Evidence that ecdysteroids and methyl farnesoate control allometric growth and differentiation in a crustacean.

Allometric, or disproportionate, growth of body parts is a basic problem in morphogenesis. Male spider crabs, Libinia emarginata, have several forms or morphotypes. During the terminal molt, the propodus enlarges disproportionately, exceeding the carapace length by as much as 35%. Even though shorter clawed males are reproductive, the large-clawed males become primary reproductives. We stimulated penultimate stage males to molt by eyestalk ablation, which removes molt inhibiting hormone (MIH) and mandibular organ inhibiting hormone (MIOH), and measured ecdysones by radioimmunoassay and methyl farnesoate (MF) in hemolymph by high-performance liquid chromatography (HPLC) using an internal standard. Eyestalk ablation accelerated molting and increased ecdysteroids to peak at 150 ng/ml before the molt. In control animals the ecdysteroids peaked at 90 ng/ml 3 days before the molt, with MF remaining less than 0.5 ng/ml. These became large males with large allometric claws. In contrast, the ablated ones, with increased MF (1 to 1.5 ng/ml), increased carapace size, but retained shorter non-allometric claws, with length shorter than the carapace. The results are consistent with experiments that we have performed with MF administration (Abdu et al., Biol. Bull., Woods Hole, MA 195 (1998) 112; Laufer et al., Gen. Comp. Endocrinol. 111 (1998) 113; Laufer et al., in: IV Symp. Aquaculture in Central America: Focusing on Shrimp and Tilapia, (1997a), p. 161; Laufer et al., Invert. Reprod. Dev. 31 (1997b) 63) which led to the interpretation that ecdysteroids and low MF concentrations promote allometric growth, while ecdysones with relatively higher concentrations of MF inhibited allometric growth. These results indicate and support the conclusion that MF and ecdysteroids determine the control of morphogenesis in allometric growth of Crustacea.

Arachidyl amido cholanoic acid (Aramchol) is a cholesterol solubilizer and prevents the formation of cholesterol gallstones in inbred mice.

We have recently synthesized fatty acid bile acid conjugates (FABAC) that were able to reduce and retard cholesterol crystallization in model and human biles. When given orally, they prevented the formation of cholesterol crystals in the bile of hamsters. The aim of the present study was to determine whether the FABAC are cholesterol solubilizers, whether they can dissolve pre-existing crystals, whether they can prevent the formation of cholesterol gallstones, and to investigate the optimal type of bond between the fatty acid and bile acid. The presence of cholesterol crystals was determined by light microscopy, and the total crystal mass of precipitated crystals was measured by chemical means. Inbred (C57J/L) mice on a lithogenic diet were used to evaluate cholesterol crystal formation, dissolution, and gallstone formation in vivo. Arachidyl amido cholanoic acid (Aramchol) was the FABAC used in the present experiments. At equimolar amounts, the cholesterol-solubilizing capacity of Aramchol was higher than that of taurocholate and similar to that of phosphatidylcholine. The addition of Aramchol dissolved approximately 50% of pre-existing crystals in model bile solutions. The same phenomenon was demonstrated in human bile ex vivo, with a dose-response effect. All inbred mice developed cholesterol crystals in bile after 10-14 d on the lithogenic diet. Thereafter, supplementation of the diet with Aramchol progressively reduced the proportion of mice with crystals to 25% after 28 d. On the lithogenic diet, 100% of inbred mice developed cholesterol gallstones in the gallbladder by day 21. None of the mice whose diet was supplemented with 0.5 mg or 1.0 mg of Aramchol/d developed stones or crystals. FABAC are a new class of molecules that are cholesterol solubilizers and which are able to dissolve cholesterol crystals in bile. Upon oral administration, they dissolve pre-existing cholesterol crystals and prevent the formation of gallstones in gallstone-susceptible mice.

Settlement and Metamorphosis of Capitella Larvae Induced by Juvenile Hormone-Active Compounds Is Mediated by Protein Kinase C and Ion Channels.

