Mito-SinCe2 Approach to Analyze Mitochondrial Structure-Function Relationship in Single Cells.

The cross talk between mitochondrial dynamic structure, determined primarily by mitochondrial fission and fusion events, and mitochondrial function of energetics, primarily ATP and ROS production, is widely appreciated. Understanding the mechanistic details of such cross talk between mitochondrial structure and function needs integrated quantitative analyses between mitochondrial dynamics and energetics. Here we describe our recently designed approach of mito-SinCe2 that involves high resolution confocal microscopy of genetically expressed ratiometric fluorescent probes targeted to mitochondria, and its quantitative analyses. Mito-SinCe2 analyses allows for quantitative analyses of mitochondrial structure-function relationship in single cells toward understanding the role of mitochondria and their heterogeneity in various physiological and pathological conditions.

Interplay of mitochondrial fission-fusion with cell cycle regulation: Possible impacts on stem cell and organismal aging.

Declining mitochondrial function and homeostasis is a hallmark of aging. It is appreciated that the role of mitochondria is much more complex than generating reactive oxygen species to cause aging-related tissue damage. More recent literature describes that the ability of mitochondria to undergo fission or fusion events with each other impacts aging processes. A dynamic balance of mitochondrial fission and fusion events is required to sustain critical cellular functions including cell cycle. Specifically, cell cycle regulators modulate molecular activities of the mitochondrial fission (and fusion) machinery towards regulating cell cycle progression. In this review, we discus literature leading to our understanding on how shifts in the dynamic balance of mitochondrial fission and fusion can modulate progression through, exit from, and re-entry to the cell cycle or in undergoing senescence. Importantly, core regulators of mitochondrial fission or fusion are emerging as crucial stem cell regulators. We discuss the implication of such regulation in stem cells in the context of aging, given that aberrations in adult stem cells promote aging. We also propose a few hypotheses that may provide direction for further understanding about the roles of mitochondrial fission-fusion dynamics in aging biology.

Substance P and capsaicin release prostaglandin E2 from rat intrapulmonary bronchi.

We hypothesized that substance P and capsaicin would cause the release of prostaglandin E2 (PGE2) from intrapulmonary bronchi isolated from Sprague-Dawley rats. Substance P (1 microM) caused the release of PGE2, measured with enzyme immunoassay, from the isolated airway segments; PGE2 release was inhibited by the neurokinin (NK)1-receptor antagonist, RP-67580, by inhibition of cyclooxygenase with meclofenamate, and by removal of the epithelium. The release of PGE2 caused by capsaicin (1 microM) was similar in magnitude to that caused by substance P. The capsaicin-induced release of PGE2 was inhibited by desensitization of sensory nerves with capsaicin and by RP-67580, meclofenamate, and epithelial denudation. We conclude that activation of NK1 receptors on epithelium causes release of PGE2, which most likely represents the ultimate mediator of airway smooth muscle relaxation, produced by exogenous neuropeptides and by activation of the sensory nerve inhibitory system. Epithelial damage, such as that seen in asthmatic airways, would disrupt this protective system in the lungs, which could contribute to the development of airway disease.

Sensory nerve-mediated inhibitory responses in airways of F344 rats.

We recently described a sensory nerve inhibitory system that mediates relaxation in the airways of Sprague-Dawley rats. Results of several studies have shown that this system protects the lungs against injury induced by toxic stimuli. Whether a similar inhibitory system exists in the airways of Fischer 344 (F344) rats is unknown. Because this rat strain is used extensively in lung toxicological research, the purpose of this study was to determine whether a sensory nerve inhibitory system exists in intrapulmonary bronchi and tracheae isolated from F344 rats. In intrapulmonary bronchi at resting tone, substance P (1.0 microM) evoked a transient contraction that was inhibited by the 5-HT2A receptor antagonist, ketanserin. Exposing airway segments to compound 48/80 to degranulate mast cells also abolished substance P-induced contractions. Inhibition of cyclooxygenase with meclofenamate augmented markedly the contraction to substance P in the intrapulmonary bronchi. In intrapulmonary bronchi that were contracted with bethanechol, substance P evoked a biphasic response characterized by an increase in tension above that induced by bethanechol followed by relaxation. Incubation of the airways with ketanserin abolished the contractile portion of the response; relaxation responses were augmented after ketanserin. In contracted intrapulmonary bronchi that had been treated with compound 48/80, substance P and capsaicin caused relaxation responses that were inhibited markedly or were nearly abolished by the NK1 receptor antagonist, RP67580, by meclofenamate, and by denuding the epithelium. Capsaicin-induced relaxation responses also were abolished by desensitization of C-fibers with capsaicin. Only ketanserin-sensitive contractile responses were observed in response to substance P in tracheal segments. We conclude that a sensory nerve inhibitory system exists in the intrapulmonary airways of F344 rats. The presence of this inhibitory system in F344 rat airways may play a protective role against lung injury induced by inhaled toxicants.

