Hepato-pancreato-biliary fat: the good, the bad and the ugly

Obesity has become epidemic in the United States, in Europe, and in many urban areas in the developing world. The globalization of certain 'fast foods' and 'soft drinks' may, in part, be contributing to this epidemic. Diets high in saturated fatty acids and trans fats as well as drinks that have high fructose corn syrup levels may be particularly harmful. Recent research suggests that fat is a dynamic endocrine organ and that visceral fat is associated with the metabolic syndrome. Central obesity leads to organ steatosis and altered serum adipokines including reduced adiponectin and markedly elevated leptin. This abnormal adipokine milieu results in increased tissue infiltration of monocytes and macrophages which produce proinflammatory cytokines that alter organ function. Over many years, the combination of steatosis and local inflammation leads to fibrosis and eventually to cancer. Nonalcoholic fatty liver disease (NAFLD) is a precursor for nonalcoholic steatohepatitis (NASH). NAFLD and NASH (1) lead to cirrhosis and hepatocellular carcinoma, (2) increase the risk of liver resection, and (3) compromise the outcome of liver transplantation. Similarly, in the pancreas nonalcoholic fatty pancreas disease (NAFPD) may lead to nonalcoholic steatopancreatitis (NASP). NAFPD and NASP may (1) promote the development of chronic pancreatitis and pancreatic cancer, (2) exacerbate the severity of acute pancreatitis, and (3) increase the risk of pancreatic surgery. In the gallbladder nonalcoholic fatty gallbladder disease (NAFGBD, cholecystosteatosis) may lead to steatocholecystitis. Cholecystosteatosis may be an explanation for (1) the increased incidence of chronic acalculous cholecystitis and (2) the increased number of cholecystectomies.     

Further Studies on the Noradrenergic and the Renin Angiotensin Systems in the Brain of the Rat

Previous investigations have shown that depletion of brain norepinephrine (NE) induced by chemical sympa thectomy resulted in significant changes in the central renin-angiotensin system. The purpose of the present work was to increase the NE concentration in the central nervous system (CNS) in order to analyze its effect on the peptidergic complex and on the blood pressure (BP) levels. Treated rats were given the following drugs in the drinking water: 1-dopa (12 mg/rat/day), carbidopa (6 mg/rat/day) and pargyline (10 mg/rat/day) during 25 days. BP was determined, blood and cerebrospinal fluid (CSF) samples were obtained. The CNS was dissected into several areas. NE, angiotensinogen (AoC) and renin concentration (RC) were determined in the brain parenchyma; AoC was evaluated in CSF and plasma samples. Pharmaco-logical treatment resulted in an hypotensive effect and, at the same time, an increase of NE in the CNS (about 100 %; pCO .0005). These changes were accompanied by a significant decrease in the peripheral and central AoC. These results add new evidence to the postulated relationship between these two important regulatory systems involved in cardiovascular control.     

Consequences of RAS and MAPK activation in the ovary: the good, the bad and the ugly

This review summarizes studies providing evidence (1) that endogenous RAS activation regulates important physiological events during ovulation and luteinization (2) that expression of the mutant, active KRAS(G12D) in granulosa cells in vivo causes abnormal follicle growth arrest leading to premature ovarian failure and (3) that KRAS(G12D) expression in ovarian surface epithelial (OSE) cells renders them susceptible to the pathological outcome of transformation and tumor formation. These diverse effects of RAS highlight how critical its activation is linked to cell- and stage-specific events in the ovary that control normal processes and that can also lead to altered granulosa cell and OSE cell fates.     

