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K W Lam

Publications and source records attributed to K W Lam.

At least 19 recordsLinked to original sources
PubMed1992

Heterogeneity of hairy cell tartrate-resistant acid phosphatase.

The human nonerythrocytic acid phosphatases (AcP) are composed of seven distinct activity bands in nondenaturing polyacrylamide gel electrophoresis (PAGE) when stained using either 1-naphthyl phosphate or naphthol ASBI phosphate as substrate. They are numbered 0, 1, 2, 3, 3b, 4, and 5 according to their increasing mobility toward the cathode in acidic conditions. Of these, only the most cationic "band 5" is tartrate resistant (TRAcP). When naphthol ASBI phosphate is used as substrate, AcP activity can also be stained in situ. In the presence of tartrate, activity remains strong in the hairy cells (HC) of hairy cell leukemia (HCL). Thus, the TRAcP stain has remained a reliable marker for HC. To investigate the function of TRAcP in HC, we purified two isoforms of TRAcP from HCL spleen tissue and found them to have similar substrate specificities and inhibitor sensitivities. In this report, we describe in detail the methods for TRAcP purification and compare some of the structural properties of the two isoforms to reinforce the concept that human TRAcP is a heterogeneous group of related enzymes. Band 5 represented only 15-20% of the total TRAcP extracted from HCL spleen. The remaining 80% of TRAcP hydrolyzed p-nitrophenyl phosphate but not naphthol ASBI phosphate and was not detectable in acidic, nondenaturing PAGE gels. Band 5 was solubilized from tissue using 500 mmol/L NaCl after previous extraction with 0.5% (v/v) NP-40 removed most other AcP and TRAcP activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1992

Comparison of ascorbic acid levels in the eye and remainder of the chicken embryo during development.

Ascorbic acid was not detectable in egg white and egg yolk of fertilized eggs prior to incubation. It was demonstrated in the embryo at 5 days of incubation and its content in the eyes and other parts of the embryo increased with the embryo's age. Ascorbic acid of the eyes rose rapidly after day 6 during maximal eye growth and reached a peak by days 10-14 when the expansion of the eyes began to level off. Ascorbic acid content of the eyes decreased thereafter, while ascorbic acid in the head and the body tissues continued to rise up to the time of hatching. Whether this rapid increase of ascorbic acid concurrent with the increase of eye diameter indicates a role for ascorbic acid in the growth of the embryonic eye remains to be determined.

Animals↗
PubMed1992

Oxidation of ascorbic acid as an indicator of photooxidative stress in the eye.

When whole retinal pigmented epithelium (RPE) cells isolated from bovine eyes are incubated with 14C-labeled ascorbic acid and exposed to a visible laser, the ascorbic acid is oxidized to dehydro-L-ascorbic acid (DHA). The amount of ascorbic acid which is oxidized is proportional to the radiant exposure of the sample (i.e. the total amount of radiation per unit area delivered over the exposure time). Blue light is more effective than red light in driving the reaction. The amount of label appearing in the DHA fraction is increased if unlabeled DHA is present in the reaction mixture, indicating that some redox cycling of ascorbate is occurring in the RPE cells. The ascorbic acid oxidizing activity does not depend on intact cells, is not inactivated by heating the cells to 80 degrees C, and appears to reside mainly in the subcellular fraction which contains melanin pigment granules. The ascorbic acid oxidation may be caused by free radicals formed when melanin is illuminated with light. This reaction appears to be a useful method for quantifying the production of free radicals during photooxidative stress.

Animals↗
PubMed1992

Protein-tyrosine phosphatase activity of hairy cell tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAcP) is a reliable cytochemical marker for the diagnosis of hairy cell leukemia (HCL). The enzyme has been the subject of much biochemical investigation yet its function in the hairy cells (HC) is still unknown. Two TRAcPs have been purified from HCL spleen tissues by a series of chromatographic separations. The two enzymes, provisionally called peak 1 and peak 2, had specific activities of greater than 600 U/mg and 800 U/mg respectively when p-nitrophenyl phosphate (p-NPP) was used as substrate and had Km values in the range of 1 to 5 mM p-NPP. The two TRAcPs had the same substrate specificities and inhibitor sensitivities, therefore could be isoforms of the same enzyme. Their pH optima were between 5 and 6 for all substrates tested including the phosphotyrosine-containing peptide, Raytide, which was still hydrolyzed efficiently at neutral pH. Neither phosphoserine nor phosphoserine-containing casein were hydrolyzed by either enzyme. The TRAcPs of HC may thus be capable of functioning as protein-tyrosine phosphatases (PTP). High activity of a PTP could regulate the activities of protein-tyrosine kinases and thereby influence the growth and differentiation of the hairy cells.

Acid Phosphatase↗
PubMed1991

Amino acid composition of a neutrophil respiratory burst stimulant. Evidence for a protein, noncollagenous source.