The signal transduction pathway by which juvenile hormone-active compounds induce settlement and metamorphosis of metatrochophore larvae of the polychaete annelid Capitella sp. 1 was investigated. The known protein kinase C (PKC) activator phorbol-12, 13-dibutyrate was an active inducer of settlement and metamorphosis, whereas H-7, an inhibitor of PKC, inhibited settlement and metamorphosis in response to juvenile hormone III (JH III). JH III and methyl farnesoate (MF) also directly activated, in vitro, both a PKC-like enzyme present in Capitella homogenates and PKC purified from rat brain. In addition, binding studies using the fluorescent PKC inhibitor RIM-1 revealed the presence of a PKC-like enzyme in intact Capitella larvae and juveniles. Settlement and metamorphosis of the larvae was also stimulated by membrane-depolarizing concentrations of KCI. This response to KCl was inhibited by tetraethylammonium. The potassium channel blocker 4-aminopyridine induced settlement and metamorphosis, whereas settlement and metamorphosis in response to JH III was inhibited by the potassium channel ionophore nigericin. Settlement and metamorphosis induced by JH III was inhibited by the calcium channel blockers Ni2+, Zn2+, and verapamil, whereas settlement and metamorphosis was induced by the calcium ionophore A23187. These results suggest that in mediating this response, juvenile hormones may cause activation of PKC, leading to subsequent modulation of potassium and calcium channels.

Stimulation of ovarian maturation in the crayfish Procambarus clarkii by methyl farnesoate.

Stimulation of ovarian maturation in the red swamp crayfish Procambarus clarkii was accomplished in three different trials by administration of methylfarnesoate (MF). After 30 days of treatment, the ovaries of prereproductive females were 2- to 10-fold larger and in later stages of vitellogenesis than those of the controls. Similar and statistically significant results were observed in a second 30-day trial, which was begun during the middle of the vitellogenic cycle. In this experiment the ovaries of the controls and the treated groups were in late vitellogenesis, and the oocyte diameters of both groups were similar, suggesting that MF stimulated more oocytes to mature. In a third experiment of 60 days duration, the ovary was again enlarged in the treated animals. In addition, MF levels in the hemolymph of treated females undergoing vitellogenesis were found to be twice as great as those of the controls. We conclude that exogenous MF can stimulate and enhance ovarian maturation. These experiments strongly support the concept that MF acts as a gondatropin in crustaceans.

Effect of Methyl Farnesoate on Late Larval Development and Metamorphosis in the Prawn Macrobrachium rosenbergii (Decapoda, Palaemonidae): A Juvenoid-like Effect?

Methyl farnesoate (MF), the unepoxidated form of insect juvenile hormone III, was detected in larvae of the freshwater prawn Macrobrachium rosenbergii, which metamorphose to post-larvae following 11 larval stages. The possible role of MF as a morphogen was studied by administering the compound to M. rosenbergii larvae via an Artemia vector. Higher MF levels caused earlier retardation of late larval growth, and the highest dose retarded larval development. Furthermore, MF significantly affected the patterns of metamorphosis and the appearance of intermediate individuals exhibiting both larval and post-larval morphology and behavior. Three intermediate types were defined, two of which were found only at the MF-treated groups and one that was exclusive to the higher dose treatments. The relative abundance of intermediate specimens increased from 2% in the control to 32% in the high MF concentration, which suggests that MF has a juvenoid-like effect in this decapod crustacean.

Calcium and the anionic polypeptide fraction (APF) have opposing effects on cholesterol crystallization in model bile.

BACKGROUND/AIMS: Cholesterol gallstones contain both calcium and biliary proteins, but their respective roles in gallstone pathogenesis are unknown. We have studied the effects of calcium and a major biliary protein, anionic polypeptide fraction, on the process of cholesterol crystallization in bile. METHODS: Anionic polypeptide fraction was purified from human bile. Model bile composed of cholesterol, egg yolk lecithin and sodium taurocholate was prepared in a lipid concentration (18 mM, 37 mM, and 120 mM, respectively) simulating lithogenic human gallbladder bile. The crystallization process was observed by phase contrast light microscopy, and sequential separation of precipitable cholesterol structures by sucrose density gradient ultracentrifugation. RESULTS: Addition of calcium, or anionic polypeptide fraction alone, or both together did not influence the crystal observation time of bile (the time which elapsed from initiation of supersaturation to the first appearance of crystals). However, the rate and quantity of cholesterol precipitation and crystal formation were affected by both. Calcium increased in a dose-dependent manner the cholesterol monohydrate crystal mass before apparent equilibrium was reached. This effect was inhibited by anionic polypeptide fraction, which increased the amount of cholesterol within precipitable phospholipid vesicles, and decreased the rate of crystal formation. Fluorescence-labeled anionic polypeptide fraction revealed that anionic polypeptide fraction (with and without calcium) was primarily associated with vesicle aggregates. CONCLUSIONS: Our data demonstrate that calcium and anionic polypeptide fraction have opposing effects on the process of cholesterol crystallization and the resultant crystal mass without influencing the crystal observation time of bile. These findings suggest that biliary proteins, in addition to being crystallization effectors by themselves, may further influence cholesterol crystallization and gallstone formation by interacting with calcium and possibly other elements that coexist in bile.

