Long-term melatonin administration protects brain mitochondria from aging

Abstract:  We tested whether chronic melatonin administration in the drinking water would reduce the brain mitochondrial impairment that accompanies aging. Brain mitochondria from male and female senescent prone (SAMP8) mice at 5 and 10 months of age were studied. Mitochondrial oxidative stress was determined by measuring the levels of lipid peroxidation and nitrite, glutathione/glutathione disulfide ratio, and glutathione peroxidase and glutathione reductase activities. Electron transport chain activity and oxidative phosphorylation capability of mitochondria were also determined by measuring the activity of the respiratory chain complexes and the ATP content. The results support a significant age-dependent mitochondrial dysfunction with a diminished efficiency of the electron transport chain and reduced ATP production, accompanied by an increased oxidative/nitrosative stress. Melatonin administration between 1 and 10 months of age completely prevented the mitochondrial impairment, maintaining or even increasing ATP production. There were no major age-dependent differences between males in females, although female mice seemed to be somewhat more sensitive to melatonin treatment than males. Thus, melatonin administration as a single therapy maintained fully functioning brain mitochondria during aging, a finding with important consequences in the pathophysiology of brain aging.     

Novel bromomelatonin derivatives suppress osteoclastic activity and increase osteoblastic activity: implications for the treatment of bone diseases

Abstract:  The teleost scale is a calcified tissue that contains osteoclasts, osteoblasts, and bone matrix, all of which are similar to those found in mammalian membrane bone. Using the goldfish scale, we recently developed a new in vitro assay system and previously demonstrated that melatonin suppressed both osteoclastic and osteoblastic activities in this assay system. In mammals, 2-bromomelatonin possesses a higher affinity for the melatonin receptor than does melatonin. Using a newly developed synthetic method, we synthesized 2-bromomelatonin, 2,4,6-tribromomelatonin and novel bromomelatonin derivatives (1-allyl-2,4,6-tribromomelatonin, 1-propargyl-2,4,6-tribromomelatonin, 1-benzyl-2,4,6-tribromomelatonin, and 2,4,6,7-tetrabromomelatonin) and then examined the effects of these chemicals on osteoclasts and osteoblasts. All bromomelatonin derivatives, as well as melatonin, had an inhibitory action on osteoclasts. In particular, 1-benzyl-2,4,6-tribromomelatonin (benzyl-tribromomelatonin) possessed a stronger activity than melatonin. At an in vitro concentration of 10⁻¹⁰  m , benzyl-tribromomelatonin still suppressed osteoclastic activity after 6 hr of incubation. In reference to osteoblasts, all bromomelatonin derivatives had a stimulatory action, although melatonin inhibited osteoblastic activity. In addition, estrogen receptor mRNA expression (an osteoblastic marker) was increased in benzyl-tribromomelatonin (10⁻⁷  m )-treated scales. Taken together, the present results strongly suggest that these novel melatonin derivatives have significant potential for use as beneficial drug for bone diseases such as osteoporosis.     

Evaluation of immunotoxins containing single-chain ribosome-inactivating proteins and an anti-CD22 monoclonal antibody (OM124): in vitro and in vivo studies

Immunotoxins were prepared with three ribosome-inactivating proteins (RIP), momordin, pokeweed antiviral protein from seeds (PAP-S) and saporin-S6, linked to the anti-CD22 monoclonal antibody OM124. These immunotoxins inhibited protein synthesis by CD22-expressing cell lines Daudi, EHM, BJAB, Raji and BM21 with IC₅₀ (concentration causing 50% inhibition) ranging from < 5 × 10⁻¹⁵ to 7.6 × 10⁻¹¹  M as RIP, and IC₉₀ (concentration causing 90% inhibition) ranging from 5 × 10⁻¹⁴ to 5 × 10⁻⁸  M , with no effect on a CD22-negative HL60 cell line at the highest concentration tested (5 × 10⁻⁸  M ). Apoptosis was induced in sensitive cells. The formation of bone marrow colonies was inhibited by no more than 40% by the immunotoxins at concentrations up to 10⁻⁹  M . Treatment with the immunotoxins, alone or in combination, significantly extended the survival time of mice bearing transplanted Daudi cells. A treatment with cyclophosphamide and OM124/saporin immunotoxin was particularly effective in SCID mice transplanted with a low number of cells (3 × 10⁻⁶), when 60% of the animals remained tumour-free.     

