Stroke burden in Guinea: Results from the Conakry Ignace Deen Hospital stroke registry.

Sub-Saharan Africa has extremely high stroke prevalence and case fatality. Most Sub-Saharan African regions are uncharted in terms of stroke characteristics, epidemiology, and burden. We report here the results from the first stroke registry in Guinea.

Detecting episomal or integrated human papillomavirus 16 DNA using an exonuclease V-qPCR-based assay.

Screening for human papillomavirus (HPV) integration into host cell chromosomes typically requires large amounts of time and reagents. We developed a rapid and sensitive assay based on exonuclease V (ExoV) and quantitative polymerase chain reaction (qPCR) to determine HPV genome configurations in cell lines and tissues. We established the assay using genomic DNA from cell lines known to harbor integrated or episomal HPV16. DNA was incubated with ExoV, which is specific for linear DNA, and the DNA fraction resistant to digestion was measured by qPCR. The percent of DNA resistant to ExoV digestion was calculated relative to undigested DNA for determination of episomal or integrated HPV16. The ExoV assay was accurate, capable of distinguishing episomal from integrated HPV16 in cell lines and tissues. Future applications of the ExoV assay may include screening of HPV genome configurations in the progression of HPV-associated cancers.

Inhibition of Epstein-Barr Virus Replication in Human Papillomavirus-Immortalized Keratinocytes.

Epstein-Barr virus (EBV) is implicated in the pathogenesis of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OSCC). EBV-associated cancers harbor a latent EBV infection characterized by a lack of viral replication and the expression of viral oncogenes. Cellular changes promoted by HPV are comparable to those shown to facilitate EBV latency, though whether HPV-positive cells support a latent EBV infection has not been demonstrated. Using a model of direct EBV infection into HPV16-immortalized tonsillar cells grown in organotypic raft culture, we showed robust EBV replication in HPV-negative rafts but little to no replication in HPV-immortalized rafts. The reduced EBV replication was independent of immortalization, as human telomerase-immortalized normal oral keratinocytes supported robust EBV replication. Furthermore, we observed reduced EBV lytic gene expression and increased expression of EBER1, a noncoding RNA highly expressed in latently infected cells, in the presence of HPV. The use of human foreskin keratinocyte rafts expressing the HPV16 E6 and/or E7 oncogene(s) (HPV E6 and E7 rafts) showed that E7 was sufficient to reduce EBV replication. EBV replication is dependent upon epithelial differentiation and the differentiation-dependent expression of the transcription factors KLF4 and PRDM1. While KLF4 and PRDM1 levels were unaltered, the expression levels of KLF4 transcriptional targets, including late differentiation markers, were reduced in HPV E6 and E7 rafts compared to their levels in parental rafts. However, the HPV E7-mediated block in EBV replication correlated with delayed expression of early differentiation markers. Overall, this study reveals an HPV16-mediated block in EBV replication, through E7, that may facilitate EBV latency and long-term persistence in the tumor context.IMPORTANCE Using a model examining the establishment of EBV infection in HPV-immortalized tissues, we showed an HPV-induced interruption of the normal EBV life cycle reminiscent of a latent EBV infection. Our data support the notion that a persistent EBV epithelial infection depends upon preexisting cellular alterations and suggest the ability of HPV to promote such changes. More importantly, these findings introduce a model for how EBV coinfection may influence HPV-positive (HPV-pos) OSCC pathogenesis. Latently EBV-infected epithelial cells, as well as other EBV-associated head-and-neck carcinomas, exhibit oncogenic phenotypes commonly seen in HPV-pos OSCC. Therefore, an HPV-induced shift in the EBV life cycle toward latency would not only facilitate EBV persistence but also provide additional viral oncogene expression, which can contribute to the rapid progression of HPV-pos OSCC. These findings provide a step toward defining a role for EBV as a cofactor in HPV-positive oropharyngeal tumors.

[Investigation of epidemiology of neurotrauma in the Republic of Guinea].

