CO2 is the first species formed upon CO oxidation by CO dehydrogenase from Pseudomonas carboxydovorans

The active species of CO₂, i.e. CO₂ or HCO ₃ ^- , formed in the CO dehydrogenase reaction was determined using the pure enzyme from the carboxydotrophic bacterium Pseudomonas carboxydovorans. Employing an assay system similar to that used to test for carbonic anhydrase, data were obtained which are quite compatible with those expected if CO₂ is the first species formed. In addition, carbonic anhydrase activity was not detected in P. carboxydovorans.     

CD44 standard and variant isoform expression in normal human skin appendages and epidermis

 CD44 isoforms have been implicated in tumor progression and metastasis formation. This study presents a thorough immunohistochemical analysis of CD44 standard and isoform expression in normal human skin appendages and epidermis applying monoclonal antibodies against CD44s, CD44v3, -v4, -v5, -v6, and -v9. An improved immunohistochemical protocol with microwave-based antigen retrieval in paraffin sections and heavy metal amplification of the diaminobenzidine reaction product provided enhanced resolution and sensitivity as compared to studies on frozen sections. The hair follicle, the seborrheic and eccrine sweat glands were strongly positive for all CD44 isoforms studied. In the latter, the clear cells but not the dark (intercalated) cells were positive. The sudoriferous ducts adjacent to the glands were weakly positive for all CD44 isoforms and strongly positive near the skin surface. In the apocrine glands, the basal cells showed only a moderate positivity. The myoepithelial cells expressed only CD44s. In the epidermis, all CD44 isoforms were detectable, with strongest CD44 immunostaining in the lower third of the stratum spinosum and weaker staining in the stratum basale and the upper two-thirds of the stratum granulosum. The stratum granulosum and corneum were unreactive. Thus, a regional and cell type-specific CD44 expression was revealed. Accepted: 10 May 1996     

Identification of single base-pair mutation on uidA gene of Escherichia coli O157:H7 by Peptide Nucleic Acids (PNA) mediated PCR clamping

Peptide Nucleic Acids (PNA) is a new type of DNA analogue with a peptide backbone. We developed a rapid identification system of Escherichia. coli O157:H7 using PNA mediated PCR clamping. Firstly, we confirmed a single nucleotide alteration in the uidA gene (T93G), which is specific to E. coli O157: H7. We designed forward mutant DNA primer, wild type PNA, and a reverse DNA primer corresponding to the uidA sequence. PCR cycle consisted of four steps including dual annealing temperatures, 57 degrees C and 45 degrees C. Among 20 E. coli strains with various serotypes and 4 neighboring strains, the amplified bands (517 bp) were detected only in E. coli O157:H7 strains. PNA has specifically inhibited the PCR amplification from a wild type uidA gene. We successfully developed a multiplex PCR system, which detects both shigatoxin (stx) and uidA genes at once, to get reliable results by easier and rapid operation. We also analyzed kinetic parameters of PNA/DNA association using surface plasmon resonance and melting temperature using fluorescence resonance energy transfer (FRET). We discussed a selection mechanism of PCR clamping from these results.     

Comparative study of the genotoxic response of freshwater mussels Unio tumidus and Unio pictorum to environmental stress

Genotoxic response of freshwater mussels U. tumidus and U. pictorum to environmental stress was studied using comet assay on hemocytes and gill cells. The mussels were acclimated to controlled laboratory conditions for 10 days, and then exposed at 4 sites in the Sava and Danube rivers in the area of the city of Belgrade. Samples of each species were taken after 7, 14, and 30 days of exposure. The mussels sampled immediately after acclimation served as controls. Genotoxic response in both species was induced earlier at sites receiving untreated wastewaters from the city’s main collectors (7 days), than at the site receiving only domestic wastewaters from small settlements located upstream from the city (30 days). There was a correlation between the comet tail intensity values in tissues of exposed mussels and the concentrations of zinc, copper, iron, and arsenic at the exposure sites. The genotoxic responses in both tissues of U. pictorum and in hemocytes of U. tumidus were similar, while U. tumidus gill cells failed to exhibit significant genotoxic response at two sites. These findings, together with higher mortality of U. tumidus at the most polluted sites, promote U. pictorum as a model for genotoxicity monitoring in freshwater environments.     

Factors affecting distribution pattern of dominant macroinvertebrates in Mantovo Reservoir (Republic of Macedonia)

In the present study, we analysed spatial and temporal heterogeneity of the limnological characteristics to provide more detailed information about the processes taking place within Mantovo Reservoir (Republic of Macedonia). The relationship between principal macroinvertebrate species and environmental variables was analysed in order to explore factors that dominantly affect community distribution pattern. Unlike the most reservoirs, strong longitudinal gradient for suspended organic matter and nutrients (total phosphorous, nitrates and nitrites) along the reservoir doesn??t exist. However, the process of thermal stratification has a strong influence on the metabolism and structure of the Mantovo ecosystem, which can be demonstrated by the vertical and longitudinal distribution of dissolved oxygen (DO), CO₂, pH and metals concentrations. Canonical Correspondence Analysis (CCA) indicated that the main factors controlling spatial distribution of Limnodrilus hoffmeisteri and Chironomus plumosus group were temperature, dissolved oxygen and manganese, including sulphates for C. plumous group. Chaoborus crystallinus showed opposite distribution pattern. Cladotanytarsus mancus group was strongly associated with shallower part (littoral and sublittoral) of Mantovo Reservoir characterized by favourable oxygen condition. None of the environmental variables included in CCA showed any relationship with density of Procladius sp.     