Accumulation of catalytically active PKC-ζ into the nucleus of HL-60 cell line plays a key role in the induction of granulocytic differentiation mediated by all-trans retinoic acid

The effect of differentiating doses of all- trans retinoic acid (ATRA, 10⁻⁶  m ) and vitamin D3 (10⁻⁷  m ) was investigated on the nuclear levels of endogenous ceramide and protein kinase C-ζ (PKC-ζ) catalytic activity in HL-60 myeloid cells. ATRA induced a parallel increase of ceramide and catalytically active PKC-ζ into the nuclear compartment of HL-60 cells (peak at 72 h). On the other hand, vitamin D3 increased the levels of nuclear ceramide and PKC-ζ activity to a lesser extent and with a delayed kinetics compared to ATRA (peak at 96 h).
Pretreatment of HL-60 cells with high pharmacological concentrations of exogenously-added C₂-ceramide (10⁻⁶  m ) completely blocked the ATRA-mediated activation of nuclear PKC-ζ. Exogenous C₂-ceramide (10⁻⁶  m ) also inhibited the granulocytic differentiation induced by ATRA, whereas it did not affect monocytic differentiation mediated by vitamin D3.
Transient transfection experiments performed with a plasmid construct containing a constitutively active mutated form of the PKC-ζ cDNA fused in 3' to a fluorescent tag protein (pEGFP-PKC-ζ) demonstrated that the overexpression of catalytically active PKC-ζ was not accompanied by the appearance of a differentiated morphology. These findings suggest that nuclear PKC-ζ is necessary but not sufficient to induce granulocytic differentiation of HL-60 myeloid malignant cells.     

Visualization of the antioxidative effects of melatonin at the mitochondrial level during oxidative stress-induced apoptosis of rat brain astrocytes

Oxidative stress-induced mitochondrial dysfunction has been shown to play a crucial role in the pathogenesis of a wide range of diseases. Protecting mitochondrial function, therefore, is vital for cells to survive during these disease processes. In this study, we demonstrate that melatonin, a chief secretory product of the pineal gland, readily rescued mitochondria from oxidative stress-induced dysfunction and effectively prevented subsequent apoptotic events and death in rat brain astrocytes (RBA-1). The early protection provided by melatonin in mitochondria of intact living cells was investigated by the application of time-lapse conventional, confocal, and multiphoton fluorescent imaging microscopy coupled with noninvasive mitochondria-targeted fluorescent probes. In particular, we observed that melatonin effectively prevented exogenously applied H2O2-induced mitochondrial swelling in rat brain astrocytes at an early time point (within 10 min) and subsequently reduced apoptotic cell death (150 min later). Other early apoptotic events such as plasma membrane exposure of phosphatidyl serine and the positive YOPRO-1 staining of the early apoptotic nucleus were also prevented by melatonin. A mechanistic study at the mitochondrial level related to the early protection provided by melatonin revealed that the indole molecule significantly reduced mitochondrial reactive oxygen species (ROS) formation induced by H2O2 stress. Melatonin also prevented mitochondrial ROS generation caused by other organic hydroperoxides including tert-butyl hydroperoxide and cumene hydroperoxide. This antioxidative effect of melatonin is more potent than that of vitamin E. Via its ability to reduce mitochondrial ROS generation, melatonin prevented H2O2-induced mitochondrial calcium overload, mitochondrial membrane potential depolarization, and the opening of the mitochondrial permeability transition (MPT) pore. As a result, melatonin blocked MPT-dependent cytochrome c release, the downstream activation of caspase 3, the condensation and karyorrhexis of the nucleus and apoptotic fragmentation of nuclear DNA. Thus, the powerful mitochondrial protection provided by melatonin reinforces its therapeutic potential to combat a variety of oxidative stress-induced mitochondrial dysfunctions as well as mitochondria-mediated apoptosis in various diseases.     

