Recent progress in the discovery of macrocyclic compounds as potential anti-infective therapeutics.

Novel therapeutic strategies are urgently needed for the treatment of serious diseases caused by viral, bacterial and parasitic infections, because currently used drugs are facing the problem of rapidly emerging resistance. There is also an urgent need for agents that act on novel pathogen-specific targets, in order to expand the repertoire of possible therapies. The high throughput screening of diverse small molecule compound libraries has provided only a limited number of new lead series, and the number of compounds acting on novel targets is even smaller. Natural product screening has traditionally been very successful in the anti-infective area. Several successful drugs on the market as well as other compounds in clinical development are derived from natural products. Amongst these, many are macrocyclic compounds in the 1-2 kDa size range. This review will describe recent advances and novel drug discovery approaches in the anti-infective area, focusing on synthetic and natural macrocyclic compounds for which in vivo proof of concept has been established. The review will also highlight the Protein Epitope Mimetics (PEM) technology as a novel tool in the drug discovery process. Here the structures of naturally occurring antimicrobial and antiviral peptides and proteins are used as starting points to generate novel macrocyclic mimetics, which can be produced and optimized efficiently by combinatorial synthetic methods. Several recent examples highlight the great potential of the PEM approach in the discovery of new anti-infective agents.

Plasma membrane Ca2+-atpase isoforms 2b and 4b interact promiscuously and selectively with members of the membrane-associated guanylate kinase family of PDZ (PSD95/Dlg/ZO-1) domain-containing proteins.

Spatial and temporal regulation of intracellular Ca(2+) signaling depends on localized Ca(2+) microdomains containing the requisite molecular components for Ca(2+) influx, efflux, and signal transmission. Plasma membrane Ca(2+)-ATPase (PMCA) isoforms of the "b" splice type contain predicted PDZ (PSD95/Dlg/ZO-1) interaction domains. The COOH-terminal tail of PMCA2b isolated the membrane-associated guanylate kinase (MAGUK) protein SAP97/hDlg as a binding partner in a yeast two-hybrid screen. The related MAGUKs SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bound to the COOH-terminal tail of PMCA4b, whereas only the first three bound to the tail of PMCA2b. Coimmunoprecipitations confirmed the interaction selectivity between PMCA4b and SAP102 as opposed to the promiscuity of PMCA2b and 4b in interacting with other SAPs. Confocal immunofluorescence microscopy revealed the exclusive presence and colocalization of PMCA4b and SAP97 in the basolateral membrane of polarized Madin-Darby canine kidney epithelial cells. In hippocampal neurons, PMCA2b was abundant throughout the somatodendritic compartment and often extended into the neck and head of individual spines where it colocalized with SAP90/PSD95. These data show that PMCA "b" splice forms interact promiscuously but also with specificity with different members of the PSD95 family of SAPs. PMCA-SAP interactions may play a role in the recruitment and maintenance of the PMCA at specific membrane domains involved in local Ca(2+) regulation.

Plasma membrane Ca2+ ATPase isoform 4b binds to membrane-associated guanylate kinase (MAGUK) proteins via their PDZ (PSD-95/Dlg/ZO-1) domains.

Plasma membrane Ca2+ ATPases are P-type pumps important for intracellular Ca2+ homeostasis. The extreme C termini of alternatively spliced "b"-type Ca2+ pump isoforms resemble those of K+ channels and N-methyl-D-aspartate receptor subunits that interact with channel-clustering proteins of the membrane-associated guanylate kinase (MAGUK) family via PDZ domains. Yeast two-hybrid assays demonstrated strong interaction of Ca2+ pump 4b with the PDZ1 + 2 domains of several mammalian MAGUKs. Pump 4b and PSD-95 could be co-immunoprecipitated from COS-7 cells overexpressing these proteins. Surface plasmon resonance revealed that a C-terminal pump 4b peptide interacted with the PDZ1 + 2 domains of hDlg with nanomolar affinity (KD = 1.6 nM), whereas binding to PDZ3 was in the micromolar range (KD = 1.2 microM). In contrast, the corresponding C-terminal peptide of Ca2+ pump 2b interacted weakly with PDZ1 + 2 and not at all with PDZ3 of hDlg. Ca2+ pump 4b bound strongly to PDZ1 + 2 + 3 of hDlg on filter assays, whereas isoform 2b bound weakly, and the splice variants 2a and 4a failed to bind. Together, these data demonstrate a direct physical binding of Ca2+ pump isoform 4b to MAGUKs via their PDZ domains and reveal a novel role of alternative splicing within the family of plasma membrane Ca2+ pumps. Alternative splicing may dictate their specific interaction with PDZ domain-containing proteins, potentially influencing their localization and incorporation into functional multiprotein complexes at the plasma membrane.

mRNA expression of the four isoforms of the human plasma membrane Ca(2+)-ATPase in the human hippocampus.

