Prothrombin cleavage by human vascular smooth muscle cells: A potential alternative pathway to the coagulation cascade

Thrombin is a potent mitogen for human vascular smooth muscle cells (HVSMC) and its enzymatic activity is required for this function. The present study demonstrates that prothrombin is also mitogenic for HVSMC due to the generation of enzymatically active thrombin which occurs upon incubation of prothrombin with the cells. Analysis by SDS-PAGE, immunoblotting, and amino acid sequencing revealed that prothrombin incubated with HVSMC undergoes limited proteolysis. Prethrombin 1 was formed through cleavage at R¹⁵⁵-S¹⁵⁶. Cleavage at R²⁷¹-T²⁷² generated fragment 1.2 and prethrombin 2 whilst cleavage at R²⁸⁴-T²⁸⁵ yielded truncated prothrombin 2 (prethrombin 2′). However, cleavage at R³²⁰-I³²¹ which, during prothrombin activation produces two-chain α-thrombin, was not detectable. Studies on HVSMC-conditioned medium revealed that a similar pattern of prothrombin cleavage occurred by a cell-secreted factor(s). Amidolytic activity analysis indicated that 1–3% catalytically active thrombin-like activity was generated upon incubation of prothrombin with HVSMC-conditioned medium. By treating conditioned medium with various classes of proteinase inhibitors or hirudin, it was determined that prothrombin is cleaved by a cell-derived serine proteinase-like factor(s) at R²⁷¹-S²⁷² and by α-thrombin at R¹⁵⁵-S¹⁵⁶ and R²⁸⁴-T²⁸⁵. Antibodies neutralising the activity of either urokinase, tissue plasminogen activator, or factor Xa failed to alter the prothrombin cleaving activity of conditioned medium. This activity which may catalyse an alternative pathway for the generation of thrombin, was eluted from a gel filtration column as a single peak with apparent molecular mass of 30–40 kDa. © 1995 Wiley-Liss, Inc.     

Identification,characterization, and developmental profile of a high molecular weight,juvenile hormone-binding protein in the hemolymph of the migratory grasshopper,Melanoplus sanguinipes

In the hemolymph of Melanoplus sanguinipes, a high molecular weight juvenile hormone binding protein (JHBP) was identified by photoaffinity labelling and found to have a M_r of 480,000. The JHBP, purified using native gel electrophoresis followed by electroelution, has an equilibrium dissociation constant for JH III of 2.1 nM and preferentially binds JH III over JH I. Antibody raised against JHBP recognized only the 480,000 band. Under denaturing conditions the native JHBP gave a single band with a M_r 78,000. The antibody against native JHBP recognized only the 78,000 protein in SDS-treated hemolymph samples, indicating that JHBP is a hexamer in this species. The concentration of JHBP fluctuates in both the sexes during nymphal and adult development in parallel with total protein content of hemolymph. © 1995 Wiley-Liss, Inc.     

Genetic regulation of gibberellin deactivation in Pisum

The regulation of gibberellin (GA) deactivation was examined using the sin (slender) mutation in the garden pea (Pisum sativum L.). This mutation blocks the deactivation of GA₂₀, the precursor of the bioactive GA₁. Firstly, crosses were made to combine sin with the GA biosynthesis mutations na, lhⁱ and le-3. The combination sin na produced a novel phenotype, with long (‘slender’) basal internodes and extremely short (‘nana’) upper internodes. In contrast, the double mutant sin lhⁱ was phenotypically dwarf. The mutation sin causes an accumulation of GA₂₀ in maturing seeds, and this was unaffected by na, since the na mutation is not expressed in seeds. In contrast, lhⁱ seeds did not accumulate GA₂₀, since lhⁱ imposes an early block on GA biosynthesis. Secondly, the effects of sin on several steps in GA deactivation were investigated. In maturing seeds, the mutation sin blocks two steps in GA₂₀ metabolism, namely, GA₂₀ to GA₂₉, and GA₂₉ to GA₂₉-catabolite. In the vegetative plant, on the other hand, sin blocked the step GA₂₀ to GA₂₉, but not GA₂₉ to GA₂₉-catabolite; the steps GA₂₀ to GA₈₁ and GA₂₀ to GA₁ were also not impaired in this mutant. It is clear that the effects of sin, like those of na, are strongly organ-specific. The presence of separate enzymes for the steps GA₂₀ to GA₂₉ and GA₂₉ to GA₂₉-catabolite was suggested by the observation that GA₈ inhibited the latter step, but not the former, and by the inability of GA₂₀ and GA₂₉ to inhibit each other's metabolism. It is suggested that the Sin gene may be a regulatory gene controlling the expression of two structural genes involved in GA deactivation.     

