Can co-culturing of two white-rot fungi increase lignin degradation and the production of lignin-degrading enzymes?

The aim of this work was to investigate the poorly understood effects of co-culturing of two white rot fungi on the production of lignin-degrading enzyme activities. Four species, Ceriporiopsis subvermispora, Physisporinus rivulosus, Phanerochaete chrysosporium and Pleurotus ostreatus were cultured in pairs to study the degradation of aspen wood and the production of lignin-degrading enzymes. Potential of co-culturing for biopulping was evaluated. Chemical analysis of decayed aspen wood blocks showed that co-culturing of C. subvermispora with P. ostreatus could significantly stimulate wood decay, when compared to monocultures. Based on the fungi tested here, however, this effect is species-specific. Other combinations of fungi were slightly stimulating or not stimulatory. The pattern of lignin degradation was altered towards the acid insoluble part of lignin especially in co-cultures where P. ostreatus was included as a partner. The use of agar plates containing the polymeric dye Poly R-478 showed elevated dye decolourization at the confrontation zone between mycelia. Laccase was significantly stimulated only in the co-culture of P. ostreatus with C. subvermispora. Manganese peroxidase activity was stimulated in co-cultures of P. ostreatus with C. subvermispora or with P. rivulosus. Immunoblotting indicated changes in lignin-degrading enzymes and/or their isoform composition in response to co-culturing. This is the first report on the effects of co-culturing of potential biopulping fungi on wood degradation, and gives basic knowledge on fungal interactions during wood decay that can be utilized in practical applications.     

Conversion of 17α-hydroxypregnenolone to 17α-hydroxyprogesterone by the canine testis in vivo

17α-Hydroxypregnenolone labeled with tritium in position 7 has been infused at a constant rate by the left spermatic artery of anesthetized dogs. Testosterone, δ⁴-androstenedione, dehydroepiandrosterone and 17α-hydroxyprogesterone containing tritium were isolated from the venous blood of the left testis.

• 1.1. 17α-Hydroxypregnenolone is capable of being converted to 17α-hydroxyproegsterone by the canine testis in vivo.
• 2.2. When the testis is stimulated with human chrionic gonadotropin there is an increased conversion of 17α-hydroxypregnenolone to 17α-hydroxyprogesterone as well as an increase in testosterone production.
• 3.3. Infusion of large amounts of unlabeled pregnenolone may also increase the conversion of 17α-hydroxypregnenolone to 17α-hydroxyprogesterone.
     

Postnatal development of uterine response to estradiol-17β in the rat

The gradual acquisition of uterine responsiveness to a single injection of estradiol-17β was studied in rats aged 5–30 days with respect to wet weight and the content of DNA, RNA, and protein. No significant response in any of these parameters was found in 5- or 10-day-old rats. In 15- and 20-day-old rats all the parameters, except DNA content, were elevated by the hormone treatment; only in 30-day-old rats was there a significant increase in DNA content.     

Retroperitoneal white adipose tissue mitochondrial function and adiponectin expression in response to ovariectomy and 17β-estradiol replacement

Sexual dimorphism has been previously found both in mitochondrial biogenesis and function and in adiponectin expression of retroperitoneal WAT. However, little is known about the E2 effects on WAT mitochondrial function. Accordingly, the aim of this study was to examine in greater depth the role of estrogens in sexual dimorphism. This was accomplished by studying the effects of ovariectomy and E2 replacement on retroperitoneal WAT mitochondrial function. Fourteen-week-old female and ovariectomized (OVX) female Wistar rats were used in this study. The ovariectomy was performed at 5 weeks of age and at 10 weeks of age OVX rats were divided into two experimental groups: OVX, and OVX treated with 17β-estradiol (E2) (OVX+E2). Subcutaneous injections of E2 (10 μg/kg/48 h) were administered to the OVX+E2 rats for 4 weeks previous to the sacrifice whereas OVX rats were treated only with the vehicle. Levels of the main markers for mitochondrial biogenesis and function and those representatives of the antioxidant defense system and insulin sensitivity were determined. Additionally, the mRNA levels of the α and β estrogen receptors and of some adipocyte differentiation markers were studied. Our results indicate that retroperitoneal WAT was able to adapt itself to ovariectomy without any changes in mitochondrial function markers or for the adiponectin levels. However, E2 supplementation led to an unexpected decrease in: TFAM protein levels, in LPL, PPARγ and adiponectin gene expression and in the systemic HMW adiponectin levels. This decrease is probably due to the down-regulation of the ERα mRNA expression to avoid an over-stimulation by E2.     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

