Combining the influence of two low O2 affinity-inducing chemical modifications of the central cavity of hemoglobin

HexaPEGylated hemoglobin (Hb), a non-hypertensive Hb, exhibits high O2 affinity, which makes it difficult for it to deliver the desired levels of oxygen to tissues. The PEGylation of very low O2 affinity Hbs is now contemplated as the strategy to generate PEGylated Hbs with intermediate levels of O2 affinity. Toward this goal, a doubly modified Hb with very low O2 affinity has been generated. The amino terminal of the beta-chain of HbA is modified by 2-hydroxy, 3-phospho propylation first to generate a low oxygen affinity Hb, HPPr-HbA. The oxygen affinity of this Hb is insensitive to DPG and IHP. Molecular modeling studies indicated potential interactions between the covalently linked phosphate group and Lys-82 of the trans beta-chain. To further modulate the oxygen affinity of Hb, the alpha alpha-fumaryl cross-bridge has been introduced into HPPr-HbA in the mid central cavity. The doubly modified HbA (alpha alpha-fumaryl-HPPr-HbA) exhibits an O2 affinity lower than that of either of the singly modified Hbs, with a partial additivity of the two modifications. The geminate recombination and the visible resonance Raman spectra of the photoproduct of alpha alpha-fumaryl-HPPr-HbA also reflect a degree of additive influence of each of these modifications. The two modifications induced a synergistic influence on the chemical reactivity of Cys-93(beta). It is suggested that the doubly modified Hb has accessed the low affinity T-state that is non-responsive to effectors. The doubly modified Hb is considered as a potential candidate for generating PEGylated Hbs with an O2 affinity comparable to that of erythrocytes for developing blood substitutes.     

Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH.

The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide (1), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide-receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1-Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible beta-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first beta-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second beta-turn. Furthermore, the presence of a beta-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH-NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I beta-turn structure is also supported by CD spectral analysis. The cIBR peptide (1) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.     

Plant regeneration from embryogenic cell suspension cultures of wild sorghum (Sorghum dimidiatum Stapf.)

A simple and efficient protocol is described for regeneration of wild sorghum (Sorghum dimidiatum) from cell suspension cultures. Fast-growing cell suspensions were established from shoot-meristem-derived callus. Plating of the suspension on Murashige and Skoog agar medium supplemented with 2.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in the formation of embryogenic calli. High-frequency (80%) somatic embryogenesis from small cell clusters (300–400 μm) was observed when the cultures were initially maintained in liquid medium with reduced levels of 2,4-D (0.25 mg l^–1), followed by transfer to regeneration medium. Direct plating of these small clusters on regeneration medium or transfer to liquid regeneration medium containing kinetin and 6-benzylaminopurine resulted in the development of mature somatic embryos and plantlets. The regenerants developed to maturity and were all phenotypically and cytologically normal. Received: 20 May 1998 / Revision received: 1 September 1998 / Accepted: 23 September 1998     

Production and evaluation of transgenic sorghum for resistance to stem borer

Transgenic sorghum plants were produced through particle bombardment and Agrobacterium methods in two elite, but recalcitrant genotypes of Sorghum bicolor L. Moench. Use of target cells from developing tissues (immature embryos and multiple shoot buds), pre-culture of target tissue, small size of target tissue (2–3 mm), and regular subculture improved the selection and regeneration efficiencies. Addition of amino acid l-cysteine during co-cultivation and blotting sheet interface was helpful for complete decontamination of Agrobacterium tumefaciens and regeneration of transgenic plants. We demonstrated production of transgenic sorghum plants expressing a Bacillus thuringiensis lepidopteran toxin, through tailored in vitro protocols. Our results showed that decontamination of agrobacteria employing subtle treatments aided recovery of transgenic plants in recalcitrant genotypes. We generated 14 independent transgenic lines carrying different classes of B. thuringiensis toxin genes, cry1Aa and cry1B. Many single copy events were generated in two elite parental lines, CS3541 and 296B. Accumulation of the B. thuringiensis protein in leaves during the susceptible period of plant growth ranged from 35 to 500 ng/g fresh leaf tissue. Comprehensive insect bioassays for tolerance to spotted stem borer (Chilo partellus) were conducted through leaf disk and whole plant assays. Transgenic progeny plants showed 20–30% of damage as compared to 70–80% in non-transformed controls.     

