omentum was adherent to the cords within 24 hours, and there were vascular communications between them after 72 hours, as shown by the presence of dye in the cord vessels, which had been injected into the omental vessels. The histologic destructions appeared in these cords were obviously less severe than those in the control ones. Motor functions and SEP, demonstrated in the animals in the second group, were favorable in the cords of cats with omental transplantation (P less than 0.05). According to the findings in these experiments, we tend to believe that omental transplantation to the traumatized cord is of some benefit.

Animals↗