Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells.     

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.     

Albino inflorescence proliferation of Dendrocalamus latiflorus

Summary Inflorescence proliferation is a plant tissue culture technique that, can be used to obtain in vitro inflorescences year-round without the intervening development of vegetative organs. In this study, we used albino mutant inflorescences of Dendrocalamus latiflorus as the original explant material to investigate, the effect of plant growth regulators on long-term inflorescence proliferation. The albino inflorescences proliferated on solidified Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ), and the optimal concentration for successful long-term inflorescence proliferation was 0.45 μM TDZ. A combination of α-naphthaleneacetic acid (NAA) with 0.45 μM TDZ inhibited the inflorescence proliferation. Inflorescences cultured on a TDZ-free medium supplemented with 26.82 μM NAA rooted in 21 d, vegetative shoots formed by 42 d and, in one case, flowering occurred after 63 d. The auxins 2,4-dichlorophenoxyacetic acid (2,4-D, 4.52 μM) and pieloram (4.14 μM) induced shoot formation. The protocol described can be used to produce large numbers of mutant inflorescences within a relatively short period of time.     

Tumor necrosis factor induces acute phase proteins in rats

Inoculation of WAG rats with recombinant mouse tumor necrosis factor results in a rapid and marked increase in several acute phase proteins in the serum (haptoglobin, alpha 1 acid glycoprotein, alpha 2 macroglobulin) and in the plasma (fibrinogen). We conclude that TNF may play an important role in the inflammatory response in vivo and possibly in the pathogenesis of inflammatory disorders.     

Hepatocellular Carcinoma in Hepatitis B Surface Antigen Carriers in Eight Institutions

We reviewed records of all persons dying between 1979 and 1986 in eight California institutions for the mentally retarded. Autopsies had been done in 71% of the 1,181 deaths. Nine deaths were due to hepatocellular carcinoma, which invariably developed in carriers of hepatitis B surface antigen (HBsAg) and was fatal within four months of diagnosis. The mean age at death was 32.7 years. The incidence of hepatocellular carcinoma in HBsAg carriers was 140 times greater than in the US population. Persistent hepatitis B infection was probably etiologically related to hepatocellular carcinoma in this population, which is relatively free of exposure to other hepatocarcinogens.     

A new experimental approach for the study of cardiopulmonary physiology during early development

In this report, an experimental approach and newly designed apparatus for liquid ventilation of preterm animals are described. Findings of age-related changes in cardiopulmonary function of this animal preparation are presented. Thirty-one lambs, 102-137 days gestation (term 147 +/- 3 days), were studied. The carotid artery, jugular vein, and trachea of the exteriorized fetus were cannulated under local anesthesia. Immediately after cesarean section delivery, ventilation commenced; warmed (39 degrees C) and oxygenated (PIO2 greater than 500 Torr) liquid fluorocarbon (RIMAR 101) was delivered to the lung by a mechanically assisted liquid ventilation system. Skeletal muscle paralysis, low-dose exogenous buffering, and thermal support were maintained during the 3-h experiment. Pulmonary gas exchange, acid-base status, and cardiopulmonary and metabolic function were assessed. By utilizing these techniques, effective arterial oxygenation, CO2 elimination, acid-base status, and cardiovascular stability were supported independent of gestational age. The results demonstrate a developmental increase in specific lung compliance and mean arterial pressure and decrease in heart rate and systemic O2 consumption per kilogram with advancing gestational age. These findings demonstrate that liquid ventilation negates the dependency of effective pulmonary gas exchange on surfactant development, thereby extending the limits of viability of the immature extrauterine lamb. As such this new experimental approach is useful for the study of physiological development over an age range previously limited to fetal animal preparations and, therefore, may provide insight regarding adaptation of the premature to the extrauterine environment.     

Autonomous DNA replication in human cells is affected by the size and the source of the DNA.

We previously developed short-term and long-term assays for autonomous replication of DNA in human cells. This study addresses the requirements for replication in these assays. Sixty-two random human genomic fragments ranging in size from 1 to 21 kb were cloned in a prokaryotic vector and tested for their replication ability in the short-term assay. We found a positive correlation between replication strength and fragment length, indicating that large size is favored for efficient autonomous replication in human cells. All large fragments replicated efficiently, suggesting that signals which can direct the initiation of DNA replication in human cells are either very abundant or have a low degree of sequence specificity. Similar results were obtained in the long-term assay. We also used the same assays to test in human cells a random series of fragments derived from Escherichia coli chromosomal DNA. The bacterial fragments supported replication less efficiently than the human fragments in the short-term and long-term assays. This result suggests that while the sequence signals involved in replication in human cells are found frequently in human DNA, they are uncommon in bacterial DNA.