Evidence that small molecule enhancement of ß-hexosaminidase activity corrects the behavioral phenotype in Dutch APP(E693Q) mice through reduction of ganglioside-bound Aß.

Certain mutant Alzheimer's amyloid-ß (Aß) peptides (that is, Dutch mutant APP(E693Q)) form complexes with gangliosides (GAß). These mutant Aß peptides may also undergo accelerated aggregation and accumulation upon exposure to GM2 and GM3. We hypothesized that increasing ß-hexosaminidase (ß-hex) activity would lead to a reduction in GM2 levels, which in turn, would cause a reduction in Aß aggregation and accumulation. The small molecule OT1001 is a ß-hex-targeted pharmacological chaperone with good bioavailability, blood-brain barrier penetration, high selectivity for ß-hex and low cytotoxicity. Dutch APP(E693Q) transgenic mice accumulate oligomeric Aß as they age, as well as Aß oligomer-dose-dependent anxiety and impaired novel object recognition (NOR). Treatment of Dutch APP(E693Q) mice with OT1001 caused a dose-dependent increase in brain ß-hex levels up to threefold over those observed at baseline. OT1001 treatment was associated with reduced anxiety, improved learning behavior in the NOR task and dramatically reduced GAß accumulation in the subiculum and perirhinal cortex, both of which are brain regions required for normal NOR. Pharmacological chaperones that increase ß-hex activity may be useful in reducing accumulation of certain mutant species of Aß and in preventing the associated behavioral pathology.

Prevalence of an ulcerative colitis-associated CD8+ T cell receptor beta-chain CDR3-region motif and its association with disease activity.

The normal human intestinal mucosa contains clonal T cell expansions. Clonal populations of T cells can be determined through evaluation of the idiotypic, hypervariable region of their T cell receptor (TCR). We have previously reported that there exists a highly conserved TCR pattern among intestinal CD8+ T cells in the majority of ulcerative colitis (UC) patients undergoing colectomy that was not present in normal control individuals. This TCR pattern, or motif, was characterized by particular beta-chain usage (TCRBV3 and TCRBJ1S6) and a defined length in the hypervariable third complementarity determining region (CDR3). The aim of this study was to assess the motif's relationship to disease activity. Subjects were 66 with UC, 19 with Crohn's disease, 14 inflammatory controls, and 6 normal controls. cDNA and gDNA were prepared from colonic biopsies and paraffin blocks, respectively, obtained from study subjects and used to assess TCRBV CDR3 region length and usage, as well as for cloning and sequencing of TCRs. The TCRBV CDR3 region was present in 25 of a series of 48 UC subjects but only 3 of 19 Crohn's disease patients and 3 of 14 inflammatory controls. The motif was more common in UC than either Crohn' s disease or inflammatory controls (chi2 = 7.5, P = 0.006, and chi2 = 4.1, P = 0.04, respectively). The motifs presence was not dependent upon histologic disease activity (either active or inactive UC). Clinical UC disease activity was also not significantly associated with an increased presence of the motif in 14 paired biopsies, which were taken during times of clinical activity or inactivity. There was a trend toward persistence of the motif, as it was present in 6 of 14 subjects over a 3- to 6-month time period. The previously described UC-associated TCRBV CDR3 region motif located in the intestinal CD8+ T-cell subset is found in a significant proportion of UC subjects. The TCR motif does not significantly discriminate active from inactive disease states. The persistent and diffuse nature of this TCR-associated motif in UC suggests that an ongoing T-cell response to a particular antigen(s) is occuring in this disorder.

Clinical, biological, and histologic parameters as predictors of relapse in ulcerative colitis.

