Have we overestimated the benefit of human(ized) antibodies?

The infusion of animal-derived antibodies has been known for some time to trigger the generation of antibodies directed at the foreign protein as well as adverse events including cytokine release syndrome. These immunological phenomena drove the development of humanized and fully human monoclonal antibodies. The ability to generate human(ized) antibodies has been both a blessing and a curse. While incremental gains in the clinical efficacy and safety for some agents have been realized, a positive effect has not been observed for all human(ized) antibodies. Many human(ized) antibodies trigger the development of anti-drug antibody responses and infusion reactions. The current belief that antibodies need to be human(ized) to have enhanced therapeutic utility may slow the development of novel animal-derived monoclonal antibody therapeutics for use in clinical indications. In the case of murine antibodies, greater than 20% induce tolerable/negligible immunogenicity, suggesting that in these cases humanization may not offer significant gains in therapeutic utility. Furthermore, humanization of some murine antibodies may reduce their clinical effectiveness. The available data suggest that the utility of human(ized) antibodies needs to be evaluated on a case-by-case basis, taking a cost-benefit approach, taking both biochemical characteristics and the targeted therapeutic indication into account.Key words: immunogenicity, human anti-mouse antibody, cytokine release syndrome     

Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria

Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have several positive implications with respect to future development of a transformation system for cellulolytic thermophiles. Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280. Received 12 September 2000/ Accepted in revised form 20 November 2000     

Titin isoform changes in rat myocardium during development

Developmental changes in the alternative splicing patterns of titin were observed in rat cardiac muscle. Titin from 16-day fetal hearts consisted of a single 3710 kDa band on SDS agarose gels, and it disappeared by 10 days after birth. The major adult N2B isoform (2990 kDa) first appeared in 18-day fetal hearts and its proportion in the ventricle increased to approximately 85% from 20 days of age and older. Changes in three other intermediate-sized N2BA isoform bands also occurred during this same time period. The cDNA sequences of fetal cardiac, adult ventricle, and adult soleus were different in the PEVK and alternatively spliced middle Ig domain. Extensive heterogeneity in splice patterns was found in the N2BA PEVK region. The extra length of the fetal titin isoforms appeared to be due to both a greater number of middle Ig domains expressed plus the inclusion of more PEVK exons. Passive tension measurements on myocyte-sized fragments indicated a significantly lower tension in neonate versus adult ventricles at sarcomere lengths greater than 2.1 microm, consistent with the protein and cDNA sequence results. The time course of the titin isoform switching was similar to that occurring with myosin and troponin I during development.     

Characteristics of troponin C binding to the myofibrillar thin filament: extraction of troponin C is not random along the length of the thin filament.

Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin responsible for initiating the cascade of events resulting in contraction of striated muscle. This protein can be readily extracted from myofibrils with low-ionic-strength EDTA-containing buffers. The properties of TnC extraction have not been characterized at the structural level, nor have the interactions of TnC with the native myofibrillar thin filament been studied. To address these issues, fluorescein-labeled TnC, in conjunction with high-resolution digital fluorescence microscopy, was used to characterize TnC binding to myofibrils and to determine the randomness of TnC extraction. Fluorescein-5-maleimide TnC (F5M TnC) retained biological activity, as evidenced by reconstitution of Ca(2+)-dependent ATPase activity in extracted myofibrils and binding to TnI in a Ca(2+)-sensitive manner. The binding of F5M TnC to highly extracted myofibrils at low Ca2+ was restricted to the overlap region under rigor conditions, and the location of binding was not influenced by F5M TnC concentration. The addition of myosin subfragment 1 to occupy all actin sites resulted in F5M TnC being bound in both the overlap and nonoverlap regions. However, very little F5M TnC was bound to myofibrils under relaxing conditions. These results suggest that strong binding of myosin heads enhances TnC binding. At high Ca2+, the pattern of F5M TnC binding was concentration dependent: binding was restricted to the overlap region at low F5M TnC concentration, whereas the binding propagated into the nonoverlap region at higher levels. Analysis of fluorescence intensity showed the greatest binding of F5M TnC at high Ca2+ with S1, and these conditions were used to characterize partially TnC-extracted myofibrils. Comparison of partially extracted myofibrils showed that low levels of extraction were associated with greater F5M TnC being bound in the nonoverlap region than in the overlap region relative to higher levels of extraction. These results show that TnC extraction is not random along the length of the thin filament, but occurs more readily in the nonoverlap region. This observation, in conjunction with the influence of rigor heads on the pattern of F5M TnC binding, suggests that strong myosin binding to actin stabilizes TnC binding at low Ca2+.     