Substitution rate of the hepatitis B virus surface gene

Summary.  Molecular epidemiology of hepatitis B virus (HBV) often relies on the comparison of HBV surface (S) gene sequences, although little is known about the substitution rate of the HBV S-gene. In this study, we compared HBV S-gene sequences in longitudinal sample pairs of 40 untreated, chronically HBV-infected patients, spanning 210 years of cumulative follow-up. The 40 patients included HBV e-antigen positive and negative persons; with HBV DNA levels ranging from 10³ to 10⁹ cps/mL and belonging to HBV genotypes A, B, C, D and E. In the 40 sample pairs, 70 nucleotide changes occurred in the HBV S-gene (0–8 per patient), resulting in an average substitution rate of 5.1 × 10⁻⁴ nucleotide changes/site/year (range: 0–1.3 × 10⁻²). Surprisingly, the number of substitutions was strongly associated with the inverse level of viremia; and only weakly with the duration of follow-up: in 11 highly viremic patients (HBV DNA ≥10⁸ cps/mL), only four substitutions occurred despite a cumulative observation period of 56 years (substitution rate: 1.1 × 10⁻⁴), while in the 10 patients with viremia below 10⁴ cps/mL, 29 substitutions occurred during 30 years of follow-up (substitution rate: 14.6 × 10⁻⁴). We conclude that in chronic hepatitis B virus infection the rate of nucleotide substitution in the HBV S-gene is inversely related to the level of viremia and thus varies widely from person to person; hampering the phylogenetic analysis of possible chains of HBV infection.     

Cytoprotective effects of melatonin on C6 astroglial cells exposed to glutamate excitotoxicity and oxidative stress

Abstract:  To preserve the central nervous system (CNS) function after a traumatic injury, therapeutic agents must be administered to protect neurons as well as glial cells. Cell death in CNS injuries and diseases are attributed to many factors including glutamate toxicity and oxidative stress. We examined whether melatonin, a potent anti-oxidant and free radical scavenger, would attenuate apoptotic death of rat C6 astroglial cells under glutamate excitotoxicity and oxidative stress. Exposure of C6 cells to 500 μ m l -glutamic acid (LGA) and 100 μ m hydrogen peroxide (H₂O₂) for 24 hr caused significant increases in apoptosis. Apoptosis was evaluated by Wright staining and ApopTag assay. Melatonin receptor 1 appeared to be involved in the protection of these cells from excitotoxic and oxidative damage. Cells undergoing excitotoxic and oxidative stress for 15 min were then treated with 150 n m melatonin, which prevented Ca²⁺influx and cell death. Western blot analyses showed alterations in Bax and Bcl-2 expression resulting in increased Bax:Bcl-2 ratio during apoptosis. Western blot analyses also showed increases in calpain and caspase-3 activities, which cleaved 270 kD α-spectrin at specific sites to generate 145 kD spectrin breakdown product (SBDP) and 120 kD SBDP, respectively. However, 15-min post-treatment of C6 cells with melatonin dramatically reduced Bax:Bcl-2 ratio and proteolytic activities, decreasing LGA or H₂O₂-induced apoptosis. Our data showed that melatonin prevented proteolysis and apoptosis in C6 astroglial cells. The results suggest that melatonin may be an effective cytoprotective agent against glutamate excitotoxicity and oxidative stress in CNS injuries and diseases.     

Glucosamine-induced insulin resistance in L6 muscle cells

Background:  Glucosamine increases flux through the hexosamine pathway, causing insulin resistance and disturbances similar to diabetic glucose toxicity.
Aim:  This study examines the effect of glucosamine on glucose uptake by cultured L6 muscle cells as a model of insulin resistance.
Methods:  Glucose uptake by L6 myotubes was measured using the non-metabolized glucose analogue 2-deoxy- d -glucose after incubation with glucosamine for 4 and 24 h, with and without insulin and several other agents (metformin, peroxovanadium and d -pinitol) that improve glucose uptake in diabetic states.
Results:  After 4 h, high concentrations of glucosamine (5 × 10⁻³ and 10⁻² M) reduced basal and insulin-stimulated glucose uptake by up to 50%. After 24 h, the effect of insulin was completely abolished by 10⁻² M glucosamine and reduced over 50% by 5 × 10⁻³ M glucosamine. Lower concentrations of glucosamine did not significantly alter glucose uptake. The effect of glucosamine could not be attributed to cytotoxicity assessed by the Trypan Blue test. Metformin, peroxovanadium and d -pinitol, each of which increased glucose uptake by L6 cells, did not prevent the decrease in glucose uptake with glucosamine.
Conclusion:  Glucosamine decreased insulin-stimulated glucose uptake by L6 muscle cells, providing a potential model of insulin resistance with similarities to glucose toxicity. Insulin resistance induced by glucosamine was not reversed by three agents (metformin, peroxovanadium and d -pinitol) known to enhance or partially mimic the effects of insulin.     

