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We developed a method to follow the metabolic fate of [(14)C]-labeled Euglena gracilis protein amino acids in Aedes aegypti mosquitoes under three different adult nutritional regimes. Quantitative analysis of blood meal protein amino acid metabolism showed that most of the carbon of the amino acids was either oxidized to CO(2) or excreted as waste. Under the three different adult nutritional regimes, no significant differences in the metabolism of amino acids were found, which indicated that the female A. aegypti mosquitoes possess a substantial capacity of maintaining metabolic homeostasis during a gonotrophic cycle. The amount of maternal glycogen and lipid after egg laying were significantly lower in the mosquitoes that underwent a partial starvation before a blood meal and/or starvation after the blood meal. The content of egg lipid or protein or the number of eggs laid did not show a significant difference among the three different regimes, which indicates that stable fecundity of A. aegypti under the partial starvation before a blood meal and/or starvation after the blood meal seemed to result from a trade-off between current fecundity and future survival after the eggs laid. The methods described in this paper can be applied to a wide range of questions about the effects of environmental conditions on the utilization of blood meal amino acids.

The octahedral Ru(II) amine complexes [TpRu(L)(L')(NH(2)R)][OTf] (L = L' = PMe(3), P(OMe)(3) or L = CO and L' = PPh(3); R = H or (t)Bu) have been synthesized and characterized. Deprotonation of the amine complexes [TpRu(L)(L')(NH(3))][OTf] or [TpRu(PMe(3))(2)(NH(2)(t)Bu)][OTf] yields the Ru(II) amido complexes TpRu(L)(L')(NH(2)) and TpRu(PMe(3))(2)(NH(t)Bu). Reactions of the parent amido complexes or TpRu(PMe(3))(2)(NH(t)Bu) with phenylacetylene at room temperature result in immediate deprotonation to form ruthenium-amine/phenylacetylide ion pairs, and heating a benzene solution of the [TpRu(PMe(3))(2)(NH(2)(t)Bu)][PhC(2)] ion pair results in the formation of the Ru(II) phenylacetylide complex TpRu(PMe(3))(2)(C[triple bond]CPh) in90% yield. The observation that [TpRu(PMe(3))(2)(NH(2)(t)Bu)][PhC(2)] converts to the Ru(II) acetylide with good yield while heating the ion pairs [TpRu(L)(L')(NH(3))][PhC(2)] yields multiple products is attributed to reluctant dissociation of ammonia compared with the (t)butylamine ligand (i.e., different rates for acetylide/amine exchange). These results are consistent with ligand exchange reactions of Ru(II) amine complexes [TpRu(PMe(3))(2)(NH(2)R)][OTf] (R = H or (t)Bu) with acetonitrile. The previously reported phenyl amido complexes TpRuL(2)(NHPh) [L = PMe(3) or P(OMe)(3)] react with 10 equiv of phenylacetylene at elevated temperature to produce Ru(II) acetylide complexes TpRuL(2)(C[triple bond]CPh) in quantitative yields. Kinetic studies indicate that the reaction of TpRu(PMe(3))(2)(NHPh) with phenylacetylene occurs via a pathway that involves TpRu(PMe(3))(2)(OTf) or [TpRu(PMe(3))(2)(NH(2)Ph)][OTf] as catalyst. Reactions of 1,4-cyclohexadiene with the Ru(II) amido complexes TpRu(L)(L')(NH(2)) (L = L' = PMe(3) or L = CO and L' = PPh(3)) or TpRu(PMe(3))(2)(NH(t)Bu) at elevated temperatures result in the formation of benzene and Ru hydride complexes. TpRu(PMe(3))(2)(H), [Tp(PMe(3))(2)Ru[double bond]C[double bond]C(H)Ph][OTf], [Tp(PMe(3))(2)Ru=C(CH(2)Ph)[N(H)Ph]][OTf], and [TpRu(PMe(3))(3)][OTf] have been independently prepared and characterized. Results from solid-state X-ray diffraction studies of the complexes [TpRu(CO)(PPh(3))(NH(3))][OTf], [TpRu(PMe(3))(2)(NH(3))][OTf], and TpRu(CO)(PPh(3))(C[triple bond]CPh) are reported.

The venoms of Loxosceles spiders cause severe dermonecrotic lesions in human tissues. The venom component sphingomyelinase D (SMD) is a contributor to lesion formation and is unknown elsewhere in the animal kingdom. This study reports comparative analyses of SMD activity and venom composition of select Loxosceles species and representatives of closely related Haplogyne genera. The goal was to identify the phylogenetic group of spiders with SMD and infer the timing of evolutionary origin of this toxin. We also preliminarily characterized variation in molecular masses of venom components in the size range of SMD. SMD activity was detected in all (10) Loxosceles species sampled and two species representing their sister taxon, Sicarius, but not in any other venoms or tissues surveyed. Mass spectrometry analyses indicated that all Loxosceles and Sicarius species surveyed had multiple (at least four to six) molecules in the size range corresponding to known SMD proteins (31–35 kDa), whereas other Haplogynes analyzed had no molecules in this mass range in their venom. This suggests SMD originated in the ancestors of the Loxosceles/Sicarius lineage. These groups of proteins varied in molecular mass across species with North American Loxosceles having 31–32 kDa, African Loxosceles having 32–33.5 kDa and Sicarius having 32–33 kDa molecules.

