A systematic approach to evaluate the modification of lens proteins by glycation-induced crosslinking

Normal gait requires the dynamic integration of central and peripheral nervous systems acting on an intact musculoskeletal framework. A number of specific disease processes, as well as aging, may compromise this interaction. Despite the complexity of human gait, most common gait disorders can be identified by the experienced clinician, using the fundamental tools of history and physical examination.     

Glutathione levels of the human crystalline lens in aging and its antioxidant effect against the oxidation of lens proteins

This paper reports the role of glutathione (GSH) in the crystalline lens as an antioxidant against the oxidation of lens protein. GSH levels in normal lenses decreased gradually with increasing age, from approximately 5 mumol per g lens (wet weight) to 3 mumol per g lens (wet weight). On the other hand, levels of oxidized GSH in the lenses increased until the age of 40. After that, it remained almost constant at the level of approximately 0.9 mumol per g lens. Protein-bound GSH levels in both soluble and insoluble lens proteins dropped noticeably in the 50-year and older age groups, although there were significant differences in levels between both fractions. A decrease of tryptophan and tyrosine residues in lens proteins was proportional to a decrease in GSH levels in the lens as a result of aging. Those residue levels in the cataractous lenses were approximately half those in the normal lens proteins, and GSH levels in such lenses were almost one-tenth that in the normal lens. This study revealed that GSH may play an important role in preventing the oxidation of lens proteins from various oxidants. Furthermore, it is conceivable that these normal changes in GSH levels in the lenses increase the vulnerability of the lens to senile cataractogenesis.     

Hierarchy of lens proteins requiring protection against heat-induced precipitation by the alpha crystallin chaperone

Tooth to denture base discrepancy (the discrepancy) is the difference between the dental arch length and the sum of crown diameters of teeth in the jaw, a concept which was originally developed in orthodontics. Since the cause-effect relationship between a soft diet and the discrepancy has been demonstrated, the size of the discrepancy should indicate the amount of load on the masticatory system from chewing foods in jaws from archaeological periods. The dietary condition of 71 citizens compared to that of 186 slaves from the Yin-Shang period of China was reconstructed through a study of the discrepancy. The prevalence of the discrepancy in the Yin-Shang period was around 15%, almost the same as it was during the later Jomon to Yayoi (3000-2000 BP) periods, when rice agriculture was introduced into Japan, and also the same as for present-day pastoralists around Lake Turkana, Kenya. Although the frequency of the discrepancy was slightly higher in male citizens, there were no significant differences in the frequencies between male citizens and female citizens or slaves. The differences in diet may not have been fundamental since the Yin-Shang period would be at the very beginning of the age in which differences of diet according social class began to appear, with implications for the load on the masticatory system. At that time agriculture may not been sufficiently intensified in variety or quantity to have produced a differentiation of the diet between social classes.     

Discrimination against contact lens wearers

Employers' attitudes toward the use of contact lenses at work have become less discriminatory as lenses have improved and numerous studies have demonstrated their safety, provided that additional personal protective equipment is used when necessary. In 1994, the Occupational Safety and Health Administration published its relevant Standard (29 CFR 1910), stating that "contact lenses do not pose additional hazards to the wearer...". Accommodations required by wearers of contact lenses must comply with Title I of the Americans with Disabilities Act. However, many companies still oppose their use. The recently published policy of the American College of Occupational and Environmental Medicine and the American Academy of Ophthalmology on the use of contact lenses should lead to their wider acceptance. Elements of a corporate contact lens policy are outlined. International aspects are summarized as well.     