BACKGROUND & AIMS: Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease. We aimed to assess whether clinical, biological, and histologic parameters in quiescent UC predict time to clinical relapse. METHODS: Seventy-four patients with clinically and endoscopically determined inactive UC were followed up for 1 year or for a shorter period if they had a relapse. Serum erythrocyte sedimentation rate; C-reactive protein, interleukin (IL)-1beta, IL-6, and IL-15 values; anti-neutrophil cytoplasmic antibody titers; and rectal biopsy specimens were obtained at baseline, at 6 and 12 months, and/or at relapse. Multivariate survival analysis was performed to determine independent predictors of clinical relapse. RESULTS: Twenty-seven patients relapsed (19/42 women; 8/32 men). Multivariate Cox regression analysis retained younger age (P = 0.003; hazard ratio, 0.4 per decade), greater number of prior relapses in women (P < 0.001; hazard ratio, 1.6 per prior relapse), and basal plasmacytosis (P = 0.003; hazard ratio, 4.5) on rectal biopsy specimens as predictors of shorter time to clinical relapse. Kaplan-Meier survival curves showed the 20-30-year-old age group and women with more than 5 prior relapses to be groups with shorter times to relapse. CONCLUSIONS: Younger age, multiple previous relapses (for women), and basal plasmacytosis on rectal biopsy specimens were independent predictors of earlier relapse. These findings may help identify patients with inactive UC who will require optimal maintenance medical therapy.

The role of cyclooxygenase 2 in ulcerative colitis-associated neoplasia.

Cyclooxygenase 2 (COX-2) overexpression has been described in sporadic colonic neoplasia, but its role in ulcerative colitis (UC) neoplastic progression remains unexplored. Although the specific role of cyclooxygenase in colonic neoplasia is uncertain, its inhibition by nonsteroidal anti-inflammatory drugs decreases the risk of sporadic colonic adenocarcinoma and causes regression of adenomas in familial adenomatous polyposis. To investigate the role of COX-2 in UC-associated neoplasia, we assessed COX-2 protein and mRNA expression throughout the spectrum of UC-associated neoplastic lesions in four total colectomy specimens, using immunocytochemistry and a novel TaqMan reverse transcriptase-polymerase chain reaction assay. The findings were correlated with DNA ploidy and inflammatory activity. We found COX-2 overexpression throughout the neoplastic spectrum in UC (P: < 0.0001, R:(2)=0.53), even in diploid samples that were negative for dysplasia. Overall, neoplastic change explained 53% of the variation in COX-2 expression, whereas inflammatory activity explained only 11%. COX-2 was overexpressed in all aneuploid samples and in 38% of diploid samples (P: = 0.0074). cDNA representational difference analysis was also performed and revealed that COX-2 mRNA was an up-regulated cDNA representational difference analysis difference product. COX-2 overexpression occurs early in UC-associated neoplasia, and the increase cannot be explained by inflammatory activity alone. The data suggest that COX-2-specific inhibitors may have a chemopreventative role in UC but the possibility that they could exacerbate UC inflammatory activity needs to be tested.

Improved two-dimensional gel electrophoresis representation of serum proteins by using ProtoClear.

ProtoClear is a proprietary technique for clearing albumin and immunoglobulin G (IgG) from human serum samples. Albumin constitutes 57-71% of total serum protein and IgG ranges from 8-26%. Removal of these two proteins alone clears approximately 75% of the total protein present in serum and allows the detection of the remaining proteins that are present in far lower concentrations. ProtoClear effectively removed >95% of human serum albumin (HSA) and >97% of human IgG as measured by an anti-HSA competitive immunoassay and a radial immunodiffusion assay, respectively. ProtoClear was far more specific at removing albumin and IgG than Cibracon Blue Dye chromatography (Cibracon Blue), the typically utilized alternative. Comparing two-dimensional (2-D) gels of serum cleared by either Cibracon Blue or by ProtoClear, it was apparent that Cibracon Blue removed a number of proteins in addition to albumin. Following removal of albumin and IgG from serum, we found a significant improvement in the resolution of polypeptide spots detected on two-dimensional gels.

Evidence of T cell receptor beta-chain patterns in inflammatory and noninflammatory bowel disease states.

T cell activation, as defined by expression of relevant cell surface molecules, such as the interleukin-2 receptor (CD25), is increased in many chronic relapsing diseases, including inflammatory bowel disease (IBD). These T cells are generally activated through contact of their clonotypic T cell receptor (TCR) with a peptide antigen presented by a major histocompatibility complex molecule. One of the putative antigenic contact sites for the TCR is the third complementarity determining region (CDR3) of the TCR beta-chain variable region (TCRBV). Therefore, analysis of the TCRBV CDR3 provides insight into the diversity of antigens encountered by a given T cell population. This study evaluated the TCRBV CDR3 usage of the activated intestinal lymphocytes from human subjects with IBD, diverticulitis (inflammatory control), and a normal tissue control. Public patterns, as demonstrated by shared TCRBV CDR3 amino acid sequences of activated intestinal T cell subpopulations, were observed. In particular, a public pattern of TCRBV22, a conserved valine in the fifth position, and use of TCRBJ2S1 or TCRBJ2S5 was present in three of four Crohn's disease subjects while not present in the ulcerative colitis subjects. However, the private patterns of TCRBV CDR3 region amino acid sequences were far more striking and easily demonstrated in all individuals studied, including a normal noninflammatory control. Thus we conclude that selective antigenic pressures are prevalent among an individual's activated intestinal lymphocytes.

