Monoclonal antibodies against transferrin. Precipitating mixtures and lack of inter-species cross-reactivity

Five stable hybridoma lines were prepared using the myeloma cell line P3-X63-Ag.653 and spleen cells of mice hyperimmunized by pig transferrin. All hybridomas grew well in mouse peritoneal cavity and produced antibodies of the IgG₁ subclass. Antibody preparations obtained from ascitic fluids tested for their capacity of antigen precipitation. No precipitation was obtained with single antibodies and with pairs of antibodies. Three out of 10 possible triads gave clear and sharp precipitation zones and rings in immunodiffusion tests performed in agar gel. All 5 antibodies were shown by quantitative enzyme-immunoassay to be specific for pig transferrin: no cross-reaction was obtained with mouse, human, horse and sheep transferrins.     

Release of prolactin and luteinizing hormone by histamine agonists in ovariectomized,steroid-treated rats under ether anesthesia

Responses to histamine agonists administered intraventricularly under ether anesthesia were analyzed to evaluate receptor mediation in histamine stimulation of prolactin and LH release in ovariectomized, estradiol-progesterone-treated rats (OVX-E2P-treated rats). Prolactin release was markedly increased by the H2-histamine agonists, 4-methyl histamine and Dimaprit. These effects were antagonized by metiamide, an H2-blocking agent. The H1-histamine agonist, 2-(2-pyridyl)ethylamine (PEA) in high doses released prolactin and its effect was partially prevented by metiamide. Mepyramine, and H1-antagonist, did not exert any effect on the release of prolactin enhanced by the histamine agonists. LH release was significantly increased after 4-methyl histamine administration. Its effect was weak and was blocked by metiamide. Neither Dimaprit nor PEA exhibited action on plasma LH levels. The results obtained with histamine agonists suggest that histamine evokes prolactin release in OVX,E2P-treated rats through H2-receptors. At present, conclusions on H2-receptor mediation in LH release induced by histamine cannot be drawn from these results. The above-mentioned data, however, conclusively discard a significant participation of H1-receptors.     

Acute effect of endogenous inhibitors and exogenous cytostatics on the ultrastructure of bone marrow cells. I. Single dose of 1,2 : 5,6-dianhydrogalactitol (DAD).

The ultrastructural effects of the endogenous inhibitor, granuloid crude extract (GCE), known to control the proliferation of myeloid cells, and of the current hexitol derivative, 1,2 : 5,6-dianhydrogalactitol (DAD) were compared on the rat bone marrow. A single intraperitoneally injected LD50 dose of DAD induces the following changes in the fine structure: The mitochondria become swollen, the matrix less electron-dense, the cristae fragmentate, the ribosomes aggregate, anomalies appear in the perinuclear the cell membranes, and myelin figures and intranuclear bodies develop. Autophagy, degeneration and the phagocytotic activity of the reticulum cells is appreciable in 4 hours after treatment and increase by the 24th hour. The toxic effect of DAD is cell aspecific but in the ultrastructure its myelotropic effect manifests earlier than in erythropoiesis. In contrast, the arrest caused by a single dose of the endogenous granuloid inhibitor [2] is cell-specific and non-toxic.     

Dopamine enhancement of NMDA currents in dissociated medium-sized striatal neurons: role of D1 receptors and DARPP-32

Dopamine (DA), via activation of D1 receptors, enhances N-methyl-D-aspartate (NMDA)-evoked responses in striatal neurons. The present investigation examined further the properties of this enhancement and the potential mechanisms by which this enhancement might be effected. Dissociated medium-sized striatal neurons were obtained from intact rats and mice or mutant mice lacking the DA and cyclic adenosine 3',5' monophosphate (cAMP)-regulated phosphoprotein of M(R) 32,000 (DARPP-32). NMDA (10-1,000 microM) induced inward currents in all neurons. In acutely dissociated neurons from intact rats or mice, activation of D1 receptors with the selective agonist, SKF 81297, produced a dose-dependent enhancement of NMDA currents. This enhancement was reduced by the selective D1 receptor antagonist SKF 83566. Quinpirole, a D2 receptor agonist alone, produced small reductions of NMDA currents. However, it consistently and significantly reduced the enhancement of NMDA currents by D1 agonists. In dissociated striatal neurons, in conditions that minimized the contributions of voltage-gated Ca(2+) conductances, the D1-induced potentiation was not altered by blockade of L-type voltage-gated Ca(2+) conductances in contrast to results in slices. The DARPP-32 signaling pathway has an important role in D1 modulation of NMDA currents. In mice lacking DARPP-32, the enhancement was significantly reduced. Furthermore, okadaic acid, a protein phosphatase 1 (PP-1) inhibitor, increased D1-induced potentiation, suggesting that constitutively active PP-1 attenuates D1-induced potentiation. Finally, activation of D1 receptors produced differential effects on NMDA and gamma aminobutyric acid (GABA)-induced currents in the same cells, enhancing NMDA currents and inhibiting GABA currents. Thus simultaneous activation of D1, NMDA, and GABA receptors could predispose medium-sized spiny neurons toward excitation. Taken together, the present findings indicate that the unique potentiation of NMDA receptor function by activation of the D1 receptor signaling cascade can be controlled by multiple mechanisms and has major influences on neuronal function.     