Ca2+ dyshomeostasis is a contributing factor to the development and progression of neurodegenerative disease. Plasma membrane Ca(2+)-ATPases (PMCAs) are responsible for setting intracellular Ca2+ levels and may be involved in the dynamic processing of Ca2+ loads in normal and pathological conditions. In situ hybridization was employed to determine the expression pattern of the four human PMCA isoforms in the human hippocampus. PMCA1 and 3 mRNAs were weakly expressed throughout the hippocampal formation, whereas PMCA2 and 4 mRNA expression showed distinct regional differences, with increased levels in CA2 and the dentate gyrus. Differential expression of PMCA isoforms may reflect cellular differences in Ca(2+)-handling properties and provide a partial explanation for the differential susceptibility of hippocampal neurons to Ca(2+)-mediated cell death.

Application of a chimeric green fluorescent protein to study protein-protein interactions.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is an emerging tool to monitor gene expression in situ and in vivo. Because of its fluorescence properties, when GFP is fused in-frame to a specific protein of interest, various aspects of the behavior of this protein can be analyzed noninvasively. Here we describe a fusion between GFP and human calmodulin-like protein (CLP) and show that this protein retains fluorescence and known characteristics of CLP, including Ca(2+)-dependent interaction with phenyl-Sepharose and interaction with a specific cellular target protein. The results suggest a novel application for GFP fusion proteins in the rapid, nonradioactive detection of interacting proteins on gel overlays.

Primary structure of human plasma membrane Ca(2+)-ATPase isoform 3.

The complete coding sequence of the human plasma membrane calcium ATPase (PMCA) isoform 3 was determined from overlapping genomic and cDNA clones. The cDNAs for the two major alternative splice variants 3a (3CII) and 3b (3CI) code for proteins of 1173 and 1220 amino-acid residues, respectively, which show 98% identity with the corresponding rat isoforms. On a multiple human tissue Northern blot, a major PMCA3 transcript of about 7 kb was detected exclusively in the brain, demonstrating the highly restricted pattern of expression of this isoform to human neuronal tissues. With the elucidation of the human PMCA3 primary structure, complete sequence information is now available for the entire family of human PMCA isoforms.

The organization of the suprachiasmatic circadian pacemaker of the rat and its regulation by neurotransmitters and modulators.

The long-term goal of our research is to understand how cells of the suprachiasmatic nucleus (SCN) are organized to form a 24-hr biological clock, and what roles specific neurotransmitters and modulators play in timekeeping and resetting processes. We have been addressing these questions by assessing the pattern of spontaneous neuronal activity, using extracellular and whole-cell patch recording techniques in long-lived SCN brain slices from rats. We have observed that a robust pacemaker persists in the ventrolateral region of microdissected SCN, and have begun to define the electrophysiological properties of neurons in this region. Furthermore, we are investigating changing sensitivities of the SCN to resetting by exogenous neurotransmitters, such as glutamate, serotonin, and neuropeptide Y, across the circadian cycle. Our findings emphasize the complexity of organization and control of mammalian circadian timing.

An unusual complication of laparoscopic cholecystectomy.

Thus far, the morbidity and mortality of laparoscopic cholecystectomy compares favorably to that of open cholecystectomy. Neither procedure, however, is without complications. Laparoscopic cholecystectomy combines diagnostic laparoscopy as well as cholecystectomy. It is the purpose of this article to briefly review these procedures and report an interesting variation: penetration of small bowel as a result of an unusual variation of a Meckel's Diverticulum.

Femoral nerve dysfunction after retroperitoneal hemorrhage: pathophysiology revealed by computed tomography.

In three patients receiving anticoagulation therapy who developed retroperitoneal hemorrhage computed tomography (CT) clearly localized the resulting hematoma in each case. Three distinct syndromes are described. A hemorrhage within the iliacus muscle resulted in femoral nerve dysfunction. A large hemorrhage within the iliacus muscle which extended into the psoas muscle produced both femoral and obturator nerve dysfunction. A retroperitoneal hemorrhage extrinsic to both the iliacus and psoas muscles did not produce peripheral nerve dysfunction. The pathophysiology of peripheral nerve dysfunction in retroperitoneal hemorrhage is reviewed in detail.

Obturator hernia: report of a case and brief review of its status.

The only case of obturator hernia in over 230,000 admissions is discussed. In the review of the recent English literature, a total of 30 cases, including our own, was collected. Characteristics of the condition were analyzed. The median age of the patients was 67 years; the majority were females in a ratio of 9:1 and both sides were equally affected. This entity occurs with the signs and symptoms of small bowel obstruction and, in almost one half of the patients, the pathognomonic Howship-Romberg sign.

Postpartum hemorrhage into a large nonfunctioning islet cell tumor of the pancreas.

A case of postpartum hemorrhage into a nonfunctioning islet cell tumor of the pancreas is presented. The manifestations and surgical management of this tumor are discussed. The incidence and pathological significance of such a tumor are examined.