The paper focuses on analysis of incidence of neurotrauma in economically underdeveloped country such as Republic of Guinea. It is found that leading etiology of central nervous system injuries are road accidents and indoor traumatism. Investigation of system of medical care revealed its poor condition and severe defects which prevent practical application of evidence-based recommendations for management of traumatic brain injury in underdeveloped countries including Republic of Guinea. Development of multiplanar strategy of control of neurotrauma is required which can be achieved only in case of massive governmental and international aid.

HPV infection in women with and without cervical cancer in Conakry, Guinea.

BACKGROUND: Cervical cancer incidence in western Africa is among the highest in the world. METHODS: To investigate human papillomavirus (HPV) infection in Guinea, we obtained cervical specimens from 831 women aged 18-64 years from the general population of the capital Conakry and from 77 locally diagnosed invasive cervical cancers (ICC). Human papillomavirus was detected using a GP5+/6+ PCR-based assay. RESULTS: Among the general population, the prevalence of cervical abnormalities was 2.6% by visual inspection and 9.5% by liquid-based cytology. Fourteen of 15 high-grade squamous intraepithelial lesions were visual inspection-negative. Human papillomavirus prevalence was 50.8% (32.1% for high-risk types) and relatively constant across all age groups. Being single or reporting > or =3 sexual partners was significantly associated with HPV positivity. HPV16 was the most common type, both among the general population (7.3%) and, notably in ICC (48.6%). HPV45 (18.6%) and HPV18 (14.3%), the next most common types in ICC, were also more common in ICC than in HPV-positive women with normal cytology from the general population. CONCLUSION: The heavy burden of HPV infection and severe cervical lesions in Guinean women calls for new effective interventions. Sixty-three per cent of cervical cancers are theoretically preventable by HPV16/18 vaccines in Guinea; perhaps more if some cross-protection exists with HPV45.

Matrix metalloproteinases MMP-3 and MMP-1 levels in sera and synovial fluids in patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVES: To investigate whether serum levels of matrix metalloproteinases (MMP-3, stromelysin) and (MMP-1, collagenase) are specifically elevated in joint disease as rheumatoid arthritis (RA) compared to osteoarthritis (OA), and to assess how these markers reflect the clinical activity of RA compared to circulating cytokine as tumor necrosis factor-alpha (TNF-alpha) as well as established variables as [C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR)]. SUBJECTS AND METHODS: This study included 22 patients with RA, 10 patients with OA and 10 healthy control subjects matched for age and sex. Patients with superimposed infection were excluded. Serum levels of MMP-3, MMP-1, TNF-alpha and CRP were assayed. Synovial fluid (SF) levels of MMP-3 and MMP-1 were also assayed. RESULTS: Serum levels of TNF-alpha and CRP in RA patients were significantly higher than normal subjects. Serum MMP-1 was significantly elevated in patients with RA and OA, compared to healthy controls but there were no significant differences between patients with RA and those with OA. Serum MMP-3 levels did not differ between OA patients and normal sera. However, RA patients displayed significantly elevated levels of this enzyme, compared to OA and control sera. Levels of MMP-3 and MMP-1 in the SF of RA patients were significantly higher than in OA fluids. CRP, ESR, TNF-alpha and MMP-3 correlated significantly with the swollen joint count. The strongest positive correlations existed between rheumatoid activity as assessed by the levels of CRP and circulating levels of MMP-3. Similar correlations between TNF-alpha concentration and CRP, MMP-1 and MMP-3 were observed in RA patients. Serum levels of MMP-3 correlated significantly with serum concentrations of MMP-1 in RA patients (r = 0.487, p < 0.05). There was close correlation between serum and SF concentrations of MMP-3 in RA patients (r = 0.619, p < 0.01). In the same patients there was highly significant correlation between SF concentrations of MMP-3 and MMP-1 (r = 0.732, p < 0.001). CONCLUSIONS: Our data suggested that elevated MMP-3 levels reflected disease activity of RA better than cytokine levels. However, MMP-3 levels do not exceed the association of CRP with clinical activity.

