The G-protein inhibitor, pertussis toxin, inhibits the secretion of brain-derived neurotrophic factor.

Secretion of neurotrophins is critical for the delivery of neurotrophic support. Brain-derived neurotrophic factor is targeted to a regulated secretory pathway in neurons as well as the neurosecretory AtT-20 cells. Here, we show that pertussis toxin, which inactivates Gi and Go G proteins, inhibits up to 50% of the regulated release of brain derived neurotrophic factor by AtT-20 cells. To determine whether pertussis toxin-sensitive G proteins may regulate brain-derived neurotrophic factor release in vivo, the effect of intraocular pertussis toxin was assessed on the isthmo-optic nucleus in the developing chick visual system. The isthmo-optic nucleus projects axons from the midbrain to innervate retinal amacrine cells and depends on target-derived brain-derived neurotrophic factor between embryonic days 13 and 17 (E13-17). During this period approximately 50% of isthmo-optic neurons are eliminated by programmed cell death. Intraocular pertussis toxin administered at E13 increased cell death of isthmo-optic neurons by 42%, whereas injections at E19 had no effect. Co-injection of brain-derived neurotrophic factor with pertussis toxin rescued approximately 50% of isthmo-optic neurons from enhanced cell death, although overall retinal brain derived neurotrophic factor protein levels were unaffected by pertussis toxin. Retrograde transport of exogenous 125I-labeled brain derived neurotrophic factor from the retina to the midbrain was increased by co-administration of pertussis toxin, possibly owing to diminished competition from endogenously released brain-derived neurotrophic factors for the receptors that mediate retrograde axonal transport. These data suggest that the release of a major fraction of brain-derived neurotrophic factor in the secretory pathway in vitro and in vivo is regulated by the activity of pertussis toxin-sensitive G proteins.

Neuronal activity induction of the stathmin-like gene RB3 in the rat hippocampus: possible role in neuronal plasticity.

Synaptic activity induces a rapid transcriptional response that is essential for the establishment of long-term neuronal plasticity. Using a differential cloning technique, we have identified a gene induced by seizure activity in the brain as RB3. RB3 is a recently cloned gene belonging to the stathmin family of phosphoproteins. Like SCG10, RB3 is brain-specific, although in situ hybridization results show that the expression of RB3 is more ubiquitous than is that of SCG10. Using genomic DNA sequencing, we show that the 27 amino acid sequence unique to the RB3" transcript is encoded by an alternatively spliced exon, exon 2'. Using a peptide antibody raised against exon 2' to detect RB3" and an anti-Flag antibody to detect an epitope-tagged version of RB3, we show that both proteins are localized to the Golgi apparatus of transfected COS7 cells. Of particular interest, RB3 mRNA, but not SCG10 mRNA, is rapidly induced in the dentate gyrus granule layer of the hippocampus after electrically induced seizure activity as well as stimuli leading to long-term potentiation (LTP). In addition, RB3 mRNA is induced in pheochromocytoma (PC12) cells treated with 250 ng/ml NGF. These results suggest that RB3 may play a role in activity-induced neuronal plasticity and neuronal differentiation.

Heat stability of two candidate international reference preparations for recombinant human tissue factor.

Adequate heat stability of international reference preparations (IRP) for thromboplastin (tissue factor) is an essential requirement. Accelerated degradation testing was performed by three laboratories on two candidate IRP for recombinant human tissue factor. Heat treatment of these candidates resulted in slight shortening of the PT, contrasting with heat-induced prolongation of the PT observed with a conventional human brain derived IRP. Heat stability of these candidates was improved when compared with the stability of previous recombinant human tissue factor preparations. The PT-ratio did not change significantly when the candidates were stored for 28 days at 44 degrees C. It can therefore be concluded that both candidates are acceptable with regard to stability.

Regulated release and polarized localization of brain-derived neurotrophic factor in hippocampal neurons.

The site and regulation of neurotrophic factor release from neurons is poorly understood. We used a combination of model cell lines and primary culture systems to study the polarity of BDNF sorting and the regulation of its release from hippocampal neurons. Transfection and expression of a human BDNF cDNA in a mouse pituitary cell line, AtT20, resulted in the colocalization of BDNF with the secretory granule marker, chromogranin A. Furthermore, stimulation of these cells with 56 mM KCl or with 5 mM 8-bromo-cAMP increased the release of BDNF approximately 10-to 15-fold within 30 min. To study BDNF release from primary cultures of hippocampal neurons, cells were infected with a defective Herpes Simplex Viral (HSV) vector expressing human BDNF. Depolarizing conditions increased the release of BDNF 5-fold from these cells, further verifying that secretion is regulated. Immunocytochemical analysis using highly specific antibodies determined that endogenous BDNF was predominantly localized to the somatodentritic domain of hippocampal neurons. These findings support the view that BDNF functions as a target-derived signal for afferents to hippocampal pyramidal cells and that it may serve as a regulator of hippocampal plasticity.

