Guanylyl cyclase is a heat-stable enterotoxin receptor.

Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors. We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin. GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains. Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity. In addition, heat-stable enterotoxin from E. coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C. The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors. These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.     

Specificity, stability, and potency of monocyclic beta-lactam inhibitors of human leucocyte elastase.

Stable, potent, highly specific, time-dependent monocyclic beta-lactam inhibitors of human leucocyte elastase (HLE) are described. The heavily substituted beta-lactams are stable under physiological conditions including in the presence of enzymes of the digestive tract. The beta-lactams were unstable in base. At pH 11.3 and 37 degrees C they were hydrolyzed with half-lives of 1.5-2 h. Hydrolysis produced characteristic products including the substituent originally at C-4 of the lactam ring, a substituted urea, and products resulting from decarboxylation of the acid after ring opening. The most potent beta-lactam displayed only 2-fold less activity versus HLE than alpha 1PI, the natural proteinaceous inhibitor. The compounds were more potent against the human and primate PMN elastases than versus either the dog or rat enzymes. Differences in the structure-activity relationships of the human versus the rat enzymes suggest significant differences between these two functionally similar enzymes. The specificity of these compounds toward HLE versus porcine pancreatic elastase (PPE) is consistent with the differences in substrate specificity reported for these enzymes [Zimmerman & Ashe (1977) Biochim. Biophys. Acta 480, 241-245]. These differences suggest that the alkyl substitutions at C-3 of the lactam ring bind in the S1 specificity pocket of these enzymes. The dependence of the stereochemistry at C-4 suggests additional differences between HLE and PPE. Most of the compounds do not inhibit other esterases or human proteases. Weak, time-dependent inhibition of human cathepsin G and alpha-chymotrypsin by one compound suggested a binding mode to these enzymes that places the N-1 substitution in the S1 pocket.     

Haemophilia B (sixth edition): a database of point mutations and short additions and deletions.

The sixth edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1380 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on factor IX activity, factor IX antigen in circulation and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.     

Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein.

Splicing of pre-mRNAs occurs via a lariat intermediate in which an intronic adenosine, embedded within a branch point sequence, forms a 2'',5''-phosphodiester bond (RNA branch) with the 5'' end of the intron. How the branch point is recognized and activated remains largely unknown. Using site-specific photochemical cross-linking, we have identified two proteins that specifically interact with the branch point during the splicing reaction. U2AF65, an essential splicing factor that binds to the adjacent polypyrimidine tract, crosslinks to the branch point at the earliest stage of spliceosome formation in an ATP-independent manner. A novel 28-kDa protein, which is a constituent of the mature spliceosome, contacts the branch point after the first catalytic step. Our results indicate that the branch point is sequentially recognized by distinct splicing factors in the course of the splicing reaction.     

The molecular analysis ofbrown eye color mutations isolated from geographically discrete populations ofDrosophila melanogaster

A large proportion of spontaneous mutations inDrosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessivebrown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caughtDrosophila melanogaster females,bw mutations were isolated from seven separate geographic sites distributed among Japan, California, Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in thebw gene. The remainder are inseparable from wild-typebw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneousbw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.     

Technical considerations for assessing alterations in skeletal muscle sarcoplasmic reticulum Ca++-sequestration functionin vitro

A multiple measurement system for assessing sarcoplasmic reticulum (SR) Ca⁺⁺-ATPase activity and Ca⁺⁺-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle.In vitro measurements were performed on whole muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96±0.1 and 0.99±0.1 mg/g in WG and RG, respectively. The percent Ca⁺⁺-ATPase recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca⁺⁺-activated Ca⁺⁺-ATPase activity was not affected by quick freezing of HOM or CM, but basal ATPase was reduced (P<0.05) in frozen HOM (5.12±0.18–3.98±0.20 mole/g tissue/min in WG and from 5.39±0.20–4.48±0.24 mole/g tissue/min in RG). Ca⁺⁺-uptake was measured at a range of physiological free [Ca⁺⁺] using the Ca⁺⁺ fluorescent dye Indo-1. Maximum Ca⁺⁺-uptake rates when corrected for initial [Ca⁺⁺]_f were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca⁺⁺ was reduced (P<0.05) in quick frozen HOM (1.30±0.1–0.66±0.1 mole/g tissue/min in WG and 1.04±0.2–0.60±0.1 mole/g tissue/min in RG). Linear correlations between Ca⁺⁺-uptake and Ca⁺⁺-ATPase activity measured in the presence of the Ca⁺⁺ ionophore A23187 were r=+0.25, (P<0.05) and r=+0.74 (P<0.05) in HOM and CM preparations, respectively, and were not altered by freezing. The linear relationships between HOM and CM maximum Ca⁺⁺-uptake (r=+0.44, P<0.05) and between HOM and CM Ca⁺⁺-ATPase activity (r=+0.34, P<0.05) were also not altered by tissue freezing. These data suggest that alterations in maximal SR Ca⁺⁺-uptake function and maximal Ca⁺⁺-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage. (Mol Cell Biochem139, 41–52, 1994)     