A neurohormone regulating both methyl farnesoate synthesis and glucose metabolism in a crustacean.

Methyl farnesoate (MF) has been identified as a juvenile hormone-like compound in crustacea which has central roles in the regulation of development and reproduction. To study the regulation of MF synthesis, we isolated a neuropeptide which inhibits MF synthesis from the neurohemal organ-sinus gland X-organ complex of the spider crab Libinia emarginata. The primary structure of this neuropeptide has been determined. It has 72 amino acid residues (deduced molecular mass 8490.5 Da) with pyroglutamic acid at the N-terminus and NH3 at the C-terminus. It shares a high percentage of sequence identity with other sinus gland neuropeptids which form the unique family of CHH neuropeptides of crustacea. Activity studies showed that this neurohormone has dual effects: it inhibited MF synthesis in vitro and had hyperglycemic activity when injected into crabs.

Monitoring cholesterol crystallization from lithogenic model bile by time-lapse density gradient ultracentrifugation.

BACKGROUND/AIMS: Cholesterol crystallization in a dilute, bile salt-rich model bile is a multiphase process in which early filamentous crystals gradually transform to classical cholesterol monohydrate plates. The pertinence of similar transformations in more complex model systems or native bile is, however, unclear. The aim of the present study was to characterize and monitor cholesterol crystallization in a model bile of physiological relevance. METHODS: A supersaturated model bile was prepared with a lipid composition (18 mM cholesterol, 37 mM lecithin, 120 mM taurocholate) that was derived from analyzing 10 gallbladder biles from cholesterol gallstone patients. Cholesterol crystallization was followed by light and electron microscopy, and sequential density gradient analysis of cholesterol-containing precipitates. RESULTS: During cholesterol crystallization a reproducible sequence of events was recorded. First (T<18 h), cholesterol-rich vesicular and multilamellar structures (density 1.005-1.015 g/ml) were observed. Later, (T>60 h) filamentous, helical, tubular (density 1.015-1.04 g/ml) and plate-like (density 1.04-1.06 g/ml) cholesterol crystals appeared. The concentration of crystals increased gradually, while bilayer structures became desaturated with cholesterol and disappeared, and early crystal forms were replaced by plates. Eventually (T>25 days) only classical plate-like cholesterol monohydrate crystals were present. Exposure of cholesterol-containing precipitates to micellar (100 mM) deoxycholate dissolved the bilayer structures but not the crystals. CONCLUSIONS: These data demonstrate that cholesterol crystallization in a physiologically relevant model bile is a multiphase process consisting of a sequence of transitions from vesicular and multilamellar structures to early crystal forms and to classical plate-like cholesterol monohydrate crystals. These transitions are associated with increasing density and decreasing phospholipid content of cholesterol precipitates. We suggest that time-lapse density gradient ultracentrifugation is a useful method for investigating and quantitating the process of cholesterol crystallization and factors that influence this process in bile.

cDNA cloning of a mandibular organ inhibiting hormone from the spider crab Libinia emarginata.

