Structural Changes in Rat Hepatocyte Nucleolus under Nucleolar Stress Caused by Hypothermia.

Structural changes in rat hepatocyte nucleoli were studied during deep hypothermia simulated by immersion in water at 5°C for 40 min (ambient air temperature 7°C). In comparison with the control, phenomena of nucleolar stress occurred in rats during hypothermia: the number of fibrillar centers (FC) per nucleus (by 1.7 times) and per nucleolus (by 1.6 times), nucleolonemal nucleoli per nucleus (by 2.8 times), and the relative content of nucleolonemal nucleoli per nucleus (by 2.6 times) significantly decreased (p=0.0000001); the number of FC per nucleolonemal nucleolus also decreased by 1.4 times (p=0.01). In the hepatocyte nuclei, we observed an increase in the relative content of transitional type nucleoli per nucleus (by 1.3 times; p=0.01), the number of FC per transitional type nucleolus (by 1.4 times; p=0.003), the content of free FC per nucleus (by 3 times; p=0.00004), and the percentage of free FC per nucleus (by 3.5 times; p=0.00004). These changes can be considered as compensatory and adaptive reactions, and transitional type nucleoli can be attributed to the "reserve" nucleolar pool.

Effect of Carnosine on the Activity of Matrix Metalloproteinase-2 and Oxidative Stress in the Kidneys in Experimental Urate Nephrolithiasis.

The effect of carnosine on MMP-2 activity and oxidative stress in the kidneys in experimental urate nephrolithiasis was studied. Urate nephrolithiasis was modeled in Wistar rats by intragastric administration of a mixture of oxonic and uric acids. Carnosine was administered intragastrically through a tube in a dose of 15 mg/kg. In rats treated with carnosine, the concentration of MMP-2 in the urine decreased by 3.7 times, and the excretion of MMP-2 with urine decreased by 4.3 times. In the homogenate of the kidneys from rats treated with carnosine, the concentration of TBA-reactive substances decreased by 5 times and the concentration of MMP-2 decreased by 12.7%. After treatment with carnosine, the number of histologically confirmed cases of urate nephrolithiasis decreased by 2 times, while the mean size of urate deposits decreased by 2.7 times. Thus, carnosine inhibits MMP-2 and reduces the intensity of oxidative stress in the kidneys, which prevents the development of urate nephrolithiasis.

Nephroprotective Effect of Leu-Ile-Lys Tripeptide in Experimental Diabetes Mellitus.

We studied the effect of tripeptide Leu-Ile-Lys on kidney function in rats with experimental diabetes mellitus modeled by single intraperitoneal administration of streptozotocin (65 mg/kg). The tripeptide was intragastrically administrated in a dose of 11.5 mg/kg from week 5 to week 8 of the pathological process. The concentrations of glucose, protein, and creatinine in the urine were measured before and then weekly throughout the experiment. In 8 weeks, the markers of activity of the free radical oxidation process (concentration of TBA-reactive substances, total prooxidant activity, and total antioxidant activity, as well as activities of catalase, superoxide dismutase, and glutathione peroxidase) were assayed and a morphological study was conducted. After administration of the tripeptide Leu-Ile-Lys for 4 weeks, glucose concentration in the urine decreases by 3-44 times (p<0.001) and protein concentration by 2.3-3.7 times (p=0.007). The concentration of TBA-reactive substances decreased by 1.3 times (p<0.001), and the total antioxidant activity increased by 2.3 times (p<0.001). Administration of the tripeptide Leu-Ile-Lys to animals with experimental diabetes mellitus led to significant improvement of the renal function against the background of significant alleviation of oxidative damage and an increase in the antioxidant protection of the renal tissues. Improvement of the morphofunctional state of tissues and cells of the renal glomerulus was confirmed histologically, in particular, an increase in the number of podocytes by 1.5 times was observed.

Effect of Carnosine on the Course of Experimental Urate Nephrolithiasis.

