A "virtual gland" method for quantifying epithelial fluid secretion

We developed a new apparatus, the virtual gland (VG), for measuring the rate of fluid secretion (Jv), its composition, and the transepithelial potential (TEP) in cultured epithelial cells under open circuit. The VG creates a 10-microl chamber above the apical surface of epithelial cells on a Costar filter with a small hole leading to an oil-filled reservoir. After the chamber is primed with a fluid of choice, secreted fluid is forced through the hole into the oil, where it forms a bubble that is monitored optically to determine Jv and collected for analysis. Calu-3 cells were mounted in the VG with a basolateral bath consisting of Krebs-Ringer bicarbonate buffer at 37 degrees C. Basal Jv was 2.7 +/- 0.1 microl x cm(-2) x h(-1) (n = 42), and TEP was -9.2 +/- 0.6 mV (n = 33); both measures were reduced to zero by ouabain (n = 6) x Jv and TEP were stimulated 64 and 59%, respectively, by 5 microM forskolin (n = 10), 173 and 101% by 1 mM 1-ethyl-2-benzimidazolinone (n = 5), 213 and 122% by 333 nM thapsigargin (n = 5), and 520 and 240% by forskolin + thapsigargin (n = 6). Basal Jv and TEP were inhibited to 82 and 63%, respectively, with 10 microM bumetanide (n = 5), 71 and 82% with 100 microM acetazolamide (n = 5), and 47 and 56% with 600 microM glibenclamide (n = 4). Basal Jv and TEP were 52 and 89% of control values, respectively, after HCO3- replacement with HEPES (n = 16). The net HCO3- concentration of the secreted fluid was close to that of the bath (25 mM), except when stimulated with forskolin or VIP, when it increased (approximately 80 mM). These results validate the use of the VG apparatus and provide the first direct measures of Jv in Calu-3 cells.     

Effect of contrast ventriculography of the left ventricle on coronary hemodynamics in patients with ischemic heart disease]

The effect of left ventricular (LV) contrast ventriculography (VG) on the state of intracardiac hemodynamics during the administration of a contrast medium and 30-40 sec. after it was investigated in 48 CHD patients. It followed retrograde catheterization of the left ventricle by a parallel use of two catheters that permitted recording intraventricular pressure directly during LV VG. The performance of left VG with 76% urografin administered in a dose of 0.6-0.8 ml/kg with the rate of 13-15 ml/s produced no significant change in the state of intracardiac hemodynamics, relaxation and pumping function of the LV in the course of the first three contrasted cardiocycles. Transitory disorder of hemodynamics observed in the patients after contrast VG was restored by itself in 15-20 min.     

A comparative assessment of the effect of Omnipaque and Verografin on ECG indices and myocardial contractility in patients with unstable angina during coronary ventriculography

Altogether 30 patients with unstable angina (UA) were examined. In 15 patients omnipaque was used during coronography and ventriculography (VG), in the rest of 15 patients--verografin at common dosages. A study was made of the changes in the indices of central hemodynamics and myocardial contractility resulting from the action of these radiopaque media during VG, and of ECG changes during coronography. VG with verografin was accompanied by more noticeable disorders of left ventricular contractility, an increase in myocardial oxygen consumption; coronography with omnipaque was characterized by later ECG changes, less noticeable disorders of intraventricular conductivity. It is advisable that coronaroventriculography in patients with UA should be performed using a new group of radiopaque media, in particular omnipaque.     

Mechanics of the left ventricular myocardial interstitium: effects of acute and chronic myocardial edema

Myocardial interstitial edema forms as a result of several disease states and clinical interventions. Acute myocardial interstitial edema is associated with compromised systolic and diastolic cardiac function and increased stiffness of the left ventricular chamber. Formation of chronic myocardial interstitial edema results in deposition of interstitial collagen, which causes interstitial fibrosis. To assess the effect of myocardial interstitial edema on the mechanical properties of the left ventricle and the myocardial interstitium, we induced acute and chronic interstitial edema in dogs. Acute myocardial edema was generated by coronary sinus pressure elevation, while chronic myocardial edema was generated by chronic pulmonary artery banding. The pressure-volume relationships of the left ventricular myocardial interstitium and left ventricular chamber for control animals were compared with acutely and chronically edematous animals. Collagen content of nonedematous and chronically edematous animals was also compared. Generating acute myocardial interstitial edema resulted in decreased left ventricular chamber compliance compared with nonedematous animals. With chronic edema, the primary form of collagen changed from type I to III. Left ventricular chamber compliance in animals made chronically edematous was significantly higher than nonedematous animals. The change in primary collagen type secondary to chronic left ventricular myocardial interstitial edema provides direct evidence for structural remodeling. The resulting functional adaptation allows the chronically edematous heart to maintain left ventricular chamber compliance when challenged with acute edema, thus preserving cardiac function over a wide range of interstitial fluid pressures.     

