Influence of melatonin and serotonin on the number of rat pineal “synaptic” ribbons and spherules in vitro

Summary Previous studies have shown that the synaptic ribbons (SR) and spherules (SS) of the mammalian pineal gland may respond differently under physiological and various experimental conditions. The aim of the present study was to gain insight into the mechanisms that may be responsible for the numerical changes of these organelles during a 24-h cycle. As the possibility exists that the structures are influenced by substances synthesized within the pinealocyte, rat pineal glands were cultured with and without added melatonin or serotonin, using an experimental protocol such that the addition of melatonin and serotonin mimicks the circadian changes of the respective substances within the pineal. The tissue was processed for electron microscopy and the numbers of SR and SS were counted in a unit area of pineal tissue. The results obtained indicate that melatonin added to the incubation medium increases the number of SR in the first half of the night; serotonin decreases SR numbers in the morning. SS numbers, by contrast, decrease following melatonin administration in the afternoon, and increase in the morning following serotonin administration. It thus appears that the numbers of SR and SS are influenced by melatonin and serotonin and that the two structures are regulated by differential, but nevertheless biochemically closely related mechanisms.Financial support of the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm Neuroendokrinologie, Vo 135/8-4), the Polish Academy of Sciences (Research Program 10.4.04.6), and the Freunde der Universität Mainz e.V. is gratefully acknowledged.On leave from Department of Anatomy, University Medical School, Pécs, Hungary     

Isolation and long-term serial cultivation of endothelial cells from the microvessels of the adult human dermis

A method to isolate and maintain microvascular endothelial cells from the cutaneous vessels of adult human skin in long-term culture has been developed. Endothelial cells lining the microvessels of the papillary dermis are released from surrounding tissue during a brief trypsin incubation (0.3% trypsin, 1% EDTA). Cells are plated onto a fibronectin substrate and maintained in Leibovitz (L15) culture medium containing pooled human serum (50%) and antibiotics. Proliferation is dependent upon the presence of several additional growth factors, cholera enterotoxin (1 X 10(-9) M), isobutyl methylxanthine (3.3 X 10(-5) M), and medium conditioned by explant culture of the mouse EHS sarcoma. Using this supplemented medium, cells proliferate readily and can be cultivated serially for more than 6 passages (3 months in vitro). These cells retain their characteristic endothelial cell morphology, stain positively for Factor VIII antigen, and contain Weibel-Palade bodies.     

Protoveratrines A and B increase sleep apnea index in Sprague-Dawley rats

Trbovic, Sinisa M., Miodrag Radulovacki, and David W. Carley. Protoveratrines A and B increase sleep apneaindex in Sprague-Dawley rats. J. Appl.Physiol. 83(5): 1602-1606, 1997.The action ofprotovertarines A and B, which stimulate carotid sinus baroreceptorsand vagal sensory endings in the heart as well as pulmonary bed, wereassessed on spontaneous and postsigh central sleep apneas in freelymoving Sprague-Dawley rats. During the 6-h recording period, animalswere simultaneously monitored for sleep by using electroencephalogramand electromyogram recordings, for respiration by single-chamberplethysmography, and for blood pressure and heart period by usingradiotelemetry. After administration of 0.2, 0.5, or 1 mg/kg sc ofprotoveratrines, cardiopulmonary changes lasting at least 6 h wereobserved in all three behavioral states [heart period increasedup to 23% in wakefulness, 21% in non-rapid-eye-movement (non-REM)sleep, and 20% in REM sleep; P < 0.005 for each]. At the same time, there was a substantial increase in the number of spontaneous (375% increase;P = 0.04) and postsigh (268%increase, P = 0.0002) apneas. Minuteventilation decreased by up to 24% in wakefulness, 25% in non-REM,and 35% in REM sleep (P < 0.05 foreach). We conclude that pharmacological stimulation of baroreflexespromotes apnea expression in the sleeping rat.

     

Growth characteristics of human esophageal epithelial cells in primary explant and serial culture

Summary Growth characteristics of human esophageal epithelial cells have been determined in primary explant and serial culture. Normal human esophagus was obtained from donor patients in a heart/lung transplantation program; tissue obtained at autopsy (6 to 22 h after death) was not viable. When mucosal specimens (1.5 mm²) were explanted on a plastic surface and attached with a plasma clot, 35% of explants detached from the surface within 48 h. The addition of epsilon amino caproic acid (EACA) to the culture medium increased explant attachment of 93% (P<0.001). Outgrowth kinetics were similar in both the presence and absence of EACA. No advantage of human serum over nonhuman sera was observed in primary culture. Esophageal epithelium could be frozen in 10% dimethyl sulfoxide without affecting growth kinetics. Addition of dexamethasone (DEX) significantly altered esophageal cell morphology in primary culture and increased viability on serial culture. Studies of pH revealed an optimum at pH 7.4 with significantly decreased growth occuring at 6.8 and no growth at 6.2. Esophageal cells in primary explant cultures could be released by trypsin and passaged two additional times with an eightfould increase in total number. An increased rate of attachment and multiplication was observed for cells plated on a collagen substrate compared to platic. The addition of EACA and DEX to the culture media and the subculture on a collagen substrate provide a method for the isolation and serial cultivation of human esophageal cells from biopsy-sized specimens of normal esophageal epithelium. Supported in part by Grant AM—14121 of the United States Public Health Service. A preliminary report of this work appeared in Clin. Res. 30: 93A; 1982.     