The objective of this study was to characterize the transfer of cholesterol from Manduca sexta larvae fat body to high-density lipophorin. [3H]–Cholesterol–labeled fat body was incubated with lipophorin under different conditions and cholesterol transfer was determined. Transfer rate exhibited a hyperbolic dependency on lipophorin concentration with an apparent Km of 3.6 mg/ml, which is consistent with either an aqueous diffusion mechanism of cholesterol transfer or a receptor-mediated process. Several results, including the high Km, the high activation energy, and the lack of Ca2+ dependence favor aqueous diffusion model. In addition, anti-lipid transfer particle antibodies had only a small inhibitory effect, suggesting it is not involved in cholesterol transfer. However, the transfer was inhibited in the presence of suramin, which would be consistent with a receptor-mediated process. The effects of suramin may be complex because it can change membrane properties when bound to the lipophorin receptor and affect the rate of cholesterol desorption. The preponderance of data suggests that the export of cholesterol from fat body to lipophorin follows a simple aqueous diffusion pathway. Although we cannot completely exclude some contribution from a receptor-mediated pathway, it seems that if such a pathway were present, it represents a minor route.

Midgut extracts from Aedes aegypti females exhibited hydrolytic activities against synthetic substrates for carboxypeptidase A, carboxyopeptidase B and leucine-aminopeptidase. The three activities showed a broad pH optimum, with maximum activities at pH between 6.5 and 8.5. Enzymatic activities were further characterized by testing the effects of a variety of protease inhibitors. Captopril and 1-10-phenantroline inhibited the activities of carboxypeptidases A and B, while leuhistin, amastatin and bestatin inhibited aminopeptidase activity. Exopeptidase activities were induced by a blood meal and the highest activities were found during the peak of trypsin activity, about 20–24 h after feeding. An amino acid meal failed to induce significant increases in any of the three exopeptidase activities. The amounts of exopeptidase activities induced were proportional to the protein concentration of the meal. The addition of soy-trypsin inhibitor to the protein meal blocked the post-feeding induction of exopeptidases. The features of the induction of synthesis of the three exopeptidase activities resembled the induction of synthesis of late trypsin during the second phase of digestion.  2002 Elsevier Science Ltd. All rights reserved.

The objective of this study was to characterize the transfer of diacylglycerol (DAG) and cholesterol from larval Bombyx mori lipophorin to ovarioles. Transfer studies were carried out by incubating pupal ovarioles (5-day) with [3H]-cholesterol and [3H]-DAG-labeled lipophorin under different conditions. Transfer of both cholesterol and DAG exhibited hyperbolic dependency on lipophorin concentration with apparent Km values of 0.83 ± 0.17 mg/ml and 0.74 ± 0.16 mg/ml, respectively. Pretreatment of ovarioles with anti-lipid transfer particle (LTP) IgG significantly inhibited transfer of labeled DAG to ovarioles (75%) and not cholesterol. Injection of B. mori pupae (day 4) with anti-LTP IgG significantly affected the weight (65%), number of eggs (49%), amount of lipid (74%), and protein (65%) of the adult ovaries. Matured eggs had a very faint yellow color and deformed shape compared to controls. The inhibitory effect demonstrates the active role LTP plays in growth of ovaries, development, and oogenesis. The effect on vitellogenin shortage on egg development and maturation was determined by implanting ovaries in male recipients that lack vitellogenin. An 80% decline in egg production was observed. However, the mature eggs were normal in shape, color, and lipid content. Thus, restricting lipid or protein delivery to developing ovaries would dramatically affect choriogenesis.

Comparison of the genomes and proteomes of the two diptera Anopheles gambiae and Drosophila melanogaster , which diverged about 250 million years ago, reveals considerable similarities. However, numerous differences are also observed; some of these must reflect the selection and subsequent adaptation associated with different ecologies and life strategies. Almost half of the genes in both genomes are interpreted as orthologs and show an average sequence identity of about 56%, which is slightly lower than that observed between the orthologs of the pufferfish and human (diverged about 450 million years ago). This indicates that these two insects diverged considerably faster than vertebrates. Aligned sequences reveal that orthologous genes have retained only half of their intron/exon structure, indicating that intron gains or losses have occurred at a rate of about one per gene per 125 million years. Chromosomal arms exhibit significant remnants of homology between the two species, although only 34% of the genes colocalize in small “microsyntenic” clusters, and major interarm transfers as well as intra-arm shuffling of gene order are detected.

While providing osteopathic manipulative treatment to patients with Parkinson's disease at the clinic of the New York College of Osteopathic Medicine of New York Institute of Technology, physicians noted that these patients may exhibit particular cranial findings as a result of the disease. The purpose of this study was to compare the recorded observations of cranial strain patterns of patients with Parkinson's disease for the detection of common cranial findings. Records of cranial strain patterns from physician-recorded observations of 30 patients with idiopathic Parkinson's disease and 20 age-matched normal controls were compiled. This information was used to determine whether different physicians observed particular strain patterns in greater frequency between Parkinson's patients and controls. Patients with Parkinson's disease had a significantly higher frequency of bilateral occipitoatlantal compression (87% vs. 50%; P.02) and bilateral occipitomastoid compression (40% vs. 10%; P.05) compared with normal controls. Over subsequent visits and treatments, the frequency of both strain patterns were reduced significantly (occipitoatlantal compression, P.01; occipitomastoid compression, P.05) to levels found in the control group.

Lipophorin is the major hemolymph protein responsible for lipid transport between tissues of insects. Lipophorins from several insect species in order Diptera (the fruit fly Drosophila melanogaster from the suborder Brachycera, the mosquito Aedes aegypti; the phantom midges Chaoborus maximus and minimus; the black fly Simulium vittatum; the crane fly Nephrotoma abbreviata, all from the suborder Nematocera) were isolated and characterized. All lipophorins consisted of two protein subunits of approximately 240 and 75 kDa each. The density of each lipophorin was in the high-density lipoprotein range (1.112 to 1.128 g/ml). The predominant neutral lipid carried by lipophorin from insects belonging to the infraorder Culicomorpha was triacylglycerol. Lipophorin from the crane fly Nephrotoma abbreviata, which belongs to the infraorder Tipulomorpha, carried approximately equivalent amounts of diacylglycerol and triacylglycerol. Lipophorin from D. melanogaster was found to carry diacylglycerol as the predominant neutral lipid.

