The isolation and characterization of a glycoprotein from human thoracic aorta

1. A glycoprotein extracted by cold alkali from the walls of human aorta was purified by chromatography on DEAE-cellulose. 2. The compound was electrophoretically homogeneous and essentially so by chromatography on DEAE-cellulose. Ultracentrifugal examination revealed two components, and it is suggested that the faster-sedimenting component represents an aggregated form of the glycoprotein. 3. Glycoprotein preparations contained approx. 8% of carbohydrate. Digestion with Pronase yielded a glycopeptide fraction containing all the carbohydrate of the glycoprotein. The glycopeptide, of molecular weight about 7800, contained sialic acid, galactose, mannose, fucose and hexosamine in the approximate molar proportions 5:10:5:2:11. Sialic acid was terminal with respect to the polysaccharide chains. 4. Both elastase and elastomucoproteinases exhibited proteolytic activity towards the glycoprotein. Studies by other investigators have led to the conclusion that elastomucoproteinases attack protein-carbohydrate complexes occurring in intimate association with elastin in aorta and other tissues, and it is suggested that the glycoprotein may be identified with one of these compounds.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs

1. After the administration of labelled proline to guinea pigs deprived of ascorbic acid for 15 days, the dorsal skin was examined 5 days later in an attempt to detect the presence of hydroxyproline-deficient collagen (protocollagen). The extent of incorporation of proline into skin collagens indicated a severe impairment of collagen synthesis. 2. A comparison of proline and hydroxyproline specific radioactivities in diffusible peptides obtained by treatment with collagenase of either purified skin collagens or direct hot-trichloroacetic acid extracts of skin failed to indicate the presence of protocollagen. Possible reasons for this are discussed. 3. The incorporation results did not indicate an inability of normal collagen, i.e. collagen hydroxylated to the normal degree, to cross-link in scurvy. 4. Incorporation of labelled proline into aortic elastin isolated from the same animals did not indicate a decrease in elastin biosynthesis in ascorbic acid deficiency, beyond that attributable to the inanition accompanying the vitamin deficiency. The proline/hydroxyproline specific-radioactivity ratio in elastin from scorbutic guinea pigs was about 6:1 in contrast with the 1:1 ratio in control groups. It is concluded that the formation of elastin hydroxyproline was ascorbate-dependent and that a hydroxyproline-deficient elastin is formed and retained in scurvy. The formation of desmosines was unimpaired in scorbutic animals. 5. Studies with chick embryos confirmed the formation of elastin hydroxyproline from free proline. Incorporation of free hydroxyproline into elastin hydroxyproline was negligible. 6. Digestion of solubilized samples with collagenase indicated that the hydroxyproline in guinea-pig aortic elastin preparations was not derived from contamination by collagen. It is suggested that most if not all of the hydroxyproline in the guinea pig elastin preparations investigated can be considered an integral part of the elastin molecule.     

A tribute to Dr. Gordon Hisashi Sato (December 24, 1927–March 31, 2017)

Gordon H. Sato, an innovator in mammalian tissue culture and integrated cellular physiology, passed away in 2017. In tribute to Dr. Sato, In Vitro Cellular and Developmental Biology—Animal presents a collection of invited remembrances from six colleagues whose associations with Dr. Sato spanned more than 40 years. Dr. Sato was a past president of the Tissue Culture Association (now the Society for In Vitro Biology), editor-in-chief of In Vitro Cellular and Developmental Biology (1987–1991), and the recipient of the lifetime achievement award from the Society for In Vitro Biology (2002). He was elected to the US National Academy of Sciences in 1984.     

A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies

The conserved glycosylation site Asn²⁹⁷ of a monoclonal antibody (mAb) can be decorated with a variety of sugars that can alter mAb pharmacokinetics and recruitment of effector proteins. Antibodies lacking the core fucose at Asn²⁹⁷ (afucosylated mAbs) show enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and increased efficacy. Here, we describe the development of a robust platform for the manufacture of afucosylated therapeutic mAbs by engineering a Chinese hamster ovary (CHO) host cell line to co-express a mAb with GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose to a dead-end product, resulting in the production of afucosylated mAb (GlymaxX? Technology, ProBioGen). Expression of the mAb and RMD genes was coordinated by co-transfection of separate mAb and RMD vectors or use of an internal ribosome entry site (IRES) element to link the translation of RMD with either the glutamine synthase selection marker or the mAb light chain. The GS-IRES-RMD vector format was more suitable for the rapid generation of high yielding cell lines, secreting afucosylated mAb with titers exceeding 6.0 g/L. These cell lines maintained production of afucosylated mAb over 60 generations, ensuring their suitability for use in large-scale manufacturing. The afucosylated mAbs purified from these RMD-engineered cell lines showed increased binding in a CD16 cellular assay, demonstrating enhancement of ADCC compared to fucosylated control mAb. Furthermore, the afucosylation in these mAbs could be controlled by simple addition of L-fucose in the culture medium, thereby allowing the use of a single cell line for production of the same mAb in fucosylated and afucosylated formats for multiple therapeutic indications.     

