Early pregnancy termination in rats immunized with denatured chicken riboflavin carrier protein

Immunoneutralization of the maternal riboflavin carrier protein in the pregnant rat with antibodies to chicken egg vitamin carrier has earlier been shown to terminate their pregnancies. In order to understand the nature of the epitopic conformations capable of eliciting antibodies bioneutralizing the endogenous riboflavin carrier protein in the pregnant rat, we compared pregnancy progression in the fertile rodents following active immunization with either the native, SDS-denatured, reduced-carboxymethylated or SDS-treated reduced carboxymethylated avian egg white riboflavin carrier protein. The data revealed that despite the total antibody titers being higher in the animals immunized with the native protein, the antibodies elicited against the denatured avian vitamin carrier exhibited relatively better potencies to bioneutralize the endogenous maternal protein as evidenced by higher rates of early fetal resorption.

A Hybrid Multilevel Inverter Topology for an Open-End Winding Induction-Motor Drive Using Two-Level Inverters in Series With a Capacitor-Fed H-Bridge Cell

In this paper, a new five-level inverter topology for open-end winding induction-motor (IM) drive is proposed. The open-end winding IM is fed from one end with a two-level inverter in series with a capacitor-fed H-bridge cell, while the other end is connected to a conventional two-level inverter. The combined inverter system produces voltage space-vector locations identical to that of a conventional five-level inverter. A total of 2744 space-vector combinations are distributed over 61 space-vector locations in the proposed scheme. With such a high number of switching state redundancies, it is possible to balance the H-bridge capacitor voltages under all operating conditions including overmodulation region. In addition to that, the proposed topology eliminates 18 clamping diodes having different voltage ratings compared with the neutral point clamped inverter. On the other hand, it requires only one capacitor bank per phase, whereas the flying-capacitor scheme for a five-level topology requires more than one capacitor bank per phase. The proposed inverter topology can be operated as a three-level inverter for full modulation range, in case of any switch failure in the capacitor-fed H-bridge cell. This will increase the reliability of the system. The proposed scheme is experimentally verified on a four-pole 5-hp IM drive.

Effect of amino acid substitutions at the subunit interface on the stabiity and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the dimer interface of thymidylate synthase

The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation. While the R178F mutant showed apparent stability to thermal denaturation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5 degrees C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions. Circular dichroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS. The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 A crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution. The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein. Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures.

A new method for modulating the activity of parvalbumin-positive neurons and cerebellar Purkinje cells in vivo

Neurons can be divided into various classes according to their location, morphology, neurochemical identity and electrical properties. They form complex interconnected networks with precise roles for each cell type. GABAergic neurons expressing the calcium-binding protein parvalbumin (Pv) are mainly interneurons, which serve a coordinating function. Pv-cells modulate the activity of principal cells with high temporal precision. Abnormalities of Pv-interneuron activity in cortical areas have been linked to neuropsychiatric illnesses such as schizophrenia. Cerebellar Purkinje cells are known to be central to motor learning. They are the sole output from the layered cerebellar cortex to deep cerebellar nuclei. There are still many open questions about the precise role of Pv-neurons and Purkinje cells, many of which could be answered if one could achieve rapid, reversible cell-type specific modulation of the activity of these neurons and observe the subsequent changes at the whole-animal level. The aim of these studies was to develop a novel method for the modulation of Pv-neurons and Purkinje cells in vivo and to use this method to investigate the significance of inhibition in these neuronal types with a variety of behavioral experiments in addition to tissue autoradiography, electrophysiology and immunohistochemistry. The GABA(A) receptor γ2 subunit was ablated from Pv-neurons and Purkinje cells in four separate mouse lines. Pv-Δγ2 mice had wide-ranging behavioral alterations and increased GABA-insensitive binding indicative of an altered GABA(A) receptor composition, particularly in midbrain areas. PC-Δγ2 mice experienced little or no motor impairment despite the lack of inhibition in Purkinje cells. In Pv-Δγ2-partial rescue mice, a reversal of motor and cognitive deficits was observed in addition to restoration of the wild-type γ2F77 subunit to the reticular nucleus of thalamus and the cerebellar molecular layer. In PC-Δγ2-swap mice, zolpidem sensitivity was restored to Purkinje cells and the administration of systemic zolpidem evoked a transient motor impairment. On the basis of these results, it is concluded that this new method of cell-type specific modulation is a feasible way to modulate the activity of selected neuronal types. The importance of Purkinje cells to motor control supports previous studies, and the crucial involvement of Pv-neurons in a range of behavioral modalities is confirmed.