Novel phosphoserine phosphatase inhibitors

Phosphoserine phosphatase (EC 3.1.1.3) catalyzes the final step in the major pathway of L-serine biosynthesis in brain. This enzyme may also regulate the levels of glycine and D-serine, the known and putative co-agonists for the glycine site of the N-methyl-D-aspartate receptor in caudal and rostral brain regions, respectively. Using L-phosphoserine as substrate, the rank order potency for inhibition of phosphoserine phosphatase was p-chloromercuriphenylsulfonic acid (CMPSA) > glycerophosphorylcholine > hexadecylphosphocholine > or = phosphorylcholine > N-ethylmaleimide > or = L-serine > fluoride > D-2-amino-3-phosphonopropionic acid (D-AP3). Glycerylphosphorylcholine (IC50 18 microM) was found to be an uncompetitive inhibitor of phosphoserine phosphatase. Glycerylphosphorylcholine probably binds a novel site on the enzyme since the known allosteric inhibitor L-serine is highly selective for its feedback regulatory site, indicated by the inactivity of 25 L-serine analogs. Fluoride ion (IC50 770 microM) may bind the active site as has been shown for other Mg2+-dependent enzymes. The sulfhydryl reagent CMPSA is a potent, noncompetitive inhibitor of the enzyme using L-phosphoserine as substrate (IC50 9 microM) but is > 300-fold less potent using D-phosphoserine as substrate. Substrate-dependent differences are also observed with the sulfhydryl alkylator N-ethylmaleimide, which inhibits L-phosphoserine, but stimulates D-phosphoserine hydrolysis. These sulfhydryl reagents may dissociate multimeric forms of the enzyme to form monomers; the multimeric forms and monomers may preferentially cleave L- and D-phosphoserine, respectively. Phosphorylcholine esters and sulfhydryl reagents may prove useful in determining the contribution of phosphoserine phosphatase to the biosynthesis of glycine and D-serine in neuronal tissue in vitro.     

Microdomains of GPI-anchored proteins in living cells revealed by crosslinking

There is some discussion as to whether glycosyl-phosphatidylinositol(GPI)-anchored proteins occur in microdomains in the cell membrane. These putative microdomains have been implicated in processes such as sorting in polarized cells and signal transduction. Complexes enriched in GPI-anchored proteins, cholesterol and glycosphingolipids have been isolated from cell membranes by using non-ionic detergents: these complexes were thought to represent a clustered arrangement of GPI-anchored proteins. However, results obtained when clustering of GPI-anchored proteins induced by antibodies or by detergents was prevented support the idea of a dispersed surface distribution of GPI-anchored proteins at steady state. Here we use chemical crosslinking to show that membrane microdomains of a GPI-anchored protein exist at the surface in living cells. This clustering is specific for the GPI-anchored form, as two transmembrane forms bearing the same ectodomain do not form oligomers. Depletion of membrane cholesterol causes the clustering of GPI-anchored proteins to break up, whereas treatment of cells with detergent substantially increases the size of the complexes. We find that in living cells these GPI-anchored proteins reside in microdomains consisting of at least 15 molecules, which are much smaller than those seen after detergent extraction.     

Novel bisphosphonate inhibitors of phosphoglycerate kinase

PURPOSE: Gamma-amino butyric acid (GABA) is a major inhibitory neurotransmitter in the retina, possibly participating in its normal development. The distribution of gamma-aminobutyric acid receptors was therefore examined in mature and developing rabbit retina by GABA(A) receptor alpha1 and beta2/3 subunit immunocytochemistry. RESULTS: Beta2/3 subunits appeared already at the E25 stage (25 days after gestation) although only weakly and irregularly. Alpha1 subunit immunoreactivity was first observed at birth. On the fifth postnatal day, immunostaining was clearly seen in the inner plexiform layer, and weaker, in the outer plexiform layer. There were at this stage no well-delineated sublayers in either the inner or the outer plexiform layer, but three clearly defined sublayers appeared in the inner plexiform layer at PN10. Amacrine cell bodies now also appeared, labelling for both the GABA(A) receptor alpha1 and the beta2/3 chains. A punctuate labelling appeared in the outer plexiform layer. From the 20th postnatal day, the immunoreactivity was similar to that seen in adult rabbits. CONCLUSIONS: Like in the adult, the alpha1 and beta2/3 subunits of the GABA(A) receptor thus colocalize predominantly in certain amacrine cells and in processes of the inner plexiform layer during the development of the retina. The GABA(A) receptors appear later than GABA during the development of the rabbit retina, and functioning GABA neurotransmitter circuits appear to be assembled primarily after the 3rd to 5th postnatal day. Our results support the hypothesis that GABA may have other functions than mediating classic synaptic neurotransmission before the formation of receptors containing the alpha1 and beta2/3 subunits. Like in the brain, the alpha subunits may have more selective functions during the development than the beta subunits.     