Notes on parasitism by Amblyomma humerale (Acari: Ixodidae) in the state of Rondônia,western Amazon,Brazil

The tick Amblyomma humerale Koch is endemic to South America. All host records refer to the adult stage parasitizing tortoises, mostly yellow-footed tortoise, Geochelone denticulata (L.), and red-footed tortoise, Geochelone carbonaria (Spix). The current study reports the presence of A. humerale in the state of Rond?nia, Brazil. A total of 215 adult ticks (201 males, 14 females) was collected from six G denticulata in an Indian reserve and nine Geochelone sp. in rural Monte Negro County, giving an overall mean infestation of 14.3 +/- 12.0 (range: 2-44) ticks per tortoise. Male ticks always outnumbered females on the host and nine tortoises had only male ticks. Male ticks were mostly attached in clusters on the ventral sides of the carapace near the anterior and posterior margins, and more rarely on the outer margin of the plastron. All females were found attached to the tortoise skin, at different sites such as head, neck, shoulders or legs. Male ticks were rarely observed attached to the body skin. Seven engorged nymphs collected on small vertebrates from Monte Negro County molted to adults of A. humerale. This included one nymph each on the seven-colored lizard, Plica plica (L), green tree climber, Plica umbra (L.), and wide-foraging lizard, Kentropyx calcarata Spix,three nymphs on the common opossum, Didelphis marsupialis L., and one nymph on the silky anteater, Cyclopes didactylus L. These constitute the first host records for the immature stages of the tick A. humerale.     

A comparative study of the JAM test and 51Cr-release assay to assess the cytotoxicity of dendritic cells on hematopoietic tumor cells

Dendritic cells (DCs) are potent antigen presenting cells and possess a direct anti-tumor cytotoxic ability. Nevertheless, the mechanism of anti-tumor cytotoxicity by DCs and the methods for its evaluation are not fully elucidated. In order to clarify this mechanism of cytotoxicity, we examined the ability of DCs 1) to suppress [³H] thymidine (³H-TdR) uptake by tumor cells; 2) to induce cytolysis on ⁵¹Cr-labeled tumor cells; 3) and to induce DNA fragmentation on ³H-TdR labeled tumor cells (JAM test). Cytolysis and DNA fragmentation are markers of necrotic and apoptotic mechanisms of cytotoxicity in vitro, respectively. DCs inhibited approximately 38.6% to 54.8% of the growth of B4D6, NB4, U937, and Daudi cells as evaluated by the uptake of ³H-TdR. However no cytolysis was verified by ⁵¹Cr-release assay. On the other hand, cytotoxicity rates found using the JAM test ranged from 3 to 81% depending on the cell line and the effector to target cell ratio. The discrepancy of cytotoxicity between ⁵¹Cr-release assay and the JAM test may be due to the phagocytosis of apoptotic tumor cells or the absorption of released ⁵¹Cr by DCs surrounding the target cells. In conclusion, the JAM test was more sensitive than the 4-h and the 10-h ⁵¹Cr-release assay to investigate cytotoxicity mediated by DCs toward hematopoietic tumor cell lines in vitro.     

