Activation of Classical Complement Pathway by Naturally Occurring Heteroantibodies in Normal Rabbit Serum. Effect on Subpopulations of Human Lymphoid Cells

Heteroantibodies present in normal rabbit serum (NRS) are toxic to human B lymphocytes, T lymphocytes, and monocytes. Even NRS, which exhibits little back ground cytotoxicity for human lymphoid cells in conventional HLA or B-cell lymphocytotoxic assays, can be shown to contain considerable activity by making two modifications in usual procedures: by washing cells in saline or balanced salt solutions devoid of protein or sugar substances, and by increasing incubation time for 1 h to 3--4 h. Using such modifications, the cytotoxic activity of NRS towards human lymphoid cells was investigated and was found to involve activation of the classical complement pathway rather than activation of the alternate complement pathway. Residual unwanted background cytotoxicity of NRS toward human lymphoid cells can be decreased without loss of desired complement activity either by heating NRS for 15 min at 50 degrees C or by mixing NRS with small amounts of normal human serum.     

“Porcupine” polymers and double-star polymers

Anionic block copolymerization of styrene and divinylbenzene is known to lead to the formation of star-shaped macromolecules. This “arm-first” method has been widely used and studied. The present paper is devoted to two special aspects of this method: The first is concerned with the efficiency of the protection exerted by the arms on the crosslinked core, preventing gelation of the reaction medium. A number of “porcupine” polymers involving bulky cores, fitted with a large number of arms, were synthesized and characterized. The second deals with the possibility of using the “living” carbanionic sites present in the cores, either for purpose of functionalization, or to grow new branches from the core. The presence of remaining unsaturations in the core was evidenced. This is a drawback, since the possibility for “transverse” bonds to be formed results in couplings, inducing gelation.     

Protective hinge in insulin opens to enable its receptor engagement

Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal B-chain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated “microreceptors” that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 β-turn, coupling reorientation of Phe^B24 to a 60° rotation of the B25-B28 β-strand away from the hormone core to lie antiparallel to the receptor''s L1–β₂ sheet. Opening of this hinge enables conserved nonpolar side chains (Ile^A2, Val^A3, Val^B12, Phe^B24, and Phe^B25) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.How insulin engages the insulin receptor has inspired speculation ever since the structure of the free hormone was determined by Hodgkin and colleagues in 1969 (1, 2). Over the ensuing decades, anomalies encountered in studies of analogs have suggested that the hormone undergoes a conformational change on receptor binding: in particular, that the C-terminal β-strand of the B chain (residues B24–B30) releases from the helical core to expose otherwise-buried nonpolar surfaces (the detachment model) (3–6). Interest in the B-chain β-strand was further motivated by the discovery of clinical mutations within it associated with diabetes mellitus (DM) (7). Analysis of residue-specific photo–cross-linking provided evidence that both the detached strand and underlying nonpolar surfaces engage the receptor (8).The relevant structural biology is as follows. The insulin receptor is a disulfide-linked (αβ)₂ receptor tyrosine kinase (Fig. 1A), the extracellular α-subunits together binding a single insulin molecule with high affinity (9). Involvement of the two α-subunits is asymmetric: the primary insulin-binding site (site 1^*) comprises the central β-sheet (L1–β₂) of the first leucine-rich repeat domain (L1) of one α-subunit and the partially helical C-terminal segment (αCT) of the other α-subunit (Fig. 1A) (10). Such binding initiates conformational changes leading to transphosphorylation of the β-subunits’ intracellular tyrosine kinase (TK) domains. Structures of wild-type (WT) insulin (or analogs) bound to extracellular receptor fragments were recently described at maximum resolution of 3.9 Å (11), revealing that hormone binding is primarily mediated by αCT (receptor residues 704–719); direct interactions between insulin and L1 were sparse and restricted to certain B-chain residues. On insulin binding, αCT was repositioned on the L1–β₂ surface, and its helix was C-terminally extended to include residues 711–714. None of these structures defined the positions of C-terminal B-chain residues beyond B21. Support for the detachment model was nonetheless provided by entry of αCT into a volume that would otherwise be occupied by B-chain residues B25–B30 (i.e., in classical insulin structures; Fig. 1B) (11).Open in a separate windowFig. 1.Insulin B-chain C-terminal β-strand in the μIR complex. (A) Structure of apo-receptor ectodomain. One monomer is in tube representation (labeled), the second is in surface representation. L1, first leucine-rich repeat domain; CR, cysteine-rich domain; L2, second leucine-rich repeat domain; FnIII-1, -2 and -3; first, second and third fibronectin type III domains, respectively; αCT, α-subunit C-terminal segment; coral disk, plasma membrane. (B) Insulin bound to μIR; the view direction with respect to L1 in the apo-ectodomain is indicated by the arrow in A. Only B-chain residues indicated in black were originally resolved (11). The brown tube indicates classical location of residues B20-B30 in free insulin, occluded in the complex by αCT. (C) Orthogonal views of unmodeled 2F_obs-F_calc difference electron density (SI Appendix), indicating association of map segments with the αCT C-terminal extension (transparent magenta), insulin B-chain C-terminal segment (transparent gray), and Asn^A21 (transparent yellow). Difference density is sharpened (B_sharp = −160 Ų). (D–F) Refined models of respective segments insulin B20–B27, αCT 714–719, and insulin A17-A21 within postrefinement 2F_obs-F_calc difference electron density (B_sharp = −160 Ų). D is in stereo.We describe here the structure and interactions of the detached B-chain C-terminal segment of insulin on its binding to a “microreceptor” (μIR), an L1–CR domain-minimized version of the α-subunit (designated IR310.T) plus exogenous αCT peptide 704–719 (11). Our analysis defines a hinge in the B chain whose opening is coupled to repositioning of αCT between nonpolar surfaces of L1 and the insulin A chain. To understand the role of this hinge in holoreceptor binding and signaling, we designed three insulin analogs containing structural constraints (d-Ala^B20, d-Ala^B23]-insulin, ∆Phe^B25-insulin, and ∆Phe^B24-insulin, where ∆Phe is (α,β)-dehydrophenylalanine (Fig. 2) (12). The latter represents, to our knowledge, the first use of ∆Phe—a rigid “β-breaker” with extended electronic conjugation between its side chain and main chain (SI Appendix, Fig. S1)—as a probe of induced fit in macromolecular recognition. In addition, a fourth analog, active but with anomalous flexibility in the B chain (5, 6) (

