Expression and distribution patterns of spermine, spermidine, and putrescine in rat hair follicle.

No expression and distribution patterns of polyamines (PAs), spermine, spermidine, and their precursor putrescine in mammalian hair follicle are available, although polyamines are known to correlate well with hair growth and epidermal tumor genesis. Immunohistochemistry (IHC) using our original two monoclonal antibodies (mAbs) ASPM-29 specific for spermine or spermidine, and APUT-32 specific for putrescine allowed us to detect immunoreactivity for polyamines in hair follicles from normal adult rats. A wide range of immunoreactivity for the total spermine and spermidine was observed in the compartments of hair follicle: The highest degree of immunoreactivity for polyamines was observed in the matrix, in the Huxley's layer, in the deeper Henle's layer, and in the cuticle of the inner root sheath/the hair cuticle, while moderate immunoreactivity existed in the lower-to-mid cortex and the companion layer, followed by lower immunoreactivity in the outer root sheath, including the bulge region and in the deeper medulla, in which the immunoreactivity was also evident in their nuclei. In addition, somewhat surprisingly, with IHC by APUT-32 mAb, we detected significant levels of putrescine in the compartments, in which the immunostaining pattern was the closely similar to that of the total spermine and spermidine. Thus, among these compartments, the cell types of the matrix, the Huxley's layer, the deeper Henle's layer, and the cuticle of the inner root sheath/the hair cuticle seem to have the biologically higher potential in compartments of anagen hair follicle, maybe suggesting that they are involved more critically in the biological event of hair growth. In addition, we noted sharp differences of immunostaining by IHCs between ASPM-29 mAb and APUT-32 mAb in the epidermis cells and fibroblast. ASPM-29 mAb resulted in strong staining in both the cell types, but APUT-32 mAb showed only very light staining in both types. Consequently, the use of the two IHCs could be extremely useful in further studies on hair cycle and epidermal tumor genesis experimentally or clinically.

Technical note: Hapten synthesis, antibody production and development of an enzyme-linked immunosorbent assay for detection of the natural steroidal alkaloid Dendrogenin A.

We have recently discovered the existence of 5α-Hydroxy-6ß-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3ß-ol, called Dendrogenin A (DDA), as the first endogenous steroidal alkaloid ever described in mammals. We found that the DDA content of tumors and cancer cell lines was low or absent compared with normal cells showing that a deregulation in DDA biosynthesis was associated with cancer and therefore suggesting that DDA could represent a metabolomic cancer biomarker. This prompted us to produce antibodies that selectively recognize DDA. For this purpose, the hapten 5α-hydroxy-6ß-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3ß-o-hemisuccinate with a carboxylic spacer arm attached to the 3ß-hydroxyl group of DDA was synthesized. The hapten was coupled to bovine serum albumin and keyhole limpet hemocyanin for antibody production to develop an enzyme-linked immunosorbent assay (ELISA). The protein conjugates were injected into BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the hybridoma cell lines established were assessed with indirect ELISA by competitive assays using dilutions of a DDA standard. The antibodies from the selected hybridomas had an IC(50) value ranging from 0.8 to 425 ng/ml. Three antibodies showed no cross-reactivity with structurally related compounds including histamine, cholesterol, ring B oxysterols and a regio-isomer of DDA. In this study, high-affinity and selective antibodies against DDA were produced for the first time, and a competitive indirect ELISA was developed.

Motor neuronal protection by L-arginine prolongs survival of mutant SOD1 (G93A) ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to motor neuron degeneration. Despite the fact that many different therapeutic strategies have been applied to prevent disease progression, no cure or effective therapy is currently available for ALS. We found that L-arginine protects cultured motor neurons from excitotoxic injury. We also found that L-arginine supplementation both prior to and after the onset of motor neuron degeneration in mtSOD1 (G93A) transgenic ALS mice significantly slowed the progression of neuropathology in lumbar spinal cord, delayed onset of motor dysfunction, and prolonged life span. Moreover, L-arginine treatment was associated with preservation of arginase I activity and neuroprotective polyamines in spinal cord motor neurons. Our findings show that L-arginine has potent in vitro and in vivo neuroprotective properties and may be a candidate for therapeutic trials in ALS.

