Canine splenic lymphoid nodules are currently classified as indolent lymphomas (marginal zone lymphoma [MZL], mantle cell lymphoma [MCL]) or nodular hyperplasia (lymphoid [LNH] or complex [CNH] type). Their differentiation can be difficult on morphology, because of similar histologic appearance and poorly defined diagnostic criteria. Thirty-five surgical samples of splenic lymphoid nodules were reviewed in order to assess the diagnostic contribution of immunophenotyping, proliferative activity and clonality (PARR) in differentiating between hyperplastic and neoplastic lesions. Proliferative activity was evaluated by double immunolabeling for Ki-67 and CD79a, in order to separately assess the proliferative activity of B cells and non-B cells. Definitive diagnoses were MZL ( n = 11), MCL ( n = 4), LNH ( n = 10), and CNH ( n = 10). The overall concordance between histology and PARR was above 90%. Lymphomas had a significantly higher percentage of CD79a-positive areas (mean, 36.30%; P = .0004) and a higher B-cell proliferative activity (median Ki-67 index, 5.49%; P = .0012). The threshold value most accurately predicting a diagnosis of lymphoma was ≥28% of B-cell areas, with a Ki-67 index above 3%. Dogs were monitored for a median follow-up time of 870 days (IQR, 569-1225), and no relapses were documented. Overall median survival time was 1282 days. The combination of histology, immunohistochemistry and PARR can improve the diagnostic accuracy for canine splenic lymphoid nodules, although the long-term behavior of these lesions appears similar.

Aliphatic polyamines are ubiquitous compounds classified as plant growth substances (). They act mainly in processes based on cell division. It is known that exogenous polyamines can induce cell division in plant tissues temporarily lacking in polyamines (). Many authors have suggested the possible utilization of these compounds as tumor markers because in general they increase during the early stage of the disease, but more detailed studies demonstrate that this increase is positively related in plant and animal tissues to growth rate rather than tumorigenesis per se, both in normal and tumor tissues. Even though polyamines alone seem not specifically related with morphogenic effects (i.e., embryogenesis and organogenesis), in association with other plant hormones (notably auxins and cytokinins) they can modify and/or regulate this phenomenon. Their involvement has been reported in the growth of "crown gall" caused by Agrobacterium tumefaciens (), and more recently in the genetic transformation induced by Agrobacterium rhizogenes, the so-called "hairy root disease"; in this context the big interest about polyamines concerns the transfer and expression of genetic information from Ri plasmid to host plant cell. Results obtained up to now are in favor of changes in polyamine levels (both free and conjugated forms, trichloroacetic acid [TCA]-soluble and -insoluble) during the growth cycle of transgenic tobacco plants both in vivo and in vitro (), and of isolated hairy roots in culture (). In particular, changes in free polyamine levels and in the activities of related enzymes seem to be linked to different growth kinetics (cell division and/or root elongation) depending on the different degree of expression of T-DNA genes. In addition, because some "unusual" polyamines can serve as taxonomic markers in microorganisms, and also in the different species of the genus Agrobacterium (), it is of interest to analyze polyamine pattern and distribution in the A. rhizogenes strains (), also in relation to the different virulence properties.

The effect of several inhibitors of polyamine synthesis on root growth and morphology and on polyamine titres in normal and Agrobacterium rhizogenes-transformed hairy root cultures of Hyoscyamus muticus was investigated. Five millimolar cyclohexylamine (CHA) and 1 mM methylglyoxal-bis-guanylhydrazone (MGBG) significantly inhibited both root growth and lateral root formation. One millimolar alpha-difluoromethylarginine (DFMA) significantly reduced while 1 mM alpha-difluoromethylornithine (DFMO) stimulated elongation growth of primary and lateral roots without affecting lateral root number. Both normal and hairy roots treated with DFMO, alone or in combination with DFMA, displayed a very evident lack of root hairs. Free polyamines constituted only a small part of the total polyamines present in hairy roots; of these, spermidine was the most abundant. A conspicuous accumulation of trichloracetic acid-soluble and -insoluble conjugated putrescine was also found. Although DFMO did not significantly alter total putrescine content, DFMA reduced it dramatically. This effect was further accentuated when DFMO and DFMA were supplied together. CHA and MGBG did not provoke the expected depletion in spermidine and/or spermine, but both inhibitors caused a marked accumulation in free and conjugated putrescine.

The enzymes involved in putrescine synthesis and oxidation were studied in transformed tobacco plants micropropagated in vitro and arising from Agrobacterium rhizogenes inoculation. They showed different degrees of phenotypic alterations, particularly evident in supertransformed plants. Diamine oxidase activity in the leaves was generally higher in transformed and even more in supertransformed plants than in controls. Arginine and ornithine decarboxylase activities, determined in both apices and leaves, were in agreement with data on endogenous polyamine levels in apices, but not in the leaves. This was probably due to a different compartmentation of enzymes, substrates and products.

Clones of transgenic tobacco plants, regenerated from transformed roots arising from Agrobacterium rhizogenes inoculation, were analyzed both in vivo under greenhouse conditions and in in vitro culture. Transformed plants displayed phenotypic alterations, referred to as hairy root syndrome. These traits were particularly accentuated in some plants, designated supertransformed, and seemed to be modulated by in vivo versus in vitro culture conditions. The comparison of protein, amino acid and polyamine levels between normal and transformed plants revealed alterations in nitrogen metabolism. In fact, under in vivo conditions apices and leaves of transformed plants displayed lower levels of glutamine and asparagine. The evolution in total polyamine content was generally an increase from 6-13 weeks, followed by a decrease at 20 weeks, the transformed tissues always containing lower polyamine levels than the normal ones. The major difference in vivo consisted in a strong reduction in the TCA-soluble and - insoluble bound polyamines, particularly evident in the leaves of transformed and supertransformed plants. On the whole, significant differences were found between in vivo and in vitro conditions: TCA-soluble bound polyamines were undetectable in both transformed and untransformed in vitro plantlets; in addition, supertransformed in vitro plantlets contained unexpectedly high levels of chlorophyll, proteins, amino acids and free polyamines compared with their counterparts grown in soil.