Immunological reactions against PMMA lens material?

We investigated the kind of reactions that occur after injection of a PMMA lens powder into the back skin of rabbit. The lens powder was suspended in NaCl and incomplete Freund's adjuvant to reinforce the immunological reaction. The ELISA test was carried out to detect antibodies against the lens material as a whole and against the UV absorber Tinuvin in particular. We also performed histological and immunohistochemical examinations of the back skin. We did not detect antibodies against either the lens material or against Tinuvin. Histological examination showed a foreign-body or delayed allergic reaction against the lens material. Lymphocytes surrounding the PMMA were found to be mainly of the T-type, which supports the results of the ELISA test.     

Novel disaccharide inhibitors of human glioma cell division

Several alpha-L-Fuc-(1-->3)-alpha-D-GlcNAcOC8H17 disaccharide derivatives bearing different hydroxylated alkyl chains, with or without sulfate groups at C-4 and/or C-6 positions of the GlcNAc unit, have been synthesized and tested as inhibitors of human astrocytoma lines U-373 and U-118. The antimitotic activity was dependent on the structure and position of the hydroxylated chain linked to the disaccharide. The compounds with a pentaerythritol or L-glyceryl chain at the C-6 position showed the best inhibitory properties, with an ID50 value of ca. 200 microM. On the contrary, sulfated disaccharide derivatives were inactive. The antimitotic activities of the compounds tested were essentially independent of the mitogen used to stimulate cell division.     

Novel delivery systems for coagulation proteins

Interferon-alpha (IFN-alpha) is an important molecule in the antiviral response, but cells from HIV-1-infected individuals show a reduced ability to secrete IFN-alpha. We investigated an association between an imbalance of type 1/type2 cytokines and the production of IFN-alpha in HIV-1 infection. We used whole blood culture to study the cytokine production profile, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), in response to HIV-1 antigens and to study the Sendai Virus and HSV-1-induced-production of IFN-alpha in seven HIV-1-infected patients. An impaired synthesis of IFN-alpha was obtained in patients with a predominant IL-4 production (IL-4 > IFN-gamma), and we found a positive correlation between the ex vivo production of IFN-alpha and the IFN-gamma/IL-4 ratio but not with the HIV RNA copy number in plasma. We investigated the role of T-cell-derived cytokines in the in vitro production of IFN-alpha by PBMC from eight healthy donors, activated with Sendai Virus or HSV-1. Whereas type 2 cytokines (IL-4, IL-13) inhibited virus-induced IFN-alpha synthesis, on the contrary, type 1 cytokines (IL-2, IFN-gamma) enhanced it. A disarray in the T-cell-derived cytokine response may play a role in the defect of IFN-alpha production in HIV-1-infected individuals. Further investigations are needed to explore this hypothesis.     

Lens glutathione, lens protein glycation and electrophoretic patterns of lens proteins in STZ induced diabetic rats

As diabetes is a very complex disease, with the pathological symptoms varying with age, diabetic type and means of control, it still warrants many in vivo and in vitro studies. During hyperglycaemia, increases in the sorbitol pathway, nonenzymatic glycosylation of lens proteins and damage to antioxidant systems have been reported to cause opacification of the lens leading to cataract formation. In this study, intracapsular extracts of lenses from STZ induced diabetic female rats were examined. Total protein, glutathione and nonenzymatic glycosylation were determined by the Lowry, Ellman reagent and thiobarbituric acid methods respectively. Laemmli protein electrophoresis was also carried out on the lens homogenates. After a period of as short as 5 weeks, a decrease in lens glutathione, and an increase in nonenzymatic glycosylation of lens proteins were found. The electrophoresis showed an increase in proteins of high molecular weight.     

