[Controllable systemic destruction of old collagen fibers as a novel approach for rejuvenating biological aging in mammals.]

Aging of extracellular proteins colloidal systems is one of major synchronizing mechanism in mammal`s «biological clock¼. We hypothesized that induced controllable modification of connective tissue composition could reverse aging. In murine experimental models collagenase was used for selective destruction of old collagen. Oxygen consumption, urine hydroxyproline excretion, density and distribution of mature and old collagen and elastine fibers in dermal biopsies were determined. Collagenase injections significantly increased hydroxyproline excretion. We observed reduced density of mature and old collagen fibers and increased oxygen consumption in dermal biopsies after course of collagenase injections. Collagenase treatment intensified the destruction of mature and old collagen matrix and enhanced synthesis of new collagen and elastine fibers. Furthermore oxygen consumption increased. Our findings can be considered as indicator of collagenase systemic anti-aging (rejuvenation) activity.

Transthoracic monitoring of the impedance of the right lung in patients with cardiogenic pulmonary edema.

OBJECTIVE: To evaluate the suitability of the new electrical impedance monitor RS-205 for monitoring of cardiogenic pulmonary edema (CPE). DESIGN: Prospective, controlled study. SETTING: A department of internal medicine in a 1,200-bed university-affiliated, teaching hospital. PATIENTS: Sixty patients, aged 52-80 yrs, 30 without CPE (controls) and 30 with or at high risk for CPE. INTERVENTIONS: Internal thoracic impedance (ITI) was monitored by the RS-205. The RS-205 is approximately three times more sensitive than the Kubicek monitor, and it eliminates the effect of the drift of skin-to-electrode impedance. This is achieved by eliminating skin electrode impedance by a special algorithm, thus allowing measurement of ITI rather than total transthoracic impedance. Measuring ITI, the main component of which is lung impedance, is a noninvasive and safe method. CPE was diagnosed in accordance with well-accepted clinical and roentgenological criteria. MEASUREMENTS AND MAIN RESULTS: The controls' initial ITI was 68.3 +/- 12.38 ohms. During 6 hrs of monitoring, the ITI attained a minimum average value of -1.3 +/- 2.08% and a maximum average value of 4.6 +/- 3.56% relative to baseline. In all patients entering CPE, ITI decreased by 14.4 +/- 5.42% on the average (p <.001) 1 hr before the appearance of clinical symptoms. In patients with evolving CPE, ITI decreased significantly compared with controls (22.25 +/- 9.82%, p <.001). In patients at the peak of pulmonary edema, ITI was 2.1 times lower than in the control group (33.1 +/- 10.90 ohms, p <.001). In the last hour before the resolution of CPE, ITI increased in all patients by 17.7 +/- 19.74% compared with the peak of disease (p <.05). After the resolution of pulmonary edema, ITI increased in all patients by 44.14 +/- 26.90% compared with the peak of disease (p <.001). Importantly, the trend in ITI in all patients changed in accordance with the dynamics of CPE. A mixed general linear model shows that ITI values correlated well with the degree of crepitation, a direct characteristics of CPE. CONCLUSIONS: The RS-205 is suitable for monitoring patients at high risk of CPE development. It enables detection of CPE and the monitoring of patients at all stages of CPE.

Alleviating constipation in the elderly improves lower urinary tract symptoms.

BACKGROUND: Constipation and lower urinary tract symptoms (LUTS) very frequently occur in the elderly, and several reports have suggested that dysfunction in either one of these systems may affect the other. Most studies correlating rectal and bladder dysfunction, however, have been carried out in children or young women. OBJECTIVE: To examine the effect of alleviating constipation on LUTS in the elderly. METHODS: Fifty-two patients aged 65-89 (mean 72 +/- 13) years with chronic constipation and LUTS participated in this prospective cohort study. Before treatment of constipation was initiated and on their monthly visits, patients completed a questionnaire regarding their constipation pattern, urinary symptoms, sexual function and mood, and underwent urinalysis. Urinary tract anatomy and residual urine were evaluated by abdominal ultrasound at the commencement and completion of the study. Patients were followed up for 4 months. RESULTS: Treatment of constipation increased the number of weekly defecations from 1.5 +/- 0.9 to 4.7 +/- 1.2 (p < 0.001). Patients spent less time on the toilet (25 +/- 2.1 versus 63 +/- 1.9 min, p < 0.0001). Fewer patients reported urgency (16 versus 34, p < 0.001), frequency (25 versus 47, p < 0.001) and burning sensation during urination (6 versus 17, p < 0.05). There was improvement in the scoring of urgency, frequency and burning sensation (from a baseline of 52 to 126, 131 and 95, respectively, p < 0.001). Urinary stream disturbances improved in 32 of the 52 patients (p < 0.001). Residual urine volume decreased from 85 +/- 39.5 to 30 +/- 22.56 ml (p < 0.001). There was also a significant decrease in the number of patients with bacteriurial events (5 versus 17, p < 0.001), and an improvement in sexual activity and mood (p < 0.05). CONCLUSIONS: Our data demonstrated that medical relief of constipation significantly improves LUTS in the elderly which, in turn, improves the patient's mood, sexual activity and quality of life.

