Purification of L-Isoleucyl t-rna Synthetase by Affinity Chromatography

L-Isoleucyl t-RNA synthetase was purified to 97% purity in a single step by affinity chromatography on a column carrying L-isoleucinyl 5′-adenylate as the insolubilized ligand. Recovery was 50–60%.     

Absorption Changes in Bacterial Chromatophores

The magnitude and kinetics of photo-induced absorption changes in bacterial chromatophores (R. rubrum, R. spheroides and Chromatium) have been studied as a function of potential, established by added redox couples. No photochanges can be observed above +0.55 v or below -0.15 v. The loss of signal at the higher potential is centered at +0.439 v and follows a one-electron change. The loss of signal at the lower potential is centered at -0.044 v and is also consistent with a one-electron change. Both losses are reversible. A quantitative relationship exists between light-minus-dark and oxidized-minus-reduced spectra in the near infrared from +0.30 to +0.55 v. Selective treatment of the chromatophores with strong oxidants irreversibly bleaches the bulk pigments but appears to leave intact those pigments responsible for the photo- and chemically-induced absorption changes. Kinetic studies of the photochanges in deaerated samples of R. rubrum chromatophores revealed the same rise time for bands at 433, 792, and 865 mμ (t_½ = 50 msec.). However, these bands had different decay rates (t_½ = 1.5, 0.5, 0.15 sec., respectively), indicating that they belong to different pigments. Analysis of the data indicates, as the simplest interpretation, a first-order (or pseudo first-order) forward reaction and two parallel first-order (or pseudo first-order) decay reactions at each wavelength. These results imply that all pigments whose kinetics are given are photooxidized and the decay processes are dark reductions. These experiments are viewed as supporting and extending the concept of a bacterial photosynthetic unit, with energy migration within it to specific sites of electron transfer.     

Purification of animal immunoglobulin G (IgG) using peptoid affinity ligands

The availability of highly pure animal antibodies is critical in the production of diagnostic tools and biosensors. The peptoid PL16, previously isolated from an ensemble of peptoid variants of the IgG-binding peptide HWRGWV, was utilized in this work as affinity ligand on WorkBeads resin for the purification of immunoglobulin G (IgG) from a variety of mammalian sources and chicken immunoglobulin Y (IgY). The chromatographic protocol initially optimized for murine serum and ascites was subsequently employed for processing rabbit, goat and sheep, donkey, llama, and chicken sera. The PL16-WorkBeads resin proved able to recover all antibody targets with values of yield between 50 and 90%, and purity consistently above 90%. Notably, PL16 not only binds a broader spectrum of animal immunoglobulins than the reference ligands Protein A and G, but it also binds equally well with all their subclasses. Unlike the protein ligands, in fact, PL16 afforded excellent values of yield and purity of mammalian polyclonal IgG, namely murine (47 and 94%), rabbit (66.5 and 91.7%), caprine IgG (63 and 91–95%), donkey, and llama (93 and 97%), as well as chicken IgY (42 and 92%). Of notice, it is also the ability of PL16 to target monomeric IgG without binding aggregated IgG; when challenged with a mixture of monomeric and aggregated murine IgG, PL16 eluted <3% of fed aggregates, against 11–13% eluted by Protein A and G. Collectively, these results prove the potential of the proposed peptoid ligand for large-scale purification of animal immunoglobulins.     

The mutant “proboscipedia” in Drosophila melanogaster — A case of hereditary homoösis

Summary The recessive mutant proboscipedia, found byBridges April 27, 1931, has its locus in the third chromosome at approximately 5.3 units to the right of Dichaete, or at 45.7.In proboscipedia the oral lobes are converted into a labium-like organ, which also resembles a pair of antenna-like or tarsus-like appendages. This change, as well as the modifications of the labrum, maxillae, maxillary palpi, and other structures give to the mouth parts of proboscipedia a resemblance to those of the biting type found in insects of lower orders.The four-fold resemblance of the modified oral lobes in proboscipedia to the labrum, the maxillary palpi, the antenae and the tarsi is comprehensible in view of the homology of these three head-appendages with each other and with the walking legs.

