The first clinical trial for determination of alpha 1 fetoprotein by means of Sevatest-ELISA AFP Kit (micro I)

At the Institute of Sera and Vaccines, Praha, was invented and tested on clinical samples a kit for detection and quantification of alpha 1 fetoprotein in human serum. It is a heterogeneous EIA on the "sandwich" principle. Rabbit antibody to alpha 1 fetoprotein (further AFP) was used for coating the solid surface and goat horse-radish peroxidase labelled antibody to AFP was used as the tracer. Microtitration plate of Czechoslovak manufacture (KOH-I-NOOR, Dalecín) type P with 96 wells was used as the solid phase. The range of an approximately linear part of the calibration curve was intentionally chosen between 10 and 400 ng/ml, since in this way it fills the detection gap in AFP determination between 10 and 200 ng/ml, which is, on the one hand, a physiological value of AFP in human serum and, on the other hand, the bottom limit of sensitivity of counter immunoelectrophoresis (CIEP). Attention was devoted both to reproducibility of the method, i.e. results of intra- and interassays, and comparability with other foreign ELISA Kits. According to the correlation analysis, the kit was ascertained to be very well comparable with kits of foreign provenance. The coefficient of variation (CV) for the interassays varied between 11 and 16% and for intraassays it equalled 15%.     

The time course of biochemical and histological changes following carbon tetrachloride-induced liver damage in rats of both sexes

A detailed analysis is presented of the time changes in the development of liver damage 6, 12, 24, 48 and 72 hours after i.p. administration of carbon tetrachloride [CCl4] in a dose of 0.75 ml, i.e. 1 200 mg/kg body weight to rats of both sexes. The severity of liver damage was assessed from the histological and biochemical changes of AST, ALT, alkaline phosphatase and GMT serum activity. From our experiments it follows that in male rats the level of transaminases increases earlier than in female rats, as early as 6 h after the administration of CCl4, reaching a maximum 12 h later. These changes prevail for a longer time period, the level of transaminases remaining increased even 72 h after CCl4 administration. In female rats the biochemical changes occur later reaching the maximum elevation of AST and ALT 24 h after CCl4 administration. The values slowly return to normal after 48 h, and after 72 h the levels of transaminases are identical with the control group. The above given biochemical results are in good agreement with the histological findings demonstrating a higher regenerative activity in female rats. This finding was also proved by specific liver DNA activity assay.     

Big flies, small repeats: the "Thr-Gly" region of the period gene in Diptera

The region of the clock gene period (per) that encodes a repetitive tract of threonine-glycine (Thr-Gly) pairs has been compared between Dipteran species both within and outside the Drosophilidae. All the non- Drosophilidae sequences in this region are short and present a remarkably stable picture compared to the Drosophilidae, in which the region is much larger and extremely variable, both in size and composition. The accelerated evolution in the repetitive region of the Drosophilidae appears to be mainly due to an expansion of two ancestral repeats, one encoding a Thr-Gly dipeptide and the other a pentapeptide rich in serine, glycine, and asparagine or threonine. In some drosophilids the expansion involves a duplication of the pentapeptide sequence, but in Drosophila pseudoobscura both the dipeptide and the pentapeptide repeats are present in larger numbers. In the nondrosophilids, however, the pentapeptide sequence is represented by one copy and the dipeptide by two copies. These observations fulfill some of the predictions of recent theoretical models that have simulated the evolution of repetitive sequences.      

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.      

Excitation of System I and System II in Photosynthesis as a Possible Control Mechanism of Glycolate Metabolism in the Blue-Green Alga Anacystis nidulans

Photosynthetic enhancement studies performed at 619 nm (excitation of Systems I and II) and at 446 nm (mainly excitation of System I) revealed an 18% photosynthetic enhancement simultaneously with a 31% reduction in glycolate excretion. This observation supports the hypothesis that some glycolate may be consumed in an oxidation process associated with System I when System II is poorly excited and the supply of electrons from the water splitting process of photosynthesis is low.     