The possibility of a relation between the expression of root inducing (Ri) T‐DNA genes of Agrobacterium rhizogenes and changes in polyamine metabolism has been explored in fast‐growing tobacco hairy roots. Transformed root cultures have been established on hormone‐fee medium; they came from transgenic plants of Nicotiana tabacum L. cv. Xanthi with different altered phenotypes, designated transformed (T) and supertransformed (T'). T and especially T′ roots developed more rapidly both by elongation and lateral branching, and showed a higher growth rate than the untransformed control. After 3 weeks in culture, normal roots showed a very reduced meristematic zone, and flow cytometric analysis indicated that 2C nuclei were predominant in the apical parts in contrast to T and T′ roots, in which endopolyploidisation also appeared. Putrescine, spermidine and traces of spermine were present in all the samples, both in free and in conjugated forms. Putrescine was the major polyamine detected in controls and in transformed roots. At the time of excision, the polyamine levels were similar in normal, T and T′ roots. Significant differences were found during the progression of growth, particularly in the TCA‐insoluble fraction in which polyamines varied differently according to the type of roots, increasing considerably in T roots on day 8, then decreasing. The lower polyamine contents found in growing transformed roots were concomitant to low arginine (EC 4.1.1.19) and ornithine (EC 4.1.1.17) decarboxylase activities. It is suggested that polyamine levels and related enzyme activities are linked to growth kinetics rather than being a consequence of foreign gene expression.

Growth parameters of Agrobacterium rhizogenes (strains 1855 and 8196) were measured under different culture conditions. Yeast-mannitol medium supported better growth, compared with synthetic and polyamine-free 199 medium. Homospermidine was the major polyamine in both strains; putrescine was present in bacteria in both culture conditions, while spermidine and spermine were undetectable in bacteria grown on 199 medium. Arginine and ornithine decarboxylase activities were detected in both strains in the exponential phase of growth on yeast-mannitol and 199 medium. Generally polyamine synthesizing enzyme activities were significantly higher in A. rhizogenes strains grown on 199 medium. The relative flux through the arginine or ornithine decarboxylase pathways was controlled by culture conditions. Compared with arginine decarboxylase, ornithine decarboxylase was higher in bacteria grown on 199 medium. The results are discussed in relation to the intracellular contents of free arginine and ornithine.

A comparative study of free polyamine levels, arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) activities was carried out in the different stages of somatic embryos of Vitis vinifera cv. Grenache noir, and in zygotic embryos of the same cultivar, before and after germination. Somatic embryos showed a high level of free polyamines and a high putrescine/spermidine ratio. On a per unit basis, an accumulation of putrescine (Put) and spermidine (Spd) occurred in the late torpedo stage, which coincided with the beginning of abnormal growth and disorganized cell proliferation. In globular, heart-shaped and torpedo somatic embryos, ADC activity was higher than ODC activity. Later, in giant somatic embryos, a very high ADC and an even higher ODC activity occurred, except when expressed on a dry weight basis. By contrast, zygotic embryos showed a low level of free polyamines and a Put/Spd ratio approximately equal to one. These data suggest that the abnormal behaviour of grape somatic embryos and their low rate of development into plantlets (< 5%) could be due to the high free polyamine content and/or to an inadequate Put/Spd ratio.

Polyamine content, ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19) activities, as well as the effects of two inhibitors of putrescine synthesis, were studied during the preembryogenic phase and different stages of somatic embryogenesis in suspension cultures carrots (Daucus carota L.). In the preembryogenic phase α-difluoromethylornithine at 5mM, an irreversible inhibitor of ornithine decarboxylase, does not reduce growth but causes inhibition of putrescine accumulation; canavanine, a competitive inhibitor of arginine decarboxylase, completely blocks growth in the same phase. Difluoromethylornithine seems to prolong the commitment of the cells to embryogenesis during the preembryogenic phase, while canavanine inhibits the subsequent embryo differentiation. In general, a decrease in polyamine content and higher ornithine decarboxylase as compared to arginine decarboxylase activity is observed during the preembryogenic phase. Difluoromethylornithine does not affect ornithine decarboxylase in vivo, while both difluoromethylornithine and canavanine inhibit ornithine and arginine decarboxylase, respectively, in vitro. Spermidine and spermine contents, expressed on a per embryo basis, increase during the progression from globular to heart and torpedo stages of embryogenesis. While putrescine is the main polyamine in the heart stage, spermidine is more abundant in the torpedo stage. Embryos treated with difluoromethylornithine for 36h develop into normal plantlets at a higher frequency, and they are consistently larger than controls; the canavanine treatment blocks any further development of the embryos, which are always abnormal.

The biochemical characterization of a carrot cell line repeatedly subcultured on a medium containing 5 mM α-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, is reported. The cells grown on DFMO had an identical growth rate to the controls but a larger surface area that decreased with increasing numbers of subcultures. During the exponential phase of growth the DFMO treated cells displayed an increase in polyamine and protein content and a decrease in arginine and ornithine decarboxylase activities. The uptake of the inhibitor was slower in treated cells, and occurred against a concentration gradient. After the 12th subculture the amount of 14C-DFMO-bound to the protein fraction of crude cell extracts was followed over a 50 h period after the 12th subculture. The ratio between free and bound labelled DFMO was initially the same in both control and treated cells but increased with time in the latter probably as a result of the high endogenous pool of DFMO accumulated.