In vivo function and membrane binding properties are correlated for Escherichia coli lamB signal peptides.

Wild-type and pseudorevertant signal peptides of the lamB gene product of Escherichia coli interact with lipid systems whereas a nonfunctional deletion mutant signal peptide does not. This conclusion is based on interaction of synthetic signal peptides with a lipid monolayer-water surface, conformational changes induced by presence of lipid vesicles in an aqueous solution of signal peptide, and capacities of the peptides to promote vesicle aggregation. Analysis of the signal sequences and previous conformational studies suggest that these lipid interaction properties may be attributable to the tendency of the functional signal peptides to adopt alpha-helical conformations. Although the possibility of direct interaction between the signal peptide and membrane lipids during protein secretion is controversial, the results suggest that conformationally related amphiphilicity and consequent membrane affinity of signal sequences are important for function in vivo.

To build a virus capsid. An equilibrium model of the self assembly of polyhedral protein complexes.

The capsids of spherical (icosahedral) viruses are constructed of multiples of 60 subunits. The question of how these polymers assemble is basic to understanding the viral life cycle. A formalism describing virus assembly as an equilibrium between coat protein subunits, assembly intermediates and intact virus is presented. This equilibrium model of virus assembly is consistent with experimental observations of virus assembly. At equilibrium, either intact virus or free subunits are dominant species, assembly intermediates are predicted to be found only in trace concentrations. The concentration of assembled virus at equilibrium is expected to be extremely concentration-dependent and resemble a highly cooperative reaction although the model does not explicitly include cooperativity. For statistical assembly of a polyhedron, a nucleus is not necessarily required and polymerization can proceed through a cascade of bimolecular reactions rather than a single higher order reaction. Thus, kinetics of assembly do not necessarily show the extreme concentration dependence typical of nucleated protein polymerization. Modest intersubunit interaction energies result in a very stable capsid; consequently, a small change in this interaction energy can result in a considerable change in the capsid-subunit equilibrium. Some possible effects of nucleation and protein-nucleic acid interactions on virus assembly and capsid morphology are considered.

An alpha-helical peptide model for electrostatic interactions of proteins with DNA. The N terminus of RecA.

A series of synthetic peptides have been studied as models for non-specific protein-DNA interactions. In an alpha-helical conformation, the charged amino acid residues of the N-terminal 24 residues of RecA protein are asymmetrically distributed; at neutral pH there is a +4 charge on one face of the helix and a -3 charge on the other face. Modeling suggests that the positive face of the helix can bind five DNA phosphate groups by electrostatic interactions. Circular dichroism (c.d.) spectra indicate that the analogous peptide, Rec24 (AIDENKQKALAAALGQIEKQFGKG-amide), is largely unstructured in water but becomes highly helical in the presence of DNA. Peptide titrations of fluorescent etheno-DNA confirm that the changes in the c.d. spectrum of the peptide are associated with binding, although a dependence of the c.d. signal on the degree of DNA saturation is observed, indicating that peptide can be bound in more than one conformation. At saturation the peptide binds to 5.0(+/- 0.5) DNA phosphate groups as predicted and the electrostatic nature of the binding is confirmed by a strong dependence on salt concentration. A "mutant" peptide where an acidic glutamate residue replaces an alanine on the basic face of the Rec24 helix exhibits weaker binding to single-stranded DNA, also consistent with the electrostatic nature of the proposed peptide-DNA interaction. Extending Rec24 by ten amino acid residues, where the additional residues do not participate in the helical motif, does not noticeably affect binding. Thus, we show experimentally that an asymmetric charge distribution on an alpha-helix can represent an important element for binding nucleic acids.

RecA protein self-assembly. II. Analytical equilibrium ultracentrifugation studies of the entropy-driven self-association of RecA.

