High rates of multiple antibiotic resistance in Streptococcus pneumoniae from healthy children living in isolated rural communities: association with cephalosporin use and intrafamilial transmission.

OBJECTIVE: Streptococcus pneumoniae is one of the most clinically significant pathogens with emerging antibiotic resistance. We performed a surveillance study in isolated rural populations of healthy children to estimate the prevalence of pneumococcal resistance and to contrast factors that predict pneumococcal carriage with those that specifically predict resistant pneumococcal carriage. METHODS: The study was conducted in 1998 in 2 rural communities in Utah. Families were recruited directly for participation through community canvassing. Surveillance nasopharyngeal cultures were obtained from children who were younger than 8 years. Antibiotic usage and information on other potential risk factors were obtained from questionnaires and local pharmacy records. Resistance was determined by testing isolates for susceptibility to penicillin, cefaclor, trimethoprim-sulfamethoxazole, erythromycin, ceftriaxone, and trovafloxacin. Selected resistant isolates were characterized further by serotyping, pulsed field gel electrophoresis, and Southern blot with DNA probes specific for the pneumococcal lytA gene and for antibiotic resistance genes. RESULTS: In April 1998, surveillance nasopharyngeal cultures were obtained from 368 children aged

Functional analysis of Streptococcus pneumoniae MurM reveals the region responsible for its specificity in the synthesis of branched cell wall peptides.

The recently identified murMN operon of Streptococcus pneumoniae encodes enzymes involved in the synthesis of branched structured muropeptides of the pneumococcal cell wall peptidoglycan. Its inactivation was shown to cause production of a peptidoglycan composed exclusively of linear muropeptides and a virtually complete loss of resistance in penicillin-resistant strains. The studies described in this communication follow up these observations in several directions. The substrate of the MurM-catalyzed reaction (addition of alanine or serine) was identified as the lipid-linked N-acetylglucosamine-muramyl pentapeptide. Different murM alleles from several penicillin-resistant S. pneumoniae strains, each with a characteristic branched peptide pattern, were cloned into pLS578, a pneumococcal plasmid capable of replicating in S. pneumoniae, and transformed into the penicillin-susceptible laboratory strain R36A. All transformants remained penicillin-susceptible; however, their cell wall composition changed in directions corresponding to the muropeptide pattern of the strain from which the murM allele was derived. This suggests that the muropeptide composition of the pneumococcal cell walls is determined by the particular murM allele carried by the cells. A 30-amino acid long sequence within the MurM protein was shown to be the main determinant of the specificity of the reaction (addition of alanine versus serine).

Penicillin-resistant Streptococcus pneumoniae in metropolitan New York hospitals: case control study and molecular typing of resistant isolates.

During the 4-month period from January to April, 1998, 476 patients with Streptococcus pneumoniae infections were detected in 12 metropolitan New York hospitals and 112 penicillin-resistant (PRP) isolates (24%) were identified in 11 institutions. A case control study of 100 patients with penicillin-resistant and susceptible pneumococci from four of the widely dispersed hospitals revealed a high incidence of underlying medical illnesses in adult patients (74%), a preponderance of patients with pneumonia (63%), and a majority of patients who had underlying risk factors for pneumonia or invasive disease (51%). In this limited case control study, no difference was noted between cases and controls regarding known risk factors for penicillin-resistant pneumococcal infections. The percentage of single-patient PRP isolates varied among individual hospitals but the mean percentages of PRP from the four participating University Medical Centers and seven community hospitals were similar: 26% and 22% respectively. By E-test, 60% and 26% were high-level penicillin and ceftriaxone resistant, respectively. Pulsed-field gel electrophoresis identified 26 chromosomal macrorestriction patterns among the 103 PRP isolates available for analysis, but almost half (50 isolates or 48%) of these belong to two drug-resistant internationally spread clones, SP(23)-1 and SP(9/14)-3, that were detected in all hospitals and were recovered from invasive and noninvasive sites in both children and adults.