Dopamine increases cyclic AMP concentration in the rat spleen lymphocytes in vitro

The effect of dopamine on the cyclic AMP concentration in the rat spleen lymphocytes has been investigated $\text{in}\text{vitro}$. It has been shown that dopamine in concentration above 10^?6M induces a significant increase of cyclic AMP level. The maximal stimulatory effect was observed after 10 minutes of the lymphocytes incubation with dopamine. These data suggest that the dopamine receptor in lymphocyte belongs to D-1 category.     

Limitations in the use of [3H]thymidine incorporation into DNA as an indicator of epidermal keratinocyte proliferation in vitro

The validity of using the incorporation of [3H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [3H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [3H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [14C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [3H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

Growth characteristics of human epidermal kerationcytes from newborn foreskin in primary and serial cultures

Summary Using gels of acid-soluble, collagen as a culture surface, trypsin-released keartinocytes from 0.1-mm, split-thickness sections of newborn foreskin may be plated with high efficiency and subcultured at a 1∶5 split a 2- to 3-week intervals for three subpassages. When plated at a density of 3.2×10⁴ cells per cm², keratinocytes attach to the gel with an efficiency of over 70%; after a lag phase of 3 days, the cells multiply exponentially with a doubling time of 60 hr. Cultures reach a growth-plateau phase at a density of 47.7×10⁴ cells per cm². Both hydrocortisone and epidermal growth factor (EGF) stimulate slightly the growth of primary cultures; both factors are required for proliferation of the 2nd and further passage of keratinocytes. As the cultures reach, confluence multilayers, of stratified cells are formed and cells of squamous morphology are spontaneously released from the surface. When the released cells and the attached cells are pulsed with [³H]-histidine and [¹⁴C]-leucine, a higher ratio of histidine to leucine is observed in the released cells indicating the biochemical onset of maturation. Orange G-Aniline Blue staining of the released cells show some of the cells to be completely keratinized. Fibrous proteins extracted from the cultured cells and analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis display the characteristic stratum corneum proteins of 60,000 and 66,000 daltons. Supported in part by Grants AM 14121 and AM 19595 of the U.S. Public Health Service.     

Human Ikaros function in activated T cells is regulated by coordinated expression of its largest isoforms

The Ikaros gene is alternately spliced to generate multiple zinc finger proteins involved in gene regulation and chromatin remodeling. Whereas murine studies have provided important information regarding the role of Ikaros in the mouse, little is known of Ikaros function in human. We report functional analyses of the two largest human Ikaros (hIK) isoforms, hIK-VI and hIK-H, in T cells. Abundant expression of hIK-H, the largest described isoform, is restricted to human hematopoietic cells. We find that the DNA binding affinity of hIK-H differs from that of hIK-VI. Co-expression of hIk-H with hIk-VI alters the ability of Ikaros complexes to bind DNA motifs found in pericentromeric heterochromatin (PC-HC). In the nucleus, hIK-VI is localized solely in PC-HC, whereas the hIK-H protein exhibits dual centromeric and non-centromeric localization. Mutational analysis defined the amino acids responsible for the distinct DNA binding ability of hIK-H, as well as the sequence required for the specific subcellular localization of this isoform. In proliferating cells, the binding of hIK-H to the upstream regulatory region of known Ikaros target genes correlates with their positive regulation by Ikaros. Results suggest that expression of hIK-H protein restricts affinity of Ikaros protein complexes toward specific PC-HC repeats. We propose a model, whereby the binding of hIK-H-deficient Ikaros complexes to the regulatory sequence of target genes would recruit these genes to the restrictive pericentromeric compartment, resulting in their repression. The presence of hIK-H in the Ikaros complex would alter its affinity for PC-HC, leading to chromatin remodeling and activation of target genes.     

Food For Thought: Short-Term Fasting Upregulates Glucose Transporters in Neurons and Endothelial Cells,But Not in Astrocytes

Neurochemical Research - Our group previously reported that 6-h fasting increased both insulin II mRNA expression and insulin level in rat hypothalamus. Given that insulin effects on central...