Retrograde intramedullary nailing of the femur using a tibial nail--the adjunctive use of an existing implant: a case report.

A 74-year-old male involved in a pedestrian-automobile collision sustained a comminuted supracondylar-diaphyseal femur fracture. The fracture was stabilized by retrograde intramedullary fixation with a Synthes unreamed tibial nail. Knee motion reached 0 degree-120 degrees by the sixth postoperative day and the fracture healed within twelve weeks. Twelve months after his injury, his knee motion was symmetric to his uninjured side and he had resumed full preinjury activities, including martial arts training. Although antegrade intramedullary nailing remains the treatment of choice for fractures of the femur, this case highlights the usefulness of retrograde nailing and demonstrates the adjunctive application of an existing implant, the tibial nail, in certain special trauma situations.

A survey of furin substrate specificity using substrate phage display.

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.

Improving cross-cultural skills of medical students through medical school-community partnerships.

Postulating that a program integrating language skills with other aspects of cultural knowledge could assist in developing medical students' ability to work in cross-cultural situations and that partnership with targeted communities was key to developing an effective program, a medical school and two organizations with strong community ties joined forces to develop a Spanish Language and Hispanic Cultural Competence Project. Medical student participants in the program improved their language skills and knowledge of cultural issues, and a partnership with community organizations provided context and resources to supplement more traditional modes of medical education.

Autoproteolytic activation of the mouse prohormone convertase mPC1.

In this study the activation of the prohormone convertase mPC1 was determined. Expression and characterization of catalytic domain mutations (Ser382 to Ala or His208 to Ala) in the prohormone convertase mPC1, unequivocally demonstrated that pro-region cleavage proceeds by an autocatalytic mechanism. Furthermore, these results suggest that autoproteolysis may be the result of an intramolecular reaction, since proregion processing of the active-site mutant could not be complemented by the overexpression of active furin or PC1. Additionally coexpression of a cleavage-site mutant (Arg110-Ala) with the substrate prorelaxin further demonstrated that autoproteolysis is required for the full activity of PC1.

In vitro activity of new quinoxaline compounds against Campylobacter species and Clostridium difficile.

SC-44914 and SC-44942-A are two new quinoxaline compounds with a spectrum of activity similar to that of metronidazole. We studied the activity of SC-44914 and SC-44942-A against 35 Campylobacter jejuni, 30 C. coli, and 20 Clostridium (Cl.) difficile and compared it with that of metronidazole by utilizing an agar dilution method. The quinoxalines had little activity against the C. jejuni and C. coli [minimum inhibitory concentration (MIC)90 > or = 64 micrograms/ml]. SC-44914 and SC-44942-A had excellent activity against Cl. difficile (MIC90 < or = 0.06 micrograms/ml for SC 44914, and 0.5 micrograms/ml for SC-44942A).

Evaluation of an outbreak of foodborne illness initiated in the emergency department.

STUDY OBJECTIVES: For many foodborne outbreaks, the pathogen and food vehicle never are identified. Delayed collection of epidemiologic and microbiologic information may contribute to this. We postulated that collection of this information from ill persons as they presented to the emergency department during a recent outbreak might contribute to earlier identification of the pathogen and vehicle. DESIGN: At least 690 of 1,900 conventioneers developed gastrointestinal symptoms after attending a banquet. A questionnaire was developed to collect information on specific food histories, incubation periods, symptoms, physical findings, and demographics. These results were compared with results of investigations by the city and state departments of public health. SETTING: The ED of Rush-Presbyterian-St Luke's Medical Center, a tertiary care university hospital in Chicago, Illinois. TYPE OF PARTICIPANTS: Adults (24 men and nine women) presenting to the ED with gastrointestinal symptoms after eating a common meal. MEASUREMENTS AND MAIN RESULTS: The clinical syndrome suggested an invasive pathogen. Based on this, clinical microbiology laboratory procedures were modified (isolation plates were reviewed during the evening shift). This led to early identification of the first isolates (Salmonella enteritidis) from the outbreak. The questionnaire also narrowed the vehicle to one of two foods served. Investigations by the departments of public health subsequently identified one of these, bread pudding with a raw egg based-sauce, as the vehicle. CONCLUSION: Outbreak evaluations can begin in the ED or any other patient care facility. This evaluation need not always add significantly to the expenditure of time, manpower, or laboratory studies. The evaluation of even a small percentage of ill persons from a large outbreak may provide useful epidemiologic information and be particularly important in settings with limited public health resources.

