[Morphology and diagnosis of the oral protozoans Trichomonas tenax and Entamoeba gingivalis using the Giemsa-Romanovsky stain]. / Morfológia a diagnostika ústnych prvokov Trichomonas tenax a Entamoeba gingivalis zafarbených podl'a Giemsu-Romanovského.

In the microscopic diagnosis of Trichomonas tenax and Entamoeba gingivalis is the technically and time not demanding native preparation of a culture, in which both protozoans can be detected according to their typical motility, determining. In the permanent preparation of the culture stained according to Giemsa-Romanovsky, which has also documentary character, are all of the characteristic cell organelles stainable, enabling so their detection without their typical motility. Staining according to Giemsa-Romanovsky is technically simple and not time consuming, not very laborious, low cost and the coloration is permanent, that means optimal for the diagnostic of oral protozoans in permanent preparations. (Fig. 5, Ref. 4.)

Selective oxidation of methionyl residues in the human recombinant secretory leukocyte proteinase inhibitor. Effect on the inhibitor binding properties.

Binding of the human recombinant secretory leukocyte proteinase inhibitor (SLPI) [native and with the methionyl residues at positions 73, 82, 94 and 96 of domain 2 oxidized to the sulfoxide derivative (Met(O) SLPI)] to bovine alpha-chymotrypsin (alpha-chymotrypsin) [native and with the Met192 residue converted to the sulfoxide derivative (Met(O) alpha-chymotrypsin)] as well as to native bovine beta-trypsin (beta-trypsin), which does not contain methionyl residues, has been investigated between pH 4.0 and 8.0, and between 10.0 degrees C and 30.0 degrees C, from thermodynamic and/or kinetic viewpoints. By increasing the number of oxidized methionyl residues present at the proteinase:inhibitor contact interface (from 0 to 3), the adducts investigated are increasingly destabilized and the relaxation time of the complexes into conformers less stable is enhanced. On the other hand, the selective oxidation of methionyl residues of SLPI and alpha-chymotrypsin, by reaction with chloramine T, does not affect the proteinase inhibition recognition mechanism. Therefore, even though conformational changes may occur in the conversion of native SLPI and native alpha-chymotrypsin to their Met(O) derivatives, a localized steric hindrance can be considered as the main structural determinant accounting for the reported results.

Selective oxidation of Met-192 in bovine alpha-chymotrypsin. Effect on catalytic and inhibitor binding properties.

Catalytic and inhibitor binding properties of bovine alpha-chymotrypsin, in which the Met-192 residue has been converted by treatment with chloramine T to the sulfoxide derivative (Met(O)192 alpha-chymotrypsin), have been examined relative to the native enzyme (alpha-chymotrypsin), between pH 4.5 and 8.0 (mu = 0.1), and/or 5.0 degrees C and 40.0 degrees C. Values of kcat, k+2 and/or k+3 for the hydrolysis of all the substrates examined (i.e., tMetAcONp, ZAlaONp, ZLeuONp, ZLysONp and ZTyrONp) catalyzed by native and Met(O)192 alpha-chymotrypsin are similar, as well as values of Km for the hydrolysis of ZLeuONp, ZLysONp and ZTyrONp. On the other hand, Ks and Km values for the hydrolysis of ZAlaONp and tMetAcONp are decreased by about 5-fold. Met-192 oxidation does not affect the kinetic and thermodynamic parameters for the (de)stabilization of the complex formed between the proteinase and the bovine basic pancreatic trypsin inhibitor. On the other hand, the recognition process between between alpha-chymotrypsin and the recombinant proteinase inhibitor eglin c from the leech Hirudo medicinalis is influenced by the oxidation event. Considering known molecular models, the observed catalytic and inhibitor binding properties of native and Met(O)192 alpha-chymotrypsin were related to the inferred stereochemistry of the proteinase-substrate and proteinase-inhibitor contact region(s).

Hemoglobin Dallas (alpha 97(G4)Asn-->Lys): functional characterization of a high oxygen affinity natural mutant.

Hemoglobin Dallas, an alpha-chain variant with a substitution of lysine for asparagine at position 97(G4), was found to have increased oxygen affinity (p1/2 = 1 mmHg at pH 7.3 and 20 degrees C), diminished cooperativity (n, the Hill coefficient = 1.7) and reduced Bohr effect (about 50%). Addition of allosteric effectors (such as 2,3-diphosphoglycerate, inositol hexakisphosphate and bezafibrate) led to a decrease in oxygen affinity and increase in cooperative energy. Kinetic studies at pH 7.0 and 20 degrees C revealed that (i), the overall rate of oxygen dissociation is 1.4-fold slower than that for HbA and (ii), the carbon monoxide dissociation rate is unaffected. The abnormal properties of this hemoglobin variant can be attributed to a more 'relaxed' T-state.

[Occurrence of the protozoa, Entamoeba gingivalis and Trichomonas tenax in the mouths of children and adolescents with hyperplastic gingivitis caused by phenytoin]. / K výskytu prvokov Entamoeba gingivalis a Trichomonas tenax v ústach detí a mládeze s hyperplastickou gingivitídou po fenytoíne.