Antagonism of cholinergic nerve-mediated contractions by the sensory nerve inhibitory system in rat bronchi.

The purpose of this study was to examine the potential functional significance of the sensory nerve inhibitory system in modulating contraction. Tension development in response to electrical field stimulation (EFS) and exogenous acetylcholine was monitored in segments of intrapulmonary bronchi isolated from male Sprague-Dawley rats. Contractile responses to EFS were enhanced by desensitization of sensory nerves with capsaicin, by antagonizing neurokinin NK1 receptors with RP-67580, and by inhibition of cyclooxygenase with meclofenamate. Except for RP-67580, which had a slight inhibitory effect on acetylcholine-induced contractions, these interventions were without effect on contraction to acetylcholine. Incubation of capsaicin-desensitized airway segments with substance P attenuated contractions evoked by a half-maximal frequency of EFS by approximately 92%, whereas contractions elicited by a half-maximal concentration of acetylcholine were not affected. Contractile responses elicited by a lower concentration of acetylcholine were inhibited by approximately 50% by substance P. The inhibitory effect of substance P was blocked by RP-67580, meclofenamate, and epithelial denudation. We conclude that the sensory nerve inhibitory system modulates cholinergic contractions and thus plays a role in the regulation of airway smooth muscle tone.

Sensory nerve- and neuropeptide-mediated relaxation responses in airways of Sprague-Dawley rats.

We examined the role of sensory nerves in mediating nonadrenergic inhibitory responses in airway segments isolated from male Sprague-Dawley rats. In the presence of adrenergic blockade, capsaicin (Cap; 1 microM) elicited marked relaxation responses in isolated bronchi precontracted with bethanechol (Beth). Cap-induced inhibitory responses were unaffected by tetrodotoxin (TTX), were attenuated by incubation of the airway with indomethacin (Indo), phosphoramidon, or RP-67580, but were abolished by previous exposure of the airway to Cap and by denuding the epithelium. Substance P (SP; 1 microM), neurokinins A and B (1 microM), and calcitonin gene-related peptide (0.1 microM) relaxed Beth-contracted airway segments to a similar extent. The SP-induced responses were unaffected by adrenergic blockade or by pretreatment with either TTX, phosphoramidon, or Cap, but were attenuated by RP-67580 and abolished by Indo and by denuding the epithelium. In anesthetized mechanically ventilated rats, Cap (50 and 100 micrograms/kg i.v.) elicited a dose-dependent reversal of the increase in lung resistance induced by an infusion of Beth. The Cap-induced bronchodilation was unaffected by pretreatment with propranolol alone or in combination with hexamethonium. SP (44 nmol/kg iv) also evoked bronchodilatory responses in intact animals, which were unaffected by propranolol and hexamethonium but were abolished by treatment of the animals with Indo. Electrical-field stimulation (EFS) evoked nonadrenergic noncholinergic relaxation responses in contracted airway segments. These EFS-induced inhibitory responses were markedly attenuated by treatment of the airway segment with TTX, Cap, or RP-67580. We conclude that neuropeptides released from Cap-sensitive sensory nerves have potent inhibitory effects in rat airways that are mediated, in part, by activation of neurokonin NK1 receptors on epithelium and subsequent release of an inhibitory prostaglandin(s).

Biochemical profile of African American women during three trimesters of pregnancy and at delivery.

