BDNF increases release probability and the size of a rapidly recycling vesicle pool within rat hippocampal excitatory synapses

Exerting its actions pre-, post- and peri-synaptically, brain-derived neurotrophic factor (BDNF) is one of the most potent modulators of hippocampal synaptic function. Here, we examined the effects of BDNF on a rapidly recycling pool (RRP) of vesicles within excitatory synapses. First, we estimated vesicular release in hippocampal cultures by performing FM4-64 imaging in terminals impinging on enhanced green fluorescent protein (eGFP)-labelled dendritic spines – a hallmark of excitatory synapses. Consistent with a modulation of the RRP, BDNF increased the evoked destaining rate of FM4-64 only during the initial phase of field stimulation. Multiphoton microscopy in acute hippocampal slices confirmed these observations by selectively imaging the RRP, which was loaded with FM1-43 by hyperosmotic shock. Slices exposed to BDNF showed an increase in the evoked and spontaneous rates of FM1-43 destaining from terminals in CA1 stratum radiatum, mostly representing excitatory terminals of Schaffer collaterals. Variance-mean analysis of evoked EPSCs in CA1 pyramidal neurons further confirmed that release probability is increased in BDNF-treated slices, without changes in the number of independent release sites or average postsynaptic quantal amplitude. Because BDNF was absent during dye loading, imaging, destaining and whole-cell recordings, these results demonstrate that BDNF induces a long-lasting enhancement in the probability of transmitter release at hippocampal excitatory synapses by modulating the RRP. Since the endogenous BDNF scavenger TrkB-IgG prevented the enhancement of FM1-43 destaining rate caused by induction of long-term potentiation in acute hippocampal slices, the modulation of a rapidly recycling vesicle pool may underlie the role of BDNF in hippocampal long-term synaptic plasticity.     

Optimization of peptide linker length in production of MHC class II/peptide tetrameric complexes increases yield and stability,and allows identification of antigen-specific CD4+T cells in peripheral blood mononuclear cells

Reliable, efficient systems for producing soluble HLA-DR molecules, suitable for multimerization and use as staining reagents, have proved elusive. We found that the addition of a flexible linker between peptide and N terminus of the DRB1*0101-chain (Crawford, F., Kozono, H., White, J., Marrack, P. and Kappler, J., Immunity 1998. 8: 675-682.), results in greater in vitro folding efficiency of Escherichia coli-expressed alpha- and beta-chains, and increases both the yield and stability of the DRA1*0101/DRB1*0101/peptide complexes. Although a 10-amino acid linker functioned efficiently for a 20mer epitope from HIV p24, a longer linker was required to produce a DR1 MHC class II tetramer with the influenza hemagglutinin epitope (HA(306-318)). The DR1-HA tetramer was able to stain positively over 98% of a specific clone (HA 1.7) with only a brief 30-min incubation. The tetrameric complexes detected clone cells diluted into PBMC, with high sensitivity, coupled with low background staining in CD4(+) cells. It was possible to detect antigen-specific CD4(+) T cells within a population of PBMC stimulated with the HA peptide. This demonstrates the potential to monitor CD4(+) T cell responses in peripheral blood in a number of clinical scenarios.     

Antibody response of humans to the circumsporozoite protein of Plasmodium vivax.

We studied the interaction of sera from residents of an area in northern Peru where vivax malaria is endemic with four recombinant DNA-derived circumsporozoite (CS) proteins of Plasmodium vivax. The antigens used in the enzyme-linked immunosorbent assay included one Escherichia coli-produced and three Saccharomyces cerevisiae-produced recombinant proteins. Three of the proteins (NS1(81)V20, Vivax-1, and Vivax-2) contain the entire central repeat region of the P. vivax CS protein, and one protein (Vivax-3) contains only two repeat sequences. Vivax-1, Vivax-2, and Vivax-3 contain different lengths of sequences flanking the repeats. A higher percentage of the sera had antibodies to Vivax-2 and Vivax-3, the two proteins containing the longest nonrepeat sequences, than to NS1(81)V20 or Vivax-1. Children less than 5 years of age did not have immunoglobulin G antibodies to NS1(81)V20; however, they had antibodies to Vivax-1, Vivax-2, and Vivax-3. The finding that individuals living in a malaria-endemic area produce antibodies to peptides containing nonrepeat regions of the CS protein emphasizes the need to characterize the immune response to these regions in naturally exposed and experimentally immunized humans.     