In Memoriam

The Patient Self-Determination Act was implemented in December 1991. Before and after its implementation, we used a structured interview of 302 randomly selected patients to determine their awareness, understanding, and use of advance directives. Implementation of the Act did not have a major effect on these. Although more than 90% of patients were aware of the living will, only about a third selected the correct definition or the correct circumstances in which it applied, and less than 20% of patients had completed one. About a third of patients were aware of a Durable Power of Attorney for Health Care and chose the correct definition, and about half identified the correct circumstances in which it applies; less than 10% had completed such a document. Surprisingly, patients who said they had completed advance directives did not demonstrate better understanding of these documents. Our results indicate that many patients, including some who have completed advance directives, do not fully understand them. It may be unwise to regard these documents as carefully considered, compelling statements of patients'' preferences. Appropriate responses to our findings include increased public education, revising state statutes to bring them into congruence with public perception, and expanding the dialogue between physicians and patients.     

A method for α-helical integral membrane protein fold prediction

Integral membrane proteins (of the α-helical class) are of central importance in a wide variety of vital cellular functions. Despite considerable effort on methods to predict the location of the helices, little attention has been directed toward developing an automatic method to pack the helices together. In principle, the prediction of membrane proteins should be easier than the prediction of globular proteins: there is only one type of secondary structure and all helices pack with a common alignment across the membrane. This allows all possible structures to be represented on a simple lattice and exhaustively enumerated. Prediction success lies not in generating many possible folds but in recognizing which corresponds to the native. Our evaluation of each fold is based on how well the exposed surface predicted from a multiple sequence alignment fits its allocated position. Just as exposure to solvent in globular proteins can be predicted from sequence variation, so exposure to lipid can be recognized by variable-hydrophobic (variphobic) positions. Application to both bacteriorhodopsin and the eukaryotic rhodopsin/opsin families revealed that the angular size of the lipid-exposed faces must be predicted accurately to allow selection of the correct fold. With the inherent uncertainties in helix prediction and parameter choice, this accuracy could not be guaranteed but the correct fold was typically found in the top six candidates. Our method provides the first completely automatic method that can proceed from a scan of the protein sequence databanks to a predicted three-dimensional structure with no intervention required from the investigator. Within the limited domain of the seven helix bundle proteins, a good chance can be given of selecting the correct structure. However, the limited number of sequences available with a corresponding known structure makes further characterization of the method difficult. © 1994 John Wiley & Sons, Inc.     

Cell-substrate adhesion and metastatic potential of cultured mesothelioma cells induced by asbestos

Cell-substrate adhesion was quantified for two cultured mesothelioma cell lines (epitheliomatus and sarcomatous) on glass, fibronectin and laminin substrates. Interference reflection microscopy (IRM) was used to image the adhesion patterns of cells and a grey level analysis was employed to quantify adhesion. Sarcomatous cells demonstrated marked adhesion to glass and fibronectin-coated substrates but not to laminin-coated substrate, with the greatest adhesion occurring on the fibronectin-coated surface. This adhesion was accompanied by cytoplasmic spreading. By contrast, epitheliomatous cells showed little tendency to adhere to any of the substrates and only showed significant spreading when in contact with the laminin substrate (P < 0.01). A bioassay was used to determine the metastatic potential of each of the cell lines. Via the intravenous route, the sarcomatous cells killed the host rats in 24.7 ± 1.5 (S.D.) days compared to 27.3 ± 0.9 (S.D.) days for the epitheliomatous cells (P < 0.01). After subcutaneous inoculation of tumour cells, the sarcomatous cells killed the host rats in 54.7 ± 0.7 (S.D.) days compared to 48.5 ± 0.5 (S.D.) days for the epitheliomatous cells (P < 0.01). We conclude that the results of the metastasis bioassays were consistent with the predicted behavior of these cell lines based on their ability to adhere to substrates in the in vitro adhesion assays.