Copper distribution in the normal human brain

Copper concentration was determined in samples from 38 areas of 7 normal human brains. The grey matter contained higher concentrations of copper than the white matter. Identical areas of the grey and white matter of the cerebral cortex showed significant differences between individuals. In the caudate nucleus the highest concentrations of copper were found in the tail followed by the body and the head, respectively. A negative linear regression between age and brain copper levels was demonstrated.     

Acclimation to temperature and death at changing lethal temperatures in the freshwater fish Chalcalburnues chalcoides

1. 1.|In the freshwater fish Chalcalburnus chalcoides, an increase in the body (standard) size caused decreases in the upper LT-50 from 36.6° to 36.0°C and lower LT-50 from 6.3° to 5.3°C

2. 2.|The fish acclimated to constant temperatures between 10°C and 30°C showed reasonable heat acclimation and also reasonable cold acclimation. Thus, an increase in the acclimation temperature from 10°C to 30°C caused increases in the upper LT-50 from 34° to 36.2°C and the lower LT-50 from 1.25 to 6.5°C.

3. 3|The mean survival time — temperature curves of 10°, 20° and 30°C acclimated fish at various constant temperatures showed decreased in the survival tim ewith increasing lethal temperatures. Furthermore, an increase in the acclimation temperature causes a shift in the survival duration-temperature curve to the right, i.e., the fish become more heat resistant. Thus, the mean survival duration of 10°, 20° and 30°C acclimated fish at 35°C were 7.5, 79.6 and 530 minutes, respectively.

4. 4.|The effect of the thermal experience to changing lethal temperatures depends on the first lethal temperature to which the fish were exposed as well as the sequence of temperature changes. In the experiments in which the first lethal temperatures were between 32° and 34°C and the temperature was varied in an ascending order, their thermal resistance was increased and the fish required 114 to 174% of the expected lethal doses to die while in the experiments in which the starting temperature were between 38° and 40°C and the temperature varied in descending order, the fish become more sensitive to the upper lethal temperature and they died after receiving only 62 to 81% of the expected lethal doses. Thus, with a gradual increase in the lethal temperature, the fish show additional acclimation in the zone of resistance which in turn causes an increase in the thermal resistance. This may have ecological significance in nature.

A comparative study on the formation of citric acid and polyols and on morphological changes of three strains of free and immobilized Aspergillus niger

Summary The formation of citric acid, oxalic acid, erythritol and glycerol by three strains of Aspergillus niger immobilized in calcium alginate was investigated and compared with that of free cells when cultivated in shake flasks under phosphate limitation. Morphological changes were followed using an electron microscope. The production of acids and polyols, the consumption of glucose and fructose, and also the morphological changes were strain-dependent. The results also reflected the influence of long storage of a strain on productivity, morphological behaviour and phosphate consumption. Offprint requests to: H.-J. Rehm     

Internode length inPisum: Genelkc

A new single gene-recessive internode length mutant inPisum, lkc, is characterized. The internodes oflkc plants are 30–40% shorter than those of comparableLkc plants, and this is attributable to reductions in both cell length and the number of cells per internode. Dwarfism in the mutant is not due to modified gibberellin (GA) levels, as determined by gas chromatography-selected ion monitoring (GC-SIM) for GA₁ and GA₂₀, and bioassay (rice cv. Tan-ginbozu). Furthermore,lkc plants are not as responsive as the wild-type to applied GA₁. The diminished stature oflkc plants appears to result from a direct or indirect interference with the transduction of the GA₁ signal.     

Cell surface T antigen in cells infected with simian virus 40 or an adenovirus-simian virus 40 hybrid, Ad2+D2.

Cell surface T antigen, detected by a radioimmune assay that uses 125I-labeled Staphylococcus aureus protein A and antibodies against either authentic T antigen or D2 hybrid T antigen, was found in simian virus 40-transformed and -infected cells and in cells infected with an adenovirus-simian virus 40 hybrid, Ad2+D2. In simian virus 40 lytic infection, the surface T antigen appeared at the same time as the nuclear T antigen.     

Physiology and characterization of a fungal milk-clotting enzyme from Aspergillus flavus.

Aspergillus flavus produced extracellularly an active rennin-like enzyme when grown aerobically in whey media. The enzyme was detected at early stages of growth reaching a maximum after three to four days at 25 degrees. The activity was destroyed by heating to temperatures higher than 50 degrees, whereas the presence of skim milk during heating preserved the enzyme activity, at least, up to 70 degrees. Calcium chloride significantly stimulated the milk-clotting activity up to 1% final concentration. The clotting time was inversely proportional to protein concentration in the range 0.2-0.6 mg/ml and the enzyme exhibited marked stability when stored at 37 degrees at pH 6.