Isolation of a phosphoryl choline-binding protein from the hemolymph of the snail, Achatina fulica

A phosphorylcholine-binding protein from the hemolymph of the snail Achatina fulica was purified to near homogeneity using a Sepharose phenylphosphorylcholine affinity column. The protein bound to the affinity column was eluted with 5 mM phosphorylcholine as a single symmetrical peak. The purified protein (400 Kda) contained 35–40% carbohydrate. On SDS-PAGE the protein separated into two bands of 20 and 24 Kda, and had a pI of 5.9. On immunodiffusion, antiserum to the snail phosphorylcholine binding protein did not cross-react against other phosphorylcholine binding proteins, like rat serum phosphorylcholine-binding protein (PCBP), limulus C-reactive protein (CRP), or human CRP. On pretreatment of the snail hemolymph with this antiserum, the hemagglutination titer of the hemolymph was markedly decreased. The purified snail phosphorylcholine binding protein agglutinated rabbit erythrocytes in the absence of divalent cation (Ca+2) but trace amount of Ca+2 increased its binding. The strongest inhibitor of the agglutination reaction was lactose, followed by melibiose and 2-deoxygalactose. The relationships of the snail phosphorylcholine binding protein to other hemolymph agglutinins and to CRPs are discussed in light of common phylogeny.

Nucleosomes on linear duplex DNA allow homologous pairing but prevent strand exchange promoted by RecA protein

To understand the molecular basis of gene targeting, we have studied interactions of nucleoprotein filaments comprised of single-stranded DNA and RecA protein with chromatin templates reconstituted from linear duplex DNA and histones. We observed that for the chromatin templates with histone/DNA mass ratios of 0.8 and 1.6, the efficiency of homologous pairing was indistinguishable from that of naked duplex DNA but strand exchange was repressed. In contrast, the chromatin templates with a histone/DNA mass ratio of 9.0 supported neither homologous pairing nor strand exchange. The addition of histone H1, in stoichiometric amounts, to chromatin templates quells homologous pairing. The pairing of chromatin templates with nucleoprotein filaments of RecA protein-single-stranded DNA proceeded without the production of detectable networks of DNA, suggesting that coaggregates are unlikely to be the intermediates in homologous pairing. The application of these observations to strategies for gene targeting and their implications for models of genetic recombination are discussed.

A simplified method for the purification of riboflavin-binding protein from hen's egg yolk

A simple and efficient procedure for the purification of the riboflavin-binding protein from hen's egg yolk is described. This method involves the removal by exclusion of lipoproteins and subsequent fractionation of soluble yolk proteins held on a DEAE-cellulose column by a salt gradient which is followed by purification by gel filtration on Sephadex G-100. The protein thus isolated is homogeneous by various physicoehemical, immunological, and functional criteria.

Riboflavin carrier protein from carp (C. carpio) eggs: comparison with avian riboflavin carrier protein

A protein exhibiting immunological cross-reactivity with the chicken egg-white riboflavin carrier protein was detected by radioimmunoassay in the eggs and serum of the fresh water fish Cyprinus carpio and subsequently purified to homogeneity by use of affinity chromatography. Fish riboflavin carrier protein resembled chicken riboflavin carrier protein with respect to most of its physicochemical characteristics. The major epitopes of chicken riboflavin carrier protein were shown to be conserved in the fish protein as probed with monoclonal antibodies to the avian vitamin carrier.

Quantitative and compositional changes in myelin of undernourished and protein malnourished rat brains

Effects of undernutrition and protein malnutrition on the quantitative and qualitative changes in myelin isolated from rat brain at 3 and 8 weeks of age were investigated. Undernutrition during suckling period was induced by increasing the litter size, and continued from the 3rd to the 8th week by limited food intake, or the rats were rehabilitated with adequate food. Protein malnutrition was induced by feeding the lactating dams 5% protein diet as against 25% protein diet in controls. The protein malnourished rats were rehabilitated from the 3rd to the 8th week with the normal 25% protein diet. Undernutrition produced 16% and 35% reductions in the myelin content at 3 and 8 weeks of age, respectively, and was only partially restored on rehabilitation. Protein malnutrition caused more drastic reduction of 27% in the myelin content at 3 weeks, which was also partially restored on rehabilitation. The specific activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase was not affected by undernutrition, whereas protein malnutrition caused a 25% reduction at 3 weeks, which was totally reversed by rehabilitation. Undernutrition had not altered the relative composition of myelin proteins, but protein malnutrition resulted in a significant reduction in the proteolipid protein at 3 weeks of age, which could be reversed by rehabilitation.