Perturbation of the Intermolecular Contact Regions (Molecular Surface) of Hemoglobin S by Intramolecular, Low-O2-Affinity-Inducing Central Cavity Cross-Bridges

The general assumption among researchers on hemoglobin is that the intramolecular central cavity cross-bridging of Hb does not result in any generalized perturbations at the protein surface. A corollary of this is that central cavity cross-bridges are unlikely to influence the polymerization of deoxy HbS, since polymerization is a protein surface phenomenon involving the participation of multiple protein surface amino acid residues. In an attempt to evaluate this experimentally, we have introduced two low-O₂-affinity-inducing central cavity cross-bridges into HbS, ββ-sebacyl [between the two Lys-82(β) residues] and αα-fumaryl [between the two Lys-99(α) residues], and investigated their influence on the polymerization of the deoxy protein. The O₂ affinities of the cross-bridged HbS exhibited sensitivity toward the buffer ions and pH in a cross-link-specific fashion. The modulation of the O₂ affinity of these cross-bridged HbS in the presence of allosteric effectors, DPG and L-35, is also very distinct, reflecting the differences in the conformational features these two cross-bridges induce within the central cavity at the respective effector-binding domains. In addition, the αα-fumaryl cross bridge inhibited the polymerization, reflecting the perturbation of the microenvironment of one or more intermolecular contact residues, protein surface residues, as a consequence of the central cavity cross-bridge. On the other hand, the ββ-sebacyl cross-bridge exerted a slight potentiating effect on the polymerization of HbS. This reflects the fact that the perturbations at the protein surface are limited and favor polymerization. The results presented demonstrate that the structural changes induced by the central cavity cross-bridges are very specific and not simply restricted to the sites of modification, but are propagated to distant sites/domains, both within and outside the central cavity. It is conceivable that other surface regions that are not involved in the polymerization could also experience similar structural/conformational consequences. These results should be taken into consideration in designing intramolecularly cross-bridged asymmetric hybrid HbS for mapping the contribution of the intermolecular contact residues in the cis and trans dimers of deoxy HbS during polymerization.     

Design of novel lipidated peptidomimetic conjugates for targeting EGFR heterodimerization in HER2 + cancer

The human epidermal growth factor receptor (EGFR) family is known to be involved in cell signaling pathways. The extracellular domain of EGFR consists of four domains, of which domain II and domain IV are known to be involved in the dimerization process. Overexpression of these receptors is known to play a significant role in heterodimerization of these receptors leading to the development of cancer. We have designed peptidomimetic molecules to inhibit the EGFR heterodimerization interaction that have shown antiproliferative activity and specificity for HER2-positive cancer cell lines. Among these, a peptidomimetic, compound 5, exhibited antiproliferative activity at low nanomolar concentrations in HER2-overexpressing cancer cell lines. To improve the stability of this peptidomimetic, we have designed and synthesized a novel conjugate of peptidomimetic compound 5 with a lipid, stearic acid. The antiproliferative activity of this conjugate was evaluated in HER2-positive cancer cell lines. Results suggested that the conjugate exhibited selective antiproliferative activity in HER2-overexpressing breast and lung cancer cell lines and was able to block HER2:HER3 heterodimerization. Also, the conjugate showed improved stability with a half-life of 5?h in human serum compared to the half-life of 2?h for parent compound 5. The binding affinity of the conjugate to HER2 protein was evaluated by SPR analysis, and the mode of binding of the lipid conjugate to domain IV of HER2 protein was demonstrated by docking analysis. Thus, this novel lipid conjugate can be used to target HER2-overexpressing cancers.     

Conjugation of Multiple Copies of Polyethylene Glycol to Hemoglobin Facilitated Through Thiolation: Influence on Hemoglobin Structure and Function