Overexpression of the human VPAC2 receptor in the suprachiasmatic nucleus alters the circadian phenotype of mice

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) belong to a superfamily of structurally related peptide hormones that includes glucagon, glucagon-like peptides, secretin, and growth hormone-releasing hormone. Microinjection of VIP or PACAP into the rodent suprachiasmatic nucleus (SCN) phase shifts the circadian pacemaker and VIP antagonists, and antisense oligodeoxynucleotides have been shown to disrupt circadian function. VIP and PACAP have equal potency as agonists of the VPAC(2) receptor (VPAC(2)R), which is expressed abundantly in the SCN, in a circadian manner. To determine whether manipulating the level of expression of the VPAC(2)R can influence the control of the circadian clock, we have created transgenic mice overexpressing the human VPAC(2)R gene from a yeast artificial chromosome (YAC) construct. The YAC was modified by a strategy using homologous recombination to introduce (i) the HA epitope tag sequence (from influenza virus hemagglutinin) at the carboxyl terminus of the VPAC(2)R protein, (ii) the lacZ reporter gene, and (iii) a conditional centromere, enabling YAC DNA to be amplified in culture in the presence of galactose. High levels of lacZ expression were detected in the SCN, habenula, pancreas, and testis of the transgenic mice, with lower levels in the olfactory bulb and various hypothalamic areas. Transgenic mice resynchronized more quickly than wild-type controls to an advance of 8 h in the light-dark (LD) cycle and exhibited a significantly shorter circadian period in constant darkness (DD). These data suggest that the VPAC(2)R can influence the rhythmicity and photic entrainment of the circadian clock.     

pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA

We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNA(fMet) to the aa-tRNAs Phe-tRNA(Phe), Ala-tRNA(Ala), Gly-tRNA(Gly), Pro-tRNA(Pro), Asn-tRNA(Asn), and Ile-tRNA(Ile), selected to cover a large range of intrinsic pK(a)-values for the α-amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and displayed different pH-dependence, quantified as the pH-value, pK(a)(obs), at which the rate was half maximal. The pK(a)(obs)-values were downshifted relative to the intrinsic pK(a)-value of aa-tRNAs in bulk solution. Gly-tRNA(Gly) had the smallest downshift, while Ile-tRNA(Ile) and Ala-tRNA(Ala) had the largest downshifts. These downshifts correlate strongly with molecular dynamics (MD) estimates of the downshifts in pK(a)-values of these aa-tRNAs upon A-site binding. Our data show the chemistry of peptide bond formation to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and indicate rate limiting chemistry for all six aa-tRNAs.     

On the problems of the antituberculous drug resistance]

In this part, we will discuss exclusively about the technique of measurement. It is desirable especially for the proportion method that two inoculations with the same doses give the same number of colonies and when diluted, the colonies decrease exactly parallel to it degree of dilution. But this ideal is not necessarily realized and especially the difference between the dilution of 10(-3) and 10(-4) mg/ml on the drug free medium and that of 10(-2) and 10(-3) on the drug containing medium is fairly remarkable: the former is not so large but the latter sometimes varies tremendously with the drugs and their concentrations. So it is necessary to use always two inoculations for all specimens and the resistance is calculated on the medium inoculated with the same doses, for that, colonies on the control must be countable and suitable number that is 50-300. Further too concentrated solution like 10(-2) mg/ml must not be used for the proportion method. The selection of the most suitable two dilutions is specially important for this method. Original one is 10(-3) and 10(-5). Unfortunately we did not follow this formula and at first we adopted the combination of 10(-3) and 10(-4), then 10(-2) and 10(-4) finally 10(-2) and 10(-3) which was the worst. Now we searched for the dilution which gave the suitable number of colonies from our several thousand cases and found that 10(-4) was the most excellent and gave this number in 63%, next 10(-5) in 14%, and then 10(-3) in 7.2%, 10(-2) in only 1.0%. With the combination of 2 solutions 10(-3) and 10(-5)--original one--was most excellent (94.8%) then 88.4% with 10(-4) and 10(-5). The combination of 10(-2) and 10(-3) was worst, it gave only 8.9%. As the proportion method demands the most strict procedures for all cases, it is necessary as simplify as possible, thus in every day practice, only the drugs most frequently used (3 or 4 drugs) and one concentration for each drug could be examined. Further we discussed on the difference between DH-SM and SM, direct and indirect method and problem of plug.     