The prospect of using the antioxidant dipeptide carnosine for the treatment of urate nephrolithiasis was evaluated. Urate nephrolithiasis was modeled in rats by intragastric administration of a mixture of oxonic and uric acids. Carnosine was administered intragastrically through a tube in a dose of 15 mg/kg. In rats treated with carnosine, the concentration of TBA-reactive products decreased by 1.4 times, the total antioxidant activity increased by 1.4 times, and catalase activity increased by 1.3 times. By the end of the experiment, the lactate dehydrogenate level in experimental rats was 2-fold lower than in the control, and the number of urate deposits decreased by 1.6 times with a concomitant alleviation of the inflammatory processes. Thus, the use of direct peptide antioxidant carnosine attenuated the manifestations of urate nephrolithiasis.

Experience of Using Tripeptide Leu-Ile-Lys for Experimental Therapy of Chronic 16-Week Oxalate Nephrolithiasis in Rats.

We studied the effect of tripeptide Leu-Ile-Lys on the course of chronic 16-week oxalate nephrolithiasis in rats modeled by administration of 1% ethylene glycol solution in drinking water for 16 weeks. The tripeptide Leu-Ile-Lys obtained by chemical synthesis (sample purity ≥98%) was administered intragastrically through a probe in a dose of 11.5 mg/kg in 1 ml saline. It was found that during tripeptide Leu-Ile-Lys significantly alleviated the course of experimental pathology, which was confirmed by characteristic biochemical and morphological indicators. We observed a decrease in the concentration of calcium ions by 4.4 times, weakening of oxidative stress in the renal tissue due to a decrease in the total prooxidant activity by 1.2 times, normalization of increased catalase activity, and reduction of superoxide dismutase activity by 2.4 times relative to disease control. Histological signs of nephrolithiasis were recorded in 9% cases (vs. 75% cases in disease control).

Effect of Tripeptide Leu-Ile-Lys on the Course of Experimental Nephrolithiasis.

We studied the effect of Leu-Ile-Lys tripeptide on the course of experimental oxalate nephrolithiasis modeled in rats by administration of 1% ethylene glycol solution instead of drinking water for 6 weeks. The Leu-Ile-Lys tripeptide obtained by chemical synthesis (purity ≥98%) was administered through a gastric tube (11.5 mg/kg in 1 ml saline). Administration of Leu-Ile-Lys tripeptide against the background of experimental oxalate nephrolithiasis significantly alleviated the course of experimental pathology, which was confirmed by typical biochemical and morphological changes: decrease in urinary activity of γ-glutamyltransferase (by 2.1 times in comparison with the initial level) and intensity of oxidative stress (the content of TBA-reactive products decreased by 1.3 times in comparison with that in untreated animals) and increase in glutathione peroxidase activity by 1.8 times; no histological signs of nephrolithiasis were found in animals treated with the tripeptide.

Relationship between Cooling Medium and Hepatocyte Ploidometric Parameters in Albino Rats.

Relationship between the method of hypothermic exposure and ploidometric characteristics of hepatocyte nuclei of rats were studied immediately after hypothermic exposure and during the posthypothermic period. The following cooling modes were used: single submersion into 5°C water (until rectal temperature drop to 20-25°C; deep hypothermia) and single exposure at air temperature of -25°C (until rectal temperature drop to 30°C; moderate hypothermia). A manifest destructive effect of deep hypothermia on rat liver was detected: the exposure caused manifest alteration of hepatocytes (alteration index increased 81-fold immediately after the exposure and remained 16-fold higher than normally after 14 days) and an increase of DNA accumulation index with predominance of hepatocytes with 5c-8c nuclei (60% of the population) during the entire period of observation, which indicated intensification of the compensatory and adaptive processes in the liver with predominating high-ploid population of hepatocytes. Hepatocyte alteration processes in response to moderate hypothermia were less intense (hepatocyte alteration index increased 19-fold immediately after the exposure and remained 3-fold higher than normally after 14 days). The DNA accumulation index increased moderately immediately after the exposure and returned to normal after 14 days, with emergence of 2c hepatocytes and an increase in the level of 4c hepatocytes (51% of the population), which indicated realization of a different strategy of liver regeneration - recovery of the cell population intrinsic to intact animals.