Multichannel quartz crystal microbalance.

Arrays of quartz crystal resonators are fabricated on a single quartz wafer as a multichannel quartz crystal microbalance (MQCM). Three types of four-channel array of 10-MHz resonators are prepared and tested. Mechanical oscillation of each channel is entrapped within the channel almost completely, so that the interference between the channels via the quartz crystal plate is almost negligible. A mass change on each channel is quantitatively evaluated on the basis of Sauerbrey's law. Thus, each channel of a MQCM device can be used as an independent QCM. Influence from a longitudinal wave generated from another channel is also negligible compared to the influence from the wave from the monitored channel itself. The simultaneous oscillation of channels is also possible. The potential applicability of MQCM to the two-dimensional mapping of mass changes is demonstrated.

[Pharmacokinetic analysis of scavenger receptor-mediated uptake of mucopolysaccharides in various cells].

Although the scavenger receptor-mediated uptake has been qualitatively investigated in the research fields of biochemistry and pathology, pharmacokinetic characteristics of the scavenger receptors are poorly understood. In this review, we summarized basic findings on scavenger receptors reported in available literature, and introduced our recent studies on the quantitative characteristics of the scavenger receptor-mediated uptake. High molecular weight fractionated [3H]heparin (HMWFH, 16,000-24,000 Da), one of the model mucopolysaccharides, was investigated to elucidate its uptake mechanism into isolated rat Kupffer cells, isolated peritoneal macrophages and liver parenchymal cells in primary culture. The equilibrium bindings of HMWFH to isolated Kupffer cells and peritoneal macrophages were concentration-dependent with the respective dissociation constants (Kd) of 5.7 and 6.0 nM and with the respective maximum binding capacities (Bmax) of 1.5 and 1.9 pmol/10(6) cells. Several ligands of scavenger receptors inhibited the binding of HMWFH to macrophages, suggesting the involvement of scavenger receptors in the uptake of HMWFH by these macrophages. It was also suggested that the scavenger receptor-mediated uptake is different from the receptor-mediated endocytosis of polypeptides and phagocytosis, based on the evidence of the now inhibitory effects of an inhibitor of receptor-mediated endocytosis of polypeptides(phenylarsine oxide) and phagocytosis inhibitors (cytochalasine B and colchicine) on the internalization. The involvement of scavenger-like receptors was also suggested in the uptake of HMWFH by liver parenchymal cells in primary culture by demonstrating inhibitory effects of ligands for scavenger receptors. The internalization into liver parenchymal cells by scavenger-like receptors was not affected by an inhibitor of receptor-mediated endocytosis of polypeptides and phagocytosis inhibitors, similarly to the results in the macrophage scavenger receptors. The Kd of 53.5 nM and Bmax of 32.8 pmol/10(6) cells in parenchymal cells were both in the order of magnitude larger than those in isolated Kupffer cells, suggesting the binding of HMWFH to scavenger-like receptors in parenchymal cells with lower affinity and higher capacity. On the other hand, an apparent internalization rate constant (kint, app) of 0.0056 min-1 was comparable with that in Kupffer cells (0.0118 min-1). Thus, we demonstrated the involvement of scavenger receptors in the uptake of HMWFH by rat Kupffer cells, peritoneal macrophages and liver parenchymal cells, and succeeded in characterizing the uptake kinetically. These findings should provide useful information for not only establishing the rational clinical use of mucopolysaccharides but also developing new drugs such as antiatherosclerotic agents and peptides delivered to cells with scavenger receptors.

Involvement of nuclear binding sites for melatonin in the regulation of IL-2 and IL-6 production by human blood mononuclear cells.

