Insulin stimulates GDP release from G proteins in the rat and human liver plasma membranes

Plasma membranes (1–2 mg protein) prepared from the livers of adult male rats and human organ donors were incubated with 0.6 μM [α-³²P] guanosine triphosphate (GTP) in an adenosine triphosphate (ATP)-regenerating buffer at 37°C for 1 h; during this incubation, the [³²P]GTP is hydrolyzed and the nucleotide that is predominantly bound to the membranes is [³²P] guanosine diphosphate (GDP). [³²P]GDP release from the liver membranes was proportional to the protein concentration and increased as a function of time. At 5 mM, Ca²⁺, Mg²⁺, Mn²⁺, and Zn²⁺ maximally inhibited GDP release by 80–90%, whereas, 5 mM Cu²⁺ maximally stimulated the reaction by 100%. Therefore, cations were not included in the buffer used in the GDP release step. One μM Gpp(NH)p (5′-guanylylimidodiphosphate), a nonhydrolyzable analog of GTP, maximally stimulated [³²P]GDP release in the liver membranes by up to 30%. Although 10 nM Gpp(NH)p had no effect on GDP release, it appeared to stabilize the hormonal effect by blocking further GDP/GTP exchange. In the rat membranes, 1–100 nM glucagon (used as a positive control) stimulated [³²P]GDP release by about 17% (P < .05); similarly, 0.1–100 nM insulin stimulated [³²P]GDP release by 10–13% (P < .05). In the human membranes, 10 pM to 100 nM insulin stimulated [³²P]GDP release by 7–10%. In the rat membranes, 10 nM insulin stimulated [³²P]GDP release by 17 and 24% at 2 and 4 min, respectively (P < .05); in the human membranes, 10 nM insulin stimulated [³²P]GDP release by about 9% at 2 and 4 min. Normal rabbit IgG (used as a control for insulin receptor antibody) by itself stimulated the GDP release by rat and human membranes. However, the stimulation of the GDP release by insulin receptor antibody was consistently higher than that observed with normal rabbit IgG. Four to 15 μg of insulin receptor antibody stimulated [³²P]GDP release by 12–22% (P < .05) and 7–14% in rat and human membranes, respectively. These results indicate that ligand binding to the insulin receptor results in a functional interaction of the receptor with a guanine nucleotide-binding transducer protein (G protein) and activation of GTP/GDP exchange.     

Cell growth stimulation by EGF: inhibition through antisense-oligodeoxynucleotides demonstrates important role of casein kinase II

Casein kinase II (CKII) is a highly conserved ubiquitous serine/threonine kinase composed of two catalytically active subunits (alpha and/or alpha') and two presumably regulatory subunits (beta). CKII has numerous cellular functions including a possible role in mitogenic signaling. To address this question, growth-arrested primary human fibroblasts (IMR-90) were exposed prior growth stimulation by epidermal growth factor (EGF) to oligodeoxynucleotides complementary to the translation start region of mRNAs coding for CKII alpha and beta subunits. A significant inhibition of growth stimulation (up to 60%) was observed with both antisense-alpha and antisense-beta. The inhibition was reversible, became decreased with mutated antisense-oligodeoxynucleotides, and neutralized by simultaneous presence of respective sense-oligodeoxynucleotides. The expected down-regulation of CKII protein due to hybrid formation of antisense-oligodeoxynucleotides with target mRNAs was investigated by determination of the intracellular protein level of CKII beta-subunit by immunofluorescence and quantitative image analysis. The protein was revealed to be localized predominantly in the nucleus and to become significantly decreased due to antisense-beta treatment of cells. The maximum decrease coincided with the early phase (first several hours) of growth stimulation by EGF when antisense-beta incubation was started 6-2 h before growth stimulation, the period within which application of antisense-alpha and antisense-beta caused the maximum of inhibition of growth stimulation. Thus CKII obviously plays, with both subunit alpha and subunit beta, an important role in the early phase of mitogenic stimulation.     

