Effects of phosphorylation by CAK on cyclin binding by CDC2 and CDK2.

The cyclin-dependent protein kinases (CDKs) are activated by association with cyclins and by phosphorylation at a conserved threonine residue by the CDK-activating kinase (CAK). We have studied the binding of various human CDK and cyclin subunits in vitro, using purified proteins derived from baculovirus-infected insect cells. We find that most CDK-cyclin complexes known to exist in human cells (CDC2-cyclin B, CDK2-cyclin A, and CDK2-cyclin E) form with high affinity in the absence of phosphorylation or other cellular components. One complex (CDC2-cyclin A) forms with high affinity only after CAK-mediated phosphorylation of CDC2 at the activating threonine residue. CDC2 does not bind with high affinity to cyclin E in vitro, even after phosphorylation of the CDC2 subunit. Thus, phosphorylation is of varying importance in the formation of high-affinity CDK-cyclin complexes.     

Electron probe X-ray microanalysis of calcium and phosphorus distribution in developing hamster tooth germs in vitro and in vivo

The aim of the present study was to investigate the spatial distribution of Ca and P in dentin and enamel of developing first (M1) and second (M2) maxillary hamster molars (age: 3-5 days) in comparison with cultured molars. For culturing the germs were dissected from 3-day-old hamsters and incubated for 1 and 2 days, respectively, in a modified BGJb medium. Electron probe X-ray measurements were carried out on 3 regions extending in a vertical axis from cusp tip over cusp middle to cusp base next to the cervical loop region. Neither the in vivo nor the in vitro group was statistically different in the Ca and P concentration in the regions of dentin. In both groups the measurements in enamel showed a gradient with an increase in Ca and P from enamel surface towards dentin-enamel junction and a gradient with an increase from cusp base towards cusp tip. Direct comparison of the in vivo group with the in vitro group did not demonstrate a statistical difference between the mineral content of the 4-day-old germs and the 1-day culture germs, respectively the 5-day-old germs and the 2-day culture germs. The results indicate a high correspondence between the mineralization process of in vitro and in vivo tooth germ development.     

Introduction to the Special Issue on Savanna Chimpanzees

International Journal of Primatology -     

Adsorptive membranes for bilirubin removal

In this study, we employed ethylene vinyl alcohol (EVAL) adsorptive membranes with bovine serum albumin (BSA) as bioligand for affinity supports for bilirubin (BR) retention. Microfiltration membranes were prepared from ternary or quaternary water/(1-octanol)/DMSO/EVAL systems. To obtain active binding sites for BSA, the EVAL membranes were either chemically functionalized in aqueous and organic medium and by plasma dischargement or physically activated by entrapping of active particles. Static BR removal was determined for all EVAL-BSA membranes. BR retentions relevant for human plasma were gained for the mixed adsorber membranes and additionally investigated in the dynamic mode.     

Strain specificity and binding affinity requirements of neutralizing monoclonal antibodies to the C4 domain of gp120 from human immunodeficiency virus type 1.

The binding properties of seven CD4-blocking monoclonal antibodies raised against recombinant gp120 of human immunodeficiency virus type 1 strain MN (HIV-1MN) and two CD4-blocking monoclonal antibodies to recombinant envelope glycoproteins gp120 and gp160 of substrain IIIB of HIVLAI were analyzed. With a panel of recombinant gp120s from seven diverse HIV-1 isolates, eight of the nine antibodies were found to be strain specific and one was broadly cross-reactive. Epitope mapping revealed that all nine antibodies bound to epitopes located in the fourth conserved domain (C4) of gp120. Within this region, three distinct epitopes could be identified: two were polymorphic between HIV-1 strains, and one was highly conserved. Studies with synthetic peptides demonstrated that the conserved epitope, recognized by antibody 13H8, was located between residues 431 and 439. Site-directed mutagenesis of gp120 demonstrated that residue 429 and/or 432 was critical for the binding of the seven antibodies to gp120 from HIV-1MN. Similarly, residues 423 and 429 were essential for the binding of monoclonal antibody 5C2 raised against gp120 from HIV-1IIIB. The amino acids located at positions 423 and 429 were found to vary between strains of HIV-1 as well as between molecular clones derived from the MN and LAI isolates of HIV-1. Polymorphism at these positions prevented the binding of virus-neutralizing monoclonal antibodies and raised the possibility that HIV-1 neutralization serotypes may be defined on the basis of C4 domain sequences. Analysis of the binding characteristics of the CD4-blocking antibodies demonstrated that their virus-neutralizing activity was directly proportional to their gp120-binding affinity. These studies account for the strain specificity of antibodies to the C4 domain of gp120 and demonstrate for the first time that antibodies to this region can be as effective as those directed to the principal neutralizing determinant (V3 domain) in neutralizing HIV-1 infectivity.     