The role of murMN operon in penicillin resistance and antibiotic tolerance of Streptococcus pneumoniae.

The recently identified murMN operon is essential for the production of branched-structured muropeptides in the cell wall and also for the expression of the resistant phenotype in penicillin-resistant strains of Streptococcus pneumoniae. The purpose of studies described in this communication was to understand better the role of murMN in penicillin resistance. Deletion of murM in the penicillin-resistant strain Pen6, which causes reduction in the penicillin MIC from 6.0 to 0.03 microg/ml, was successfully complemented to recover the original high level of penicillin resistance in transformants that received functional murM alleles cloned in plasmid pLS578. Inactivation of penicillin resistance was not accompanied by any detectable change in the low affinity or abnormal molecular size pattern of the penicillin-binding proteins (PBPs) nor in the mosaic sequence of PBP2X typical of resistant strain Pen6. Exposure of strain Pen6 with inactivated murM to 0.05 microg/ml of penicillin (i.e., a concentration more than 100 times below the MIC of the parental strain) initiated a phenotypic response typical of penicillin-susceptible strains of pneumococci: inhibition of growth followed by rapid and extensive loss of viability and lysis. Unexpectedly, inactivation of murMN also caused hypersensitivity to lysis by low concentrations of a variety of cell wall active antibiotics such as fosfomycin, D-cycloserine, and nisin, suggesting that the murMN operon may perform an important regulatory role in the control of the irreversible antimicrobial effects of cell wall inhibitors.

Distribution of the mosaic structured murM genes among natural populations of Streptococcus pneumoniae.

The presence and sequence variation of the murM gene were studied in a large collection (814 strains) of genetically diverse Streptococcus pneumoniae isolates, which included 27 different serogroups and both penicillin-resistant (423 isolates, 67 pulsed-field gel electrophoretic [PFGE] types) and intermediately penicillin-resistant (165 isolates, 66 PFGE types) and penicillin-susceptible (226 isolates, 135 PFGE types) strains. Diversity of the murM sequences was tested by hybridization with mainly two kinds of probes: one derived from the amplification of the nucleotide sequence between nucleotides 201 and 624 in the penicillin-susceptible laboratory strain R36A (murMA probe) and a second probe that amplified the comparable, highly divergent sequence in the penicillin-resistant strain Pen6 (murMB probe). The great majority of the strains (761 of 814), including both penicillin-susceptible and penicillin-resistant isolates, reacted exclusively with the murMA probe. A smaller group of penicillin-resistant strains (48 of 814 isolates) reacted only with the murMB DNA probe, and an additional 5 isolates reacted with both probes. High-pressure liquid chromatography analysis of the peptidoglycan of strains hybridizing with murMB showed that they invariably contained an increased proportion of branched peptides. Complete sequencing of murM from a group of penicillin-resistant isolates allowed the identification of a number of different murMB alleles that differed in the length and exact position of the divergent (Pen6 type) sequences within the particular murM. The close similarity of these divergent sequences in the various murM alleles suggests a possible common heterologous origin.

Prophage carriage as a molecular epidemiological marker in Streptococcus pneumoniae.

The great majority of clinical isolates of Streptococcus pneumoniae carry prophages that may be identified through their hybridization with a DNA probe specific for the pneumococcal lytA gene (M. Ramirez, E. Severina, and A. Tomasz, J. Bacteriol. 181:3618-3625, 1999). We now show that the lytA hybridization pattern of chromosomal SmaI digests is stable for a given strain during extensive serial culturing in the laboratory; the pattern is specific for the strain's clonal type, as defined by pulsed-field gel electrophoretis (PFGE) pattern, and variations in PFGE subtypes may be explained by changes in the number and chromosomal localization of this prophage(s). These observations indicate that the lytA hybridization pattern may be used as a molecular epidemiological marker that offers additional resolution of the genetic background of S. pneumoniae strains.