Refined structure of chicken skeletal muscle troponin C in the two-calcium state at 2-A resolution

The structure of troponin C has been refined at 2A resolution to an R value of 0.172 using a total of 8,100 reflections. Troponin C has an unusual dumbbell shape with only the two C-domain high affinity sites III and IV occupied with metals, while the pair of N-domain low affinity sites I and II are devoid of metals. The coordination of the Ca2+ approaches seven with the last glutamic acid residue in each site forming an asymmetric bidentate ligand. The flanking helices in the metal-bound EF hands are in similar orientation (both 113 degrees) while in the apo sites they are more obtuse (134 and 149 degrees). The EF hands of holo sites III and IV are similar while the apo sites I and II are less similar (rms for backbone atoms, 0.78 and 1.44). The half-loops of the 12-residue holo and apo sites show better agreement than the full loops themselves, suggesting a hinge motion at the midpoint of the loops. The long central helix is stabilized by electrostatic interactions and salt bridges between charged side chains spaced at 3 or 4 residues along the helix. A cluster of water molecules encircle the long helix and hydrogen bond to the backbone carbonyls. At the beginning of the B-helix, a water molecule is interposed at each of two consecutive backbone NH...OC hydrogen bonds. The terminal pair of helices A/D (apo) match with E/H (holo), and the internal pair of helices B/C (apo) match with F/G (holo). Thus, muscle contraction may be triggered by Ca2+ binding to loops I and II which results in a concerted rearrangement of residues in the loops, including the essential Gly at position 6 in each loop. This rearrangement than causes a reorientation of helices B and C along with the BC linker.     

Interactions of Photobleaching and Inorganic Nutrients in Determining Bacterial Growth on Colored Dissolved Organic Carbon

Abstract Bacteria are key organisms in the processing of dissolved organic carbon (DOC) in aquatic ecosystems. Their growth depends on both organic substrates and inorganic nutrients. The importance of allochthonous DOC, usually highly colored, as bacterial substrate can be modified by photobleaching. In this study, we examined how colored DOC (CDOC) photobleaching, and phosphorus (P) and nitrogen (N) availability, affect bacterial growth. Five experiments were conducted, manipulating nutrients (P and N) and sunlight exposure. In almost every case, nutrient additions had a significant, positive effect on bacterial abundance, production, and growth efficiency. Sunlight exposure (CDOC photobleaching) had a significant, positive effect on bacterial abundance and growth efficiency. We also found a significant, positive interaction between these two factors. Thus, bacterial use of CDOC was accelerated under sunlight exposure and enhanced P and N concentrations. In addition, the accumulation of cells in sunlight treatments was dependent on nutrient availability. More photobleached substrate was converted into bacterial cells in P- and N-enriched treatments. These results suggest nutrient availability may affect the biologically-mediated fate (new biomass vs respiration) of CDOC.     

Calcium alone does not fully activate the thin filament for S1 binding to rigor myofibrils.

Skeletal muscle contraction is regulated by calcium via troponin and tropomyosin and appears to involve cooperative activation of cross-bridge binding to actin. We studied the regulation of fluorescent myosin subfragment 1 (fS1) binding to rigor myofibrils over a wide range of fS1 and calcium levels using highly sensitive imaging techniques. At low calcium and low fS1, the fluorescence was restricted to the actin-myosin overlap region. At high calcium and very low fS1, the fluorescence was still predominantly in the overlap region. The ratio of nonoverlap to overlap fluorescence intensity showed that increases in the fS1 level resulted in a shift in maximum fluorescence from the overlap to the nonoverlap region at both low and high calcium; this transition occurred at lower fS1 levels in myofibrils with high calcium. At a fixed fS1 level, increases in calcium also resulted in a shift in maximum fluorescence from the overlap region to the nonoverlap region. These results suggest that calcium alone does not fully activate the thin filament for rigor S1 binding and that, even at high calcium, the thin filament is not activated along its entire length.     

