Search

Patients with genetically-based hyperlipidemias exhibit a wide phenotypic variability. Investigation of clinical and biochemical features is important for identifying genetically-based hyperlipidemias, determining disease prognosis, and initiating timely treatment. We analyzed genetic data from 3374 samples and compared clinical data, lipid levels (low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol, triglycerides, and lipoprotein (a)), frequency, age at onset of coronary heart disease (CHD), and the severity of carotid and femoral atherosclerosis (plaque number, maximum stenosis, total stenosis, maximum plaque height, and plaque score) among patients with familial hypercholesterolemia (FH), familial dysbetalipoproteinemia (FD), polygenic hypercholesterolemia (HCL), severe HCL, and those without lipid disorders (n = 324). FH patients exhibited the highest LDL-C (median 8.03 mmol/L, p < 0.001). FD patients had elevated triglyceride levels (median 4.10 mmol/L), lower LDL-C (median 3.57 mmol/L), and high-density lipoprotein cholesterol (median 1.03 mmol/L) compared to FH, polygenic HCL, and severe HCL, p < 0.05. FH and FD patients had similar early onset of CHD, with a median age of 44 and 40 years and comparable frequencies of 29.5% and 31.0%, respectively. They were more likely to develop CHD than subjects without lipid disorders (p = 0.042 and p < 0.001, respectively). Additionally, FH patients had higher a carotid plaque number, total carotid stenosis, and carotid plaque score. This study presents the first simultaneous comparison of clinical and biochemical features among FD, FH, polygenic, and severe HCL, along with the first comprehensive evaluation of carotid and femoral atherosclerosis ultrasound parameters in FD patients. The results highlight distinct phenotypic features unique to each hyperlipidemia analyzed and underscore FH and FD as the most atherogenic hyperlipidemias., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Blokhina et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Familial dysbetalipoproteinemia (FD) is a highly atherogenic, prevalent genetically based lipid disorder. About 10% of FD patients have rare APOE variants associated with autosomal dominant FD. However, there are insufficient data on the relationship between rare APOE variants and FD. Genetic data from 4720 subjects were used to identify rare APOE variants and investigate their pathogenicity for autosomal dominant FD. We observed 24 variants in 86 unrelated probands. Most variants were unique (66.7%). Five identified APOE variants (p.Glu63ArgfsTer15, p.Gly145AlafsTer97, p.Lys164SerfsTer87, p.Arg154Cys, and p.Glu230Lys) are causal for autosomal dominant FD. One of them (p.Lys164SerfsTer87) was described for the first time. When we compared clinical data, it was found that carriers of pathogenic or likely pathogenic APOE variants had significantly higher triglyceride levels (median 5.01 mmol/L) than carriers of benign or likely benign variants (median 1.70 mmol/L, p = 0.034) and variants of uncertain significance (median 1.38 mmol/L, p = 0.036). For the first time, we estimated the expected prevalence of causal variants for autosomal dominant FD in the population sample: 0.27% (one in 619). Investigating the spectrum of APOE variants may advance our understanding of the genetic basis of FD and underscore the importance of APOE gene sequencing in patients with lipid metabolism disorders.