A high incidence of prophage carriage among natural isolates of Streptococcus pneumoniae.

The majority (591 of 791, or 76%) of Streptococcus pneumoniae clinical isolates examined showed the presence of two or more chromosomal SmaI fragments that hybridized with the lytA-specific DNA probe. Only one of these fragments, frequently having an approximate molecular size of 90 kb, was shown to carry the genetic determinant of the pneumococcal autolysin (N-acetylmuramic acid-L-alanine amidase). Strains carrying multiple copies of lytA homologues included both antibiotic-susceptible and -resistant isolates as well as a number of different serotypes and strains recovered from geographic sites on three continents. Mitomycin C treatment of strains carrying several lytA-hybridizing fragments caused the appearance of extrachromosomal DNA hybridizing to the lytA gene, followed by lysis of the bacteria. Such lysates contained phage particles detectable by electron microscopy. The findings suggest that the lytA-hybridizing fragments in excess of the host lytA represent components of pneumococcal bacteriophages. The high proportion of clinical isolates carrying multiple copies of lytA indicates the widespread occurrence of lysogeny, which may contribute to genetic variation in natural populations of pneumococci.

Molecular epidemiologic characterization of penicillin-resistant Streptococcus pneumoniae invasive pediatric isolates recovered in six Latin-American countries: an overview. PAHO/Rockefeller University Workshop. Pan American Health Organization.

The Pan American Health Organization (PAHO) has conducted a study of Streptococcus pneumoniae in six Latin-American countries: Argentina, Brazil, Chile, Colombia, Mexico, and Uruguay. Sterile site isolates from children aged < or =5 years showing clinical symptoms of pneumonia (as defined by the clinical criteria of WHO), meningitis, sepsis or bacteremia (without infectious foci), arthritis, and peritonitis were the source of most of the invasive pneumococcal isolates collected between the end of 1993 and 1996 in the six participating countries. Partial characterization of these isolates (antibiotic resistance and serotyping) have already been described (Microbial Drug Resistance 3:(2):131-163, 1997). In the next phase of the study, 326 S. pneumoniae isolates with reduced penicillin susceptibility were transferred to the Laboratory of Microbiology at The Rockefeller University for molecular characterization, and a summary and overview of the findings is described in this article. Some of the most interesting findings were as follows: (1) There was a surprisingly high representation of two internationally spread clones, which made up >80% of the strains with penicillin MIC of 1 microg/ml or higher; most of these isolates were recovered in large cities, supporting the likelihood that the source of these clones is through international travel. (2) The frequency of resistance to trimethoprim/sulfamethoxazole was extremely high (present in 85% of all isolates with decreased penicillin susceptibility). (3) None of these isolates was resistant to ofloxacin, and macrolide resistance was rare (present in 6.4% of the isolates). (4) There was an apparent inverse relationship between level of penicillin resistance and genetic diversity. (5) There were striking differences in the "microbiologic profiles" of the six different Latin-American countries.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in 12 New York hospitals. MRSA Collaborative Study Group.

Consecutive single-patient methicillin-resistant Staphylococcus aureus (MRSA) isolates (270) from 12 hospitals (8217 beds) in metropolitan New York City were collected during May 1996. In 11 of 12 hospitals, MRSA was most frequent in the general medical services. DNA typing ("fingerprinting") revealed that mecA:Tn554:PFGE (pulsed-field gel electrophoresis) type I:A:A accounted for 113 (42%) of 270 isolates, was detected in all hospitals, and was the predominant clone in 9. Thirteen of 15 I:E:F isolates were from 1 hospital, and the remaining 2 were from another hospital of the same health system. Type V:NH:E was isolated from 22 (79%) of the 28 patients with AIDS, including 8 of 9 patients from an additional hospital. Subtype V:NH:E2 was recovered from 11 patients, 9 of whom had AIDS, including all 5 AIDS patients from one floor of a nursing home affiliated with a third hospital. By using both mecA:Tn554 probes and PFGE, MRSA clusters and outbreaks may be detected and provide a rationale for appropriate infection control intervention.

