Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy.

A method is described for the cryofixation of biological specimens for ultrastructural analysis and immunocytochemical detection studies. The method employs plunge freezing of specimens in a sealed capillary tube into a cryogen such as liquid propane or liquid nitrogen. Using this method a number of single-cell test specimens were well preserved. Also multicellular organisms, such as Caenorhabditis elegans, could be frozen adequately in low ionic strength media or even in water. The preservation of these unprotected specimens is comparable to that achieved with high-pressure freezing in the presence of cryoprotectant. The results are explained by the fact that cooling of water in a confined space below the melting point gives rise to pressure build-up, which may originate from the conversion of a fraction of the water content into low-density hexagonal ice and/or expansion of water during supercooling. Calculations indicate the pressure may be similar in magnitude to that applied in high-pressure freezing. Because the specimens are plunge cooled, suitable cryogens are not limited to liquid nitrogen. It is shown that a range of cryogens and cryogen temperatures can be used successfully. Because the pressure is generated inside the specimen holders as a result of the cooling rather than applied from an external source as in high-pressure freezing, the technique has been referred to as self-pressurized rapid freezing.

GeneSeeker: extraction and integration of human disease-related information from web-based genetic databases.

The identification of genes underlying human genetic disorders requires the combination of data related to cytogenetic localization, phenotypes and expression patterns, to generate a list of candidate genes. In the field of human genetics, it is normal to perform this combination analysis by hand. We report on GeneSeeker (http://www.cmbi.ru.nl/GeneSeeker/), a web server that gathers and combines data from a series of databases. All database searches are performed via the web interfaces provided with the original databases, guaranteeing that the most recent data are queried, and obviating data warehousing. GeneSeeker makes the same selection of candidate genes as the human geneticists would have performed, and thus reducing the time-consuming process to a few minutes. GeneSeeker is particularly well suited for syndromes in which the disease gene displays altered expression patterns in the affected tissue(s).

Ethylenediamine as active site probe for Na+/K+-ATPase.

(1) Ethylenediamine is an inhibitor of Na+- and K+-activated processes of Na+/K+-ATPase, i.e. the overall Na+/K+-ATPase activity, Na+-activated ATPase and K+-activated phosphatase activity, the Na+-activated phosphorylation and the Na+-free (amino-buffer associated) phosphorylation. (2) The I50 values (I50 is the concentration of inhibitor that half-maximally inhibits) increase with the concentration of the activating cations and the half-maximally activating cation concentrations (Km values) increase with the inhibitor concentration. (3) Ethylenediamine is competitive with Na+ in Na+-activated phosphorylation and with the amino-buffer (triallylamine) in Na+-free phosphorylation. Significant, though probably indirect, effects can also be noted on the affinity for Mg2+ and ATP, but these cannot account for the inhibition. (4) Inhibition parallels the dual protonated or positively charged ethylenediamine concentration (charge distance 3.7 A). (5) Direct investigation of interaction with activating cations (Na+, K+, Mg+, triallylamine) has been made via binding studies. All these cations drive ethylenediamine from the enzyme, but K+ and Mg+ with the highest efficiency and specificity. Ethylenediamine binding is ouabain-insensitive, however. (6) Ethylenediamine neither inhibits the transition to the phosphorylation enzyme conformation, nor does it affect the rate of dephosphorylation. Hence, we provisionally conclude that ethylenediamine inhibits the phosphoryl transfer between the ATP binding and phosphorylation site through occupation of cation activation sites, which are 3-4 A apart.

Ultrastructural localization of a peroxisomal protein in rat liver using the specific antibody against the non-specific lipid transfer protein (sterol carrier protein 2).

An antibody against the non-specific lipid transfer protein from rat liver was purified by immunoabsorbent affinity chromatography. This antibody in conjunction with protein A-colloidal gold was used to localize the transfer protein in rat liver by electron microscopy. Labeling by this immunocytochemical technique was found to be mainly restricted to the peroxisomes; low labeling was observed in the cytoplasm. Subsequent analysis of isolated peroxisomes by immunoblotting indicated that the non-specific lipid transfer protein (mol. wt. 14800) was absent from this organelle and that a protein of molecular weight 58000 was responsible for the immunological response. Immunoblotting of the membrane-free cytosol showed the presence of both proteins. It remains to be established to what extent the non-specific lipid transfer protein in the cytosol and the high-molecular weight protein in the peroxisomes are related.

Cloning and sequence analysis of a hypothalamic cDNA encoding a D1c dopamine receptor in tilapia.