We have investigated the self-association of RecA protein from Escherichia coli by equilibrium ultracentrifugation. Monomeric RecA (Mr = 37,842) was observed in reversible equilibrium with trimers, hexamers and dodecamers in the presence of 1.5 M-KCl, 5 mM-Hepes, 1 mM-EDTA, 2 mM-ATP (pH 7.0) at 1 degrees C. The equilibrium was strongly temperature-dependent, with polymerization being favored as the temperature was raised from 1 degrees C 21 degrees C, and was reversible with respect to temperature. The values of both the standard enthalpy and entropy of self-association were positive, indicating that it is an entropy-driven process under these conditions. In the absence of KCl, in 50 mM-citrate, 5 mM-ATP, 5% (v/v) glycerol (pH 6.0) at 4 degrees C, only small amounts of RecA monomer could be detected, while in 10 mM-Tris-acetate, 10% glycerol (pH 7.5) at 4 degrees C, the smallest species present in significant concentration appeared to be the trimer. The majority of the species observed had molecular weights between 228,000 and 456,000, suggesting dominant stoichiometries of six to 12 monomers per oligomer. At pH 6.0, in the absence of ATP, much larger oligomers containing at least 24 monomers also appeared to be present. The data are consistent with an equilibrium mixture of monomers, trimers, hexamers, dodecamers, 24-mers and higher oligomers, with the distribution of oligomers being dependent on solution conditions. Thermodynamic analysis indicates that these oligomeric species are in reversible equilibrium with each other. It is not certain whether trimers assemble directly into hexamers, or whether disassembly into monomers is a prerequisite for the formation of higher oligomers. The possible role of higher-order RecA oligomers in the formation of RecA nucleoprotein filaments is discussed.

RecA protein self-assembly. Multiple discrete aggregation states.

Light scattering, sedimentation and electron microscopy have been used to investigate the aggregation states of highly purified RecA protein in solution. We show that RecA protein will self-assemble into a discrete series of quaternary structures depending upon protein concentration, ionic environment, and nucleotide cofactors. In a stock solution at moderate concentration (10 to 50 microM) RecA protein exists as small particles approximately 4 nm in diameter, larger particles approximately 12 nm in diameter (most probably rings of RecA protein), 10 nm diameter rods varying from 50 to 200 nm in length, and finally as much larger bundles of rods. The addition of monovalent salt shifts the distribution of RecA protein between its various oligomeric states. Increasing protein concentration favors more highly aggregated structures. At a given protein concentration, addition of mM levels of MgCl2 promotes the rapid formation of rods and slow formation of bundles. Under conditions typical of in vitro strand exchange reactions, RecA protein was found to exist as a mixture of rods and 12 nm particles with relatively few monomers.

Hepatitis B virus capsid: localization of the putative immunodominant loop (residues 78 to 83) on the capsid surface, and implications for the distinction between c and e-antigens.

Hepatitis B virus capsid protein comprises a 149 residue "assembly" domain that polymerizes into icosahedral particles, and a 34 residue RNA-binding "protamine" domain. Recently, the capsid structure has been studied to resolutions below 10 A by cryo-electron microscopy, revealing much of its alpha-helical substructure and that it appears to have a novel fold for a capsid protein; however, the resolution is still too low for chain-tracing by conventional criteria. Aiming to establish a fiducial marker to aid in the process of chain-tracing, we have used cryo-microscopy to pinpoint the binding site of a monoclonal antibody that recognizes the peptide from residues 78 to 83. This epitope resides on the outer rim of the 30 A long spikes that protrude from the capsid shell. These spikes are four-helix bundles formed by the pairing of helix-turn-helix motifs from two subunits; by means of a tilting experiment, we have determined that this bundle is right-handed. Variants of the same protein present two clinically important and non-crossreactive antigens: core antigen (HBcAg), which appears early in infection as assembled capsids; and the sentinel e-antigen (HBeAg), a non-particulate form. Knowledge of the binding site of our anti-HBcAg antibody bears on the molecular basis of the distinction between the two antigens, which appears to reflect conformational differences between the assembled and unassembled states of the capsid protein dimer, in addition to epitope masking in capsids.

Renal pathologic findings associated with monoclonal gammopathies.

The myeloma kidney is characterized by casts in the distal and collecting tubules. The glomeruli are hardly affected unless amyloidosis is present. When the glomeruli are involved, the proteinuria is nonselective and, in some cases, the whole paraprotein is excreted in the urine. Nephrocalcinosis may be present and focal myeloma cell infiltration in the interstitium is a characteristic, but inconstant, finding. The nephrotic syndrome is extremely rare; if it exists, amyloidosis should be suspected. In contrast to multiple myeloma, the glomeruli are frequently involved in macroglobulinemia of Waldenstrom. Hyaline intracapillary deposits consisting of pure IgM are a characteristic finding as is infiltration of the kidney with lymphoid cells. No characteristic lesion of the kidney has been described in the heavy-chain diseases. Mixed cryoglobulinemia associated with an IgM paraprotein can produce glomerulonephritis that is due to the deposition in the glomeruli of an immune complex consisting of IgG, IgM, and complement.

