Direct interaction of the 170 kDa isoform of synaptojanin 1 with clathrin and with the clathrin adaptor AP-2.

Synaptojanin 1, a polyphosphoinositide phosphatase, is expressed as two major alternatively spliced isoforms of 145 kDa (SJ145) and 170 kDa (SJ170) [1] [2], which are thought to have pleiotropic roles in endocytosis, signaling and actin function [3] [4] [5]. SJ145 is highly enriched in nerve terminals where it participates in clathrin-dependent synaptic vesicle recycling [1] [5]. SJ170, which differs from SJ145 by the presence of a carboxy-terminal extension, is the predominant isoform in developing neurons and is expressed in a variety of tissues [2]. The carboxy-terminal domain unique to SJ170 was previously shown to bind Eps15 [6], a protein involved in receptor-mediated endocytosis. Here, we show that the same domain also binds clathrin and the clathrin adaptor AP-2. These interactions occur both in vitro and in vivo and are direct. Binding of AP-2 is mediated by the ear domain of its alpha-adaptin subunit and binding of clathrin by the amino-terminal domain of its heavy chain. Overexpression in chinese hamster ovary (CHO) cells of full-length SJ170 or its unique carboxy-terminal region caused mislocalization of Eps15, AP-2 and clathrin, as well as inhibition of clathrin-dependent transferrin uptake. These findings suggest a close association of SJ170 with the clathrin coat and provide new evidence for its physiological role in the regulation of clathrin coat dynamics.

The epsins define a family of proteins that interact with components of the clathrin coat and contain a new protein module.

Epsin (epsin 1) is an interacting partner for the EH domain-containing region of Eps15 and has been implicated in conjunction with Eps15 in clathrin-mediated endocytosis. We report here the characterization of a similar protein (epsin 2), which we have cloned from human and rat brain libraries. Epsin 1 and 2 are most similar in their NH(2)-terminal region, which represents a module (epsin NH(2) terminal homology domain, ENTH domain) found in a variety of other proteins of the data base. The multiple DPW motifs, typical of the central region of epsin 1, are only partially conserved in epsin 2. Both proteins, however, interact through this central region with the clathrin adaptor AP-2. In addition, we show here that both epsin 1 and 2 interact with clathrin. The three NPF motifs of the COOH-terminal region of epsin 1 are conserved in the corresponding region of epsin 2, consistent with the binding of both proteins to Eps15. Epsin 2, like epsin 1, is enriched in brain, is present in a brain-derived clathrin-coated vesicle fraction, is concentrated in the peri-Golgi region and at the cell periphery of transfected cells, and partially colocalizes with clathrin. High overexpression of green fluorescent protein-epsin 2 mislocalizes components of the clathrin coat and inhibits clathrin-mediated endocytosis. The epsins define a new protein family implicated in membrane dynamics at the cell surface.

The functional role of positively charged amino acid side chains in alpha-bungarotoxin revealed by site-directed mutagenesis of a His-tagged recombinant alpha-bungarotoxin.

A polyhistidine tag was added to the N-terminus of alpha-bungarotoxin (Bgtx) recombinantly expressed in E. coli. The His-tagged Bgtx was identical to native, venom-derived Bgtx in its apparent affinity for the nicotinic acetylcholine receptor (nAChR) in Torpedo electric organ membranes. Furthermore, in a physiological assay involving mouse muscle nAChR expressed in Xenopus oocytes, the His-tagged Bgtx was as effective as authentic Bgtx at blocking acetylcholine-evoked currents. Ala-substitution mutagenesis of His-tagged Bgtx was used to evaluate the functional contribution of Arg36, a residue that is invariant among all alpha-neurotoxins. Replacement with Ala resulted in a 90-fold decrease in the apparent affinity for the Torpedo nAChR and a corresponding 150-fold increase in the IC50 for block of heterologously expressed mouse muscle nAChR, demonstrating the critical importance of this positive charge for the binding and functional activity of a long alpha-neurotoxin. The observed decrease in affinity corresponds to a DeltaDeltaG of 2.7 kcal/mol and indicates that Arg36 makes a major contribution to complex formation. This finding is consistent with the proposal that Arg36 mimics the positive charge found on acetylcholine and directs the toxin to interact with receptor sites normally involved in acetylcholine recognition. In comparison, Ala-substitution of the highly conserved Lys26 resulted in only a 9-fold decrease in apparent affinity. Truncation of the His-tagged Bgtx following residue 67 produces a toxin lacking the seven C-terminal residues including the two positively charged residues Lys70 and Arg72. Truncation leads to a 7-fold decrease in apparent binding affinity.

Waldenström's macroglobulinemia resulting from localized gastric lymphoplasmacytoid lymphoma.