Direct and indirect evidence of size-selective grazing on pelagic bacteria by freshwater nanoflagellates

Size-selective grazing of three heterotrophic nanoflagellates (with cell sizes of 21, 44, and 66 mum) isolated from Lake Arlington, Texas was examined by using a natural mixture of fluorescence labelled lake bacteria. Sizes of ingested bacteria in food vacuoles were directly measured. Larger bacterial cells were ingested at a frequency much higher than that at which they occurred in the assemblage, indicating preferential flagellate grazing on the larger size classes within the lake bacterioplankton. Water samples were collected biweekly from June through September, 1989, fractionated by filtration, and incubated for 40 h at in situ temperatures. The average bacterial size was always larger in water which was passed through 1-mum-pore-size filters (1-mum-filtered water) (which was predator free) than in 5-mum-filtered water (which contained flagellates only) or in unfiltered water (in which all bacterivores were present). The increase of bacterial-cell size in 1-mum-filtered water was caused by a shift in the size structure of the bacterioplankton population. Larger cells became more abundant in the absence of flagellate grazing.     

Adequate enzymatic substitution in treating exocrine pancreatic insufficiency.

The chymotrypsin in the stool test was used to monitor adequate enzymatic substitution in treating exocrine pancreatic insufficiency with 18 patients (16 suffering from chronic pancreatitis and 2 having passed duodenopancreatectomy due to pancreatic cancer). This test helps to identify pancreatic insufficiency and can be successfully used in monitoring the adequate amount of pancreatic substitute, which, we have found, differs from patient to patient. The dosage can be higher in cases of chronic pancreatitis than in those required after duodenopancreatectomy.     

The anabolic action of intermittent parathyroid hormone on cortical bone depends partly on its ability to induce nitric oxide‐mediated vasorelaxation in BALB/c mice

There is strong evidence that vasodilatory nitric oxide (NO) donors have anabolic effects on bone in humans. Parathyroid hormone (PTH), the only osteoanabolic drug currently approved, is also a vasodilator. We investigated whether the NO synthase inhibitor L‐NAME might alter the effect of PTH on bone by blocking its vasodilatory effect. BALB/c mice received 28 daily injections of PTH[1–34] (80 µg/kg/day) or L‐NAME (30 mg/kg/day), alone or in combination. Hindlimb blood perfusion was measured by laser Doppler imaging. Bone architecture, turnover and mechanical properties in the femur were analysed respectively by micro‐CT, histomorphometry and three‐point bending. PTH increased hindlimb blood flow by >30% within 10 min of injection (P < 0.001). Co‐treatment with L‐NAME blocked the action of PTH on blood flow, whereas L‐NAME alone had no effect. PTH treatment increased femoral cortical bone volume and formation rate by 20% and 110%, respectively (P < 0.001). PTH had no effect on trabecular bone volume in the femoral metaphysis although trabecular thickness and number were increased and decreased by 25%, respectively. Co‐treatment with L‐NAME restricted the PTH‐stimulated increase in cortical bone formation but had no clear‐cut effects in trabecular bone. Co‐treatment with L‐NAME did not affect the mechanical strength in femurs induced by iPTH. These results suggest that NO‐mediated vasorelaxation plays partly a role in the anabolic action of PTH on cortical bone. © 2016 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.     

Repeated immunostaining of the same tissue section using alkaline phosphatase as a reporter

One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80^o C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier.     

Barley microgreen incorporation in diet-controlled diabetes and counteracted aflatoxicosis in rats

Increased environmental pollution and unhealthy lifestyle are blamed for escalated chronic diseases. Exposure to aflatoxins was recently suggested to have a role in the increased incidence of type 2 diabetes mellitus. Diet modification and consumption of different functional food are now gaining attention, especially in diabetes management. This study investigates the effect of a diet containing barley microgreen against diabetes induced by streptozotocin with or without aflatoxin administration in rats. Barley microgreen was rich in 3′-Benzyloxy-5,6,7,4′-tetramethoxyflavone (48.8% of total) followed by 5β,7βH,10α-Eudesm-11-en-1α-ol (18.46%). Streptozotocin injection and/or aflatoxin administration significantly elevated glucose level, decreased insulin level, decreased β-cell function, deteriorated liver and kidney function parameters, and induced oxidative stress in the liver. Histopathology revealed irregular small-sized islets and decreased area % of insulin-positive beta cells in the pancreas, hepatic degeneration, nephropathy, and neuropathy in diabetic and/or aflatoxin administered rats compared to control. Barley microgreen diet fed to diabetic rats with or without aflatoxin alleviated all evaluated parameters. Barley microgreen diet also ameliorated the toxic effect of aflatoxin. In conclusion, exposure to aflatoxin aggravated diabetes and its complication. The incorporation of barley microgreen in the diet was able to control type 2 diabetes mellitus and the improved outcomes observed with barley microgreen treatments involved or occurred in conjunction with improved biomarkers of oxidative stress.