Effect of dietary nitrogen on gypsy moth larval nutritional indices, development and lipid content

Nutritional indices, development rates, percent dry weights and total lipids were determined in gypsy moth larvae (Lymantria dispar L.) reared on a high wheat germ (HWG) diet or diets prepared from lyophilized, ball-milled oak or pine foliage as the only source of dietary nitrogen (N). With regard to both total and proteinaceous N content, HWG diet>oak diet>pine diet. All nutritional indices measured were significantly lower in second instars fed pine diet vs. oak diet. Protein supplementation of pine diet with either casein or ovalbumin to bring total N up to the level present in oak diet resulted in small increased in approximate digestibility (AD) and effciency of conversion of ingested food (ECI), but relative growth rate (RGR) remained unaffected. The low RGR of larvae fed pine diet (unsupplemented or protein supplemented), as compared to those fed HWG or oak diet, was accompanied by significantly lower larval percent dry weight and percent total lipid. In contrast, RGR, larval percent dry weight and total lipid values were comparable in second instars fed HWG or oak diet. Insects reared from the first through the final instar on oak diet exhibited lower pupal weights compared to those reared on HWG. Casein addition to oak diet generally resulted in even more extended larval development times and further reduced pupal weights, but wheat germ addition to oak diet did not alter development rates and caused an increase in pupal weights.     

Differing macrophage and lymphocyte roles in resistance to Legionella pneumophila infection.

Similar to guinea pig macrophages and human monocytes, macrophages from the peritoneal cavity of thioglycolate pretreated A/J mice are permissive for growth of Legionella pneumophila. In contrast, macrophages from BDF1 mice are not permissive for L. pneumophila. Lymphocytes from A/J and BDF1 mice proliferated in response to Legionella Ag but guinea pig lymphocytes did not. Also, splenocyte cultures from A/J mice treated with either Con A or Legionella vaccine produced supernatants which induced A/J macrophages to restrict Legionella growth, but guinea pig splenocyte culture supernatants obtained after stimulation with L. pneumophila vaccine did not induce Legionella growth restriction activity by guinea pig macrophages. Murine rIFN-gamma but not rIFN-alpha markedly inhibited growth of Legionella in A/J mouse macrophages and monoclonal anti-IFN-gamma antibody neutralized the anti-Legionella activity of culture supernatants from A/J mouse splenocytes responding to Legionella Ag. From these data, IFN-gamma appears to be an important factor in anti-Legionella activity of Ag-activated mouse splenocyte culture supernatants. Cyclosporin A, when given to either A/J or BDF1 mice, reduced the proliferation responses of splenocytes to T cell mitogens and also decreased the IFN production of A/J spleen cells to Legionella Ag. In addition, drug treatment decreased the resistance of A/J mice to Legionella infection as shown by an increase in the number of viable bacteria in the liver. However, injection of drug treated mice with lymphokine-rich splenocyte culture supernatant reconstituted the resistance of these animals. These results suggest an important role for lymphocyte activation and lymphokine production in the resistance of A/J mice to Legionella infection. The greater resistance of BDF1 mice, however, may result from nonpermissive macrophages and responsive lymphocytes. In the case of guinea pigs, susceptibility to Legionella infections may result from both the permissive nature of the macrophages and the relatively unresponsive nature of the lymphocytes in these animals.     

Structural basis of C3b binding by glycoprotein C of herpes simplex virus.

Glycoproteins C (gC) from herpes simplex virus type 1 (HSV-1) and HSV-2, gC-1 and gC-2, bind the human complement fragment C3b, although the two glycoproteins differ in their abilities to act as C3b receptors on infected cells and in their effects on the alternative complement pathway. Previously, we identified three regions of gC-2 (I, II, and III) which are important for C3b binding. In this study, our goal was to identify C3b-binding sites on gC-1 and to continue our analysis of gC-2. We constructed a large panel of mutants by using the cloned gC-1 and gC-2 genes. Most of the mutant proteins were transported to the surface of transiently transfected L cells and reacted with one or more monoclonal antibodies to discontinuous epitopes. By using 31 linker insertion mutants spread across the coding region of gC-1, we identified four regions in the ectodomain of gC-1 which are important for C3b binding, three of which are similar in position to C3b-binding regions I, II, and III of gC-2. Region III shares some similarities with the short consensus repeat found in CR1, the human complement receptor. These were, in part, the targets for construction of 20 single amino acid changes in region III of gC-1 and gC-2. These mutants identified similarities and differences in the C3b-binding properties of gC-1 and gC-2 and suggest that the amino half of region III is more important for C3b binding. However, our results do not support the concept of a structural relationship between the short consensus repeat of CR1 and gC, since mutations of some of the conserved residues, including three of four cysteines in region III, had no effect on C3b binding. Finally, we constructed four deletion mutants of gC-1, including one which lacked residues 33 to 123, as well as residues 367 to 449. This severely truncated molecule, lacking four cysteines and five potential N-linked glycosylation sites, was transported to the cell surface and retained its ability to bind monoclonal antibodies as well as C3b. Thus, the four distinct C3b-binding regions of gC-1 and several epitopes within two different antigenic sites are localized within residues 124 to 366.     

