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Previous research has indicated that patients with semantic short-term memory (STM) deficits demonstrate unusual intrusions of previously presented material during serial recall tasks (Martin and Lesch, 1996). These intrusions suggest excessive proactive interference (PI) from previous lists. Here, we explore one such patient's susceptibility to PI. Experiment 1 demonstrated patient M.L.'s extreme susceptibility to PI using a probe recognition task that manipulates the recency of negative probes (the recent negatives task). When stimuli consisted of letters, M.L. showed greatly exaggerated effects of PI, well outside of the range of healthy control participants. Experiment 2 used a variation of the recent negatives task to examine the relative contribution of semantic and phonological relatedness in PI. This task manipulated semantic and phonological relatedness of probes and recently presented list items. Relative to healthy control participants, patient M.L. showed exaggerated interference effects for both phonological and semantically related probes, both for probes related to the current list and for probes related to the previous list. These data have important implications for theories of semantic STM deficits. Specifically, these data suggest that it is not the rapid decay of semantic representations that is responsible for difficulties in short-term recall, but rather the abnormal persistence of previously presented material. We propose that this susceptibility to PI is the result of a deficit in control processes acting on STM.

Recent theories of working memory have emphasized the role of inhibition in suppressing irrelevant information. Moreover, psychometric studies have reported that several inhibition tasks with very diverse requirements load on a single inhibition factor. A patient with left inferior frontal damage, Patient M.L., previously reported to have a semantic short-term memory deficit (R. C. Martin & He, 2004), showed evidence of difficulty with inhibition on short-term memory tasks. We investigated whether he would show evidence of inhibition difficulty on two verbal tasks (a Stroop task and a recent-negatives task) and two nonverbal tasks (a nonverbal spatial Stroop task and an antisaccade task). M.L. was impaired on both verbal tasks but performed normally on the nonverbal tasks. M.L.'s data also represent a dissociation between Stroop and antisaccade performance, two tasks that load on a single factor in factor-analytic studies. The implications of these data for theories of inhibition and executive function are discussed.

We have recently described a biochemical detection method for peptide products of enzymatic reactions based on the formation of PDZ domain*peptide ligand complexes. The product sensor is based on using masked or cryptic PDZ domain peptide ligands as enzyme substrates. Upon enzymatic processing, a PDZ-binding motif is exposed, and the product sequence bound specifically by a Eu(3+)chelate-labeled GST-PDZ ([Eu(3+)]GST-PDZ). The practical applicability of this PDZ-based detection method is determined by the affinity of the PDZ domain*peptide ligand interaction, and the efficiency of the enzyme to process the masked peptide ligand. To expand the use of this PDZ-based detection strategy to a broader range of enzymatic assays, we have taken advantage of the plasticity in ligand recognition by the variety of PDZ domains found in nature. In the original work, the PDZ3 of PSD-95 was used, which preferentially recognizes the consensus sequence Ser-X-Val-COOH. Here, we show that NHERF PDZ1, which binds to the consensus sequence Thr/Ser-X-Leu-COOH, can be used to extend the flexibility in the recognition of the carboxy-terminal amino acid of the ligand, and monitor the enzymatic activity of HIV protease. The choices of detection format, for example, TRET or ALPHA, were also investigated and influenced assay design.

A time-resolved fluorescence resonance energy transfer (TR-FRET) detection method based on the formation of a PDZ domain.peptide ligand complex has been developed for enzymatic assays as an alternative to immuno-based detection strategies. The enzyme substrate is a "masked" biotinylated PDZ domain peptide ligand containing the consensus sequence Ser-X-Val-COOH. The critical residues in the binding consensus sequence of the ligand have been modified, for example, by phosphorylation of Ser or C-terminal extensions, providing binding-incompetent PDZ domain peptides. On processing by the corresponding enzyme, the binding epitope is exposed, and the product sequence is recognized specifically by Eu(3+) chelate-labeled GST-PDZ ([Eu(3+)]GST-PDZ) (GST-PDZ-glutathione S-transferase fused to PDZ domain). A ternary complex is subsequently formed by addition of allophycocyanin-labeled streptavidin ([XL665]SA), which binds to the biotinylated N terminus of the peptide, and detected by TR-FRET. Reported here are examples of the applicability of this detection strategy to three enzymatic systems, an endoprotease, an exoprotease, and a Ser/Thr phosphatase., (©2002 Elsevier Science (USA).)