Decreased fecal bile acid output in patients with coronary atherosclerosis.

In most patients with atherosclerosis, the underlying metabolic derangement remains undefined. Animal experiments have suggested that the ability to produce and excrete large amounts of bile acids may be an adaptation mechanism to cholesterol overload protecting against the atherogenic effects of cholesterol. However, there are very few data on bile acid excretion in human atherosclerosis. In the present study, we have investigated fecal bile acid secretion in subjects with and without coronary artery disease. The target group consisted of 30 patients with proven coronary artery disease and the control group consisted of 27 matched subjects without clinical or laboratory evidence of coronary atherosclerosis. Fecal bile acids were measured by gas-liquid chromatography from 24-hr stool collections under a controlled diet. The patients excreted significantly less bile acids than the controls (325+/-135 vs. 592+/-223 mg/day, respectively, p < 0.0001). The difference was primarily due to a reduced excretion of secondary bile acids. Less than 50% of deoxycholate was excreted by patients (180+/-81 mg/day) as compared to controls (367+/-168 mg/day, p < 0.0002), while lithocholic acid excretion was 111+/-62 mg/day in patients vs. 190 +/-70 mg/day in controls (p < 0.005). The fecal output of the two primary bile acids, cholic and chenodeoxycholic acid, did not differ significantly between patients and controls. The fecal output of total bile acids correlated with that of both secondary bile acids in patients as well as in controls. These findings suggest that patients with coronary heart disease are unable to excrete adequate amounts of bile acids to rid themselves of excess cholesterol, even if they are able to maintain a plasma cholesterol level comparable to that of healthy controls.

Characterization of liposome-mediated gene delivery: expression, stability and pharmacokinetics of plasmid DNA.

We have characterized a new synthetic gene delivery system, termed DLS, which may be suitable for systemic gene therapy. DLS constitutes a lipopolyamine and a neutral lipid and associated plasmid DNA in the formation of lamellar vesicles (DLS-DNA). The ratio of lipids and lipid to DNA as well as the method of preparation were optimized to yield a high in vitro transfection efficiency compared with that previously reported for cationic lipid systems. DLS-DNA showed a rapid cellular uptake and distribution in the cytoplasmic and nuclear (especially in the nucleoli) compartments as determined by laser-assisted confocal microscopy. There was little or no plasmid DNA degradation over a period of 20 min, relatively slow plasma clearance, and effective and rapid cellular uptake of DLS-DNA following intravenous administration in mice. Supercoiled plasmid DNA could be detected in blood cells up to 1 h after injection. Systemic administration of DLS-DNA yielded transgene expression in mouse tissues, such as in lung or liver. The ratio of DLS:DNA and the procedure used to form DLS-DNA affected both the level and cellular specificity of expression of a luciferase reporter gene showing that in vitro transfection efficiency of DLS-DNA formulations cannot be easily extrapolated to an in vivo setting. Optimization of the formulation of a DNA delivery system was critical to obtain a defined structure resulting in a preparation with high reproducibility and stability, greater homogeneity of particle size and high efficacy following systemic gene transfer. In addition, the DLS system may be formulated for specific target tissues and may have a wide range of applications for gene therapy.

Systemic gene therapy: biodistribution and long-term expression of a transgene in mice.