Physiological and pharmacological studies have indicated that during acid stress a D1-like dopamine receptor becomes functional on intermediate pituitary melanocyte-stimulating hormone cells of tilapia (Oreochromis mossambicus). As a first step towards physiological expression studies we isolated a D1-like dopamine receptor from a tilapia hypothalamus cDNA library. Construction of a phylogenetic tree of most of the D1-like receptors known in human, rat, Xenopus, goldfish and Drosophila revealed that the here presented clone is most likely the tilapia equivalent of the Xenopus D1c dopamine receptor.

Contractile adaptations preserving cardiac output predispose the hypertrophied canine heart to delayed afterdepolarization-dependent ventricular arrhythmias.

BACKGROUND: In dogs, chronic complete atrioventricular block (CAVB) results in structural (biventricular hypertrophy) and electrical (delayed repolarization) remodeling, which predisposes the heart to torsade de pointes arrhythmias. We assessed the contractile alterations in the CAVB dog and tested the hypothesis that these adaptations increase delayed afterdepolarization (DAD)-dependent triggered arrhythmias. METHODS AND RESULTS: Steady-state and dynamic (fast pacing: 1 to 68 stimuli) left and right ventricular systolic and diastolic parameters were determined by positive and negative inotropic interventions at acute AVB and CAVB. Concomitantly, left and right ventricular endocardial monophasic action potentials were registered. In CAVB, all systolic contractile parameters were markedly increased, resulting in preserved cardiac output. The increase was most pronounced at low heart rates, altering the force-frequency response. At both acute AVB and CAVB, the degree of potentiation of cardiac function with pacing was dependent on the number of stimuli and showed a maximum at 8 to 13 stimuli. With CAVB, this potentiation curve was shifted upward, and it was only then that pacing resulted in DADs (in 8 of 10 dogs) and ectopic beats (EBs, in 6 of 10 dogs). The incidence of EBs in relation to the number of stimuli also had a maximum at 8 to 13 stimuli. Ouabain increased the incidence of DADs and EBs, whereas the negative inotropic interventions prevented them completely. CONCLUSIONS: The alterations responsible for improvement in systolic contractile function in CAVB dogs predispose the hypertrophied heart to DAD-dependent triggered arrhythmias during positive inotropic interventions.

Downregulation of delayed rectifier K(+) currents in dogs with chronic complete atrioventricular block and acquired torsades de pointes.

BACKGROUND: Acquired QT prolongation enhances the susceptibility to torsades de pointes (TdP). Clinical and experimental studies indicate ventricular action potential prolongation, increased regional dispersion of repolarization, and early afterdepolarizations as underlying factors. We examined whether K(+)-current alterations contribute to these proarrhythmic responses in an animal model of TdP: the dog with chronic complete atrioventricular block (AVB) and biventricular hypertrophy. METHODS AND RESULTS: The whole-cell K(+) currents I(TO1), I(K1), I(Kr), and I(Ks) were recorded in left (LV) and right (RV) ventricular midmyocardial cells from dogs with 9+/-1 weeks of AVB and controls with sinus rhythm. I(TO1) density and kinetics and I(K1) outward current were not different between chronic AVB and control cells. I(Kr) had a similar voltage dependence of activation and time course of deactivation in chronic AVB and control. I(Kr) density was similar in LV myocytes but smaller in RV myocytes (-45%) of chronic AVB versus control. For I(Ks), voltage-dependence of activation and time course of deactivation were similar in chronic AVB and control. However, I(Ks) densities of LV (-50%) and RV (-55%) cells were significantly lower in chronic AVB than control. CONCLUSIONS: Significant downregulation of delayed rectifier K(+) current occurs in both ventricles of the dog with chronic AVB. Acquired TdP in this animal model with biventricular hypertrophy is thus related to intrinsic repolarization defects.

Chronic amiodarone evokes no torsade de pointes arrhythmias despite QT lengthening in an animal model of acquired long-QT syndrome.

