Novel monoclonal antibodies to putative selectin carbohydrate ligands that inhibit selectin binding to myeloid cells

Four newly developed monoclonal antibodies (MAbs) are characterized using flowcytometry, enzyme-linked immunoadsorbent assay (ELISA), immunoprecipitation and Western blots, carbohydrate epitope mapping, glycosidase cleavage, and competition binding assays. Their effects on selectin binding to myeloid cells was tested. These MAbs react only with myeloid cells. MAbs CI-1, BU60, and HIM95 recognize epitopes expressed by CD11/CD18 (beta2) integrins, while HI247 and CSLEX1 do not. The epitopes require Lewis x [Galbeta1-4 (Fucalpha1-3)GlcNAc] based on reactivity with oligosaccharide-polyacrylamide-biotin or oligosaccharide-BSA conjugates. MAb HI247 recognizes a related structure, sialyl-Lewis x, NeuAcalpha2-3GaLbeta1-4(Fucalpha1-3)GlcNAc. The three MAbs against Lewis x show some minor differences in their reactivity such as recognizing their antigens on CD11/CD18 integrins after endo-beta-galactosidase treatment and recognizing free Lewis x. The hydroxyl group on C-3 of the terminal galactose is important for recognition by MAb CI-1, BU60, and HIM95 as its substitution with sulfo group of sialic acid abolishes the binding of these MAbs. The C-3 sialic acid is crucial for the binding of MAb HI247. Its replacement by sulphate or its cleavage by sialidase eliminates recognition by this MAb. MAbs HI247 and CSLEX-1 did not react in ELISA with immobilized CD11/CD18, suggesting that the majority of sialyl Lewis x on CD11/CD18 molecules may have sialic acid 6-linked rather than 3-linked to galactose. Unexpectedly, MAb BU60 inhibited binding of P-selectin mu chain chimera to HL-60 or U937 cells, while CI-1, HIM95 and three other defined anti-Lewis x MAbs (6C7, M6-1 and LeuM1) did not. MAb HI247 inhibited binding of both E- and P-selectin chimeras to these cell lines more effectively than several characterized MAbs (CSLEX-1, FH6, HECA-452) to sialyl Lewis x and related oligosaccharides. Certain combinations of these anticarbohydrate MAbs had additive inhibitory effects on selectin binding, suggesting a potential application of these new MAbs in cell adhesion/migration and tumor metastasis studies.     

Investigation of spectral reproducibility in direct analysis of bacteria proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) can be used for rapid detection of bacteria proteins in a crude mixture. It can potentially be used as a tool for bacterial identification based on the mass spectral patterns or the appearance of some characteristic mass peaks. However, there are many experimental parameters that can potentially have a strong effect on the observed mass spectra. The objective of this work is to address the mass spectral reproducibility issue. Several experimental parameters that may affect the MALDI spectra are systematically investigated. Results of spectral comparison from two laboratories with different operators and instrumentation are presented. It is demonstrated that minor variations in the sample/matrix preparation procedures for MALDI and in the experimental conditions used for bacterial protein extraction can result in a significant change in the observed spectra, though a number of peaks are conserved in the spectra obtained under different experimental conditions from the same bacterial sample. These conserved peaks may potentially be used as the biomarkers for bacterial identification. It is stressed that this type of investigation on spectral reproducibility should be carried out for different bacterial species in order to identify the mass spectral peaks that are consistently detected regardless of operator and nominal variations in sample preparation approach.     

Deficient transport of dehydroascorbic acid in the glucose transporter protein syndrome

The glucose transporter protein syndrome (GTPS) is caused by defective transport of glucose across the blood-brain barrier via the glucose transporter GLUT1, resulting in hypoglycorrhachia, infantile seizures, and developmental delay. Recent reports indicated that GLUT1 is a multifunctional transporter. We investigated the transport of vitamin C in its oxidized form (dehydroascorbic acid) via GLUT1 into erythrocytes of 2 patients with GTPS. In both patients, uptake of oxidized vitamin C was 61% of the mothers' values. Our findings are consistent with recent observations that vitamin C is transported in its oxidized form via GLUT1. We speculate that impaired transport of this substrate and perhaps other substrates in GTPS might contribute to the pathophysiology of this condition.     

Circadian expression of tryptophan hydroxylase mRNA in the chicken retina

Many aspects of retinal physiology are controlled by a circadian clock located within the eye. This clock controls the rhythmic synthesis of melatonin, which results in elevated levels during the night and low levels during the day. The rate-limiting enzyme in melatonin biosynthesis in retina appears to be tryptophan hydroxylase (TPH)[G.M. Cahill and J.C. Besharse, Circadian regulation of melatonin in the retina of Xenopus laevis: Limitation by serotonin availability, J. Neurochem. 54 (1990) 716-719]. In this report, we found that TPH mRNA is strongly expressed in the photoreceptor layer and the vitread portion of the inner nuclear layer; the message is also expressed, but to a lesser extent, in the ganglion cell layer. The abundance of retinal TPH mRNA exhibits a circadian rhythm which persists in constant light or constant darkness. The phase of the rhythm can be reversed by reversing the light:dark cycle. In parallel experiments we found a similar pattern of expression in the chicken pineal gland. However, whereas a pulse of light at midnight suppressed retinal TPH mRNA by 25%, it did not alter pineal TPH mRNA, suggesting that there are tissue-specific differences in photic regulation of TPH mRNA. In retinas treated with kainic acid to destroy serotonin-containing amacrine and bipolar cells, a high amplitude rhythm of TPH mRNA was observed indicating that melatonin-synthesizing photoreceptors are the primary source of the rhythmic message. These observations provide the first evidence that chick retinal TPH mRNA is under control of a circadian clock.     