The ability of 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) and methyl-beta-cyclodextrin (MbetaCD) to promote cholesterol efflux from [3H]cholesterol-labeled larval Manduca sexta fat body and midgut was tested. In fat body, both beta-cyclodextrins induced a two-phase efflux of cholesterol. The first rapid phase depended on cyclodextrin concentration and was more rapid for MbetaCD than for HPbetaCD. The second, slower, phase was independent of cyclodextrin concentration and type. In midgut, only the concentration-dependent phase was observed; the rate constants are approximately 85% slower than for fat body. In both cases, a low activation energy for transfer was observed, consistent with a collision mechanism where cyclodextrin interacts directly with cholesterol in plasma membrane to affect transfer. In fat body, the second slower phase is suggestive of a second pool of exchangeable cholesterol and most likely represents transfer of cholesterol from internal membranes or different lateral domains of the plasma membrane. The lack of this second phase in midgut suggests that midgut has only a single pool of exchangeable cholesterol. Although the rates are somewhat different, the overall kinetic pattern for cyclodextrin-mediated cholesterol transfer in insect fat body closely resembles that for vertebrate cells, while the single pool behavior of the midgut is not found in vertebrate cells.

In Manduca sexta larvae, radioactive free cholesterol is absorbed directly from the midgut into mucosal cells where it is stored both in the free form (87% in males and 93% in females) and esterified form (13% in males and 7% in females). Subsequently, cholesterol is transported to fat body via lipophorin in the hemolymph exclusively in the free form. In fat body, the distribution of cholesterol between the free and esterified form varied significantly between genders and developmental stages. Except for the larval stage, males and females were able to store cholesterol in both free and esterified forms in the fat body and in the adult stage cholesterol ester accounted for more than 75% of the stored cholesterol. Placement of radioactive cholesterol at different locations in the gut—foregut, midgut, and hindgut—demonstrated that the midgut is the site where cholesterol is absorbed and released into the hemolymph. Cholesterol-labeled lipophorin injected into larval hemolymph was cleared from the hemolymph with a half-life of 10.2 h. After 17 h, most of the cleared radioactivity was recovered in the fat body (38%). Arch. Insect Biochem. Physiol. 49:167–175, 2002. © 2002 Wiley-Liss, Inc.

To determine whether patients with Parkinson's disease who are enrolled in a resistance training program can gain strength similar to that of normal control subjects and whether these gains in strength would improve their gait function.Subjects included 14 patients with mild-to-moderate Parkinson's disease of either gender and six normal control subjects of similar age. The training consisted of an 8-wk course of resistance training twice per week, focused primarily on the lower limbs. The primary outcome measures consisted of exercise performance monitoring and quantitative gait analysis before and after the training course.Both the patients with Parkinson's disease and normal control subjects significantly increased their performance with resistance training. Subjects with Parkinson's disease had gains in strength similar to those of normal elderly adults. Patients with Parkinson's disease also had significant gains in stride length, walking velocity, and postural angles compared with pretreatment values.Patients with mild-to-moderate Parkinson's disease can obtain increases in performance or strength similar to that of normal adults of the same age in a resistance training program. Resistance training can produce functional improvements in gait and may, therefore, be useful as part of a physical rehabilitation and/or health maintenance program for patients with Parkinson's disease.

In this work, we describe the ability of living hemocytes from an insect ( Manduca sexta, Lepidoptera) to hydrolyze extracellular ATP. In these intact cells, there was a low level of ATP hydrolysis in the absence of any divalent metal (8.24 ± 0.94 nmol of P i /h × 10 6 cells). The ATP hydrolysis was stimulated by MgCl 2 and the Mg 2+ -dependent ecto-ATPase activity was 15.93 ± 1.74 nmol of P i /h × 10 6 cells. Both activities were linear with cell density and with time for at least 90 min. The addition of MgCl 2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.33 mM MgCl 2 . This stimulatory activity was not observed when Ca 2+ replaced Mg 2+ . The apparent K m values for ATP −4 and Mg-ATP 2− were 0.059 and 0.097 mM, respectively. The Mg 2+ -independent ATPase activity was unaffected by pH in the range between 6.6 and 7.4, in which the cells were viable. However, the Mg 2+ -dependent ATPase activity was enhanced by an increase of pH. These ecto-ATPase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A 1 , ouabain, furosemide, vanadate, sodium fluoride, tartrate, and levamizole. To confirm the observed hydrolytic activities as those of an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid), as well as suramin, an antagonist of P 2 -purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg 2+ -independent and the Mg 2+ -dependent ATPase activities to different extents. Interestingly, lipopolysaccharide, a component of cell walls of gram-negative bacteria that increase hemocyte aggregation and phagocytosis, increased the Mg 2+ -dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg 2+ -independent ecto-ATPase activity.

The pathway for the synthesis of diacylglycerol in larval Manduca sexta midgut was studied. Fifth instar larvae were fed with [9, 10-(3)H]-oleic acid-labeled triolein and the incorporation of the label into lipid intermediates was analyzed as a function of time. The results showed that the triacylglycerol was hydrolyzed to fatty acids and glycerol in the midgut lumen. In midgut tissue, the labeled fatty acids were rapidly incorporated into phosphatidic acid, diacylglycerol and triacylglycerol, but no significant labeling of monoacylglycerol was observed. Dual-labeling experiments were performed in order to characterize the kinetics of diacylglycerol biosynthesis in the midgut, its incorporation into hemolymph lipophorin and its clearance from hemolymph. The results were best described by a model in which the rate-limiting step in diacylglycerol biosynthesis was the uptake of fatty acid from the lumen of the midgut. Once in the cell the fatty acid was rapidly incorporated in phosphatidic acid and diacylglycerol. Diacylglycerol was converted to triacylglycerol or exported into hemolymph. The interconversion of diacylglycerol and triacylglycerol was fairly rapid, suggesting that triacylglycerol serves as a reservoir from which diacylglycerol can be produced. This mechanism permits the cell to maintain a low steady-state concentration of diacylglycerol and yet efficiently absorb fatty acids from the lumen of the midgut.