Mechanisms of isoproterenol‐induced cardiac mitochondrial damage: protective actions of melatonin

Mitochondrial dysfunction due to oxidative damage is the key feature of several diseases. We have earlier reported mitochondrial damage resulting from the generation of oxidative stress as a major pathophysiological effect of isoproterenol (ISO)‐induced myocardial ischemia in rats. That melatonin is an antioxidant that ameliorates oxidative stress in experimental animals as well as in humans is well established. We previously demonstrated that melatonin provides cardioprotection against ISO‐induced myocardial injury as a result of its antioxidant properties. The mechanism of ISO‐induced cardiac mitochondrial damage and protection by melatonin, however, remains to be elucidated in vitro. In this study, we provide evidence that ISO causes dysfunction of isolated goat heart mitochondria. Incubation of cardiac mitochondria with increasing concentrations of ISO decreased mitochondrial succinate dehydrogenase (SDH) activity, which plays a pivotal role in mitochondrial bioenergetics, as well as altered the activities of other key enzymes of the Kreb's cycle and the respiratory chain. Co‐incubation of ISO‐challenged mitochondria with melatonin prevented the alterations in enzyme activity. That these changes in mitochondrial energy metabolism were due to the perpetration of oxidative stress by ISO was evident from the increased levels of lipid peroxidation and decreased reduced glutathione/oxidized glutathione ratio. ISO‐induced oxidative stress also altered mitochondrial redox potential and brought about changes in the activity of the antioxidant enzymes manganese superoxide dismutase and glutathione peroxidase, eventually leading to alterations in total ATPase activity and membrane potential. Melatonin ameliorated these changes likely through its antioxidant abilities suggesting a possible mechanism of cardioprotection by this indole against ISO‐induced myocardial injury.     

Melatonin improves cardiovascular function and ameliorates renal, cardiac and cerebral damage in rats with renovascular hypertension

Abstract:  The effect of melatonin was investigated in an angiotensin II-dependent renovascular hypertension model in Wistar albino rats by placing a renal artery clip (two-kidney, one-clip; 2K1C), while sham rats did not have clip placement. Starting either on the operation day or 3 wk after the operation, the rats received melatonin (10 mg/kg/day) or vehicle for the following 6 wk. At the end of the nineth week, after blood pressure (BP) and echocardiographic recordings were obtained, plasma samples were obtained to assay lactate dehydrogenase (LDH), creatine kinase (CK), antioxidant capacity (AOC), asymmetric dimethylarginine (ADMA), and nitric oxide (NOx) levels. In the kidney, heart and brain tissues, malondialdehyde (MDA) and glutathione (GSH) levels, superoxide dismutase (SOD), catalase (CAT), myeloperoxidase (MPO) and Na⁺-K⁺ ATPase activities were determined. 2K1C caused an increase in BP and left ventricular (LV) dysfunction. In hypertensive animals LDH, CK, ADMA levels were increased in plasma with a concomitant reduction in AOC and NOx. Moreover, hypertension caused a significant decrease in tissue SOD, CAT, and Na⁺, K⁺-ATPase activities and glutathione content, while MDA levels and MPO activity were increased in all studied tissues. On the other hand, both melatonin regimens significantly reduced BP, alleviated oxidative injury and improved LV function. In conclusion, melatonin protected against renovascular hypertension-induced tissue damage and improved cardiac function presumably due to both its direct antioxidant and receptor-dependent actions, suggesting that melatonin may be of therapeutic use in preventing oxidative stress due to hypertension.     

Efficacy of artemisinin and mefloquine combinations against Plasmodium falciparum. In vitro simulation of in vivo pharmacokinetics