Many functional studies show that melatonin plays a fundamental role in neuroimmunomodulation. In this paper, we have extended our studies on the influence of melatonin on IL-2 and IL-6 production by human peripheral blood mononuclear cells (PBMCs) by comparing the effects of the specific membrane receptor agonist S 20098, the RZR/ROR(alpha) receptor agonist CGP 52608, and structurally related thiazolidinediones. Melatonin bound to membranes as well as to nuclei of human PBMCs with about the same affinity (IC50 values around 5 nM). S 20098 bound to PBMC membranes but not to PBMC nuclei, although the affinity was at least 100 times lower than that of melatonin; this compound did not stimulate cytokine production. In contrast, all four CGP compounds did not bind to PBMC membranes, while binding to nuclei exhibited IC50 values comparable to those of melatonin. The thiazolidinediones activating the RZR/ROR(alpha) receptor (CGP 52608, CGP 53079) also increased IL-2 and IL-6 production. CGP 55644 had no effect on cytokine production and antagonized the effects of CGP 52608 on IL-2 and IL-6 production; moreover, CGP 55644 decreased the enhanced IL-2 production caused by melatonin. Results obtained in monocyte cultures resembled closely those shown in PBMCs. The results reported in this paper confirm the involvement of a nuclear mechanism in the melatonin effects on cytokine production in human PBMCs. We have also shown a synergistic effect of S 20098 and CGP 52608, suggesting a possible link between nuclear and membrane melatonin receptors in PBMCs.

Melatonin enhances IL-2, IL-6, and IFN-gamma production by human circulating CD4+ cells: a possible nuclear receptor-mediated mechanism involving T helper type 1 lymphocytes and monocytes.

This paper shows that melatonin is able to activate human Th1 lymphocytes by increasing the production of IL-2 and IFN-gamma in vitro. Th2 cells appear not to be affected by melatonin, since IL-4, which is mostly produced by Th2 cells, is not modified by the hormone. Melatonin also enhances IL-6 production by PBMCs. The activation by melatonin of IL-6 production is apparently related to the presence of monocytes, rather than to Th2 cells, in the cell preparation, since PBMCs depleted of monocytes (CD14+ cells) were not activated. Activation of PBMCs by melatonin was dependent on the dose and, measured by cytokine production, was observed only when cells were either not activated or only slightly activated by low concentrations of PHA, or when cell activation was achieved by incubating the cells with previously irradiated cells. Using a different approach to identify what type of cells among the PBMC subsets was activated by melatonin, the expression of CD69, a marker of cell activation, was studied. Melatonin increased the percentage of cells expressing the CD69 Ag in CD4+ but not in CD8+ cells. We have also achieved enhanced production of IL-2 and IL-6 using CGP 52608, a specific ligand of the putative nuclear melatonin receptor RZR/ROR, raising the possibility of direct effects of melatonin on gene regulation in both Th1 cells and monocytes. The results suggest that melatonin may be involved in the regulation of human immune functions by modulating the activity of Th1 cells and monocytes via nuclear receptor-mediated transcriptional control.

Kinetic characterization of binding and internalization of fractionated [3H]heparin in rat liver parenchymal cells in primary culture.

The binding and internalization of fractionated [3H]heparin (FH) was kinetically analyzed in rat liver parenchymal cells to clarify its cellular uptake mechanism. The binding of FH to the cell surface was saturable with the dissociation constant (Kd) of 53.5 nM and a maximum binding capacity (Bmax) of 19.9 pmol/mg protein. The binding of FH to the cell surface was competitively inhibited not only by heparan sulfate, a polyanion analogous to heparin, but also by rose bengal, an organic anion, suggesting the binding is based on an electric interaction requiring an anionic charge for substrates and consistent with the earlier suggestion of the involvement of the scavenger-like receptor. According to kinetic model analysis, the rate constants of association (K(on)), dissociation (k(off)), and internalization (k(int).app) were estimated to be 0.0005 nM-1 min-1, 0.0112 min-1, and 0.0056 min-1, respectively. Although both Kd and Bmax were larger than those reported in Kupffer cells, suggesting lower affinity and higher capacity in liver parenchymal cells, the apparent internalization rate constant was similar to that in Kupffer cells. We thus provided additional evidence suggesting that a scavenger-like receptor exists in rat liver parenchymal cells, and then kinetically characterized the surface binding and internalization of fractionated heparin by this receptor.