A second locus (GLC3B) for primary congenital glaucoma (Buphthalmos) maps to the 1p36 region

Primary congenital glaucoma (gene symbol: GLC3) is an ocular disorder that occurs for 0.01-0.04% of blind people. In the majority of familial cases reported so far, this condition is inherited as an autosomal recessive trait. We have recently used a group of 17 GLC3 families with a minimum of two affected offspring and consanguinity in most of the parental generation and mapped the first GLC3 locus (GLC3A) to the 2p21 region. Six families did not show any linkage to the GLC3A locus and thus provided evidence for genetic heterogeneity of this disorder. A total of eight families unlinked to the 2p21 region were used to search for the chromosomal location of the second GLC3 locus. Herein, we describe mapping of a new locus (designated GLC3B) for primary congenital glaucoma to the short arm of chromosome 1 (1p36.2-36.1) that is situated centromeric to the neuroblastoma and Charcot-Marie-Tooth type 2A (CMT2A) loci. A total of 17 DNA markers were genotyped from this region of chromosome 1. Four families showed no recombination with the two markers D1S2834 and D1S402 with a maximum lod score of 4.510 and 4.157 respectively. Pairwise and multipoint linkage analysis and inspection of the haplotypes revealed that the remaining four families are not linked to this part of chromosome 1, thus providing further evidence that at least one more locus for the autosomal recessive form of GLC3 must exist in the genome. Based on the recombination events, the overall linkage map of this region is: tel-D1S1192-D1S1635-D1S1193 - (D1S1597/-D1S489/D1S228)- [GLC3B/D1S2834/D1S402] - (D1S1176/D1S507/D1S407) - D1S2728-(MFAP2/D1S170) - D1S1368 - D1S436- D1S1592-cen.      

The role of estrogen and progesterone in the control of preputial gland sex attractant odors in the female rat

Sexually experienced male rats were used to test the attractiveness of body odors of female rats. The attractiveness of these odors varied with the estrous cycle. Odors from female rats in proestrus were the most attractive to male rats and those from female rats during the darkness hours of diestrus the least attractive. The preputial glands appeared to be the source of these odors for the male rats showed no preference for the odors of proestrous female rats that had been preputialectomized. Administration of 1 μg estrdiol benzoate (EB) for 5 days increased the attractiveness of body odors of ovariectomized rats. A higher dose of EB (5 μg) had the same effect when administered for 1 or 5 days although the increase that occurred after 3 days was not significant. A single dose of progesterone (P) (500 μg) on the other hand, decreased the attractiveness of ovariectomized female odors although no change was seen after 3 days of treatment. A single injection of P also decreased the attractiveness of odors of ovariectomized females that had received EB for 3 days. However, P failed to decrease the attractiveness of odors in ovariectomized females after preputialectomy. We conclude that the preputial glands are an important source of sex attractant odors in the female rat and that the changes in the release of these odors that occur throughout the estrous cycle and pregnancy are controlled by ovarian steroids. While estrogen acts to stimulate the production and release of these odors P appears to inhibit their release.     

A photoetched cell relocation matrix for long-term, quantitative observations of selected individual neurons in culture

A photoetched matrix of indium tin oxide (ITO) on glass has been developed and tested as a tool to assist in the relocation and identification of individual neuronal cells in culture. The matrix is formed by 10-15 micron wide and 300 A thick ITO lines which subdivide a 1-cm2 area into 625 smaller squares. Each of the smaller squares measures 400 micron on a side and contains a photoetched two-letter "address". The address code allows precise relocation of specific regions of a culture as well as verification of the identities of individual neurons selected for repeated observation. Marks at 50 micron intervals along the sides of the address squares permit quantitative analysis of morphological changes, cell migration, reaggregation, etc. The ITO is transparent and does not interfere with visualization of even fine details of cells with high power microscopy.     