The activity of artemisinin in combination with mefloquine was tested in vitro against a chloroquine‐sensitive (F32) strain of Plasmodium falciparum . A method of repetitive dosing and extending the culture observation period to 28–30 days was used to mimic the in vivo pharmacokinetic situation. Plasmodium falciparum was exposed to artemisinin from 10⁻⁸ to 10⁻⁵  m , mefloquine from 3×10⁻⁹ to 10⁻⁵  m and their combinations. The exposure time for artemisinin was 3 hours twice daily and for mefloquine 24 hours. The drug‐dosing duration was 3 days.
Neither artemisinin nor mefloquine alone provided radical clearance of P. falciparum , even when maximum concentrations (10⁻⁵  m ) were applied. The antiparasitic activity of artemisinin and mefloquine were significantly higher when dosed alone. Effective concentrations for different degrees of inhibition (EC 50, 90 and 99) of both artemisinin and mefloquine respectively were significantly lower when used in combination. At concentrations normally reached in vivo , this effect was clearly synergistic ( P =0.016)
Our in vitro model of intermittent dosing of artemisinin and mefloquine combinations for 3 days provides significant evidence of positive interaction between the two compounds. Lower combination concentrations around the MIC‐values for the individual compounds showed synergistic effect, and high concentrations showed additive effect. This indicates that such drug combinations may provide radical clearance at concentrations lower than those required for single‐drug treatment.     

Anti-CD30 (BER-H2) immunotoxins containing the type-1 ribosome-inactivating proteins momordin and PAP-S (pokeweed antiviral protein from seeds) display powerful antitumour activity against CD30+ tumour cells in vitro and in SCID mice

The anti-CD30 immunotoxin (IT) Ber-H2/saporin is effective in patients with refractory Hodgkin's disease. However, responses are short and partial, one of the main reasons being the inability to repeat IT doses because of formation of human antibodies against the murine antibody and/or the toxin. To overcome this problem, we constructed two new anti-CD30 ITs by covalently linking the mouse monoclonal antibody Ber-H2 to the type 1 ribosome-inactivating proteins (RIPs) momordin (MOM) and pokeweed antiviral protein from seeds (PAP-S), which do not cross-react with each other or with saporin. Both ITs inhibited protein synthesis by Hodgkin's disease and anaplastic large-cell lymphoma (ALCL)-derived CD30⁺ target cell lines with a very high efficiency (IC₅₀ ranging from < 5 × 10⁻¹³  m to 2.75 × 10⁻¹¹ m , as RIP). In a SCID mouse model of xenografted CD30⁺ human ALCL, a 3 d treatment with non-toxic doses of Ber-H2/MOM (50% LD₅₀), started 24 h after transplantation, prevented tumour development in about 40% of the animals and significantly delayed tumour growth rate in the others. Main toxicity signs in mice and rabbits were a dose-related increase of serum transaminases (AST and ALT) and creatine phosphokinase (CPK). LD₅₀ (as RIP) in Swiss mice was 7 mg/kg for Ber-H2/MOM and 0.45 mg/kg for Ber-H2/PAP-S. Sequential administration of two anti-CD30 ITs (Ber-H2/MOM and Ber-H2/saporin) was well tolerated and did not result in formation of antibodies cross-reacting with the two plant toxins. The results presented in this paper suggest that, in the future, sequential administration of anti-CD30 humanized antibodies linked to antigenically distinct type 1 RIPs (saporin, MOM, PAP-S) should be feasible.     

Adriamycin stimulates proliferation of human lymphoblastic leukaemic cells via a mechanism of hydrogen peroxide (H2O2) production

It is becoming clear that adriamycin cytotoxicity may be mediated by semiquinone-free radicals derived from the drug itself and reactive oxygen species (ROS). Recent evidence supports the concept that low concentrations of ROS are able to stimulate cell proliferation, and, based on the observation that subtoxic concentrations of adriamycin can also induce cell proliferation, we hypothesize that low concentrations of adriamycin stimulate cell proliferation by a ROS generation mechanism. We have employed spin-trapping and electron spin resonance (ESR) spectroscopy to investigate the nature of the adriamycin-generated ROS. The spin trap 3,5-dibromo-4-nitrosobenzenesulphonate (DBNBS), which is oxidized in the presence of H₂O₂ and peroxidase enzymes, was used to produce a characteristic three-line spectrum, and it was found that an identical spectrum was produced by human lymphoblastic leukaemic cells (CCRF-CEM cells) after exposure to adriamycin. We tested our hypothesis further by exposing CCRF-CEM cells to subtoxic concentrations of adriamycin (10⁻⁸, 10⁻⁹ and 10⁻¹⁰  M ) and low concentrations of H₂O₂ (10⁻⁸, 10⁻⁹ and 10⁻¹⁰  M ) and subsequently monitored cell proliferation. We found that low concentrations of both adriamycin and H₂O₂ significantly stimulate CCRF-CEM cell proliferation. We therefore conclude that subtoxic concentrations of adriamycin are likely to induce cell proliferation via an H₂O₂ mediated mechanism.     