Trafficking of wild-type and an endoproteolytic-site mutant of the mouse mammary tumor virus glycoprotein.

We have utilized a mouse mammary tumor virus (MMTV) glycoprotein gene containing a mutation in the endoproteolytic cleavage site to investigate the biological significance of processing, the structural requirements for and the events involved in the proteolytic maturation of MMTV. Using oligonucleotide-directed mutagenesis, the endoproteolytic cleavage site within the MMTV glycoprotein was mutated from Arg-Ala-Lys-Arg to Arg-Ala-Asn-Gln and both the wild-type and mutated genes were transfected and expressed in HTC rat hepatoma cells. Indirect immunofluorescence, steady state radiolabeling and pulse-chase kinetic experiments demonstrated that this mutated glycoprotein was transported to the cell surface with the same efficiency as the wild-type maturation products; however, proteolytic cleavage and fusogenic activity were almost completely abolished. Consistent with the lack of cleavage, the endoglycosidase H-resistant precursor, gp78, accumulated on the cell surface and in the extracellular environment. When HTC cells expressing the wild-type MMTV glycoprotein were treated with the Golgi mannosidase I and II inhibitors, deoxymannojirimycin and swainsonine respectively, the resulting endogycosidase H-sensitive glycoproteins were processed efficiently. Taken together, these results suggest that proteolytic processing of the MMTV glycoprotein most likely occurs in the trans Golgi or at a later step in the exocytic pathway and occurs after the formation of an endoglycosidase H-resistant, terminally sialylated intermediate. Moreover, the acquisition of endoglycosidase H-resistant oligosaccharides is not a prerequisite for recognition by the cellular proteases to generate the viral maturation products. Our evidence also suggests that the processing of the MMTV envelope glycoprotein is required for the functional exposure of the fusion domain which is involved in viral infectivity.

Glucocorticoid-regulated stability of a constitutively expressed mouse mammary tumor virus glycoprotein.

Glucocorticoids regulate the transport and processing of mouse mammary tumor virus (MMTV) glycoproteins in viral-infected HTC rat hepatoma cells. To begin to determine the role of cellular components involved in this steroid-mediated response, a constitutively expressed MMTV glycoprotein gene containing a mutation in the endoproteolytic cleavage site was used to simplify the viral maturation products. Expression of the uncleavable MMTV glycoprotein gene in transfected HTC rat hepatoma cells demonstrated that treatment with the synthetic glucocorticoid dexamethasone resulted in a 5-fold increase in the steady state level of the intracellular and cell surface MMTV glycoproteins. Under these conditions, dexamethasone did not alter the level of MMTV glycoprotein transcripts. Pulse-chase radiolabeling with [35S]methionine demonstrated that dexamethasone did not affect the apparent rate of MMTV glycoprotein translation, and an analysis of oligosaccharide side-chain structure by endoglycosidase-H digestion revealed that glucocorticoids did not alter the 45-min endoplasmic reticulum to Golgi transit time. Pulse-chase kinetic analysis of 4-h pulse-labeled cells revealed that the half-life of the mature glycosylated MMTV polyprotein, gp78, was 105 min in glucocorticoid-treated cells and 45 min in untreated cells. Taken together, our results suggest that glucocorticoids increase the stability of MMTV glycoproteins by a posttranslational mechanism and that this effect may be occurring relatively early in the exocytic pathway.

Multiplane fracture of the distal humerus.

A unique but complex intraarticular fracture of the distal humerus is described. The term multiplane fracture has been applied to highlight a fracture pattern that features not only the well recognized T fracture lines in the sagittal and horizontal planes but also a coronal fracture of the trochlea. The Herbert screw was used in five cases to achieve stable fixation of the purely articular coronal fracture. At a follow-up of 20 months, on average, all fractures healed with an acceptable functional result. No radiographic evidence of avascular necrosis was seen in the follow-up radiographs.