Affinity requirements for induction of sequential phases of human B cell activation by membrane IgM-cross-linking ligands

The affinity of Ag interaction with a B cell's membrane IgM (mIgM) receptors has long been considered to play a critical role in the in vivo clonal selection of B lymphocytes. This study has examined a possible basis for this affinity selection at the level of Ag induction of sequential B cell activation phenomena, i.e., elevated membrane class II MHC expression (G0* excitation), G1 entry, and S phase entry. Functional experiments with model bivalent Ag, i.e., a group of murine mAb of diverse intrinsic binding affinities for human IgM, revealed that the minimal affinity requisites for inducing the above phenomena vary significantly. At a ligand concentration of 100 micrograms/ml, the induction of increased class II MHC expression, G1 entry, and S phase had minimal affinity thresholds of Ka approximately 0.2 to 2 x 10(6) M-1; approximately 7 x 10(6) M-1; and approximately 1 x 10(8) M-1, respectively. Pulsing studies revealed that whereas high affinity ligand was essential at later periods in the prolonged (greater than 24 h) signaling period that leads to S phase entry, mAb with significantly lower affinity were competent at signaling during the first 24 h. Because all but the lowest affinity ligand (Ka = 2 x 10(5) M-1) could effectively modulate mIgM, and furthermore, because B cells show a substantial increase in surface area during activation, it appears likely that one factor contributing to the higher affinity requirements for induction of late activation phenomena is a progressive decrease in the density of mIgM on the responsive B cells. These studies suggest that whereas only a small proportion of B cells, i.e., those with relatively high affinity for an antigenic epitope, will be triggered to clonally expand on encountering a paucivalent Ag in the absence of T cell help, a much wider spectrum of the B cell repertoire will be triggered to a state of partial activation. How the presence of ancillary T cells and cytokines may facilitate the full clonal expansion of these latter cells is discussed.     

Accurate determination of mean cell volume by isotope dilution in erythrocyte populations with variable deformability.

Variations in erythrocyte deformability and morphology lead to artifacts in electronic determinations of mean cellular volume (MCV) by the aperture-impedance method. The micropipette-aspiration technique loses accuracy when applied to severely aberrant cells such as dense sickle cells. A new light-scattering technique requires that the cells be capable of undergoing isovolumetric sphering. In contrast, the isotope-dilution (ID) method measures absolute mean volume and is free of artifacts associated with abnormal deformability or morphology. It does not depend on any algorithms or correction factors and does not subject the cells to any stringent processing, not even centrifugation. The ID method can be used to determine the mean volume of red cells in hypo- or hypertonic media or in the presence of pharmacologic agents. It requires no more than a 1-ml aliquot of suspended cells at a hematocrit of at least 30%. The cells can be readily recovered, washed, and reused. Using EDTA labeled with 57Co as an extracellular space marker we have used ID to determine the MCV of fractionated normal human red blood cells (RBC), unfractionated RBC containing SS hemoglobin, and RBC from four other mammalian species. In the case of human RBC obtained from eight normal donors, we obtained mean MCV values (+/- SD) of 83.6 +/- 3.0, 87.5 +/- 3.9, and 76.5 +/- 5.3 fl for unfractionated and top and bottom 10% density fractions, respectively. The value 83.6 is significantly lower than the generally accepted range of 89-91 indicated by electronic analyzers calibrated against spun microhematocrits. The discrepancy of about 7% can account for the difference between mean cell hemoglobin concentration (MCHC) data determined by a calibrated Coulter Counter and corresponding data obtained with paired samples using a cyanmethemoglobin procedure specified in NCCLS Standard H15-A and corrected for trapped plasma.     

Development and evaluation of the polymerase chain reaction to detect Mycoplasma genitalium

The polymerase chain reaction (PCR) was developed to detect Mycoplasma genitalium. Oligonucleotide primers were used to amplify a 374 bp region of the attachment protein of the mycoplasma. DNA from three strains of M. genitalium tested gave a characteristic PCR product which was not seen with DNA from any other source. As little as 10(-15) g of M. genitalium DNA could be detected and it was found in the vagina of progesterone-treated BALB/c mice inoculated with M. genitalium organisms later than they could be cultured from this site, but not in mice that never became colonised vaginally.     

Change in energy metabolism of ascites cancer cells with a decrease in pH]

In Hank's balanced salt solution EL-4 ascites thymoma cells possessed endogenous respiration which was sufficient for the maintenance of their ATP level: pH decrease down to 6.0 had no effect either on endogenous respiration or the ATP level. Glucose had no influence on the respiration of EL-4 cells but inhibited that of Ehrlich ascites carcinoma (EAC) cells by 40% (Crabtree effect); respiration of the both cell lines was strongly (4-fold) inhibited after simultaneous addition of glucose, lactate and pH decrease. EL-4 cells had no endogenous glycolysis; EAC cells showed a low level of glycolysis only after pH decrease. Glucose addition led to activation of glycolysis (both inhibited 2-fold after a decrease of pH down to 6.0. The respiration inhibition at pH 7.3 and 6.0 caused no decrease of ATP depletion when glucose was present in the medium; this result may be due to suppression of ATP consumption. Incubation of EL-4 cells under respiration and glycolysis deficiency conditions resulted in a sharp ATP depletion; pH decrease delayed this depletion.