Genetic study of Andalusia's ovine and caprine breeds.

SUMMARY: Two different breeds of Andalusian sheep, 'Grazalema Merino' and 'Lebrijan Churro', and two different breeds of Andalusian goats, 'Andalusian White' and 'Andalusian Black', chosen by previous studies (Rodero et al. 1992a) as priority breeds for conservation, were studied. The systems used corresponded to ethnozootechnic characteristics, as well as the different biochemical-polymorphism variables. Farms were differentiated within breeds, or between themselves, and different tests were used of genetic and genotypic frequencies: Wright's indices, medium heterozygosities, Whalund's variances, G test of probability of reason, etc. Also Cavalli-Sforza's genetic distance was obtained. In the Andalusian Black and Grazalema Merino breeds, the Whalund's variances obtained were a result of selection, that has divided the breeds into distinct populations differentiated spatially. Medium heterozygosities of each breed do not differ much within themselves, but when each system is considered alone, discrepancies between ethnic groups are relevant. Wright's F indices demonstrated in the Andalusian White and Grazalema Merino breeds, genetic heterozygosities between populations or studied herds can be deduced, but this is not possible in the Andalusian Black. The F(IS) values indicated, despite the small size of the populations, that inbreeding has been avoided, probably because of the entry of foreign sires. In none of the breeds is there a significant excess of heterozygosis. The genetic distances between flocks within breeds do not differ from those found between breeds. RÉSUMÉ: On a travallé avec, differents troupeau des races de montons de l'Andalusie, Grazalema Merino et Lebrija Churro, et avec les races caprines Andalusian White et Andalusian Black, choisie entre les races Andaluciennes comme prioritaires pour la conservation, dans un etudie avant (Rodero et col. 1992a). Les sistémes utilicés dans cette travaille correspondent á charactérés etnozootechniques et á differents variables de polymorphism biochimique. Lorsque on fait differences entre troupeau, dedans de races, ou entre elles, on a utilicés differents preuves, á partir des fréquences géniques et génotipiques: l'index de Wright, hétérozygotie moyennes, variances de Whalund, preuve G de raison de probabilité, etc. Aussi le distance de Cavalli-Sforza. Comme conclusion, dans les races Andalusian Black et Grazalema Merino les variances de Whalund obtenues sont cosequences de l'action de la selection, donant different populations avec differentiation spaciale. Les hétérozygoties moyennes de chaque race sont parus, mais lorsque on considérent chaque systéme separé, les differences entre groupes ethniques sont importantes. Les indexes F de Wright demonstrent que, dans le races Andalusian White et Grazalema Merino on peuvent déduire d'heterozygoties génétique entre les populations ou troupeau analicées, dans le race Andalusian Black les differences valeurs de FIS indiquent que, malgré les petites dimensions des populations, on a evité la consanguinitée, due, probablement, á l'entrée d'étalons externes. Il n'y a pas, chez auqune race, d'un signifivative accroissement d'hétérizygosis. Les distances génétiques entre troupeau, dedans des races, en different pas des distances obtenues entre races. RESUMEN: Se ha trabajado con diferentes ganaderias de las razas ovinas andaluzas Merino de Grazalema y Churra Lebrijana, y con las caprinas