BACKGROUND: Amiodarone is an effective antiarrhythmic drug rarely associated with torsade de pointes arrhythmias (TdP). The noniodinated compound dronedarone could resemble amiodarone and be devoid of the adverse effects. In the dog with chronic complete atrioventricular (AV) block (CAVB) and acquired long-QT syndrome, the electrophysiological and proarrhythmic properties of the drugs were compared after 4 weeks of oral treatment. METHODS AND RESULTS: Amiodarone (n=7, 40 mg. kg(-1). d(-1)) and dronedarone (n=8, 20 mg/kg BID) were started at 6 weeks of CAVB (baseline). Six dogs served as controls. Surface ECGs and endocardially placed monophasic action potential catheters in the left (LV) and right (RV) ventricles were recorded to assess QTc time, action potential duration (APD), interventricular dispersion (DeltaAPD=LV APD minus RV APD), early afterdepolarizations (EADs), ectopic beats, and TdP. Both amiodarone (+21%) and dronedarone (+31%) increased QTc time. Amiodarone showed no increase in DeltaAPD in 4 of 7 dogs, whereas dronedarone augmented DeltaAPD in 7 of 8 animals. After dronedarone, TdP occurred in 4 of 8 dogs with the highest DeltaAPD (105+/-20 ms). TdP was never seen with amiodarone, not even in the dogs that had DeltaAPD values comparable to those with dronedarone. Furthermore, a difference existed in EADs and ectopic activity incidence (dronedarone 3 of 8; amiodarone 0 of 7), which was also seen during an epinephrine challenge. CONCLUSIONS: In the CAVB dog model, both amiodarone and dronedarone prolong QT time (class III effect). The absence of TdP with amiodarone seems to be related to homogeneous APD lengthening in the majority of dogs and the lack of EADs and/or ventricular ectopic beats in all.

Biogenesis of F71 and F72 fimbriae of uropathogenic Escherichia coli: influence of the FsoF and FstFG proteins and localization of the Fso/FstE protein.

The F71 and F71 P-fimbriae of Escherichia coli are encoded by the fso (F seven one) and fst (F seven two) gene clusters, respectively (Van Die et al., 1984; 1985). With the immunocytochemical gold-labelling technique it was demonstrated that both the FsoE and FstE proteins are non-adhesive minor fimbrial sub-units located at the tip of the fimbrial structure. The FsoF and FstFG proteins play an important role in the initiation of polymerization of the minor and major subunits into the fimbrial structure.

Electrophysiologic parameters and predisposing factors in the generation of drug-induced Torsade de Pointes arrhythmias.

When a new (cardiovascular) drug shows signs of QT interval prolongation on the ECG (delay in repolarization time), the regulatory agencies demand screening of its possible proarrhythmic potential before approving it for clinical practice. In this review, identified predisposing factors have been related to specific electrophysiological parameters, allowing quantification of their contribution to Torsade de Pointes arrhythmias. In addition, arrhythmogenic mechanisms involved in the initiation and perpetuation of drug-induced Torsade de Pointes are discussed.

Average orientation of aromatic residues in proteins determined from linear dichroism spectroscopy. A comparison of results on bovine gamma-crystallins with X-ray data.

The structures of the two very closely related proteins, bovine gamma II- and gamma IVa-crystallin have been studied by means of near-ultra-violet linear dichroism spectroscopy on squeezed polyacrylamide gel systems. The crystallin spectra are discussed in terms of the spectra of the aromatic chromophores present in these proteins and provide detailed information on the average orientation of these residues in the proteins. A comparison of our results with information based on crystallographic X-ray experiments shows excellent agreement, reflecting even some of the minor differences between the two proteins studied. Since linear dichroism measurements as performed here take a few days only, and can be done on most aqueous protein solutions, linear dichroism spectroscopy may give a valuable contribution to structural studies on proteins.

Periplasmic accumulation of truncated forms of outer-membrane PhoE protein of Escherichia coli K-12.

In order to localize the information within PhoE protein of Escherichia coli K-12 required for export of the protein to the outer membrane, we have generated deletions throughout the phoE gene. Immunocytochemical labelling on ultrathin cryosections revealed that the polypeptides encoded by the mutant alleles are transported to, and accumulate in, the periplasm. These results show that, except for the signal sequence, there is no specific sequence within the PhoE protein that is essential for transport through the cytoplasmic membrane. The overall structure of the protein, rather than a particular sequence of amino acids, seems to be important for assembly into the outer membrane.

R56865, an antiarrhythmic drug with class III effects that terminates ouabain induced ventricular tachycardia in an inverse rate dependent manner.