In this paper we assessed the ability of modulators of the activity of glycogen phosphorylase b from the fat body of larval Manduca sexta to stabilize the enzyme against thermal denaturation. This approach has allowed us to distinguish between modulators that stabilize the enzyme, presumably through some conformational effect, from those that do not affect thermal stability. For example, 5'-AMP and 5'-IMP are both positive modulators of the enzyme and the K(m)s for AMP and IMP were similar, 0.71 and 1.09 mM, respectively. However, the V(max) for AMP (123 nmol/mg/min) was 10 times higher than the value found for IMP (12.5 nmol/mg/min) and AMP increased the thermal stability of glycogen phosphorylase b, however IMP did not increase the enzyme's thermal stability. Indeed, IMP decreased both the allosteric activation of the enzyme by AMP and the thermal protection conferred by AMP. The allosteric inhibitors ADP and ATP, which in vertebrate phosphorylase bind to the same site as AMP, both increased the thermal stability of the enzyme, however with less efficiency than AMP. Inorganic phosphate increased thermal stability, but glycogen and amylose did not. Glycerol, at 600 mM, protected the enzyme against thermal inactivation, whereas sorbitol at the same concentration did not show any effect. Among the polyols tested, trehalose was the most effective in conferring thermal stability. In fact, in the presence of 20 mM AMP and 600 mM trehalose, 90% of the enzyme activity remained after 20 min at 60 degrees C.

Lipophorin binding to the midgut of Manduca sexta larvae was characterized in a midgut membrane preparation, using iodinated larval high-density lipophorin ((125)I-HDLp-L). The iodination procedure did not change the affinity of the preparation for lipophorin. In the presence of increasing concentrations of membrane protein, corresponding increases in lipophorin binding were observed. The time-course of lipophorin binding to the membranes was affected by the lipophorin concentration in the medium, and at a low lipoprotein concentration, a longer time was required for equilibrium to be reached. The specific binding of lipophorin to the midgut membrane was a saturable process with a K(d) = 1.5+/-0.2x10(-7) M and a maximal binding capacity = 127+/-17 ng lipophorin/microg of membrane protein. Binding did not depend on calcium, was maximal around pH 5.5, was strongly inhibited by an increase in the ionic strength, and abolished by suramin. However, suramin did not completely displace lipophorin that was previously bound to the membrane preparation. The lipid content of the lipophorin did not significantly affect the affinity of the membrane preparation for lipoprotein.

The study of the changes in the steady-state levels of the early trypsin (ET) messenger RNA (mRNA) was used as a sensitive assay for measuring the effects of recombinant juvenile hormone esterase (rJHE) on juvenile hormone (JH)-dependent gene expression in mosquitoes. ET is a female-specific protease present in the midgut of the yellow fever mosquito Aedes aegypti during the first few hours after ingestion of a blood meal. Transcription of the early trypsin gene is part of the normal postemergence maturation of the midgut in the adult female, and it is regulated by JH. JHE was cloned from Heliothis virescens and expressed in a baculovirus vector. Injection of rJHE into mosquitoes resulted in an increase of JHE activity in the haemolymph. Injection of rJHE into newly emerged adult females delayed the normal increase in steady-state levels of ET mRNA observed in controls. Topically applied methoprene (a JH analogue) reversed the effect of rJHE. Injection of increasing concentrations of rJHE into 3-day-old unfed females resulted in a dose-dependent decrease in the steady-state levels of ET mRNA after 24 h. The effect of rJHE was transient, once the enzyme was cleared (72 h after injection), the steady-state levels of ET mRNA were restored. The injection of rJHE is an effective tool for modifying JH-dependent expression of the early trypsin gene in mosquitoes.

Patients with Parkinson's disease exhibit a variety of motor deficits which can ultimately result in complete disability. The primary objective of this study was toquantitatively evaluate the effect of osteopathic manipulative treatment (OMT)on the gait of patients with Parkinson's disease. Ten patients with idiopathicParkinson's disease and a group of eight age-matched normal control subjects ~eresubjected to an analysis of gait before and after a single session of an OMT protocol. A separate group of 10 patients with Parkinson's disease was given asham-control procedure and tested in the same manner. In the treated group ofpatients with Parkinson's disease, statistically significant increases were observedin stride length, cadence, and the maximum velocities of upper and lower extremitiesafter treatment. There were no significant differences observed in the controlgroups. The data demonstrate that a single session of an OMT protocol hasan immediate impact on Parkinsonian gait. Osteopathic manipulation may be aneffective physical treatment method in the management of movement deficits inpatients with Parkinson's disease.

Thrombin is inhibited by its cognate plasma inhibitor antithrombin, through the formation of covalent thrombin-antithrombin (TAT) complexes that are found as ternary complexes with vitronectin (VN-TAT). To determine whether the metabolism of VN-TAT ternary complexes is different from that previously reported for binary TAT complexes, plasma clearance studies were done in rabbits using human VN-TAT. 125I-VN-TAT was shown to be cleared rapidly from the circulation (t1/2alpha = 3.8 min) in a biphasic manner mainly by the liver. 125I-TAT had a similar initial clearance (t1/2alpha = 5.3 min) but had a significantly faster beta-phase clearance (t1/2beta = 42.8 min versus 85.4 min for VN-TAT; p = 0.005). Protamine sulfate and heparin abolished the rapid initial alpha-phase of 125I-VN-TAT clearance and reduced its liver-specific association and in vivo degradation. Heparin also reduced the alpha-phase clearance of 125I-TAT and was associated with the appearance of high molecular weight complexes, suggesting enhanced complex formation between VN and TAT. 125I-VN-TAT binding to HepG2 cells was reduced by competition with VN-TAT or heparin but to a much lesser extent in the presence of TAT. The binding of VN-TAT to HepG2 cells was not inhibited by competition with the low density lipoprotein receptor-related protein ligand, methylamine-alpha2-macroglobulin. 125I-VN-TAT binding was also inhibited by treating HepG2 cells with heparinase or by growing the cells in the presence of beta-D-xyloside. Finally, both heparin and chloroquine, but not methylamine-alpha2-macroglobulin, reduced the internalization and degradation of VN-TAT by HepG2 cells. Taken together, these data indicate the importance of VN in TAT metabolism and demonstrate that VN-TAT binds to liver-associated heparan sulfate proteoglycans, which mediate its internalization and subsequent intracellular degradation.