Age-related alterations in Ca2+ signals and mitochondrial membrane potential in exocrine cells are prevented by melatonin

Abstract:  Information regarding age-induced Ca²⁺ signal alterations in nonexcitable cells is limited. In addition, little evidence exists on the ability of melatonin to palliate the effects of aging on Ca²⁺ signals and mitochondrial potential, a parameter involved in both Ca²⁺ signaling and aging. We studied the ability of melatonin to prevent the effects of aging on intracellular Ca²⁺ homeostasis and mitochondrial potential in exocrine cells. Pancreatic acinar cells were obtained from adult (3 months old) and aged (22–24 months old) mice by collagenase dispersion. Ca²⁺ signals, in situ mitochondrial potential and in vitro amylase secretion were determined. Secretion in response to increasing levels of the secretagogues, acetylcholine and cholecystokinin (CCK), were impaired in aged pancreatic acini. This decrease was accompanied by an inhibition in the amplitude of the peak response to maximal concentrations of the agonists, and by a decrease in the pattern of Ca²⁺ oscillations induced by postprandial levels of CCK. Both the size of the calcium pools, assessed by low levels of ionomycin, and capacitative calcium entry, induced by depletion of the stores with thapsigargin, were diminished in aged cells. These changes in Ca²⁺ homeostasis were associated with depolarization of intracellular mitochondria. Oral administration of melatonin for 3 months to aged mice restored the secretory response, the amplitude and frequency of Ca²⁺ responses, the size of intracellular calcium pools, the capacitative calcium entry, and the mitochondrial potential. In conclusion, melatonin restores secretory function, Ca²⁺ signals and mitochondrial potential of aged exocrine cells.     

Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate

Abstract:  We investigated the relationship between oxidative stress and poor oocyte quality and whether the antioxidant melatonin improves oocyte quality. Follicular fluid was sampled at oocyte retrieval during in vitro fertilization and embryo transfer (IVF-ET). Intrafollicular concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in women with high rates of degenerate oocytes were significantly higher than those with low rates of degenerate oocytes. As there was a negative correlation between intrafollicular concentrations of 8-OHdG and melatonin, 18 patients undergoing IVF-ET were given melatonin (3 mg/day), vitamin E (600 mg/day) or both melatonin and vitamin E. Intrafollicular concentrations of 8-OHdG and hexanoyl-lysine adduct were significantly reduced by these antioxidant treatments. One hundred and fifteen patients who failed to become pregnant with a low fertilization rate (50%) in the previous IVF-ET cycle were divided into two groups during the next IVF-ET procedure; 56 patients with melatonin treatment (3 mg/day) and 59 patients without melatonin treatment. The fertilization rate was improved by melatonin treatment compared to the previous IVF-ET cycle. However, the fertilization rate was not significantly changed without melatonin treatment. Oocytes recovered from preovulatory follicles in mice were incubated with H₂O₂ for 12 hr. The percentage of mature oocytes with a first polar body was significantly reduced by addition of H₂O₂ (300  μ m ). The inhibitory effect of H₂O₂ was significantly blocked by simultaneous addition of melatonin. In conclusion, oxidative stress causes toxic effects on oocyte maturation and melatonin protects oocytes from oxidative stress. Melatonin is likely to improve oocyte quality and fertilization rates.     

Diagnosis of hospital-acquired pneumonia and methods of testing for pathogens

• Hospital-acquired pneumonia is diagnosed in patients who, in addition to abnormal shadowing on chest radiography, have ≥2 of the following: fever, abnormal white blood cell count and purulent discharge.
• Treatment effect is judged from clinical symptoms and microorganism test results 2–3 days after the start of treatment, and reassessment is made with regard to change, addition or discontinuation of antimicrobial agents.
• Coordination with the microbiology laboratory is extremely important in diagnosing infectious diseases.
• Microorganisms isolated from tracheal aspirate at 10⁶ cfu/mL (3+), from BAL at 10⁴–10⁵ cfu/mL (2+) and from a protected specimen brush at 10³ cfu/mL (1+) have a high possibility of being the causative microorganisms.
• Pneumonia can almost be ruled out when no significant microbes are detected from the lower respiratory tract in patients with suspected ventilator-assisted pneumonia (when no change has been made to antimicrobial administration within 72 h).
• When MRSA or Pseudomonas aeruginosa are not detected in sputum tests, involvement of these drug-resistant bacteria may be considered unlikely, and the case treated accordingly.
• Involvement of aspiration is suspected when a number of pathogens are observed in lower respiratory tract specimens.
• When antimicrobials are administered with reference to breakpoint concentrations in Western countries, differences in dosage between these countries and Japan need to be considered.     