An interleukin 12-related cytokine is up-regulated in ulcerative colitis but not in Crohn's disease.

BACKGROUND & AIMS: Interleukin 12 (IL-12) is a heterodimeric, macrophage-derived cytokine that is elevated in Crohn's disease (CD). Epstein-Barr virus-induced gene 3 (EBI3) is a recently characterized human glycoprotein that is homologous to the 40-kilodalton chain of IL-12 and forms a heterodimer with the 35-kilodalton chain of IL-12. We investigated the expression of EBI3 in colonic mucosa of normal control subjects, patients with ulcerative colitis (UC), and patients with CD. METHODS: Colonic tissue was analyzed for messenger RNA (mRNA) expression by quantitative polymerase chain reaction and for protein expression by immunohistology and Western blotting. RESULTS: EBI3 mRNA was present in intestinal biopsy specimens from healthy subjects and patients with CD but was elevated only in active UC. EBI3 levels in UC specimens correlated with histological scores of activity and T-cell infiltration. EBI3-positive cells that had a shape consistent with that of macrophages were identified in the lamina propria, and protein was detected by Western blotting. CONCLUSIONS: EBI3 is a novel IL-12-related cytokine that is expressed by macrophage-like cells in normal intestine and CD and has enhanced expression in active UC but not in active CD.

Targeting the IL-15 receptor with an antagonist IL-15 mutant/Fc gamma2a protein blocks delayed-type hypersensitivity.

Owing to shared receptor components, the biologic activities of IL-15 are similar to those of IL-2. However, the patterns of tissue expression of IL-2/IL-2R alpha and IL-15/IL-15R alpha differ. The development of agents targeting the receptor and signaling elements of IL-15 may provide a new perspective for treatment of diseases associated with expression of IL-15/IL-15R. We designed, genetically constructed, and expressed a receptor site-specific IL-15 antagonist by mutating glutamine residues within the C terminus of IL-15 to aspartic acid and genetically linked this mutant IL-15 to murine Fc gamma2a. These mutant IL-15 proteins specifically bind to the IL-15R, competitively inhibit IL-15-triggered cell proliferation, and do not activate the STAT-signaling pathway. Because the receptor site-specific antagonist IL-15 mutant/Fc gamma2a fusion proteins had a prolonged t(1/2) in vivo and the potential for destruction of IL-15R+ leukocytes, we examined the immunosuppressive activity of this agent. An IL-15 mutant/Fc gamma2a fusion protein markedly attenuated Ag-specific delayed-type hypersensitivity responses and decreased leukocyte infiltration within the delayed-type hypersensitivity sites. These findings suggest that 1) IL-15/IL-15R+ cells are crucial to these T cell-dependent immune responses, and 2) treatment with IL-15 mutant/Fc gamma2a protein may ameliorate T cell-dependent immune/inflammatory diseases.

Interleukin-2 fusion protein (DAB389IL-2) selectively targets activated human peripheral blood and lamina propria lymphocytes.

Activated mucosal T lymphocytes correlate with the intestinal inflammation of inflammatory bowel disease. Activated T cells elaborate interferon-gamma (IFN-gamma) and express high-affinity interleukin-2 (IL-2) receptors. The IL-2/diphtheria toxin fusion protein (DAB389IL-2) has been shown to specifically kill high affinity IL-2 receptor-bearing cells. We tested whether DAB389IL-2 could specifically target activated lamina propria lymphocytes. Lymphocytes were activated in vitro with phytohemagglutinin and IL-2 for 24-48 hr. Toxin efficacy was determined by the [14C]leucine incorporation, IFN-gamma ELISA, and flow cytometry. DAB389IL-2 (10(-11) M) inhibited protein synthesis by 80% in activated lamina propria lymphocytes. This inhibition was blocked by coculture of either excess IL-2 or a nonfunctional IL-2 diphtheria toxin mutant protein. DAB389IL-2 (10(-12) M) also significantly reduced the numbers of activated helper T cells and IFN-gamma levels in 24-hr cultures. DAB389IL-2 specifically targets activated IL-2 receptor-positive lamina propria lymphocytes and is a potential new therapeutic agent for patients with active inflammatory bowel disease.