Construction of an encapsulated ESAT-6-based anti-TB DNA vaccine and evaluation of its immunogenic properties.

Experimental preparations based on a DNA vaccine encoding the ESAT-6 antigen of Mycobacterium tuberculosis have been obtained (KpONE6) and studied for immunogenic effects in the murine model. The core of the preparation contains DNA of the recombinant plasmid pONE6 encapsulated within a spermidine-polyglucin conjugate, thereby protecting the DNA vaccine from degradation. KpONE6 induces a proliferative T-cell immune response in mice upon intramuscular immunization.

Definition of the fine specificity of the monoclonal antibody 81D4: its reactivity with lysine and polyamine isopeptide cross-links.

The 81D1C2 monoclonal antibody (Mab) directed against the Nepsilon-(gamma-L-glutamyl)-L-lysine isopeptide was found to cross-react on Enzyme Immuno Assay (EIA) with acylated lysines. Using a differential screening EIA procedure, a new Mab 81D4 was selected, which did not cross-react with acylated lysines but exhibited strong reactivity with Nepsilon-(gamma-L-glutamyl)-L-lysine formed by covalently coupling the gamma-carboxyl of NalphaCBZ OtBu glutamic acid to epsilon-NH2 derivatized microtiter plates. When Nepsilon-(gamma-L-glutamyl)-L-lysine isopeptides were generated on gamma-carboxyl derivatized plates, only lysine isopeptides with blocked alpha-amines were reactive, regardless of whether the bond formed by the amine blocking agent was a carbamate with carbobenzyloxychloride or an amide with acetic anhydride. The 81D4 Mab showed little or no affinity for free Nepsilon-(gamma-L-glutamyl)-L-lysine (IC50>5 mM), for N1 or N4 mono(gamma-Poly L-glutamyl)putrescine, and for N1 mono(gamma-Poly L-glutamyl)spermidine (IC50>5 mM). However, when these same isopeptides were synthesized as cross-links between two protein chains--Nepsilon-(gamma-L-glutamyl)-L-lysine between Poly L-glutamate and Poly L-lysine; N1N4 -bis(gamma-Poly L-glutamyl)putrescine, N1N8 -bis(gamma-Poly L-glutamyl)spermidine between Poly-L-glutamate chains--very good reactivity was observed (IC50 400 microM for lysine; 80 microM for putrescine and spermidine). In addition to the chemically synthesized isopeptide cross-links that were recognized by this Mab, the naturally occurring Nepsilon-(ã-L-glutamyl)-L-lysine isopeptide cross-links in D-dimer, which are formed by the action of plasma transglutaminase (Factor XIII) on fibrin, were also detected on immunoblots using 81D4 as the primary antibody.

Immunoelectron microscopy study of polyamines using a newly prepared monoclonal antibody against spermidine: use of a mixture of glutaraldehyde and paraformaldehyde as a cross-linking agent in the preparation of the antigen.

We developed a mouse monoclonal antibody (ASPD-19, IgG3 sub-isotype mAb) against spermidine (Spd) conjugated to bovine serum albumin (BSA) using a mixture of glutaraldehyde (GA) and paraformaldehyde (PFA)-sodium borohydride for applications in immunoelectron microscopic studies. The antibody specificity was evaluated by an ELISA binding test simulating the immunocytochemistry (ICC) of tissue sections. The ASPD-19 mAb is highly specific for Spd and Spm, almost the same degree to each, and can distinguish alterations in the chemical structure of other polyamine (PA) analogs, showing less than 3.2% cross-reaction with N(1)-acetylspermidine, acetylspermine, or N(8)-acetylspermidine. By an indirect immunoperoxidase method using the ASPD-19 mAb, PA-like immunoreactivities were observed in different tissues fixed with Karnovsky fixative (a mixture of GA and PFA) in combination with borohydride reduction. In contrast, immunoreactivity was very low in tissues when the borohydride reduction step was omitted. The PA-like immunoreaction was completely abolished by the adsorption of the ASPD-19 mAb with 100 microg/ml of Spd or Spm, but was inhibited little or none by other PA-related compounds or amino acids. A light microscopic ICC method using ASPD-19 produced immunostaining of PAs in certain cells in rat tissues with high biosynthetic activities (small intestine, pancreas and spinal cord). A pre-embedding immunoelectron microscopic study using rat spinal cord showed PA immunoreactivity located predominantly on free (polysomes) and attached ribosomes of the rough endoplasmic reticulum (Nissl bodies) in the cytoplasm of motor neurons. These results are in complete agreement with the results obtained by our recent ICC method using another mAb (ASPM-29) produced against GA-conjugated Spm.