---

Zusammenfassung Die rezessive Mutante Proboscipedia, die vonBridges am 27. 4. 31 gefunden wurde, ist im 3. Chromosom ungefähr 5,3 Einheiten rechts von Dichaete, oder bei 45,7 lokalisiert.Bei Proboscipedie sind die oralen Lappen zusammengebogen zu einem lippenartigen Organ, das einem Paar antennenähnlicher oder tarsusähnlicher Anhänge gleicht. Diese Abart und ebenso die Modifikationen des Labrum, der Maxillen, der Maxillargliedmaßen und anderer Strukturen geben den Mundteilen von Proboscipedia eine Ähnlichkeit mit denen des kauenden Typus, wie er bei Insekten niederer Ordnung gefunden wurde.Die vierfache Ähnlichkeit der modifizierten oralen Lappen bei Proboscipedia hinsichtlich des Labrum, der Maxillargliedmaßen, der Antennen und der Tarsi ist verständlich da diese drei Kopfanhänge und die Laufbeine homolog sind.

     

Expressing and purifying membrane transport proteins in high yield

Structural analysis of native or recombinant membrane transport proteins has been hampered by the lack of effective methodologies to purify sufficient quantities of active protein. We addressed this problem by expressing a polyhistidine tagged construct of the cardiac sodium-calcium exchanger (NCX1) in Trichoplusia ni larvae (caterpillars) from which membrane vesicles were prepared. Larvae vesicles containing recombinant NCX1-his protein supported NCX1 transport activity that was mechanistically not different from activity in native cardiac sarcolemmal vesicles although the specific activity was reduced. SDS-PAGE and Western blot analysis demonstrated the presence of both the 120 and 70 kDa forms of the NCX1 protein. Larvae vesicle proteins were solubilized in sodium cholate detergent and fractionated on a chelated Ni(2+) affinity chromatography column. After extensive washing, eluted fractions were mixed with soybean phospholipids and reconstituted. The resulting proteoliposomes contained NCX1 activity suggesting the protein retained native conformation. SDS-PAGE revealed two major bands at 120 and 70 kDa. Purification of large amounts of active NCX1 via this methodology should facilitate biophysical analysis of the protein. The larva expression system has broad-based application for membrane proteins where expression and purification of quantities required for physical analyses is problematic.     

Aneuploid spermatozoa in infertile men: teratozoospermia.

We and others have demonstrated that infertile men who are candidates for intracytoplasmic sperm injection (ICSI) have an increased frequency of chromosomal abnormalities in their sperm. Reports based on prenatal diagnosis of ICSI pregnancies have confirmed the increased frequency of chromosomal abnormalities in offspring. Most studies to date have lumped various types of infertility together. However, it is quite likely that some subsets of infertility have an increased risk of sperm chromosomal abnormalities whereas others do not. We have studied nine men with severe teratozoospermia (WHO, 1992 criteria, 0-13% morphologically normal forms) by multicolour fluorescence in situ hybridisation (FISH) analysis to determine if they have an increased frequency of disomy for chromosomes 13, 21, XX, YY, and XY, as well as diploidy. All of the men also had aesthenozoospermia (< 50% forward progression) but none of the men had oligozoospermia (<20 x 10(6) sperm/ml). The patients ranged in age from 20 to 49 years (mean 33.2 years) in comparison to 18 normal control donors who were 23 to 58 years (mean 35.6 years). The control donors had normal semen parameters and no history of infertility. A total of 180,566 sperm were scored in the teratozoospermic men with a minimum of 10,000 sperm analyzed/donor/chromosome probe. There was a significant increase in the frequency of disomy in teratozoospermic men compared to controls for chromosomes 13 (.23 vs.13%), XX (.13 vs.05%), and XY (.50 vs.30%) (P <.0001, 2-tailed Z statistic). This study indicates that men with teratozoospermia and aesthenozoospermia but with normal concentrations of sperm have a significantly increased frequency of sperm chromosomal abnormalities.