Photodynamic Effect of some Phthalocyanines on Enveloped and Naked Viruses.

Activity of three photosensitizing phthalocyanine derivatives was tested for photodynamic inactivation towards two coated and two non-enveloped viruses - bovine viral diarrhea virus (BVDV), influenza virus A(H3N2), poliovirus type 1 (PV-1) and human adenovirus type 5 (HAdV5). In the case of coated viruses, a combination of virucidal and irradiation effects was registered by octa-methylpyridyloxy-substituted Ga phthalocyanine (Ga8) toward BVDV, whereas tetra-methylpyridyloxy-substituted Ga phthalocyanine (Ga4) revealed a marked photoinactivation only. No such effect was observed towards influenza A virus. In contrast, the photoinactivating potential of Ga4 and Ga8 marked very high values on naked viruses, especially on HAdV5, at which both the virucidal as well as the irradiation effects became combined.

Reliable optical characterization of e-beam evaporated TiO2 films deposited at different substrate temperatures.

We studied e-beam evaporated TiO2 films deposited at two different substrate temperatures between 120°C and 300°C. We reliably characterized the film samples on the basis of in situ and ex situ measurements. We carried out annealing on the samples and studied the induced changes in the properties of the films. The results can be useful for further laser-induced damage threshold investigations.

Measurements of the group delay and the group delay dispersion with resonance scanning interferometer.

We developed a method for group delay and group delay dispersion measurements, based on location of interference resonance peaks. Such resonance peaks can be observed in transmittance or in reflectance when two mirrors are placed parallel to each other and separated by a thin air spacer. By using a novel approach, based on simultaneous processing of the data acquired for different spacer distances we obtained reliable results with high resolution. Measurements were performed both in transmittance and reflectance layouts depending on the reflectivity of the mirror to be measured. The developed method allows dispersion measurements of ultraviolet mirrors and ultra-broadband mirrors spanning more than one optical octave to be performed.

High-dispersive mirrors for high power applications.

We report on the development and manufacturing of two different types of high-dispersive mirrors (HDM). One of them provides a record value for the group delay dispersion (GDD) of -4000 fs2 and covers the wavelength range of 1027-1033 nm, whereas the other one provides -3000 fs2 over the wavelength range of 1020-1040 nm. Both of the fabricated mirrors exhibit a reflectance of >99.9% and are well suited for intracavity applications. Mirrors of the second type have been successfully employed in a Kerr-lens mode-locked Yb:YAG thin-disk oscillator for the generation of 200-fs pulses with multi-10-W average power.

Laser induced autofluorescence studies of animal skin used in modeling of human cutaneous tissue spectroscopic measurements.

BACKGROUND/PURPOSE: Laser-induced autofluorescence spectroscopy provides excellent possibilities for medical diagnostics of different tissue pathologies including cancer. However, to create the whole picture of pathological changes, investigators collect spectral information from patients in vivo or they study different tumor models to obtain objective information for fluorescent properties of every kind of healthy and diseased tissue. Therefore, it is very important to find the most appropriate, and close to the human skin, animal samples from the fluorescence point of view, which will allow the extrapolation of the animal data to human spectroscopic diagnostics. METHODS: In the present work, we examined the autofluorescence properties of different animal skin tissues, which are considered as the most common skin models. A nitrogen laser was used as an excitation source. Samples of healthy mouse, chicken and pig skin in vivo and/or ex vivo were studied and were compared with results obtained from investigations of healthy human skin in vivo. RESULTS AND CONCLUSION: Specific features of the recorded spectra are discussed and the possible origin of the obtained fluorescence signals is proposed. Quantitative evaluation of data extrapolation for each skin type is also depicted.

The tissue concentration of UDP-N-acetylglucosamine modulates the stimulatory effect of insulin on skeletal muscle glucose uptake.