An obligatory requirement for the heterotrimeric G protein Gi3 in the antiautophagic action of insulin in the liver

Heterotrimeric G proteins of the G(i) class have been implicated in signaling pathways regulating growth and metabolism under physiological and pathophysiological conditions. Knockout mice carrying inactivating mutations in both of the widely expressed Galpha(i) class genes, Galpha(i2) and Galpha(i3), demonstrate shared as well as gene-specific functions. The presence of a single active allele of Galpha(i3) is sufficient for embryonic development, whereas at least one allele of Galpha(i2) is required for extrauterine life. Mice lacking both Galpha(i2) and Galpha(i3) are massively growth-retarded and die in utero. We have used biochemical and cell biological methods together with in situ liver perfusion experiments to study Galpha(i) isoform-specific functions in Galpha(i2)- and Galpha(i3)-deficient mice. The subcellular localization of Galpha(i3) in isolated mouse hepatocytes depends on the cellular metabolic status. Galpha(i3) localizes to autophagosomes upon starvation-induced autophagy and distributes to the plasma membrane upon insulin stimulation. Analysis of autophagic proteolysis in perfused mouse livers showed that mice lacking Galpha(i3) are deficient in the inhibitory action of insulin. These data indicate that Galpha(i3) is crucial for the antiautophagic action of insulin and suggest an as-yet-unrecognized function for Galpha(i3) on autophagosomal membranes.     

Drugs in the Pipeline for the Obesity Market

Obesity is a major public health problem. For many obese patients, diet and exercise are an inadequate treatment and bariatric surgery may be too extreme of a treatment. As with many other chronic diseases, pharmacologic treatment may be an attractive option for selected obese patients. Antiobesity drugs may potentially work through one of three mechanisms: (1) appetite suppression, (2) interference with absorption of nutrients, and (3) increased metabolism of nutrients. The three most widely prescribed drugs approved to treat obesity are phentermine, sibutramine, and orlistat. Drugs approved for treating obesity usually result in an additional weight loss of approximately 2–5 kg in addition to placebo. For pharmacologic therapy in obesity to be widely utilized, greater effectiveness and safety will be needed. Four types of single-agent drugs are in late stage development, including (1) selective central cannabinoid-1 receptor blockers, (2) selective central 5-hydroxytryptamine 2C serotonin receptor agonists, (3) an intestinal lipase blocker, and (4) central-acting incretin mimetic drugs. Four combination agent compounds in late stage development include (1) Contrave, which combines long-acting versions of naltrexone and bupropion; (2) Empatic, which combines long-acting bupropion and long-acting zonisamide; (3) Qnexa, which combines phentermine with controlled release topiramate; and (4) an injectable combination of leptin and pramlintide. Peptide YY and melanin-concentrating hormone receptor-1 antagonists are centrally acting agents in early stage development. It is expected that several new drug products for obesity will become available over the next few years. Their role in managing this disease remains to be determined.     

Weight reduction decreases the circulating concentration of the N-terminus of the ANF prohormone.

The N-terminus of the atrial natriuretic factor (ANF) prohormone (ie, proANF 1-98) contains two vasodilatory peptides consisting of amino acids (aa): aa 1-30 (ie, proANF 1-30) and aa 31-67 (ie, proANF 31-67) of the 126 aa prohormone. The relationship of this N-terminus to the renin-aldosterone axis and blood pressure reduction was investigated in 18 obese subjects (5 hypertensive and 13 normotensive) placed on a 12-week, low sodium (40 mmol), weight reducing diet. The N-terminus of the ANF prohormone and proANF 31-67, which circulates as a distinct entity after being proteolytically cleaved from the N-terminus, were significantly (p less than 0.001) higher (767 +/- 1.01 and 816 +/- 135 fmol/ml) in the obese hypertensive group compared with the obese normotensive group (377 +/- 24 and 356 +/- 17 fmol/ml, respectively) prior to beginning the weight reduction program. There was a dramatic fall in the N-terminus and in proANF 31-67 after 1 week of weight reduction in both obese groups, which correlated with the decrease in mean arterial pressure during the first week and throughout the 12 weeks of weight reduction (r = .54, p less than 0.001 and r = .59, p less than 0.001, respectively). ProANF 1-98 had a significant (p less than 0.01) inverse correlation with plasma renin in both obese groups. ProANF 31-67, likewise, had an inverse correlation with plasma renin in the hypertensive (p less than 0.002), as well as the normotensive (p less than 0.03) subjects. ProANF 31-67 did not significantly correlate with aldosterone in either group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preferential transfer of the complete glycan is determined by the oligosaccharyltransferase complex and not by the catalytic subunit