IgA deficiency and autoimmune hemolytic disease.

A case of familial selective IgA deficiency associated with autoimmune hemolytic disease is reported that illustrates the therapeutic implications when these two entities coexist. Immunoglobulin screening is recommended for patients with AHD who require blood transfusions, in order to identify IgA-deficient patients who may have anti-IgA anaphylactic reactions.

The extracellular domain of immunodeficiency virus gp41 protein: expression in Escherichia coli, purification, and crystallization.

The env gene of SIV and HIV-1 encodes a single glycoprotein gp 160, which is processed to give a noncovalent complex of the soluble glycoprotein gp120 and the transmembrane glycoprotein gp41. The extracellular region (ectodomain), minus the N-terminal fusion peptide, of gp41 from HIV-1 (residues 27-154) and SIV (residues 27-149) have been expressed in Escherichia coli. These insoluble proteins were solubilized and subjected to a simple purification and folding scheme, which results in high yields of soluble protein. Purified proteins have a trimeric subunit composition and high alpha-helical content, consistent with the predicted coil-coil structure. SIV gp41 containing a double cysteine mutation was crystallized. The crystals are suitable for X-ray structure determination and, preliminary analysis, together with additional biochemical evidence, indicates that the gp41 trimer is arranged as a parallel bundle with threefold symmetry.

Shared motifs of the capsid proteins of hepadnaviruses and retroviruses suggest a common evolutionary origin.

The structure of the dimeric C-terminal domain of the HIV-1 capsid protein (CA), recently determined by X-ray crystallography (Gamble et al. (1997)), has a notable resemblance to the structure of the hepatitis B virus (HBV) capsid protein (Cp) dimer, previously determined by cryo-electron microscopy (Conway et al. (1997), Böttcher et al. (1997)). In both proteins, dimerization is effected by formation of a four-helix bundle, whereby each subunit contributes a helix-loop-helix and most of the interaction between subunits is mediated by one pair of helices. These are the first two observations of a motif that is common to the capsid proteins of two enveloped viruses and quite distinct from the eight-stranded anti-parallel beta-barrel found in most other virus capsid proteins solved to date (Harrison et al. (1996)). Motivated by the structural resemblance, we have examined retroviral and HBV capsid protein sequences and found weak but significant similarities between them. These similarities further support an evolutionary relationship between these two virus families of great medical importance -- the hepadnaviruses (e.g. HBV) and retroviruses (e.g. HIV).

Intraoperative detection of pulmonary thromboemboli with epicardial echocardiography.

We report a novel intraoperative use of epicardial echocardiography in detecting and guiding the removal of pulmonary arterial thromboemboli. We describe a patient with a right atrial thrombus that could not be visualized with intraoperative transesophageal echocardiography. Because we suspected acute pulmonary embolization, epicardial echocardiography was used to visualize the right and left pulmonary arteries. Pulmonary thromboemboli were identified, and pulmonary thromboembolectomy was successfully performed.

The efficacy of intraoperative internal intercostal nerve block during video-assisted thoracic surgery on postoperative pain.

BACKGROUND: Video-assisted thoracic surgery (VATS) is widely used for many thoracic surgical procedures. Post-operative pain is less after VATS than after conventional thoracic surgery, but is still significant. The objective of this study was to assess the efficacy of thoracoscopic, internal intercostal nerve block in alleviating immediate postoperative pain. METHODS: Thirty-two patients underwent VATS bilateral sympathectomy for the treatment of hyperhidrosis. The patients were randomly divided into two groups with similar demographic and preoperative physiologic parameters. Group A (n = 16) was submitted to thoracoscopic, internal intercostal nerve blocks performed at T2, T3, and T4 intercostal levels using 3 cc of 0.5% bupivacain in each intercostal space. The injections were performed bilaterally, immediately after the sympathectomy, through the same port. Group B (n = 16) underwent bilateral thoracic sympathectomy without the block. During the immediate postoperative period, heart rate, blood pressure, respiratory rate, pain score, and analgesic requirements were monitored every 30 minutes. RESULTS: No morbidity was recorded in association with the thoracoscopic, internal intercostal nerve block. The mean heart rates (77 +/- 6 vs 89 +/- 12 beats per minute, p < 0.001), respiratory rates (15 +/- 2 vs 18 +/- 3 respirations per minute, p < 0.01), pain score (1.9 +/- 0.6 vs 2.7 +/- 0.5, p < 0.01), and postoperative analgesic requirements (20 +/- 18 vs 50 +/- 21 mg pethidine HCL, p < 0.001) were significantly lower in group A. There was no significant difference in blood pressures. CONCLUSIONS: Thoracoscopic, internal intercostal nerve block with bupivacain 0.5% during VATS is safe and effectively reduced the immediate postoperative pain and analgesic requirements.