Protection against cell death and sustained tyrosine hydroxylase phosphorylation in hydrogen peroxide- and MPP+-treated human neuroblastoma cells with melatonin

Abstract:  Neuroprotective effects of melatonin against oxidative stress-induced neuronal cell degeneration in human SH-SY5Y neuroblastoma cells were investigated in this report. The results demonstrate that exogenous administration of H₂O₂ and 1-methyl, 4-phenyl, pyridinium ion (MPP⁺) significantly decreased cell viability in SH-SY5Y cultured cells. Desipramine, a monoamine uptake blocker was able to abolish the toxic effects of MPP⁺ but not H₂O₂ in reduction of cell viability. Conversely, melatonin reversed the toxic effects of H₂O₂ and MPP⁺ on cell viability. In addition, the reduction of phosphorylation of tyrosine hydroxylase, the rate limiting enzyme in dopamine synthesis, and phosphorylation of cyclic AMP responsive element-binding protein by H₂O₂ and MPP⁺ was also diminished by melatonin. These results demonstrate some effective roles of melatonin on neuroprotection and its action on the modulation of tyrosine hydroxylase phosphorylation.     

Application of the intravenous glucose tolerance test and the minimal model to patients with insulinoma: insulin sensitivity (Si) and glucose effectiveness (Sg) before and after surgical excision

Background  The unmodified frequently sampled intravenous glucose tolerance test (FSIGT) has not previously been used to assess insulin/glucose kinetics in patients with insulinoma.
Objective  To measure insulin sensitivity (Si) and glucose effectiveness (Sg) by means of the FSIGT in patients with insulinoma, before and after surgical removal of the tumour.
Subjects and methods  FSIGTs were performed in five patients, before and approximately 3 months post-surgery, and in 11 controls. Si and Sg were estimated using Minimal Model computer analysis of dynamic glucose and insulin data.
Results  Si was lower in insulinoma patients before, compared with after surgery (3·37 ± 0·62 vs. 6·24 ± 1·09 SE [×10⁻⁴] min⁻¹µU⁻¹ ml, P  < 0·05). Sg was similar in patients pre- and post-surgery (3·0 ± 0·67 vs. 2·4 ± 0·6 [×10⁻²] min⁻¹, NS).
Conclusions  Insulin sensitivity improves after excision of an insulinoma. Glucose effectiveness is not influenced by chronic hyperinsulinaemia and hypoglycaemia.     

PMA-induced megakaryocytic differentiation of HEL cells is accompanied by striking modifications of protein kinase C catalytic activity and isoform composition at the nuclear level

We investigated whether members of the protein kinase C (PKC) family of enzymes could play a role in the nuclear events involved in megakaryocytic differentiation. PKC activity was analysed using a serine substituted specific peptide, which enabled us to evaluate the whole catalytic activity in the pluripotent haemopoietic HEL cell line treated with 10⁻⁷  m phorbol myristate acetate (PMA) or haemin. In parallel, the subcellular distribution of different PKC isoforms (α, βI, βII, γ, δ, ε, θ, η, ζ) was evaluated by Western blot. PKC catalytic activity in the nuclei of HEL cells showed a peak after acute (30 min) treatment with PMA, followed by a significant ( P  < 0.05) decline after prolonged exposure (72 h) to the same agonist, when most HEL cells had acquired a differentiated megakaryocytic phenotype. Western blot analysis of nuclear lysates consistently showed a significant increase of PKC-α, -βI, -ε, -θ and -ζ isoforms after 30 min of PMA treatment, followed by a drastic decline of all but PKC-ζ isoforms. Moreover, PKC-δ appeared in HEL nuclei only after 72 h of exposure to PMA. On the other hand, neither the catalytic activity nor the immunoreactivity of the different PKC isoforms showed remarkable variations in nuclei of HEL cells induced to differentiate along the erythroid lineage with 10⁻⁷  m haemin.
The possible implications of these findings for a better understanding of the molecular events underlying the process of megakaryocytic differentiation are discussed.