Determination of the ejection fraction and cardiac volumes using magnetic resonance tomography

The results of NMR-tomography and radiocontrast ventriculography (VG) were compared in 20 CHD patients to determine ejection fraction (EF), and left ventricular (LV) end-diastolic and end-systolic volumes (EDV and ESV). NMR-tomography of the heart was performed at the field power of 0.23 T using the synchronization with ECG. Two methods of orientation of NMP-section on the LV long axis were employed. A sufficiently high correlation of NMR-tomography and VG findings in the determination of EF (r = 0.9) was obtained, low and insignificant--in the determination of EDV and ESV. Possible causes of differences between the results of both methods are under discussion.     

Characterisation of a Colombian granulovirus (Baculoviridae: Betabaculovirus) isolated from Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae

A strategy for biological control of the fall armyworm, Spodoptera frugiperda, has included the use of baculoviruses principally the nucleopolyhedrovirus SfMNPV, which have been extensively characterised. In contrast, the granulovirus of S. frugiperda (SfGV) has been poorly studied even though it is able to enhance the infectivity and virulence of NPVs. In this work, a Colombian SfGV isolate (VG008) was characterised in comparison with a reference isolate from Brazil (VG014). The viral morphology was characterised by ovoidal-shaped occlusion bodies (OB) that contained one single internal virion. Median lethal concentrations (LC₅₀) and mean times to death (MTD) were 4.5 × 10⁵ OBs/mL and 29 days for VG008 and 1.6 × 10⁵ OBs/mL and 33 days for VG014. Both isolates reduced their insecticidal activity by 94%, after one hour of direct irradiation with ultraviolet light type B. The most prominent protein had an apparent molecular mass of 27 kDa and corresponded with the Granulin. Genomic comparison among isolates from Colombia and Brazil generated by restriction profiles showed differences in the number and size of fragments. Partial sequences of lef-8 and lef-9 genes and complete sequence of gran gene of Colombian SfGV isolate (VG008) showed high similarity values with VG014 and SfGV A12-4 homologous sequences, showing genetic distance lower than 0.015 (Kimura 2-parameter model), which confirmed that the three isolates belong to the same viral species. The characterisation of VG008 isolate demonstrated its high genomic and biological similarity with the Brazilian isolate.     

A perfusable 3D cell-matrix tissue culture chamber for in situ evaluation of nanoparticle vehicle penetration and transport

A key factor in gene or drug therapy is the development of carriers that can efficiently reach targeted cells from a distal administration. In many gene/drug delivery studies, results obtained in 2D cultures fail to translate to similar results in vivo. In this work, we developed a perfusable 3D chamber for studying nanoparticle penetration and transport in cell-gel soft tissue cultures. The compartmented chamber is made of a polydimethylsiloxane (PDMS) top layer with the chamber features, created using micromachined lithography, bonded to a bottom glass coverslip. A solution of cells embedded in a hydrogel is loaded in the chamber between PDMS posts that serve as anchors to the cell-matrix at the gel-media interface. The chamber offers the following unique features: (i) rapid fabrication and simplicity in assembly, (ii) direct in situ cell imaging in a plane normal to the direction of flow or action, (iii) an easily configurable and controllable environment conducive cell culture under static or interstitial flow conditions, and (iv) facile recovery of live cells from chambers for post-experimental analysis. To assess the chamber, we delivered fluorescently labeled nanoparticles of three distinct sizes to cells-embedded Matrigels in the 3D chamber under flow and static conditions. Penetration of nanoparticles were enhanced under interstitial flow while live cell imaging and flow cytometry of recovered cells revealed particle size restrictions to efficient delivery. Although designed for delivery studies, the chamber is versatile and can be easily modified. Thus it may have broad applications for biological, tissue engineering, and therapeutic studies.     

