Uptake and metabolism of [3H]retinoic acid delivered to human foreskin keratinocytes either bound to serum albumin or added directly to the culture medium

Retinoic acid (RA), a potent modulator of cell proliferation and differentiation is present in plasma bound to serum albumin. The biologic significance or source of plasma RA is not clear. Although most cellular RA is believed to be made in situ via the oxidation of retinol, plasma RA could potentially provide target cells with a source of preformed RA. To investigate RA uptake, we have used a model system of human foreskin keratinocytes (HKc) cultured in serum-free media to compare the uptake and metabolism of [3H]RA added directly to the culture medium in ethanol to that delivered bound to bovine serum albumin (BSA). [3H]RA added directly to the culture medium was rapidly taken up by HKc during the first 10 min of incubation (25-35% of the applied RA), no further accumulation occurred between 10 min and 90 min, and then cell-associated radioactivity rapidly decreased to about 3-5% of the applied dose by 12 h. In contrast, when [3H]RA was delivered to HKc bound to BSA, total cell-associated radioactivity reached about 2.5% of the applied dose by 5 min, increased to 3-5% of the applied radioactivity by 1 h, and no further accumulation or loss occurred over the next 23 h. The uptake by HKc of [3H]RA delivered bound to BSA or added directly to the culture medium was not influenced by pre-treatment of the cells for 72 h with unlabeled RA or by excess unlabeled RA added at the time of uptake. Analysis of the cells and media by high-performance liquid chromatography for RA metabolites found that [3H]RA added directly to the medium is rapidly converted by HKc to polar compounds that are subsequently excreted back into the medium. Also, RA added directly to the medium was susceptible to degradation in the absence of cells. In marked contrast, [3H]RA added to the media bound to BSA was much less susceptible to degradation in the absence of cells, and few [3H]RA metabolites were found in the media even after exposure to HKc for 24 h. The binding of RA to albumin clearly protects RA from conversion to polar metabolites, and also provides for a controlled delivery of RA from the aqueous extracellular environment to the cell surface.     

The transfer of retinol from serum retinol-binding protein to cellular retinol-binding protein is mediated by a membrane receptor

The hypothesis that the cellular uptake of retinol involves the specific interaction of a plasma membrane receptor with serum retinol-binding protein (RBP) at the extracellular surface followed by ligand transfer to cytoplasmic cellular retinol-binding protein (CRBP) has been investigated. The experimental system consisted of the [3H]retinol-RBP complex, Escherichia coli-expressed recombinant apo-CRBP containing the 10 amino acid long streptavidin-binding peptide sequence at its C terminus (designated as CRBP-Strep) and permeabilized human placental membranes. [3H]Retinol transfer from RBP to CRBP-Strep was monitored by measuring the radioactivity associated with CRBP-Strep retained by an immobilized streptavidin resin. Using this assay system, we have demonstrated that optimal retinol uptake is achieved with holo-RBP, the membrane receptor and apo-CRBP. The effects are specific: other binding proteins, including beta-lactoglobulin and serum albumin, despite their ability to bind retinol, failed to substitute for either RBP or apo-CRBP. The process is facilitated by membranes containing the native receptor suggesting that this protein is an important component in the transfer mechanism. Taken together, the data suggest that the RBP receptor, through specific interactions with the binding proteins, participates (either directly or via associated proteins) in the mechanism which mediates the transfer of retinol from extracellular RBP to intracellular CRBP.     

Interactions of transthyretin (TTR) and retinol-binding protein (RBP) in the uptake of retinol by primary rat hepatocytes

The mechanism by which cells take up retinol from retinol-binding protein (RBP) and the role of the RBP-transthyretin (TTR) complex remain unclear. Here we report on retinol uptake through the RBP-TTR complex by primary cultured rat hepatocytes (parenchymal cells, PC) and nonparenchymal cells (NPC) following incubation with [3H]retinol-RBP or the [3H]retinol-RBP-TTR complex under several conditions. The cellular accumulation of retinol was time and temperature dependent in both PC and NPC. Analysis by HPLC showed that the incorporated [3H]retinol in NPC was mainly converted to retinyl ester, although in PC it remained mainly as unesterified retinol. However, the amount of retinol taken up from the RBP-TTR complex was nearly twofold greater than that from RBP alone. The uptake of [3H]retinol from protein-bound retinol was inhibited by an excess of either retinol-RBP or retinol-RBP-TTR complex. Moreover, retinol uptake through the RBP-TTR complex was inhibited by an excess of free TTR. From these results we postulate that TTR may take part as a positive regulator in the delivery of RBP-bound retinol from plasma, possibly by a membrane receptor, and that retinol uptake takes place preferentially from the RBP-TTR complex into both PC and NPC. The uptake of [3H]retinol (2 microM) by PC was saturated, whereas uptake by NPC was not. These results indicate that the physiological importance of TTR in retinol delivery may be especially important to vitamin A-storing stellate (Ito) cells in the NPC fraction.     