Effects of a non-divalent cation binding mutant of myosin regulatory light chain on tension generation in skinned skeletal muscle fibers.

Each myosin molecule contains two heavy chains and a total of four low-molecular weight light chain subunits, two "essential" and two "regulatory" light chains (RLCs). Although the roles of myosin light chains in vertebrate striated muscle are poorly understood at present, recent studies on the RLC have suggested that it has a modulatory role with respect to Ca2+ sensitivity of tension and the rate of tension development, effects that may be mediated by Ca2+ binding to the RLC. To examine possible roles of the RLC Ca2+/Mg2+ binding site in tension development by skeletal muscle, we replaced endogenous RLC in rabbit skinned psoas fibers with an avian mutant RLC (D47A) having much reduced affinity for divalent cations. After replacement of up to 80% of the endogenous RLC with D47A RLC, maximum tension (at pCa 4.5) was significantly reduced compared with preexchange tension, and the amount of decrease was directly related to the extent of D47A exchange. Fiber stiffness changed in proportion to tension, indicating that the decrease in tension was due to a decrease in the number of tension-generating cross-bridges. Decreases in both tension and stiffness were substantially, although incompletely, reversed after reexchange of native RLC for D47A. RLC exchange was also performed using a wild-type RLC. Although a small decrease in tension was observed after wild-type RLC exchange, the decrease was not proportional to the extent of RLC exchange and was not reversed by reexchange of the native RLC. D47A exchange also decreased the Ca2+ sensitivity of tension and reduced the apparent cooperativity of tension development. The results suggest that divalent cation binding to myosin RLC plays an important role in tension generation in skeletal muscle fibers.     

Cerebellar neurones: Differentiation and modulation of sensitivity to excitotoxic treatment

The neurite outgrowth and adhesion complex (NOAC), isolated from rabbit sera has been dissociated in its major components by reverse-phase chromatography in HPLC by using a C₁₈ column. SDS-PAGE analisys of the active fractions revealed the presence of three major bands of approximately 100, 70 and 50 kDa. Studies on the biological activity of NOAC were carried out on rat cerebellar granule cells. NOAC-cultured cells exhibit a marked resistance to excitotoxic stimuli carried by glutamate.     

Chicken cardiac myofibrillogenesis studied with antibodies specific for titin and the muscle and nonmuscle isoforms of actin and tropomyosin

Summary Myofibrillogenesis was studied in cultured chick cardiomyocytes using indirect immunofluorescence microscopy and antibodies against - and -actin, muscle and nonmuscle tropomyosin, muscle myosin, and titin. Initially, cardiomyocytes, devoid of myofibrils, developed variable numbers of stress fiber-like structures with uniform staining for anti-muscle and nonmuscle actin and tropomyosin, and diffuse, weak staining with anti-titin. Anti-myosin labeled bundles of filaments that exhibited variable degrees of association with the stress fiber-like structures. Myofibrillogenesis occurred with a progressive, and generally simultaneous, longitudinal reorganization of stress fiber-like structures to form primitive sarcomeric units. Titin appeared to attain its mature pattern before the other major contractile proteins. Changes in the staining patterns of actin, tropomyosin, and myosin as myofibrils matured were interpreted as due to longitudinal filament alignment occurring before ordering in the axial direction. Non-muscle actin and tropomyosin were found with sarcomeric periodicity in the initial stages of sarcomere myofibrillogenesis, although their staining patterns were not identical. The localization of the sarcomeric proteins -actin and muscle tropomyosin in stress fiber-like structures and the incorporation of non-muscle proteins in the initial stages of sarcomere organization bring into question the meaning of sarcomeric proteins in regard to myofibrillogenesis.