Inhibition of voltage-gated Na(+) current by nanosecond pulsed electric field (nsPEF) is not mediated by Na(+) influx or Ca(2+) signaling

In earlier studies, we found that permeabilization of mammalian cells with nsPEF was accompanied by prolonged inhibition of voltage-gated (VG) currents through the plasma membrane. This study explored if the inhibition of VG Na(+) current (I(Na)) resulted from (i) reduction of the transmembrane Na(+) gradient due to its influx via nsPEF-opened pores, and/or (ii) downregulation of the VG channels by a Ca(2+)-dependent mechanism. We found that a single 300 ns electric pulse at 1.6-5.3 kV/cm triggered sustained Na(+) influx in exposed NG108 cells and in primary chromaffin cells, as detected by increased fluorescence of a Sodium Green Dye. In the whole-cell patch clamp configuration, this influx was efficiently buffered by the pipette solution so that the increase in the intracellular concentration of Na(+) ([Na](i)) did not exceed 2-3 mM. [Na](i) increased uniformly over the cell volume and showed no additional peaks immediately below the plasma membrane. Concurrently, nsPEF reduced VG I(Na) by 30-60% (at 4 and 5.3 kV/cm). In control experiments, even a greater increase of the pipette [Na(+)] (by 5 mM) did not attenuate VG I(Na), thereby indicating that the nsPEF-induced Na(+) influx was not the cause of VG I(Na) inhibition. Similarly, adding 20 mM of a fast Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) into the pipette solution did not prevent or attenuate the inhibition of the VG I(Na) by nsPEF. These findings point to possible Ca(2+)-independent downregulation of the VG Na(+) channels (e.g., caused by alteration of the lipid bilayer) or the direct effect of nsPEF on the channel.     

An in vivo analysis of the vestigial gene in Drosophila melanogaster defines the domains required for Vg function

Considerable evidence indicates an obligate partnership of the Drosophila melanogaster Vestigial (VG) and Scalloped (SD) proteins within the context of wing development. These two proteins interact physically and a 56-amino-acid motif within VG is necessary and sufficient for this binding. While the importance of this SD-binding domain has been clearly demonstrated both in vitro and in vivo, the remaining portions of VG have not been examined for in vivo function. Herein, additional regions within VG were tested for possible in vivo functions. The results identify two additional domains that must be present for optimal VG function as measured by the loss of ability to rescue vg mutants, to induce ectopic sd expression, and to perform other normal VG functions when they are deleted. An in vivo study such as this one is fundamentally important because it identifies domains of VG that are necessary in the cellular context in which wing development actually occurs. The results also indicate that an additional large portion of VG, outside of these two domains and the SD-binding domain, is dispensable in the execution of these normal VG functions.     

Evidence that both Ca(2+)-specific sites of skeletal muscle TnC are required for full activity

To investigate the role of the Ca2(+)-specific (I and II) sites of fast skeletal muscle troponin C (TnC) in the regulation of contraction, we have produced two TnC mutants which have lost the ability to bind Ca2+ at either site I (VG1) or at site II (VG2). Both mutants were able to partially restore force to TnC-depleted skinned muscle fibers (approximately 25% for VG1 and approximately 50% for VG2). In contrast, bovine cardiac TnC (BCTnC), which like VG1 binds Ca2+ only at site II, could fully reactivate the contraction of TnC-depleted fibers. Higher concentrations of both mutants were required to restore force to the TnC-depleted fibers than with wild type TnC (WTnC) or BCTnC. VG1 and VG2 substituted fibers could not bind additional WTnC, indicating that all of the TnC-binding sites were saturated with the mutant TnC's. The Ca2+ concentration required for force activation was much higher for VG1 and VG2 substituted fibers than for WTnC or BCTnC substituted fibers. Also, the steepness of force activation was much less in VG1 and VG2 versus WTnC and BCTnC substituted fibers. These results suggest cooperative interactions between sites I and II in WTnC. In contrast, BCTnC has essentially the same apparent Ca2+ affinity and steepness of force activation as does WTnC. Thus, cardiac TnC must have structural differences from WTnC which compensate for the lack of site I, while in WTnC, both Ca2(+)-specific sites are probably crucial for full functional activity.     