Blanca Serrana y Negra Serrana, elegidas entre el resto de las razas de Andalucia como prioritarias pra la conservación, por estudio previo (Rodero y col., 1992a). Los sistemas utilizados en este trabajo corresponden tanto a caracteres etnozootécnicos como a diferentes variables de polimorfismos bioquimicos. Cuando se han diferenciado las ganaderias dentrde razas, o las ganaderias entre si, se han utilizado diferentes pruebas, a partir de las frecuencias genéticas y genotipicas: indices de Wright, heterocigosidades media, varianzas de Whalund, prueba G de razón de probabilidad, etc. También se obtuvieron las distancia genéticas de Cavalli-Sforza. Se concluye que en las razas Negra Serrana y Merino de Grazalema las varianzas de Whalund obtenidas son consecuencia de la acción de la selección que ha actuado dividiendo las razas en distintas poblaciones con diferenciación espacial. Las heterocigosidades medias de cada raza no difieren mucho entre si, pero cuando se considera cada sistema aisladamente, las discrepancias entre grupos étnicos son acusadas. Los indices F de Wright ponen de manifiesto que, mientras en las razas Blanca Serrana y Merino de Grazalema se pueden deducir heterocigosidades genéticas entre las problaciones o ganaderias estudiadas, no ocurre otro tanto en la raza Negra Serrana. Los valores de F(IS) parecen indicar que, a pesar del tamaño pequeño de las poblaciones, se ha evitado la consanguinidad, probablemente por la entrada de sementales externos. No se produce en ninguna de las razas un exceso significativo de heterocigosis. Las distancias genéticas entre ganaderias dentro de razas no difieren de lashalladas entre razas. ZUSAMMENFASSUNG: Es wurde mit verschiedenen andalusischen Zuchten der Schafrassen 'Grazalema Merino' and 'Lebrijan Churro' und der Ziegenrassen 'Andalusian White' und 'Andalusian Black' gearbeitet, die man von den andalusichen Rassen im Hinblick auf Erhaltung ausgewählt hat. Die benutzten Systeme in dieser Forschungsarbeit entsprechen Merkmalen, die sich sowohl auf ethnisch wie auch auf die verschiedenen Variablen des biochemischen Polimorfismius bezichen. Zur Unterschlidung von Zuchten innerhalb die Rassen oder von zuchten untereinander wurden verschiedene Tests benutzt, die von den genetischen und genotypischen Frequenzen ausgehen: Wright-Index, Durchschnittsheterozygositäten, Wahl und Varianz G-Test der Wahrscheinlichkeit, etc. Außerdem wurden die genetischen Distanzen nach Cavalli-Sforza errechnet. Man Kommt zum Schluß, daß in den Andalusian Black und Grazalema Merino die Wahl und Varianz das Ergebnis einer Selektionsaktivität ist, die die Rassen in Verschiedenen Populationen und unterschiedlichen Räumen aufgeteilt hat. Die durchschnittlichen Heterozygositäten jeder Rasse unterscheiden sich wenig voneinander, aber wenn man jedes System für sich betrachtet, stell man doch erhebliche Diskrepanzen zwischen den ethnischen Gruppen fest. Der Wright-Index offenbart, daß man in den Rassen Andalusian White und Grazalema Merino genetische Heterozygositäten ableiten kann zwischen den Populationen oder den untersuchten Zuchten; dies ist nicht der Fall inder Rasse Andalusian Black. Die F(IS) werte scheinen anzugeben, da trotz der kleinen Größe der Populationen die Blutsverwandschaft vermieden wurde, wahrscheinlich durch von außerhalb kommenden Böcken. In keiner der Rassen existiert übermässige Heterozygotie Die genetischen Distanzen zwischer der Zuchten unterscheiden sich nicht van dener der Rassen.

