Simultaneous assessment of Asp isomerization and Asn deamidation in recombinant antibodies by LC-MS following incubation at elevated temperatures

The degradation of proteins by asparagine deamidation and aspartate isomerization is one of several chemical degradation pathways for recombinant antibodies. In this study, we have identified two solvent accessible degradation sites (light chain aspartate-56 and heavy chain aspartate-99/101) in the complementary-determining regions of a recombinant IgG1 antibody susceptible to isomerization under elevated temperature conditions. For both hot-spots, the degree of isomerization was found to be significantly higher than the deamidation of asparagine-(387, 392, 393) in the conserved CH3 region, which has been identified as being solvent accessible and sensitive to chemical degradation in previous studies. In order to reduce the time for simultaneous identification and functional evaluation of potential asparagine deamidation and aspartate isomerization sites, a test system employing accelerated temperature conditions and proteolytic peptide mapping combined with quantitative UPLC-MS was developed. This method occupies the formulation buffer system histidine/HCl (20 mM; pH 6.0) for denaturation/reduction/digestion and eliminates the alkylation step. The achieved degree of asparagine deamidation and aspartate isomerization was adequate to identify the functional consequence by binding studies. In summary, the here presented approach greatly facilitates the evaluation of fermentation, purification, formulation, and storage conditions on antibody asparagine deamidation and aspartate isomerization by monitoring susceptible marker peptides located in the complementary-determining regions of recombinant antibodies.     

Investigation of the mechanism of protein turnover in HeLa S-3 cells by incubation at elevated temperatures

An apparent paradox relating to the degradation of endogenous proteins in HeLa S-3 cells occurs at 45 degrees C, at which their proteolysis is considerably enhanced in vitro but completely inhibited in vivo. No significant differences in rates of degradation of short-lived (nascent) and long-lived ('existing') proteins synthesised at 37 degrees C were found when chased at temperatures up to 43 degrees C, but at 45 degrees C degradation of both categories was reduced to zero in vivo. Synthesis of protein was suppressed at temperatures above 41 degrees C, being reduced by up to 60% at 43 degrees C. Proteolysis in vitro proceeded 1.6-1.7 times faster at 45 degrees C than at 37 degrees C and neutral pH. Evidence is presented for the involvement of the basal system; the findings both in vivo and in vitro do not seem to implicate the lysosomal system, no firm indication being obtained of its 'induction' at elevated temperatures. The results are discussed in terms of the arrest of intracellular circulation at elevated temperatures, thereby reducing the delivery rate of proteins as substrates of the intracellular basal proteolytic enzyme system to negligible levels (i.e., to the frequency of encounters due solely to the diffusion of protein molecules with the cytoplasm).     

A morphological analysis of the first cleavage mitotic cytoskeleton isolated from sea urchin embryos (Strongylocentrotus droebachiensis)

Morphological changes in the mitotic cytoskeleton (MC) that occurred through the course of the first cleavage of Strongylocentrotus droebachiensis, cultured at 8°C or at 0°C, temperatures within the natural range for this species, have been investigated. Electron microscopy of MCs isolated from zygotes has revealed that they consisted largely of microtubules (mts). Thus, the morphology of these MCs is derived from the arrangement of the mts which form them. During anaphase, astral rays elongated while kinetochore fibers shortened. Asters enlarged during anaphase as a result of two events: astral ray lengthening and centrosphere enlargement. At the end of anaphase, asters of 8°C MCs filled the entire cell volume. The pattern of changes that occurred in 8°C mitotic apparatuses (MAs) also occurred in 0°C MCs. The observation of asters in 0°C MCs is contradictory to that of Stephens ('72b), who reported that 0°C MCs in this species were anastral. However, in 0°C metaphase MCs, the astral rays and spindle fibers were not as long as those in 8°C MCs. Also in 0°C MCs, the centrosphere was largely filled with dense material, whereas the centrosphere in 8°C MCs was larger and contained little dense material. Asters of 0°C MCs did not attain a large enough size to fill the egg volume completely, as did asters of 8°C MCs.     