Experimental hydroxyapatite cement cranioplasty.

Hydroxyapatite cement is a calcium phosphate-based material that when mixed with water forms a dense paste that sets within 15 minutes and isothermically converts in vivo to a microporous hydroxyapatite implant. This cement was used to reconstruct bilateral 2.5-cm-diameter full-thickness critical-sized parietal skull defects in six cats. One side was reconstructed with 100 percent hydroxyapatite cement, and the other with a mixture of 50 percent hydroxyapatite cement and 50 percent ground autogenous bone by weight. These animals were sacrificed at 6 and 12 months after implantation. Positive and negative controls also were prepared. The anatomic contour of the soft tissue overlying all hydroxyapatite cement implants was well maintained, there were no wound infections or structural failures, and the implants were well tolerated histologically. None of the negative (unreconstructed) control defects was completely filled with repair bone, and all positive (methyl methacrylate) controls demonstrated foreign-body giant-cell formation and fibrous encapsulation of the implants. Examination of decalcified and undecalcified sections revealed progressive but variable replacement of the cement by new bone and soft tissue without a change in the shape or volume of the hydroxyapatite cement-reconstructed areas. New bone comprised 77.3 and 64.7 percent of the tissue replacing the hydroxyapatite cement and hydroxyapatite cement-bone implants, respectively. Replacement of the hydroxyapatite cement implants by new bone is postulated to occur by a combination of osteoconduction and implant resorption. These results indicate that further experimental research leading to the possible application of hydroxyapatite cement for full-thickness calvarial defect reconstruction in humans is warranted.     

Pericentric inversion of the X chromosome: presentation of a case and review of the literature.

A large pericentric inversion of the X chromosome [inv(X)(p22.31q26.3)] was found to be transmitted in four generations through phenotypically normal males and females. In one female carrier, the inv(X) was late replicating in 70% of lymphocytes and 46% of skin fibroblasts. Steroid sulfatase (STS), an enzyme which normally escapes inactivation has been located to Xp22.32 and, in our case, has been moved to an aberrant position. We have assayed its activity in clones with the inv(X) inactive or the normal X inactive and found no significant differences. Thus, the STS locus escaped X inactivation in both the normal and the inverted X chromosomes. A review of the literature shows that almost half of the breakpoints on the short arm are found at region p22 and we propose that low-copy repetitive DNA segments along the X chromosome are responsible for non-homologous pairing and production of inversions.     

Waardenburg syndrome (WS): the analysis of a single family with a WS1 mutation showing linkage to RFLP markers on human chromosome 2q.

Waardenburg syndrome type I (WS1; MIM 19350) is caused by a pleiotropic, autosomal dominant mutation with variable penetrance and expressivity. Of individuals with this mutation, 20%-25% are hearing impaired. A multilocus linkage analysis of RFLP data from a single WS1 family with 11 affected individuals indicates that the WS1 mutation in this family is linked to the following four marker loci located on the long arm of chromosome 2: ALPP (alkaline phosphatase, placental), FN1 (fibronectin 1), D2S3 (a unique-copy DNA segment), and COL6A3 (collagen VI, alpha 3). For the RFLP marker loci, a multilocus linkage analysis using MLINK produced a peak lod (Z) of 3.23 for the following linkage relationships and recombination fractions (theta i): (ALPP----.000----FN1)----.122----D2S3----.267----CO L6A3. A similar analysis produced a Z of 6.67 for the following linkage relationships and theta i values among the markers and WS1: (FN1----.000----WS1----.000----ALPP)----.123----D2S 3----.246----COL6A3. The data confirm the conclusion of Foy et al. that at least some WS1 mutations map to chromosome 2q.     