[The features of the pathologic changes in chronic oxalate stone disease after 16 week of experimental phase].

AIM: to study the pathologic features of chronic oxalate stone disease. MATERIALS AND METHODS: a model of the experimental oxalate stone disease was done in rats that drank the 1% ethylene glycol solution for 16 weeks. The pathologic analysis of the rats kidneys was done. RESULTS: After 16 weeks of oxalate stone disease modeling in all parts of kidneys the large crystals deposits were found, which plugged renal tubules. Moreover, purulent calculous pyelonephritis developed complicated by fibrosis.

Morphological Evaluation of the Influence of the Peptide Complex from Tissue of Porcine Kidneys on the Experimental Urolithiasis.

We performed morphological analysis of the effect of the peptide complex from porcine kidneys on the course of experimental urolithiasis modeled in rats by treatment with 1% ethylene glycol solution (in drinking water) for 6 weeks. The peptide complex obtained by acetic acid extraction was administered in a dose of 15 mg. Administration of the peptide complex to animals with experimental kidney stone disease leads to 100% destruction of large and medium stones to the "dust" granularity.

Mast Cells in Renal Cancer: Clinical Morphological Correlations and Prognosis.

Morphofunctional activity of mast cells in renal cancer is studied on specimens from the center of the tumor, peritumorous zone (with an obligatory fragment of the pseudocapsule), and from renal tissues maximally distant from the tumor. The counts and areas of tumor cells were maximum in the peritumorous zone in comparison with intact kidney tissue and central zone of the tumor. Analysis of correlations revealed a direct correlation between the tumor cell count in the peritumorous zone and some clinical morphological parameters of the tumor (anaplasia degree, size, presence of invasion and metastases, and angiogenesis). The count of mast cells in the renal peritumorous zone inversely correlated with 5-year survival: 100% cumulative 5-year survival in patients with mast cell count <6.7±1.6 per field of view.

Expression of B23/Nucleophosphamine Nonribosomal Nucleolar Protein in Smooth Muscle Tumors of the Corpus Uteri.

Immunohistochemical study of B23/nucleophosmine (NPM), the key argyrophilic nonribosomal protein associated with the nucleolus-producing regions, in smooth muscle formations of the body of the uterus and myometrium was carried out. The uterus index, number of positively stained nucleoli in the nucleus, integral optical density, and area of positively stained nucleoli were studied by computer-aided image analysis. The results indicated significant differences in the integral optical density and less so in the areas of NPM+ nucleoli in leiomyosarcomas, but not in intact myometrium and leiomyomas. Coefficients of variations in the integral optical density and NPM area proved to be significant parameters for differentiating leiomyosarcomas by size (≤7 cm or larger), evaluating the malignancy grade and stage of the process. NPM parameters in leiomyomas and leiomyosarcomas differed significantly, depending on the levels of Ki-67 and p53 expression and the receptor status of leiomyosarcoma. The index of area and the integral optical density of NPM could serve a accessory objective differential diagnostic criteria in cellular and mitotically active liomyomas and well-differentiated leiomyosarcoma of the corpus uteri.

Correlation of HER-2/Neu protein expression and HER2 gene amplification with clinical morphological parameters of nonsmall cell lung cancer.

A total of 218 operation specimens from patients with nonsmall cell lung cancer were examined. The expression of HER-2 protein was studied by the immunohistochemical method, amplification of HER2 gene and centromere part of chromosome 17 (CEP17) - by the in situ chromogenic hybridization method. Moderate correlation (r=0.38, p<0.001) between the immunohistochemical status and HER2 gene amplification was detected. A relationship between TNM clinical morphological parameters and HER-2 protein content and chromosome 17 polysomia (for N parameter), HER-2 protein content, and HER2 gene amplification (for tumor histogenesis) was detected. The content of HER-2 protein and amplification of HER2 and CEP17 correlated with the clinical morphological parameters of patients with nonsmall cell lung cancer.