To delineate the biochemical mechanism by which increased availability of GlcN impairs insulin action on skeletal muscle glucose uptake, we replenished the uridine pool during GlcN administration. Co-infusion of uridine with GlcN prevented the GlcN-induced fall in skeletal muscle UDP-glucose levels (24.9 +/- 5. 3 versus 10.1 +/- 2.9 nmol/g; p < 0.01) and further increased the skeletal muscle UDP-GlcNAc levels (198.4 +/- 26.3 versus 96.0 +/- 8. 4 nmol/g; p < 0.01). Greater reductions in the rates of glucose infusion ( approximately 53%), glucose uptake ( approximately 43%), and glycogen synthesis ( approximately 60%) were observed with the addition of uridine. Similarly, the infusion of uridine alone markedly increased the skeletal muscle levels of both UDP-glucose (55.2 +/- 14.2 versus 17.8 +/- 6.1 nmol/g; p < 0.01) and UDP-GlcNAc (86.8 +/- 8.8 versus 35.9 +/- 8.4 nmol/g; p < 0.05) and induced marked insulin resistance. The decrease in insulin action on peripheral glucose uptake was highly correlated with the increase in skeletal muscle UDP-GlcNAc levels. Finally, immunoisolation of GLUT4-containing vesicles revealed that the rate of labeled GlcN incorporation was approximately 100-fold greater following GlcN compared with saline infusions (p < 0.01). We suggest that the marked reduction in insulin action induced by GlcN and uridine is mediated by increased accumulation of muscle UDP-N-acetylhexosamines, perhaps via altered glycosylation of protein(s) in GLUT4-containing vesicles.

Fluence rate effects on photodynamic therapy of B16 pigmented melanoma.

In vivo experiments were performed to evaluate the effect of fluence rate on the efficiency of Zn(II)-2,3 naphthalocyanine (ZnNc) photosensitization of B16 pigmented melanoma subcutaneously transplanted in C57B1/6 mice. The tumour was irradiated with 774 nm light at 24 h after an injection of liposome--which incorporated ZnNc (0.5 mg kg-1 b.w.). A total light dose of 360 J cm-2 was delivered at fluence rates of 260, 320, 380, 440 and 500 mW cm-2. Separate groups of mice utilized to monitor tumour temperature changes during irradiation without or after anaesthesia. Tumour response was determined by measuring the mean tumour diameter of the treated towards the untreated animals for a period of 21 days following PDT, as well as the percentage of cured animals. The most promising result (40% complete tumour response) was obtained with anaesthetized mice following 380 mW cm-2. Higher dose rates (440 and 500 mW cm-2) led to less promising results for both anaesthetized and non anaesthetized mice. These results outline the potential of PDT with longer wavelengths for the treatment of highly pigmented tumour tissues. The optimal fluence rate for tumour treatment should be chosen in order to avoid inflammatory effects (tissue swelling) and oxygen suppression with sublethal injury to the tumour cells.

Increased hexosamine availability similarly impairs the action of insulin and IGF-1 on glucose disposal.