Enhanced inhibitory avoidance learning prevents the memory-impairing effects of post-training hippocampal inactivation

Rats were trained on an inhibitory avoidance task to study the effects of post-training administration of tetrodotoxin (TTX, which temporarily inactivates neural activity) on memory consolidation. During training, independent groups of rats received either a mild foot shock (0.8 mA) or a stronger (1.0 mA) foot shock. TTX was administered bilaterally into the dorsal hippocampus immediately after training, and memory of the task was measured 48 h later. We corroborated the typical amnesic effect of intrahippocampal infusions of TTX in those rats trained with the mild-intensity foot shock. More importantly, with the stronger foot shock, the same treatment was ineffective in producing amnesia. These results suggest that, after an enhanced learning experience, other brain regions are also activated, which may compensate for the amnesic effect of TTX infusions into the hippocampus.Due to an error in the citation line, this revised PDF (published in December 2003) deviates from the printed version, and is the correct and authoritative version of the paper.     

Analysis of CDKN1A polymorphisms: markers of cancer susceptibility?

The CDKN1A (TP21) gene encodes a 21-kD protein that is a critical downstream mediator of wild-type TP53 and an important regulator of the cell cycle. Failure in the function of this gene would be expected to result in abnormal cell proliferation and transformation. Tumor-associated mutations of the coding region of the TP21 are rare. On the other hand, some TP21 polymorphisms have been identified and characterized by single base substitutions. In the present study, we investigated the potential role of TP21 gene polymorphisms in skin, head, and neck tumorigenesis. A total of 261 samples were examined by polymerase chain reaction single-strand conformational analysis, and one mutation at codon 31 and four polymorphisms in exons 2 (codon 55) and 3 [nucleotide (nt)590] and in promoter region (nt2298) were identified. In conclusion, this investigation confirmed the rarity of mutations in this gene, arguing against a role for TP21 mutations in skin, head, and neck cancers. Also, our results show significant differences in nt2298 allele frequencies between normal individuals and skin malignant tumors (P < 0.05). The results suggest that this polymorphism affects TP21 transactivator binding and may be important during the pathogenesis of skin cancer.     

First case of compound heterozygous BHLHA9 variants in mesoaxial synostotic syndactyly with phalangeal reduction

Mesoaxial synostotic syndactyly with phalangeal reduction (MSSD) is an extremely rare autosomal recessive limb abnormality characterized by the fusion of third and fourth fingers. To date, only homozygous missense and frameshift mutations have been reported in BHLHA9 associated to MSSD. In this study, we report a patient who presented with clinical and radiological features of MSSD. A customized skeletal dysplasia NGS panel revealed the presence of two novel compounds heterozygous variants in BHLHA9: NM_001164405.1: c.[226A>T][269G>C]; p.[(Lys76*)][(Arg90Pro)]. Thus, this is the first case of MSSD in a nonconsanguineous family.     

Effect of polyphenolic compounds on the renal Na+,K(+)-ATPase during development and persistence of hypertension in rats

It has been suggested that polyphenolic substances provide protection against the risk factors of cardiovascular diseases. The present study was designed to investigate whether application of red wine polyphenols influences the kinetic properties of the renal Na+,K(+)-ATPase in rats with hypertension (164 +/- 8 mmHg) that was experimentally induced by the NO synthase inhibitor N(G.) -nitro-L- arginine methyl ester (L-NAME). Polyphenols in a dose of 40 mg kg(-1) day(-1) in drinking fluid induced different effects on the properties of the renal Na+,K(+)-ATPase depending on the mode of their administration. Preventive application of polyphenols during the development of hypertension (144 +/- 5 mmHg) partially protected the Na+,K(+)-ATPase molecule against hypertension-induced deterioration via increased capability of the enzyme to bind ATP and/or Na+ as suggested by decrease of Km and KNa, respectively, even to values lower than in controls. However, polyphenols did not prevent the hypertension-induced reduction of the number of active Na+,K(+)-ATPase molecules as shown by similar V(max) values as compared to the hypertensive L-NAME group. The above protection is probably secured by a NO-dependent mechanism as suggested by 150% increase of the NO synthesis. Additional treatment of already hypertensive animals with polyphenols (153 +/- 8 mmHg) resulted in partial restoration of the Na+,K(+)-ATPase affinities especially for sodium as indicated by significant diminution of KNa. However, polyphenols in this mode of application did not slow down the L-NAME-induced decrease in the number of Na+,K(+)-ATPase molecules in the kidney as suggested by additional significant decrease in V(max) values when comparing this group with the control group and also the hypertensive L-NAME group. In this case the polyphenols affected the Na,K-ATPase molecule in a NO-independent way as indicated by the fact that polyphenols failed to restore normal NO synthesis.