Mandibular organs (MO) produce a crustacean juvenile hormone, methyl farnesoate (MF). MO activity is negatively regulated by factors, called mandibular organ inhibiting hormones (MOIHs), from the crustacean sinus gland X-organ complex in the eyestalks. Three MOIHs have been isolated previously from the spider crab Libinia emarginata and are characterized as members of the crustacean hyperglycemic hormone (CHH) neuropeptide family. In the research reported here, a full length cDNA sequence of 972 bp of a MOIH was isolated by screening a cDNA library constructed from the eyestalks of Libinia emarginata. This cDNA sequence encodes a preprohormone peptide with 137 amino acid residues, including a 26-amino acid long signal peptide, a 34-amino acid long precursor peptide, a dibasic peptide, the full length of 72-amino acid long MOIH, and a tri-peptide Gly-Lys-Lys which designates the potential amidation site at the C-terminus of the mature peptide.

Characteristics of gallbladder bile of infants and children.

In the past, biliary lipids of infants and children were studied in duodenal aspirates. This study was performed on original bile aspirated from the gallbladder. The analysis of lipids in bile demonstrated a significantly lower total lipid content in the bile of infants than in children (3.3 g/dl vs. 9.1 g/dl). The most prominent difference was demonstrated in the bile salt concentration (43.2 mM vs. 126.7 mM) and thereafter in the phospholipid content. Infants had a shorter nucleation time and a higher cholesterol saturation index than did children. These results may explain the increased tendency of infants to produce sludge and gallstones during total parenteral nutrition.

Lithogenicity of human bile is reduced by freezing and thawing.

We examined the effects of freezing and thawing upon the nucleation time and the distribution of cholesterol between micelles and vesicles in 9 human gallbladder and 7 hepatic biles. The nucleation time was significantly longer after freezing when compared to fresh samples (22.4 +/- 2.6 vs. 7.4 +/- 1.9 days, respectively). Concomitantly, a substantial shift of cholesterol from vesicles to micelles was noted, with the proportion of vesicular cholesterol decreasing from 26.5% +/- 6.0% in fresh biles to 8.6% +/- 2.3% after freezing. These effects were observed in all types of human biles, regardless of origin, cholesterol saturation or initial presence of cholesterol crystals, and were most notable after the first week of freezing. The decrease in vesicular cholesterol in all biles and the increase in nucleation time of gallbladder biles correlated with the time the samples had been in a frozen state. It is concluded that the lithogenic properties of human bile are not maintained during storage at -20 degrees C. Freezing results in a shift of cholesterol from vesicles to micelles and reduces the tendency of cholesterol to crystallize from bile samples.

Methyl farnesoate levels in male spider crabs exhibiting active reproductive behavior.

The concentration of methyl farnesoate (MF) in the hemolymph and its synthesis by the mandibular organs (MOs) were investigated to determine whether this compound is related to the differences in the size of the reproductive system and the mating behavior among male morphotypes of the spider crab, Libinia emarginata. Large-claw abraded males displayed mating behavior under competitive conditions. They have the largest reproductive systems, their MOs synthesize large amounts of MF in vitro, and the concentration of MF in their hemolymph is high. Small-claw abraded males displayed mating behavior with receptive females only when isolated. These smaller crabs have intermediate-sized reproductive systems, their MOs synthesize the most MF, and they have the highest circulating level of MF relative to their body size. The unabraded males did not display mating behavior; their reproductive systems are smaller; their MO activity is low, as is their circulating level of MF. The strong relationship between MF levels and the intensity of reproductive behavior suggests that MF may be one of the driving forces behind mating behavior in Crustacea.

Regional Differences in Methyl Farnesoate Production by the Lobster Mandibular Organ.

Visual examination of the mandibular organ (MO) from the lobster, Homarus americanus, disclosed two distinct morphological regions: a fan-folded region along one edge of the gland, and a smooth, unfolded region comprising the rest of the gland. Because MOs produce methyl farnesoate (MF), the MF content of both regions was measured. In freshly dissected glands, more than 95% of the MF was found in the fan-folded region of the gland. In MO sections incubated with [3H-methyl]methionine (a radiolabeled precursor of MF), more than 90% of MF synthesis was found in the fan-folded region. Eyestalk ablation, a procedure that increases MO activity, caused the MF content of MOs to increase more than 130-fold, but had little effect on the regional distribution of MF. Histological observations indicated that these two regions had different cellular compositions. The fan-folded region contained two cell types (A and B). The A cells were mitotically active and appeared to be undifferentiated. The B cells contained a large number of small vacuoles. The unfolded region was largely composed of a third cell type (C). The C cells were large and morphologically complex, containing many mitochondria and large vacuolar-like structures. They contained relatively few small vacuoles. On the basis of appearance and location, B cells appear to be the likely site of MF synthesis. The physiological importance of C cells is unknown.