Prolonged glucosamine (GlcN) infusion increases the skeletal muscle hexosamine concentration and induces peripheral insulin resistance in conscious rats. IGF-1 and insulin share common steps in signal transduction, and the action of IGF-1 on carbohydrate metabolism is preserved in certain insulin-resistant states. In our study, we attempted to delineate whether increased GlcN availability also impairs the effects of IGF-1 on glucose uptake (Rd), glycolysis, and glycogen synthesis. We performed euglycemic IGF-1 (5 and 15 microg x kg(-1) x min(-1)) and insulin (3 and 18 mU mg x kg(-1) x min(-1)) clamp studies at 0-2 h and 5-7 h in conscious rats (n = 44) during saline or GlcN infusions. GlcN infusion raised plasma GlcN levels to approximately 2.0 mmol/l and skeletal muscle uridinediphospho-n-acetylglucosamine to 80-150 nmol/g (approximately three- to fivefold over basal). During physiological hyperinsulinemia (3 mU x kg(-1) x min(-1), plasma insulin approximately 50 microU/ml), GlcN infusion caused comparable decreases in Rd (15.7 +/- 1.0 [5-7 h] vs. 21.7 +/- 2.3 [0-2 h] mg x kg(-1) x min(-1); P < 0.01) and glycogen synthesis (5.4 +/- 0.5 [5-7 h] vs. 10.4 +/- 1.9 [0-2 h] mg x kg(-1) x min(-1); P < 0.005). Furthermore, GlcN markedly decreased Rd by 7.8 +/- 1.2 mg x kg(-1) x min(-1) (18.7 +/- 0.7 [5-7 h] vs. 26.5 +/- 1.3 [0-2 h] mg x kg(-1) x min(-1); P < 0.001 vs. control) during IGF-1 (5 microg x kg(-1) x min(-1)) clamp studies. This decline was associated with a 26% decrease in the steady-state concentration of skeletal muscle Glc-6-P (286 +/- 45 vs. 386 +/- 36 nmol/g; P < 0.01) and was primarily caused by impaired glycogen synthesis (6.7 +/- 0.5 [5-7 h] vs. 13.9 +/- 0.9 [0-2 h] mg x kg(-1) x min(-1); P < 0.005). The effects of GlcN infusion on glucose disposal (percentage decrease in Rd) were correlated (r2 = 0.803; P < 0.01) with the skeletal muscle concentration of UDP-GlcNAc. To investigate whether IGF-1 can overcome GlcN-induced insulin resistance, GlcN and insulin (18 mU x kg(-1) x min(-1)) were infused for 7 h during euglycemic clamps, and IGF-1 (15 microg x kg(-1) x min(-1)) was superimposed during the final 2 h. GlcN infusion induced severe impairment of insulin action on Rd (39.4 +/- 3.2 [4-5 h] vs. 49.8 +/- 3.6 [1-2 h] mg x kg(-1) x min(-1); P < 0.05), which the addition of IGF-1 failed to improve (35.9 +/- 2.3 [6-7 h] vs. 39.4 +/- 3.2 [4-5 h] mg x kg(-1) x min(-1); P > 0.1). In summary, GlcN induced severe resistance to the actions of both insulin and IGF-1 on glucose uptake and glycogen synthesis, and IGF-1 was unable to overcome GlcN-induced insulin resistance. Thus, it is likely that GlcN causes peripheral insulin resistance acting at a site common to both IGF-1 and insulin signaling pathways.

Glucosamine-induced inhibition of liver glucokinase impairs the ability of hyperglycemia to suppress endogenous glucose production.

Although the kinetic characteristics of hepatic glucokinase (GK) suggest its potential role as the hepatic "glucose sensor," its impact on the regulation of in vivo hepatic glucose production (HGP) is still controversial. Since decreased GK activity has been linked to experimental and human diabetes, we examined whether a moderate and transient inhibition of GK activity diminishes the ability of hyperglycemia to suppress HGP. We first determined the concentration of the competitive inhibitor, glucosamine (GlcN), which decreases hepatic GK activity by approximately 60% in vitro. GlcN was then infused into conscious rats to achieve a similar inhibition of the in vivo GK activity (plasma GlcN levels = approximately 2 mmol/l; rats infused with saline served as control, n = 20). To maintain equal plasma insulin and glucagon concentrations throughout the studies, somatostatin and insulin (basal replacement) were infused for 4 h. [3-(3H)]-glucose and [U-(14C)]-lactate were infused to measure HGP, gluconeogenesis, and glucose cycling (GC) during 2 h of euglycemia (glucose approximately 8 mmol/l) followed by 2 h of hyperglycemia (glucose approximately 18 mmol/l). Our results support the notion that hepatic GK activity is indeed decreased by GlcN in vivo. In fact, in response to hyperglycemia the "direct" pathway of hepatic glucose-6-phosphate (G-6-P) formation was approximately 40% lower with GlcN compared with saline infusion (37 +/- 3 vs. 63 +/- 3%; P < 0.001). Furthermore, while hyperglycemia stimulated GC by approximately 2.5-fold during saline infusion (from 3.0 +/- 0.6 to 7.7 +/- 1.4 mg.kg-1.min-1, P < 0.001, euglycemia vs. hyperglycemia), this increase was blunted in the presence of GlcN (4.6 +/- 0.6 mg.kg-1.min-1, P = NS). Finally, in the presence of GlcN, the hepatic concentration of G-6-P was decreased by approximately 40% compared with saline (234 +/- 38 and 390 +/- 24 nmol/g, P < 0.01). During the euglycemic studies, HGP was similar (12.6 +/- 0.6 and 11.3 +/- 0.2 mg .kg-1.min-1 with GlcN or saline infusion, respectively). However, while hyperglycemia per se suppressed HGP by approximately 65%, HGP was inhibited by approximately 38% and it was approximately twofold higher than in the saline-infused rats (7.8 +/- 0.8 and 4.0 +/- 0.3 mg.kg-1.min-1, P < 0.01) in the presence of GlcN-induced inhibition of hepatic GK. This increase in HGP was largely accounted for by the decreased inhibition of hepatic net glycogenolysis by hyperglycemia (3.3 +/- 0.8 and 1.1 +/- 0.3 mg.kg-1.min-1 with GlcN or saline infusion, respectively, P < 0.01). We conclude that intact GK activity is required for the normal suppression of HGP by hyperglycemia and its impairment may contribute to increased HGP in experimental and human diabetes.