Non-alcoholic fatty liver disease (NAFLD) is considered the most common chronic liver disease worldwide, affecting nearly 25% of the global adult population. Increasing evidence suggests that functional and compositional changes in the gut microbiota may contribute to the development and promote the progression of NAFLD. 16S rRNA gene next-generation sequencing is widely used to determine specific features of the NAFLD microbiome, but a complex system such as the gut microbiota requires a comprehensive approach. We used three different approaches: MALDI-TOF-MS of bacterial cultures, qPCR, and 16S NGS sequencing, as well as a wide variety of statistical methods to assess the differences in gut microbiota composition between NAFLD patients without significant fibrosis and the control group. The listed methods showed enrichment in Collinsella sp. and Oscillospiraceae for the control samples and enrichment in Lachnospiraceae (and in particular Dorea sp.) and Veillonellaceae in NAFLD. The families, Bifidobacteriaceae , Lactobacillaceae , and Enterococcaceae (particularly Enterococcus faecium and Enterococcus faecalis ), were also found to be important taxa for NAFLD microbiome evaluation. Considering individual method observations, an increase in Candida krusei and a decrease in Bacteroides uniformis for NAFLD patients were detected using MALDI-TOF-MS. An increase in Gracilibacteraceae , Chitinophagaceae , Pirellulaceae , Erysipelatoclostridiaceae , Muribaculaceae , and Comamonadaceae , and a decrease in Acidaminococcaceae in NAFLD were observed with 16S NGS, and enrichment in Fusobacterium nucleatum was shown using qPCR analysis. These findings confirm that NAFLD is associated with changes in gut microbiota composition. Further investigations are required to determine the cause-and-effect relationships and the impact of microbiota-derived compounds on the development and progression of NAFLD.

Background: Left ventricular noncompaction (LVNC) cardiomyopathy is a disorder that can be complicated by heart failure, arrhythmias, thromboembolism, and sudden cardiac death. The aim of this study is to clarify the genetic landscape of LVNC in a large cohort of well-phenotyped Russian patients with LVNC, including 48 families (n=214)., Methods: All index patients underwent clinical examination and genetic analysis, as well as family members who agreed to participate in the clinical study and/or in the genetic testing. The genetic testing included next generation sequencing and genetic classification according to ACMG guidelines., Results: A total of 55 alleles of 54 pathogenic and likely pathogenic variants in 24 genes were identified, with the largest number in the MYH7 and TTN genes. A significant proportion of variants -8 of 54 (14.8%) -have not been described earlier in other populations and may be specific to LVNC patients in Russia. In LVNC patients, the presence of each subsequent variant is associated with increased odds of having more severe LVNC subtypes than isolated LVNC with preserved ejection fraction. The corresponding odds ratio is 2.77 (1.37 -7.37; p <0.001) per variant after adjustment for sex, age, and family., Conclusion: Overall, the genetic analysis of LVNC patients, accompanied by cardiomyopathy-related family history analysis, resulted in a high diagnostic yield of 89.6%. These results suggest that genetic screening should be applied to the diagnosis and prognosis of LVNC patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Meshkov, Myasnikov, Kiseleva, Kulikova, Sotnikova, Kudryavtseva, Zharikova, Koretskiy, Mershina, Ramensky, Zaicenoka, Vyatkin, Kharlap, Nikityuk, Sinitsyn, Divashuk, Kutsenko, Basargina, Barskiy, Sdvigova, Skirko, Efimova, Pokrovskaya and Drapkina.)

To continue progress in the treatment of cardiovascular disease, there is a need to improve the overall understanding of the processes that contribute to the pathogenesis of cardiovascular disease (CVD). Exploring the role of gut microbiota in various heart diseases is a topic of great interest since it is not so easy to find such reliable connections despite the fact that microbiota undoubtedly affect all body systems. The present study was conducted to investigate the composition of gut microbiota in patients with atherosclerotic cardiovascular disease (ASCVD) and heart failure syndromes with reduced ejection fraction (HFrEF) and HF with preserved EF (HFpEF), and to compare these results with the microbiota of individuals without those diseases (control group). Fecal microbiota were evaluated by three methods: living organisms were determined using bacterial cultures, total DNA taxonomic composition was estimated by next generation sequencing (NGS) of 16S rRNA gene (V3-V4) and quantitative assessment of several taxa was performed using qPCR (quantitative polymerase chain reaction). Regarding the bacterial culture method, all disease groups demonstrated a decrease in abundance of Enterococcus faecium and Enterococcus faecalis in comparison to the control group. The HFrEF group was characterized by an increased abundance of Streptococcus sanguinus and Streptococcus parasanguinis . NGS analysis was conducted at the family level. No significant differences between patient's groups were observed in alpha-diversity indices (Shannon, Faith, Pielou, Chao1, Simpson, and Strong) with the exception of the Faith index for the HFrEF and control groups. Erysipelotrichaceae were significantly increased in all three groups; Streptococcaceae and Lactobacillaceae were significantly increased in ASCVD and HFrEF groups. These observations were indirectly confirmed with the culture method: two species of Streptococcus were significantly increased in the HFrEF group and Lactobacillus plantarum was significantly increased in the ASCVD group. The latter observation was also confirmed with qPCR of Lactobacillus sp. Acidaminococcaceae and Odoribacteraceae were significantly decreased in the ASCVD and HFrEF groups. Participants from the HFpEF group showed the least difference compared to the control group in all three study methods. The patterns found expand the knowledge base on possible correlations of gut microbiota with cardiovascular diseases. The similarities and differences in conclusions obtained by the three methods of this study demonstrate the need for a comprehensive approach to the analysis of microbiota.