The dynamic tear test is used extensively for characterization of the fracture toughness properties of both ferrous and nonferrous metals. This report describes a method for dynamic tear testing of titanium plate using a nonstandard 1-inch dynamic tear specimen. The dimensions and preparation of the specimen, a description of the apparatus and details of the testing procedure are given. The results of dynamic tear tests demonstrated that the nonstandard specimen accurately measures the fracture energy of titanium alloy plate. Use of this method is recommended for characterization of the fracture toughness properties of alpha-beta titanium alloys in cases of limited material availability.

wound bed. As the thermoplastic cools, it becomes inflexible and maintains the shape of the graft. Once hardened, it can be trimmedwith scissors to extend 5 mm beyond the graft margins. Next, a piece of nonadherent dressing covered in ointment is applied to the graft, and the thermoplastic dressing is sutured or stapled into place (Figs 2 and 3). An absorbent dressing can be placed over the thermoplastic dressing. The thermoplastic bolster is removed in 5 to 7 days. In summary, the thermoplastic dressing increases surface adherence between a full-thickness skin graft and its recipientwoundbed.Once sutured into place, it immobilizes the graft against mechanical forces. One of its major advantages is that the moldable plastic can be shaped to fit the convex and concave surfaces of the nose and ear. In contrast to traditional gauze bolsters, the perforated thermoplastic dressing allows the patient to shower normally. A potential complication is the possibility of contact dermatitis in sensitive individuals. Expense may be another disadvantage based on the size, color, and number of dressings purchased. A package of 10 (33 3-in), ivory thermoplastic splints costs $46. In our clinic, we have not observed graft failure or any other adverse outcome related to this dressing. We have found it tobeapractical tool forsecuring full-thickness skingrafts.

During experiments to identify putative hepatic receptors for thrombin-antithrombin (TAT) complexes, a 45-kDa protein was identified by ligand blotting. Following gel purification, amino acid sequencing revealed the 45-kDa TAT-binding polypeptide to be cytokeratin 18 (CK18). The presence of CK18 on the surface of intact rat hepatoma cells was demonstrated by binding of 125I-anti-CK18 antibodies. Anti-CK18 antibodies reduced the binding and internalization of 125I-TAT by rat hepatoma cells. Immunocytochemical analysis, to determine the location of CK18 in vivo, revealed a periportal gradient of CK18 staining; with hepatocytes around the portal triads demonstrating striking pericellular staining. In addition, anti-CK18 IgG associated with perfused livers to a significantly greater extent than preimmune IgG. Taken together, these data provide evidence that CK18 is found on the extracellular surface of hepatocytes and could play a role in TAT removal. Finally, these data, in conjunction with recent reports of CK8 (Hembrough, T. A., Li, L., and Gonias, S. L. (1996) J. Biol. Chem. 271, 25684-25691) and CK1 cell membrane surface expression (Schmaier, A. H. (1997) Thromb. Hemostasis 78, 101-107), indicate a novel role for these proteins as putative cellular receptors or cofactors to cellular receptors.

Canatoxin is a toxic protein isolated from the jackbean, Canavalia ensiformis. The toxin injected intraperitoneally is lethal for mice and rats; however, it is inactive if given orally. In this study. Manduca sexta (L.) (Lepidoptera), Schistocerca Americana (Drury) (Orthoptera), Drosophila melanogester (L.) (Diptera), Aedes aegypti (L.) (Diptera), Rhodnius prolixus (Stal)(Hemiptera), and Callosobruchus maculatus (F.) (Coleoptera) were fed on canatoxin- containing diets. No effects were seen in M. sexta, S. Americana, D. melanogaster or A. aegypti . No traces of canatoxin were found in their feces, suggesting that the protein was digested completely by these insects, which characteristically have a trypsin-based digestion. In contrast, canatoxin was lethal for insects displaying cathepsin-based digestion. Thus, for C. maculatus , a diet containing 0.25% wt:wt canatoxin caused complete inhibition of larval growth. When R. prolixus were fed on canatoxin, 2 effects were seen: impairment of water excretion and increased lethality 48-96 h after feeding. The lethal effect of canatoxin in R. prolixus was blocked partially or completely when the digestion of the toxin by R. prolixus midgut enzymes was impaired. The data showed that canatoxin is highly toxic when ingested by some species of insects but not affecting others, probably in correlation with the characteristics of the digestive process of the insect.