Principle of a Dual Current Source Converter for AC Motor Drives

The principle of operation of a dual current source converter is briefly explained. The combination of two single current source converters (SCSC) to form a ``dual (duplex) current source converter'' (DCSC) is proposed. The DCSC is shown to have the following merits: 1) it retains all the advantages of the SCSC; 2) it reduces the harmonic content of the current waveform considerably; and 3) since the load current is shared equally between two current source converters, ratings of the individual components employed in the circuit are considerably lowered. A DCSC can be an attractive choice for sophisticated large horsepower drives where a good performance of the drive rather than cost is a prime factor. An open-loop control scheme employing the DCSC for an ac motor drive has been successfully implemented in the laboratory. Oscillograms of the improved load current waveforms are shown.

Isolation and characterization of thiamin-binding protein from chicken egg white

A thiamin-binding protein was isolated and characterized from chicken egg white by affinity chromatography on thiamin pyrophosphate coupled to aminoethyl-Sepharose. The high specificity of interaction between the thiamin-binding protein and the riboflavin-binding protein of the egg white, with a protein/protein molar ratio of 1.0, led to the development of an alternative procedure that used the riboflavin-binding protein immobilized on CNBr-activated Sepharose as the affinity matrix. The thiamin-binding protein thus isolated was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, double immunodiffusion and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, had a mol.wt. of 38,000 +/- 2000 and was not a glycoprotein. The protein bound [14C]thiamin was a molar ratio of 1.0, with dissociation constant (Kd) 0.3 micrometer.

A Deterministic Optimization Approach to Protein Sequence Design Using Continuous Models

Determining the sequence of amino acid residues in a heteropolymer chain of a protein with a given conformation is a discrete combinatorial problem that is not generally amenable for gradient-based continuous optimization algorithms. In this paper we present a new approach to this problem using continuous models. In this modeling, continuous "state functions" are proposed to designate the type of each residue in the chain. Such a continuous model helps define a continuous sequence space in which a chosen criterion is optimized to find the most appropriate sequence. Searching a continuous sequence space using a deterministic optimization algorithm makes it possible to find the optimal sequences with much less computation than many other approaches. The computational efficiency of this method is further improved by combining it with a graph spectral method, which explicitly takes into account the topology of the desired conformation and also helps make the combined method more robust. The continuous modeling used here appears to have additional advantages in mimicking the folding pathways and in creating the energy landscapes that help find sequences with high stability and kinetic accessibility. To illustrate the new approach, a widely used simplifying assumption is made by considering only two types of residues: hydrophobic (H) and polar (P). Self-avoiding compact lattice models are used to validate the method with known results in the literature and data that can be practically obtained by exhaustive enumeration on a desktop computer. We also present examples of sequence design for the HP models of some real proteins, which are solved in less than five minutes on a single-processor desktop computer Some open issues and future extensions are noted.

A hypothesis on a specific sequence-dependent conformation of DNA and its relation to the binding of the lac-repressor protein

The structure and properties of the double-helical form of the alternating copolymer poly(dA-dT) are considered. Different lines of evidence are interpreted in terms of a structure in which every second phosphate-diester linkage has a conformation different from that of the normal B form. A rationale for this “alternating-B” structure is given which provides an explanation for the effects of chemical modifications of the T residues on the binding of the poly(dA-dT)· poly(dA-dT) to the lac repressor of Escherichia coli.

Homeostatic regulation of free retinol-binding protein and free thyroxine pools of plasma by their plasma carrier proteins in chicken

The mechanism underlying homeostatic regulation of the plasma levels of free retinol-binding protein and free thyroxine, the systemic distribution of which is of great importance, has been investigated. A simple method has been developed to determine the rate of dissociation of a ligand from the binding protein. Analysis of the dissociation process of retinol-binding protein from prealbumin-2 reveals that the free retinol-binding protein pool undergoes massive flux, and the prealbumin-2 participates in homeostatic regulation of the free retinol-binding protein pool. Studies on the dissociation process of thyroxine from its plasma carrier proteins show that the various plasma carrier proteins share two roles. Of the two types of protein, the thyroxine-binding globulin (the high affinity binding protein) contributes only 27% of the free thyroxine in a rapid transition process, despite its being the major binding protein. But prealbumin-2, which has lower affinity towards thyroxine, participates mainly in a rapid flux of the free thyroxine pool. Thus thyroxine-binding globulin acts predominantly as a plasma reservoir of thyroxine, and also probably in the �buffering� action on plasma free thyroxine level, in the long term, while prealbumin-2 participates mainly in the maintainance of constancy of free thyroxine levels even in the short term. The existence of these two types of binding protein facilitates compensation for the metabolic flux of the free ligand and maintenance of the thyroxine pool within a very narrow range.