Two enzyme-linked immunosorbent assay (ELISA) systems for N1,N8-diacetylspermidine and N1,N12-diacetylspermine using monoclonal antibodies.

We obtained monoclonal antibodies against N(1),N(12)-diacetylspermine (DiAcSpm) and N(1),N(8)-diacetylspermidine (DiAcSpd), and developed two systems of competitive ELISA that utilize the antibodies and a common enzyme-labeled antigen to measure these di-acetylpolyamines. Cross-reactions with N(1)-acetylspermidine in the assay of DiAcSpm and with N8-acetylspermidine in the assay of DiAcSpd were as low as 0.26 and 0.6%, respectively, and were judged to be insignificant in clinical use for measuring urinary diacetylpolyamines. These assays were used to assess diurnal variations in diacetylpolyamine excretion in urine to show that the excretion of diacetylpolyamines after normalization for the concentration of creatinine is stable over a day with only minimal diurnal variation.

Recombinant anti-polyamine antibodies: identification of a conserved binding site motif.

Polyamines are small linear polycations found ubiquitously in eukaryotic cells. They are involved in nucleic acid and protein synthesis and rises in cellular polyamine levels have been correlated with cell proliferation. Antibodies to these molecules have potential as prognostic indicators of disease conditions and indicators of treatment efficacy. Antipolyamine monoclonal antibodies of differing but defined specificities have been generated in our laboratory using polyamine ovalbumin conjugates as immunogens. These antibodies show small but significant cross reactivities with other polyamine species; IAG-1 cross reacts with spermidine (8%), JAC-1 with spermine (6%) and JSJ-1 with both putrescine (11%) and spermine (6%). We have rescued and sequenced the heavy and light chain variable regions of all three of these antibodies. While the light chains of two antibodies, IAG-1 and JSJ-1, were 93% homologous at the amino acid level, none of the heavy chains displayed any significant sequence homology. However, computer-generated models of all three antibody binding sites revealed a three-dimensionally conserved polyamine binding site motif. The polyamine appears to bind into a negatively charged cleft lined with acidic and polar residues. The cleft is partially or completely closed at one end and the specificity of the interaction is determined by placement of acidic residues in the cleft. Aromatic residues contribute to polyamine binding interacting with the carbon backbone. The polyamine-binding motif we have identified is very similar to that observed in the crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia coli.

Preparation of antibodies highly specific to N1,N8-diacetylspermidine, and development of an enzyme-linked immunosorbent assay (ELISA) system for its sensitive and specific detection.

N8-Acetylspermidine was coupled to mercaptosuccinylated BSA using a bifunctional cross-linker, N-(4-maleimidobutyryloxy)succinimide, and the resulting conjugate was used to raise N1,N8-diacetylspermidine (DiAcSpd)-specific antibodies in rabbits. DiAcSpd-specific antibodies were enriched from crude sera through a series of affinity-based fractionations using ligands with structures mimicking those of DiAcSpd and monoacetylspermidines. With the N8-acetylspermidine-BSA conjugate as a solid phase antigen in a competitive ELISA system, the selectivity for DiAcSpd over other polyamine species was high, but competition by DiAcSpd added to the fluid phase was too weak for the system to be applicable to measurement of the concentration of DiAcSpd in human urine. In contrast, with the N1-acetylspermidine-BSA conjugate adsorbed on the ELISA plate, DiAcSpd efficiently competed for the same antibody, thus yielding a sensitive competitive ELISA system for measuring DiAcSpd. The Ki value for DiAcSpd with the latter competitive ELISA system was 54 nM, and the cross-reactivity with DiAcSpd, N1,N12-diacetylspermine, N8-acetylspermidine, N1-acetylspermidine, and acetylputrescine was 100, 1.2, 0.74, 0.12, and 0.08%, respectively. The DiAcSpd-specific antibodies and the competitive ELISA system developed in this study will prove to be useful for analyzing the urinary level of DiAcSpd, that was recently shown to be a promising diagnostic and prognostic indicator of malignant disorders.