A patient with a high level serum monoclonal IgM lambda paraprotein (3,850 mg/dL) was found to have a mass infiltrating the gastric mucosa. Gastric biopsy with immunohistochemical stains showed a B-cell lymphoplasmacytoid infiltrate expressing IgM lambda, consistent with Waldenström's macroglobulinemia. The patient's response to gastric radiation indicated that the primary source of the macroglobulinemia was the stomach. This is an extremely rare presentation, with only six other reported cases of this entity with gastric involvement.

A longitudinal study of special-needs adoptive families.

This longitudinal study of special-needs adoptive families assesses change on several measures of adoption outcome. Outcomes were somewhat more negative at the second administration of the survey than at the initial administration, presumably because many of the study children had entered adolescence. On balance, however, the outcomes were still positive at the second administration. Findings support the need for enhanced postadoptive services.

Functional expression and site-directed mutagenesis of a synthetic gene for alpha-bungarotoxin.

In order to explore the structure-function relationships of the curare mimetic alpha-neurotoxins we have constructed and cloned a synthetic gene for Bungarus multicinctus alpha-bungarotoxin which is expressed in Escherichia coli. The recombinant alpha-bungarotoxin is expressed as a fusion protein with alpha-bungarotoxin linked to the COOH-terminal end of the T7 Gene 9-encoded coat protein. After treatment of the fusion protein with Factor Xa protease, a recombinant alpha-bungarotoxin is released that co-migrates with authentic alpha-bungarotoxin upon reverse-phase high performance liquid chromatography and ion-exchange chromatography. Final yields of active recombinant alpha-bungarotoxin were about 0.4 mg/liter of starting bacterial culture. The recombinant alpha-bungarotoxin contains 10 additional residues linked to the NH2-terminal Ile of the alpha-bungarotoxin sequence due apparently to the inaccessibility of the engineered cleavage site to Factor Xa. Nevertheless, the recombinant alpha-bungarotoxin is capable of binding to the nicotinic acetylcholine receptor with an apparent affinity that is only decreased approximately 1.7-fold from that of authentic alpha-bungarotoxin. Alanine substitution of a residue, Asp30, highly conserved among alpha-neurotoxins and previously suggested to play a key role in receptor recognition, resulted in a recombinant alpha-bungarotoxin whose receptor binding activity is indistinguishable from authentic alpha-bungarotoxin.

Adoption outcomes for children with handicaps.

About 800 families who had adopted children with special needs responded to a mailed survey. Most reported good adoption outcomes. The presence of a handicap--vision, hearing, or physical impairment, mental retardation, or serious medical condition--was not an important factor influencing outcome.

A descriptive study of abuse and neglect in out-of-home-placement.

Selected characteristics of 290 reports of physical abuse, sexual abuse and neglect in foster homes, group homes, residential treatment centers, and institutions are described. At each type of setting physical abuse reports were most common, and neglect reports were least common. Quantitative and qualitative methods demonstrate that a significant percentage of confirmed reports are of a serious nature. Injuries occurred most frequently because of physical abuse while sexual abuse reports were most likely to be confirmed. Prior allegations of abuse or neglect regarding the perpetrator were indicated in 27% of reports. Factors contributing to abuse and neglect and the role of a state institutional review team in developing a systematic approach to this problem are discussed.

Patterns of reported child abuse and neglect.

Confirmed reports of abuse and neglect logged in a large state registry file from 1977 to 1984 are analyzed. Boys tend to sustain more frequent and more serious injuries. Victims of male perpetrators tend to sustain more serious injuries. A modest same-sex perpetrator/victim pattern is revealed for physical abuse; males are more likely to physically abuse boys while females are more likely to physically abuse girls. Among younger victims (aged 12 and younger) boys outnumber girls in all reporting categories except sexual abuse. Among adolescent victims, female victims greatly outnumber male victims in all reporting categories. Similarities between adolescent physical abuse and spouse abuse are noted; males are are the predominant perpetrators and females the predominant victims. As expected, victim age emerged as a strong predictor of severity of physical abuse injury. Several other factors including social isolation, mental health problems, lower income, and unemployment of the father are identified as modest predictors of severity.

Correction techniques for a multilevel reflection scanner.

This paper discusses the fundamental problems and design solutions for a multilevel reflection scanner. The scanner consists of a computer, a cathode ray tube light source, a system for imaging the light on the scanned document, two photomultiplier tubes, and a system for digitizing the PMT signals. One PMT measures cathode ray tube radiance while the other intercepts reflected light from the document. The computer controls the intensity focus and position of the spot and processes the digitized photomultiplier tube signals. The three fundamental problems associated with this type of scanner are tonal distortion due to the placement of the PMTs, the differing reflection properties of papers, and PMT drift. Procedures for reducing the effects of these problems, utilizing a computer correction table, are discussed. In addition, a detailed analysis is made of the most serious problem of tonal distortion due to the geometry of the system, and the results of this analysis are compared with experimental results. Finally, a noncomputer associated scanner is described.