Multivesicular bodies play a key role in vitellogenin endocytosis by Xenopus oocytes

A combination of electron microscopic tracers and subcellular fractionation has been used to examine the endocytic pathway of the yolk protein precursor, vitellogenin (VG), in Xenopus oocytes. VG was adsorbed to colloidal gold, and the organelles traversed by newly internalized ligand were examined at various time intervals after endocytosis. VG-Au enters oocytes via coated pits and vesicles and then appears rapidly in tubular endosomes and multivesicular bodies (MVBs). MVBs play a central role in VG processing for storage; the large majority of newly internalized VG enters this compartment, remaining there for up to several hours. Condensation of VG into crystalline bodies begins in MVBs, and continues with growth of the crystals until typical platelets are formed. When oocytes are exposed to high [VG], MVBs containing large amounts of internalized VG are morphologically indistinguishable from the primordial yolk platelets described earlier (Dumont, 1978). The use of VG-Au particles of two sizes demonstrates that gold particles in early MVBs were generally associated with the limiting membrane of these organelles, while older MVB compartments have gold particles well separated from the limiting membranes, suggesting that dissociation of VG from its receptor occurs in this compartment. Newly internalized ligand preferentially forms a new MVB, rather than fusing and mixing with previously formed MVBs. Progressive yolk protein condensation gradually transforms MVBs into yolk platelets over a period of several hours. Analysis of 125I-VG-Au behavior after sucrose gradient fractionation of oocytes allowed correlation of biochemical compartments with those observed in the electron microscope. MVBs containing yolk in progressive stages of condensation were found at densities from 1.16 up to 1.21 g/cc. The final, rate-limiting step in VG transport is a shift of ligand from light (1.21 g/cc) to heavy (1.23 g/cc) platelet compartments (Wall and Meleka, 1985). The morphological correlate of this process is movement of VG-Au from small (less than 3-4 microns diameter) to large (greater than 4 microns diameter) platelets.     

Selection of strains of Streptomyces griseus Kr., the producer of a streptothricin antibiotic grisin, using the method of protoplast fusion

The effect of protoplasting on antibiotic activity of the grisin-producing organism was shown. High frequency of Grn- mutants after strain VG307f protoplasting and no capacity in these mutants for reversion to the initial Grn+ phenotype were shown. The reversion frequency was less than 10(-8). Moreover, it was shown that all the Grn- mutants lost their stability (GrnR) to the effect of their own antibiotic. With respect to strain VG212 there was noted a significant increase in the number of both the minus and the plus variants after the protoplast formation and regeneration. Fusing of protoplasts of strains VG307f and VG212 belonging to the divergent lines in selection of S. griseus Kr. yielded the phage stable strain VG7849 with high levels of the antibiotic production and improved technological properties.     

The Effect of Local Delivery Doxycycline and Alendronate on Bone Repair

The aim of the present study was to investigate the local effect of 10% doxycycline and 1% alendronate combined with poly(lactic-co-glycolic acid) (PLGA) on bone repair. Thirty rats were divided into three groups, as follows: control group (CG), drug group (DG), and vehicle-PLGA group (VG). Bone defect was created in the right femur and filled with the following: blood clot (CG); PLGA gel, 10% doxycycline and 1% alendronate (DG); or vehicle-PLGA (VG). The animals were euthanized 7 or 15 days after surgery. Bone density, bone matrix and number of osteoclasts were quantified. At 7 days, the findings showed increased density in DG (177.75?±?76.5) compared with CG (80.37?±?27.4), but no difference compared with VG (147.1?±?41.5); no statistical difference in bone neoformation CG (25.6?±?4.8), VG (27.8?±?4), and DG (18.9?±?7.8); and decrease osteoclasts in DG (4.6?±?1.9) compared with CG (26.7?±?7.4) and VG (17.3?±?2.7). At 15 days, DG (405.1?±?63.1) presented higher density than CG (213.2?±?60.9) and VG (283.4?±?85.8); there was a significant increase in percentage of bone neoformation in DG (31.5?±?4.2) compared with CG (23?±?4), but no difference compared with VG (25.1?±?2.9). There was a decreased number of osteoclasts in DG (20.7?±?4.7) and VG (29.5?±?5.4) compared with CG (40?±?9.4). The results suggest that the association of 10% doxycycline and 1% alendronate with PLGA-accelerated bone repair.     

DPP4 inhibition improves functional outcome after renal ischemia-reperfusion injury

