Melatonin Reduces Apoptosis Induced by Calcium Signaling in Human Leukocytes: Evidence for the Involvement of Mitochondria and Bax Activation

We have evaluated the effect of melatonin on apoptosis evoked by increases in [Ca²⁺] _c in human leukocytes. Our results show that treatment of neutrophils with the calcium mobilizing agonist FMLP or the specific inhibitor of calcium reuptake thapsigargin induced a transient increase in [Ca²⁺] _c . Our results also show that FMLP and thapsigargin increased caspase-9 and -3 activities and the active forms of both caspases. The effect of FMLP and thapsigargin on caspase activation was time-dependent. Similar results were obtained when lymphocytes were stimulated with thapsigargin. This stimulatory effect was accompanied by induction of mPTP, activation of the proapoptotic protein Bax and release of cytochrome c. However, when leukocytes were pretreated with melatonin, all of the apoptotic features indicated above were significantly reversed. Our results suggest that melatonin reduces caspase-9 and -3 activities induced by increases in [Ca²⁺] _c in human leukocytes, which are produced through the inhibition of both mPTP and Bax activation.     

Sterol efflux to apolipoprotein A-I originates from caveolin-rich microdomains and potentiates PDGF-dependent protein kinase activity

The kinetics of sterol efflux from human aortic smooth muscle cells equilibrated with a [(3)H]benzophenone-modified photoactivable free cholesterol analogue ((3)H-FCBP) did not differ significantly from those labeled with free cholesterol ((3)H-FC). Trypsin digestion of caveolin cross-linked by photoactivation of FCBP led to association of radiolabel in a single low molecular weight fraction, indicating relative structural homogeneity of caveolin-bound sterol. These findings were used to investigate the organization of sterols in caveolae and the ability of these domains to transfer sterols to apolipoprotein A-I (apo A-I), the major protein of human plasma high-density lipoproteins (HDL). During long-term (4-5 h) incubation with apo A-I, caveolin-associated (3)H-FC and (3)H-FCBP decreased, in parallel with an increase in apo A-I-associated sterol. Assay of caveolin-associated labeled sterols indicated that caveolae were a major source of sterol lost from the cells during HDL formation. Short-term changes of sterol distribution in caveolae were assayed using platelet-derived growth factor (PDGF). PDGF was without effect on FC efflux in the absence of apo A-I, but when apo A-I was present, PDGF increased FC efflux approximately 3-fold beyond the efflux rate catalyzed by apo A-I alone. At the same time, caveolin-associated FC decreased, and PDGF-dependent protein kinase activity was stimulated. Parallel results were obtained with (3)H-FCBP-equilibrated cells, in which apo A-I potentiated a PDGF-mediated reduction of radiolabel cross-linked to caveolin following photoactivation. These results suggest that sterols within caveolae are mobile and selectively transferred to apo A-I. They also suggest a novel role for sterol efflux in amplifying PDGF-mediated signal transduction.     

Case fatality among infants with congenital malformations by lethality

OBJECTIVE: Infant mortality rates continue to show that congenital anomalies are the leading cause of infant death in the United States. However, studies of factors contributing to increased mortality across different types of congenital anomalies have been limited. The objective of this study was to assess whether the likelihood of infant mortality varied by maternal race and ethnic group while considering the severity of the birth defect. METHODS: A retrospective cohort analysis was conducted using data from Colorado's statewide, population-based birth defects surveillance system (CRCSN). The cohort included infants, born between 1995 and 2000 to Colorado resident mothers, who were diagnosed with major congenital malformations stratified by degree of lethality. Multiple logistic regression was performed for each level of lethality, and included the following potential explanatory variables: maternal race/ethnicity, clinical gestation, birth weight, maternal education level, maternal age, and sex of child. RESULTS: Within the low/very low lethality cohort, maternal race/ethnicity of Black/non-Hispanic was associated with increased risk of infant mortality, OR 2.81 (1.41-5.19), as were low and very low birth weight, OR 2.21 (1.12-4.04) and 19.31 (11.84-31.01), respectively. Maternal race/ethnicity was not a significant risk factor in either high or very high lethality groups; however, the interaction between birth weight and gestational age significantly increased the risk of mortality. CONCLUSIONS: Through the use of statewide, population-based birth defects surveillance data, a disparity in infant mortality has been identified in a specific subset of the population that could be investigated further and targeted for prevention activities.     

Differential response of pineal melatonin levels to light at night in laboratory-raised and wild-captured 13-lined ground squirrels (Spermophilus Tridecemlineatus)

Pineal melatonin levels were compared in laboratory-raised or wild-captured 13-lined ground squirrels (Spermophilus tridecemlineatus) that were either exposed to 10 h of darkness at night or to light which had an irradiance of 400 μW/cm². In laboratory-born squirrels the period of darkness was associated with a gradual rise in pineal melatonin levels with peak values being reached at 0200 h, 6 h after darkness onset. Thereafter, melatonin levels decreased and were back to low daytime levels by 0800 h, 2 h after light onset. The exposure of laboratory-raised animals to an irradiance of 400 μW/cm² during the night totally prevented the nocturnal rise in pineal melatonin levels in these animals. In wild-captured ground squirrels the period of darkness at night was associated with a rapid rise in pineal melatonin such that by 2200 h, 2 h after lights out, peak melatonin values were already attained; additionally, melatonin levels remained high throughout the period of darkness but returned to daytime values by 0800 h. Exposure of wild-captured squirrels to a light irradiance of 400 μW/cm² during the normal dark period was completely incapable of suppressing pineal melatonin levels. The difference in the sensitivity of the pineal gland of laboratory-raised and wild-captured ground squirrels may relate to their previous lighting history.     