Time-lapse videography of human oocytes following intracytoplasmic sperm injection: Events up to the first cleavage division

A total of 341 fertilized and 37 unfertilized oocytes from 63 intracytoplasmic sperm injection (ICSI) treatment cycles were included for retrospective assessment using the Embryoscope™ time-lapse video system. The second polar body (pb2) extrusion occurred at 2.9 ± 0.1 h (range 0.70–10.15 h) relative to sperm injection. All oocytes reduced in size following sperm injection (p < 0.05) with shrinkage ceasing after 2 h in the unfertilized and at pb2 extrusion in the fertilized oocytes. Pb2 extrusion was significantly delayed for women aged >38 years compared to those <35 years (3.4 ± 0.2 vs. 2.8 ± 0.1, p < 0.01) or 35–38 years (3.4 ± 0.2 vs. 2.8 ± 0.1, p < 0.01), but timing was not related to the Day 3 morphological grades (1–4) of subsequent embryos (2.9 ± 0.1, 2.9 ± 0.1, 2.8 ± 0.2 and 3.0 ± 0.1; p > 0.05 respectively). A shorter time of first cleavage division relative to either sperm injection or pb2 extrusion is associated with both top grade (AUC = 0.596 or 0.601, p = 0.006 or 0.004) and usable embryos (AUC = 0.638 or 0.632, p = 0.000 respectively) on Day 3. In summary, (i) pb2 of human oocytes extrudes at various times following sperm injection, (ii) the timing of pb2 extrusion is significantly delayed when female age >38 years, but not related to subsequent embryo development, (iii) all human oocytes reduce in size following sperm injection, (iv) completion of pb2 extrusion in the fertilized oocytes is a pivotal event in terminating shrinkage of the vitellus, and (v) time to first cleavage division either from sperm injection or pb2 extrusion is a significant predictive marker for embryo quality on Day 3.     

Relationships between the completion of first cleavage and the chromosomal complement,sex, and developmental rates of bovine embryos generated in vitro

One thousand eighty-four two-cell bovine embryos produced from 1,574 oocytes matured and fertilized in vitro were cultured as groups separated according to the time when they completed their first cleavage (24,30,40,48, or 62 hr postinsemination; hpi). At 5 days after insemination, the proportions of each group that had progressed to the eight-cell stage or beyond were determined and the 350 that had done so were fixed and examined cytogenetically for cell number, chromosomal abnormalities, and sex. Embryos in the “early” cleaving (24 and 30 hpi) and “late” cleaving (40–62 hpi) groups were compared. Early cleaving embryos were more likely to have developed to the eight-cell stage or beyond (52.2% vs. 20%), contained more cells (22 vs. 17), and were more likely to be male (3.6:1 vs. 0.93:1). It is suggested that these phenotypic differences between the sexes begin before the embryonic genome is generally thought to become activated and are due either to differential processing of X- and Y-bearing sperm within the zygote or to very early differential expression of genes derived from X- and Y-bearing sperm. © 1993 Wiley-Liss, Inc.     

Symbiosis and host morphological variation: Symbiodiniaceae photosynthesis in the octocoral Briareum asbestinum at ambient and elevated temperatures

Intra-species morphological variation may occur in sessile organisms, such as corals, living in different habitats. Conversely, the octocoral Briareum asbestinum exhibits both encrusting and upright branching morphologies at the same shallow water habitat, enabling studying physiological differences uncoupled from habitat variation due to depth or reef location. We investigated the mutualism between endosymbiotic dinoflagellate algae, Breviolum spp. (previously clade B Symbiodinium), and these B. asbestinum morphologies at ambient and elevated temperatures. Based on msh1 gene sequences, the host morphologies were genetically similar although they differed in protein content, polyp expansion behavior, and associated Breviolum (B19 for encrusting and B21 for branching B. asbestinum). Due to colony orientation, polyps in encrusting B. asbestinum experienced irradiance levels nearly three times higher than polyps in the branching morph, which probably contributed to the lower photochemical and light absorption efficiencies of the Breviolum in encrusting fragments. The light-limited portion of photosynthesis–irradiance curves and the intracellular chlorophyll concentrations, however, indicated that Breviolum in both morphologies were acclimated to similar internal irradiances. Encrusting B. asbestinum exhibited higher Breviolum density, areal chlorophyll a, and greater photosynthetic rates cm⁻² compared to branching B. asbestinum. Notably, elevated temperature did not cause bleaching in either morphology, as Breviolum and chlorophyll densities did not differ significantly from ambient temperature, although the two morphologies adjusted some of the measured parameters, indicating coping with the stressor. In the face of continued ocean warming, the high thermal tolerance of octocorals may reinforce the shift of Caribbean reefs from scleractinian coral to octocoral dominance.