Deletion-mutant epidermal growth factor receptor in human gliomas: effects of type II mutation on receptor function.

Malignant human glioma D-298 MG amplifies a rearranged epidermal growth factor receptor (EGFR) gene (c-erbB proto-oncogene), resulting in an in-frame deletion of 83 amino acids in domain IV of the extracellular domain of the EGFR. EGF and transforming growth factor-a (TGF-a) bound to the mutant EGFR with high affinity and enhanced the intrinsic mutant EGFR kinase activity. The mutant EGFR was capable of transducing EGF-stimulated glioma cell proliferation and invasiveness in an in vitro three-dimensional spheroid model. The deletion-mutant EGFR in D-298 MG is capable of being activated by growth factor; this suggests that overexpression of this mutant EGFR protein rather than structural alteration may be the more significant biologic event.     

Carboxy Mb at pH 3. Time-resolved resonance Raman study at cryogenic temperatures.

Cryogenic samples of MbCO at pH3 are studied using nanosecond and picosecond time-resolved resonance Raman spectroscopy. It is observed that under excitation conditions sufficient to completely photodissociate MbCO at pH7, the pH3 sample at 10 ns remains substantially unphotolyzed even at 15 K. The similarity in the optical and resonance Raman spectra of MbCO at pH3 with that of pH7 indicates that at pH3 the iron remains six-coordinate and low-spin. The Fe-CO stretch frequency is consistent with a more upright CO orientation. The absence of the v(Fe-His) band in the 30 ps photoproduct Raman spectrum suggests that the Fe-His(F8) bond is broken within 30 ps of photodissociation. Other Raman bands, though, are not consistent with a normal four-coordinate heme for the photoproduct, Mb*. Suggested possible interpretations include a four-coordinate heme highly perturbed by the close lying protonated proximal histidine or a five-coordinate heme with the Fe-His bond significantly weakened. The partial photolysis monitored at 30 ps and 100 K indicates either a significant amount of geminate recombination within 30 ps or low quantum yield or photolysis. The time course for CO recombination is monitored via the Raman spectra from 30 ps to 3 ns at 100 K and 160 K. Of the fraction of protein-ligand pairs that remain photodissociated at 30 ps, 50% recombine by approximately 250 ps at 100 K and 160 K, supporting the flash photolysis rebinding data of Cowen et al. (Cowen, B. R. 1990. Ph. D. thesis. University of Illinois at Urbana-Champaign; Cowen, B. R., D. Braunstein, H. Frauenfelder, P. J. Steinbach, and R. D. Young. 1989. Biophys. J. 55:55a. [Abstr.].) The conclusions from these resonance Raman studies are extended to solution phase studies at ambient temperatures.     

Dynamics and reactivity of HbXL99 alpha. A cross-linked hemoglobin derivative

Resonance Raman spectroscopy, transient absorption, and fluroescence techniques have been employed to investigate the structure and dynamics of the alpha-cross-linked hemoglobin derivative, HbXL99 alpha. The resonance Raman spectra of the deoxy form of HbXL99 alpha are identical to those of native NbA (VFe-His approximately 222 cm-1), which exhibit a T-state (low affinity) structure regardless of solvent conditions. The resonance Raman spectra of the transient heme photoproduct resulting from CO photolysis from HbXL99 alpha appear to have structures intermediate between deoxy-T and ligand-bound R structures (VFe-His approximately 222 cm-1). Time-resolved resonance Raman data of HbXL99 alpha-CO show that complete CO recombination occurs after approximately 5 ms, with only a small amount of the CO-bound species reforming within approximately 200 ns (geminate recombination). Transient absorption spectra of HbXL99 alpha-O2 indicate that the extent of sub-nanosecond geminate recombination of O2 is also reduced in the cross-linked derivative relative to native HbA. The decrease in tryptophan fluorescence of HbXL99 alpha upon oxygenation further indicates that tertiary structural changes at the alpha 1-beta 2 interface upon ligation are apparently reduced, but not eliminated in the cross-linked derivative relative to HbA.