Argyrophilic proteins of nucleolar organizer regions and proliferative activity of cells in squamous cell carcinoma of the lung.

Argyrophilic proteins associated with nucleolar organizer regions (Ag-NOR proteins) and Ki-67 antigen were analyzed in 118 samples of squamous cell carcinoma of the lungs. Tumors with low and high content of Ag-NOR proteins and Ki-67 index were selected. It was found that the content of Ag-NOR proteins correlated with some clinical and morphological parameters (indexes T and N, tumor size less and more that 3 cm, stage of the disease, and tumor differentiation degree) and survival rate. High survival was associated with low content of Ag-NOR proteins and Ki-67 index and low survival correlated with high content of Ag-NOR proteins and Ki-67 index, while intermediate survival was associated with opposite values of Ag-NOR protein content and Ki-67 index. The tumor size, parameter N, and the content of Ag-NOR proteins had independent effects on patient's survival. In patients with squamous cell carcinoma of the lung without metastases in lymph nodes, survival correlated with the content of Ag-NOR proteins; in patients without metastases, survival correlated with tumor size.

[Topoisomerase IIa expression in correlation with clinical and morphological parameters and proliferation (based on argyrophilic proteins of nucleolar organizer regions and Ki-67 antigen) in lung adenocarcinoma].

Investigated topoisomerase IIalpha (TopoII alpha), argyrophilic proteins associated with nucleolar organizer regions (Ag-NOR) and antigen Ki-67 in lung adenocarcinoma. Defined tumor with low and high TopoII alpha, Ag-NOR and Ki-67. TopoII alpha had a relationship with clinical and morphological parameters (greatest dimension, tumor differentiation) and proliferation markers (Ag-NOR, Ki-67) . Survival of patients with lung adenocarcinoma with low content of TopoII alpha is better as compared with high content of TopoII alpha. Survival of patients with lung adenocarcinoma depends on the mutual content of TopoII alpha and clinical and morphological parameters (value T, greatest dimension, value N, stage, tumor differentiation), proliferation markers (Ag-NOR). TopoII alpha content in lung adenocarcinoma is an independent prognostic factor. Mutual research of TopoII alpha with clinical and morphological parameters and Ag-NOR has prognostic value in lung adenocarcinoma.

Relationship between the argyrophilic proteins of the nucleolar organizer region and stage of squamous-cell carcinoma of the lung.

Argyrophilic proteins associated with chromosome nucleolar organizer regions (Ag-NOR proteins) were studied in lung squamous-cell carcinoma cells. After routine staining with silver nitrate, the area index and coefficient of variations of Ag-NOR protein were estimated by computer-aided image analysis. The Ag-NOR protein area indexes of tumor cells differed in clinical morphological variants of squamous-cell cancer (differing by T and N parameters, size of the primary node <3 cm or larger, by disease stage, and differentiation degree). Ag-NOR protein variation coefficient can be used for discriminating the tumors by size (<3 cm and larger), presence/absence of metastases, and evaluation of squamous-cell cancer differentiation degree. Ag-NOR protein area index and variation coefficient can be used as additional objective differential diagnostic criteria of clinical and morphological forms of squamous-cell carcinoma of the lung.

[Age changes of the connective tissue structures of human penis].

This investigation was aimed at the study of age changes of penis connective tissue structures. Tissue fragments of penis were obtained from 20 cadavers of men at the age of 20-38 years in group I, and from 20 cadavers of men at the age of 41-59 years in group II. The criteria for the exclusion of material from the research were arterial hypertension, diabetes mellitus, atherosclerosis of internal iliac arteries, Peyronie's disease, and anomalies of genital organ development. It was shown that in the cavernous body of penis, aging was associated with the increased amount and thickening of collagen and argyrophilic fibers, decreased content and thinning of elastic fibers, and the reduced amount of smooth muscle cells (SMC). The average area of fibroblast and SMC nucleolus was not different in both groups studied. The average area of endotheliocyte nucleolus was equal to 1.9+/-0.9 microm2 in group II, being lower than that one in group I, in which this index was equal to 2.1+/-0.9 microm2. No differences in the content of type III and IV collagen were found between the study groups. Age-associated decrease in the average area of endothelial cell nucleolus in the cavernous bodies may reflect the reduction of the activity of these cells and may indicate the development of endothelial dysfunction, which is one of the most important steps in the morphogenesis of age-related male erectile dysfunction.