Parasitofauna of freshwater fishes in the Serbian open waters: a checklist of parasites of freshwater fishes in Serbian open waters

As a result of freshwater fish parasitofauna investigations, throughout the past 75 years, the number of parasitic species is presented. This paper reviews the history, current state, and tendencies of the fish parasitofauna studies in Serbian open waters. Up to now, in total 170 parasitic species have been reported. Systematic parasitofauna investigations have been carried out on 54 freshwater fish species in waters of the Danube River Basin in Serbia, also in hill-mountanious watercourses, lakes and salmonid fishpond of Sjenicko-Pesterska plateau (south-west region of Serbia), Homolje area (east Serbia), and in Vlasina lake (south-east Serbia). This data review pointed that it is necessary to continue the investigation on fish parasites in Serbia. The data on freshwater fish parasites are important for evaluation of general influence on the community structure.     

Macroinvertebrates along the Serbian section of the Danube River (stream km 1429–925)

Results of the investigation of the aquatic macroinvertebrate fauna along a 504 km stretch of the Danube River in Serbia are presented. A total of 74 macroinvertebrate taxa were observed during a 2001 survey. Oligochaeta and Mollusca were the principal components of the community with regard to species richness and abundance. Based on data on the qualitative composition of the macroinvertebrate fauna, a correspondence analysis divided the investigated stretch in three sectors — upper (Pannonian), Iron Gate sector and entrance sector to the Iron Gate stretch. The distribution patterns of certain species supported the division of sectors defined by correspondence analysis.     

Characterization of recombinant human insulin-like growth factor binding proteins 4, 5, and 6 produced in yeast.

The insulin-like growth factor binding protein (IGFBP) family comprises six structurally distinct, but highly homologous proteins. They have been identified in serum and other biological fluids, tissue extracts, and cell culture media. We have recently cloned cDNAs encoding human IGFBP-4, -5, and -6 and have now expressed these BPs in yeast as ubiquitin (Ub)-IGFBP fusion proteins. Western ligand blotting with 125I-IGF II under nonreducing conditions of recombinant human (rh) IGFBP-containing yeast lysates revealed specific binding bands for IGFBP-4, -5, and -6 at apparent molecular masses of 24-26, 30-32, and 24-26 kDa, respectively, indicating processing of the fusion proteins. High-performance liquid chromatography-purified rhIGFBPs had virually the same amino acid composition, amino acid number, and NH2-terminal sequences as the native BPs. Except for the affinity of rhIGFBP-6 for IGF I (Ka = 8.5 x 10(8) M-1), the affinity constants of the three IGFBPs for IGF I and II lie between 1.7 and 3.3 x 10(10) M-1, i.e. 25-100 times higher than the IGF I and II affinities of the type I IGF receptor. When present in excess, rhIGFBP-4, -5, and -6 inhibited IGF I- and II-stimulated DNA and glycogen synthesis in human osteoblastic cells, but rhIGFBP-6 had only a weak inhibitory effect on IGF I in agreement with its relatively lower IGF I affinity constant. The results of this study show that the primary effect of the three rhIGFBPs is the attenuation of IGF activity and suggest that IGFBPs contribute to the control of IGF-mediated cell growth and metabolism.     

Options in uraemia therapy for diabetics with end-stage renal disease

In many countries, diabetic renal disease has become, or will soon become, the single most common cause of end-stage renal disease (ESRD). End-stage renal failure (ESRF) in type-2 diabetic patients is increasing worldwide. Incidence of ESRF caused by diabetic nephropathy (DN) in 1996 in the USA was 41.7% and prevalence was 32.4%. ESRD and ESRF caused by DN was 10%, 5-15% in different haemodialysis centres in adults in the year 2000 in the Republic of Macedonia. In this review article we discuss options in uraemia therapy for diabetics with ESRD. Assessment and treatment of a diabetic with ESRD must be highly individualized. Haemodialysis (HD) has emerged as the most common treatment for all forms of renal failure including diabetic nephropathy. In diabetics patients with ESRD, dialysis is started early at creatinine clearance as high as 15-20 ml/min, at serum creatinin levels as low as 3-5 mg/dl. The first choice of HD access in diabetics is an autologous a-v fistula of the Cimino-Brescia type. The A-V fistula should be created several months before starting HD when creatinine clearance is above 20-25 ml/min. When peritoneal dialysis (PD) is selected, advance planning should ensure that a suitable peritoneal catheter is in situ 2-4 weeks before starting dialysis. HD procedures should be with low ultrafiltration rates and prolonged duration of dialysis sessions. The ultrafiltration in diabetics should not exceed more than 500-600 ml/h on HD. This means dialysis sessions of more than 4h and, in larger patients, of more than 5h HD three times per week. Renal transplantation (RT) is a safe and effective treatment modality for diabetic subjects with ESRD. Cardiovascular disease and serious infections are the major causes of death in haemodialysed and transplanted diabetics. Despite recent improvement, rehabilitation of HD diabetics continues to be inferior to that of non-diabetics. Improvement of survival is a matter of reduction of cardiovascular death and infection.