Cystic fibrosis, phenylketonuria, alpha-1 antitrypsin deficiency, and sensorineural hearing loss are among the most common autosomal recessive diseases, which require carrier screening. The evaluation of population allele frequencies (AF) of pathogenic variants in genes associated with these conditions and the choice of the best genotyping method are the necessary steps toward development and practical implementation of carrier-screening programs. We performed custom panel genotyping of 3821 unrelated participants from two Russian population representative samples and three patient groups using real-time polymerase chain reaction (PCR) and next generation sequencing (NGS). The custom panel included 115 known pathogenic variants in the CFTR , PAH , SERPINA1 , and GJB2 genes. Overall, 38 variants were detected. The comparison of genotyping platforms revealed the following advantages of real-time PCR: relatively low cost, simple genotyping data analysis, and easier detection of large indels, while NGS showed better accuracy of variants identification and capability for detection of additional pathogenic variants in adjacent regions. A total of 23 variants had significant differences in estimated AF comparing with non-Finnish Europeans from gnomAD. This study provides new AF data for variants associated with the studied disorders and the comparison of genotyping methods for carrier screening.

We performed a targeted sequencing of 242 clinically important genes mostly associated with cardiovascular diseases in a representative population sample of 1,658 individuals from the Ivanovo region northeast of Moscow. Approximately 11% of 11,876 detected variants were not found in the Single Nucleotide Polymorphism Database (dbSNP) or reported earlier in the Russian population. Most novel variants were singletons and doubletons in our sample, and virtually no novel alleles presumably specific for the Russian population were able to reach the frequencies above 0.1-0.2%. The overwhelming majority (99.3%) of variants detected in this study in three or more copies were shared with other populations. We found two dominant and seven recessive known pathogenic variants with allele frequencies significantly increased compared to those in the gnomAD non-Finnish Europeans. Of the 242 targeted genes, 28 were in the list of 59 genes for which the American College of Medical Genetics and Genomics (ACMG) recommended the reporting of incidental findings. Based on the number of variants detected in the sequenced subset of ACMG59 genes, we approximated the prevalence of known pathogenic and novel or rare protein-truncating variants in the complete set of ACMG59 genes in the Ivanovo population at 1.4 and 2.8%, respectively. We analyzed the available clinical data and observed the incomplete penetrance of known pathogenic variants in the 28 ACMG59 genes: only 1 individual out of 12 with such variants had the phenotype most likely related to the variant. When known pathogenic and novel or rare protein-truncating variants were considered together, the overall rate of confirmed phenotypes was about 19%, with maximum in the subset of novel protein-truncating variants. We report three novel protein truncating variants in APOB and one in MYH7 observed in individuals with hypobetalipoproteinemia and hypertrophic cardiomyopathy, respectively. Our results provide a valuable reference for the clinical interpretation of gene sequencing in Russian and other populations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ramensky, Ershova, Zaicenoka, Kiseleva, Zharikova, Vyatkin, Sotnikova, Efimova, Divashuk, Kurilova, Skirko, Muromtseva, Belova, Rachkova, Pokrovskaya, Shalnova, Meshkov and Drapkina.)