We have investigated the in vivo efficacy of a systemic gene transfer method, which combines a liposomal delivery system (DLS liposomes) with episomally replicative DNA plasmids to effect long-term expression of a transgene in cells. A single i.v. injection of a plasmid DNA vector containing the luciferase gene as a marker was administered with the DLS liposomes in BALB/c mice. The luciferase gene and its product were found in all mouse tissues tested as determined by PCR analysis and immunohistochemistry. Luciferase activity was also detected in all tissues tested and was present in lung, liver, spleen, and heart up to 3 months postinjection. In contrast to the nonepisomal vectors tested (pRSV-luc and pCMVintlux), human papovavirus (BKV)-derived episomal vectors showed long-term transgene expression. We found that these episomal vectors replicated extrachromosomally in lung 2 weeks postinjection. Results indicated that transgene expression in specific tissues depended on the promoter element used, DNA/liposome formulation, dose of DNA per injection, and route of administration.

[Purification and properties of GTP-cyclohydrolase from Bacillus subtilis]. / Ochistka i svoistva GTP-tsikogidrolazy Bacillus subtilis.

Highly purified GTP-cyclohydrolase was obtained by fractionation of cell extracts with ammonium sulfate, ion-exchange and hydrophobic chromatography. The N-terminal amino acid sequence and amino acid composition of the protein were determined. According to SDS-PAGE data, the molecular weight of the enzyme is 45 kDa. The active enzyme has several isoforms separable by native electrophoresis. The maximal enzyme activity is determined at 1.5 mM Mn2+; 70% of enzymatic activity is detected with Mg2+. The enzyme is inhibited by heavy metal ions and chelators and is inactive in the absence of thiol-reducing agents. The enzyme activity is detected in a broad range of pH with a maximum at pH 8.2. The pyrimidine product of the GTP-cyclohydrolase reaction. 2.5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate was purified and identified. Another product of this reaction is pyrophosphate.

[Various features of the formation and secretion of bile acids in spontaneous atherosclerosis]. / Nekotorye osobennosti obrazovaniia i vydeleniia zhelchnykh kislot pri spontannom ateroskleroze.

Main pathways of cholesterol catabolism were found to be dissimilar in patients with spontaneous coronary atherosclerosis accompanied by hyperlipoproteinemia as compared with healthy persons of the same age. In the patients, independently of the hyperlipoproteinemia type, a longer pathway prevailed which terminated at the step of cholic acid formation, while in healthy persons the shorter pathway was found to be discontinuous after chenodeoxycholic acid production. The specific property of cholesterol catabolism in healthy persons appears to be due to a decrease in activity of V12 alpha-hydroxylase thus protecting against development of spontaneous atherosclerosis.

[Subcloning and study of the GTP-cyclohydrolase gene of Bacillus subtilis]. / Subklonirovanie i issledovanie gena GTP-tsiklogidrolazy Bacillus subtilis.

Bacillus subtilis GTP-cyclohydrolase gene and its deletion derivatives were subcloned in Escherichia coli cells. The position of the gene within the riboflavine operon was defined. The deletion of the 14 kDa fragment from the N-end of GTP-cyclohydrolase gene did not affect the enzyme activity.

[The proteoglycan content of the duodenal juice in peptic ulcer patients]. / Soderzhanie proteoglikanov v duodenal'nom soke u bol'nykh iazvennoi bolezn'iu.

Examined were 21 patients with duodenal ulcer during exacerbation, 18 patients at the stage of clinical remission and 37 patients with exacerbation of chronic cholecystitis with the purpose of evaluating the content of glycosaminoglycans in the duodenal juice. In was established that patients with duodenal ulcer during exacerbation showed a marked reduction of the release of glycosaminoglycans while the content of these substances was increased at the stage of clinical remission and patients with exacerbation of chronic cholecystitis. The authors discuss the role of glycosaminoglycans in the pathogenesis, sanogenesis and diagnosis of duodenal ulcer.

[Classification of peptic ulcer]. / O klassifikatsii iazvennoi bolezni.

To make more simple and useful classification of peptic ulcer the following criteria of the above classification are proposed: nosological entity (ulcer), form (primary, recurrent), site (subcardial, mediogastric, antral, pyloric. duodenal bulbar and duodenal retrobulbar), activity (A-III--a severe exacerbation, A-II--a moderate exacerbation, A-I--clinical remission, A-0--complete remission), clinical course (severe, moderate, light, latent), complications (perforation, hemorrhage, penetration, malignant transformation, pyloric stenosis compensated, pyloric stenosis decompensated).

