Tuning coordination modes of pyridine/thioether Schiff base (NNS) ligands to mononuclear manganese carbonyls.

We have investigated the coordination modes of NNS Schiff base, thioether ligands to manganese(I) carbonyls. The ligands contain ortho substituted pyridines (H, CH3, OCH3, fluorophenyl) and varying substituents (H, CH3) at the Schiff base linkage. In general, reaction of [Mn(CO)5Br] with a tridentate NNS ligand in CH2Cl2 affords species in which the thioether-S may be bound or unbound to the manganese center, depending on the steric and electronic substitution in the ligand framework; as a result, the complexes exhibit two or three carbonyl ligands, respectively. Aldehyde-derived ligand frames ((R1)N(H)NS) generally afford complexes of type [((R)NNS)Mn(CO)3Br] (1(CO), 2(CO), 3(CO); R = H, OCH3, CH3) that exhibit incomplete ligation of the chelate (S not bound) in X-ray structures. In contrast, use of the iminomethyl ligand (N(Me)NS) affords a complex of formula [(N(Me)NS)Mn(CO)2Br] (4(CO)), in which the mixed N/thioether-S stabilizes the {Mn(CO)2}(+) fragment. In solid state IR spectra, complexes of type [((R)NNS)Mn(CO)3Br] (1(CO) through 3(CO)) afford three ν(CO) in the range ~2060-1865 cm(-1); the dicarbonyl complex [(N(Me)NS)Mn(CO)2Br] (4(CO)) exhibits two carbonyl stretches in the range ~1920-1845 cm(-1). Prolonged storage of the tricarbonyl [((Me)NNS)Mn(CO)3Br] (3(CO)) in presence of trace dioxygen affords the dibromide species [((Me)NNS)Mn(Br)2] (3(Br)), in which the thioether S reliably binds to the Mn(II) center. Complexes 1(CO)-3(CO) exhibit simple, diamagnetic (1)H NMR spectra in CD2Cl2. The S-ligated complex 4(CO) exhibits spectra consistent with a mixture of an S-bound (6-coordinate) and S-unbound (5-coordinate) species as represented by [(N(Me)NS)Mn(CO)2Br] ↔ [(N(Me)NS)Mn(CO)2Br]. Lastly, we obtained crystal structures of the S-bound and S-unbound conformers derived from the same ligand--the fluorophenyl derived (FPh)NNS, namely [((FPh)NNS)Mn(CO)3Br] (5(CO-a)) and [((FPh)NNS)Mn(CO)2Br] (5(CO-b)). This report represents several examples of a thioether-stabilized {Mn(CO)2}(+) fragment, a deviation from the usual 'piano stool' Mn(I) tricarbonyl motif. We highlight that coordination of these NNS ligands to Mn(I) carbonyls occurs on a soft conformational landscape, and that ligand substituents can be rationally employed to favor the desired coordination mode.

Immunobiological role of llama heavy-chain antibodies against a bacterial beta-lactamase.

In 1993, a fraction of antibodies (Abs) devoid of L chain was found naturally occurring in the Camelidae. They were found to lack L chains, as well as the first constant heavy-chain domain (CH(1)) and therefore they were named "heavy-chain Abs" (HCAbs). Subsequent studies focused on the functional, structural and biochemical properties of recombinant variable fragments (rVHHs) of HCAbs. It was stated that rVHHs have an augmented capacity to interact with "partially hidden" epitopes, like enzymes active sites, and have an increased stability to thermal and chemical aggression. It has been suggested that these unconventional Abs could represent an evolutionary advantage, being more efficient than conventional Abs to inhibit microbial enzymes, and thus exerting a more protective immune response against pathogens. The present work focuses on the immunobiological role of HCAbs, in their capacity to inhibit microbial enzymes. Two animal models were selected, comprising a model for common vertebrates without HCAbs (rabbits), and a model for vertebrates with both conventional and unconventional Abs (Lama glama). A recombinant bacterial beta-lactamase (CTX-M-2) was selected as the microbial enzymatic antigen. After conventional immunization schedules, neither serum titers nor serum inhibitory capacity showed significant differences when rabbits and llamas were compared. These results indicate that the a priori assumption that the adaptive immune system of camelids could be better "prepared" to respond to bacterial enzymes because of the presence of HCAbs, is not always accurate. Furthermore, when the different llama antibody isotypes and subclasses were purified, it was demonstrated that the inhibitory capacity of total serum was due exclusively to IgG(1). HCAbs not only failed to inhibit CTX-M-2, but instead they activated its enzymatic activity. Altogether, these results indicate that the hypotheses extrapolated from the rVHHs properties need to be revised; the real role of HCAbs in vivo remains unknown, as well as their evolutionary cause.