The research biobanking field is developing rapidly in Russia. Over the course of the last decade, numerous biobanks were created or formed from existing collections of human and environmental biospecimens. The Russian National Association of Biobanks and Biobanking Specialists (NASBIO) was established in December 2018, aiming to: (1) unite professionals and research centers to create and develop a network of biobanks in Russia; (2) provide services and expertise in the field of biobanking; (3) execute various research projects utilizing biobanks' infrastructure; and (4) facilitate integration of Russian biomedical research centers into global research activities. The organizational structure, aims, and plans of this newly formed national association are reviewed in this article. The founders of NASBIO hope that the association will promote further development of biobanks and their networking in Russia, which is critically important for the success of national biomedical, pharmaceutical, and biotechnological research, and can facilitate international biobanking projects on a global scale.

Genetic screening is an advanced tool for reducing recessive disease burden. Nowadays, it is still unclear as to the number of genes or their variants that are necessary for effective screening. This paper describes the development of a carrier screening custom panel for cystic fibrosis, phenylketonuria, alpha-1 antitrypsin deficiency, and sensorineural hearing loss consisting of 116 variants in the CFTR , PAH , SERPINA1, and GJB2 genes. The approach is based on the cheapest and fastest method, on using a small number of genes, and on the estimation of the effectiveness of carriers' detection. The custom panel was tested on a population-based cohort that included 1244 participants. Genotypes were determined by the TaqMan OpenArray Genotyping platform on the QuantStudio 12K Flex Real-Time PCR System. The frequency of heterozygotes in the Russian population was 16.87% or 1:6 (CI95%: 14.76-19.00% by Clopper-Pearson exact method): in CFTR -2.81% (1:36), PAH -2.33% (1:43), SERPINA1 -4.90% (1:20), and GJB2 -6.83% (1:15). The data on allele frequencies were obtained for the first time on a Russian population. The panel allows us to identify the vast majority of carriers of recessive diseases in the population. It is an effective approach to carrier screening for common recessive diseases.

Taking into account the impact of shipment method of biosamples is necessary for obtaining high-quality biological samples in biobanking and laboratory research. The impact of liquid nitrogen, dry ice and cold accumulators on the quality of biological markers was considered, as well as recommendations to reduce the impact of these methods of shipment. The liquid nitrogen provides the best preservation of samples, however, dry ice is used much more often during their transportation. When transporting certain types of cells using dry ice, there is the way to use CryoStor CS1 and Cell Banker 1 cryoprotectors. The dry ice has a significant effect on both the pH of liquid biological samples and the coagulological parameters of plasma samples. The penetration of CO2 into the sample leads to changes in the parameters of PTT and APPT, as well as to decrease the protein C and fibrinogen level under certain conditions. Serum and plasma samples exposed to dry ice for more than 16 hours should be thawed open at room temperature, or instead of it should be kept at -80 °C for 24 hours to avoid changes in coagulation parameters, The use of cold accumulators is unacceptable for long-term shipment of serum and plasma containing unstable biomarkers because of insufficiently low temperature (increase over time to -25 °C and above). Besides, metal pellets can be used as cold storage batteries at low temperatures (up to -80 ° C), but they are not as effective as dry ice, since it is able to hold the required temperature for much longer., Competing Interests: The authors declare no conflict of interest.