Antibacterial efficacy of nisin against multidrug-resistant Gram-positive pathogens.

The rapid spread of multidrug-resistant bacterial pathogens necessitates the search for alternative antibacterial agents. We examined the efficacy of the antibiotic nisin against 56 multidrug-resistant isolates of Streptococcus pneumoniae, 33 Staphylococcus aureus and 29 vancomycin-resistant Enterococcus faecium and Enterococcus faecalis isolates. The test strains represented a large variety of clonal types (as determined by a combination of DNA fingerprints) isolated from a variety of geographic sources, and included some of the major internationally-spread multiresistant epidemic clones of S. pneumoniae and methicillin-resistant S. aureus (MRSA), MRSA strains resistant to over 16 generically distinct antibacterial agents, and enterococcal strains resistant to all currently available chemotherapeutic agents including glycopeptides. In the overwhelming majority of cases, treatment of growing cultures with nisin at 1 mg/L (S. pneumoniae) or 10-20 mg/L (in MRSA and enterococci) caused extensive (10(3)- to 10(4)-fold) loss of viable titre accompanied by various degrees of loss in the optical density of the cultures, which was most extensive in pneumococci (>90%) and least extensive (40-50%) in enterococci. Nevertheless, extensive variation in rates of nisin-induced autolysis was observed in each bacterial species. Serial exposure of a penicillin-susceptible strain of S. pneumoniae to nisin (1 mg/L) in liquid culture resulted in the rapid appearance of stable nisin-resistant mutants in which the MIC increased from 0.4 to 6.4 mg/L and the resistance trait was transferable by genetic transformation.

Molecular characterization of penicillin-resistant Streptococcus pneumoniae isolates causing respiratory disease in the United States.

Three hundred twenty-eight (328) penicillin-resistant Streptococcus pneumoniae isolates collected in 39 states of the United States between October, 1996, and March, 1997, from (mostly adult) patients with respiratory disease were characterized by microbiological, serological, and molecular fingerprinting techniques, including determination of chromosomal macrorestriction pattern with pulsed-field gel electrophoresis (PFGE) and hybridization with DNA probes specific for various antibiotic resistance genes. The overwhelming majority of the isolates were in five serogroups (23, 6, 19, 9, 14). All isolates had penicillin MIC values of at least 2 microg/ml, but the collection also included isolates with MIC values as high as 16 microg/ml. Virtually all isolates (96.6%) were resistant to trimethoprim/sulfamethoxazole (SXT) and many isolates were also resistant to chloramphenicol (43%), tetracycline (55%), and erythromycin (65%). Resistance to levofloxacin was extremely rare. The molecular fingerprinting methods showed that a surprisingly large proportion (167 out of 328, or 50.9%) of the isolates belonged to two international epidemic clones of S. pneumoniae: clone A (127, or 38.7%) with properties indistinguishable from that of the 23F multiresistant "Spanish/USA" clone widely spread in Europe, Asia, Latin America, and South Africa, and clone B (40, or 12.2%) belonging to the "French" serogroup 9/14 clone widely spread in Europe and South America. Virtually all members of clone A were also resistant to chloramphenicol (cat+), tetracycline (tetM+), and SXT, and about 75% were also resistant to erythromycin (mefE+ or ermB+). Close to 30% (39 out of 127) of the clone A isolates expressed anomalous serotypes (primarily serotypes 19 and 14, and nontypable) and most likely represented spontaneous capsular transformants. Most of the 40 isolates (35/40) belonging to clone B expressed serotype 9, with five of the isolates expressing serotypes 14 or 19, or were nontypable. All members of this clone were resistant to penicillin and SXT with only occasional isolates showing resistance to macrolides, tetracycline, and chloramphenicol. The combination of microbiological tests and DNA hybridizations also allowed the identification of unusual strains, for instance, isolates that reacted with the tetM or mefE DNA probes without showing phenotypic antibiotic resistance, an isolate showing phenotypic macrolide resistance without hybridizing with either the ermB or mefE DNA probes, or isolates that hybridized with both of these DNA probes. In addition to clones A and B, another large portion of the S. pneumoniae isolates (112 of 328, or 34.1%) was represented by eight clusters, each with a unique PFGE type. These clusters, together with the clone A and clone B isolates, made up 85% of all the penicillin-resistant isolates identified in this survey in the United States. Both international clones and the unique clusters showed wide geographic dispersal: Clone A was present in 30 of the 39 states and clone B in 18. The data suggest that the major mode of spread of penicillin-resistant pneumococci in the United States is by clonal expansion and that the most significant components (clones A and B) have been imported into the United States from abroad.