     

Comparison of the size of paternal and maternal homologous chromosomes during the first 2 cleavage divisions in mouse embryos

The mouse metaphase chromosomes of the 1st and 2nd cleavage divisions were prepared without colchicine and stained with trypsin-Giemsa. Both the homologues had the same pattern of differential staining (position and number of bands and interbands) in all pairs of chromosomes. The measurements of homologues of the 1st, 2nd, 3rd, 4th and 5th pairs of autosomes have shown that at the first cleavage division metaphase the paternal chromosomes are 1.2 times, on the average longer than the maternal ones, whereas at the second division metaphase no reliable differences in the length of homologues were found. In mice, thus, the heterocyclic pattern of the paternal and maternal sets of chromosomes manifested itself during the 1st cleavage division only and disappeared fully beginning from the 2nd division. This appears to be due to the early functional activity of chromosomes, i.e. to the fact that already in the 2-cell embryos both the maternal and paternal genes are expressed.     

Analysis of structural and numerical chromosomal aberrations at the first and second mitosis after X irradiation of two-cell mouse embryos

Two-cell mouse embryos were X-irradiated in the late G2 phase in vivo. The first and second postradiation mitoses were analyzed for chromosomal anomalies. The majority of structural aberrations visible at the first mitosis after irradiation were chromatid breaks and chromatid gaps; only a few interchanges and dicentrics were observed. The aberration frequency resulted in a dose-effect relationship which was well described by a linear model. At the second mitosis 29% of the structural aberrations of the first mitosis were counted; the aberration quality changed only slightly. It is discussed whether these aberrations are to be considered "new," "derived," or unchanged transmitted aberrations. Contrary to the results obtained after irradiation of one-cell embryos, little chromosome loss was induced by radiation in two-cell embryos.     

Chromosome damage and non-disjunction measured at the first cleavage division in normal and chromosomally mutant female mice irradiated at the diakinesis stage of female meiosis

We have irradiated primary murine oocytes at the diakinesis stage of the first meiotic division with 0.6 Gy X-rays. Fertilized oocytes were cultured overnight to arrest the first cleavage division and display pronuclear chromosomes. All preparations were preferentially stained for centric constitutive heterochromatin and analyzed for structural and numerical radiation effects. Females of 3 different karyotypes were irradiated (all on a Swiss random-bred genetic background): +/+ (221 female pronuclei analyzed), Rb(11.13)4Bnr T(1;13)70H/Rb(1.13)4Bnr T(1;13)70H with 11.13(1) and 1(13) large and small marker bivalents (RbT/RbT, 242 zygotes analyzed) and the same karyotype but with a 1(13)H;1(13) Wa heteromorphic bivalent (RbT/RbT*, 126 zygotes analyzed). Hyperploid chromosome counts were encountered with frequencies of 11.8% (+/+), 11.9% (RbT/RbT) and 16.6% (RbT/RbT*). In this order of karyotypes, the frequencies of dicentrics per zygote were 0.07, 0.16 and 0.11 and the frequencies of fragments 0.13, 0.18 and 0.31. In about half of the supernumerary chromosome spreads, a dicentric chromosome was included. The long marker bivalent 11.13(1) had a non-disjunction frequency of 2.5 times its control value, partially because it was involved in dicentric formation as well. For the RbT/RbT karyotype, the spontaneous maternal non-disjunction level was 5.4%. For the RbT/RbT* karyotype, it can be assumed to be the same or slightly higher because of the 1(13)H;1(13) Wa heteromorphic bivalent. This increased intrinsic sensitivity for non-disjunction was not expressed as an increased sensitivity for aneuploidy after irradiation. This fact and the numerical association between hyperploidy and dicentric formation, both for normal bivalents and for the 11.13(1) marker bivalent, lead us to suppose that in the female mouse, irradiation-caused aneuploidy is effectuated via chromatid exchange. The data presented do not rule out the existence of another mechanism.     