Unilateral arthritis was produced in rats by use of methylated bovine serum albumin in a model of antigen-induced arthritis. The progression of arthritis was measured by computerized motion analysis, bilateral joint circumference, voluntary extension force of the hindlegs, and length of ankle extension. Animals with induced arthritis were assigned to treated and untreated groups on the basis of approximately equal deficits by the parameters measured. A third group of rats, which did not have arthritis induced and received no treatment, served to establish mean normal parameters. Modified techniques of muscle energy, passive movement of the ankle and knee, and passive myofascial stretch were applied to the animals, and the animals were exercised in a mechanized exercise wheel. Parameters associated with gait were examined by computerized motion analysis of walking. Animals treated with manipulation and exercise improved significantly relative to untreated animals with . antigen-induced arthritis in vertical ankle lift, ankle-based and foot-based stride lengths, knee circumference, and normalized extension of the ankle. The results demonstrate that the parameters identified can be used to detect functional deficits and significant improvement from those deficits can be derived from a nonpharmacologic treatment paradigm that includes osteopathic manipulation and exercise in an animal model of arthritis. These parameters may be useful in the identification of the relative benefits of independent treatment variables including frequency of osteopathic manipulation and exercise and the relative benefits of each in this model. Also, they may elucidate how these treatments produce their beneficial effects clinically.

Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first few hours after ingestion of a blood meal. The enzymatic activity of early trypsin plays an essential role in the transcriptional activation of the late trypsin gene, which encodes the major midgut endoprotease involved in blood meal protein digestion. Transcription of the early trypsin gene is part of the normal post-emergence maturation of the midgut in the adult female. Abdominal ligation within 1 h of emergence completely prevented the transcription of the early trypsin gene. Topically applied JH III or methoprene induced transcription of the early trypsin gene in ligated abdomens to levels similar to those observed in non-ligated females. The induction of early trypsin transcription by JH is dose-dependent and 'head-independent', suggesting that factors coming from the neuro-secretory axis are not required.

Motor-evoked potentials elicited from transcranial magnetic stimulation were recorded from both cranial tibial muscles of 10 Lewis rats. One sciatic nerve was then transected and surgically reanastomosed and allowed to regenerate for 6 weeks. Walking function was tested weekly using a footprint test giving a functional sciatic nerve index. At the end of the study motor-evoked potentials were again performed, as were nerve conduction velocities and supramaximal stimulation of operated and unoperated sciatic nerves. The nerve was evaluated for myelinated axon numbers. Functional sciatic index, numbers of myelinated axons, nerve conduction velocities, and supramaximal stimulation demonstrated the regenerated nerve to be functional, but to a lesser extent then the unoperated nerve. In all cases the amplitude of the compound muscle action potential evoked from transcranial magnetic stimulation of the motor cortex was greater in the operated motor pathway. This finding demonstrates hyperexcitability of the motor pathway to magnetic stimulation during peripheral nerve regeneration.

California's Mediterranean ecosystems include shrubland and grassland vegetation types that are fire-prone. Dotted within this landscape are ephemeral wetlands called vernal pools. Since surrounding upland vegetation is adapted to survive fire, it is expected that vernal pool organisms should be able to survive as well. One group of animals common to vernal pools are anostracan crustaceans that survive the pool's dry period as encysted embryos. We hydrated anostracan cysts from the soil of a recently burned pool and from soil samples intentionally burned in a prescribed fire. We also sampled burned pools when refilled the next rainy season. We found that anostracan cysts in the soil can survive fire and that shrimp occur in pools in the first post-burn season. This information is important from a management perspective concerning fire effects, controlled or natural, on vernal pools and their rare and endangered species.

A 10 138 bp cDNA from the fat body of the tobacco hornworm, Manduca sexta, which encodes the precursor protein for apolipophorin aPOLP-1 and -II, the structural apolipoproteins of the insect lipoprotein, lipophorin, has been cloned and sequenced. The cDNA has a single 9915 bp open reading frame beginning at an initiating ATG at bp 59 and extending to a stop codon at position 9974. This open reading frame encodes a 3305 amino acid protein with a molecular mass of 366 812 Da. Signal peptide cleavage is predicted to occur after residue 23, leaving a 3,282 amino acid precursor protein. The precursor protein is arranged with apoLp-II at the amino terminal end and apoLp-I at the car☐y terminal end. At present, the site of cleavage of the precursor protein to generate apoLp-I and -II is unknown.

A cDNA encoding mitochondrial cytochrome c oxidase subunit I (mt COI) from Manduca sexta (Lepidoptera: Sphingidae) was cloned and sequenced. AT (adenine-thymine) content is high and codon usage is biased and likely reflects the role of mt COI in electron transport. The encoded protein is 514 amino acids long, contains seven invariant His residues observed in COIs in all organisms and would be predicted to be composed of 12 transmembrane regions.

Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first hours after ingestion of a blood meal. Early trypsin gene expression was studied by Northern blot analysis. The early trypsin mRNA, absent in larvae, pupae and newly emerged females, reaches detectable levels at 24 h post-emergence and attains a maximum level at an adult age of 4-7 days. After the first week there is a decrease in the steady-state level of the transcript, but it remains readily detectable for up to a month after emergence. Despite the high levels of early trypsin mRNA present in the midgut of the unfed female, translation of the early trypsin mRNA occurs only after a blood or a protein meal. Early trypsin mRNA levels rapidly decrease during the first 24 h after feeding, but the steady-state level of the transcript rises again at the end of the blood digestion cycle (60 h), as the mosquito prepares for a second blood meal.

This study has examined cellular and molecular aspects of glial cell function in a newly described long-lived myelin deficient rat mutant. In contrast to the shorter-lived mutants which died at 25-30 days, the longer-lived mutant rats lived to 75-80 days of age. Despite living longer, these mutants had a similar frequency of seizures to their younger counterparts. In the spinal cord and optic nerves of the older mutants, myelinated fibres in similar numbers to those seen in the younger myelin deficient rats were present. However, the total glial cell numbers were markedly reduced with few remaining normal appearing oligodendrocytes, and very few microglia compared to the younger mutants. In addition, little or no cell death or division was seen in the longer-lived rats. However, there was some evidence of ongoing myelination and the persistence of immature oligodendrocytes or their progenitors in the older mutant. There was some continued myelin gene expression, although this was at much reduced levels compared to normal, with proteolipid protein and myelin basic protein being most affected. In situ hybridization analysis for proteolipid protein mRNA showed that few proteolipid protein expressing oligodendrocytes remained in the 70-80-day-old mutant. Polymerase chain reaction analysis of exon 3 of the long-lived mutant revealed the same point mutation as described in the younger myelin deficient rat.

