Quantitative immunoassay of total cellular GAP junction protein connexin32 during liver regeneration using antibodies specific to the COOH-terminus.

A radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were used to determine relative concentrations of liver connexin32 (CX32) in rats. The RIA and ELISA utilize synthetic peptides corresponding to regions of the carboxyl-terminus and antibodies raised in rabbits against these peptides. Assuming that affinities of antisera are similar for peptide and native CX32, total cellular CX32 was found to exceed the amount of gap junction protein at the cell surface calculated from morphometric analyses by 1.5-2.0 fold. This finding raises the possibility that some of the protein is present in cytoplasmic compartments or as occult precursors in the plasma membrane. Studies of CX32 content in regenerating rat liver support this conclusion and show a time course of loss and recovery of CX32 that agrees with those reported in studies using other techniques.     

Enzymatic sulfation of gastrin in rat gastric mucosa

An enzyme which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to gastrin (G17) was identified in rat gastric mucosal cells. The enzyme activity was detected in the 105,000xg supernatant fraction. Formation of gastrin sulfate was shown by using 125I-gastrin and non-radioactive PAPS. The product was sensitive to acid hydrolysis, arylsulfatase treatment and removed by gastrin antibody, but not changed by treatments with chondro-4-sulfatase and chondro-6-sulfatase. The product had a molecular weight of 2050 daltons, close to the molecular weight of G17 sulfate, and, therefore, indicating the sulfated product is not APS derived from the degradation of PAPS. The enzyme activity showed a Km value of 5 microM for PAPS and a pH optimum of 6.0. The activity was not detected in the liver preparation.     

The form II fructose 1,6-bisphosphatase and phosphoribulokinase genes form part of a large operon in Rhodobacter sphaeroides: primary structure and insertional mutagenesis analysis

Fructose 1,6-bisphosphatase (FBPase) and phosphoribulokinase (PRK) are two key enzymes of the reductive pentose phosphate pathway or Calvin cycle of photosynthetic carbon dioxide assimilation. Early studies had indicated that the properties of enzymes isolated from photosynthetic bacteria were clearly distinct from those of enzymes obtained from the chloroplasts of higher plants [for a review, see Tabita (1988)]. The eucaryotic enzymes, which are light activated by the thioredoxin/ferredoxin system (Buchanan, 1980), were each shown to contain a putative regulatory amino acid sequence (Marcus et al., 1988; Porter et al., 1988). The enzymes from photosynthetic bacteria are not controlled by the thioredoxin/ferredoxin system but exhibit complex kinetic properties and, in the case of PRK, there is an absolute requirement of NADH for activity. In the photosynthetic bacterium Rhodobacter sphaeroides, the structural genes of the Calvin cycle, including the genes that encode FBPase (fbp) and PRK (prk), are found in two distinct clusters, and the fbp and prk genes are closely associated in each cluster. In the present investigation, we have determined the nucleotide sequence of the fbpB and prkB genes of the form II cluster and have compared the deduced amino acid sequences to previously determined sequences of light-activated enzymes from higher plants and from other eucaryotic and procaryotic sources. In the case of FBPase, there are several regions that are conserved in the R. sphaeroides enzymes, including a protease-sensitive area located in a region equivalent to residues 51-71 of mammalian FBPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Using group testing to estimate a proportion, and to test the binomial model

Group testing has been extensively studied as an efficient way to classify units as defective or satisfactory when the proportion (p) of defectives is small. It can also be used to estimate p, often substantially reducing the mean squared error (MSE) of p and cost per unit information. Group testing is useful for larger p in the estimation problem than in the classification problem, but for larger p more care must be taken in choosing the group size (k); k being too large not only increases MSE (p), but adversely affects the robustness of p to both errors in testing (misclassification) and errors in the assumed binomial model. Procedures that retest units from defective groups, if even feasible, are shown to reduce cost per unit information very little in the estimation problem, but can provide useful information for testing the model. Methods are given for using data from tests of unequal-sized groups to estimate p and for testing the validity of the binomial model.     