The data presented are the results from a prospective observational study which was conducted to investigate the effects of nutrition and other related factors on the outcome of pregnancy in nulliparous African American women 16-35 years old. Fasting blood samples were collected from the women during the first, second and third trimesters of pregnancy. At delivery, both maternal and cord samples were collected. Biochemical variables such as, serum folate, vitamin B12, ascorbic acid, vitamin E, ferritin, selected minerals as well as complete blood count (CBC) and red cell folate were analyzed in the blood samples. The concentrations of hematocrit, hemoglobin, white blood cells, red blood cells and vitamin B12 were below the reference non-pregnant ranges throughout gestation. Maternal concentrations of folate and vitamin E increased sequentially with increased gestational age. Serum ferritin, during the third trimester, declined to 58% of the first trimester concentration. Maternal levels of ferritin at delivery were one third of the values found in the infant (cord) sample. Cord levels of folate, ascorbic acid and vitamin B12 were higher than the concentrations in the maternal delivery samples. The data suggest that among this group of pregnant women, major physiological changes, such as plasma volume expansion which alters blood chemistry and maternal to fetal transfer of nutrients, were similar to the findings of other investigators. In this population however, the findings for serum and whole blood folate are contrary to those reported by other researchers, and the sequential increase in the maternal concentration of the vitamin during pregnancy could be attributed to the use of vitamin supplements.

Relationships of serum illicit drug concentrations during pregnancy to maternal nutritional status.

Findings reported are for a subset of African American subjects, residing in the urban area of Washington, D. C., who participated in a Program Project designed to study nutrition, other factors, and the outcome of pregnancy. Fasting blood samples, drawn during each trimester of pregnancy and at delivery, were screened for concentrations of cocaine, phencyclidine (PCP) and marijuana. Since substance abusers are expected to consume inadequate diets, these samples were also analyzed for serum folate, vitamin B12, ferritin and ascorbic acid. Data for these biochemical variables were compared for subjects whose serum values for drugs were either above or below the drug screening threshold concentrations established by ADAMHA/NIDA. Pearson's correlations were used to determine relationships between pregnancy outcome variables and maternal serum drug concentrations. Blood samples drawn at delivery showed higher maternal: cord ratios (mean +/- SEM) for marijuana (3.3 +/- 2.2) and PCP (2.9 +/- 1.0) than for cocaine (1.0 +/- 0.2). The subjects whose serum values were above the ADAMHA/NIDA ranges for marijuana, PCP and cocaine had concentrations of folate and ferritin that were significantly less than those of subjects with lower serum drug levels (P < or = 0.05). High maternal serum concentrations of illicit drugs were accompanied by a significant increase in leukocyte count (P < or = 0.05). The level of maternal cocaine during the third trimester was inversely correlated with birthweight (r = -0.29; n = 52; P = 0.038) and head circumference (r = -0.28; n = 52; P = 0.047).(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal low level lead and pregnancy outcomes.

We examined the relationship between the concentrations of blood lead and pregnancy outcomes in a subset of 349 African American women who enrolled in the program project, "Nutrition, Other Factors, and the Outcome of Pregnancy." Vitamin-mineral supplement users had significantly higher serum levels of ascorbic acid and vitamin E. Also, in supplement users, there were significantly lower mean concentrations of maternal blood lead. Inverse correlations were found between maternal levels of lead and the antioxidant vitamins, vitamin E and ascorbic acid. In addition, significant Pearson's correlations were observed between maternal blood lead levels and the following variables: positive correlations with calcium, phosphorus, mean corpuscular volume; inverse correlations with gestational age, Ponderal Index, infant orientation, and hematologic values. In the total subset, the three trimester sample means for maternal blood lead concentrations were not significantly different for mothers of infants who weighed less than 2500 g (low birth weight) and those who were delivered infants who weighed 2500 g or more. Clinically, nutrition may play a role in the reduction of potentially adverse effects from lead during pregnancy, i.e. protection of the fetus against lead toxicity and/or free radical damage through the antioxidant actions of vitamin E and ascorbic acid. Even when maternal blood lead levels are within the so-called "safe" range, maternal/use of a vitamin supplement supplying vitamin E and ascorbic acid during pregnancy may offer protection.