Abnormal physiological properties and altered cell wall composition in Streptococcus pneumoniae grown in the presence of clavulanic acid.

Subinhibitory concentrations of clavulanate caused premature induction of stationary-phase autolysis, sensitization to lysozyme, and reductions in the MICs of deoxycholate and penicillin for Streptococcus pneumoniae. In the range of clavulanate concentrations producing these effects, this beta-lactam compound was selectively bound to PBP 3. Cell walls isolated from pneumococci grown in the presence of clavulanate showed increased sensitivity to the hydrolytic action of purified pneumococcal autolysin in vitro. High-performance liquid chromatography analysis of the peptidoglycan isolated from the clavulanate-grown cells showed major qualitative and quantitative changes in stem peptide composition, the most striking feature of which was the accumulation of peptide species carrying intact D-alanyl-D-alanine residues at the carboxy termini. The altered biological and biochemical properties of the clavulanate-grown pneumococci appear to be the consequences of suppressed D,D-carboxypeptidase activity.

Testing the efficacy of a molecular surveillance network: methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF) genotypes in six hospitals in the metropolitan New York City area. The BARG Initiative Pilot Study Group. Bacterial Antibiotic Resistance Group.

Molecular fingerprinting techniques are rapidly becoming indispensable tools for hospital epidemiology. On the other hand, the relative complexity and unfamiliarity of these techniques to most hospital diagnostic laboratories limit their usefulness. In an attempt to provide a solution for this dilemma, we tested the feasibility and efficacy of a cooperative venture in which molecular typing of isolates recovered from patients in six hospitals was performed at two microbiology research laboratories with expertise in these techniques. In a small preliminary study, 30 methicillin-resistant Staphylococcus aureus (MRSA) and 30 vancomycin-resistant Enterococcus faecium (VREF) isolates were collected over a 3-week period from six hospitals in the metropolitan New York area and transported to the Laboratory of Microbiology at The Rockefeller University during the summer months of 1994. Nineteen of the 27 confirmed MRSA isolates were closely related strains carrying the same mecA and the same Tn554 polymorphs in a pulsed-field gel electrophoresis (PFGE) background represented by closely related subtypes of a single pattern, indicating the wide distribution of this MRSA clone among the participating hospitals. Typing of the same 27 MRSA isolates was also performed at the Tuberculosis Center of the Public Health Research Institute and identical results were obtained. The 29 confirmed VREF isolates were highly heterogeneous and belonged to as many as 23 distinct clonal types as defined by PFGE patterns and probing with vanA. Characterization of the 60 isolates by these methods was completed in one month of full-time effort by a single experienced laboratory assistant guided by a doctoral-level expert in molecular fingerprinting techniques. The collection of samples for both MRSA and VREF was not intended to address epidemiological questions but to determine the feasibility of a multicenter study. On the basis of our preliminary findings we are encouraged that a larger cooperative effort is possible and with the correct sampling method we believe that epidemiological and surveillance studies could be accomplished that would provide a tracking system to assist hospitals, clinics, and chronic care facilities in controlling the spread of multidrug-resistant pathogens.