OBJECTIVE: R56865 has been described as a substance that protects cells from intracellular Na+ and Ca2+ overload. The aim of this study was to investigate its mechanism of action, which is at present unknown. METHODS: The haemodynamic and (rate dependent) electrophysiological effects of R56865 (0.48 mg.kg-1) were examined and compared with its antiarrhythmic effect on ouabain-induced ventricular tachycardia (n = 10), and ventricular tachycardia occurring within 24 h of occlusion of the left anterior descending artery (n = 8). The experiments were all performed in dogs. RESULTS: In anaesthetised dogs R56865 increased (p < 0.05) the cycle length of the sinus rhythm, the corrected QT duration (+8%) and the effective refractory period (+16%) of the right ventricle. No rate dependency was found. R56865 had no effect on blood pressure, conduction, or refractoriness of the AV node, nor on conduction in the ventricle. In conscious dogs, R56865 did not change the cycle length of the sinus rhythm, but it did increase the QT duration (+5%, p < 0.05). The cycle length of the slower ouabain induced ventricular tachycardias which were terminated by R56865 increased to a greater extent (+55%) than that of the non-suppressible, faster ventricular tachycardias (+16%): 335(SD 30) ms, n = 5 v 285(10) ms, n = 5. The effect of R56865 on ventricular tachycardias 24 h after infarction was considered to be of minor antiarrhythmic importance. CONCLUSIONS: R56865 has (1) class III effects, (2) a partial effect in terminating ouabain induced ventricular tachycardias which is inverse rate dependent, and (3) a weak effect on ventricular tachycardias 24 h after infarction.

Observations on the onset of torsade de pointes arrhythmias in the acquired long QT syndrome.

OBJECTIVE: Premature ectopic beats may create a specific sequence of events (e.g. short-long-short) preceding Torsade de Pointes arrhythmias (TdP) in the long QT syndrome. The relevance of this sequence for the initiation of TdP is not clear. In our dog model of TdP, interventricular dispersion (DeltaAPD=left-right ventricular monophasic action potential duration: APD) is associated with TdP, therefore we tested the hypothesis that the ectopic beats contributes to DeltaAPD. METHODS: In 17 anaesthetized dogs with chronic AV-block, which showed spontaneous TdP after class III medication, APD was analyzed to 1. quantitate the alterations due to (multiple) ectopic beats on the left and right APD (measured with endocardial catheters) and 2. compare the DeltaAPD prior to the occurrence of premature beats (steady state) in dogs with non-sudden onset of TdP (n=10) and sudden onset TdP (n=7). Three phases were distinguished: phase 1: steady state beats prior to ectopic beats, phase II: the beat(s) belonging to the dynamic phase, and phase III: the beat causing TdP. Because the coupling interval of premature beats in this condition often falls within the APD, the DeltaAPD(50) was validated as an alternative for the previously applied DeltaAPD(100) (r=0.51, P<0.01). RESULTS: In steady state (phase I) DeltaAPD(50) is longer in the sudden onset TdP (130+/-35 ms) as in the non-sudden onset TdP (65+/-40 ms). In the non-sudden TdP group the dynamic phase II contribute to the heterogeneity in APD, i.e. LV-APD increases more than RV-APD leading to a DeltaAPD(50) increase to 130+/-100 ms (P<0.01) just preceding TdP (phase III). CONCLUSION: The synergism between ectopic beats (short-long-short sequence) and DeltaAPD create the circumstances for TdP initiation.

Evidence for limited within-person evolution of the V3 domain of the HIV-1 envelope in the amsterdam population.

OBJECTIVE: To study the development of the V3 region of the HIV-1 envelope over time, both within subjects and population-wide. METHODS: Direct V3 sequences were obtained from viral RNA from seroconversion samples of 138 individuals [32 intravenous drug users (IVDU), 106 homosexual men], as well as from 5-year follow-up samples of 45 of these individuals (11 IVDU, 34 homosexual men). RESULTS: The population-wide variation of the V3 region in both the seroconversion samples and the 5-year samples steadily increased over consecutive years and were of similar magnitude in each calendar year. The variation in the IVDU group was slightly lower (presumably lagging behind) than in the homosexual group, but also increased over time. The consensus sequence, representing the centre of the swarm of variants, remained almost stationary in 10 years of evolution. The V3 sequences from virions in serum collected 5 years after seroconversion still resembled those from the seroconversion sample, either in overall similarity or in specific (signature) amino acids. Seroconversion and late sequences from a donor-recipient pair were also very similar. CONCLUSIONS: The variation in V3 sequences from seroconversion samples is as large as that in 5-year follow-up samples from the same calendar year, suggesting that there is no strong selection for a particular V3 genotype at transmission. The HIV-1 subtype B quasispecies in a naive population appears to evolve through unbiased expansion around a stationary consensus sequence. Despite its large variability, the V3 region retains many of its individual characteristics after 5 years of infection. This indicates that the sampling moment (relative to the seroconversion data) will not greatly influence the results of phylogenetic analyses.

Subtilases: the superfamily of subtilisin-like serine proteases.