[Topoisomerase IIalpha activity and tumor cell proliferation level from the degree of bromodeoxyuridine label in uterine body leiomyosarcoma].

The investigation was undertaken to estimate the expression of the enzyme gyrase Topoisomerase IIalpha (TOPOIIalpha) and proliferative activity from the level of bromodeoxyuridine (Brdu) in uterine body leiomyosarcoma (UBL) (n=66) versus the unchanged myometrium (UM), a tumor of uncertain malignancy potential (STUMP) (n=12), and leiomyoma (LM) (n=46). An immunohistochemical technique was used to reveal TopOIIalpha and Brdu. The findings indicated that Brdu was undetectable in UM and simple LM and that TOPOIIalpha activity was low. At the same time there was an exponential rise in the activity of the enzyme and cells during the S phase of the cell cycle from cellular LM to UBL. An increase was also noted in the study markers as sarcoma malignancy progressed. Thus, it can be said that the estimation of the number of cell S fractions and the activity of TOPOIIalpha in UM, LM, STUMP, and UBL may serve as a relatively simple and reliable method for the differential diagnosis of benign and malignant smooth muscle tumors of the uterine body and the determination of the activity of the enzyme may be used to define the degree of tumor chemoresistance in the complex treatment of patients with UBL.

[Expression of sex hormones receptors in leiomyoma with uncertain potential of malignancy and endometrial leiomyosarcoma: differentiatial diagnosis and prognostic significance].

A retrospective evaluation of progesterone (PR) and alpha-estrogen (ER) expression was carried out in 16 patients with smooth muscle tumors with uncertain potential of malignancy (STUMP) and 76 patients with uterine leiomyosarcoma (ULMS). PR expression was observed in 100% of intact myometrium and leiomyoma samples, while ER was expressed only in 37.1 +/- 4.2% and 23.5 +/- 4.1% of samples. In STUMP samples PR expression was observed in 66.7 +/- 11.7% of cases, in ULMS samples in 50.5 +/- 5.7% of cases. However, there was an inverse correlation between malignancy grade and sex hormones expression, although the number of positive cells and staining intensity didn't prove to be independent risk factors. The patients with ULMS G1 and STUMP expressing sex hormones have favorable prognosis, while negative receptor status of STUMP tumors is the marker of the worse prognosis.

[Argyrophilic nucleolar organizer region associated proteins in adenomas with varying colonic dysplasia and adenocarcinoma].

Argyrophilic nucleolar organizer region associated (Ag-NOR) proteins were studied in rectosigmoid epithelial tumors for diagnostic purposes. Silver nitrate staining was carried out in accordance with the standardized protocol. The quantity, area, area index of Ag-NOR proteins were automatically determined by computer image analysis. There were statistically significant differences in the quantity, area, area index of Ag-NOR proteins between the groups: the mucosa (lower 2/3 crypts), mild, moderate, and severe dysplasia of adenoma. Only the estimation of the area index of Ag-NOR proteins indicated statistically significant differences between severe dysplasia and highly differentiated adenocarcinoma. Colonic adenomas showed a correlation of the activity of Ag-NOR proteins with epithelial dysplasia, but no correlation with the size and histological type of their structure.

[Modification of a histochemical technique for detection of nucleolar organizer regions in histological specimens].

The authors modified the method for silver staining of argyrophilic nucleolar organizer regions (AgNORs). This modification eliminates defects of an original technique. The method accelerates the staining and prevents fallout of deposits. The modification is recommended for wide application.