Culturable and nonculturable bacterial symbionts in the toxic benthic dinoflagellate Ostreopsis lenticularis.

The toxic benthic dinoflagellate Ostreopsis lenticularis hosts a variety of symbiont bacterial flora. Laboratory cultured Ostreopsis clones require the presence of symbiotic Pseudomonas/Alteromonas bacterial strains for growth and toxicity development. Three culturable bacterial strains associated with Ostreopsis were identified as Pseudomonas/Alteromonas strain 1, Pseudomonas/Alteromonas strain 2 and Acinetobacter. Denaturing gradient gel electrophoresis (DGGE) analyses of extracted Ostreopsis associated bacterial DNAs indicated that there were three culturable and four non-culturable associated bacterial strains. The results presented here are the first report of the presence of unculturable bacterial symbionts in a toxic benthic dinoflagellate. Ostreopsis lost toxicity when exposed to elevated temperatures in the field and laboratory culture and subsequently recovered toxicity at reduced temperatures. Ostreopsis associated culturable Pseudomonas/Alteromonas bacterial strains were significantly reduced in dinoflagellate cultures exposed to elevated temperatures. The decreased toxicity of O. lenticularis exposed to elevated temperatures and their subsequent recovery of toxicity in periods of reduced thermal stress may have resulted from the effects of elevated temperature on the spectrum of culturable and unculturable bacterial species interacting with their Ostreopsis host.     

Exercise test in patients with permanent pacemakers

From June 1988 to June 1990 we studied fifty patients who had implantation of a pacemaker. (31 females and 19 males). All of them underwent stress test with Bruce's protocol. Patients were divided in two groups; pacemaker-independent (PI) and pacemaker-dependent (PD). Over 50% of the patients inhibited the pacemaker with their own rhythm, most of them had sinus dysfunction. Complete A-V block was predominant in PD. The group of PI achieved more mets and had more oxygen consumption. Blood pressure response was similar in both groups.     

Neurodegeneration in the Central Nervous System of apoE-Deficient Mice

Apolipoprotein E (apoE) is involved in the development and regeneration of the central nervous system (CNS). ApoE may also be necessary to maintain the integrity of the synapto-dendritic complexity. We analyzed the synaptic alterations in the CNS of apoE-deficient (knockout) mice during the aging process. In apoE-deficient homozygous mice, there was an age-dependent 15 to 40% loss of synaptophysin-immunoreactive nerve terminals and microtubule-associated protein 2-immunoreactive dendrites in the neocortex and hippocampus, when compared to controls. Dendritic alterations were observed as early as 4 months of age. Ultrastructural analysis revealed extensive dendritic vacuolization and disruption of the endomembrane system and cytoskeleton in apoE-deficient homozygous mice. Further immunocytochemical studies of the neuronal cytoskeleton showed that in apoE-deficient mice there was a decrease in the immunoreactivity of α and β tubulin (but not kinesin) in the cell bodies and processes. These results support the contention that apoE might play an important role in maintaining the stability of the synapto-dendritic apparatus and that altered or deficient functioning of this molecule could underlie the synaptic and cytoskeletal alterations in Alzheimer's disease.     

Synthesis of apolar ecdysone esters by ovaries of the cockroach Periplaneta americana

Ecdysteroid levels detected by RIA in extracts of mature ovaries from Periplaneta americana increased approximately fourfold (53 +/- 10 to 184 +/- 38 ng/g; +/- SEM, n = 3) on treatment with Helix pomatia "sulphatase" enzymes. HPLC analysis showed that this increase in immunoreactivity resulted from the hydrolysis of six apolar compounds that cochromatographed with the ecdysteroid esters previously shown to be present in newly laid oothecae (A1, A2, A3, A4, A5, and A6; A. J. Slinger, L. N. Dinan, and R. E. Isaac (1986). Insect Biochem. 16 (i), 115-119). Intact ovaries cultured in saline were able to take up [3H]ecdysone from the medium and synthesize ecdysone esters, most of which cochromatographed with immunoreactive peaks from ovaries and oothecae. Crude homogenates and membranes prepared from mature ovaries were also able to esterify ecdysone in vitro. The enzyme activity associated with a high-speed pellet was greatly enhanced by the addition of coenzyme A fatty acyl esters, each reaction resulting in the synthesis of a single major metabolite. The three esters formed on incubating ecdysone with coenzyme A-palmitate, -lineate, and -oleate could be characterized by their retention times on HPLC which were identical to compounds A2, A5, and A6, respectively. These compounds were the three quantitatively important immunoreactive esters found in ovaries and newly laid oothecae. The data presented indicates that ovaries can esterify ecdysone with palmitic, linoleic, and oleic acids and that these apolar derivatives are transferred to the egg. The esters appear to be different from the ecdysone 22-fatty acyl esters that have been isolated from ticks and other insects.     