PEGylation induced changes in molecular volume and solution properties of HbA have been implicated as potential modulators of its vasoconstrictive activity. However, our recent studies with PEGylated Hbs carrying two PEG chains/Hb, have demonstrated that the modulation of the vasoconstrictive activity of Hb is not a direct correlate of the molecular volume and solution properties of the PEGylated Hb and implicated a role for the surface charge and/or the pattern of surface decoration of Hb with PEG. HbA has now been modified by thiolation mediated maleimide chemistry based PEGylation that does not alter its surface charge and conjugates multiple copies of PEG5K chains. This protocol has been optimized to generate a PEGylated Hb, (SP-PEG5K)₆-Hb, that carries ~six PEG5K chains/Hb – HexaPEGylated Hb. PEGylation increased the O₂ affinity of Hb and desensitized the molecule for the influence of ionic strength, pH, and allosteric effectors, presumably a consequence of the hydrated PEG-shell generated around the protein. The total PEG mass in (SP-PEG5K)₆-Hb, its molecular volume, O₂ affinity and solution properties are similar to that of another PEGylated Hb, (SP-PEG20K)₂-Hb, that carries two PEG20K chains/Hb. However, (SP-PEG5K)₆-Hb exhibited significantly reduced vasoconstriction mediated response than (SP-PEG20K)₂-Hb. These results demonstrate that the enhanced molecular size and solution properties achieved through the conjugation of multiple copies of small PEG chains to Hb is more effective in decreasing its vasoconstrictive activity than that achieved through the conjugation of a comparable PEG mass using a small number of large PEG chains.     

Influence of native disulfide bonds of globular proteins on their retention behavior on reverse phase supports

The reduction of the disulfide bonds of globular proteins, for example, those of lysozyme or ribonuclease-A, results in an increase in the hydrodynamic volume of the polypeptide chain. This is reflected in an earlier elution of the reduced protein on gel filtration compared to that of the native disulfide-bonded form. The reduction of the four disulfide bonds of ribonuclease-A increased its retention time on reverse phase support, suggesting an increase in the apparent hydrophobicity of the protein molecule on reduction. Performic acid-oxidized ribonuclease-A eluted ahead of native disulfide-bonded ribonuclease on RP HPLC, suggesting a decrease in the hydrophobicity of the molecule. However, the hydrodynamic volume of performic acid-oxidized ribonuclease-A is similar to that of reduced protein as reflected in its gel filtration behavior. Thus, the increased retention of the reduced protein compared to that of native disulfide-bonded protein is not related to the increased hydrodynamic volume, and is a reflection of the stronger interaction of reduced protein with the reverse phase support. Reoxidation of the reduced ribonuclease-A regenerated the original chromatographic behavior of the protein on the reverse phase support. Similar results were also obtained with hen egg white lysozyme. The results of the present study are interpreted as indicating that the native disulfide bonds of a globular protein restrict the exposure of the hydrophobic amino acid residues of the polypeptide chain with a consequent lower retention on the reverse phase support compared to its reduced form.     

Effects of exogenous growth hormone on milk production and nutrient uptake by muscle and mammary tissues of dairy cows in mid-lactation

Responses to exogenous growth hormone were measured in lactating dairy cows surgically prepared to allow measurement of nutrient exchanges across mammary and hind-limb muscle tissues. Cows were injected daily with either saline or growth hormone, at a dose of 0.1 mg/kg liveweight, over periods of 6 days. During administration of growth hormone milk yield, milk fat content and yields of milk fat protein and lactose increased. Arterial plasma concentrations of glucose and non-esterified fatty acids were increased, uptake of glucose by leg muscle tissue decreased, lactate release from leg muscle tended to increase, mammary uptake of non-esterified fatty acids increased, blood flow to leg muscle tended to increase and blood flow to mammary tissue increased during injection of growth hormone. The results show that growth hormone affects supply to and utilization of key nutrients by tissues, resulting in the supply to the mammary gland of additional precursors for milk synthesis.     

Regulation of hepatic glycine catabolism by glucagon

Glucagon stimulates 14CO2 production from [1-14C] glycine by isolated rat hepatocytes. Maximal stimulation (70%) of decarboxylation of glycine by hepatocytes was achieved when the concentration of glucagon in the medium reached 10 nM; half-maximal stimulation occurred at a concentration of about 2 nM. A lag period of 10 min was observed before the stimulation could be measured. Inclusion of beta-hydroxybutyrate (10 mM) or acetoacetate (10 mM) did not affect the magnitude of stimulation suggesting that the effects of glucagon were independent of mitochondrial redox state. Glucagon did not affect either the concentration or specific activity of intracellular glycine, thus excluding the possibilities that altered concentration or specific activity of intracellular glycine contributes to the observed stimulation. The stimulation of decarboxylation of glycine by glucagon was further studied by monitoring 14CO2 production from [1-14C]glycine by mitochondria isolated from rats previously injected with glucagon. Glycine decarboxylation was significantly stimulated in the mitochondria isolated from the glucagon-injected rats. We suggest that glucagon is a major regulator of hepatic glycine metabolism through the glycine cleavage enzyme system and may be responsible for the increased hepatic glycine removal observed in animals fed high-protein diets.