Andrea Cavaggioni

Acceleration of puberty onset in female mice by male urinary proteins.

1. Puberty onset in female mice is accelerated by exposure to conspecific adult male urine, which acts through the vomeronasal organ and the accessory olfactory system. A distinctive component of adult male mouse urine is the major urinary protein complex (MUP), which is a lipocalin; it has a hydrophobic pocket that binds small endogenous volatile molecules. The MUP gene family also codes for a hexapeptide, which has four residues in common with the N‐terminal region of MUP. 2. MUP, the volatiles bound to MUP and the MUP‐related hexapeptide have been tested for the induction of puberty acceleration by measuring the increase in uterus weight related to the first pro‐oestrus phase. MUP, together with its bound volatiles, induces puberty acceleration. Its activity is retained even when the volatiles have been removed either by organic extraction or competition displacement with a high‐affinity ligand. 3. MUP‐related hexapeptide also induces puberty acceleration in female mice. In contrast, the odorants bound to MUP do not exert this effect. 4. It is proposed that the vomeronasal organ contains receptors that recognize the short N‐terminal consensus sequence, N‐Glu‐Glu‐Ala‐X‐Ser (where X is a polar residue), common to both MUP and the hexapeptide.

Cyclic GMP and the permeability of the disks of the frog photoreceptors.

1. The diffusion of sodium, potassium and rubidium (not chloride) ions across the disk membrane is increased by cyclic guanosine monophosphate (cyclic GMP). 2. The increase is greater for sodium than for rubidium in the 0.01‐0.1 mM concentration range. 3. Cyclic adenosine monophosphate (cyclic AMP) is less efficient than cyclic GMP; GMP and guanosine triphosphate are without effect. 4. The effect is present with either 1.8 mM calcium ions or 4 mM‐EGTA in the perfusion fluid. 5. The presence of the cyclic GMP phosphodiesterase on the disk membranes is not needed for this effect. 6. The effect is present in both unbleached and fully bleached membranes.

Ligand binding characteristics of homologous rat and mouse urinary proteins and pyrazine-binding protein of calf

1. The binding affinities of three classes of homologous proteins, alpha 2u protein from rat urine, major urinary protein of mouse and pyrazine-binding protein of calf nasal mucosa have been determined for a panel of ligands. 2. Best ligands were low threshold odorants for man, but chemically unrelated. 3. The binding spectra of the homologous proteins were different.

Homology between the pyrazine-binding protein from nasal mucosa and major urinary proteins.

Sequence analysis of the pyrazine‐binding protein from bovine olfactory mucosa reveals marked homology with a family of proteins of unknown function found in the urine of the adult male mouse and rat. In view of the dramatic biological responses to odorants transmitted in male rodent urines, it is proposed that these proteins play important roles in some aspects of odor transmission and reception.

Light-activated hydrolysis of GTP and cyclic GMP in the rod outer segments.

1. The hydrolysis of guanosine triphosphate (GTP) and the consequent formation of guanosine diphosphate (GDP) and phosphate (P1) are activated by light in a suspension of broken retinal rods: the hydrolysis rate with GTP in the micrometer concentration range is 2.5‐3.5 n‐mole/min per mg of rhodopsin in the preparation. 2. The ionic composition of the medium suspending the rods is not critical: the hydrolysis is present in NaCl saline solution with MG2+ as well as in Tris‐HC1 buffer solution, and with the chelating agent EDTA. 3. The ionic strength is critical: the effect is reduced when the broken rods are suspended in a low salt mannitol solution, and is altogether abolished when they are separated from the mannitol solution; it reappears when the mannitol solution is added again in the presence of salts. An element essential for the effect is thus reversibly released in the mannitol solution. No hydrolytic activity on GTP, however, is found in the mannitol soluble fraction. 4. The cyclic nucleotide phosphodiesterase is eluted from the rods in the mannitol solution, and is reaggregated to the rods in the presence of salts; once recombined with the rods, it can be activated by light. 5. The activation of the phosphodiesterase by light is present in the absence of added nucleotide triphosphates.