Currently one of the most important problems facing biobanking specialists is the standardization of biobanks operation. Close attention is paid to this issue by international biobanking organizations, such as ISBER and BBMRI-ERIC, which develop regulatory documentation in this area. The article provides examples of standardization tools - implementation of the ISO 9001 quality standard and ISBER Best Practices. General information about the development, scope, and structure of the ISO 20387 standard is provided. The standard does not provide ready-made solutions and does not contain specific requirements for storage temperature or biosamples processing in biobanks, allowing each biobank to adapt its own management system to existing conditions and needs. The standard contains requirements for both the organization of the biobanking and the supporting processes - personnel competence; requirements for biological safety; infrastructure management, including equipment used by the biobank, environmental parameters that affect the storage of biomaterial. The standard contains requirements for the quality management system of biobank, as a necessary element of the organization of any biorepository. At the initiative of the Russian National Association of biobanks and biobanking specialists (NASBIO), development of the Russian standard GOST R ISO 20387 «Biotechnology. Collection and storage of biological samples in biobanks. General requirements» is included in the plan of the National Standardization Program for 2020 by order of Rosstandart No. 2612 of 11/01/2019. Implementing quality standards is a long and painstaking process that requires the involvement of all employees and certain resources. However, the effectiveness of strict compliance exceeds the cost of developing, implementing and maintaining management systems, as it significantly increases the confidence of researchers in the work of biobanks, guarantees high quality of biospecimens and associated data, and creates opportunities for cooperation, both at the national and international level, based on the application of common quality standards in the work., Competing Interests: The authors declare no conflict of interest.

The National Medical Research Center for Preventive Medicine of Russia (NMRCPM) conducts epidemiological and clinical research for the development of personalized medicine. This is why NMRCPM has faced the problem of how to standardize preanalytical conditions for all biospecimens from various scientific projects and of how to provide long-term responsible standardized regulated safe storage of blood and its derivatives. This article describes various aspects of establishing a biobank in a large medical center dedicated to integrating the biomarkers research activities of different departments. To date, >205,000 serum/plasma/whole blood specimens have been stored. Collaboration with >25 scientific projects as well as the biobank's own research project has been organized. The availability of this biobank became a platform for the establishment of the Personalized Medicine Center in NMRCPM.

The biobank is a structure established with the goal of long-term responsible storage of biological samples and the associated data for their further use in scientific and clinical research. The objectives of biobanking are the creation of unified recommendations on: the planning of premises and the selection of equipment for storage; development of management methods and staff training; standardization of methods for the collection, shipping, processing and storage of biomaterial of various origins, as well as methods for quality control and validation of the applied methods; creation and use of databases of information accompanying biospecimens. The lack of common standards for conducting the preanalytical phase has been the cause of low accuracy and poor reproducibility of research results. To date, a large number of guidelines and best practices have been published that provide an answer to a wide range of problems in organizing the biobanking process. The article provides an overview of the most famous biobanking guidelines that can be used to solve various research problems. Biobanking in Russia is actively developing. Since 1996 there is a work on the legislative regulation of biobanking activities, as a result of which a number of regulatory documents have been issued. An important stage in the development of biobanking in Russia was the establishment of the "National Association of Biobanks and Biobanking Specialists" (NASBio) in 2018, which included representatives of medical and research institutions, commercial firms, and qualified specialists in the field of biobanking. One of the key tasks of NASBio is the adaptation and implementation of the best biobanking practices in Russian research institutes and centers. The use of modern guidelines and best practices on biobanking will lead to an increase in the quality of research and publications., Competing Interests: The authors declare no conflict of interest.

This paper provides several definitions of the term "biobank"; a list of standards developed by the International Organization for Standardization (ISO) applicable to the activities of biobanks; analyzes the legal and ethical requirements; the Russian legal framework in the field of biobanking, the best international practices and recommendations; describes the experience of the development and implementation of quality management systems according to ISO 9001 in Biobanks established in different countries, and the experience of the Bank of Biological Material of the National Medical Research Center for Preventive Medicine of the Ministry of Healthcare of Russia. The ISO 20387 Biobanking Standard, released in August 2018, combined the knowledge and experience of specialists from around the world and defined the general requirements that must be fulfilled by biobanks and repositories wishing to guarantee their customers the high quality of the preanalytical stage of scientific research, biological samples and associated data. In 2019, the Russian version of this standard is expected. Documents of the quality management system provide the reproducibility of activities on the main storage processes and facilitates the process of incorporating a new employee; conducting internal and external audits; Biobank knowledge management - continuous staff education. The introduction of an effective quality management system into biobank activity warrants the high quality of biological samples, the standardized pre-analytical stage, reliable, regulated long-term storage of biomaterial and related information for use in research purposes today and in future., Competing Interests: The authors declare no conflict of interest.