Activation of the neutrophil respiratory burst by the supernatant fraction from an alkali-treated collagen preparations (SAC) was enhanced by longer durations of exposure to alkali (1 N NaOH for 0.5-24 hr). The concentrate obtained from ultrafiltration (greater than 30,000 molecular weight) of SAC (1 N NaOH for 24 hr) retained the stimulatory factor. Fractionation of this ultraconcentrate by high-performance liquid chromatography showed that the stimulatory activity resided in the void volume (highest molecular weight). The amino acid composition of this active fraction revealed that this proteinaceous stimulant was not derived from the collagen molecule. Treatment of the SAC with ultrapure bacterial collagenase increased its stimulatory capacity, confirming its noncollagenous nature. Alkali treatment of whole cornea also released a similar large molecular weight, noncollagenous protein that stimulated the respiratory burst of polymorphonuclear leukocytes. Enhanced stimulation after prolonged NaOH treatment of the collagen preparation or collagenase treatment of SAC suggests that the stimulant might reside between collagen fibrils and then be released as the matrix is degraded.

Amino Acids↗
PubMed1990

Comparison of tartrate resistant acid phosphatase in a giant cell bone tumor and a spleen infiltrated with hairy cells.

Acid phosphatase (E.C.3.1.3.2) in a giant cell bone tumor and a spleen infiltrated with hairy cells was extracted by citrate buffer and then by 0.3 mol/L NaCl. The cationic acid phosphatase in the crude extract was isolated by CM-cellulose chromatography, and further separated by high pressure liquid chromatography. The majority of the tartrate resistant acid phosphatase in the hairy cell spleen was unabsorbed on CM-cellulose and was insensitive to iron. A much larger portion of the acid phosphatase in the bone tumor, than in the spleen, was cationic and was eluted from the column by 0.8 mol/L NaCl. The cationic acid phosphatase was further separated into consecutive peaks of acid phosphatases with different sensitivity to iron. A major portion of acid phosphatase in the giant cell bone tumor was enhanced by iron, while the amounts of iron-enhanced and iron-insensitive acid phosphatase were about the same in the spleen. The differences of the phosphatases in these two types of pathologic specimens indicate the occurrence of two types of enzymes with different biological significance.

Acid Phosphatase↗
PubMed1990

Ascorbate-enhanced copper toxicity on bovine corneal endothelial cells in vitro.

The present results indicate that 30 micrograms/ml copper was toxic to bovine corneal endothelial cells (BCEC) cultured in a serum-free medium (SFM) when the duration of treatment was 72 hours or more. Copper at 10 micrograms/ml, if mixed with 50 micrograms/ml ascorbate 2 to 3 hours before treatment, caused a transient decrease in the number of nuclei/mm2 at 72 hour, whereas 10 micrograms/ml copper alone was apparently non-toxic. When 10 micrograms/ml copper was added to 50 micrograms/ml ascorbate at the time of treatment, the toxicity was increased. All the treated cells failed to survive beyond 24 hours, and copper at a lower concentration of 1 microgram/ml could inhibit the proliferation of BCEC. We propose that copper toxicity on BCEC is augmented by ascorbate possibly through the increased replenishment of Cu+ and the subsequent enhanced production of free radicals by copper auto-oxidation.

Analysis of Variance↗
PubMed1989

Improved immunohistochemical detection of prostatic acid phosphatase by a monoclonal antibody.

A monoclonal antibody with high affinity to acid phosphatase isoenzyme 2 (Ab-AcP2) was selected to examine its binding to different normal and tumor tissues using the indirect immunohistochemical method. Both mature prostatic epithelial cells in the prostate and the highly dedifferentiated prostatic cancer cells in the bone marrow showed strong binding to the antibody. Among nonprostatic tissues, only bone marrow, breast, and kidney showed trace staining in some specimens. The specificity of Ab-AcP2 was much better than that of the polyclonal antibody to acid phospatase previously reported. When the antibody to the prostate-specific antigen (Ab-PSA) was used, weak background staining was often encountered, and weak to moderate stains were seen in the prostatic stroma, bone marrow, lung, skin, and melanoma.

Acid Phosphatase↗
PubMed1989

Generation of monoclonal antibodies to prostatic acid phosphatase isoenzyme 2 and application in solid-phase enzyme immunoassay.

Monoclonal antibodies specific to prostatic acid phosphatase (PAP) isoenzyme 2 were generated by using an improved hybridoma technique. After three subcutaneous immunizations and three intravenous boosters, cell fusion experiments were performed. The hybrid cells were first cultured in a semisolid medium containing methylcellulose and later transferred to a liquid medium for further subculture. Out of a total of 600 colonies recovered after two cell fusion experiments, 13 were shown to exhibit affinity to PAP isoenzyme 2 by radioimmunoassay. Nine hybrid cell lines which showed high affinity and specificity were established for further evaluation. Their immunoglobulin subclass was determined to be immunoglobulin G. The association constants between PAP isoenzyme 2 and each monoclonal antibody were determined by titration curve in radioimmunoassay (RIA). Three of them (PAP 1, PAP 03, and PAP 019) were shown to be over 1 X 10(9) M-1. From the results of a matrix cross-matching procedure, a pair of antibodies (PAP 03 and PAP 1) reacting with discrete antigenic determinants were identified for preparing a solid phase sandwich enzyme immunoassay (EIA) kit. The designed EIA procedure could be performed within 40 min in a one-stage incubation protocol. The assay time was shorter than that of other commercial RIA or EIA kits, and the sensitivity was 0.4 ng/ml which was comparable to that of RIA kits. The EIA kit was shown not to cross-react with human thyroid stimulating hormone, alpha-fetoprotein, carcinoembryonic antigen, and acid phosphatases derived from tissues other than prostate. Therefore, this design was a simple and rapid method with high sensitivity and specificity for determining PAP isoenzyme 2 in human serum.

