Reversible Adsorption of Soluble Hexameric Insulin onto the Surface of Insulin Crystals Cocrystallized with Protamine: An Electrostatic Interaction

Mixing pharmaceutical preparations of soluble neutral regular insulin solution (NRI) and neutral protamine Hagedorn (NPH) crystalline insulin suspension leads to a reduction in the measurable amount of soluble insulin in the formulation supernatant. However in spite of the loss in soluble insulin, the time-actions of these components have been shown, in clinical trials, to be unaffected. The interaction between these different physical forms of insulin has been studied using reversed-phase HPLC, isothermal titrating calorimetry, and Doppler electrophoretic light scattering analysis. Sorbent surface and solution perturbation studies revealed that the NRI adsorbs to the surface of the NPH crystal with an equilibrium constant ranging from 10⁴ M^–1 to 10⁷ M^–!, depending on the protamine concentration, pH, ionic strength, and temperature. This adsorption behavior suggests that the binding is mediated by electrostatic interactions arising between the positively-charged NPH crystal and the negatively-charged NRI hexamer. Doppler electrophoretic light scattering results, used to probe the pH-dependent surface charge of NPH and soluble insulin hexamer, support the conclusion that electrostatic interactions mediate the adsorption process. Adsorption studies under physiological conditions indicate that the elevated temperature and ionic strength, in a subcutaneous depot, are sufficient to lead to the dissociation of the NRI/NPH complex that exists in these NPH mixture formulations.     

Diabetic neuropathies

Diabetic neuropathies are the most common type of neuropathies seen in clinical practice. These neuropathies can range clinically from asymptomatic to manifesting symptoms caused by motor, sensory, and autonomic nerve dysfunction. These neuropathies can affect the peripheral nervous system, pain receptors, cardiovascular, urogenital, and gastrointestinal systems. This monograph presents an overview of the different types of diabetic neuropathies, their presentations, diagnostic tools, and strategies for management.     

Genomic donor cassette sharing during VLRA and VLRC assembly in jawless vertebrates

Lampreys possess two T-like lymphocyte lineages that express either variable lymphocyte receptor (VLR) A or VLRC antigen receptors. VLRA⁺ and VLRC⁺ lymphocytes share many similarities with the two principal T-cell lineages of jawed vertebrates expressing the αβ and γδ T-cell receptors (TCRs). During the assembly of VLR genes, several types of genomic cassettes are inserted, in step-wise fashion, into incomplete germ-line genes to generate the mature forms of antigen receptor genes. Unexpectedly, the structurally variable components of VLRA and VLRC receptors often possess partially identical sequences; this phenomenon of module sharing between these two VLR isotypes occurs in both lampreys and hagfishes. By contrast, VLRA and VLRC molecules typically do not share their building blocks with the structurally analogous VLRB receptors that are expressed by B-like lymphocytes. Our studies reveal that VLRA and VLRC germ-line genes are situated in close proximity to each other in the lamprey genome and indicate the interspersed arrangement of isotype-specific and shared genomic donor cassettes; these features may facilitate the shared cassette use. The genomic structure of the VLRA/VLRC locus in lampreys is reminiscent of the interspersed nature of the TCRA/TCRD locus in jawed vertebrates that also allows the sharing of some variable gene segments during the recombinatorial assembly of TCR genes.The only two extant taxa of jawless vertebrates (agnatha), lampreys and hagfishes, occupy a unique position in chordate phylogeny and thus are a focal point for studies in comparative immunology. Although jawless vertebrates were shown to reject skin allografts and to produce serum agglutinins to mammalian red blood cells and bacteria (1, 2), the cellular and molecular bases for these adaptive responses remained elusive until the recent identification of their alternative adaptive immune system (3). In contrast to the antigen receptors of jawed vertebrates, whose structural framework is the Ig-domain, the basic building block of agnathan antigen receptors is the leucine-rich repeat (LRR) (4–6). In analogy to the situation in jawed vertebrates, mature genes of so-called variable lymphocyte receptors (VLRs) are combinatorially assembled from different types of genomic donor LRR cassettes; their sequences are inserted into incomplete germ-line VLR genes (4–6). A gene conversion-like process is postulated to underlie the VLR gene assembly (7, 8), through the activity of orthologs of mammalian activation-induced cytidine deaminase (AID), termed cytidine deaminases 1 and 2 (CDA1 and CDA2) (7, 9). As is the case for T-cell receptors (TCRs) and B-cell receptors (BCRs) of jawed vertebrates, combinatorial VLR assembly generates vast repertoires of diverse anticipatory receptors (4–6).Three VLR genes, VLRA, VLRB and VLRC, have been identified in lampreys and hagfishes (3, 9–12). The three VLR isotypes are differentially expressed by three distinct populations of lymphocytes in lampreys (9, 13). The two types of T-like cells of lamprey, VLRA⁺ and VLRC⁺ lymphocytes, are generated in the thymoid, a lymphoepithelial tissue equivalent to the thymus (13–15); this situation is analogous to the development in the thymus of the two distinct αβ and γδ T-cell lineages in jawed vertebrates. The VLRB⁺ cells appear to be generated in hematopoietic tissues outside of the thymoid (14), much like B cells in jawed vertebrates. These findings suggest that these basic pathways of lymphocyte differentiation already existed in a common ancestor of jawed and jawless vertebrates (4–6, 16).The close developmental relationship of the two principal lineages of T lymphocytes in jawed vertebrates is reflected in the unique genomic organization of the TCR genes that encode the four chains of the two different heterodimeric αβ and γδ TCRs (17). Here, we examine the sequence diversity and genomic organization of VLRA and VLRC receptor genes to gain insight into their functional and evolutionary relationship.     

Adsorbed proteins influence the biological activity and molecular targeting of nanomaterials.

The possible combination of specific physicochemical properties operating at unique sites of action within cells and tissues has led to considerable uncertainty surrounding nanomaterial toxic potential. We have investigated the importance of proteins adsorbed onto the surface of two distinct classes of nanomaterials (single-walled carbon nanotubes [SWCNTs]; 10-nm amorphous silica) in guiding nanomaterial uptake or toxicity in the RAW 264.7 macrophage-like model. Albumin was identified as the major fetal bovine or human serum/plasma protein adsorbed onto SWCNTs, while a distinct protein adsorption profile was observed when plasma from the Nagase analbuminemic rat was used. Damaged or structurally altered albumin is rapidly cleared from systemic circulation by scavenger receptors. We observed that SWCNTs inhibited the induction of cyclooxygenase-2 (Cox-2) by lipopolysaccharide (LPS; 1 ng/ml, 6 h) and this anti-inflammatory response was inhibited by fucoidan (scavenger receptor antagonist). Fucoidan also reduced the uptake of fluorescent SWCNTs (Alexa647). Precoating SWCNTs with a nonionic surfactant (Pluronic F127) inhibited albumin adsorption and anti-inflammatory properties. Albumin-coated SWCNTs reduced LPS-mediated Cox-2 induction under serum-free conditions. SWCNTs did not reduce binding of LPS(Alexa488) to RAW 264.7 cells. The profile of proteins adsorbed onto amorphous silica particles (50-1000 nm) was qualitatively different, relative to SWCNTs, and precoating amorphous silica with Pluronic F127 dramatically reduced the adsorption of serum proteins and toxicity. Collectively, these observations suggest an important role for adsorbed proteins in modulating the uptake and toxicity of SWCNTs and nano-sized amorphous silica.