Brain and testis selective expression of the glutathione S-transferase Yb3 subunit is governed by tandem direct repeat octamer motifs in the 5'-flanking region of its gene.

To gain insight into mechanisms of cell type-specific transcription of class mu-glutathione S-transferase genes, the gene encoding the Yb3 subunit was cloned. Yb3 subunits are selectively expressed at high levels in rat brain and testis but not in liver or kidney. The Yb3 subunit gene spans over 6 kb and consists of 8 exons and 7 introns and a sequence consisting of tandem direct repeat consensus octamer DNA binding motifs separated by a 6 base pair (bp) spacer was identified in its 5'-flanking region. Gel shift assays with a 40 bp segment of DNA containing the two consensus octamer sequences, revealed the presence of specific binding proteins in nuclear extracts of rat brain, testis and C6 glioma cells. DNA binding activity was greatly reduced in liver, kidney and HTC cells. Reporter vectors carrying segments of the 5'-flanking region of the Yb3 subunit gene fused to a luciferase gene were introduced into C6 glioma cells which express high levels of Yb3 subunits, and into HTC cells which do not. The plasmids consisting of the Yb3 gene promoter up to, but not including, the octamer motifs did not support luciferase transcription in the C6 glioma cells, but larger fragments that included the octamer repeat sequences, effectively directed transcription in the C6 glioma cells. With mutated octameric sequences transcriptional activity was greatly reduced, and none of the same Yb3 constructs directed substantial luciferase transcription in the HTC cells. The results show that octamer motifs in the 5'-flanking region of the Yb3 subunit gene are functional and are the principal cis-acting elements that account for its discrete cell type-selective expression. This gene is one of the few known targets for octamer DNA binding transcription factors in brain.

Characterization of a proline-directed casein kinase from bovine brain.

A messenger-independent ser/thr casein kinase, p45 casein kinase (p45 CK), was purified to homogeneity from bovine brain. The enzyme is specific for ATP with a Km value of 3.50 microM, one of the lowest values identified for protein kinases, p45 casein kinase is active over broad NaCl concentrations from 30 to 300 mM. The enzyme activity is inhibited by polylysine, spermine, transition metal ions, ADP, and AMP. The kinase completely lost its activity in the presence of 1 mM p-chloromercuric benzoate in a reaction that is reversed by 1 mM dithiothreitol. The enzyme prefers serine over threonine in its substrate bradykinin, the Vmax/Km ratio for the serine peptide (RPPGFSPFR) being 7.5-fold higher than for the threonine analog (RPPGFTPFR). Assays, performed by utilizing synthetic peptides, suggest that p45 CK prefers serine/threonine residues with a proline residue immediately carboxy-terminal to the site of phosphorylation. Distinction between p45 CK and other protein kinases found to contain a proline residue within their substrate recognition sites can be made based on phosphorylation site specificity and chromatographic and biochemical behavior. It is concluded that p45 CK is a proline-directed protein kinase recognizing the sequence X-Ser/Thr-Pro-X or Ser/Thr-Pro-X.

Identification and purification of a novel serine/threonine messenger-independent growth-related protein kinase from lactating goat mammary gland.