Individualizing glycemic targets in type 2 diabetes mellitus: implications of recent clinical trials

One of the first steps in the management of patients with type 2 diabetes mellitus is setting glycemic goals. Professional organizations advise setting specific hemoglobin A(1c) (HbA(1c)) targets for patients, and individualization of these goals has more recently been emphasized. However, the operational meaning of glycemic goals, and specific methods for individualizing them, have not been well-described. Choosing a specific HbA(1c) target range for a given patient requires taking several factors into consideration, including an assessment of the patient's risk for hyperglycemia-related complications versus the risks of therapy, all in the context of the overall clinical setting. Comorbid conditions, psychological status, capacity for self-care, economic considerations, and family and social support systems also play a key role in the intensity of therapy. The individualization of HbA(1c) targets has gained more traction after recent clinical trials in older patients with established type 2 diabetes mellitus failed to show a benefit from intensive glucose-lowering therapy on cardiovascular disease (CVD) outcomes. The limited available evidence suggests that near-normal glycemic targets should be the standard for younger patients with relatively recent onset of type 2 diabetes mellitus and little or no micro- or macrovascular complications, with the aim of preventing complications over the many years of life. However, somewhat higher targets should be considered for older patients with long-standing type 2 diabetes mellitus and evidence of CVD (or multiple CVD risk factors). This review explores these issues further and proposes a framework for considering an appropriate and safe HbA(1c) target range for each patient.     

Safety and patient selection of totally implantable hearing aid surgery: Envoy system,Esteem

Patient’s low compliance of conventional hearing aids has lead to innovation of totally implantable hearing devices such as Esteem, Envoy system. This study was designed to evaluate safety of device implantation, patient’s hearing gain, importance of anatomic landmarks, and to describe suitable criteria for patient selection. Via a non-randomized controlled clinical trial, ten patients with moderate-to-severe sensorineural hearing loss were implanted from 2007 to 2009. Mean follow-up period was 29.4 months. Correlation of pre-operative temporal bone CT scan anatomy with postoperative outcome was evaluated. Except one, all other implanted devices are active and patients’ overall average hearing gain are similar to conventional hearing aids (10–22 dB), but patients reported relatively better subjective sound quality compared with their pre-operative conventional hearing aids. One implanted device was explanted in a patient due to facial weakness and low-hearing gain. Revision surgery was done successfully in another patient secondary to excessive bone growth. Totally implantable hearing device surgery seems to be relatively safe and correct patient selection could lead to good outcomes. Lateral location of facial nerve, sclerotic mastoid air cells and narrow facial recess space seems to be related to postoperative complications.     

Prognostic implication of histopathological, immunohistochemical and clinical features of oligodendrogliomas: a study of 89 cases

Histopathological, immunohistochemical and clinical parameters were correlated with survival in 89 cases of oligodendroglioma (65 patients with grade II and 24 patients with grade III of the WHO classification). Median survival time and 5-year survival rate were 3.5 years and 76% for patients with oligodendroglioma grade II and 0.875 years and 23% for patients with oligodendroglioma grade III. The tumor biopsy specimens were immunohistochemically analyzed for Ki 67 (MIB-1), vimentin, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE) and synaptophysin. MIB-1 nuclear labeling index ranged from 0.0% to 33.4%; vimentin-immunoreactive tumor cells were found in 25 cases. MIB-1 nuclear labeling index and vimentin immunoreaction showed a significant statistical correlation to the 5-year survival rate of the patients. Tumors with vimentin expression (n = 25) and/ or high MIB-1 labeling index (n = 26) had a poorer prognosis than tumors lacking vimentin expression (n = 57) and/or displaying a low MIB-1 labeling index (n = 56). The expression of immunoreactivity for GFAP (n = 53), NSE (n = 23) and synaptophysin (n = 15) appeared to be of no prognostic relevance. Patients with gross total tumor resection (n = 47) had a median survival time and 5-year survival rate of 3.3 years and 84% compared to 1.2 years and 42% for patients with subtotal resection (n = 41). The comparison between patients who underwent surgery alone (n = 53) and those who had surgery plus postoperative radiation therapy showed no significant survival benefit from postoperative radiation therapy. In conclusion, tumor grade, MIB-1 labeling index, expression of vimentin and the extent of surgery are shown to be of prognostic relevance for patients with oligodendroglioma. Received: 29 July 1997 /Revised: 14 October 1997, 4 December 1997 / Accepted: 14 December 1997     

Coverage of pelvis and thigh region by pedicled perforator flap like deep inferior epigastric perforator (DIEP)

The deep inferior epigastric perforator flap (DIEP) is a variation of the transverse rectus abdominis myocutaneous flap (TRAM). This flap was used as a pedicled flap to reconstruct the pelvis and thigh region after resection for cancer (four cases). Various flaps have been described for covering theses tissue defects but we prefer this perforator flap for its many advantages. This flap is very reliable and generates minimal functional sequelae on donor site. This flap is useful to cover soft tissue defects after vascular and oncologic surgery, a situation that was rarely reported to our knowledge.