Interaction of the recombinant human methylpurine-DNA glycosylase (MPG protein) with oligodeoxyribonucleotides containing either hypoxanthine or abasic sites

Methylpurine-DNA glycosylases (MPG proteins, 3-methyladenine-DNA glycosylases) excise numerous damaged bases from DNA during the first step of base excision repair. The damaged bases removed by these proteins include those induced by both alkylating agents and/or oxidizing agents. The intrinsic kinetic parameters (k(cat) and K(m)) for the excision of hypoxanthine by the recombinant human MPG protein from a 39 bp oligodeoxyribonucleotide harboring a unique hypoxanthine were determined. Comparison with other reactions catalyzed by the human MPG protein suggests that the differences in specificity are primarily in product release and not binding. Analysis of MPG protein binding to the 39 bp oligodeoxyribonucleotide revealed that the apparent dissociation constant is of the same order of magnitude as the K(m) and that a 1:1 complex is formed. The MPG protein also forms a strong complex with the product of excision, an abasic site, as well as with a reduced abasic site. DNase I footprinting experiments with the MPG protein on an oligodeoxyribonucleotide with a unique hypoxanthine at a defined position indicate that the protein protects 11 bases on the strand with the hypoxanthine and 12 bases on the complementary strand. Competition experiments with different length, double-stranded, hypoxanthine-containing oligodeoxyribonucleotides show that the footprinted region is relatively small. Despite the small footprint, however, oligodeoxyribonucleotides comprising <15 bp with a hypoxanthine have a 10-fold reduced binding capacity compared with hypoxanthine-containing oligodeoxyribonucleotides >20 bp in length. These results provide a basis for other structural studies of the MPG protein with its targets.     

Effects of dietary protein or amino acids in the perfusion medium on amino acid metabolism in perfused adult rat liver

To elucidate the response of amino acid metabolism in the liver to dietary protein and plasma amino acids, the livers of adult rats fed on diet containing 10% (control) or 3% (low-protein) egg protein for 3 weeks were perfused for 120 min with amino acid-free medium in Experiment 1 or medium containing an amino acid mixture simulating that in plasma in Experiment 2. During perfusion about 40% of the free amino acids were lost from the liver in Exp. 1, and about 30% in Exp. 2. During this period, in Exp. 1 the releases of free amino acids and urea into the medium were 140 mumol and 2.52 mg, respectively, in the control group and 207 mumol and 1.10 mg respectively, in the low-protein group. Thus release was greater than decrease in free amino acids in the liver. Essential amino acids, particularly lysine and branched chain amino acids, were released preferentially. The results suggest that the amount of breakdown of liver protein in the two groups was similar, but that the nitrogen was mainly released as free amino acids in the low-protein group, and as urea in the control group. On the contrary, in Exp. 2 the amount of nitrogen released from the liver was comparable to the decrease in amino acids in the liver, and the releases of urea were also less, being 1.83 mg in the control group and 0.54 mg in low-protein group. The results show that amino acid metabolism in the liver is greatly affected by the nutritional state of the animal and the amino acid content of the perfusion fluid.     

Effect of human uremic serum and urea or creatinine containing serum on organotypic nervous tissue culture. Light microscopy studies

The mandibular glands of Kalotermes were examined in different castes. They show sexual dimorphism in the soldiers and primary reproductives, Moreover, in female soldiers and queens, mandibular gland cells contained numerous crystalline structures of mitochondrial origin. The role of these glands (secretion of saliva or pheromone) is discussed.     