[Fucose and other neutral hexoses in the feces of patients with nonspecific ulcerative colitis]. / Fukoza i drugie neitral'nye geksozy v kale u bol'nykh nespetsificheskim iazvennym kolitom.

Secretion of glycoproteins with protective mucus of large intestine was studied by means of estimation of the carbohydrate components in feces of patients with unspecific ulcerous colitis. A decrease in daily excretion with feces of glycoproteins and glycopeptides was detected in these patients as well as in the patients with chronic pancreatitis. Under conditions of unspecific ulcerous colitis distinct from chronic pancreatitis content of fucoglycoproteins, main chemical protectors of gastrointestinal tract, was primarily decreased. The defect of large intestine mucus might be responsible for impairment of barrier function of the intestinal mucose and to contribute to ulcer development.

[Elimination of glycosaminoglycans in duodenal peptic ulcer and problems of its pathogenesis]. / Vydelenie glikozaminoglikanov pri iazvennoi bolezni dvenadtsatiperstnoi kishki i nekotorye voprosy ee patogeneza.

Changes in the excretion and composition of proteoglycans specific for duodenal ulcer were studied in 50 patients with duodenal ulcer, 30 patients with gastric ulcer, 30 patients with chronic endogenous gastroduodenitis and in 35 healthy persons. In all the examinees proteoglycans were isolated from daily urine, their carbohydrate components--glycosaminoglycans (GAG)--were separated and divided into fractions (keratan sulfate, hyaluronic acid, heparan sulfate, chondroitin sulfate-4, chondroitin sulfate-6, dermatan sulfate, and heparin) by column chromatography on unmodified cellulose. It has been established that only peptic ulcer is characterized by disorders in GAG excretion differing in the period of exacerbation and remission. Changes in the composition of proteoglycans excreted with urine resulted probably from a deficiency of chondroitin sulfate-6 in patients with chronic duodenal ulcer. The deficiency was more marked during exacerbation but did not disappear in the period of remission of duodenal ulcer either.

[Excretion of various glycosaminoglycans in patients with chronic duodenal ulcer]. / Vydelenie razlichnykh glikozaminoglikanov u bol'nykh khronicheskoi duodenal'noi iazvoi.

Excretion of individual glycosaminoglycans (GAG's) with urine was studied in patients with chronic duodenal ulcer. 31 patient with acute manifestations of the duodenal ulcer, 24 patients within the period of remission as well as 29 practically healthy persons were studied. Column chromatography on cellulose was used for separation of a mixture containing keratan sulfate, hyaluronic acid, heparan sulfate, chondroitin-4 and -6-sulfates, dermatan sulfate and heparin. During the acute period of duodenal ulcer daily excretion of all the individual GAG's with urine was decreased (especially distinct for the fraction of chondroitin-6-sulfate) as compared with excretion of these carbohydrates in healthy persons. The level of GAG excretion was increased distinctly, exceeding the values found in healthy persons, during the remission. Excretion of chondroitin-6-sulfate and keratan sulfate was markedly increased in these cases.

[Clinical value of the cholesterol test in patients with arteriosclerosis]. / Klinicheskoe znachenie testa s nagruzkoi kholesterinom u bol'nykh aterosklerozom.

Capacity for the excretion of cholesterol (CS) and bile acids was studied, using an original cholesterol load test, in atherosclerotic patients. They showed reduced fecal excretion of neutral sterols and bile acids following cholesterol load, unlike normal subjects, whose respective values were increased irrespective of age. Fecal excretion of these substances was particularly low in second-type hyperlipoproteinemia. Atherosclerosis-related changes of sterol balance may be an evidence of latent incompetence of mechanisms of hepatic evacuation of substances that may be regarded as atherosclerotic risk factors.

[Study of the 210-degree region of the Bacillus subtilis chromosome using recombinant plasmids]. / Izuchenie oblasti 210 gradusov khromosomy Bacillus subtilis s pomoshch'iu rekombinantnykh plazmid.

The 210 degrees region of Bacillus subtilis DNA containing the rib operon and genes for the first (dapA) and last (lysA) steps of lysine biosynthesis was cloned. PstI fragments of B. subtilis m.m. 4.7 MD DNA containing the lys and the proximal part of rib operon were isolated from different B. subtilis strains (SB25 and SHgW) and shown to have the same restriction and genetic maps. The restriction mapping of EcoRI fragment of B. subtilis m.m. 6.3 MD DNA containing the rib operon has been carried out.