Src can regulate carboxy terminal interactions with AFAP-110, which influence self-association, cell localization and actin filament integrity

The SH2 and SH3 binding partner AFAP-110 is a tyrosine phosphorylated substrate of Src. AFAP-110 has been hypothesized to link Src to actin filaments, which may contribute to the effects of Src upon actin filament integrity. However, it has been unclear what effect activated Src (Src527F) has upon AFAP-110 structure or function and whether AFAP-110 plays a role in actin filament integrity. We report here that the carboxy terminal 127 amino acids of AFAP-110 are comprised of an alpha-helical region that contains a leucine zipper motif. This indicated the potential of AFAP-110 to self-associate. Expression of the carboxy terminus as a fusion protein (GST-cterm) will permit affinity absorption of cellular AFAP-110. The integrity of the alpha-helical leucine zipper motif in GST-cterm is required for affinity absorption, but binding is not due to a classical leucine zipper interaction. Co-expression of Src527F, unlike cSrc, will abrogate affinity absorption of AFAP-110 with GST-cterm. These data indicate that Src527F has affected a change in the carboxy terminal structure that renders AFAP-110 unavailable for affinity absorption. Superose chromatography demonstrate that AFAP-110 will fractionate as a monomer or multimer, indicating AFAP-110 can be detected in a self-associated form in cell lysates. Co-expression of Src527F resulted in AFAP-110 fractionating with a molecular weight that predicts only a multimeric population. Deletional mutagenesis also indicate a biological role for the carboxy terminus in cellular localization and actin filament integrity. Deletion of the entire carboxy terminal alpha-helix (84 amino acids) will not permit AFAP-110 to efficiently colocalize with actin filaments or the cell membrane. Deletion of only the leucine zipper region of the carboxy terminal alpha-helix (44 amino acids) from AFAP-110 (AFAPAdeltazip) demonstrate that both AFAPdeltalzip and actin filaments are repositioned into rosette-like structures, similar to the effects of Src527F, while co-expression of AFAP-110 with cSrc will not affect actin filaments. These data indicate that AFAP-110 can play an important role in modulating actin filament integrity through carboxy terminal interactions that can be affected by Src527F.     

Haemosuccus pancreaticus as a rare initial manifestation of chronic pancreatitis

Twenty-seven painful knee replacements were evaluated arthroscopically. The diagnostic and therapeutic value of these arthroscopic procedures was studied retrospectively. In 5 of the 27 cases, the arthroscopy revealed no diagnosis for the pain. Some form of arthroscopic treatment was performed in 20 cases; in 6 of these 20 cases, however, the treatment did not reduce the pain. Based on these findings, we conclude that the indications for arthroscopic evaluation and treatment of painful knee prostheses are limited.     

Tonic regulation of excitation-contraction coupling by basal protein kinase C activity in isolated cardiac myocytes

A high-speed imaging technique was used to investigate the effects of inhibitors and activators of protein kinase C (PKC) on the [Ca2+]i transients and contraction of fura-2 loaded rat ventricular cardiac myocytes. The amplitude of the [Ca2+]i transient was reduced following treatment with 100 nM phorbol 12,13-dibutyrate (PDBu), whereas the PKC inhibitors staurosporine (0.5 microM) and calphostin C (10 microM) increased [Ca2+]i transient amplitude, elevated basal [Ca2+]i and slowed the decay of the [Ca2+]i transient. These changes were paralleled by similar alterations in the rate and extent of cell shortening. The activity of nitrendipine-sensitive Ca2+ channels was monitored indirectly as the rate of Mn2+ quench of cytosolic fura-2 in electrically-paced cells. PDBu reduced Mn2+ influx by six-fold, whereas staurosporine and calphostin C increased the influx rate by eight-fold and seven-fold over basal quench, respectively. The caffeine releasable Ca2+ pool was reduced in the presence of PDBu and increased transiently in presence of staurosporine. The effects of PKC activation and inhibition on sarcoplasmic reticulum Ca2+ content may be secondary to alterations of sarcolemmal Ca2+ influx. However, the PKC inhibitors also decreased the rate of sarcoplasmic reticulum Ca2+ uptake in permeabilized myocytes, suggesting that a direct effect of PKC on the sarcoplasmic reticulum may contribute to the prolongation of the [Ca2+]i transient under these conditions. The present work demonstrates that basal PKC activity has a potent depressant effect, mediated primarily through inhibition of sarcolemmal Ca2+ influx, which may play a key role in setting the basal tone of cardiac muscle.     

Identification and synthesis of altered peptides modulating T cell recognition of a synthetic peptide antigen

In studies of T cell responses to synthetic peptides we have observed agonist and antagonist activities associated with contaminants identified within the parent synthesis. The synthesis of two candidate analogues implied by a peptide contaminant formed during the synthesis of La 51-58 (IMIKFNRL) has been carried out. The peptide contaminant was 17-18 Da smaller than the parent peptide consistent with a modified asparagine residue at position 6 and so we synthesised both an aspartimide and a nitrile analogue, representing cyclisation or dehydration of the asparagine residue. The candidate aspartimide and nitrile analogues both bound empty MHC class I molecules to form allo determinants recognised by monoclonal antibodies. These results demonstrate that altered synthetic peptides can bind class I MHC molecules and prompt caution in the use of synthetic peptides as a source of immunising antigen.