Light-induced photoactivation of hypericin affects the energy metabolism of human glioma cells by inhibiting hexokinase bound to mitochondria

Glucose-dependent energy required for glioma metabolism depends on hexokinase, which is mainly bound to mitochondria. A decrease in intracellular pH leads to a release of hexokinase-binding, which in turn decreases glucose phosphorylation, ATP content, and cell proliferation. Thus, intracellular pH might be a target for therapy of gliomas, and a search for agents able to modulate intracellular pH was initiated. Hypericin, a natural photosensitizer, displays numerous biological activities when exposed to light. Its mechanism and site of action at the cellular level remain unclear, but it probably acts by a type II oxygen-dependent photosensitization mechanism producing singlet oxygen. Hypericin is also able to induce a photogenerated intracellular pH drop, which could constitute an alternative mechanism of hypericin action. In human glioma cells treated for 1 h with 2.5 microg/ml hypericin, light exposure induced a fall in intracellular pH. In these conditions, mitochondria-bound hexokinase was inhibited in a light- and dose-dependent manner, associated with a decreased ATP content, a decrease of mitochondrial transmembrane potential, and a depletion of intracellular glutathione. Hexokinase protein was effectively released from mitochondria, as measured by an ELISA using a specific anti-hexokinase antibody. In addition to decreased glutathione, a response to oxidative stress was confirmed by the concomitant increase in mRNA expression of gamma-glutamyl cysteine synthetase, which catalyzes the rate-limiting step in overall glutathione biosynthesis, and is subject to feedback regulation by glutathione. Hypericin also induced a dose- and light-dependent inhibition of [3H]thymidine uptake and induced apoptosis, as demonstrated by annexin V-FITC binding and cell morphology. This study confirmed the mitochondria as a primary target of photodynamic action. The multifaceted action of hypericin involves the alteration of mitochondria-bound hexokinase, initiating a cascade of events that converge to alter the energy metabolism of glioma cells and their survival. In view of the complex mechanism of action of hypericin, further exploration is warranted in a perspective of its clinical application as a potential phototoxic agent in the treatment of glioma tumors.     

New approaches to the treatment of severe burns: the transplantation of keratinocytes grown in culture

On the own experience and medical practice the authors summarize the peculiarities and advantages of a new method of human skin recovery. Keratinocyte sheets greatly improve the treatment of allogeneic cell cultures' accelerates wound healing of the second degree deep burns. The efficacy of the method can be improved by cultivation and follow-up grafting of keratinocytes on the surface of microcarriers.     

Comparison of the biodistribution and the efficacy of monoclonal antibody 323/A3 labeled with either 131I or 186Re in human ovarian cancer xenografts

The effects of arginine vasopressin (AVP) and oxytocin (OT) upon thyroid-stimulating hormone (TSH), free thyroxine (FT4) and free triiodothyronine (FT3) release were studied in euthyroid rats. Intracerebroventricular (i.c.v.) infusion of AVP in doses of 0.5 ng or 5 ng led to significant increases in plasma levels of TSH as well as FT4 and FT3. The effects of OT injected i.c.v. in similar doses were not consistent (there was no parallel between the changes of respective hormones plasma levels). It may be concluded that vasopressin modulate the pituitary-thyroid system function; AVP is probably a physiological stimulator of TSH and thyroid hormones secretion.     

Influence of serum protein and medium composition on the dynamic adhesiveness of lymphocytes and erythrocytes

Homogenates of phagocytosing polymorphonuclear leukocytes obtained from rabbit peritoneum were incubated with the prostaglandin endoperoxides PGG2 or PGH2. After 2 min at 0degree C, incubation mixtures contained an increased rabbit aorta contracting activity. Ether extracts of incubation mixtures contained a substance which contracted the superfused strips of rabbit aorta and coeliac artery and had a half life which was similar to thromboxane A2. The generation of thromboxane A2-like activity from PG endoperoxides was prevented by boiling the homogenate prior to incubation, or by pretreatment with benzydamine, a drug which blocks thromboxane formation in platelets. Production of thromboxane A2-like material by leukocyte homogenates was compared with platelet microsomal thromboxane synthetase.     

Differentiation-induced enhancement of the ability of cultured human keratinocytes to suppress oxidative stress