Macromolecule-macromolecule interaction in drug distribution. V. Effects of plasma proteins on uptake of fractionated [3H]heparin in isolated rat Kupffer cells.

Effects of plasma proteins such as alpha-globulin on the uptake of high molecular weight (HMWFH: 23000 Da) and low molecular weight fractionated [3H]heparin (LMWFH: 10000 Da) were examined in isolated rat Kupffer cells. alpha-Globulin (8 mg/ml) affected neither surface binding nor internalization of LMWFH by Kupffer cells, while it reduced both surface binding and internalization of HMWFH without affecting the fraction internalized, which was a ratio of internalized amount to the total association. The total associations of HMWFH were about four times larger than that predicted assuming only the unbound fraction is available for uptake, suggesting the participation of protein-mediated transport in the uptake of HMWFH in Kupffer cells. Based on the same assumption, the saturable initial uptake of HMWFH versus concentration profile in the presence of alpha-globulin (8 mg/ml) was also analyzed to further examine the suggested protein-mediated transport. The estimated dissociation constant of 487 nM was three times larger than that in in vitro binding experiments (168 nM) and the binding capacity of 0.155 was one third of the value in vitro (0.5), suggesting apparent reductions in both binding affinity and capacity. Thus, we demonstrated the involvement of protein-mediated transport in the uptake of fractionated heparin in Kupffer cells and kinetically characterized it as the apparent enhancement of dissociation.

Molecular weight dependency in the uptake of fractionated [3H]heparin in isolated rat Kupffer cells.

The uptake of low molecular weight fractionated [3H]heparin (LMWFH, 10000 Da) was compared with that of high molecular weight fractionated [3H]heparin (HMWFH, 23000 Da) in isolated rat Kupffer cells. Several heparin analogs, including HMWFH and ligands of scavenger receptors, inhibited both the surface binding and internalization of LMWFH, suggesting the involvement of scavenger receptors in the uptake of LMWFH in isolated rat Kupffer cells as well as HMWFH, in spite of a large difference in molecular weight. Metabolic inhibitors (2,4-dinitrophenol and rotenone), receptor-mediated and adsorptive endocytosis of polypeptides (phenylarsine oxide) and phagocytosis inhibitors (cytochalasine B and colchicine) did not inhibit the internalization of LMWFH. These results suggest that the scavenger receptor-mediated uptake of LMWFH is ATP-independent and different from receptor-mediated and adsorptive endocytosis of polypeptides and phagocytosis, in agreement with our previous results for HMWFH. The equilibrium binding of LMWFH to Kupffer cells was concentration-dependent with the dissociation constant (Kd) of 50 nM and maximum binding capacity (Bmax) of 2.3 pmol/10(6) cells. The dissociation constant of LMWFH was an order of magnitude larger than that of HMWFH (5.7 nM), suggesting a decrease in binding affinity to scavenger receptors with a decrease in the molecular weight of fractionated heparin. It was also shown that LMWFH is internalized by scavenger receptors according to first-order kinetics with an apparent internalization rate constant (Kint,app) of 0.0053 min-1, which is about half that for HMWFH (0.0118 min-1). Molecular weight thus appears to be one of dominant factors determining the uptake of fractionated heparin by scavenger receptors in Kupffer cells, and may partly explain the reported lower hepatic uptake of low molecular weight heparin than that of unfractionated heparin.

Uptake mechanism of fractioned [(3)H]heparin in isolated rat kupffer cells: involvement of scavenger receptors.

The uptake of fractionated [(3)H]heparin was examined to elucidate the uptake mechanism in isolated rat Kupffer cells. The equilibrium binding of fractionated [(3)H]heparin to Kupffer cells was concentration-dependent with the dissociation constant of 5.7 nM and the maximum binding capacity of 1.5 pmol/10(6) cells. Several ligands of scavenger receptors inhibited the binding of fractionated [(3)H]heparin to Kupffer cells competitively and also the internalization of heparin, suggesting the involvement of scavenger receptors in the uptake of fractionated [(3)H]heparin. Fractionated [(3)H]heparin was also suggested to be internalized according to first order kinetics with the apparent internalization rate constant of 0.010 min (-1). Lowering temperature from 37 to 4 degrees C reduced the fraction internalized from 33% to 6% without affecting the total association, while the fraction internalized at 25 degrees C was comparable with that at 37 degrees C. Metabolic inhibitors (2,4-dinitrophenol and rotenone), an inhibitor of receptor-mediated and adsorptive endocytosis of polypeptides (phenylarsine oxide) and phagocytosis inhibitors (cytochalasine B and colchicine) did not inhibit the internalization of fractionated [3(H)]heparin. As known inhibitors of receptor-mediated and adsorptive endocytosis of polypeptides and phagocytosis did not affect the uptake of fractionated heparin, the scavenger receptor-mediated uptake is suggested to be ATP-independent and different from receptor-mediated and adsorptive endocytosis of polypeptides and phagocytosis, although for temperature dependency it showed the typical characteristics of receptor-mediated endocytosis.

