Phosphotransfer reactions as a means of G protein activation

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) serve to transduce information from agonist-bound receptors to effector enzymes or ion channels. Current models of G protein activation-deactivation indicate that the oligomeric GDP-bound form must undergo release of GDP, bind GTP and undergo subunit dissociation, in order to be in active form (GTP bound subunits and free dimers) and to regulate effectors. The effect of receptor occupation by an agonist is generally accepted to be promotion of guanine nucleotide exchange thus allowing activation of the G protein. Recent studies indicate that transphosphorylation leading to the formation of GTP from GDP and ATP in the close vicinity, or even at the G protein, catalysed by membrane-associated nucleoside diphosphate kinase, may further activate G proteins. This activation is demonstrated by a decreased affinity of G protein-coupled receptors for agonists and an increased response of G protein coupled effectors. In addition, a phosphorylation of G protein subunits and consequent phosphate transfer reaction resulting in G protein activation has also been demonstrated. Finally, endogenously formed GTP was preferentially effective in activating some G proteins compared to exogenous GTR The aim of this report is to present an overview of the evidence to date for a transphosphorylation as a means of G protein activation (see also refs [1 and 2] for reviews). (Mol Cell Biochem 157: 593, 1996)Recipient of Servier Investigator Award     

Membrane receptors for estrogen,progesterone, and testosterone in the rat brain: Fantasy or reality

Summary 1. There are numerous circumstantial evidence supporting the concept that steroid hormones control cellular function by means other than the nuclear receptor steroid binding mechanism. It is the intent of this report to present evidence indicating that steroids bind to specific sites in neuronal membranes.2. Some of the criteria to define steroid membrane receptors using steroid-BSA conjugates that can be radioiodinated to desired specific activity have been fulfilled for each of the three sex steroids using crude synaptosomal membrane preparations (P2 fractions) from the CNS of female and male rats. Ligand binding for each of the three steroids indicate high-affinity and high-capacity sites with distinct brain selectivity and stereospecificity. For example, 17-E-6-[¹²⁵I]BSA binds hypothalamic P2 fractions (HYP-P2) with an estimatedK _d of about 3±0.7 nM (X ± SE;n=3), whereas the cerebellum P2 (CB-P2) fractions bind the ligand with aK _d of 34±7 nM and, aB _max of 3 and 42 pmol/mg protein, respectively. Estrogen and testosterone binding fit best a one-single site, while progesterone binding sites can be best represented by a two-binding site, one high-affinity (K _d=1–2 nM) and one low affinity (K _d=62 nM), in CB-P2 fractions from intact adult female rat brain. Kinetics studies for T-3-[¹²⁵I]BSA indicate that the estimatedK _d of 30±2 nM for the olfactory bulb P2 fractions (OB-P2) from male rats is in good agreement withK _d values computed from Scatchard-derived data using the LIGAND algorithm.3. 17-E-6-[¹²⁵I]BSA binding sites are stereospecific and appears to be present as early as 5 days of age in both the OB- and the CB-P2 fractions without changes during development. In contrast, P-6-[¹²⁵I]BSA binding sites are practically absent during days 5 and 12 and appear by day 22.4. Finally, membrane receptor molecules for estrogen and progesterone have been isolated and purified by affinity chromatography and characterized by PAGE and Western blot. Microsequencing of one of the membrane estrogen binding proteins indicates that the high-affinity site corresponds to the OSCP subunit of the proton ATP synthase.5. It remains to be determined if P and T also bind to this complex enzyme or if they bind to other subunits of the family of proton ATPases. Overall the data indicate that steroid hormones conjugated to BSA are important tools to study the reality of membrane steroid receptors.     

The effect of dissolved oxygen tension and the utility of oxygen uptake rate in insect cell culture

Dissolved oxygen tension and oxygen uptake rate are critical parameters in animal cell culture. However, only scarce information of such variables is available for insect cell culture. In this work, the effect of dissolved oxygen tension (DOT) and the utility of on-line oxygen uptake rate (OUR) measurements in monitoring Spodoptera frugiperda (Sf9) cultures were determined. Sf9 cells were grown at constant dissolved oxygen tensions in the range of 0 to 30%. Sf9 metabolism was affected only at DOT below 10%, as no significant differences on specific growth rate, cell concentration, amino acid consumption/production nor carbohydrates consumption rates were found at DOT between 10 and 30%. The specific growth rate and specific oxygen uptake rate followed typical Monod kinetics with respect to DOT. The calculated _max and max were 0.033 h^-1 and 3.82×10^-10 mole cell^-1h^-1, respectively, and the corresponding saturation constants were 1.91 and 1.57%, respectively. In all aerated cultures, lactate was consumed only after glucose and fructose had been exhausted. The yield of lactate increased with decreasing DOT. It is proposed, that an apparent DOT in non-instrumented cultures can be inferred from the lactate yield of bioreactors as a function of DOT. Such a concept, can be a useful and important tool for determining the average dissolved oxygen tension in non-instrumented cultures. It was shown that the dynamic behavior of OUR can be correlated with monosaccharide (fructose and glucose) depletion and viable cell concentration. Accordingly, OUR can have two important applications in insect cell culture: for on-line estimation of viable cells, and as a possible feed-back control variable in automatic strategies of nutrient addition.Abbreviations DOT Dissolved oxygen tension - OUR Oxygen uptake rate - specific oxygen uptake rate - specific growth rate - X_v viable cell concentration - C_L, C^*, and oxygen concentrations in liquid phase, in equilibrium with gas phase, and medium molar concentration, respectively - H Henry's constant - K_La volumetric oxygen transfer coefficient - P_T total pressure - oxygen partial pressure - oxygen molar fraction - i discrete element     