Novel inhibitors for multidrug resistance: 1,3,5-triazacycloheptanes

1,3,5-Triazacycloheptanes were synthesized and examined for reversal of the multidrug resistance dependent on P-glycoprotein. Most of these compounds increased the intracellular uptake of vinblastine in multidrug-resistant mouse leukemia P388/ADR cells without influence upon the vinblastine accumulation in P388/S cells. The efficacy of 1,5-dibenzyl-1,3,5-triazacycloheptanes in increasing the vinblastine accumulation was in the order of 2,4-dithioxo (5) > 2-oxo-4-thioxo (4) approximately 4-(methylthio)-2-oxo (6) > 2,4-dioxo (2). The efficacy was further increased when the benzyl group was converted to a chlorobenzyl group. Among these compounds, 6c [1,5-bis(4-chlorobenzyl)-1,5,6,7-terahydro-4-(methylthio)-2H-1,3,5 - triazepin-2-one] potentiated the in vitro cell growth-inhibitory effect of vinblastine, adriamycin, and mitomycin C on P388/ADR cells and prolonged the life span of P388/ADR-bearing mice in combined therapy with vinblastine more than vinblastine alone.     

Novel bombesin-like peptide binding proteins from lung

Our first year of experience in the use of PET scanning in the management of nine patients with ovarian cancer leads us to conclude that this promising new technique may be more sensitive than either serum CA-125 determinations or computed tomography for the detection and demonstration of residual or recurrent abdominal and pelvic tumor. Seven of these patients underwent second-look laparotomy which confirmed our impressions from preoperative PET scans in six patients, and the one other scan showed a focus of metabolic uptake coinciding with residual tumor in our retrospective review. The clinical courses of two other patients who did not undergo laparotomy confirmed the impressions gained from PET scans.     

Novel phosphate mimetics for the design of non-peptidyl inhibitors of protein tyrosine phosphatases

Benzylic alpha,alpha-difluorosulfonates, alpha,alpha-difluorotetrazoles, and alpha,alpha-difluorocarboxylates of type 5 and 6 were synthesized and examined as potential phosphate biosteres for PTP1B inhibition. The alpha,alpha-difluorosulfonates and alpha,alpha-difluorotetrazoles were found to be more effective inhibitors than the analogous compounds bearing the fluoromalonyl group, a phosphate biostere currently being used for PTP inhibition.     

Novel cytokine release inhibitors. Part II: Steroids

Steroidal derivatives as IL-1 beta release inhibitors are discussed.     

Novel cytokine release inhibitors. Part I: Triterpenes

Tripterine and closely related triterpenoid derivatives as IL-1 beta release inhibitors are discussed.     

Novel peptidyl phosphorus derivatives as inhibitors of human calpain I

Dipeptidyl phosphorus compounds were synthesized as potential bioisosteric mimics of peptide alpha-ketoesters and alpha-ketoacids. alpha-Ketophosphonate Cbz-Leu-Leu-P(O)(OCH3)2 (1b), containing an alpha-ketoester bioisostere, inhibits human calpain I with an IC50 = 0.43 microM. The potency of 1b compares very favorably with that of alpha-ketoester Cbz-Leu-Leu-CO2Et (IC50 = 0.60 microM). Monomethyl ketophosphonate Cbz-Leu-Leu-P(O)(OH)(OCH3) (1a, IC50 = 5.2 microM), an alpha-ketoacid mimic, is less potent. Dibutyl and dibenzyl alpha-ketophosphonates 1c,e,f are much less potent calpain inhibitors than dimethyl alpha-ketophosphonate 1b. alpha-Ketophosphinate 1g (IC50 = 0.37 microM) and alpha-ketophosphine oxide 1h (IC50 = 0.35 microM) are also potent calpain inhibitors.     

Benzisothiazole-1,1-dioxide alkanoic acid derivatives as inhibitors of rat lens aldose reductase

Derivatives of 4-substituted 1,2-benzisothiazole-1,1-dioxide alkanoic acids were prepared and their in vitro aldose reductase inhibitory activity was tested in rat lens enzyme. The acetic derivatives 10, 12, and 16a-d proved to be much more potent inhibitors than the propionic derivatives 11, 13, and 17a-d. The presence of an acyl moiety on the amino group in position 4 of the acetic derivatives 16a-d led to a significant increase in activity with respect to the parent compound 14. One of the most active compounds in vitro, 10, was also evaluated in vivo as an inhibitor of glutathione lens depletion in galactosemic rats, but it did not show any activity in maintaining the rat lens glutathione level, probably due to problems of ocular bioavailability or metabolism.