Isozyme variation inFaidherbia albida (Leguminosae, Mimosoideae)

Genetic variation has been assessed in 30 populations (931 families) ofFaidherbia albida (Leguminosae, Mimosoideae) from across its entire African range, using six isozyme loci identified by five enzyme systems. Among the populations studied a null allele was proposed to explain the absence ofLap-1 activity in populations from southern and eastern Africa. The mean percentage of polymorphic loci per population, the mean number of alleles per locus and the mean genetic diversity within populations were 31.7%, 1.6 and 0.127 respectively. Genetic diversity was greatest in populations from West Africa and lowest in populations from eastern/southern Africa, with Ethiopian/Sudanese populations intermediate. The overall degree of genetic differentiation between populations (G_ST) indicated that approximately 56% of the enzyme variation resided within populations. Clustering of Nei's unbaised genetic distances calculated between all populations produced a dendrogram that generally followed the geographic distribution of the populations. Two major groups were identified that may be considered the eastern/southern African and the Ethiopian/West African clusters. Within the Ethiopian/West African cluster two subclusters could be recognised, one broadly corresponding to those populations from Ethiopia/Sudan and the other to those populations from West Africa. The implications of these results for theories regarding the origin ofF. albida in Africa are discussed.     

The functional morphology of Mactrinula reevesii (Bivalvia: Mactroidea) in Hong Kong: adaptations for a deposit-feeding lifestyle

The shallow subtidal mactrid bivalve Mactrinula reevesii is a deposit-feeder in the southern and south-eastern oceanic waters of Hong Kong. Buried obliquely, large quantities of fine sediment are taken into the mantle cavity and sorted on enormous labial palps. The small ctenidia probably have little value in collecting material, amounts taken in being too large. The mid gut is long and complexly folded inside the visceral mass. It is also capable of distension, although superficial visceral muscles maintain internal tonus. The rectum is narrow and creates compact faecal pellets.
Most interest is in the ventral mantle margin which is, posterior to the pedal gape and the base of the inhalant siphon, united by a sheet of cuticle. There is no fourth pallial aperture. There are, however, two pairs of flaps extending along the posterior third of the internal ventral mantle surface. These arch over left and right mantle rejection tracts which transfer unwanted material to the base of the inhalant siphon for final expulsion. The mantle flaps prevent such material from being returned to the anterior end of the mantle cavity, for palp reprocessing, when new material arrives. They, thus, maximize sorting efficiency by separating unsorted from sorted and rejected material.
Other mactrids have similar mantle flaps which they use in different ways, including the channelling of unwanted material to a fourth pallial aperture for expulsion, as in Lutraria lutraria. The Mactridae have thus evolved a unique method of increasing the efficiency of pseudofaecal waste rejection which has thereby facilitated the deposit-feeding lifestyle by the diverse representatives of this family.     

Expression of human cathepsin K in Pichia pastoris and preliminary crystallographic studies of an inhibitor complex.

Cathepsin K is a cysteine protease of the papain family, which is predominantly expressed in osteoclasts, and is regarded as a key protease in bone remodeling. To facilitate structural studies of the protein, the wild-type sequence of the protease has been mutated so as to replace a potential N-glycosylation site. We have expressed the mutant human cathepsin K to 190 mg/5 L using the Pichia pastoris expression system. Cathepsin K was inactivated with the mechanism-based inhibitor, APC3328, and crystallized from magnesium formate. A 2.2 A X-ray data set has been collected on crystals belonging to space group P2(1)2(1)2(1), with a = 41.66 A, b = 51.41 A, and c = 107.72 A. There is most likely one molecule per asymmetric unit.     

Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein.

Repair of a uracil-guanine base pair in DNA has been reconstituted with the recombinant human proteins uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta and DNA ligase III. The XRCC1 protein, which is known to bind DNA ligase III, is not absolutely required for the reaction but suppresses strand displacement by DNA polymerase beta, allowing for more efficient ligation after filling of a single nucleotide patch. We show that XRCC1 interacts directly with DNA polymerase beta using far Western blotting, affinity precipitation and yeast two-hybrid analyses. In addition, a complex formed between DNA polymerase beta and a double-stranded oligonucleotide containing an incised abasic site was supershifted by XRCC1 in a gel retardation assay. The region of interaction with DNA polymerase beta is located within residues 84-183 in the N-terminal half of the XRCC1 protein, whereas the C-terminal region of XRCC1 is involved in binding DNA ligase III. These data indicate that XRCC1, which has no known catalytic activity, might serve as a scaffold protein during base excision-repair. DNA strand displacement and excessive gap filling during DNA repair were observed in cell-free extracts of an XRCC1-deficient mutant cell line, in agreement with the results from the reconstituted system.     

Superoxide Dismutase Catalyzes Nitration of Tyrosines by Peroxynitrite in the Rod and Head Domains of Neurofilament-L

Abstract: Superoxide dismutase (SOD) catalyzes the nitration of specific tyrosine residues in proteins by peroxynitrite (ONOO⁻), which may be the damaging gain-of-function resulting from mutations to SOD associated with familial amyotrophic lateral sclerosis (ALS). We found that disassembled neurofilament-L (light subunit) was more susceptible to tyrosine nitration catalyzed by SOD in vitro. Neurofilament-L was selectively nitrated compared with the majority of other proteins present in brain homogenates. Assembled neurofilament-L was more resistant to nitration, suggesting that the susceptible tyrosine residues were protected by intersubunit contacts in assembled neurofilaments. Electrospray mass spectrometry of trypsin-digested neurofilament-L showed that tyrosine 17 in the head region and tyrosines 138, 177, and 265 in α-helical coil regions of the rod domain of neurofilament-L were particularly susceptible to SOD-catalyzed nitration. Nitrated neurofilament-L inhibited the assembly of unmodified neurofilament subunits, suggesting that the affected tyrosines are located in regions important for intersubunit contacts. Neurofilaments are major structural proteins expressed in motor neurons and known to be important for their survival in vivo. We suggest that SOD-catalyzed nitration of neurofilament-L may have a significant role in the pathogenesis of ALS.