Macromolecule-macromolecule interaction in drug distribution. IV. Molecular weight dependency in the interaction of fractionated [3H]heparin with plasma proteins.

Molecular weight dependency in the interaction of fractionated [3H]heparin (FH) with plasma proteins was evaluated by determining the protein binding of low molecular weight fractionated [3H]heparin (LMWFH: 7000 Da) and high molecular weight fractionated [3H]heparin (HMWFH: 16000 Da) by ultrafiltration and the effects of plasma proteins on the uptake in rat hepatocytes in primary culture. The unbound fractions of LMWFH were 0.5 and 0.8 in the presence of alpha-globulin and albumin, respectively, and were about 10 times larger than those of HMWFH, 0.04 and 0.1, suggesting a reduction in binding with a decrease in molecular weight. However, while the uptake of LMWFH was reduced by these proteins by the extents similar to bound fractions of LMWFH, the uptake of HMWFH was reduced by extents far smaller than bound fractions and comparable with those for LMWFH. Thus, it seemed that, while only unbound LMWFH is available for uptake, HMWFH bound to proteins is to some extent available for uptake (protein-mediated transport). The protein-mediated transport of heparin seemed to reduce with a decrease in molecular weight. It was also shown that the extended uptake of LMWFH was smaller than that of HMWFH not only in the absence of proteins but also in the presence of alpha-globulin, the major binding protein. The lower uptake of LMWFH is consistent with in vivo suggestion of lower hepatic accumulation.

High-affinity binding of melatonin by human circulating T lymphocytes (CD4+).

This paper shows the presence of high-affinity binding sites for melatonin in human circulating T lymphocytes, but not in B lymphocytes. The binding of melatonin to T cells was dependent on time, stable, reversible, saturable, specific, and inversely correlated to the production of melatonin, expressed as the nocturnal 12 h production of its urinary metabolite 6-sulfatoxymelatonin. The affinity of these binding sites (Kd = 0.27 nM) suggests that they may recognize the physiological concentrations of melatonin in serum. Moreover, among the lymphocyte subpopulations studied, binding of melatonin was mostly found in CD4+ cells rather than in CD8+ cells. Results suggest that CD4+ cells may be the target of melatonin among the human circulating lymphocytes.

Uptake of fractionated 3H-heparin by isolated rat Kupffer cells.

PURPOSE AND METHODS: The uptake of fractionated 3H-heparin by isolated rat Kupffer cells was examined to determine the uptake mechanism. RESULTS: The association of fractionated 3H-heparin was concentration-dependent with a dissociation constant of 3.4 nM and a maximum association capacity of 1.3 pmol/10(6) cells, suggesting the involvement of a specialized mechanism. Although 2,4-dinitrophenol inhibited neither the association nor internalization of fractionated 3H-heparin, lowering the temperature from 37 degrees C to 4 degrees C reduced the internalization of fractionated 3H-heparin by 70% without affecting the association. CONCLUSIONS: It is suggested that the uptake mechanism may differ from receptor-mediated endocytosis of polypeptides and be mediated by scavenger receptors, because organic anions, and several ligands of scavenger receptors, as well as several heparin analogs, inhibit the binding of fractionated 3H-heparin to Kupffer cells, while phenylarsine oxide, which is known to inhibit the receptor-mediated or absorptive endocytosis of polypeptides, inhibits neither the association nor internalization of fractionated 3H-heparin.