Dipeptidyl peptidase 4 (DPP4) is an exopeptidase which modulates the function of its substrates, among which are insulin-releasing incretins. DPP4 inhibitors are currently used to improve glucose tolerance in type 2 diabetes patients. Inhibition of DPP4 exhibits protective effects on ischemia-reperfusion injury (IRI) of the heart and lung. As DPP4 and its substrates are also expressed in the kidney, we studied the effect of the DPP4 inhibitor vildagliptin on the outcome of IRI-induced acute kidney injury in rats in a model of 30-min unilateral renal ischemia, followed by contralateral nephrectomy. Saline, 1, or 10 mg/kg vildagliptin (VG1/VG10) was administered intravenously 15 min before the surgery. Animals were euthanized after 2, 12, amd 48 h of reperfusion. DPP4 inhibition resulted in a significant dose-dependent decrease in serum creatinine (1.31 ± 0.32 and 0.70 ± 0.19 mg/dl for VG1 and VG10, respectively, vs. 1.91 ± 0.28 mg/dl for controls at 12 h; P < 0.01). Tubular morphology (PAS-PCNA) revealed significantly reduced tubular necrosis at 12 h (62.1 ± 18.0 and 77.5 ± 22.0% in VG10 and saline, respectively). VG did not affect regeneration but decreased apoptosis, as shown by twofold decreased Bax/Bcl-2 mRNA expression and a threefold decrease in apoptotic bodies on terminal deoxynucleotidyl transferase dUTP nick-end labeling-stained sections. VG treatment significantly reduced serum malondialdehyde twofold in both VG1- and VG10-treated ischemic and sham-operated animals compared with controls and also resulted in a significant decrease in mRNA expression of the proinflammatory marker CXCL10 at 2 h of reperfusion. Through a mechanism yet to be fully understood, VG treatment results in a functional protection of the kidney against IRI. This protection was associated with antiapoptotic, immunological, and antioxidative changes.     

Comparison on Virulence and Immunogenicity of Two Recombinant Vaccinia Vaccines,Tian Tan and Guang9 Strains,Expressing the HIV-1 Envelope Gene

Background

The vaccinia virus Guang9 strain (VG9), derived from the vaccinia virus Tian Tan strain (VTT) has been found to be less virulent than VTT.

Methodology/Principal Findings

To investigate whether VG9 could be a potential replicating virus vector, the TK genes in VG9 and VTT were replaced with the HIV-1 envelope gene via homologous recombination, resulting in the recombinant viruses, VG9-E and VTT-E. The biology, virulence, humoral and cellular immunological responses of VG9-E and VTT-E were evaluated. Our results indicated no obvious difference in range of host cells and diffusion between two recombinant viruses. Neurovirulence for VG9-E in weanling and suckling mice, and skin virulence in rabbits, were lower than that of VTT-E. The humoral immune responses, including binding antibody and neutralizing antibody responses, induced by VG9-E were not significantly different from those for VTT-E whilst IFN-γ response which represented cellular immune response induced by VG9-E was significantly higher than that did by VTT-E.

Conclusions/Significance

Our results indicated that VG9-E was less virulent, yet induced higher cellular immune response than VTT-E. Therefore, it could be an ideal replicating vaccinia vector for HIV vaccine research and development.     

Vitellogenesis inhibition in Oncopeltus fasciatus females (Heteroptera: Lygaeidae) exposed to cadmium

Newly moulted females of the insect Oncopeltus fasciatus were exposed to cadmium (Cd) dissolved in the drinking water (50-400 mg l(-1) Cd) for 5 days. Cd exposure delayed ovarian maturation and inhibited egg production. Exposure to Cd, moreover, decreased hemolymph levels of the two major vitellogenin polypeptides of O. fasciatus, VG1 and VG2, in a concentration-dependent way, probably by a reduction in their synthesis. The ovarian levels of VG1 and VG2 were also decreased in Cd-exposed females. It was next investigated whether Cd effects might be a consequence of the endocrine disruption of vitellogenin synthesis, which is controlled by juvenile hormone (JH). JH replacement therapy did not restore VG1 or VG2 levels in Cd-exposed females, but did so in starved females. Our results do not therefore support a disturbance of JH production or a reduction in feeding as the cause of the reduced vitellogenin polypeptide levels, but rather point to the site of action of JH, the JH receptor, as the target of Cd effects.     

Characterization of vitellogenin in the red imported fire ant, Solenopsis invicta (Hymenoptera: Apocrita: Formicidae)

Vitellin (VN) and vitellogenin (VG) profiles were analyzed in monogyne and polygyne colonies of the red imported fire ant, Solenopsis invicta. Non-denaturing and SDS-polyacrylamide gel electrophoresis (PAGE) analyses indicated that the native VN was likely 350 kDa and comprised of two subunits in the molecular size range of 170-185 kDa. SDS-PAGE of hemolymph showed that the relative mobilities and subunit patterns of VG and VN were similar. VG was present in the hemolymph of reproductive queens; alate, virgin queens; and workers, but not in males. Anti-VN, prepared from polygyne egg homogenates, reacted with egg homogenates and with hemolymph VG from reproductive, monogyne and polygyne queens and alate, virgin polygyne queens. Analysis of circulating VG and ovarian development in alate, virgin queens showed that low levels of VG appeared by five days following adult eclosion, but egg development was not observed until seven weeks. VG was evident in newly inseminated queens, and increased steadily for the first three weeks following dealation. VG levels declined slightly near eclosion of the first workers (= nanitics) and dropped sharply after nanitic emergence at five weeks following dealation. Oocyte maturation peaked at days 15-25 following dealation, but otherwise remained low but steady. These studies provide the basis for future investigations into endocrine regulations of vitellogenesis in S. invicta queens.     

