The neuronal growth-associated protein GAP-43 interacts with rabaptin-5 and participates in endocytosis

Structural plasticity of nerve cells is a requirement for activity-dependent changes in the brain. The growth-associated protein GAP-43 is thought to be one determinant of such plasticity, although the molecular mechanism by which it mediates dynamic structural alterations at the synapse is not known. GAP-43 is bound by calmodulin when Ca2+ levels are low, and releases the calmodulin when Ca2+ levels rise, suggesting that calmodulin may act as a negative regulator of GAP-43 during periods of low activity in the neurons. To identify the function of GAP-43 during activity-dependent increases in Ca2+ levels, when it is not bound to calmodulin, we sought proteins with which GAP-43 interacts in the presence of Ca2+. We show here that rabaptin-5, an effector of the small GTPase Rab5 that mediates membrane fusion in endocytosis, is one such protein. We demonstrate that GAP-43 regulates endocytosis and synaptic vesicle recycling. Modulation of endocytosis by GAP-43, in association with rabaptin-5, may constitute a common molecular mechanism by which GAP-43 regulates membrane dynamics during its known roles in activity-dependent neurotransmitter release and neurite outgrowth.     

Increased fat intake during lactation modifies hypothalamic-pituitary-adrenal responsiveness in developing rat pups: a possible role for leptin

High fat feeding reportedly enhances hypothalamus-pituitary-adrenal (HPA) responses to stress in adult rats. The present study tested whether elevated fat intake during suckling could have short and/or long lasting consequences on HPA regulation in the offspring. Mothers were fed either a control (C; 5% fat) or high fat (HF; 20% fat) diet during the last week of gestation and throughout lactation. After weaning (day 21), pups from C and HF mothers were fed a chow diet. Offspring from both C- and HF-fed mothers were tested for ACTH and corticosterone responses to stress on postnatal days 10 and 35. We found that HF feeding produced higher lipid levels in the milk of HF compared with C lactating rat dams and that offspring of these mothers had significantly increased retroperitoneal fat pad weight and relative adipose mass on day 21 as well as elevated plasma leptin levels on days 10 and 21 of age. After weaning, pups from the HF mothers had lower plasma leptin levels than those from C mothers. Maternal dietary fat affected HPA responsiveness in the offspring in an age-related manner. Neonatal pups (day 10) from the HF mothers exhibited a reduction in the ACTH and corticosterone responses to ether stress. However, in 35-day-old offspring from HF-fed dams, stress-induced ACTH secretion was increased compared with that in pups from the C-fed mothers. These results demonstrate that maternal diet and increased fat intake through the milk are important regulators of HPA responsiveness in neonates and prepubertal rats. During neonatal life, the blunted stress responsiveness seen with elevated fat intake and the resulting high leptin levels might protect the pups from excessive HPA activation. After removal of the maternal dietary influence and reduced leptin levels, enhanced ACTH stress responses are observed as in adult rats fed a HF diet. Because of the inverse relationship between plasma levels of leptin and HPA responses in pups, the possibility exists that the effects of the HF diet on stress responsiveness are mediated by changes in leptin exposure during development.     

Poly(A) polymerase contains multiple functional domains

Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS.     

Phospholipid pool, lipid peroxidation, and superoxide dismutase activity under various types of oxidative stress of the brain and the effect of low-energy infrared laser irradiation

The effect of low-energy infrared laser irradiation on the phospholipid pool, lipid peroxidation, and superoxide dismutase activity in the brain of white rats was studied in experimental ischemia, reperfusion, and acute edema. These models are characterized by oxidative stress; the contents of tri- and diphosphoinositides and sphingomyelins were lowered, whereas the levels of phosphatidylserine and phosphatidylethanolamine did not change, and the amount of phosphatidylcholine was increased. In acute brain edema, the contents of hydroperoxides and malonic dialdehyde in enzymatic and nonenzymic lipid peroxidation systems were increased in mitochondrial and microsomal fractions and the level of arachidonic acid was significantly elevated. Infrared laser irradiation contributes to the correction of the changes in the phospholipid pool; laser irradiation lowered the increased levels of hydroperoxides and malonic dialdehyde and elevated superoxide dismutase activity in the brain during ischemia, reperfusion, and acute edema of the brain. The data suggest that low-energy infrared laser irradiation has certain neuroprotective activity in various types of oxidative stress including ischemia, reperfusion, and acute edema of the brain.     

Ultrasound evaluation of the fetal central nervous system

Evaluation of the fetal central nervous system is an integral part of any obstetric examination. Critical to the diagnosis of central nervous system abnormalities is a basic understanding of the normal fetal anatomy. Anatomic features of a normal ultrasound examination of the fetal central nervous system are discussed. In addition, the sonographic findings associated with a variety of major central nervous system anomalies are reviewed.     

