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K W Lam

Publications and source records attributed to K W Lam.

At least 37 records · Page 2Linked to original sources
PubMed1995

Identification and synthesis of chemotactic tripeptides from alkali-degraded whole cornea. A study of N-acetyl-proline-glycine-proline and N-methyl-proline-glycine-proline.

PURPOSE: To identify and synthesize the polymorphonuclear leukocyte chemoattractant(s) released from alkali-degraded corneas. METHODS: Corneas were degraded in 1.0 N NaOH, neutralized, ultrafiltered, and dialyzed. The final active ultrafiltrate was subjected to high-performance liquid chromatography on a Protein PAK I-60 column. The most active fractions were further separated on a mu-Bondapak-C18 and I-60 column in sequence. RESULTS: Fraction 38 from the final I-60 column associated with a 210-nm absorption peak and elicited a polarization and chemotactic response from polymorphonuclear leukocytes. The loss of polarization activity in fraction 38 after exposure to prolidase suggests that this peptide contains a Pro-X (X = amino acid) peptide bond. The amino acid composition of fraction 38 was 35% glycine and 53% proline. Peptide sequence analysis was unable to establish a primary sequence even though Picotag analysis showed the presence of large amounts of the two amino acids. Mass spectrometry revealed only two molecular species of 312 MWt and 284 MWt. Tripeptides were synthesized using all possible amino acid permutations of 2 Pro and 1 Gly and tested in the polarization and chemotactic assays. These techniques demonstrated that n-acetyl-Pro-Gly-Pro, and to a lesser degree n-methyl-Pro-Gly-Pro, were the only synthetic tripeptides with activity similar to the purified chemoattractant. CONCLUSIONS: The data show that the chemotactic peptides, purified from alkali-degraded whole cornea and confirmed with identical synthetic tripeptides, are N-acetyl-Pro-Gly-Pro and N-methyl-Pro-Gly-Pro. Although a number of proteins contain the Pro-Gly-Pro sequence, large amounts of collagen in the cornea suggest this as a major source. The small size and hydrophilic nature of these chemoattractants are predictive of a high degree of diffusibility. These chemoattractants are likely to play a major role in the early neutrophil response after an alkali injury.

Amino Acid Sequence↗
PubMed1994

Neuron-specific enolase expression during eye development in the chicken embryo.

Neuron-specific enolase has been used by others as a marker for the identification of cells of presumed neural crest origin in the human eye. The contributions of neural crest to the ocular tissues have been studied most definitively in the chicken embryo. The developing chicken eye was therefore chosen to further investigate the usefulness of neuron-specific enolase as a neural crest marker. Chicken embryos of various development stages were processed for the immunohistochemical localization of neuron-specific enolase with two different specific antisera. The nerve fiber layer of the neural retina and the optic nerve reacted positively. The first weak staining was found at day 10; it became rapidly stronger during the next 2 days. A positive reaction was also observed in the ciliary ganglion. In contrast, no staining was found in tissues of known neural crest origin. Cornea, sclera, trabecular tissues and iris stroma remained negative throughout the developmental stages studied. Therefore, neuron-specific enolase can not be used as a reliable marker of neural crest derived cells, at least in the chicken embryo. Unexpectedly the lens epithelium showed moderate neuron-specific enolase activity beginning on day 5. alpha-Enolase is an important crystallin in the lens of some species, particularly the turtle. It has also been demonstrated to be present in the chicken lens in a fairly high concentration. It is possible, that neuron-specific or gamma-enolase has a similar role in the chicken lens.

Animals↗
PubMed1994

Reattendance audit in an inner-city emergency department.

A study was carried out in an inner-city emergency department (ED) of patients returning for related complaints within 48 h of discharge. The incidence was around 0.7% and the majority (82%) of patients presented with persistence or progression of the original symptoms. Patient-related factors accounted for 13% of returns and only 5% were found to be the result of doctor-related factors. Of the returnees, 54% were discharged and 36% were admitted. The most common initial complaint was pain (31%) which, in 23% of patients, was localized to the abdomen. Injury (14%) and febrile illnesses (13%) came second and third. Asthma and chronic obstructive airway diseases (COAD) also accounted for 8% of cases. Children of less than 10 years of age were more likely to return within 48 h. About 18% of cases were thought to be potentially avoidable. Better patient education and an improvement in primary health care services were thought to be important in decreasing such avoidable reattendance. Reattendance audit was a useful tool in the continuous quality improvement programme of emergency department.