Effects of egg aging on in vitro fertilization and first cleavage division in the hamster

The postovulatory fertile life of mammalian eggs is remarkably short (approximately 6-36h). Anomalies of embryogenesis may result from fertilization of aged, defective eggs. Attempts to study this problem using whole-animal models are complicated by chances in the natural milieu of the gametes. In the present study, postovulatory hamster eggs were allowed to agein vivo then fertilized in vitro. Cumulus-intact eggs recovered from superovulated hamsters either 2 or 9 h after the estiated time of ovulation (12 h postHCG) were incubated for 4 h with preincubated sperm suspentions in a modified Tyrode's solution devised for in vitro fertilization. Eggs were either fixed or cultured for another 20h in fresh medium to allow cleavage to occur, then examined by light microscopy (phase and interference-contrast). No significant difference was found in the ablities of fresh and aged eggs to be penetrated by spermatozoa (94% vs 90%, respectively; 8 replicated experiments), but only 59% of penetrated aged eggs were found to undergo morphologically normally fertilization (2 polar bodies, 2 prounclei) compared with 75% of fresh eggs (difference significant, P< 0.01). About 13% of eggs were polyspermic in both categories. The most common anomaly in aged fertilied egges was failure to extrude the second polor body (23% off eggs vs 8% of fresh eggs, P < 0.01). Only 21% of aged eggs underwent first cleavaage, and only 74% of these appeared morphologically normal, compared with value of 68% and 98%, respectively, for fresh eggs. These data show that in the hamster, abnormal fertilization and cleavage failure can, in part, be directly attributed to postovulatory deterioration of eggs. We also infer that the apparently very short penetrable life of hamster eggs in vivo shown by previous investigators is an indirect effect of postovulatory changes in the female reproductive tract that are unfavorable for sperm-egg interactions.     

Sperm membrane functional integrity and response of frozen-thawed bovine spermatozoa during the hypoosmotic swelling test incubation at varying temperatures

The objective of this study was to assess the sperm membrane integrity and permeability of frozen-thawed bovine spermatozoa, processed at varying temperatures during and after thawing, by exposing the spermatozoa to standardized hypoosmotic conditions. The hypoosmotic swelling (HOS) test was employed to measure changes in sperm membrane functional status and permeability. Frozen specimens (from 5 bulls) were thawed at 37h degrees C for 10 sec and transferred to a water bath at 37 (Aliquot 1), 21 (Aliquot 2) or 5 degrees C (Aliquot 3) to complete thawing (1 to 2 min). The specimens were maintained and processed at these temperatures for additional 5 to 10 min. Specimens were slowly diluted 1:1 (v/v) and washed with Ham's F-10 media containing 3% (w/v) BSA. The HOS test was performed by adding 0.1 ml of the sperm specimen to 1.0 ml of a 100 mOsm/L HOS diluent. The following treatments were performed: 1) Aliquot 1 (control), specimens were incubated in HOS solutions at 37 degrees C for 5 min; 2) Aliquot 2, specimens were incubated in HOS solutions at 21 or 37 degrees C for 5 min; and 3) Aliquot 3, specimens were incubated in HOS solutions at 5 or 37 degrees C for 5 min. Samples were obtained from the sperm specimen-HOS diluent mixtures at 1 min intervals (during the 5 min incubation period), fixed and assessed for sperm swelling patterns. The sperm response to the HOS test for specimens processed at temperatures below 37 degrees C was higher when samples were incubated in HOS diluents at 37 degrees C. This finding indicates that the potential for sperm swelling (measurement of sperm membrane functional status) can be maintained when spermatozoa are processed at temperatures below 37 degrees C. The highest response to the HOS test was observed in spermatozoa processed at 21 degrees C and incubated in a HOS solution at 37 degrees C. The response to the HOS test was superior to the one observed in specimens maintained and processed at 37 degrees C throughout. Thawing of spermatozoa at 37 degrees C, followed by processing at 21 degrees C seems to reduce the negative effects associated with osmotic shock and results in the preservation of the sperm membrane functional status during the in vitro handling of frozen-thawed bovine spermatozoa.