Type 2 diabetes (T2D) and obesity are common chronic disorders with multifactorial etiology. In our study, we performed an exome sequencing analysis of 110 patients of Russian ethnicity together with a multi-perspective approach based on biologically meaningful filtering criteria to detect novel candidate variants and loci for T2D and obesity. We have identified several known single nucleotide polymorphisms (SNPs) as markers for obesity (rs11960429), T2D (rs9379084, rs1126930), and body mass index (BMI) (rs11553746, rs1956549 and rs7195386) ( p < 0.05). We show that a method based on scoring of case-specific variants together with selection of protein-altering variants can allow for the interrogation of novel and known candidate markers of T2D and obesity in small samples. Using this method, we identified rs328 in LPL ( p = 0.023), rs11863726 in HBQ1 ( p = 8 × 10 -5 ), rs112984085 in VAV3 ( p = 4.8 × 10 -4 ) for T2D and obesity, rs6271 in DBH ( p = 0.043), rs62618693 in QSER1 ( p = 0.021), rs61758785 in RAD51B ( p = 1.7 × 10 -4 ), rs34042554 in PCDHA1 ( p = 1 × 10 -4 ), and rs144183813 in PLEKHA5 ( p = 1.7 × 10 -4 ) for obesity; and rs9379084 in RREB1 ( p = 0.042), rs2233984 in C6orf15 ( p = 0.030), rs61737764 in ITGB6 ( p = 0.035), rs17801742 in COL2A1 ( p = 8.5 × 10 -5 ), and rs685523 in ADAMTS13 ( p = 1 × 10 -6 ) for T2D as important susceptibility loci in Russian population. Our results demonstrate the effectiveness of whole exome sequencing (WES) technologies for searching for novel markers of multifactorial diseases in cohorts of limited size in poorly studied populations.

Background: Increased arterial stiffness (AS), intima-media thickness (IMT), and the presence of atherosclerotic plaques (PP) have been considered as important aspects of vascular aging. It is well documented that the cardiovascular system is an important target organ for growth hormone (GH) and insulin-like growth factor (IGF)-1 in humans, and GH /IGF-1 deficiency significantly increases the risk for cardiovascular diseases (CVD). The telomere length of peripheral blood leukocytes (LTL) is a biomarker of cellular senescence and that has been proposed as an independent predictor of (CVD). The aim of this study is to determine the role of GH/IGF-1, LTL and their interaction cardiovascular risk factors (CVRF) in the vascular aging. Methods: The study group included 303 ambulatory participants free of known CVD (104 males and 199 females) with a mean age of 51.8 ± 13.3 years. All subjects had one or more CVRF [age, smoking, arterial hypertension, obesity, dyslipidemia, fasting hyperglycemia, insulin resistance-HOMA (homeostatic model assessment) >2.5, or high glycated hemoglobin]. The study sample was divided into the two groups according to age as "younger" ( m ≤ 45 years, f ≤ 55 years) and "older" ( m > 45 years, f > 55 years). IMT and PP were determined by ultrasonography, AS was determined by measuring the carotid-femoral pulse wave velocity (c-f PWV) using the SphygmoCor system (AtCor Medical). LTL was determined by PCR. Serum IGF-1 and GH concentrations we measured by immunochemiluminescence analysis. Results: Multiple linear regression analysis with adjustment for CVRF indicated that HOMA, GH, IGF-1, and LTL had an independent relationship with all the arterial wall parameters investigated in the younger group. In the model with c-f PWV as a dependent variable, p < 0.001 for HOMA, p = 0.03 for GH, and p = 0.004 for LTL. In the model with IMT as a dependent variable, p = 0.0001 for HOMA, p = 0.044 for GH, and p = 0.004 for IGF-1. In the model with the number of plaques as a dependent variable, p = 0.0001 for HOMA, and p = 0.045 for IGF-1. In the older group, there were no independent significant associations between GH/IGF-1, LTL, HOMA, and arterial wall characteristics. Conclusions: GH/IGF-1, IR, HOMA, and LTL were the important parameters of arterial aging in younger healthy participants.

