The mammalian brain high-affinity L-proline transporter is enriched preferentially in synaptic vesicles in a subpopulation of excitatory nerve terminals in rat forebrain.

The expression of a brain-specific high-affinity Na+-dependent (and Cl--dependent) L-proline transporter (PROT) in subpopulations of putative glutamatergic neurons in mammalian brain suggests a physiological role for this carrier in excitatory neurotransmission (). To gain insights into potential sites where PROT may function, we used a C-terminal domain antipeptide antibody to determine the regional distribution and subcellular localization of PROT in rat forebrain. PROT immunoreactivity was seen in processes having a regional light microscopic distribution comparable to that of known glutamatergic projections within the cortex, caudate putamen nucleus (CPN), hippocampal formation, and other forebrain regions. In all regions examined by electron microscopy (cortex, CPN, and the stratum oriens of CA1), PROT labeling was observed primarily within subpopulations of axon terminals forming asymmetric excitatory-type synapses. Immunogold labeling for PROT was detected in close contact with membranes of small synaptic vesicles (SSVs) and more rarely with the plasma membrane in these axon terminals. Subcellular fractionation studies confirmed the preferential distribution of PROT to synaptic vesicles. The topology of PROT in synaptic vesicles was found to be inverted with respect to the plasma membrane, suggesting that PROT-containing vesicles are generated by a process involving endocytosis from the plasma membrane. Because PROT lacks any of the known characteristics of other vesicular transporters, these results suggest that certain excitatory terminals have a reserve pool of PROT associated with SSVs. The delivery of PROT to the plasma membrane by exocytosis could play a critical role in the plasticity of certain glutamatergic pathways.

Polyamines may regulate S-phase progression but not the dynamic changes of chromatin during the cell cycle.

Several studies suggest that polyamines may stabilize chromatin and play a role in its structural alterations. In line with this idea, we found here by chromatin precipitation and micrococcal nuclease (MNase) digestion analyses, that spermidine and spermine stabilize or condense the nucleosomal organization of chromatin in vitro. We then investigated the possible physiological role of polyamines in the nucleosomal organization of chromatin during the cell cycle in Chinese hamster ovary (CHO) cells deficient in ornithine decarboxylase (ODC) activity. An extended polyamine deprivation (for 4 days) was found to arrest 70% of the odc- cells in S phase. MNase digestion analyses revealed that these cells have a highly loosened and destabilized nucleosomal organization. However, no marked difference in the chromatin structure was detected between the control and polyamine-depleted cells following the synchronization of the cells at the S-phase. We also show in synchronized cells that polyamine deprivation retards the traverse of the cells through the S phase already in the first cell cycle. Depletion of polyamines had no significant effect on the nucleosomal organization of chromatin in G1-early S. The polyamine-deprived cells were also capable of condensing the nucleosomal organization of chromatin in the S/G2 phase of the cell cycle. These data indicate that polyamines do not regulate the chromatin condensation state during the cell cycle, although they might have some stabilizing effect on the chromatin structure. Polyamines may, however, play an important role in the control of S-phase progression.

Rat homologues of yeast sec7p.

Mutations in the Saccharomyces cerevisiae sec7 locus lead to a pleiotropic secretory phenotype that is characterized by an accumulation of Golgi cisternae and a loss of secretory granules. This indicates that the corresponding gene product sec7p is involved in the budding of secretory granules from the Golgi apparatus. Here we report the primary structure of three rat homologues of sec7p, called msec7-1, -2, and -3. The mRNAs of these genes are expressed in all tissues tested. All msec7s share the same domain structure in which an N-terminal coiled-coil domain is followed by a sec7-homology domain and a pleckstrin-homology domain. On the protein level, msec7s are present in all rat tissues tested, with highest protein levels in brain and adrenal. In the adult rat brain, they are present in soluble and membrane-associated pools.

16-BAC/SDS-PAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins.

Integral membrane proteins, particularly those with more than one transmembrane domain, have traditionally been difficult to separate by two-dimensional electrophoretic methods. Here we report the adaptation of a previously published procedure [D. E. Macfarlane (1989) Anal. Biochem. 176, 457-463] for the analytical and semipreparative separation of membrane proteins. The first dimension involves discontinuous gel electrophoresis in an acidic buffer system using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC). The second dimension consists of discontinuous SDS-PAGE. Using carbonate-washed membranes of synaptic vesicles and clathrin-coated vesicles as examples, we demonstrate that complex membrane protein mixtures can be resolved with a resolution at least fivefold higher than that of one-dimensional SDS-PAGE. Protein patterns are highly reproducible and proteins with single or multiple transmembrane domains are resolved as clearly distinct spots. Smearing of bands or losses of protein are minimal. Several spots were identified by immunoblotting and internal sequencing. Thus, this method is suitable for the analytical and semipreparative characterization of membrane proteins derived from complex biological samples.

Structure of synaptogyrin (p29) defines novel synaptic vesicle protein.