Ubiquitous presence of a mecA homologue in natural isolates of Staphylococcus sciuri.

In an effort to explore the origin and/or reservoirs of the genetic determinant(s) of methicillin resistance in Staphylococcus aureus, we examined over 200 strains representing 13 different species within the genus Staphylococcus for the presence of the mecA gene, using a DNA probe internal to this gene prepared from a methicillin-resistant strain of S. aureus. Occasional mecA- positive isolates were detected among several staphylococcal species. On the other hand, each one of the 134 isolates of Staphylococcus sciuri, a species considered taxonomically the most primitive among staphylococci and found primarily on rodents and primitive mammals, gave positive reaction with the DNA probe when tested under conditions of high stringency. About two thirds (99) of these isolates, all of which belonged to S. sciuri subspecies "sciuri," as well as 9 of the 11 species carnaticum isolates, showed only marginal, if any, resistance to methicillin (minimal inhibitory concentration of 0.75-6.0 micrograms/ml), while most of the remaining isolates that belonged to the subspecies "rodentius" (13 isolates in all) expressed antibiotic resistance with a heterogeneous phenotype similar to those seen in many methicillin-resistance strains of S. aureus In SmaI digests of chromosomal DNA isolated from such "methicillin-resistant S. aureus-like" strains, the mecA probe hybridized with DNA fragments in the range of 145-180 kb, while in subspecies "sciuri" and carnaticum isolates the mecA hybridizing fragment was located in the SmaI fragment with the highest molecular size (> or = 400 kb). A DNA probe comprising an internal sequence to the regulatory gene mecI from Staphylococcus epidermidis identified the presence of sequences with low degree of homology in isolates of the three S. sciuri subspecies. The mecA-reacting sequences in these bacteria differed from mecA of S. aureus in several respects (e.g., by the absence of a ClaI restriction site from mecA of subspecies "sciuri" and carnaticum, and in some isolates of subspecies "rodentius." The uniform presence of mecA in each one of a large number of S. sciuri strains belonging to distinct ribotypes and macrorestriction patterns and recovered over a 20-year period from a wide variety of animal sources and geographic sites suggests that mecA may be a native genetic element with an as yet unidentified physiologic function in this staphylococcal species.

The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide.

The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1-60 microM and 50-370 microM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca2(+)-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca2(+)-binding affinity was 1.5-2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca2(+)-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca2(+)-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca2(+)-ATPase activity of permeabilized erythrocytes. At Ca2+ concentrations below 0.7 microM, ouabain (0.5-1 mM) activated whereas at higher Ca2+ concentrations it inhibited the Ca2(+)-ATPase activity. Taking this observation into account the Na+/K(+)-ATPase was determined as the difference of between the ATPase activities in the presence of Na+ and K+ and in the presence of K+ alone. At physiological concentration of Mg2+ (370 microM), the addition of 0.3-1 microM Ca2+ increased Na+/K(+)-ATPase activity by 1.5-3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg2+ concentration (e.g., 50 microM) only Na+/K(+)-ATPase inhibition by Ca2+ was seen. It was found that at [NaCl] less than 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca2(+)-free media. This activating effect of furosemide was enhanced with a diminution of [Na+] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na+/K(+)-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb+ influx into intact erythrocytes.

[Assessment of the role of endogenous regulators in the activation of Ca-ATPase in erythrocyte membranes]. / Otsenka roli éndogennykh reguliatorov v aktivatsii Ca-ATPazy membran éritrotsitov.