Upon injection of synthetic adipokinetic hormone, lipophorin from Lethocerus medius decreased in density and became associated with apolipophorin-III (apoLp-III). ApoLp-III isolated from hemolymph of Lethocerus medius had a M(r) = 19,000 and an amino acid composition high in methionine, in comparison with other apoLp-IIIs. Its circular dichroism spectrum was consistent with a protein with secondary structure of predominantly alpha-helix. NH2-terminal sequence alignment with apoLp-III sequences from other species showed a conservation of the hydrophobic or hydrophilic properties of residues at each position rather than of specific amino acids. ApoLp-III from Lethocerus medius has the potential to form amphipathic alpha-helices, similar to those found in the three-dimensional structure of Locusta migratoria apoLp-III. A portion of the apoLp-III molecules that are not associated with lipophorin contained the blue chromophore, biliverdin.

Trypsin activity during the first hours after feeding is essential to induce late trypsin gene expression. These results are consistent with the idea that free amino acids or other products released during digestion might be the initial signal for transcriptional activation of late trypsin. Besides early trypsin, some other factor(s) have to be translated for induction of late trypsin. This is the first case in which the proteolytic activity of a digestive enzyme is part of the signal transduction system which regulates expression of a second gene. The presence of two trypsins allows the mosquito to assess the quality of the meal and adjust the levels of late trypsin for a particular meal with remarkable flexibility.

Glycogen phosphorylase b has been purified to homogeneity from the fat body of larval Manduca sexta. The purification procedure involved ammonium sulfate precipitation, and chromatography of DEAE-cellulose, 5'-AMP-Sepharose and Q-Sepharose. The final product, which showed a single band on SDS-PAGE with a M(r) = 92,500, was purified 50-fold from the original homogenate in a yield of about 3%. The molecular mass of the native purified phosphorylase b was estimated to be 186,000 Da from gel filtration, suggesting that the native enzyme is a dimer. The apparent Km values for glycogen, phosphate and 5'-AMP were 1.4 mM, 82 mM and 1.1 mM, respectively. The enzyme had a pH optimum of 7.05, and was inhibited by ATP, ADP and glucose, but not by trehalose, even at high concentration. Conversion of phosphorylase b into the a form was achieved by incubation with rabbit phosphorylase kinase and Mg(2+)-ATP. The molecular mass of phosphorylase a was estimated to be 250,000 Da by gel filtration chromatography. The specific activity of the a form in the presence of 5'-AMP was 1.6-1.7-fold higher than the specific activity of the b form under the same conditions. Thus, 5'-AMP activates the a form by about 20%, whereas ATP has no effect on the phosphorylase a activity.

Reconciling economic development with biodiversity conservation has become one of the most important elements in the search for sustainable development. This problem is particularly acute in remote rural areas of developing countries where biodiversity is concentrated and where poverty tends to be pervasive. Facing a range of development crises with limited public funds, most developing countries have invested little in biodiversity conservation. Partly as a result, fragile and unique ecosystems are being degraded or converted to agricultural use on a large scale. This trend is exacerbated by policies which encourage land conversion and resource overexploitation, as well as a lack of information about the economic value of biodiversity conservation.

Levels of RNA transcription were examined in L5 sensory ganglion neurons after unilateral crush injuries of the sciatic nerve using an in situ autoradiographic technique. Neuronal transcription increased in a biphasic pattern on the lesioned side within the first two weeks after injury. The timing and pattern of increases in transcription corresponded to previously reported increases RNA synthesis. The data demonstrate a dynamic regulation of RNA transcription in regenerating neuronal populations. This technique may prove to be useful in the study of factors controlling transcriptional activity in injured neurons.

We have purified trypsin from the midgut of Manduca sexta and shown it has an alkaline pH optimum of 10.5. In order to clone the midgut trypsin, a DNA probe was generated using the polymerase chain reaction (PCR) with template isolated from a midgut cDNA library phage stock, a mixture of degenerate primers synthesized to code for the highly conserved region around the active site serine found in trypsins, and the T7 sequencing primer. Three different trypsin cDNAs were isolated each of which encodes a preproenzyme of 256 amino acids with a putative signal sequence of 17 amino acids, an activation peptide of seven amino acids and a mature trypsin of 232 amino acids. The encoded midgut trypsins contain the highly conserved residues, Asp, His, Ser, involved in catalysis in serine proteases, along with the residues which define the trypsin specificity pocket. Sequence comparisons show that all sequences are similar to other invertebrate and vertebrate serine proteases, but they differ in that two of the three encoded trypsins have an odd number of cysteines. Northern analysis localizes the trypsin mRNA to the middle third of the midgut. A large number of arginines (19, 20 and 21) are encoded by the three cDNAs which may stabilize the trypsin, by remaining protonated, in the alkaline midgut of M. sexta.

The amount of nerve growth factor (NGF) in the L5, L6, and cervical dorsal root ganglia of rats was examined from 1 to 30 days after a unilateral crush lesion of the sciatic nerve and adjacent branches of the lumbar plexus at the level of the sciatic notch. Unilateral nerve crush produced increases in NGF content of lumbar ganglia at 1, 4, and 7-8 days after injury, with increased NGF mRNA at 4 and 7-8 days. Increases in NGF at 1 and 4 days were most pronounced on the unlesioned side while increases at days 7 and 8 were most pronounced on the lesioned side. NGF content increased in cervical ganglia of nerve-lesioned animals at 3 and 7 days after injury and in lumbar and cervical ganglia of sham-operated animals 3-5 days after surgery, with no comparable changes in NGF mRNA. Elevations of ganglionic NGF coincide temporally with some of the alterations in metabolism and morphology which occur in dorsal root ganglion neurons after sciatic nerve crush. However, the bilateral nature of increases in NGF demonstrates that the factor(s) producing the response is not restricted to ganglia axotomized by the injury. The data suggest that ganglionic NGF may be regulated by systemic factors, produced during stress or trauma, as well as by factors from the denervated target tissue and/or regenerating axons.