A second messenger-independent serine/threonine protein kinase from lactating goat mammary gland is purified and characterized. The purification steps include: homogenization, ultracentrifugation, ammonium sulphate precipitation, DEAE-Sepharose, phosphocellulose, hydrophobic and Mono Q columns. On the final step of purification the enzyme is revealed as a single band of mol wt 45,000 on silver-stained SDS-PAGE. Mg2+ and K+ are necessary for its optimum activity. Phosvitin and casein are substrates for the enzyme but kemptide, RRREEETEEE, protamine and histone mixture are all poorly phosphorylated. The kinase is inhibited by quercetin, heparin, random tyrosine- and glutamic acid-containing polymers, Ca2+, NaF, 2,3-bis-phosphoglycerate. 1 mM Mn2+ affects positively the basal level of the kinase activity but 5 mM Mn2+ completely suppress the effect of 10 mM Mg2+. Km of this enzyme for ATP is 1.57 microM and pH optimum is from 6 to 7. Isolation of this kinase is facilitated by its unusually high affinity for phosphocellulose.

Optimization of the pumping conditions for Nd:YAG lasers.

In this paper we report on the results of comparative investigations of different pump cavities. Numerical estimations of the geometrical transfer efficiency of various cavities are presented. An experimental study of the pumping efficiency and pumping uniformity of the laser heads with respect to the cavity geometry, types of flashlamp, and reflecting coatings have been carried out. The influence of the discharge circuit on the pumping efficiency is also discussed.

Amplification of bacterial plasmids without blocking protein biosynthesis.

The effect of amino acids (presence or absence from the growth media) and metal ions on the replication of Escherichia coli plasmids in rel A+ strains was studied. It was found that: (i) The absence of one amino acid from the growth media had no effect on the plasmid copy number in prototrophic E. coli strains: (ii) The presence of only one amino acid in artificial media free of amino acids had a negligible effect on the plasmid copy number for the amino acids Ala, Arg, Glu, His, Leu, Phe, Thr, Trp, and Tyr: (iii) The combination of Met and Thr caused a rise in pBR322 plasmid copy number up to 90-100 plasmid copies per cell: (iv) The Fe3+ concentration had an amplification effect on E. coli plasmids. The pBR322 plasmid copy number for media free of amino acids and supplemented with 0.2-0.4 mM FeCl3 was 60-80 plasmid copies per cell: (v) The combination of Fe3+ with certain amino acids (Ala, Arg, Glu, Leu, Thr, and Trp) leads to a dramatic increase in the plasmid copy number reaching 180-270 plasmid copies per cell for the plasmid pBR322 and 20-24 for the plasmid pR100.

[Coagglutination test to localize a urinary infection]. / Koaglutinatsionen test za lokalizirane na uroinfektsiiata.

A method has been developed for discovering of antibody covered bacteria in the urine by means of a coagglutination test with a protein A containing strain--St. aureus Cowan's I. The test is based on the ability of staphylococcal protein A to bind Fc-fragment of IgG. As a specificity control St. aureus Wood 46 was used. 38 patients with urinary infections were examined. As pyelonephritic criteria the following were considered: febrile episodes, lumber pains, polyuria, pollakiuria, leukocyturia, proteinuria, raised arterial pressure, anemia, diminished renal function or chronic renal failure, x-ray, ultrasound and isotopic-nephrographic changes. In the presence of antibody covered bacteria the test is positive--there is coagglutination only with st. aureus Cowan's I. If the bacteria are not antibody coated no coagglutination takes place. If there is coagglutination with both strains the reaction is considered non-specific. II samples gave non-specific reaction. In 75% of the pyelonephritic patients the test was positive. In 70% of the patients with urinary infections of the lower urinary tract, i.e. with bacteria without immunoglobulins, the test was negative. In 74.1% of the cases there is a correlation between the coagglutination test and the localization of the urinary infection by means of other clinical and paraclinical methods. It is suggested that the coagglutination test should be included in the examination of patients with urinary infections, the positive test indicates renal localization of the infection.

[New trends in the control of enzootic bovine leukemia in Bulgaria]. / Novi nasoki v borbata s enzootichnata levkoza po govedata u nas.

Experimental and literature data as well as personal observations with herds infected with enzootic bovine leukosis have revealed some new aspects that could be made use of in the control of the disease. The more important ones are stated: newborn calves of cows with positive leukosis response can be given untreated colostrum which has been shown to neutralize the virus; in individual regions and Agro-Industrial Complexes where sporadic cases of leukosis have been recorded isolation premises for positively reacting animals could be designed; cows in the last two months of pregnancy do not always give reliable results with the immunodiffusion test, so such animals should not be investigated at that time with this method.