Interleukin-15 signals T84 colonic epithelial cells in the absence of the interleukin-2 receptor beta-chain.

Interleukin-15 (IL-15) shares many biological functions with interleukin-2 (IL-2) due to common receptor components. IL-15 binds to the IL-2 receptor (IL-2R) beta-chain and the common gamma-chain receptor in addition to one other IL-15 binding receptor protein (IL-15R alpha). Both IL-2R beta- and gamma-chains are required to promote cell growth in hematopoietic cells. The colonic cryptlike epithelial cell line T84 contains the common gamma-chain but lacks the IL-2R beta-chain. We report IL-15R alpha-chain mRNA in T84 cells with the use of reverse transcriptase-polymerase chain reaction. T84 and normal colonic epithelial cells bind a FLAG-IL-15 fusion protein in immunoperoxidase and flow cytometric experiments. In addition, IL-15, but not IL-2, accelerates and enhances the development of transepithelial resistance across T84 monolayers in a dose-dependent fashion. We conclude that normal and T84 colonic epithelial cells express IL-15R alpha and are able to bind IL-15. IL-15 can deliver a nonproliferative functional signal in the absence of IL-2R beta-chain in T84 cells.

A noncytolytic IL-10/Fc fusion protein prevents diabetes, blocks autoimmunity, and promotes suppressor phenomena in NOD mice.

We have been successful in our efforts to develop a long lived noncytolytic murine IL-10/Fc fusion protein. In the nonobese diabetic mouse (NOD) model, administration of IL-10/Fc from 5 to 25 wk of age completely prevented the occurrence of diabetes. Moreover, these mice remained disease-free long after cessation of IL-10/Fc therapy. Immunohistochemistry studies show that IL-10/Fc treatment inhibits expression of TNF-alpha, proinflammatory cytokine, as well as Th1-type cytokines, IL-2 and IFN-gamma, but promotes expression of IL-4 and IL-10, Th2-type cytokines, by islet-infiltrating leukocytes. In an adoptive transfer model of diabetes in NOD mice, we found that: 1) IL-10/Fc treated hosts bear leukocytes that block expression of diabetes and 2) these leukocytes persisted even 8 wk after cessation of IL-10/Fc treatment. The potent antidiabetogenic effects provided by IL-10/Fc in the NOD model, together with its apparent lack of systemic toxicity, are notable.

Partial mediation of glucocorticoid antiproliferative effects by lipocortins.

The glucocorticoids (GCs) dexamethasone (DEX) and prednisolone (PRED), in a concentration-dependent fashion, profoundly inhibit mitogen-induced proliferation of human peripheral blood mononuclear lymphocytes (PBML). This inhibition was specific for GCs, as non-GC steroids were devoid of any antiproliferative capacity. GCs enhanced the mRNA (Northern blot) and protein (Western blot) expression of the calcium and phospholipid binding proteins lipocortin I, II, and V. As a consequence of mitogenic stimulation, PBML secrete PGE2 and leukotriene B4 (LTB4). Antiproliferative concentrations of both DEX and PRED as well as recombinant lipocortin I abolished PGE2 and LTB4 production, suggesting an involvement of lipocortins in GC-mediated antiproliferative effects, possibly by inhibiting eicosanoid production and, consequently, mitogen-induced cellular proliferation. Whereas 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid mimicked DEX and PRED in inhibiting PGE2 and LTB4 production, neither 5,8,11,14-eicosatetraynoic acid nor nordihydroguaiaretic acid had any effect on mitogen-induced PBML proliferation, indicating that the GC-mediated antiproliferative effect is separate from their effects on eicosanoid release. Furthermore, neutralizing anti-lipocortin I and anti-lipocortin II mAb, while reversing the inhibitory activity of DEX and PRED on PGE2 and LTB4 production, only partially reversed DEX- and PRED-mediated antiproliferative effects. This indicates that the GC-mediated antiproliferative effect is not dependent on inhibition of eicosanoid release by lipocortins and suggests the existence of lipocortin-dependent and lipocortin-independent pathways by which GCs mediate their antiproliferative effects.