The topologic and chronologic patterns of hematopoietic cell seeding in host femoral bone marrow after transplantation.

The early stages of homing, seeding, and engraftment of hematopoietic stem and progenitor cells are poorly characterized. We have developed an optical technique that allows in vivo tracking of transplanted, fluorescent-tagged cells in the host femurs. In this study we used fluorescence microscopy to monitor the topologic and chronologic patterns of hematopoietic cell seeding in the femoral bone marrow (BM) of mice. PKH-labeled cells homed to the femur within minutes after injection into a peripheral vein. Most cells drifted within the marrow space and gradually seeded in clusters close to the endosteal surface of the epiphyseal cortex. Three days after transplantation 85% to 94% (14%) of PKH-labeled cells in the femoral marrow were located within 100 microm of the epiphyseal bone surface (P <.001 versus the more central cells), whereas labeled cells were absent in the femoral diaphysis. Primary seeding of juxtaendosteal, epiphyseal marrow occurred independently of recipient conditioning (myeloablated and nonconditioned hosts), donor-recipient antigen disparity, or the phenotype of the injected cells (whole BM and lineage-negative cells) and was consistently observed in secondary recipients of BM-homed cells. Seeding in regions close to the epiphyseal bone was also observed in freshly excised femurs perfused ex vivo and in femurs assessed without prior placement of optical windows, indicating that the site of primary seeding was not affected by surgical placement of optical windows. Four to 5 days after transplantation, cellular clusters appeared in the more central regions of the epiphyses and in the diaphyses. Centrally located cells showed decreased PKH fluorescence, suggesting that they were progeny of the seeding cells, and brightly fluorescent cells (quiescent first-generation seeding cells) were observed close to the bone surface for as long as 24 days after transplantation. These data indicate that the periphery of the femoral marrow hosts primary seeding and that quiescent cells continue to reside in the periphery for weeks and do not divide. The site of proliferation of transplanted cells is the center of the marrow space.     

Tumor Necrosis Factor Alpha Enhances Antifungal Activities of Polymorphonuclear and Mononuclear Phagocytes against Aspergillus fumigatus

Invasive aspergillosis is a serious complication in immunocompromised patients. The effects of recombinant human tumor necrosis factor alpha (TNF-α) on antifungal activities of human neutrophils (polymorphonuclear leukocytes [PMNs]), human monocytes (MNCs), and rabbit pulmonary alveolar macrophages (PAMs) against Aspergillus fumigatus were studied. The percentage of PMN-induced hyphal damage was increased after 30 min of incubation of PMNs with 0.1 ng of TNF-α per ml at 37°C (P = 0.043). At 0.1 to 10 ng/ml, TNF-α also increased superoxide anion (O₂⁻) produced by PMNs in response to phorbol myristate acetate, N-formylmethionyl leucyl phenylalanine, and unopsonized hyphae (P < 0.01) but did not exert any effect on PMN phagocytosis of conidia in the presence of serum. By comparison, TNF-α induced only a slight increase in O₂⁻ production by MNCs in response to phorbol myristate acetate (P = 0.05) and no concomitant increase in the percentage of MNC-induced hyphal damage. Incubation of MNCs with TNF-α at 0.001 to 10 ng/ml for 2 days had no effect on phagocytosis or conidiocidal activity. By contrast, incubation of PAMs with TNF-α at 0.1 to 10 ng/ml for 2 days increased phagocytosis of conidia (P = 0.03). Thus, TNF-α augments the capacity of PMNs to damage Aspergillus hyphae, possibly through enhanced oxidative mechanisms, and increases PAM phagocytic activity against conidia. As such, TNF-α may have an important role in host defense against aspergillosis, and neutralization of its activity may be complicated by increased susceptibility to aspergillosis.     

Androgenic control of porphyrin in the harderian glands of the male syrian hamster is modulated by the photoperiod,which suggests that the sexual differences in porphyrin concentrations in this gland are important functionally