Enteropathogenic yersiniae (Yersinia pseudotuberculosis and Yersinia enterocolitica) typically cause chronic disease as opposed to the closely related Yersinia pestis, the causative agent of bubonic plague. It is established that this difference reflects, in part, carriage by Y. pestis of a unique 9.6-kb pesticin or Pst plasmid (pPCP) encoding plasminogen activator (Pla) rather than distinctions between shared approximately 70-kb low-calcium-response, or Lcr, plasmids (pCD in Y. pestis and pYV in enteropathogenic yersiniae) encoding cytotoxic Yops and anti-inflammatory V antigen. Pla is known to exist as a combination of 32.6-kDa (alpha-Pla) and slightly smaller (beta-Pla) outer membrane proteins, of which at least one promotes bacterial dissemination in vivo and degradation of Yops in vitro. We show here that only alpha-Pla accumulates in Escherichia coli LE392/pPCP1 cultivated in enriched medium and that either autolysis or extraction of this isolate with 1.0 M NaCl results in release of soluble alpha and beta forms possessing biological activity. This process also converted cell-bound alpha-Pla to beta-Pla and smaller forms in Y. pestis KIM/pPCP1 and Y. pseudotuberculosis PB1/+/pPCP1 but did not promote solubilization. Pla-mediated posttranslational hydrolysis of pulse-labeled Yops in Y. pseudotuberculosis PB1/+/pPCP1 occurred more slowly than that in Y. pestis but was otherwise similar except for accumulation of stable degradation products of YadA, a pYV-mediated fibrillar adhesin not encoded in frame by pCD. Carriage of pPCP by Y. pseudotuberculosis did not significantly influence virulence in mice.

We have sequenced the lcrGVH operon from Y. pseudotuberculosis plasmid pYV995 and compared its sequence with that of Y. pestis. The sequences were highly homological, however, six base pair substitutions were found in one short 14 bp region termed variable sequence. Two oligonucleotides corresponding to variable sequence of Y. pestis (pes-V) or Y. pseudotuberculosis (ptb-V) were synthesized and were used as molecular probes in hybridization experiments with sets of Y. pestis and Y. pseudotuberculosis strains. All 17 Y. pestis strains tested were positive only with the pes-V probe, 18 of 21 Y. pseudotuberculosis strains were positive with the ptb-V probe, while three Y. pseudotuberculosis strains reacted with the pes-V probe but not the ptb-V probe. The 200 bp fragment including variable sequence was sequenced in seven Y. pseudotuberculosis strains. The Y. pseudotuberculosis strains which were positive with the pes-V probe possessed the 200 bp fragment sequence almost identical with that from Y. pestis. No correlation between the Y. pestis-like lcrV sequence and virulence was found for these strains. Moreover, the Y. pseudotuberculosis strains with Y. pestis-like sequences in contrast to Y. pestis possessed unaltered yadA gene. However, we have found the yadA frameshift mutation characteristic for Y. pestis in one Y. pseudotuberculosis strain 312.

Colchicine in a total dose of 0.6 mg/100 g body weight per day was shown to reduce the level of apical surfactant secretion by type II alveolar cells in random-bred male albino rats, thereby demonstrating that the cytoplasmic microtubules participate in the release of surfactant into the alveolar lumen. In addition, basal secretion of surface-active material was found in 51% of all the cells. In a single dose of 8 mg/100 g b.w., pilocarpine stimulated apical surfactant secretion. If injected after colchicine, it slightly increased the number of type II alveolar cells ready to release surfactant, but actual secretion was not observed; the level of basal secretion did not increase. It has been suggested that microtubular function is not completely responsible for basal secretion and is only partly responsible for apical surfactant secretion.