Persistent clonal expansions of peripheral blood CD4+ lymphocytes in chronic inflammatory bowel disease.

It is increasingly recognized that chronic Ag exposure may lead to clonal expansions of T cells, including those within the peripheral blood. Inflammatory bowel disease is a chronic, multisystemic disease of unknown origin that predominantly affects the intestine. We sought to determine whether clonal expansions of T cells are present in the peripheral blood of patients with inflammatory bowel disease by an examination of TCR usage. Positively selected CD4+ and CD8+ peripheral blood T cells were isolated from subjects with active ulcerative colitis, Crohn's disease, and diverticulitis and from normal controls. Analysis of complementarity determining region 3 lengths of 24 TCR-beta chain V region families from CD4+ and CD8+ peripheral blood T cells showed a skewed distribution in the three inflammatory groups, consistent with expansion of T cell clones, in comparison to the normally distributed pattern observed among the control donors. Random sequencing of the PCR amplification products of CD4+ peripheral blood T cells from the subjects with ulcerative colitis, Crohn's disease, and diverticulitis revealed reiterative TCR-beta chain sequences that were not found in the normal donors. In subjects with Crohn's disease, the reiterative TCR-beta chain sequences from the CD4+ peripheral blood T cells were persistent over at least a 1-yr period. The persistently expanded TCR-beta chain sequences of CD4+ peripheral blood T cells were identifiable in genomic DNA isolated from archival tissue of intestine from subjects with Crohn's disease and ulcerative colitis by Southern blotting and direct DNA sequencing. An identical twin pair, concordant for Crohn's disease, shared the same reiterative TCR-beta chain sequences in their CD4+ peripheral blood T cells. These studies show that chronic intestinal inflammation is associated with expansions of CD4+ peripheral blood T cells. Furthermore, in inflammatory bowel disease these T cell clonal expansions are persistent and shared among HLA-identical individuals, implicating a response to specific, persistent, and stimulating Ags in these diseases.

Risk and natural history of colonic neoplasia in patients with primary sclerosing cholangitis and ulcerative colitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) has been suggested as a risk factor for the development of colorectal cancer in ulcerative colitis (UC); however, previous studies of this association have been limited by small numbers of patients with PSC or have been performed retrospectively. This study prospectively evaluates the risk and natural history of colonic tumorigenesis in patients with PSC and UC and compares it with patients with UC without PSC. METHODS: Twenty patients with PSC and UC and 25 control patients with UC were followed prospectively by colonoscopic surveillance using extensive mucosal biopsy sampling. All control patients with UC had disease extending beyond the sigmoid colon of > or = 8 years' duration; patients with PSC and UC were studied regardless of disease duration. RESULTS: Forty-five percent (9 of 20) of the patients with PSC and UC had dysplasia compared with 16% (4 of 25) of the control patients with UC (P < or = 0.002). Prior liver transplantation did not affect the risk of colonic dysplasia. The time course for progression to dysplasia was similar between the patients with PSC and UC and the patients with UC; however, the patients with PSC and UC were five times more likely to develop dysplasia. CONCLUSIONS: Patients with PSC and UC represent a subset of patients with UC who are at markedly increased risk for colonic neoplasia and who need close colonoscopic surveillance with extensive biopsy sampling.

Enhanced intramucosal cytotoxic lymphocyte gene expression in ulcerative colitis.