Background: The porphyrin concentrations of the Harderian glands of Syrian hamsters show marked sexual differences, with male levels being much lower than those of females. Porphyrinogenesis is inhibited by androgens, so orchidectomy leads to elevated male porphyrin concentrations; however, a number of other procedures (some of which also lower androgen levels) prevent this. We studied the effects of short-day photoperiods and melatonin on Harderian porphyrin concentrations. Methods: Intact, castrated, or pinealectomized hamsters of both sexes were exposed to long-day or short-day photoperiods. Intact or castrated hamsters were given melatonin injections in the morning or the afternoon, or were given beeswax pellets containing melatonin. After a variable period, Harderian glands were dissected and porphyrins were measured. Results: Prolonged short-day exposure (13 weeks) led to increased Harderian porphyrin concentrations and this rise was prevented by pinealectomy. The rise in Harderian porphyrins following short-day exposure was small, compared with that following castration. Short-day photoperiods also prevented the rise in porphyrin levels associated with castration and this effect was prevented by removal of the pineal. Melatonin injections, whether given in the morning or in the afternoon, had no effect on Harderian porphyrin concentration of castrated male hamsters. Continuous release melatonin pellets reduced the postcastrational rise in porphyrin levels in one experiment, while having no effect in another. In female hamster, neither short photoperiods nor melatonin pellets influenced Harderian porphyrin concentrations. Conclusions: These results suggested that a factor from the pineal gland helps maintain the low levels of porphyrin which are characteristic of male Harderian glands, despite the decrease in androgen levels which typically results from exposure to short days. Morning and afternoon injections of melatonin and continuous release melatonin pellets failed to resolve the question of whether this pineal factor is melatonin. Our results demonstrated that low male and high female porphyrin levels are maintained in Syrian hamsters, despite seasonal variations in the hormonal milieu, suggesting that these sexual differences are important for the (still unestablished) function of the Harderian glands in this species. © 1994 Wiley-Liss, Inc.     

Esophagopleural Fistula: A Complication of Chest Intubation for Empyema

This report documents the first recorded patient in the recent literature with an esophageal perforation and an esophagopleural fistula following chest intubation for empyema. It was treated successfully by conservative method with feeding gastrostomy. It is important to realize that tube thoracostomy drainage is not an innocuous procedure and to be alert to this complication, especially in the presence of empyema.     

Polyl-histidine

Poly-l-histidine (PHSTD) of molecular weight 26,000 induced the generation of large amounts of Superoxide (O ₂ ^– ) and hydrogen peroxide (H₂O₂) in human neutrophils (PMNs). Despite its low solubility at neutral pH, PHSTD was bound very rapidly to the PMN surfaces. Maximal generation of O ₂ ^– took place with 4–5 ×10^–6 M of PHSTD, starting after a lag of about 25 sec and proceeding for 15–17 min at a rate of 150 nmol/10⁷ PMNs/min, suggesting that this polycation is one of the most potent stimulators of O ₂ ^– generation known, PHSTD was found to be non-toxic for PMNs even at millimolar concentrations. Generation of O ₂ ^– by PHSTD depended on extracellular calcium; it was inhibited by calcium channel blockers and by trifluoperazine, and it triggered a sharp rise in intracellular calcium as determined by the Quin 2 fluorescence technique. The generation of both O ₂ ^– and H₂O₂ by PHSTD was partially inhibited by cytochalasin B or (CYB, CYE). On the other hand, CYB markedly enhanced the generation of both O ₂ ^– and H₂O₂ following stimulation of PMNs either by PHSTD, polyarginine, histone, or by antibody-opsonized group A streptococci. Electron microscopic analysis and NBT reduction tests revealed that both PHSTD and PHSTD-opsonized streptococci were avidly phagocytosed by PMNs. Since CYB totally inhibited internalization of both PHSTD and the PHSTD-opsonized streptococci, it was suggested that these agents stimulated oxygen radical generation mainly on the leukocyte surfaces. Complexes (CX) formed between PHSTD and polyanethole sulfonate (a strong polyanion) or between histone and the polyanion mimicked immune CX in their ability to trigger the generation of large amounts of O ₂ ^– which were inhibited by CYB. Generation of O ₂ ^– and chemiluminescence either by PHSTD or by PHSTD-opsonized streptococci were markedly inhibited by poly-l-glutamate, suggesting that PHSTD acted as a cationic agent which interacted via electrostatic forces with some negatively charged sites in the leukocyte membrane. Generation of H₂O₂ by PHSTD was also markedly inhibited by deoxyglucose, KCN, DASA, as well as by the lipoxygenase inhibitors nordihydroguaiaretic acid, phenidone, and propylgallate. On the other hand, cyclooxygenase inhibitors such as aspirin, indomethacin, and piroxicam were inactive, suggesting that arachidonic acid metabolism via lipoxygenase pathway might have been involved in the activation by PHSTD of the NADPH oxidase in PMNs. PHSTD may mimic the effects of antibodies both as an opsonin and as a potent stimulator of the respiratory burst in PMNs and may thus serve as a model for further study of leukocyte-bacteria interactions in infectious and inflammatory sites and of the pathogenicity of immune complexes.Supported by a research grant from Dr. S. M. Robbins of Cleveland, Ohio.