Most eukaryotic cells show a strong preference for the transfer in vivo and in vitro of the largest dolichol-P-P-linked glycan (Glc(3)Man(9)GlcNAc(2)) to protein chains over that of biosynthetic intermediates that lack the full complement of glucose units. The oligosaccharyltransferase (OST) is a multimeric complex containing eight different proteins, one of which (Stt3p) is the catalytic subunit. Trypanosomatid protozoa lack an OST complex and express only this last protein. Contrary to the OST complex from most eukaryotic cells, the Stt3p subunit of these parasites transfers in cell-free assays glycans with Man(7-9)GlcNAc(2) and Glc(1-3)Man(9)GlcNAc(2) compositions at the same rate. We have replaced Saccharomyces cerevisiae Stt3p by the Trypanosoma cruzi homologue and found that the complex that is formed preferentially transfers the complete glycan both in vivo and in vitro. Thus, preference for Glc(3)Man(9)GlcNAc(2) is a feature that is determined by the complex and not by the catalytic subunit.     

Factors influencing the shape of the inspiratory flow

An index (IS), quantitating the departure of the inspiratory flow profile (IFP) from the rectangular one, i.e. the optimal profile (IS=0), was computed from pneumotachograms recorded in 26 normal, anesthetized subjects breathing spontaneously through the endotracheal tube (ETT) or face mask (FM) with or without added resistances (R) and in 27 awake subjects breathing through the mouth and FM in the supine and seated posture at rest and during exercise (40 W) on a cycloergometer, through the nose and FM, and through the mouthpiece (MP). During anesthesia, IS decreased with R both while breathing through the ETT (DeltaIS=-0.037+/-0.006 (SE); P<0.001) and FM (DeltaIS=-0.054+/-0.008; P<0.001). This indicates that (a) the change of IFP towards the optimal shape is reflex in nature and related to the dynamic inspiratory load, and (b) tracheobronchial mechanoreceptors and inspiratory muscles are involved in this response. The reflex is also operative in awake subjects, since IS decreased whenever the inspiratory dynamic load was increased, as on turning from seated to supine posture (DeltaIS=-0.024+/-0.003; P<0.001), shifting from mouth to nose breathing (DeltaIS=-0.034+/-0.003; P<0.05), from rest to mild exercise (DeltaIS=-0.066+/-0.005; P<0.001). The different IS value between FM and MP breathing (DeltaIS=0.036+/-0.004; P<0.001) indicates, however, that other factors, likely behavioral, also affect the IFP.     

The endocrine changes, the timing of ovulation and the efficacy of the Doublesynch protocol in the Murrah buffalo (Bubalus bubalis)