Human keratinocytes in culture were harvested at different stages of differentiation. Both the level of antioxidants and the response of cells to oxidative stress were measured as a function of growth and differentiation. As the keratinocyte cultures became confluent and began to differentiate, the cellular levels of glutathione, glutathione peroxidase, glutathione S transferase, and glucose-6-phosphate dehydrogenase increased. This higher level of antioxidants was maintained until the cells began to lose viability. Further, as the keratinocyte cultures began to differentiate, they became more resistant to the toxic effect of cumene hydroperoxide in terms of both of the rate of loss of cell mass and total glutathione and of the rate of decline in the activity of oxidation-sensitive enzymes. To determine how tightly the observed effects are linked to the calcium-dependent aspects of differentiation and to rule out effects related to time in culture, the cells were switched from 1.2 mM Ca++ to 0.03 mM Ca++ to suppress Ca(++)-dependent differentiation. After 4 d, these cells were then treated with 0.5 mM cumene hydroperoxide. The switch to 0.03 mM Ca++ blocked the normal increases in both glutathione peroxidase and glucose-6-phosphate dehydrogenase activities. Further, cells in 0.03 mM Ca++ had reduced resistance to cumene hydroperoxide relative to cells cultured for the same length of time in 1.2 mM Ca++. This indicates that there is a differentiation-associated, Ca(++)-specific increase in both the level of antioxidants and in tolerance to organic hydroperoxides.     

Fragile sites on human chromosomes: demonstration of their dependence on the type of tissue culture medium

The observation of heritable fragile sites on human chromosomes prepared for lymphocyte cultures has been shown to depend on the type of tissue culture medium in which the lymphocytes are grown. The sites are observed at a much greater frequency when medium 199 is used than when RPMI 1640, Ham's F10, Eagle's (basal), and CMRL 1969 are used. One site on the X chromosome is of clinical significance in that it is a marker for X-linked mental retardation.     

Different expression of the alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein in human keratinocytes and fibroblasts

BACKGROUND AND OBJECTIVES: Although partner notification has been a long-standing intervention and prevention strategy for sexually transmitted diseases (STD), variations in partner notification practices across sites have never been documented. GOALS OF THE STUDY: To describe provider-assisted partner notification practices in four STD programs in the United States. STUDY DESIGN: Eleven disease intervention specialists (DIS) in each of three urban sites and seven DIS in one rural site documented their activities and clients for 14 working days using a personal digital assistant. RESULTS: Of 2,506 recorded activity hours across sites, 37.4% of the recorded time was spent on partner notification (PN) activities with 1148 clients. Field visits to locate contacts accounted for the largest proportion of time spent on PN. Overall, PN clients were cases of or were contacts to nonprimary and secondary (P&S) syphilis (39.6%), gonorrhea (25.5%), chlamydia (18.0%), HIV/AIDS (10.4%), and P&S syphilis (6.5%). CONCLUSION: The activities which constitute PN, the diseases for which PN is used, and the time spent on each PN client vary across sites. More research is needed on the determinants of these variations and their association with the ultimate goal of disease prevention.     

Soluble or bound laminin elicit in human neuroblastoma cells short- or long-term potentiation of a K+ inwardly rectifying current: relevance to neuritogenesis

Changes in the resting potential (VREST) and in the underlying ionic conductances were measured by the patch-clamp technique in SH-SY5Y human neuroblastoma cells exposed to substrate-bound or soluble Laminin (bLN; sLN), as compared to integrin-independent substrates (polylysine (PL); bovine serum albumin (BSA)). While PL and BSA were ineffective, both forms of LN caused an early (5-15 min) activation of a peculiar type of Inwardly Rectifying K+ current (IIR) characterised by a voltage-dependent inactivation in the range of membrane potentials around -50/0 mV. IIR was blocked by Cs+ ions and by the antiarrhythmic drug E-4031, a specific inhibitor of the HERG-codified channels. In cells adherent to bLN, IIR potentiation (85%) persisted for 90-120 min and was accompanied by a similar, but transient, increase in the leakage conductance (GL). Successively, the persistence of a high IIR conductance and the decrease of GL progressively bring VREST from -12 to approximately -30 mV in about 120 min. On the other hand, in cells adherent to PL, exposure to sLN produced a similar but not persistent activation of IIR, without any increase in GL: this caused a rapid, transient hyperpolarisation of VREST. The effects of bLN and sLN were mimicked by antibodies raised against the integrin beta 1 subunit, suggesting a specific integrin-mediated mechanism. In fact, when bound to the culture dishes, these antibodies simultaneously activated the IIR and GL, whereas in soluble form they only activated IIR. Cells adherent to bLN emitted neurites, a process impaired by the block of IIR by E-4031 and Cs+. On the whole data suggest that the integrin-mediated activation of IIR plays a crucial role in the commitment to neuritogenesis of neuroblastoma cells, independently on the effects of this activation on VREST.     