Free ADP levels in transgenic mouse liver expressing creatine kinase. Effects of enzyme activity, phosphagen type, and substrate concentration

ADP is an important regulator of hepatic metabolism. Despite its importance the level of free ADP in the liver remains controversial. Recently, we engineered transgenic mice which express high levels of creatine kinase in liver. The reaction catalyzed by creatine kinase was assumed to be at equilibrium and used to calculate a free ADP level of 0.059 mumol/g wet weight. In this report we test the equilibrium assumption by studying the free ADP level as a function of enzyme activity or substrate content. Over a 5-fold range of creatine kinase activity, from 150-800 mumol/min/g wet weight, there was no change in the free ADP level. The average value of ADP for these mice was 0.061 +/- 0.016 mumol/g wet weight. Similarly, altering hepatic creatine content from 1.6 to 30 mumol/g wet weight had no effect on the calculated total free ADP level. The average value of ADP for the creatine levels was 0.048 +/- 0.015 mumol/g wet weight. Finally, the free ADP level was calculated using the equilibrium with cyclocreatine rather than creatine as substrate. The equilibrium of the reaction with cyclocreatine lies 30 times more toward phosphorylation than does the equilibrium with creatine. A free ADP level of 0.063 +/- 0.031 mumol/g wet weight was calculated using cyclocreatine. This value is not different from that found with creatine. These results show that the equilibrium assumption used to calculate free ADP levels in transgenic mouse liver is valid, and the presence of creatine kinase does not affect ADP levels.     

Transport and deamination of amino acids by a gram-positive, monensin-sensitive ruminal bacterium.

Strain F, a recently isolated ruminal bacterium, grew rapidly with glutamate or glutamine as an energy source in the presence but not the absence of Na. Monensin, a Na+/H+ antiporter, completely inhibited bacterial growth and significantly reduced ammonia production (85%), but 3,3',4',5-tetrachlorosalicylanide (a protonophore) and valinomycin had little effect on growth or ammonia production. Dicyclohexylcarbodiimide, a H(+)-ATPase, inhibitor had no effect. The kinetics of glutamate and glutamine transport were biphasic, showing unusually high rates at high substrate concentrations. On the basis of low substrate concentrations (less than 100 microM), the Km values for glutamate and glutamine were 4 and 11 microM, respectively. Strain F had separate carriers for glutamate and glutamine which could be driven by a chemical gradient of Na. An artificial delta psi was unable to drive transport even when Na was present. The glutamate carrier had a single binding site for Na with a Km of 21 mM; the glutamine carrier appeared to have more than one binding site, and the Km was 2.8 mM. Neither carrier could use Li instead of Na. Histidine and serine were also rapidly transported by Na-dependent systems, but serine alone did not allow growth even when Na was present. Because exponentially growing cells at pH 6.9 had little delta psi (-3 mV) and a slightly reversed Z delta pH (+17 mV), it appeared that the membrane bioenergetics of strain F were solely dependent on Na circulation.     

Antigen-specific clones of proliferating T lymphocytes. I. Methodology, specificity, and MHC restriction

In this report we describe in detail a new method for cloning antigen-specific, proliferating T lymphocytes directly from primed murine lymph nodes after 3 days of activation in vitro. After expansion in liquid culture the cells from the colonies were shown to be antigen specific and to require I-A histocompatible, irradiated spleen cells for stimulation. For hapten-carrier-type antigens, the T cells were shown to be carrier specific in their recognition but they were also capable of distinguishing the presence of the hapten. Recloning of small numbers of these cells in soft agar under conditions of high plating efficiency yielded true clones (i.e., populations derived from a single cell) whose antigen specificity was identical to that of cells from the original colony. The fact that a clone of T cells was I-A restricted in its antigen recognition demonstrates that suppressor T cell function cannot account for the phenomenon of major histocompatibility complex restriction.     

Brugia pahangi: Feeding and nutrient uptake in vitro and in vivo

Incubation in vitro of adult Brugia pahangi in an apparatus which permitted the separate exposure of the anterior, middle, or posterior region of the worms to medium-containing radioactively labeled d-glucose, l-leucine, and adenosine has provided evidence that these materials are taken up in physiologically significant amounts by a transcuticular route. No evidence for an oral ingestion of materials has been obtained from worms in vitro, but in vivo an oral uptake of Trypan blue has been demonstrated. The ultrastructure and cytochemical staining reactions for nonspecific esterase, acid phosphatase (EC-3.1.3.2), and leucine naphthylamidase of the gut and body wall are described.