Nutrition, genital tract infection, hematologic values, and premature rupture of membranes among African American Women.

Using a prospective comparative design, African American gravidae with and without genital tract infection were assessed with respect to dietary intakes, serum nutrient values, hematologic values, and pregnancy outcomes. Intakes of ascorbic acid, vitamin A, protein, and iron were the dietary variables while levels of ascorbic acid, protein, albumin, globulin, and ferritin were the variables measured in serum. The hematologic variables included hemoglobin, hematocrit, and red and white blood cell counts. Pregnancy outcome was defined on the basis of premature rupture of the membranes (PROM), and infant birth weight, birth length, gestational age, and head circumference. The sample consisted of 335 nulliparous women who were between 16-35 years of age, 96 of whom had genital tract infection based on laboratory reports. Findings indicated no significant differences between the mean dietary intakes as well as serum values of the infected and non-infected women, and no difference in the incidence of PROM. However, non-infected women had a better mean hematologic profile than the infected gravidae during pregnancy. Also, for the non-infected group, there were significant relationships between head circumference and protein consumption (P = .015) and serum ferritin (P = .05). For the infected women, the relationship between the hemoglobin and hematocrit measurements obtained at the first prenatal visit and infant birth weight, birth length and head circumference were statistically significant.

The vacuolated glycogen-laden leukemic lymphocytes of a T-cell lymphoproliferative disorder.

In this report, we present a case of peripheral T-cell leukemia/lymphoma having mostly small- to medium-sized cells with abundant clear cytoplasmic vacuoles. The presentation at the time of diagnosis was one of leukemia/lymphoma. The phenotype of the leukemic cells of the peripheral blood was T1+, T11+, TQ1+, interleukin-2+, T3-, T4-, and T8-. The cells in the peripheral blood as well as those obtained from lymph node biopsy were strongly periodic acid-Schiff positive; the positivity was diastase sensitive. Electron microscopy confirmed the presence of glycogen in the cytoplasmic vacuoles. Serologic tests were negative for human T-cell lymphotropic virus type 1 antibody. The data presented in this article support the existence of a vacuolated variant of peripheral T-cell leukemia/lymphoma and further expand the morphological spectrum of T-cell lymphoproliferative disorders.

Scanning electron microscopy of dysplastic Barrett's epithelium.

Twenty-six patients with Barrett's esophagus (BE) were followed prospectively by endoscopic examination and biopsy. Two biopsies were taken from each of 4 areas of BE. One was processed for light microscopy (LM) and one for scanning electron microscopy (SEM). Those in whom dysplastic BE was demonstrated by LM were reexamined at 6-mo intervals, and the others at yearly intervals. One patient had low grade dysplasia (LGD) by LM on entry, and in 2 others, LGD was recognized on the second examination. These changes have persisted in semiannual examinations over 3, 2, and 2 yr, respectively. SEM prints were examined without knowledge of LM findings, and features that might correlate with LGD by LM were sought. SEM findings were similar to those of Zwas et al. (Gastroenterology 90:1932, 1986) in that most glandular cells had surface features unlike either gastric or intestinal cells but unique to BE. In the patient with LGD on entry, there was an aggregate of very large cells covered by short microvilli with bald patches. In the other patients with LGD, there was more variation in size and shape of cells than in nondysplastic cases.

Early effects of ionizing radiation on pulmonary endothelial angiotensin-converting enzyme and 5'-nucleotidase, in vivo.