Immunologic "vaccination" for the prevention of autoimmune diabetes (type 1A).

Diabetes type 1A is an autoimmune condition characterized by lymphocytic infiltration of islets and selective destruction of insulin-secreting beta-cells. Numerous investigators have prevented diabetes in animal models with a variety of antigens and routes of administration. It is also now possible to identify high-risk individuals even before the appearance of autoantibodies. These advances have created the opportunity to design and begin human prevention trials. This review focuses on a variety of immunomodulatory approaches (including administration of adjuvants, autoantigens, T-cells, T-cell receptors, and DNA) that we have collectively termed immunologic "vaccination." In addition, we discuss the potential benefits and dangers of these approaches and issues relating to the design of human trials.

Genotypic and phenotypic characterization of six new recombinant congenic strains derived from NOD/Shi and CBA/J genomes.

Recombinant Congenic Strains (RCS) are useful for dissecting complex polygenic traits. Here, we describe genetic and phenotypic characterization of six new RCS generated from outcrosses between NOD/Shi and CBA/LsLt, followed by sib mating of first backcross progeny (to CBA) for 20 generations, whereupon genetic and phenotypic analysis commenced. Four of the RCS were selected on the basis of residual heterozygosity present at F20 in one of the three original RCS. Contrary to expectations for RCS developed at first backcross, all derived at least 50% of the polymorphic markers typed from the NOD parental strain. Development of autoimmune insulin-dependent diabetes mellitus (IDDM) in NOD is a strain-specific characteristic. The major genetic component predisposing NOD mice to IDDM, their H2(g7) haplotype, was present in all RCS. Nevertheless, the presence of variable amounts of CBA genome at non-MHC loci conferred complete resistance in all RCS to spontaneous IDDM development, and rendered them strongly resistant to cyclophosphamide-induced IDDM. Although the RCS more resemble NOD in regard to certain strain-specific characteristics, such as prolificacy, an immunologic phenotype that was significantly reduced when compared to both parental strains was the number of peripheral CD8(+) T cells. Given the genetic characterization presented, these new RCS should prove valuable to investigators interested in studying genes controlling differential susceptibilities distinguishing the NOD and CBA inbred strain backgrounds.

T cell receptor gene polymorphisms associated with anti-insulin, autoimmune T cells in diabetes-prone NOD mice.

The great majority of insulin-specific T cell clones isolated from islets of NOD mice react with insulin peptide B-(9-23) (amino acids 9-23 of the insulin B chain). The T cell receptors of these clones contain diverse beta-chains but restricted alpha-chains. The dominant alpha-chain motif is a V alpha 13 segment (10 out of 13) combined to either a J alpha 45 or a J alpha 34 segment (eight out of 13). Furthermore, nine out of 10 of these V alpha 13 segments are a product of a novel NOD TCR V alpha gene which we have termed V alpha 13.3. Analysis of V alpha 13 transcripts from splenic cDNA libraries from the NOD, BALB/c and C57BL/6 mice revealed significant differences between strains. The NOD sequences contained both V alpha 13.1 and the novel V alpha 13.3. The BALB/c contained the previously reported V alpha 13.1 and V alpha 13.2, but not the V alpha 13.3 sequence identified in the NOD anti-insulin T cell clones. The C57BL/6 had V alpha 13.1 and V alpha 13.3 plus two additional novel sequences which we have termed V alpha 13.4 and V alpha 13.5. These V alpha 13 subfamily members differed by two to four amino acids in either the CDR1 region or adjoining the CDR2 region. The frequency of utilization of the different V alpha 13 subtypes varied dramatically between strains. In the NOD spleen, V alpha 13.3 was detected 79% of the time, compared to 21% for V alpha 13.1. In contrast, the C57BL/6 spleen contained only 7% of V alpha 13.3 sequences compared to the other subfamily members present (V alpha 13.1: 27%; V alpha 13.4: 56%; V alpha 13.5: 10%). MHC polymorphisms or other unknown selective pressures may contribute to these differences in V alpha 13 utilization. We hypothesize that the presence and frequent utilization of the V alpha 13.3 T cell receptor element is involved in targeting insulin B-(9-23) and may be related to diabetes susceptibility of NOD mice.