Striated intramural gallbladder lucencies on US studies: predictors of acute cholecystitis

Ultrasound scans of 51 consecutive patients with gallbladder wall thickening were reviewed, and specific sonographic features were correlated with surgical and clinical follow-up. Two patterns of thickening were identified as specific indicators of the presence or absence of acute cholecystitis. "Striated" wall thickening, consisting of several alternating, irregular, discontinuous, lucent and echogenic bands, was seen in eight of 13 patients (62%) with acute cholecystitis. This pattern was not encountered in any of the patients who did not have acute cholecystitis. Conversely, "three-layer" thickening, consisting of a single circumferential lucent zone between two relatively uniform echogenic layers, was seen in only one of 13 patients (8%) with acute cholecystitis but in 11 of 38 patients (29%) with other diagnoses. Other abnormalities, including the presence of intramural echogenic foci and wall irregularities, were more frequently seen in patients with acute cholecystitis but were not as helpful. Use of these features may suggest or help exclude a diagnosis of acute cholecystitis in those patients in whom the cause of gallbladder wall thickening is otherwise not apparent.     

How much energy does the breast fed infant consume and expend?

Energy intake in breast fed infants is uncertain. The doubly labelled water method was used to measure, simultaneously and non-invasively, energy expenditure, energy intake, milk volume intake, energy deposition, and the energy content of breast milk in 12 "free living" breast fed babies at 5 and 11 weeks of age. The validity of this new approach was assessed in a parallel study in 12 formula fed infants. The babies who were exclusively breast fed expended 1.28 and 1.68 MJ/day at five and 11 weeks and had intakes of 1.81 and 2.22 MJ/day; these intakes were associated with normal growth but were well below those recommended previously. At five and 11 weeks the calculated energy content of breast milk was 0.24 and 0.25 MJ/100 ml, which is substantially lower than that commonly reported in milk obtained unphysiologically by expression of the breast. These data cast doubt on the widely used published standards for infant feeding.     

Clinical pharmacology of oral and intravenous 4-demethoxydaunorubicin

Summary The clinical pharmacology of 4-demethoxydaunorubicin (4-DMDNR) was studied in 28 patients with advanced breast cancer, using a sensitive reverse-phase HPLC technique. All patients had normal renal and hepatic function. The serum levels of 4-DMDNR after a single i.v. bolus injection followed a triple exponential decay curve (T1/2=9.6 min, T1/2=3.2 h and T1/2=34.7 h) and conformed to a three-compartment model. Comparison of the area under the curve (AUC) and urinary excretion for the oral and i.v routes suggests an oral bioavailability of approximately 24%. In patients treated with a schedule of weekly oral administration for periods of up to 12 months there was no significant alteration in either AUC or elimination half-life for the parent drug or its principal metabolite 13-OH4DMDNR. Moreover, there was no evidence of accumulation of the metabolite although measurable amounts were present 7 days after administration of 4-DMDNR.This work was supported by the Cancer Research Campaign and Farmitalia Carlo Erba Ltd     

Fish Hydrolysate Supplementation Containing n-3 Long Chain Polyunsaturated Fatty Acids and Peptides Prevents LPS-Induced Neuroinflammation

Neuroinflammation constitutes a normal part of the brain immune response orchestrated by microglial cells. However, a sustained and uncontrolled production of proinflammatory factors together with microglial activation contribute to the onset of a chronic low-grade inflammation, leading to neuronal damage and cognitive as well as behavioral impairments. Hence, limiting brain inflammatory response and improving the resolution of inflammation could be particularly of interest to prevent these alterations. Dietary n-3 long chain polyunsaturated fatty acids (LC-PUFAs) and low molecular weight peptides are good candidates because of their immunomodulatory and proresolutive properties. These compounds are present in a fish hydrolysate derived from marine-derived byproducts. In this study, we compared the effect of an 18-day supplementation with this fish hydrolysate to a supplementation with docosahexaenoic acid (DHA) on lipopolysaccharide (LPS)-induced inflammation in mice. In response to peripherally injected LPS, the fish hydrolysate supplementation decreased the hippocampal mRNA expression of the proinflammatory cytokines IL-6 (p < 0.001), IL-1β (p = 0.0008) and TNF-α (p < 0.0001), whereas the DHA supplementation reduced only the expression of IL-6 (p = 0.004). This decline in proinflammatory cytokine expressions was associated with an increase in the protein expression of IκB (p = 0.014 and p = 0.0054 as compared to the DHA supplementation and control groups, respectively) and to a modulation of microglial activation markers in the hippocampus. The beneficial effects of the fish hydrolysate could be due in part to the switch of the hippocampal oxylipin profile towards a more anti-inflammatory profile as compared to the DHA supplementation. Thus, the valorization of fish byproducts seems very attractive to prevent and counteract neuroinflammation.