Differential effects of human serum and cells on the growth of Plasmodium falciparum adapted to serum-free in vitro culture conditions

A single Plasmodium falciparum isolate was adapted for growth in serum-free culture medium. The parasitemia increased from 0.5% to 20% on day 7 after thawing. The asexual forms of the parasites appeared morphologically normal and pigment formation was comparable with that seen under standard conditions with serum present. Parasites were coincubated in 96-well plates with serum, peripheral blood mononuclear cells (PBMC), and PBMC in the presence of autologous serum from healthy non-immune individuals (n = 12), healthy semi-immune individuals (n = 12), and malaria patients (n = 7). Growth was monitored for six days. The concentration of interleukin-6 and interferon-gamma (IFN-gamma) in supernatants from the continuous cultures were measured by a bioassay and an enzyme-amplified sensitivity immunoassay. The results of this study showed that parasites cultured in serum-free medium in the presence of PBMC develop more rapidly, particularly with cells from malaria patients, compared with parasites cultured alone. The growth of parasites was different if 10% autologous serum was added to the culture. Parasite growth with sera from acutely infected individuals was similar with that with sera from aparasitemic, nonimmune individuals, and both supported significantly higher parasite growth over the six-day culture period compared with sera from the uninfected semi-immune individuals. Production of IFN-gamma by cells from nonimmune individuals and malaria patients was higher when cultures did not contain autologous serum. Nonimmune donor cells produced high amounts of IFN-gamma, but cells from the semi-immune donors produced little of this cytokine. There was no marked inhibition of parasite growth with any combination of serum and cells over six days of culture. A difference between the groups was observed after two days of culture, when growth with cells and serum from the uninfected, semi-immune group was significantly lower than that from the nonimmune group, but this was not subsequently sustained. The results of the study show that continuous cultivation of P. falciparum in serum-free medium provides a novel in vitro model to study mechanisms of the interplay between components of the human immune system and the malarial parasite, in which any possible influence of human serum is removed.     

No association between the serotonin transporter-linked promoter region polymorphism and either schizophrenia or density of the serotonin transporter in human hippocampus

BACKGROUND: Allelic association case-control analysis of a deletion/insertion polymorphism in the serotonin transporter-linked polymorphic region (5-HTTLPR) has suggested associations with unipolar disorder, bipolar disorder, depression, and Alzheimer's disease. Moreover, the heterozygous long form of the 5-HTTLPR has been associated with increased levels of mRNA for the serotonin transporter (5-HTT) and increased serotonin uptake in lymphoblastic cell lines. This study attempts to determine whether there is an association between 5-HTTLPR genotype and schizophrenia or the binding of [3H]paroxetine to the human hippocampal 5-HTT. MATERIALS AND METHODS: DNA from the cerebellum from 58 schizophrenic and 62 control subjects was used to genotype the 5-HTTLPR. In addition, the relationship between 5-HTTLPR genotype and the affinity and density of [3H]paroxetine binding to the hippocampal 5-HTT was assessed. RESULTS: There were no associations between 5-HTTLPR allotype or genotype and/or the parameters of [3H]paroxetine binding to the hippocampal 5-HTT. CONCLUSIONS: Our data suggest that 5-HTTLPR genotype neither confers an increased susceptibility for schizophrenia nor dictates the expression of the 5-HTT in the human hippocampus.     

The crystal structure of the hyperthermophile chromosomal protein Sso7d bound to DNA

BACKGROUND: Whether herd immunity will occur with widespread Haemophilus influenzae type b (Hib) vaccination in developing countries is dependent on whether the vaccines are capable of reducing carriage in these settings. However, few population-based studies of Hib carriage in developing countries exist. METHODS: To study Hib carriage in the Dominican Republic, we collected nasopharyngeal swab specimens from a population-based sample of 983 children 0 to 47 months old in a periurban area of Santo Domingo. RESULTS: Nasopharyngeal swabs of 76 (7.7%) children were positive for Hib. Hib carriage varied by age group with a low of 1.5% among 0 to 5 month olds, a peak of 12.5% in 6 to 11 month olds and prevalence rates of 6.0, 7.9 and 9.8% among 1-, 2- and 3-year-olds, respectively. Hib carriage was 51% lower among currently breast-fed 6 to 11 month olds than among those not currently breast-fed (18.2% vs. 9.0%; P=0.08). CONCLUSIONS: Infants and young children in Santo Domingo have high rates of Hib carriage, characterized by an early peak in carriage that corresponds with the peak of risk for Hib meningitis. The ability of Hib vaccines to diminish carriage to levels that will effectively reduce transmission and lead to herd immunity in this setting needs to be determined.