Astrocytes, not neurons, show most prominent staining for spermidine/spermine-like immunoreactivity in adult rat brain.

Polyamines are involved in a variety of basic cellular functions including proliferation and differentiation. Recent in vitro evidence suggests a role for spermidine or spermine as possible modulators of ionotropic glutamate receptors and inwardly rectifying potassium channels. However, before a functional role of spermidine or spermine in vivo can be considered, the presence of these polyamines in the mammalian central nervous system must be demonstrated. Here we report the localization of spermine/spermidine-like immunoreactivity in the major cell types of the adult rat brain, using polyclonal antibodies raised against glutaraldehyde-conjugated spermine. Neuronal staining was restricted to several discrete brain nuclei and was generally weak. In the hippocampus, immunoreactivity was found in the area of perforant path terminals and in the CA2/CA3 subfields. The CA1 region and the area of the mossy fiber terminals was largely negative. Throughout the brain, the most prominent staining was displayed by astrocytes, as confirmed by comparison with astrocyte and microglial markers, but immunolabel was also detected in oligodendrocytes and pericytes. Their intense staining for spermidine/spermine-like immunoreactivity suggests that astrocytes are the most likely source for extracellular polyamines in the rat brain.

JSJ-1, an anti-spermidine monoclonal antibody with potential clinical applications.

Polyamines have been implicated in a wide variety of functions including nucleic acid synthesis and protein synthesis. Their levels have been shown to increase in response to cell growth and differentiation. Use of polyamines as prognostic indicators of proliferative disease conditions has been hindered by the lack of suitable rapid and sensitive assays. We report the characterization of an anti-spermidine antibody, JSJ-1, with novel putrescine cross reactivity. JSJ-1 cross-reacts more strongly with putrescine (11%) than with spermine (6%). This suggests that the aminobutyl group common to both putrescine and spermidine is an important element in the antibody-antigen interaction. We have demonstrated that antibody-spermidine binding is effected by increased ionic strength. This finding is consistent with the antibody-antigen interaction being ionic. The JSJ-1 antibody has been successfully used to detect increased polyamine levels in clinical serum samples and identify those with increased polyamine levels.

Immunocytochemical localization of polyamines in the gastrointestinal tracts of rats and mice.

An immunocytochemical method using a recently produced monoclonal antibody (ASPM-29) with an antibody specificity to spermine (Spm) and spermidine (Spd) fixed in situ, was used to demonstrate an immunocytochemical localization of polyamine (PA) pools in the gastrointestinal tracts of rats and mice. High PA immunoreactivity was always found in the cytoplasm of cells not only at the cell proliferative zone or the precursor cell zone but also at the neighboring non-proliferative premature cell zone of the epithelium, and a gradient of decreasing PA levels was noticed from these cells to the fully mature differentiated gastric surface mucous cells and absorptive cells of the small and large intestines. Also, strong staining for PAs was seen in the cytoplasm of fully differentiated gastric chief cells and neurons of both the myenteric and submucous plexuses, whereas the nuclei of the cells remained virtually unstained. These results may suggest that PAs are closely associated with the high biosynthetic activity in the cells of the gastrointestinal mucosa of normal rats and mice. This seems to be consistent with the PA immunocytochemical results previously obtained for neoplastic cells and active protein- or peptide-secreting cells, including exocrine or endocrine cell types.

Production and characterization of a monoclonal antibody specific for the polyamine spermidine and its application in ELISA.