Experiments were conducted to investigate (a) the timing of ovulation and the associated endocrine changes (progesterone, estrogen and LH) during estrous cycle and (b) the efficacy, with respect to the pregnancy rate, in cycling and anestrus in Murrah buffaloes subjected to the Doublesynch protocol during the low breeding season. In experiment 1, 10 cycling buffaloes were administered PGF(2α) on day 0 (without regard to the estrous cycle stage), GnRH on day 2, a second PGF(2α) injection on day 9, and a second GnRH injection on day 11. Transrectal palpation was performed at 2-h intervals after the first and second GnRH treatments until ovulation was detected or for upto 96 h. The plasma progesterone and total estrogen concentrations were determined in blood samples collected at daily intervals starting 2 days before the onset of the protocol and continued until the day of the second detected ovulation. The plasma LH and total estrogen concentrations were measured in blood samples collected at 30-min intervals for 8h following the first and second GnRH injections and thereafter at 2-h intervals until 2h after the detection of ovulation. Ovulation occurred in 9/10 buffaloes (90%) at 22.2 ± 1.2 h (mean ± S.E.M.; range 18.0-26.0 h) and 10/10 buffaloes (100%) at 23.2 ± 1.0 h (mean ± S.E.M.; range 20.0-28.0 h) after the first and second GnRH treatments, respectively. The peak LH concentrations of 99.8 ± 28.5 ng/ml (range 37.8-320.0 ng/ml) and 62.3 ± 11.9 ng/ml (range 20.9-143.9 ng/ml) occurred 2.1 ± 0.3 h (range 1.0-3.5 h) and 2.3 ± 0.3 h (range 0.5-3.0 h) after the first and second GnRH treatments, respectively. The total estrogen concentration gradually increased from the day of both the first and second PGF(2α) administrations until the LH peak (with great variability) and then gradually declined to the basal level, which was reached at the time ovulation was detected. In experiment 2, 10 cycling and 11 non-lactating anestrus buffaloes were subjected to the Doublesynch protocol with timed artificial insemination (TAI) 16 and 24 h after the second GnRH treatment, and 55 cycling buffaloes were inseminated after spontaneous estrus was detected (control group). The pregnancy rates were 60% using TAI on cycling buffaloes (experiments 1 and 2), 55% for anestrus buffaloes (experiment 2), and 27.3% for cycling buffaloes inseminated following spontaneous estrus. The overall pregnancy success rates after the Doublesynch protocol in both cycling and anestrus buffaloes increased by 30.8% compared to spontaneous estrus (58.1% vs. 27.3%). In conclusion, the Doublesynch protocol effectively synchronized ovulation twice (after the first and second GnRH treatments) irrespective of the stage of estrous cycle in Murrah buffaloes. The study also demonstrated that the Doublesynch protocol followed by TAI significantly (P<0.005) enhanced the pregnancy rate in cycling and anestrus buffaloes in comparison to untreated controls during the low breeding season.     

In vivo validation of the coincidence of the peak and end of the T wave with full repolarization of the epicardium and endocardium in swine.

OBJECTIVES/BACKGROUND: Previous in vitro studies have suggested full repolarization of the epicardium coincides with the peak of the T wave (T(peak)) and that of the M cells coincides with the end of the T wave (T(end)). However, in vivo validation of the theory is lacking. METHODS: Monophasic action potentials (MAPs) were recorded using the CARTO mapping system from 51 +/- 10 epicardial sites and 64 +/- 9 endocardial sites of the left ventricle in 10 pigs and from 41 +/- 4 epicardial sites and 53 +/- 2 endocardial sites of the right ventricle in two of the 10 pigs. End of repolarization (EOR) times over the epicardium (EOR(epi)), endocardium (EOR(endo)), and over both (EOR(total)) were obtained. QT(peak) and QT(end) intervals were measured from simultaneously recorded 12-lead ECG. RESULTS: Minimal and maximal EOR(total) were observed in the left ventricle in all pigs. Minimal EOR(total) was on the epicardium in five pigs, and maximal EOR(total) was on the endocardium in nine pigs. Minimal, mean, and maximal QT(peak) intervals all were significantly smaller than maximal EOR(epi) (322 +/- 23 ms, P <.01). No significant difference was found between maximal QT(end) interval (338 +/- 30 ms) and maximal EOR(endo) (339 +/- 24 ms, difference = 1 +/- 19 ms, P =.92), between maximal QT(end) interval and maximal EOR(total) (341 +/- 24 ms, difference = 2 +/- 18 ms, P =.69), or between minimal QT(peak) interval (283 +/- 28 ms) and minimal EOR(total) (282 +/- 20 ms, difference = 0 +/- 15 ms, P =.95). CONCLUSIONS: In in vivo pig models, T(peak) does not coincide with full repolarization of the epicardium but coincides well with the earliest EOR, whereas the T(end) corresponds with the latest EOR. These findings suggest that not only the transmural gradients but also the apicobasal repolarization gradients contribute to genesis of the T wave.     