Melatonin as an antioxidant: biochemical mechanisms and pathophysiological implications in humans

This brief resume enumerates the multiple actions of melatonin as an antioxidant. This indoleamine is produced in the vertebrate pineal gland, the retina and possibly some other organs. Additionally, however, it is found in invertebrates, bacteria, unicellular organisms as well as in plants, all of which do not have a pineal gland. Melatonin's functions as an antioxidant include: a), direct free radical scavenging, b), stimulation of antioxidative enzymes, c), increasing the efficiency of mitochondrial oxidative phosphorylation and reducing electron leakage (thereby lowering free radical generation), and 3), augmenting the efficiency of other antioxidants. There may be other functions of melatonin, yet undiscovered, which enhance its ability to protect against molecular damage by oxygen and nitrogen-based toxic reactants. Numerous in vitro and in vivo studies have documented the ability of both physiological and pharmacological concentrations to melatonin to protect against free radical destruction. Furthermore, clinical tests utilizing melatonin have proven highly successful; because of the positive outcomes of these studies, melatonin's use in disease states and processes where free radical damage is involved should be increased.     

Inhibition of cerebellar nitric oxide synthase and cyclic GMP production by melatonin via complex formation with calmodulin

Constitutive rat cerebellar nitric oxide synthase (NOS) activity is shown to be inhibited by physiological concentrations of the pineal hormone melatonin. The inhibition was dose-dependent and was coupled to an inhibition of the cyclic GMP production activated by L-arginine. Results also show that calmodulin appears to be involved in this process because its presence in the incubation medium was able to prevent the effect of melatonin on both NOS activity and cyclic GMP production. Moreover, polyacrylamide gel electrophoresis studies suggest that melatonin can interact with calmodulin modifying the binding of the peptide to the synthetic NOS peptide encompassing the calmodulin-binding domain of constitutive NOS from rat cerebellum, the natural mechanism by which calmodulin activates cerebellar NOS. J. Cell. Biochem. 65:430–442. © 1997 Wiley-Liss, Inc.     

Isolation of three separate anaphylatoxins from complement-activated human serum

Summary Recent methodologies used in preparing anaphylatoxins from complement-activated serum are described. Activation of the alternative pathway generates C3a and C5a; however, activation of the classical pathway is required to generate the anaphylatoxin from C4. This article describes an activation scheme that simultaneously generates all three of the anaphylatoxins (e.g., C3a, C4a and C5a) in human serum and outlines a procedure for isolating each as homogeneous products. Purification of intact anaphylatoxins directly from complement-activated serum takes place only if an exopeptidase in serum, known as carboxypeptidase N (SCPN), is properly inhibited. A new series of mercapto derivatives of arginine analogs are introduced as potent and effective inhibitors of SCPN. These inhibitors permit normal complement activation but prevent degradation of the released activation fragments C3a, C4a or C5a.The SCPN inhibitor previously used was 6-aminohexanoic acid (EACA), but it required a 1 M concentration for effective inhibition, the substituted mercapto-guanido compounds prove to be effective in the mM range.     

Attachment and metamorphosis of the cheilo-ctenostome bryozoan bugula neritina (linné)

The structure, attachment and subsequent metamorphosis of larvae of the marine bryozoan Bugula neritina were studied by light and electron microscopy. Two points of larval anatomy are of special significance to proper interpretation of the metamorphosis:

• 1 Two cytologically similar blastemal tissues, each laden with free ribosomes, occur as parts of the apical organ complex. The upper blastema directly contacts the larval surface, forming the non-ciliated rows of the apical organ. The lower blastema is internal and is oral to and contiguous with the upper blastema.
• 2 The epidermal tissues of the larva are joined in the following sequence, beginning at the aboral pole: a. apical organ complex; b. apical-connecting cell; c. infolded pallial sinus epithelium; d. vesicular-connecting cell; e. aboral vesicular epithelium; f. corona; g. oral vesicular epithelium; and i., j., and k. internal sac neck, wall and roof regions.

The initial stages of metamorphosis involve a complex sequence of morphogenetic movements, including:

• 1 eversion of the internal sac, permanently attaching the larva to the substrate;
• 2 inrolling of the aboral vesicular epithelium, corona, oral vesicular and ciliated epithelia, and neck region of the internal sac into the larval interior; concomitantly the pallial sinus epithelium evaginates;
• 3 loss of connection between the invaginated tissues and the surface;
• 4 fusion of the pallial sinus epithelium with the wall region of the internal sac, maintaining the integrity of the body surface;
• 5 retraction of the apical organ complex and invagination of the pallial sinus epithelium with the simultaneous elevation of the internal sac wall region to the aboral pole.

At the conclusion of these events the preancestrular surface is covered by the wall and roof regions of the internal sac. Cells of the wall region form the epidermis of the body wall except for the attachment disc and secrete a cuticular exoskeleton that is secondarily calcified; the attachment disc is formed by the roof region of the internal sac. Internally, the ectodermal upper blastema differentiates into the lophophore and digestive tract of the ancestrular polypide, while the lower blastema forms the lining of the lophophoral coelom and the splanchnic (but not the somatic) lining of the visceral coelom. The visceral somatic peritoneum is formed from cells that may originate from the mesodermally derived pigmented cells of the larva to which they are similar in pigmentation and cytology. Such a composite derivation of a coelomic lining has not been described previously.