Bcl-2 and Bcl-X(L) belong to a family of proteins overexpressed in a variety of human cancers which inhibit apoptosis in response to a number of stimuli including chemotherapeutic agents and ionizing radiation. To better understand the role of these polypeptides in modulating the response of cancer cells to ionizing radiation we used cell lines that were engineered to overexpress the two polypeptides. Although Bcl-2 and Bcl-X(L) overexpression resulted in inhibition of radiation-induced apoptosis, it did not result in enhanced clonogenic survival. Consistent with this was the observation that Bcl-2 and Bcl-X(L) protected cells from DNA fragmentation, loss of mitochondrial membrane potential, and caspase activation for up to 72 hours after irradiation. Beyond 72 hours, there was a rapid loss in the ability of Bcl-2 and Bcl-X(L) to inhibit these markers of apoptosis. When Bcl-X(L) was analyzed at 72 hours after irradiation and beyond, a rapid accumulation of a 16-kDa form of Bcl-X(L) was observed. To test the hypothesis that cleavage of the 29-kDa form of Bcl-X(L) by caspases to a 16-kDa polypeptide results in its inability to inhibit apoptosis beyond 72 hours, we constructed a cell line that overexpressed a caspase-resistant form of Bcl-X(L) (Bcl-X(L)-deltaloop). Cells overexpressing Bcl-X(L)-deltaloop were resistant to apoptosis beyond 72 hours after irradiation and did not contain the 16-kDa form at these time points. In addition, Bcl-X(L)-deltaloop overexpression resulted in enhanced clonogenic survival compared with control or Bcl-X(L) overexpressing cells. These results provide a molecular basis for the observation that expression of Bcl-2 or Bcl-X(L) is not a prognostic marker of tumor response to cancer therapy.

Advocates for individuals with severe and long-term physical disabilities have lobbied aggressively in the U.S. and Canada for the implementation of Independent Provider Care (IPC). In this model of home support service delivery, the client assumes sole responsibility for the management and scheduling of his or her personal care and home support. This article reports on the findings of a Canadian survey that assessed case managers' beliefs regarding the efficacy, benefits, and risks to clients of an IPC program. Survey results indicated that case managers generally believed that IPC would not significantly improve their clients' well-being and quality of care, would pose risks of abuse to both the client and support attendant, and would result in greater job demands for case managers. Possible sources of their apprehension are identified, and measures that may help to alleviate their concerns are discussed.

The Salmonella typhimurium LT2 sialidase (neuraminidase, EC 3.2.1.18) structural gene, nanH, has been cloned and sialidase overproduced from multicopy plasmids in Escherichia coli. Sialidase expression was regulated positively by cAMP. In contrast, certain Tn1000 insertions located upstream of nanH coding sequences reduced sialidase activity. A nanH chromosomal insertion mutation constructed by marker exchange demonstrated a single sialidase gene copy in S. typhimurium LT2. The complete nucleotide sequence of nanH, encoding a 41,300 dalton polypeptide, was determined and the derived primary structure was similar to sialidases from Clostridium perfringens, Clostridium sordellii, Bacteroides fragilis, and Trypanosoma cruzi. Comparative sequence analysis, including codon usage and secondary structure predictions, indicated that the S. typhimurium and clostridial sialidases are homologous, strongly suggestive of an interspecies gene transfer event. At least two primary sequence motifs of the bacterial enzymes were detected in influenza A virus sialidases. The predicted secondary structure of the bacterial enzymes was strikingly similar to viral sialidase. From the population distribution of nanH detected within a collection of salmonellae, it was apparent that S. typhimurium obtained its nanH copy most recently from Salmonella arizonae. S. typhimurium LT2 is thus a genetic mosaic that differs from other strains of even the same serotype by nanH plus potentially additional characters linked to nanH. These results have relevance to the evolution and function of sialidases in pathogenic microbes, and to the origin of the sialic acids.