Efficacy of transfer factor in treating patients with recurrent ocular herpes infections

Recurrent ocular herpes is an insoluble problem for the clinician. As cellular immunity plays an important role in controlling herpes relapses, and other studies have shown the efficacy of HSV-specific transfer factor (TF) for the treatment of herpes patients, an open clinical trial was undertaken in 134 patients (71 keratitis, 29 kerato-uveitis, 34 uveitis) suffering from recurrent ocular herpetic infections. The mean duration of the treatment was 358 days, and the entire follow-up period 189121 before, and 64062 days after TF treatment. The cell-mediated immune response to the viral antigens, evaluated by the lymphocyte stimulation test (LST) and the leucocyte migration test (LMT) (P<0.001), was significantly increased by the TF treatment. The total number of relapses was decreased significantly during/after TF treatment, dropping from 832 before, to 89 after treatment, whereas the cumulative relapse index (RI) dropped, during the same period, from 13.2 to 4.17 (P<0.0001). No side effects were observed. It is concluded that patients with relapsing ocular herpes can benefit from treatment with HSV-specific TF.     

Growth and photosynthetic responses of field-grown sweetgum (Liquidambar styraciflua; Hamamelidaceae) seedlings to UV-B radiation

Very few studies have evaluated the effects of UV-B radiation on trees. especially deciduous species. In this study we evaluate the effects of supplemental UV-B radiation on the growth and photosynthetic capacity of sweetgum (Liquidambar styraciflua L.). Sweetgum seedlings were grown for 2 years in the field under either ambient or supplemental UV-B radiation. Artificial UV-B radiation was supplied by fluorescent lamps at a maximum daily supplementation of either 3.1 or 5.0 kJ of biologically effective UV-B radiation. Over the 2-year period, supplemental UV-B radiation had little effect on total plant biomass or photosynthetic capacity. However, subtle changes in leaf physiology, carbon allocation, and growth were observed. Supplemental UV-B radiation reduced photosynthetic capacity only during the first year, while leaf area and biomass were reduced in the second year. Alterations in carbon allocation included an increase in branch number and root to shoot ratio. While these data do not indicate that the productivity of sweetgum would likely be compromised by an increase in solar UV-B radiation, they do suggest that the UV-B portion of the solar spectrum contributes to the regulation of sweetgum growth and development. Therefore the possibility of significant consequences to sweetgum due to possible increases in UV-B radiation cannot be ruled out.     

Identification of Tuber magnatum Pico DNA markers by RAPD analysis

Summary Different species of truffle were studied in order to identify species-specific markers. The isolation of two Tuber magnatum Pico markers is reported. One of these could be used as a probe in dot blot hybridization, allowing the development of a rapid test able to identify Tuber magnatum species.     

Sandhoff disease in Argentina: high frequency of a splice site mutation in the HEXB gene and correlation between enzyme and DNA-based tests for heterozygote detection

The level of -hexosaminidase activity in plasma and leukocytes and the frequency of three known HEXB mutations were studied in an Argentinean deme with high incidence of infantile Sandhoff disease. Two mutations were previously identified in one of two Sandhoff patients from the region, a splice mutation, IVS-2+1 GA, and a 4-bp deletion, CTTT_782–785. These mutations, and a 16kb deletion from the 5' end of the HEXB gene common in non-Argentineans, were screened in 9 Sandhoff patients (all unrelated), 24 obligate heterozygotes, 33 additional individuals belonging to families with affected members, and 64 randomly ascertained individuals from the high risk region. Of 31 independent alleles examined, including those of the two patients previously reported, 30 had the IVS-2 splice mutation and only the originally reported patient had the CTTT deletion. The 16-kb deletion was not observed. Further, among the 57 unaffected members of families with a previous history of Sandhoff disease, and absolute correlation was found between carrier diagnosis by enzyme assay of leukocytes and the DNA-based tests for mutation. One of the 64 controls was classified as a carrier by enzyme assay but did not have one of the three mutations screened. We conclude that a single mutation predominates in this Argentinean population and that the DNA-based test can be an effective supplement or alternative to enzyme-based testing.