Distinct Reproductive Types of Male Spider Crabs Libinia emarginata Differ in Circulating and Synthesizing Methyl Farnesoate.

Levels of methyl farnesoate in the blood and in vitro rates of methyl farnesoate synthesis by the mandibular organ were investigated to determine whether this compound is related to the differences in morphology and reproductive states of distinct types of male spider crabs described by Homola et al. (1992) in winter populations. Three male types, selected from a summer population, were investigated in detail: (1) males with relatively large propoduses (claws) and worn exoskeletons (abraded), (2) males with relatively large propoduses and exoskeletons covered with epicuticle (unabraded), and (3) males with small propoduses and unabraded exoskeletons (small). All males examined had sperm, but abraded males, identical in propodus and body size to unabraded males, had a reproductive system that weighed twice as much. Large-clawed unabraded males had relatively small reproductive systems. Small-clawed males possessed a small reproductive system. Abraded males possessed larger mandibular organs, containing almost twice the total protein, and their mandibular organs synthesized significantly more methyl farnesoate in vitro than did the other types of males. Circulating levels of methyl farnesoate, in the hemolymph of the abraded males, were more then twice as high as the levels detected in any other type of male. The strong relationship between methyl farnesoate levels, male morphology, and reproductive system development calls for further studies on the role of methyl farnesoate in the regulation of reproduction and morphogenesis in male crustaceans.

Effect of phospholipids and their molecular species on cholesterol solubility and nucleation in human and model biles.

Much research in the pathophysiology of gall stones has been devoted to various molecular species of bile salts. Recent findings have shown the importance of phospholipids in biliary pathophysiology. In the present study the addition of increasing doses of egg lecithin to human and model biles progressively prolonged the nucleation time. Concurrently biliary cholesterol was shifted from the vesicular to the non-vesicular carrier(s) while the cholesterol/phospholipid ratio of the remaining vesicles was progressively lowered. Model bile solutions of identical lipid concentration were prepared using phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine as the only phospholipid. With phosphatidylethanolamine most of the cholesterol was shifted to the vesicular carrier while phosphatidylserine shifted most of the cholesterol to the non-vesicular carrier(s). With phosphatidylcholine the cholesterol was distributed in both carriers. Phosphatidyl choline species composed of various acyl fatty acids in the sn-1 and sn-2 positions were used as the sole phospholipid in otherwise identical model bile solutions. With palmitic acid in the sn-1 position and arachidonic acid in the sn-2 position most of the cholesterol was found in the non-vesicular carrier. When stearic acid was used in sn-2 position instead of arachidonic acid most of the cholesterol was found in the vesicular carrier. These and other variations in phospholipid molecular species shifted cholesterol among its carriers and also modified the nucleation time of model biles. Most of these effects were also found upon addition of the various phospholipid species to human biles. These findings show the importance of phospholipid species in biliary pathophysiology and may be useful when trying to manipulate cholesterol carriers and solubility in bile.

Gliomatosis cerebri: a case report.

We report a case of gliomatosis cerebri in a 46-year-old woman with five-year history of seizures and psychiatric disturbance. There were also two episodes of lethargy, disorientation, and headache which cleared promptly with Mannitol. A 3rd episode terminated in her death. Remarkably, between the episodes of presumed increased intracranial pressure, the neurologic examination was normal except for the patient's denial of her illness. Postmortem examination revealed the entire right cerebral hemisphere to be enlarged and infiltrated by cells resembling astrocytes. The clinical signs, symptoms, and controversial histopathologic features of this rare entity are discussed.

D-xylose absorption test. Urine or blood?