Publisher Summary A significant way of organizing a lipid transport system is provided by insect lipoproteins, which provide an interesting contrast to the mammalian lipid transport system. In insects, a single lipoprotein, lipophorin, acts as a reusable shuttle to transfer diglycerides between the fat bodies, where they are stored as triglycerides, and the peripheral tissues. This chapter describes the composition and the organization of lipids and proteins in the lipophorins and the structure of apolipophorin-III that is determined by X-ray crystallography to 2.5 A. Lipophorin is reloaded with lipid by another remarkable protein, the lipid transfer particle, which can affect a net transfer of diglycerides from the fat bodies to the depleted lipophorin particles. The major progress that is achieved in describing the lipid transport system in insects provides the results of significance for understanding lipid transport in general, an illustration of comparative biochemistry at its best. The only common major lipid component of vertebrate and insect lipoproteins is phospholipid (PL). The physiological roles of lipophorin in the biosynthesis and metabolism are also discussed.

The two most common protocols for RNA isolation, Chirgwin et al., 1979, and Chomczynski & Sacchi, 1987, were compared with a protocol using glass powder. The quality of the recovered RNA was tested using agarose gels and Northern hybridization. With the glass powder protocol, we consistently obtained intact RNA, whereas with the other protocols we often obtained degraded RNA. Using specific radioactive probes, we measured ribosomal RNA and trypsin mRNA recovered from fed adult Aedes aegypti females. Our protocol gave a 40- and a 3-fold increase in recovery when compared with the Chirgwin and Chomczynski protocol, respectively. The method using glass powder is simple, fast, reproducible and gives a high yield of intact RNA.

By cDNA cloning and sequencing we have shown that Manduca sexta larvae produce three very closely related methionine-rich storage proteins, MMR1, MMR2 and MMR3. Out of 2256 nucleotides in the coding region, the cDNAs differ by at most twenty-one bases and this leads to a single amino acid difference between MMR1 and MMR2, and between MMR2 and MMR3, whereas MMR1 and MMR3 differ by two amino acids. Using both distance and parsimony methods, similarities between the M. sexta and Bombyx mori methionine-rich and arylphorin storage proteins were examined. Homologous proteins from the two species tend to be more closely related than are the two classes of storage proteins in a single species.

The three-dimensional structure of an apolipoprotein isolated from the African migratory locust Locusta migratoria has been determined by X-ray analysis to a resolution of 2.5 {angstrom}. The overall molecular architecture of this protein consists of five long {alpha}-helices connected by short loops. As predicted from amino acid sequence analyses, these helices are distinctly amphiphilic with the hydrophobic residues pointing in toward the interior of the protein and the hydrophilic side chains facing outward. The molecule falls into the general category of up-and-down {alpha}-helical bundles as previously observed, for example, in cytochrome c{prime}. Although the structure shows the presence of five long amphiphilic {alpha}-helices, the {alpha}-helical moment and hydrophobicity of the entire molecule fall into the range found for normal globular proteins. Thus, in order for the amphiphilic helices to play a role in the binding of the protein to a lipid surface, there must be a structural reorganization of the protein which exposes the hydrophobic interior to the lipid surface. The three dimensional motif of this apolipoprotein is compatible with a model in which the molecule binds to the lipid surface via a relatively nonpolar end and then spreads on the surface in such a way as to cause themore » hydrophobic side chains of the helices to come in contact with the lipid surface, the charged and polar residues to remain in contact with water, and the overall helical motif of the protein to be maintained.« less

A 30-residue peptide [YAA(KALA)6LAA] with an amphipathic helix repeat unit of Lys-Ala-Leu-Ala (KALA) was synthesized as both the L- and the D-isomer. The peptide was shown to form alpha-helices and lyse lipid vesicles in a pH dependent fashion. The calculated helical amphipathic moment is +1.19 kcal/residue and the mean residue hydrophobicity is +0.4 kcal/residue. The formation of alpha-helices as the pH is increased is similar to poly-lysine, yielding a pK 10.2. Though not toxic when fed to insects, KALA killed Spodoptera frugiperda cells at low doses and Manduca sexta larvae when injected.

Ethanol consumption retards the hepatic regenerative response to injury. This may contribute to the pathogenesis of liver injury in alcoholic individuals. The mechanisms responsible for ethanol-associated inhibition of liver regeneration are poorly understood. To determine if the antiregenerative effects of ethanol involve modulation of polyamine metabolism, parameters of polyamine synthesis were compared before and during surgically induced liver regeneration in ethanol-fed rats and isocalorically maintained controls. After partial hepatectomy, induction of the activity of ornithine decarboxylase (ODC), the rate limiting enzyme for polyamine synthesis, was delayed in rats that had been fed ethanol. This was correlated with reduced levels of putrescine, ODC's immediate product. Increases in hepatic spermidine and spermine were also inhibited. Differences in ODC activity between ethanol-fed and control rats could not be explained by differences in the expression of ODC mRNA or by differences in ODC apoenzyme concentrations, suggesting that chronic ethanol intake inactivates ODC posttranslationally. Supplemental putrescine, administered at partial hepatectomy and 4 and 8 h thereafter, increased hepatic putrescine concentrations and markedly improved DNA synthesis and liver regeneration in ethanol-fed rats. These data suggest that altered polyamine metabolism may contribute to the inhibition of liver regeneration that occurs after chronic exposure to ethanol.