Adolescent↗
PubMed1994

Correlation of phospholipid hydroperoxide glutathione peroxidase activity to the sensitivity of rat retinas to photic injury.

We hypothesize that the differential susceptibility to photic injury among different strains of rat retinas may depend on the levels of phospholipid hydroperoxide glutathione peroxidase (PHGPX) activity, one of the endogenous antioxidant enzymes in the retina. The retinas of four inbred strains of albino rat (Fischer, Wistar, Buffalo and Lewis) were analyzed for glutathione peroxidase activity using H2O2, cumene hydroperoxide, and phospholipid hydroperoxide as assay substrates. In all four strains of rat, PHGPX was observed only in the high salt extract of the retina, while peroxidases determined by H2O2 or cumene hydroperoxide were observed mainly in the low salt extract. PHGPX was highest (66.7 mU/mg) in the most light-resistant Fischer strain and lowest in the most light-sensitive Lewis strain (31.9 mU/mg), while the activity levels in the moderately light-sensitive Buffalo and Wistar strains were 46.6 and 38.5 mU/mg, respectively. In contrast, there was no significant difference in peroxidases determined by H2O2 or cumene hydroperoxide among the four strains. These observations suggested that, in rat retina, the membrane-associated PHGPX may have an important role in the defense against light-induced free radical damage.

Animals↗
PubMed1994

Comparison of peroxidases in the retina, choroid and red blood cells.

Most of the peroxidase activity in the bovine retina is specific to glutathione (GSH) while the choroid contains both GSH peroxidases and ascorbate peroxidase. The GSH peroxidase was clearly separated from ascorbate peroxidase on a cation exchange column. The nonspecific peroxidase activity of hemoproteins accounts for the peroxidase activity detected by ascorbate oxidation. All choroidal hemoproteins contain subunits very similar to those of hemoglobin. The absence of ascorbate peroxidase in the retina indicates that the protective effect of ascorbate against photic injury is not due to its reaction with retinal peroxidase. Therefore removal of H2O2 by ascorbate peroxidase activity in the choroid could be a significant factor in studies where serum ascorbate concentrations are artificially raised far above the normal level concurrent to a very small rise in retinal ascorbate concentration.

Amino Acid Sequence↗
PubMed1994

Immunohistochemical visualization of fibrin in anterior chamber angle after YAG laser capsulotomy.

Hematoxylin and eosin staining of rabbit eyes after YAG laser anterior capsulotomy showed amorphous substances at the anterior chamber angle. The amorphous substances were identified as fibrin by immunohistochemical methods. This finding indicated that the obstruction of aqueous humor flow by fibrin could be responsible for the elevation of intraocular pressure following YAG laser anterior capsulotomy.

Animals↗
PubMed1993

Acquired immunodeficiency syndrome presenting as marrow cryptococcosis.

Disseminated cryptococcal infection is an uncommon initial manifestation in patients with the acquired immunodeficiency syndrome. The most common sites of involvement by cryptococci are the central nervous system and the lungs, and involvement of the marrow is rare. There are few descriptions in the literature on the cytologic findings of marrow cryptococcosis. We report a patient with disseminated cryptococcosis in which cytologic examination of the marrow provides the first clue to the diagnosis.

AIDS-Related Opportunistic Infections↗
PubMed1993

Detection of ascorbic acid in the eye of the early chicken embryo by silver staining.

Ascorbate is found in high concentrations in the aqueous, lens and retina of a variety of animal species. Several functions have been proposed for this, including a vital role in aqueous secretion. We investigated the possible presence of ascorbate in the early embryonic chicken eye as a marker for the differentiation of the ciliary epithelium. Embryonic chicken eyes were processed for histochemical demonstration of ascorbate by silver staining. A strongly positive reaction was found in the interstitial matrix between the lens rudiment and the optic vesicle of the 2-day chicken embryo. By 3 days, with lens and optic cup separating from each other, the silver grains were concentrated in the vitreous cavity, primarily in a band-like configuration between the lens equator and the rim of the optic cup, which is the presumptive ciliary epithelium. A weaker reaction was seen in the lens vesicle and primitive neural retina. During the second and third day of incubation the optic vesicle is just beginning to expand. The appearance of ascorbic acid in the newly formed vitreous cavity at this time may indicate its importance in the biosynthesis of the matrix that is filling the expanding optic vesicle.