The D-xylose absorption test has been used during the last four decades for evaluation of malabsorption in the small intestine. However, some disagreement still exists about the recommended method of performing this test: the 1-hr blood test, the 5-hr urine test, or both. We evaluated the test by performing 125 combined blood and urine tests in 111 patients. Normal xylose absorption was recorded in both blood and urine in 71 tests (group A, 56.8%). Abnormal test results in both blood and urine were recorded in 29 patients (group B, 23.2%). Only one patient had a pathological blood value and normal xylose excretion in the urine. Twenty-four patients (group D, 19.2%) had normal 1-hr blood xylose (greater than 25 mg/100 ml) with abnormal 5-hr urine xylose (less than 4.5 g/5 hr). Fat and/or bile salt malabsorption were documented in 21 patients (87.5%) of this group using stool fat analysis and the [14C]cholylglycine breath test. These data suggest that in adults the 5-hr urine collection more accurately reflects intestinal absorption in comparison with the 1-hr blood value.

Methyl farnesoate in the prawn Macrobrachium rosenbergii: synthesis by the mandibular organ in vitro, and titers in the hemolymph.

1. The mandibular organ of Macrobrachium rosenbergii was identified and cultured in the presence of [methyl-3H]-methionine. 2. Radiolabelled methyl farnesoate was extracted and quantitated from cultured glands and culture media of both males and females: variation in synthesis rates was observed (1558-52,652 DPM/hr per gland) between individual prawns. 3. No significant incorporation of isotope into hexane-extractable material was observed in any other tissue cultured. 4. The concentration of methyl farnesoate in the hemolymph of M. rosenbergii was determined using normal phase HPLC with cis-trans (non-biological isomer) as an internal standard. 5. Hemolymph from males contained 17.3 +/- 9.5 ng/ml methyl farnesoate. 6. Female hemolymph contained 24 +/- 13.1 ng/ml methyl farnesoate.

Can glutathione-S-transferases function as intracellular heme carriers?

The possibility that glutathione-S-transferases can serve as heme carriers in cells was studied via the following two characteristics: the ability to bind hemin reversibly and the coordination between heme and glutathione-S-transferases level in the cell. two erythroleukemic cell lines that can be induced to synthesize hemoglobin were studied, K-562 and Friend murine erythroleukemia cells. It was found that hemin-associated glutathione-S-transferase tends to lose its native structure as expressed by partial irreversible inhibition of glutathione conjugation activity. In K-562 cells, a small increase in heme synthesis was induced, but under no condition could glutathione-S-transferase be elevated. In addition, introduction of high hemin from without caused large hemoglobin production but did not induce changes in the glutathione-S-transferase content. Dimethyl sulfoxide-induced Friend murine erythroleukemia cells synthesized a large amount of endogenous hemin that had to be transported from the mitochondria for hemoglobin synthesis. Although a concomitant increase in glutathione-S-transferase level (20-40%) was observed, it was only short-lived, unlike hemin, which continued to increase. These data indicate a lack of correlation between glutathione-S-transferase and hemin or hemoglobin levels. Finally, dimethyl sulfoxide-induced cells were treated with succinyl acetone to inhibit heme synthesis. These cells showed the same increased levels and time-dependent pattern of glutathione-S-transferase as untreated cells. A similar phenomenon was observed when different substrates were used to measure the activities of glutathione-S-transferases. These results raise doubts about the possibility of glutathione-S-transferases functioning as heme carriers in cells.

Quaternary neurosyphilis in a Haitian man with human immunodeficiency virus infection.

A 22-year-old Haitian man had a 15-month course of progressive meningitis accompanied by multiple cerebral infarcts. Multiple areas of stenosis and occlusion in all branches of the circle of Willis, and hypertrophy of collateral perforating vessels at the base of the brain in a "puff of smoke" appearance typical of moyamoya disease were seen on cerebral angiogram 5 months before the patient died. At autopsy, the patient had meningovascular syphilis and a necrotizing encephalitis with massive treponemal invasion of the brain, the pathology of late-stage degenerative, "quaternary", neurosyphilis. The patient was also infected with human immunodeficiency virus (HIV). Retrovirus-like particles 100 nm in diameter with dense cores were seen by electron microscopy. Nucleic acid obtained from the patient's brain contained sequences homologous to HIV DNA as determined by dot blot hybridization. The moyamoya-like radiologic appearance of neurosyphilis has not been previously described. The autopsy finding of quaternary neurosyphilis in a patient with HIV infection supports the hypothesis that retrovirus may alter the natural history of syphilitic infection.