We investigated the early phase of pulmonary endothelial injury in rabbits exposed to a single dose (30 Gy) of ionizing radiation to the chest, by measuring endothelium-bound ectoenzyme activities. Utilizing multiple indicator-dilution techniques, the metabolism of [3H]benzoyl-Phe-Ala-Pro (BPAP) and [14C]5'-AMP by angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT), respectively, was studied during a single transpulmonary passage in conscious, chronically catheterized rabbits. From these data, the apparent kinetic constants Km and Amax were calculated. A significant (p less than 0.05) decrease in the metabolism of trace amounts of BPAP and 5'-AMP was observed at 2, 24, and 48 hr after irradiation. A similar decrease in the apparent first order rate constant (Amax/Km) of ACE was observed at 2 hr, but returned to control levels by 24 and 48 hr after irradiation. Apparent Km values of ACE for BPAP and NCT for 5'-AMP were elevated at 2, 24, and 48 hr post-treatment, whereas Amax (product of enzyme mass and the constant of product formation, kcat) of ACE was elevated at 2 and 24 hr but not at 48 hr, and Amax for NCT was elevated at 2 hr post-treatment only. Significant decreases in mean arterial blood pressure and pulmonary blood flow (Qb) at 2 hr post-treatment, and increases in Qb at 24 and 48 hr post-treatment were also recorded. No changes in endothelial structure were observed 2 hr after irradiation at the light or electron microscope level. We conclude that the early phase of radiation-induced lung injury includes changes in endothelial enzyme function in the absence of structural damage, as reflected in an apparent decrease in affinity of ACE and NCT for their substrates, allowing for the possibility that hemodynamic disturbances or their sequalae could also have contributed to the decrease in enzyme function.

Adherent platelets and surface microthrombi of the human aorta and left coronary artery: a scanning electron microscopy feasibility study.

As part of a feasibility phase of an investigator-initiated multicenter NIH supported study on the Pathobiological Determinants of Atherosclerosis in Youth (PDAY), we report observations on microthrombi and adherent platelets on the intima of the aorta and left anterior descending coronary artery. The long-term objective of this cooperative study is to define more precisely the pathogenesis of atherosclerosis during late childhood and early adulthood and to investigate the influence of selected risk factors known to be associated with clinically manifest disease in later life. Scanning electron microscopy (SEM) was utilized to survey broad areas of arterial intima. Of 109 specimens studied from 52 cases, microthrombi composed of a mixture of aggregated platelets and fibrin and measuring approximately 30-70 micron in size were observed in about 4% [corrected] of the specimens and in about 6% of the cases, while individually adherent platelets were observed in approximately 7% of the specimens and about 10% of the cases. Microthrombi and adherent platelets may be important in atherogenesis by stimulating proliferation of intimal smooth muscle cells through the release of a growth factor from platelets. This feasibility study has shown that SEM is a rapid and effective method for surveying large areas of arterial intima for the study of adherent platelets and microthrombi.

Unusual cytoplasmic inclusion bodies in medullary carcinoma of the thyroid gland.

Medullary carcinoma of the thyroid from a 43-year-old patient showed 4 types of unusual cytoplasmic inclusion bodies ultrastructurally. Type 1 inclusion is composed of rod and doughnut-shaped bodies aggregated in the cytoplasma and not enclosed by a limiting membrane. Typical neurosecretory granules are intermingled with the rod and doughnut-shaped bodies. The doughnut-shaped bodies often have an electron dense core which is similar to the neurosecretory granules. The rod-shaped bodies are bounded by trilaminar unit membrane and have a slightly electron dense matrix. A centriole is often found associated with this type of inclusion. Type 2 inclusion is composed of small secretory granules and clear vesicles. These inclusions can be found either with or without surrounding membrane. Type 3 inclusion is a large osmiophilic body, or secondary lysosome, in which some degenerated organelles are discernible. Type 4 inclusion is a fragmented nucleus with condensation and margination of the chromatin, so-called karyorrhexis. Routine hematoxylin and eosin sections did not show each type of inclusion but semithin sections stained with Paragon suggested 4 different types of inclusions. Type 3 and 4 inclusions represent forms of degeneration and cell death in the tumor cells and probably represent a remote effect of radiation. Type 1 inclusion is not commonly associated with cell death and may represent a unique organellar aberration in these tumor cells. Type 2 inclusion is simply an aggregation of secretory granules and clear vesicles.

Angiolymphoid hyperplasia with eosinophilia: report of a case with penile lesions.

A case of angiolymphoid hyperplasia with eosinophilia of the penis is reported. In addition to the proliferation of swollen endothelial cells with features of histiocytes ("histiocytoid endothelial cells"), the lesions also showed a remarkable proliferation of pericytes both in relation to formed vascular channels and away from them. The authors consider that the inflammatory component is associated with even the earliest stages in the development of the lesions and that it not be dismissed as secondary.