Animals↗
PubMed1993

Kinetic properties of light-dependent ascorbic acid oxidation by melanin.

The kinetic properties of ascorbic acid oxidation by light-activated melanin granules demonstrate the presence of a specific reactive site on the melanin granule saturable by ascorbic acid. Increased light intensity increased the Vmax and reduced the Km of this reaction, indicating increased affinity of the active site for ascorbic acid. The kinetics of this reaction are not markedly changed in a reduced-oxygen environment. Ascorbic acid oxidation is competitively inhibited by isoascorbic acid, an epimer of ascorbic acid, while other tested reducing agents are inactive. The Ki for isoascorbic acid is 1 mM, about the same as the Km of ascorbic acid.

Animals↗
PubMed1993

Alkali-degraded cells generate a respiratory burst stimulant for neutrophils.

Alkali treatment of whole cornea produces a high molecular weight polypeptide causing a respiratory burst in neutrophils. The current study was conducted to determine if this stimulant could be elicited from cultured or scraped corneal cells (epithelium, keratocytes, or endothelium). Fresh or previously frozen epithelium, endothelium, and stroma were isolated from bovine eyes, alkali treated, and tested for the presence of the respiratory burst stimulant. The same procedure was performed on cultured bovine epithelium, keratocytes, and endothelium. To determine if the stimulant is specific to corneal cells, the component parts of human blood (cellular and extracellular) were separated, treated with alkali, and tested individually. Activation of the respiratory burst of polymorphonuclear leukocytes (PMNs) was used as a marker for the presence of the stimulant. All alkali-degraded cell samples, except red blood cells, produced a respiratory burst when added to viable PMNs. Alkali-treated plasma induced weak stimulation. Bovine corneal epithelium was alkali-treated and separated by high-performance liquid chromatography (HPLC) according to molecular size. The stimulant was shown to have a high molecular weight by its recovery in HPLC fraction number 7. These results demonstrate that the PMN stimulant originates from cells. This PMN stimulant is proposed as an inflammatory mediator in the alkali-injured cornea.

Animals↗
PubMed1993

Sodium-dependent ascorbic and dehydroascorbic acid uptake by SV-40-transformed retinal pigment epithelial cells.

The present data confirmed previous studies with other cell types that ascorbic acid and dehydroascorbic acid are transported through different transporters into SV-40-transformed retinal pigment epithelial cells. These experiments were performed on cells grown on 96-well culture plates. Ascorbic acid was taken up into the cell by a high-affinity transporter with Km = 0.041 mmol/l and a low Vmax of 2.74 pmol/min/well. Dehydroascorbic acid was taken up by a low-affinity transporter with Km = 5.67 mmol/l; however, the Vmax was 325.5 pmol/min/well. The uptake of both ascorbic acid and dehydroascorbic acid was dependent on the sodium concentration. The uptake of ascorbic acid does not involve oxidation-reaction steps because the uptake of [14C]-ascorbate was unaffected by the presence of an excess amount of unlabelled dehydroascorbic acid.

Ascorbic Acid↗
PubMed1993

Purification of phospholipid hydroperoxide glutathione peroxidase from bovine retina.

A low molecular size peroxidase with a high affinity for phospholipid hydroperoxide was purified from bovine retina by sequential extraction with low and high ionic strength buffer, followed by ammonium sulfate fractionation, chromatography on an ultraspherogel column and Protein PAK-SP column. The purified enzyme has a low Km (0.011 mmol/L) for phospholipid hydroperoxide, and a high Km (1.37 mmol/L) for glutathione. Glutathione oxidation was competitively inhibited by vitamin E, Ki = 0.019 mmol/L. The retinal PHGPX is different from the PHGPX purified by others from heart and liver in molecular size. The molecular size estimated by gel filtration chromatography is below 6 kDa.

Ammonium Sulfate↗
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↗