The pyrazine-binding protein and olfaction

1. The present results provide circumstantial evidence, but not a proof, that the Pyrazine-binding Protein is an odorant carrier molecule of fundamental importance. 2. At first sight a role for a secretory protein in olfaction is not obvious. 3. Odorants freely diffuse in air, in water and in lipids, and the use of carrier proteins, would seem superfluous unless a very special combination with the odorant occurs [Gaupp E. (1902) In Anatomie des Frosches, 2nd Edn, pp. 673. Vieweg-Verlag, Braunschweig]. 4. The possibility should be considered that the Pyrazine-binding Protein and the urinary proteins belong to a large family of species-specific secretory molecules which, with the odorant bound, directly stimulate the receptor cell.

Light and gtp effects on the turbidity of frog visual membrane suspensions

SummaryThe time course of turbidity changes of frog visual membranes, dependent on osmotic shocks, on light and on nucleotide substrates or effectors of enzyme activities, were measured as absorption changes in a rapid mixing stopped-flow spectrophotometer.As a result of studies on different preparations, it is concluded that light can cause both rapid (within 50 msec) and slow (within 90 sec) changes in the turbidity of visual membranes, not associated with permeability changes, and that they are affected by GTP or its analog guanyl-5′-yl imidodiphosphate; however, the light and GTP effects are lost when a water soluble fraction containing the light-sensitive enzyme cGMP-phosphodiesterase, is removed from the rod outer segments membranes.It is suggested that the fast light and GTP-sensitive response is related to the activation of cGMP-phosphodiesterase.

Selective distribution of protein kinase A regulatory subunit RIIα in rodent gliomas

Differential diagnosis of brain tumor types is mainly based on cell morphology and could benefit from additional markers. The cAMP second-messenger system is involved in regulating cell proliferation and differentiation and is conceivably modulated during cancer transformation. The cAMP second-messenger system mainly activates protein kinases, which are in part docked to cytoskeleton, membranes, or organelles by anchoring proteins, forming protein aggregates that are detergent insoluble and not freely diffusible and that are characteristic for each cell type. The intracellular distribution of the detergent-insoluble regulatory subunits (R) of the cAMP-dependent protein kinase has been examined in mouse and rat glioma cells both in vitro and in vivo by immunohistochemistry. In normal rodent brains, the RIIalpha regulatory subunit is detergent insoluble only in ependymal cells, while in the rest of the brain it is present in soluble form. Immunohistochemistry shows that in both mouse and rat glioma cell lines, RIIalpha is mainly detergent insoluble. RIIalpha is localized close to the nucleus, associated with smooth vesicles in the trans-Golgi network area. Both paclitaxel and vinblastine cause a redistribution of RIIalpha within the cell. Under conditions that increased intracellular cAMP, apoptosis of glioma cells was observed, and it was accompanied by RIIalpha redistribution. Also in vivo, detergent-insoluble RIIalpha can be observed in mouse and rat gliomas, where it delineates the border between normal brain tissue and glioma. Therefore, intracellular distribution of detergent-insoluble RIIalpha can assist in detecting tumor cells within the brain, thus making the histologic diagnosis of brain tumors more accurate, and may represent an additional target for therapy.

Some aspects of calcium regulation in cell biology

The roles of intracellular calcium in the regulation of cell metabolism and cell membrane permeability are highlighted with examples taken from recent studies.

Search for a physiological role of cyclic GMP metabolism in the photoreceptors

Data on the time-course of the light-activation of the cyclic GMP phosphodiesterase and of the GTPase, and results on the influence of cyclic GMP on the disc membrane permeability are presented. On the basis of the kinetic data, it is not possible to separate the light-activation of these two enzymes from the early steps of photoreceptor transduction. In addition, the cyclic GMP increases the permeability of the disc membranes, indicating that a decrease of the endogenous cyclic GMP concentration, consequent to the light-activation of the phosphodiesterase, can decrease the membrane permeability shortly after illumination.