To enable the immunoassay of spermidine in tissue extracts, a monoclonal antibody, JAC-1, specific for free spermidine was raised, using a spermidine-ovalbumin conjugate as immunogen. This antibody was characterized, and found to possess a high degree of specificity for spermidine, with only 4% molar cross-reactivity with spermine. A competitive ELISA using this antibody was developed. This assay is able to detect as little as 10 pmol spermidine extracted from small, circa 10 mg, tissue samples. The assay is unaffected by the presence of up to a 4-fold molar ratio of spermine, whereas the corresponding spermine competitive ELISA is adversely affected by a 0.5-fold molar ratio of spermidine. The spermidine competitive ELISA using JAC-1 was used to estimate the spermidine content of several plant and animal tissue extracts and the results compared with HPLC data. This novel assay is a useful development in the assay of polyamines since, compared to routinely employed HPLC methods, it offers increased convenience, rapidity, and capacity for large numbers of samples.

Polyamines found in gingival fluid inhibit chemotaxis by human polymorphonuclear leukocytes in vitro.

Putrescine and spermidine occur at concentrations approaching 1 mM in gingival fluid at diseased periodontal sites. Previous work demonstrates that these polyamines potentiate Ca2+ signaling in polymorphonuclear leukocytes (PMNs), resulting in enhanced degranulation and superoxide generation. The present study extends this work by characterizing the effects of polyamines on PMN chemotaxis and phagocytosis, in which Ca2+ signaling plays a less defined regulatory role. Putrescine (1 mM) and spermidine (0.1 to 0.5 mM) significantly inhibited chemotaxis to fMet-Leu-Phe and C5a (P < 0.05). This inhibition was not strongly related to any effect polyamines have on PMN adhesion, actin polymerization, or formyl peptide receptor expression. Neither putrescine nor spermidine had a significant impact on phagocytosis of opsonized bacteria by PMNs. Thus, at concentrations similar to those found in gingival fluid, polyamines could potentially inhibit recruitment of PMNs to diseased pockets without impairing their ability to engulf invading bacteria.

A new enzyme-linked immunosorbent assay (ELISA) for spermidine using glutaraldehyde coupling of the hapten to carrier-coated microtiter plates.

An enzyme-linked immunosorbent assay (ELISA) for small molecular haptens needs conjugates of the hapten with larger protein molecules for coating the wells of microtiter plates. The formation of such conjugates is not always reproducible. This makes it difficult to evaluate hapten-protein stoichiometries and to understand the precise orientation of the hapten on the protein. In this paper we describe an assay in which the polyamine spermidine (Spd) is coupled with glutaraldehyde (GA) to carrier poly-L-lysine (PL) or human serum albumin (HSA) coated on polystyrene microtiter wells. Each step of the assay was tested for maximum efficiency. This ELISA detected Spd with excellent reproducibility (coefficient of variation = 5.9%), an EC50 of 4.3 x 10(-6) M and a detection limit of 0.1 x 10(-6) M. The present ELISA was about 100 times more sensitive in detecting Spd than a conventional ELISA for Spd using a Spd-HSA conjugate for coating microtiter wells. The Spd antiserum showed 215% cross-reaction with N1-acetyl-Spd, 30.7% with N8-acetyl-Spd, 10% with spermine, 3.3% with cadaverine, 1.7% with putrescine, and less than 0.43% with 1,3-diaminopropane in the new ELISA system. A much higher degree of hapten binding to the plate occurred with the present method than with the previously reported method using microtiter wells activated directly with GA. The hapten conjugation is simple, reproducible and should provide a general method for developing ELISAs, not only for polyamines but also for amino acid and small peptides.

A new enzyme-linked immunosorbent assay (ELISA) for studying immunocytochemical procedures using an antiserum produced against spermidine as a model.