Synaptogyrin (p29) is a synaptic vesicle protein that is uniformly distributed in the nervous system (Baumert et al., 1990). We have cloned and sequenced the cDNA encoding synaptogyrin, and the sequence predicts a protein with a molecular mass of 25,900 D with four membrane-spanning domains. The topology of the protein was confirmed by limited proteolysis using domain-specific antibodies. Database searches revealed several cDNA sequences coding polypeptides with sequence identities ranging from 32 to 46%, suggesting that synaptogyrin is a member of a multigene family. When the synaptogyrin cDNA is expressed in COS cells, the generated protein is indistinguishable from native synaptogyrin. To study intracellular sorting, synaptogyrin was expressed in CHO cells that revealed a punctate staining that was very similar to that of synaptophysin and endogenously expressed cellubrevin. Significant overlap with transferrin staining was also observed, suggesting that synaptogyrin is targeted to a recycling compartment involved in membrane traffic to and from the plasma membrane.

Synaptic targeting of rabphilin-3A, a synaptic vesicle Ca2+/phospholipid-binding protein, depends on rab3A/3C.

rab3A, a low molecular weight GTP-binding protein of synaptic vesicles with a putative function in synaptic vesicle docking, interacts in a GTP-dependent manner with rabphilin-3A, a peripheral membrane protein that binds Ca2+ and phospholipids. We now show that rabphilin-3A is an evolutionarily conserved synaptic vesicle protein that is attached to synaptic vesicle membranes via its N terminus and exhibits a heterogeneous distribution among synapses. In rab3A-deficient mice, rabphilin-3A is decreased in synapses belonging to neurons that primarily express rab3A and accumulates in the perikarya of these neurons. In contrast, neurons expressing significant levels of rab3C still contain normal levels of rabphilin-3A in a synaptic pattern, and rabphilin-3A binds rab3C in vitro. These results suggest that analogous to the membrane recruitment of raf by ras, rab3A and rab3C may function in recruiting rabphilin-3A to the synaptic vesicle membrane in a GTP-dependent manner.

Determinants of alcohol preference in the AA and ANA rat lines selected for differential ethanol intake.

A selective breeding program conducted in this laboratory has resulted in the establishment of the alcohol-preferring AA (Alko Alcohol) and alcohol-avoiding ANA (Alko Nonalcohol) rat lines. These lines have been used as a tool for attempting to identify the behavioral, neurochemical, and biochemical correlates of differential voluntary ethanol consumption. Some of the differences that have been found between the lines involve differential reinforcement: AA rats, but not ANA rats, rapidly acquire an ethanol-reinforced operant response. The AA's greater development of tolerance to the depressant effects of ethanol and their faster ethanol metabolism would also allow them to drink more. Neurochemical studies have suggested differential functioning of brain monoaminergic mechanisms. The activity of tyrosine hydroxylase and dopa decarboxylase, and the brain dopamine concentrations are higher in the AA rats than in the ANA rats, and the maximal number of dopamine D2 receptors is lower in the AA rats. The concentration of noradrenaline is higher in the brain of ANA rats than in that of AA rats, while the 5-hydroxytryptamine levels do not seem to differ greatly. The importance of these differences to the line difference in ethanol intake is not, however, clear, since there appears to be no difference in the sensitivity of monoamine systems of the two lines to ethanol.

Health for all by the year 2000: alcohol and the Nordic countries.

One of the European targets in the "Health for all by the year 2000" programme is to reduce alcohol consumption significantly by the turn of the century. This article describes how this target, and especially the 25% goal included in it, has been adopted in the Nordic countries. With the exception of Denmark, alcohol has for a long time been regarded as a serious public health problem, and the reduction of total consumption of alcohol has been held as one of the most important ways of combating alcohol problems. In the 1980s Sweden and Norway have accepted the European 25% goal with the least reservations. In Finland the target has been regarded as unrealistic. Yet Finland, like Iceland, has accepted the goal of reducing total alcohol consumption but left the amount unspecified. In Denmark, controlling total alcohol consumption has been consistently held to be an irrelevant way to reduce alcohol problems. The alcohol policy measures suggested to reach the targets are the classical ones: price increases, restrictions in alcohol availability, and more efficient information and education. One cannot, however, avoid the observation that very few concrete measures have been taken so far and that many forces work against a reduction in alcohol consumption. The European alcohol target has affected alcohol policy in the Nordic countries in terms of target setting and programme design. It remains to be seen whether the forces advocating more restrictive alcohol control policy will be strong enough to generate concrete action plans and implement the accepted targets in actual alcohol policy measures.

Immigrants and natives as mental health care recipients.

Survey results indicate more mental health problems among immigrants than in the native population in Sweden and in one country of departure, Finland. This is reflected in the high proportion of immigrants in the mental hospital studied, but not among psychiatric out-patients. The immigrant patient rate is high even in socially advantageous groups, eg. among married and employed persons. The diagnoses and problems reported in the patient case records of immigrants and natives are compared, with controls for background variables. Some problems, e.g. paranoia, somatic symtoms, and diffuseness of difficulties are clearly related to immigration status but others, eg. alcohol, work, or human relations problems are connected only with social class or sex, irrespective of immigration status.