Long-term treatment with abecarnil does not induce diazepam-like dependence in mice

Abecarnil (isopropyl-6-benzyloxy-4-methoxymethyl-beta-carboline-3-carboxylate) is a metabolically stable anxiolytic and anticonvulsant beta-carboline derivative with few sedative and muscle relaxant effects in rodents. Abecamil binds with high affinity to benzodiazepine receptors. Because long-term treatment with benzodiazepines leads to development of dependence, we evaluated in mice whether abecarnil also possesses a potential for producing dependence, using electroencephalographic and electromyographic monitoring, and behavioral assessment of anxiety to detect withdrawal responses after chronic treatment. Diazepam was used as a reference. Mice withdrawn from chronic treatment with diazepam (15 mg/kg/day for 12 days) showed a time-related evolution of anxiety, muscle rigidity and seizures between days 4 and 21 after discontinuation of the treatment. A period between withdrawal days 1 and 3 was symptom free. Mice withdrawn from chronic administration of abecarnil (6 mg/kg/day for 12 days) showed no anxiety and no changes in seizure susceptibility and muscle tone. The doses of diazepam and abecarnil used for chronic treatment were equivalent in terms of kinetics and binding to benzodiazepine receptors. These data indicate that long-term treatment with abecarnil does not induce benzodiazepine-like dependence in mice. Thus, it may be predicted that chronic treatment with abecarnil in humans may offer an important alternative to benzodiazepines in the treatment of anxiety.     

Structural and functional characterization of proteolytic fragments derived from the C-terminal regions of bovine fibrinogen

A number of new as well as previously described fragments derived from the D region of bovine fibrinogen by limited proteolysis have been characterized by sequence analysis, differential scanning calorimetry and circular dichroism. Determination of the extremities of the polypeptide chains forming individual fragments allowed the scheme of proteolysis and the borders between domains in the D region of fibrinogen to be established. It was also found that the most thermostable region of the D fragment (TSD) can be substantially reduced in size without loss of its compact structure. The alpha-helical content of the newly prepared 21-kDa TSD2 and 16-kDa TSD3 fragments were 82% and 75%, respectively, strongly supporting a coiled-coil structure for this region of the fibrinogen molecule. The DX and DZ fragments, prepared from a chymotryptic digest of the DLA fragment, were found to be similar to the DL and DY fragments, respectively, except for an internal cleavage at K393-T394 in their beta chains. This cleavage leads to destabilization of all thermolabile domains, indicating interaction between them. The DL and DY fragments, containing only one polymerization site in their beta chains, were able to inhibit fibrin polymerization at high concentration. However, these same fragments failed to bind to fibrin-Sepharose under conditions where their structural analogues, DX and DZ, were tightly bound, indicating that cleavage after K393 substantially increases the affinity of this site.     

Enhanced specificity of truncated transmembrane protein for serologic confirmation of human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 infections by western blot (immunoblot) assay containing recombinant envelope glycoproteins

Immunoassays based on the highly immunogenic transmembrane protein of human T-cell lymphotropic virus type 1 (HTLV-1) (protein 21c) are capable of detecting antibodies in all individuals infected with HTLV-1 and HTLV-2. However, because of antigenic mimicry with other cellular and viral proteins, such assays also have a large proportion of false-positive reactions. We have recently identified an immunodominant epitope, designated GD21-I located within amino acids 361 to 404 of the transmembrane protein, that appears to eliminate such false positivity. This recombinant GD21-I protein was used in conjunction with additional recombinant HTLV type-specific proteins and a whole virus lysate to develop a modified Western blot (immunoblot) assay (HTLV WB 2.4). The sensitivity and specificity of this assay were evaluated with 352 specimens whose infection status was determined by PCR assay for the presence or absence of HTLV-1/2 proviral sequences. All HTLV-1-positive (n = 102) and HTLV-2-positive (n = 107) specimens reacted with GD21-1 in the HTLV WB 2.4 assay, yielding a test sensitivity of 100%. Furthermore, all specimens derived from individuals infected with different viral subtypes of HTLV-1 (Cosmopolitan, Japanese, and Melanesian) and HTLV-2 (IIa0, a3, a4, IIb1, b4, and b5) reacted with GD21-I in the HTLV WB 2.4 assay. More importantly, HTLV WB 2.4 analysis of 81 PCR-negative specimens, all of which reacted to recombinant protein 21e in the presence or absence of p24 and p19 reactivity in the standard WB assay, showed that only two specimens retained reactivity to GD21-I, yielding an improved test specificity for the transmembrane protein of 97.5%. None of 41 specimens with gag reactivity only or 21 HTLV-negative specimens demonstrated reactivity to GD21-I. In an analysis of additional specimens (n = 169) from different geographic areas for which PCR results were not available, a substantial increase in the specificity of GD21-I detection was demonstrated, with no effect on the sensitivity of GD21-I detection among specimens from seropositive donors. Thus, the highly sensitive, GD21-I-based HTLV WB 2.4 assay eliminates the majority of false-positive transmembrane results, thereby increasing the specificity for serologic confirmation of HTLV-1 and HTLV-2 infections.