The contribution of calmodulin and protein kinases A or C to the activation of membrane Ca-ATPase was studied on saponin-permeabilized rat erythrocytes. In the presence of all endogenous regulators, the dependence of the Ca-ATPase activity of Ca2+ concentration was described by a bell-shaped curve with a maximum at 2-5 microM Ca2+; K0.5 = 0.43 microM Ca2+. Washing of erythrocyte membranes with 5-10 microM Ca2+ maintained up to 75% of the ATPase activity, while washing with EGTA (2 mM) decreased the activity, on the average, 5-fold, and increased K0.5 up to 0.54-0.6 microM Ca2+. An addition of an EGTA extract to washed membranes restored up to 75% of the original ATPase activity, while calmodulin restored about 40% of the original Ca-ATPase activity and decreased K0.5 to 0.23-0.3 microM Ca2+. The calmodulin inhibitor R24571 failed to alter the Ca-ATPase activity in permeabilized erythrocytes but slightly diminished it in reconstituted membranes. The protein kinase C inhibitors H7 and polymyxin increased the Ca-ATPase activity in permeabilized red cells and suppressed it in reconstituted membranes. The data obtained suggest that in native red cell membranes Ca-ATPase is activated by regulator(s) dependent on Ca2+ and protein kinase which are other than calmodulin.

Participation of endogenous fatty acids in Ca2+ release activation from mitochondria.

A correlation between the rate of H+/Ca2+ exchange and the content of free fatty acids in mitochondria has been found. Fatty acids were isolated from mitochondria with different activities of H+/Ca2+ exchange. It has been shown that these free fatty acids are able to induce Ca2+ release in exchange to protons after being added to freshly isolated mitochondria.

[Phospholipase A2 localization in mitochondria]. / Issledovanie lokalizatsii fosfolipazy A2 v mitokhondriiakh.

The activation of mitochondrial phospholipase A2 by Ca2+ and Sr2+ has been studied. It was shown that complete inactivation of the enzyme occurs after treatment of isolated mitochondria with ruthenium red, inhibitor of Ca transport across the inner mitochondrial membrane. It was concluded that phospholipase A2 is localized on the inner mitochondrial membrane and that its activity is stimulated by Ca2+ on the side of the mitochondrial matrix.

[Dynamics of intramembrane particles during proteolysis of certain membranes]. / Dinamika vnutrimembrannykh chastits pri proteolize nekotorykh membran.

Freeze-fracture electron microscopy has been used to study the ultrastructure of proteolytic enzymes treated of the bovine photoreceptor membranes, the rat liver microsome ghosts and the rabbit sarcoplasmic reticulum membranes. The observed increase in the intramembranous particle number in the inner fracture face suggests transmembrane dipping of amphipathic integral proteins affected by the partial proteolysis.

[Comparative study of membranes of Streptococcus faecalis and Micrococcus lysodeikticus]. / Sravnitel'noe izuchenie membran Streptococcus faecalis i Micrococcus lysodeikticus

A comparative study of ultrastructure and IR-spectroscopy of osmotic shock membranes from cells of glycolyzing (Streptococcus faecalis) and respiring (Micrococcus lysodeikticus) bacteria, was made. The S. faecalis and M. lysodeikticus membranes differ in their cross-section. Treatment of the preliminary washed membranes of S. faecalis and M. lysodeikticus with a low ionic strength solution removes 40% and 70% of their proteins respectively, decreases the membrane cross-section but does not change their fracture faces. Pre-cooling of the membrane suspensions within the temperature range of +5 degrees-10 degrees results in the appearance of large smooth areas on S. faecalis membrane fracture faces, but does not affect the ones of M. lysodeikticus membrane. Treatment of the washed suspensions with Triton X-100 results in the appearance of drastic changes of S. faecalis membrane fracture faces and does not change the fracture faces of M. lysodeikticus membranes; treatment by the detergent does not alter the IR-spectroscopy of membranes of both bacteria. Treatment of S. faecalis and M. lysodeikticus membranes with high temperature irreversibly changes the structure of 20% and 40% of protein components respectively,, but does not affect the distribution of the subparticles on their fracture faces. It is assumed that the differences found are determined by the composition of lipid components of the membranes studied and that the amount of proteins closely bound with lipids in the membranes of S. faecalis is likely to be greater than that of M. lysodeikticus membranes.