Acid Phosphatase↗
PubMed1989

Degradation of bovine corneal collagen by alkali.

This study was undertaken to demonstrate the effect of alkali on the molecular size of collagen. Type I and V collagen from bovine cornea were incubated with different concentrations of NaOH at room temperature and 37 degrees C for various times. The samples were then neutralized and analyzed by high-pressure liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Degradation of collagen molecules to a heterogeneous mixture of peptides with molecular weights less than 20,000 daltons occurred at concentrations greater than 0.25N, and the effect of alkali was much faster at 37 degrees C than at room temperature. In contrast, the molecular size of collagen was not affected by similar concentrations of HCl under similar conditions of time and temperature.

Alkalies↗
PubMed1987

Molecular cloning of cDNA for human prostatic acid phosphatase.

A human liver cDNA library in lambda gt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, lambda Hap21 and lambda Hap22 were further characterized: clone lambda Hap21 contained a 0.8-kb cDNA insert and clone lambda Hap22 a 1.8-2.0-kb insert. XbaI digestion of lambda Hap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone lambda Hap22 contained all the genes carried by lambda gt11(lac5cI857nin5Sam100) and the 2-kb insert. An Escherichia coli(lambda Hap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(lambda Hap22) lysate revealed that the non-induced lambda Hap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-beta-galactosidase and was produced only upon induction with IPTG. These results indicated that lambda Hap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.

Acid Phosphatase↗
PubMed1987

Factors affecting ascorbate oxidation in aqueous humor.

Aqueous humor from human glaucomatous eyes was divided into two groups. Group I aqueous humor contains a negligible amount of ascorbate, while group II contains ascorbate. Group I aqueous humor stimulates ascorbate oxidation. This oxidative effect is inhibited by the addition of EDTA, indicating the presence of metallic ions. The amount of copper determined by atomic spectrophotometry in these samples was 1.54 micrograms/ml. On the contrary, group II aqueous humor protects against ascorbate oxidation. The small amount of albumin present in these samples accounts for part of this protective effect.

Aqueous Humor↗
PubMed1985

Immunoelectron microscopic demonstration of prostatic acid phosphatase in human hyperplastic prostate.

Immunoelectron microscopic studies were done on prostatic tissues obtained from patients with benign hyperplasia. Rabbit IgG-peroxidase conjugate against purified human prostatic acid phosphatase band 2 (HPAP-2) was used for studies. Under the light microscope, the columnar secretory epithelia of prostatic glands showed different intensity and distribution of immunostaining whereas the basal cells were unstained. Under the electron microscope, the secretory epithelial cells often showed electron-dense reaction product in the Golgi apparatus and secretory vesicles and vacuoles, and only sparingly in the cisternae of nuclear envelope and rough ER. Sometimes, fusion of secretory vacuolar membrane and plasma membrane and discharge of the vacuolar contents into the extracellular space were noted. The surfaces of microvilli at the apical portion of the columnar epithelia and the lumen of the glandular acini always showed reaction product. These findings suggest that HPAP-2 may be synthesized in the rough ER and transported to the Golgi apparatus where it is concentrated and transferred to the secretory vesicles and vacuoles. HPAP-2 is finally discharged into the extracellular spaces through exocytosis, a secretory mechanism similar to that of other secretory proteins.

Acid Phosphatase↗
PubMed1978

An enzyme histochemical analysis of human subretinal fluid.

During rhegmatogenous retinal detachment, two major phagocytic cell populations can be identified in the subretinal fluid. Routine light and electron microscopic studies may be inadequate to assess quantitatively the free-floating retinal pigment epithelial cells and the macrophagic retinal pigment-laden cells. Enzyme histochemical staining permits a simple, rapid, reproducible method of identifying each cell type. This cytologic biochemical differentiation is an important tool for reexamination of the theories associated with subretinal fluid cell activity.

Body Fluids↗
PubMed1978

The insulin concentration in human ocular fluids.

We measured immunoreactive insulin concentrations in ocular fluids and serum of 19 patients. In rhegmatogenous retinal detachment patients without diabetes mellitus, the serum insulin level was 7 +/- 2 (SE) microunits/ml and the subretinal fluid insulin levels were mostly undetectable. By contrast, in rhegmatogenous retinal detachment patients with diabetes mellitus, serum insulin was 35 +/- 5 microunits/ml and the subretinal fluid always contained low but detectable amojnts of insulin. The data may suggest a small leak of insulin into the subretinal fluid of rhegmatogenous retinal detachment patients with diabetic retinopathy. In vitrectomy patients with diabetes mellitus there was no insulin in the vitreous in eight out of nine cases.

Body Fluids↗