Evidence for the role of alpha-MSH in the induction of pseudopregnancy in the rat

In a previous study we have demonstrated that cervical stimulation (CS) induces alpha-MSH release. The present experiments were undertaken to (1) examine the pattern of serum alpha-MSH during CS-induced pseudopregnancy (PSP) and (2) assess the possibility that alpha-MSH contributes to the induction and maintenance of PSP. Throughout PSP serum alpha-MSH fluctuated in a cyclic manner demonstrating two daily surges which occurred between 12.00 and 13.00 h (diurnal surge) and between 24.00 and 04.00 h (nocturnal surge). Chronic exposure of animals to alpha-MSH administered via minipumps (24 micrograms/day, starting on the morning of estrus), induced PSP as determined by deciduoma formation and persistence of a characteristic diestrous vaginal cytology. Furthermore, insertion of an alpha-MSH-containing minipump at diestrus 1 (D1) resulted in progesterone and prolactin (PRL) levels on the afternoon of diestrus 2 (D2) similar to those levels found on day 2 of PSP. Uterine weight was significantly decreased in alpha-MSH-treated rats and pseudopregnant rats as compared with cyclic D2 controls. alpha-MSH was found to release PRL indirectly, through stimulation of adrenal progesterone. This effect, however, necessitates the presence of the ovaries as a source of estradiol (EB) since it is demonstrable in intact and acutely ovariectomized rats, but not in chronically ovariectomized animals. EB treatment of chronically ovariectomized rats is capable of restoring the sequence. These results indicate that, as demonstrated for PRL, cervical stimulation initiates rhythmic daily surges of alpha-MSH secretion which are maintained through PSP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors affecting the determination of the serum protein-bound iodine

The protein-bound iodine (PBI) determination continues to be a valuable parameter of thyroid function, unfortunately subject to an increasing number of nonthyroidal factors which lead to spurious values. The present review collects these factors and re-emphasizes the caution that must be employed in accepting elevated and depressed PBI values in patient populations exposed to (1) large quantities of iodine in various forms, (2) natural and pharmaceutical goitrogens, (3) thyroidal and nonthyroidal hormones, and (4) drugs which affect the interaction of thyroid hormone and its carrier proteins.

It, is also pointed out that certain relatively new pharmacologic agents (such as perphenazine and triparanol) and nonthyroidal disease states (such as acute intermittent porphyria and certain metastatic tumors) may have specific influences on the PBI determination. Mercurial diuretics, often cited as a cause of spuriously low PBI values, may not affect the PBI when a particular PBI method (alkaline ash technic) is used.     

Endomyocardial biopsy in the heart transplant patient: the state of the art]

Data regarding 2176 endomyocardial biopsies (EMB) (Nov. '85-Dec. '89) performed in 164 transplanted hearts (4 etherotopic) from 158 patients (6 retransplants) are herein reported. This study was aimed to evaluate: 1) Incidence and characteristics of early ischemic myocardial damage. 2) The influence of different immunosurveillance protocols on incidence, degree and aggressiveness of acute rejection and the inflammatory infiltrate composition. 3) The immunophenotype of infiltrating cells in moderate acute rejection episodes. 4) HLA-DR antigen expression on myocyte sarcolemma. 5) Characterization of cells expressing immune response mediators. 6) Myocardial localization of opportunistic infections. 7) Useful information on chronic rejection. Our results demonstrate that: a) Mild rejection seldom progresses to moderate degree. b) Different immunosuppressive protocols can influence the incidence of acute rejection: in fact, in OKT3 protocol, the incidence of rejection episodes is higher than in other protocols as well as aggressiveness toward myocytes. c) Infiltrating cells maintain T lymphocyte prevalence with minor amounts of B lymphocytes and macrophages in the 3 different protocols. T cell subset characterization showed a slight prevalence of CD8 bearing cells over CD4 positive cells whereas CD57 cells were few and scattered. d) Class II Major Histocompatibility Complex (HLA-DR) expression never occurs on myocyte sarcolemma. e) TNF alpha is expressed in acute cardiac rejection by immunologically activated T lymphocytes and macrophages and the number of immunoreactive cells increases with progression of the rejection. f) Human cytomegalovirus infections can be primary or recurrent. Myocardial involvement has been observed in primary forms. Virus can affect endothelial cells (with no inflammatory reaction) or myocytes (myocarditis) and its diagnosis requires a combination of immunohistochemical and molecular biology techniques. Diagnosis of Toxoplasma gondii infection can be usually accomplished by routine histopathological study. g) Chronic rejection diagnosis is rarely based on biopsy derived information.