Antiserum was produced in rabbits against the polyamine spermidine (Spd) conjugated to bovine serum albumin (BSA). The reactivity of the serum to Spd and a variety of structurally related compounds was quantified by a new immunocytochemical model system incorporating an enzyme-linked immunosorbent assay (ELISA) binding test. This is based on the principle of coupling these compounds to the wells of microtiter plate activated with poly-L-lysine and glutaraldehyde and incubating the wells by the indirect immunoperoxidase method. The antiserum showed a 25% cross reaction with spermine (Spm), putrescine (Put), and cadaverine (Cad), and a 1% cross reaction with 1,3-diaminopropane (Dap), but no cross reaction with monoacetyl polyamines and amino acids. The antibody binding was inhibited most effectively by absorption of the antiserum with N1-acetylspermidine and Spd in the ELISA inhibition test. Also, immunoblot analysis of the antiserum with nitrocellulose paper gave completely identical results to the ELISA binding tests. Spd-like immunoreactivities in human melanoma BD and neuroblastoma IMR 32 cell lines are presented as examples of the staining pattern obtained with the antiserum. Absorption of the serum with N1-acetylspermidine and Spd was demonstrated to abolish the immunostaining reaction. The immunohistochemical model is simple: amines and amino acids are bound in the same way as in aldehyde-fixed tissues and, in comparison to immunoblot analysis, the immunoreactivity can be more easily and accurately quantified by assay with the antibody. The model should prove useful in assessing the specificity of other antisera.

Assay of the polyamine spermine by a monoclonal antibody-based ELISA.

Spermine-specific monoclonal antibodies were prepared by immunising mice with protein-spermine conjugates. A resulting monoclonal antibody, IAG-1, exhibited both high affinity for spermine (binding constant 5.5 x 10(7) M-1) and high specificity, cross-reacting only weakly with spermidine. Using this antibody a competitive ELISA was developed with a detection limit of 1 pmol. The assay has been used to quantify spermine content of plant tissues, without derivatization, and producing within 6 h of collection, values for the spermine content which are similar to published data obtained by HPLC.

Immunization of rabbits with spermine induces antibodies to self antigens.

Rabbits were immunized under different schedules with spermine in the free form or with random noncovalent complexes of spermine or spermidine with ovalbumin. The specificity of the induced antibodies was determined by ELISA and by dot-immunobinding assay. Our results show that in vitro conjugation of spermine and spermidine to a carrier is not an obligatory prerequisite for obtaining corresponding antibodies. Anti-spermine antibodies were found in 9 of 19 animals injected with spermine. Furthermore, all 19 rabbits produced distinct populations of IgM and IgG antibodies which reacted with histones, various synthetic peptides of histones, as well with ubiquitin, a peptide of ubiquitin, dsDNA and two 29-base 5' synthetic oligodeoxynucleotides. Even in antisera with no detectable reactivity with spermine, antibodies to some of the unrelated antigens were found. The pattern of reactivity of the antisera with the various antigens was different for each immunized animal. These findings lend support to the view that polyamines may play a role in the appearance of autoantibodies.

Establishment and preliminary application of spermidine radioimmunoassay with 125I-labelled monoclonal antibody and solid phase antigen.

Purified anti-spermidine monoclonal antibody was labelled with radioactive iodine by the Iodogen method and spermidine-bovine serum albumin (SPD-BSA) conjugate was used to coat polystyrene beads as solid phase antigen. The new solid phase 125I-labelled spermidine radioimmunoassay (RIA) depends on the competition between spermidine in the sample and the solid phase antigen for the limited amount of 125I-labelled monoclonal antibody. The sensitivity of this assay was 10 ng/ml higher than that of liquid RIA for spermidine with 14C-labelled spermidine. The coefficients of variation (CV) within and among batches were 4% and 13% respectively. The sample-batch capacity was increased from 20 (liquid RIA with 14C-labelled spermidine) to 150-200 by using this method. Because of its simplicity, the solid phase RIA kit is very convenient for population survey. This RIA could be used to determine spermidine in saliva for the diagnosis of precancerous lesions. In a preliminary study saliva spermidine levels in different populations were measured among 130 normal subjects, 202 esophageal epithelial hyperplasia cases treated with anti-tumor B for 5 years, 207 esophageal epithelial hyperplasia cases as control, and 55 esophageal cancer patients. The levels were 1,795 +/- 1,481, 3,470 +/- 6,981, 9,753 +/- 17,641 and 18,090 +/- 21,509 ng/ml, respectively, with the saliva spermidine levels in precancerous and cancer patients being significantly higher than that of normal subjects (P less than 0.001); the level in patients treated with anti-tumor B was significantly lower than that of controls (P less than 0.001). This decreased saliva spermidine content was coincident with the 47.3% reduction of canceration rate seen in precancerous patients after a 5-year treatment with anti-tumor B.