Purine metabolic enzymes in lymphocytes. I. Adenosine deaminase and purine nucleoside phosphorylase activities in mouse lymphocyte subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA-P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleen of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one-third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA-P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA-P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA-P and Con A, and that of PNP activity was enhanced by PHA-P but not by Con A.     

The role of oxygen availability in the embryonation of Heterakis gallinarum eggs.

The importance of oxygen availability in the embryonation of the infective egg stages of the gastrointestinal nematode parasite Heterakis gallinarum was studied in the laboratory. Unembryonated H. gallinarum eggs were kept under either aerobic conditions by gassing with oxygen, or anaerobic conditions by gassing with the inert gas nitrogen, under a range of constant temperatures. Oxygenated eggs embryonated at a rate influenced by temperature. Conversely, eggs treated with nitrogen showed no embryonation although when these eggs were transferred from nitrogen to oxygen gas after 60 days of treatment, embryonation occurred. This demonstrated that oxygen is an essential requirement for H. gallinarum egg development, although undeveloped eggs remain viable, even after 60 days in low oxygen conditions. The effects of climate on the biology of free-living stages studied under constant laboratory conditions cannot be applied directly to the field where climatic factors exhibit daily cycles. The effect of fluctuating temperature on development was investigated by including an additional temperature group in which H. gallinarum eggs were kept under daily temperature cycles between 12 and 22°C. Cycles caused eggs to develop significantly earlier than those in the constant mean cycle temperature, 17°C, but significantly slower than those in constant 22°C suggesting that daily temperature cycles had an accelerating effect on H. gallinarum egg embryonation but did not accelerate to the higher temperature. These results suggest that daily fluctuations in temperature influence development of the free-living stages and so development cannot be accurately predicted on the basis of constant temperature culture.     

Atomic force microscope imaging of phospholipid bilayer degradation by phospholipase A2.

We have investigated the time course of the degradation of a supported dipalmitoylphosphatidylcholine bilayer by phospholipase A2 in aqueous buffer with an atomic force microscope. Contact mode imaging allows visualization of enzyme activity on the substrate with a lateral resolution of less than 10 nm. Detailed analysis of the micrographs reveals a dependence of enzyme activity on the phospholipid organization and orientation in the bilayer. These experiments suggest that it is possible to observe single enzymes at work in small channels, which are created by the hydrolysis of membrane phospholipids. Indeed, the measured rate of hydrolysis of phospholipids corresponds very well with the enzyme activity found in kinetic studies. It was also possible to correlate the number of enzymes at the surface, as calculated from the binding constant to the number of starting points of the hydrolysis. In addition, the width of the channels was found to be comparable to the diameter of a single phospholipase A2 and thus further supports the single-enzyme hypothesis.     

Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (K_i) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.     

Isolation and characterisation of an aryl-β-D-glucoside uptake and utilisation system ( abg ) from the gram-positive ruminal Clostridium species C. longisporum

A phosphotransferase-dependent aryl-β-glucoside uptake and utilisation system (abg) was isolated from the ruminal Clostridium (“C. longisporum”). The system is composed of three genes, abgG, abgF and abgA, and a number of regulatory regions, including terminator/antiterminator type stem-loop structures preceding the abgG and abgF genes. Similarity analysis of the proteins encoded by these genes indicated that they were responsible for the regulation of the abg system through antitermination (AbgG), the uptake and phosphorylation of aryl-β-glucosides (AbgF) and the hydrolysis of the intracellular phosphorylated glycosides (AbgA). Experimental evidence for the functions of AbgF and AbgA was obtained. Although it was not possible to demonstrate any function for AbgG, a promoter 5′ to the abgG gene was identified which was responsible for expression of the downstream genes. The abg system is remarkably similar to operons from the gram negative Enterobacteriaceae, both in the coding and non-coding regulatory regions. Received: 3 April 1997 / Accepted: 8 September 1997