In situ cell retention of a CHO culture by a reverse‐flow diafiltration membrane bioreactor

Heterogeneities occur in various bioreactor designs including cell retention devices. Whereas in external devices changing environmental conditions cannot be prevented, cells are retained in their optimal environment in internal devices. Conventional reverse‐flow diafiltration utilizes an internal membrane device, but pulsed feeding causes temporal heterogeneities. In this study, the influence of conventional reverse‐flow diafiltration on the yeast Hansenula polymorpha is investigated. Alternating 180 s of feeding with 360 s of non‐feeding at a dilution rate of 0.2 h^?1 results in an oscillating DOT signal with an amplitude of 60%. Thereby, induced short‐term oxygen limitations result in the formation of ethanol and a reduced product concentration of 25%. This effect is enforced at increased dilution rate. To overcome this cyclic problem, sequential operation of three membranes is introduced. Thus, quasi‐continuous feeding is achieved reducing the oscillation of the DOT signal to an amplitude of 20% and 40% for a dilution rate of 0.2 h^?1 and 0.5 h^?1, respectively. Fermentation conditions characterized by complete absence of oxygen limitation and without formation of overflow metabolites could be obtained for dilution rates from 0.1 h^?1 – 0.5 h^?1. Thus, sequential operation of three membranes minimizes oscillations in the DOT signal providing a nearly homogenous culture over time. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1348–1355, 2014     

Synthesis and in vitro evaluation of a library of modified endomorphin 1 peptides

Endomorphin 1 (Endo-1=Tyr-Pro-Trp-Phe-NH(2)), an endogenous opioid with high affinity and selectivity for mu-opioid receptors, mediates acute and neuropathic pain in rodents. To overcome metabolic instability and poor membrane permeability, the N- and C-termini of Endo-1 were modified by lipoamino acids (Laa) and/or sugars, and 2',6'-dimethyltyrosine (Dmt) replacement of Tyr. Analogues were assessed for mu-opioid receptor affinity, inhibition of cAMP accumulation, enzymatic stability, and permeability across Caco-2 cell monolayers. C-terminus modification decreased receptor affinity, while N-terminus C8-Laa improved stability and permeability with slight change in receptor affinity. Dmt provided a promising lead compound: [C8Laa-Dmt[1]]-Endo-1 is nine times more stable (t(1/2)=43.5min), >8-fold more permeable in Caco-2 cell monolayers, and exhibits 140-fold greater mu-opioid receptor affinity (K(imu)=0.08nM).     

Light- and electron-microscopic study of the rat esophagus following intraluminal argon laser irradiation

36 rat esophagi were irradiated by argon laser via an applicator with circumferential light distribution. They were perfused with glutaraldehyde and studied by light and transmission electron microscopy immediately, 2 days and 14 days after irradiation. Immediately after irradiation the laser center showed destruction of the keratinized stratified squamous epithelium. The collagenous fibers of the connective tissue were altered; fibrocytes and fibroblasts were severely damaged, and the microvascular lumina were occluded. The smooth muscle tissue and skeletal muscle tissue showed myofilament defects and initial karyonecrosis. There was decreasing damage of both fiber types up to 4 mm from the laser center. After 2 days the morphology of the laser center was not different from that seen immediately after irradiation. At a distance of 2 mm a partly differentiated new epithelium emerged below the necrotic epithelium. An inflammatory reaction was found in the connective tissue. After 14 days the esophageal wall was replaced and the lumen was occluded by young granulation tissue in the former laser center. Peripherally the esophageal wall appeared almost normal. As the rat esophagus serves as a model for esophagotracheal fistulae in newborn children, our findings indicate that the argon laser should be capable of occluding these fistulae likewise.