Subtilases are members of the clan (or superfamily) of subtilisin-like serine proteases. Over 200 subtilases are presently known, more than 170 of which with their complete amino acid sequence. In this update of our previous overview (Siezen RJ, de Vos WM, Leunissen JAM, Dijkstra BW, 1991, Protein Eng 4:719-731), details of more than 100 new subtilases discovered in the past five years are summarized, and amino acid sequences of their catalytic domains are compared in a multiple sequence alignment. Based on sequence homology, a subdivision into six families is proposed. Highly conserved residues of the catalytic domain are identified, as are large or unusual deletions and insertions. Predictions have been updated for Ca(2+)-binding sites, disulfide bonds, and substrate specificity, based on both sequence alignment and three-dimensional homology modeling.

Laminin binding and internalization by human and murine mammary gland cell lines in vitro.

We have studied the binding and internalization of Engelbreth-Holm-Swarm mouse sarcoma laminin labeled with colloidal gold (LN-G40) by human and murine mammary gland cell lines. Interactions between the LN-G40 probe and the cells spread on a glass coverslip were monitored with video-enhanced contrast microscopy (Nanovid). Transmission electron microscopy allowed the quantitation of the LN-G40 probe at various cellular locations. During the first 15 min, a homogeneous binding of LN-G40 probe to the cell surface was observed with all cell lines. This binding did not occur with gold particles that were not conjugated to laminin. Then, the LN-G40 probe began to cluster on the cell surface and was, during the following 20 h, internalized by pits that were not coated. In the cells, the LN-G40 probe sometimes showed saltatory movements along linear tracks. The LN-G40 probe was intracellularly found in vesicles, multivesicular bodies, cisternal structures, and lysosomes, suggesting the degradation of the internalized laminin. However, not all cell surface-bound LN-G40 probe was internalized after 20 h. Differences between the cell lines were quantitative, but no clear correlation could be made between migration of cells on laminin and internalization of laminin.

Visualization of epidermal growth factor receptor in cryosections of cultured A431 cells by immuno-gold labeling.

Cryo-ultramicrotomy in combination with immuno-gold labeling has been demonstrated to present a powerful tool in the visualization of extra- and intracellular located antigens. We have applied this method to localize epidermal growth factor (EGF) receptor in cultured A431 human epidermoid carcinoma cells. However, both the labeling efficiency, maintenance of antigenicity, and the recognizability of the ultrastructure in cryosections are highly dependent upon the fixation procedures. Using 125I-EGF or a consecutive labeling with a monoclonal anti EGF-receptor antibody, rabbit-anti-mouse antibody and 125I-protein A, it was shown that maintenance of antigenicity was optimal using 2% paraformaldehyde as a fixative, whereas under these conditions also the recognizability of ultrastructure was sufficient. After appropriate fixation and labeling, gold particles were observed associated with various regions of the plasma membrane, including coated pits, and with various types of vesicles, including coated vesicles, intracellular vesicular membranes, multi-vesicular bodies and lysosomes. The results indicate that this method allows a visualization of EGF-receptors and resolution of the EGF-receptor processing pathway at the electron microscopic level, independent of the internalization process of labeled ligands.

ProSearch: fast searching of protein sequences with regular expression patterns related to protein structure and function.

This report describes the implementation of ProSearch, a computer program that can efficiently search for motifs in protein sequences. ProSearch currently uses motifs that are contained in the PROSITE database, but user-developed patterns can easily be added to any search. ProSearch can generate a report identifying the patterns present in a given protein sequence, their locations and, if desired, a short description of the identified patterns. The program is written in AWK (a small interpreted computer language), which can run on all computer platforms commonly found in laboratories. ProSearch can search a 348-amino acid protein for 690 patterns in less than 5 s on a typical workstation.

A novel procedure for pre-embedding double immunogold-silver labeling at the ultrastructural level.

Pre-embedding double immunogold-silver labeling using two ultrasmall gold conjugates has not been attempted previously because a means of distinguishing labels by conjugates of identical sizes was lacking. This study investigated the feasibility of creating a particle size segregation between two ultrasmall gold conjugates through sequential immunogold incubations and silver enhancements. Two primary antibodies, mouse anti-synaptophysin and rabbit anti-glial fibrillary acidic protein (GFAP), were used in the model system. Differentiation of the double labeling was achieved by incubating with one ultrasmall gold conjugate, followed by silver enhancement, and then incubating with the second ultrasmall gold conjugate, followed by additional silver enhancement. This resulted in two groups of silver-enhanced particles: smaller particles enhanced once and larger particles enhanced twice. Electron microscopic examination revealed two readily distinguished populations of gold-silver particles within the appropriate structures, with very little size overlap. The quality of the ultrastructure permitted identification of most subcellular organelles. This procedure provides for the first time a pre-embedding immunogold-silver labeling protocol that allows the precise subcellular co-localization of multiple antigens.