The oral protozoa Entamoeba gingivalis and Trichomonas tenax do not occur in small children and are rarely found in older ones. In adolescents their occurrence rate keeps increasing with age. They parasitize in an oral cavity changed by inflammation, yet also in a healthy mouth. Their highest occurrence rate has been recorded in adults with periodontosis and atrophy of the periodontium, a somewhat lower one in adults with gingivitis. The authors addressed the question whether the presumed low occurrence rate of oral protozoa in children becomes increased in drug-induced gingivitis after treatment with the antiepileptic 5,5-diphenylhydantoin. Cultivation for oral protozoa was performed in 231 children and adolescents. Of these 59 were epileptics. Drug-induced gingivitis was present in 66% of the epileptics. Drug-induced gingivitis did not increase the occurrence rate of oral protozoa as compared to the findings in the rest of the series studied. (Tab. 9, Ref. 7.).

[Morphology and diagnosis of Entamoeba gingivalis and Trichomonas tenax and their occurrence in children and adolescents]. / Morfológia a diagnostika Entamoeba gingivalis a Trichomonas tenax a ich výskyt u detí a mládeze.

Specimens obtained from 176 eight- to nineteen-year-old subjects were cultured for oral protozoa. Of the given series, 25 subjects (14.2%) had in their mouth only E. gingivalis, 5 (2.9%) had a mixed invasion of E. gingivalis and T. tenax, and 2 subjects (1.1%) had only T. tenax. A total of 32 subjects (18.2%) were infested by oral protozoa. The following conclusions were made: oral protozoa occur also in children and teenagers with a cured or intact dentition; their occurrence rate was higher in 11- to 19-year-old subjects than in the lower age groups; both protozoa can occur simultaneously; their occurrence rate was age dependent (increasing with age) with the rate of E. gingivalis rising significantly more rapidly with age than that of T. tenax; their occurrence was found to be sex dependent with higher rates in boys than in girls. In the light of the high recovery rate (83.3%) of E. gingivalis from dental and periodontal swabs, not only specimens of saliva should be collected but also dental nad periodontal swabs should be taken and cultured when studying the occurrence of oral protozoa in the population. (Tab.4,Fig.2,Ref.7).

Purification of the proteolytically solubilized, active catalytic subunit of adenylate cyclase from ram sperm. Inhibition by adenosine.

Ram spermatozoa adenylate cyclase is insensitive to all usual regulatory processes. The purification of its active catalytic subunit was accomplished after proteolytic solubilization of a particulate fraction by alpha-chymotrypsin. The purification (26,000-fold from the particulate fraction or 125,000-fold from the whole-sperm proteins) was achieved by conventional procedures (DEAE-Trisacryl, Ultrogel AcA 34, DEAE-Sephacel, hydroxyapatite), in the absence of detergent, and with a yield of 5-10% and a final specific activity of 19 mumol cyclic AMP formed mg protein-1 min-1 at 30 degrees C in the presence of manganese as cosubstrate. The solubilized enzyme, stable at the beginning of the purification procedure, became unstable at the later stages. After the last step (chromatography on hydroxyapatite) half-lives of 27 min, 50 min and 160 min were obtained at 30 degrees C, 20 degrees C and 4 degrees C respectively. The enzyme was stabilized by addition of bovine serum albumin and Lubrol PX, 80% of the activity remaining after 24 h at 4 degrees C. The purified enzyme exhibited a Km value similar to that of the native enzyme (Km = 1.4 mM). Unlike the native enzyme, the purified enzyme has an absolute requirement for MnATP; no significant activity was recovered in the presence of MgATP. Adenosine inhibited the activity of both the native and purified forms of the enzyme to the same extent and in a non-competitive manner. This indicates that adenosine acts on the catalytic component itself and the inhibition site and the catalytic site are different. Data obtained with adenosine analogs indicate that adenosine interacts with the cyclase catalytic subunit with a 'P-site' specificity. The purified adenylate cyclase, which had an apparent molecular mass of 38 kDa on a high-performance liquid chromatography column [Stengel, D., Guenet, L. and Hanoune, J. (1982) J. Biol. Chem. 257, 10,818-10,826], gave a doublet of 36 kDa and 34 kDa on sodium dodecyl sulfate gel electrophoresis. This represents the smallest protein entity associated with adenylate cyclase activity so far reported.

Inhibition of the catalytic subunit of ram sperm adenylate cyclase by adenosine.

Adenosine inhibits ram sperm adenylate cyclase activity which is membrane-bound and comprises only the catalytic subunit. The inhibition parameters of adenylate cyclase by adenosine were not modified when the enzyme was purified 3 to 5,000 fold. Optimal inhibition by adenosine was found to require a high concentration of manganese, and exhibited a noncompetitive pattern up to a concentration of 1 mM adenosine. Adenosine was the most potent inhibitor among various analogs tested with the following rank order of potencies: adenosine greater than 2'O-methyladenosine greater than 2'deoxyadenosine much greater than 2 chloroadenosine. Studies with agonists and antagonists of the "R"-type adenosine receptor led us to conclude that adenosine inhibits ram sperm adenylate cyclase via a "P"-site carried by the catalytic subunit itself.

Studies on the influence of ochratoxin A on rat lenses.

The influence of ochratoxin A on enzyme activities and on the content of free adenine nucleotides and intermediates of glycolysis in rat lenses was investigated. For a period of eight weeks, five times weekly, albino Wistat rats received ochratoxin A in 0.1% NaHCO3, dosed 1/100 LD50, via stomach tube. All measurements were made at the end of the experiment. Decreased contents of ATP, G-6-P, sum of fructose, and F-6-P were observed, but pyruvic acid, AMP, and DAP increased. Relatively decreased activities of enzymes MDH, ALD, HK, GK, and PK were established. The influence of ochratoxin A on the carbohydrate metabolism of rat lenses with respect to tranparency is discussed. There were no significant differences in absolute lens weight in animals treated by ochratoxin A and controls.