Innominate vein cannulation for venous drainage in minimally invasive aortic valve replacement.

Minimally invasive aortic valve or aortic root replacement may be carried out through a mini-hemisternotomy. Venous cannulation of the right atrium may be difficult, at best, and obstruct the limited operative field. We have carried out cannulation of the innominate vein with 25F or 27F thin-walled femoral venous cannulae in 20 patients. Transesophageal echocardiographic guidance is invaluable in safely passing the guidewire and subsequently the cannula into the right atrium. This approach results in an unobtrusive method of complete intrathoracic cannulation through a mini-hemisternotomy with the risks of femoral cannulation.

Monoclonal immunoglobulin disorders: a report of 154 cases.

One hundred and fifty-four patients with a monoclonal gammopathy, diagnosed between 1965-1974 in the Hadassah University Hospital are reviewed with special reference to the relative incidence of associated disorders. Most patients (63 per cent) had immunoproliferative disorders (multiple myeoloma, macroglobulinemia of Waldenstrom, chronic lymphocytic leukemia, and other non-Hodgkin lymphomata). A non-B-cell malignancy, either of blood-forming tissues or of epithelial origin, was found in 6.5 per cent. Miscellaneous nonmalignant diseases (chronic liver disease, diseases of known or suspected autoimmune origin, chronic infections, Gaucher's disease), which have been reported in the past in association with a monoclonal gammopathy, were diagnosed in 15 per cent of the patients in this series. Twelve per cent of the patients were either asymptomatic or had diseases not known to be associated with monoclonal gammopathies. Amyloidosis was diagnosed in 3.3 per cent of the patients.

Coexistent chronic myeloid leukemia and IgA monoclonal gammopathy: report of a case and review of the literature.

The coexistence of Philadelphia negative chronic granulocytic leukemia (CGL) of the neutrophilic type and IgA monoclonal gammopathy is reported in a patient. The possible relation between the two proliferative processes is discussed in the light of current knowledge on the pluripotent stem cell and the clonal origin of CGL.

Laboratory stigmata of connective tissue disorders in asymptomatic carriers of hepatitis B virus.

Sixty-nine asymptomatic HBsAg carriers have been studied for the presence of laboratory stigmata of connective tissue disorders. Anti-nuclear antibodies accompanied by a significant binding of anti-DNA were detected on one carrier only. In contrast, rheumatoid activity was detected in 10 out of 63 carriers, and lymphocytotoxins in 8 out of 33. Carriers had a significantly increased level of circulating immune complexes associated with mild hypocomplementemia. Since none of the carriers had any evidence of liver disease, it is probable that the immunologic abberations were induced by the persistent viral infection rather than liver injury. It seems that some asymptomatic carriers have an occult laboratory-wise, connective tissue disorder which, under yet unknown circumstances, gives rise to an overt immune complex disease.

Serum immunoglobulin levels in healthy adults of various ethnic groups in a rural family practice in Israel.

A study on the immunoglobulin levels of five ethnic groups in a rural population in Israel was carried out. The ethnic group comprised Yemenite, Cochin, Kurd, North African, and Ashkenazi Jews. Yemenites have a low level of IgA, Ashkenazis have a high IgM level, Cochins and North Africans have high levels of IgG and IgA, and Kurds show low IgM levels. Females have higher IgM levels than males. No positive correlation between immunoglobulin levels and age could be demonstrated. A connection between these levels and exogenous and endogenous factors in the various ethnic groups is discussed.