: The histopathology of the mucosal lesions in ulcerative colitis suggests that cellular immune hyperactivity plays a role in the pathogenesis of ulcerative colitis. The role of colonic mucosal cytotoxic lymphocytes in ulcerative colitis is highly controversial. In vitro determinations of cytotoxic lymphocyte function vary according to the experimental conditions and target cells employed. Transcription of the cytotoxic molecules granzyme B and perforin are specific for activated cytotoxic lymphocytes. We investigated whether activated cytotoxic lymphocytes are present in ulcerative colitis lesions via identification of granzyme B, perforin, and interleukin 2 transcripts. RNA was extracted from endoscopic colonic biopsies taken from normal and ulcerative colitis patients. An aliquot was reverse transcribed, followed by gene amplification in the polymerase chain reaction. A competitive template was incorporated within the reaction permitting the quantitation of specific mRNA species. Granzyme B and perforin levels of reverse transcribed cDNAs (RT-cDNAs) were significantly elevated in active ulcerative colitis as compared to normal colonic tissues (p = 0.0081 and p = 0.0018, respectively) when calculated as a ratio with the RT-cDNA of the constitutively expressed gene GAPDH. Interleukin 2 mRNA measurements did not vary between normal and UC samples to a statistically significant degree. These data suggest heightened lymphocyte cytotoxic function within ulcerative colitis mucosal lesions.

Mutations in the p53 gene: an early marker of neoplastic progression in ulcerative colitis.

BACKGROUND/AIMS: In long-term extensive ulcerative colitis, aneuploidy occurs earlier and loss of heterozygosity for p53 (p53 LOH) later during histological progression towards carcinoma. This study determined the time of onset of p53 mutation in this progression. METHODS: We developed a rapid, sensitive screening assay for p53 mutations at codon 248. The geographic distribution of this p53 mutation was mapped in two fresh colectomy specimens with mutations of codon 248 (1 cancer, 1 dysplasia) and correlated with patterns of clonal expansion, histological progression, and allelic loss. Numerous samples from throughout both colons were analyzed (216 for histology, 142 for DNA content, 104 for mutation, and 41 for p53 LOH). RESULTS: p53 mutation correlated highly with histological grade and was distributed more extensively than p53 LOH. Mutation, but not LOH, was also found in diploid, nondysplastic colonic mucosa adjacent to dysplastic areas. CONCLUSIONS: These findings suggest that p53 mutation appears to be an early genetic event that precedes p53 LOH. The very close correlation of p53 mutation with aneuploidy (P > 0.0001) emphasizes the role of normal p53 at the G1 checkpoint to help prevent entry of genetically damaged cells into the cell cycle.

Heightened intragraft CTL gene expression in acutely rejecting renal allografts.

The role of CTL in the immunopathogenesis of acute cellular rejection is controversial. To further define the relationship of activated CTLs to rejection, we analyzed gene expression of three CTL-derived effector molecules in renal allograft biopsies. CTLs are endowed with the ability to promote allograft damage through the elaboration of these highly cytopathic molecules. Intragraft gene transcript levels were determined for granzyme B, perforin, and TIA-1 and correlated with the immunologic status of the allograft as categorized by conventional clinical and histologic criteria. The categories were acute cellular rejection, chronic rejection, elements of both acute and chronic rejection, and no evidence of rejection. Biopsies were snap-frozen, total RNA extracted, and the mRNA converted to cDNA by reverse transcription. Levels were quantitated by competitive template PCR techniques. Intragraft granzyme B and perforin transcripts were highly restricted to biopsies in the acute cellular rejection category. TIA-1 expression was more ubiquitous but significantly higher transcript levels were found in the acute rejection category. The presence of these transcripts in acute cellular rejection samples implicates CTL in the pathogenesis. Moreover, intragraft CTL-specific transcript levels may serve as markers of rejection.

Synthesis of protein-reactive (aminostyryl)pyridinium dyes.

The synthesis of two protein reactive (aminostyryl)pyridinium fluorescent dyes, N-ethyl-N-[4-[2-[4-(1-methylpyridinio)]ethyl]phenyl]glycine chloride N-hydroxysuccinimide ester (SuASP, 2) and 4-[4-[2-[4-(N,N- dimethylamino)phenyl]ethenyl]pyridinio]butyrate N-hydroxysuccinimide ester (ASPSu, 3), is reported. Both form amide linkage through the activated succinimidyl ester with primary amines. The two analogues differ by the position of the pyridinium positive change relative to the activated ester. SuASP forms an amide linkage that positions the positive charge distal to the protein surface, while ASPSu places the positive charge proximal. The synthesis of SuASP utilizes a palladium coupling reaction for the arylation of 4-vinylpyridine, while the major connection for ASPSu is accomplished through an aldol condensation between 4-(N,N-dimethylamino)benzaldehyde and picoline. Both reagents are shown to label covalently bovine serum albumin.