Stochastic 16-state model of voltage gating of gap-junction channels enclosing fast and slow gates

Gap-junction (GJ) channels formed of connexin (Cx) proteins provide a direct pathway for electrical and metabolic cell-cell interaction. Each hemichannel in the GJ channel contains fast and slow gates that are sensitive to transjunctional voltage (Vj). We developed a stochastic 16-state model (S16SM) that details the operation of two fast and two slow gates in series to describe the gating properties of homotypic and heterotypic GJ channels. The operation of each gate depends on the fraction of Vj that falls across the gate (VG), which varies depending on the states of three other gates in series, as well as on parameters of the fast and slow gates characterizing 1), the steepness of each gate's open probability on VG; 2), the voltage at which the open probability of each gate equals 0.5; 3), the gating polarity; and 4), the unitary conductances of the gates and their rectification depending on VG. S16SM allows for the simulation of junctional current dynamics and the dependence of steady-state junctional conductance (gj,ss) on Vj. We combined global coordinate optimization algorithms with S16SM to evaluate the gating parameters of fast and slow gates from experimentally measured gj,ss-Vj dependencies in cells expressing different Cx isoforms and forming homotypic and/or heterotypic GJ channels.     

GABA differentially regulates the gene expression of proopiomelanocortin in rat intermediate and anterior pituitary

The GABAergic regulation of proopiomelanocortin messenger RNA (POMC mRNA) levels in rat pituitary was investigated using molecular hybridization of DNA complementary to POMC mRNA. Endogenous GABA levels increased, in vivo, by inhibiting the GABA catabolic enzyme GABA-transaminase (GAT) with ethalonamine-O-sulfate (EOS) or with vinyl-GABA (VG). Rats were treated with VG (100 mg/kg or 800 mg/kg) or EOS (100 mg/kg), administered each second day. GABA levels in the neurointermediate lobe (NIL) and anterior lobe (AL) of the hypophysis and in the hypothalamus were significantly increased following 4 days of VG treatment (800 mg/kg). All treatments resulted in a 40-60% decrease in POMC mRNA levels after 4 days in the NIL but not in the AL. A similar decrease of about 60% in POMC mRNA levels in the NIL was seen when EOS was given in the drinking water (5 mg/ml). In this set of experiments the time course of alteration of POMC mRNA in the NIL and the concentration of alpha-MSH, a POMC-derived peptide, were analysed. After one day of EOS treatment, when POMC levels had already decreased by 40%, alpha-MSH levels were significantly elevated (34% above controls), possibly reflecting an inhibition of alpha-MSH secretion. However, after 4 and 8 days, POMC mRNA levels and tissue alpha-MSH levels had significantly decreased. When tested in vitro, on primary cultures of IL cells, GABA (10 microM) reduced POMC mRNA levels by 40% after 3 days of treatment. These results show that GABA exerts a direct inhibitory effect on POMC gene expression in the intermediate lobe.     

Purification and characterization of vitellogenin from the American cockroach,Periplaneta americana

• 1.1. Vitellogenin (VG) was isolated and purified from the hemolymph of female American cockroaches.
• 2.2. The purification method used in this study comprises two steps: the first step is based on the method originally developed for purifying lipophorin from hemolymph, and the second step is the separation of VG from lipophorin by a KBr density gradient ultracentrifugation.
• 3.3. The purified VG was characterized according to molecular weight, substructure, shape and size, and lipid composition.
• 4.4. The VG molecule is almost globular in shape with the diameter of about 15.5 nm and is indistinguishable from lipophorin in shape and size.
• 5.5. The native molecular weight determined by light scattering method was 560 kDa.
• 6.6. The VG consists of four subunits with molecular weights of approximately 102, 81, 49 and 40 kDa, respectively.
• 7.7. VG is a lipoprotein and comprises 92% protein and 8% lipid.
• 8.8. Major lipid components were found to be diacylglycerol (25%) and phospholipids (71%).