Isozymes of cAMP-dependent protein kinase present in the rat corpus luteum.

Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N3[32P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RI alpha (Mr 47,000) subunits, a prominent type II holoenzyme containing RII beta (Mr 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RII beta and RII alpha (Mr 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RI alpha protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RII beta holoenzyme in very close proximity to free RI alpha and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RI alpha.     

Use of APACHE II classification to evaluate outcome of patients receiving hemodialysis in an intensive care unit.

We retrospectively reviewed the medical records of all patients who were admitted to the medical and surgical intensive care units of a university center (N = 100) and its affiliated veterans'' hospital (N = 46) between 1982 and 1986 to receive dialysis. The APACHE II severity-of-disease classification was used to identify the cases in which the prognosis was so poor that no long-term benefit would accrue from hemodialysis treatment. A "risk of death" was calculated for each patient. At a risk of death of 70% or greater, the system correctly predicted the demise of patients with 100% specificity regardless of what interventions were carried out. Sensitivity and predicted negative value were low in all cases, however, indicating a poor predictability of those who will survive. Withholding the average of 6 dialysis treatments that this group of patients received would probably have reduced patient suffering during a lingering terminal illness and led to a savings of about $4,500 per patient.     

Heterologous expression of preprosomatostatin. Intracellular degradation of prosomatostatin-II.

Somatostatin (SRIF) is a peptide hormone that is synthesized as part of a larger precursor, prepro-SRIF, consisting of a signal peptide and a proregion of 80-90 amino acids. The mature hormone exists as two different bioactive species. In addition to the most common form, which is a 14-residue peptide, there is also a 14-amino acid NH2-terminally extended form of the tetradecapeptide, SRIF-28. In mammals a single prepro-SRIF molecule undergoes tissue-specific processing to generate the mature hormone, whereas in some species of fish separate genes encode two distinct but homologous precursors, prepro-SRIF-I and -II, that give rise to SRIF-14 and -28, respectively. To investigate the molecular basis for differential processing of the prohormones, we have expressed their cDNAs in heterologous cells. Previously, we demonstrated that prepro-SRIF-I was efficiently and accurately processed in rat pituitary growth hormone (GH3) cells to generate the same hormone as synthesized in pancreatic islet D-cells, namely SRIF-14 (Stoller, T., and Shields, D. (1989) J. Biol. Chem. 264, 6922-6928). We have now compared the proteolytic processing of pro-SRIF-II to that of pro-SRIF-I in these cells. In contrast to pro-SRIF-I, pro-SRIF-II was neither processed nor secreted. Instead, greater than 70% of the precursor was degraded intracellularly in a post-trans Golgi network compartment which was inhibited by weak bases. Brefeldin A treatment prevented degradation, suggesting that turnover of the remaining pro-SRIF-II occurred after exit from the endoplasmic reticulum/intermediate compartment and prior to arrival at the trans Golgi network. The intracellular degradation of the precursor was unexpected, since heterologous cells which do not cleave prohormones generally secrete the unprocessed precursor. We speculate that unique structural domains within each precursor are recognized by the sorting apparatus in GH3 cells, thereby targeting the molecules to different intracellular organelles.     

Connective Tissue Growth Factor (CTGF/CCN2) enhances lactogenic differentiation of mammary epithelial cells via integrin-mediated cell adhesion

Background  

Connective Tissue Growth Factor (CTGF/CCN2), a known matrix-associated protein, is required for the lactogenic differentiation of mouse mammary epithelial cells. An HC11 mammary epithelial cell line expressing CTGF/CCN2 was constructed to dissect the cellular responses to CTGF/CCN2 that contribute to this differentiation program.     

A comparative analysis of nested subset patterns of species composition

We present a broad comparative assessment of nested subsets in species composition among ecological communities. We assembled presence-absence data from a broad range of taxa, geographic regions, and spatial scales; and subjected this collection of datasets to common analyses, including a variety of metrics for measuring nestedness and null hypotheses against which to evaluate them. Here we identify ecological patterns in the prevalence and strength of nested subset structure, and assess differences and biases among the available methodologies. In all, we compiled 279 presence-absence matrices, of which 163 do not overlap in their coverage of species and sites. The survey includes studies on vertebrates, arthropods, mollusks, plants, and other taxa; from north temperate, tropical, and south temperate latitudes. Our results were as follows. Statistically significant nestedness was common. Assemblages from landbridge archipelagos were strongly nested, and immigration experiments were least nested. This adds further empirical support to the hypothesis that extinction plays a major role in producing nested structure. Nestedness was positively correlated with the ratio of the areas of the largest and smallest sites, suggesting that the range in area of sites affects nestedness. Taxonomic differences in nestedness were weak. Higher taxonomic levels showed stronger nesting than their constituent lower taxa. We observed no effect of distance of isolation on nestedness; nor any effects of latitude. With regard to methodology, the metrics Nc and Ut yielded similar results, although Nc proved slightly more flexible in use, and deals differently with tied sites. Similarities also exist in the behavior of N0 (“N”) and Up, and between N1 and Ua. Standardized nestedness metrics were mostly insensitive to matrix size, and were useful in comparative analyses among presence-absence matrices. Most metrics were affected by the proportion of presences in the matrix. All analyses of nestedness, therefore, should test for bias due to matrix fill. We suggest that the factors controlling nested subset structure can be thought of as four filters that species pass to occur at a site: a sampling filter, a distance filter, a habitat filter, and an area filter – and three constraints on community homogeneity: evolutionary history, recent history, and spatial variation in the environment. The scale of examination can also have important effects on the degree of nestedness observed. Received: 13 September 1996 / Accepted: 16 September 1997     

On the definition of the compartment concept in pharmacokinetics

Current attitudes on the compartment concept, as applied in pharmacokinetics, are discussed. The concept of a relative steady state is introduced, and shown to provide the basis for a definition of a compartment. The physical significance of the compartment parameters is discussed and a method of testing compartment models by direct sampling is described.     

Binding to cell surfaces by antibodies against exoATPase

Avian oviductal secretions contain an exoATPase which has been purified up to 900-fold. Antibodies to this fraction bind to several cell types and to membrane fractions solubilized by selected detergents. The experiments indicate that a related antigen is present in plasma membranes and that exoATPase is released by a shedding process.     

Cereal protoplast recalcitrance

Summary Cereal leaf protoplasts are extremely difficult to culture (recalcitrant) in vitro. There have been few reports of division and the protoplasts typically exhibit excessive enlargement and vacuolization with reduced cell wall deposition. Inasmuch as leaf base explants are capable of callus formation in vitro, protoplasts derived from this tissue must have lost the ability to divide as a consequence of changes induced by the wall-digestion process. We review evidence suggesting that the inhibition of mitosis in these protoplasts is a consequence of a cascade of events initiated at the plasma membrane. The enzyme treatment necessary for wall removal triggers membrane depolarization and other changes that can lead to the initiation of lipid peroxidation and oxidative stress. Mitotically inactive cereal leaf protoplasts are unable to mount a protective response to these degradative processes. Consequently, the resulting membrane perturbations and permeabilization give rise to secondary effects on the cytoskeleton and the cell wall. These effects include reduced or absent microtubules as well as reduced and uneven wall deposition. Such abnormalities are observed in cereal leaf protoplasts and are sufficient to account for recalcitrance because the occurrence of mitosis is strongly dependent on a normal cell wall and cytoskeleton. This paper is NRCC number 32475.