Spermine synthesis in the uterus of the ovariectomized rat in response to oestradiol-17β

We reported that spermidine and spermine pools in the uterus both doubled within 24h after oestradiol administration to castrated rats (Russell & Taylor, 1971). Now we have studied the enzymic synthesis of spermine (by spermidine-dependent S-adenosyl-l-methionine decarboxylase) and find that the activity of the enzyme(s) involved is elevated soon after hormone administration. Enzyme activity is increased within 4h and is five times that of controls within 24h. Cycloheximide or actinomycin D administered at the time of oestradiol injection completely blocked the increase in enzyme activity. The enzyme involved in spermine synthesis, S-adenosyl-l-methionine decarboxylase, with S-adenosyl-l-methionine and spermidine as required substrates, was partially purified on Sephadex and DEAE-cellulose columns. The decarboxylation of S-adenosyl-l-methionine could not be separated from the transfer of a propylamine moiety from the decarboxylated S-adenosyl-l-methionine to spermidine to form spermine. We were unable also to separate this system from the enzyme that formed spermidine when S-adenosyl-l-methionine and putrescine are used as substrates. Spermidine-stimulated S-adenosyl-l-methionine decarboxylase has an apparent half-life of 60min, identical with the half-life reported for putrescine-stimulated S-adenosyl-l-methionine decarboxylase. These results strongly suggest that the same enzyme(s) operate in the synthesis of both spermidine and spermine.     

Conversion of 17α-pregnenolone to 17α-progesterone by rat ovarian homogenate

• 1.1. 17α-Pregnenolone, a synthetic Δ⁵-3β-hydroxy-C₂₁-steroid with a side-chain of 17α configuration, was converted by homogenates of rat ovaries to 17α-progesterone. The product was identified by paper and gas-liquid chromatography and ultraviolet spectrophotometry.
• 2.2. Isopregnenolone was oxidized at about the same rate as the naturally occurring pregnenolone: steric hindrance by the axial 17α side-chain might have been expected if attachment of the enzyme was to the α-surface of the steroid as has been proposed for a bacterial 3β-ol-dehydrogenase.
• 3.3. The question is raised whether more than one enzyme protein is involved in the oxidation of the 3β-hydroxyl group of C₁₉-and C₂₁-steroids by ovarian tissue.
• 4.4. Epimerization at C-17 did not occur during incubations of pregnenolone with ovarian homogenates. Formation of a 17α-pregnane compound de novo has been reported, however, in the human male.

     

Changes in ascorbate–glutathione pathway enzymes in response to Mycosphaerella fragariae infection in selected strawberry genotypes

Ten strawberry genotypes, resistant and moderately resistant (Joliette, Seascape, Aromas, FIN005-55 and FIN005-50) and susceptible ones (FIN00132-8, FIN00134-11, FIN00132-14, FIN005-7 and Kent) were used to assess the role of the antioxidative defence system against Mycosphaerella fragariae infection. The pathogen-induced changes of hydrogen peroxide (H₂O₂) and antioxidant enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) involved in the ascorbate–glutathione (ASC–GSH) cycle were examined in leaves of the selected genotypes. A significant different response was observed among the genotypes. A marked increase in H₂O₂ content, APX, MDHAR, DHAR and GR activities were observed in resistant and moderately resistant genotypes after inoculation by M. fragariae. In contrast, weak changes were observed in susceptible genotypes for the aforementioned enzymes and compounds. It seems that resistant genotypes capable of overproducing H₂O₂ have a higher capacity to scavenge and reduce the injury to strawberry leaves by regulating the ASC–GSH cycle. The results may be useful in future breeding programmes to select those individuals with high scavenging properties to breed new resistant lines.     

Changes in the patterns of synthesis of ribonucleic acid species in immature rat uterus in response to oestradiol-17β

1. After treatment of immature rats with diethylstilboestrol, the wet weight and RNA content of uterine tissue increased rapidly, reaching a peak at 40hr. After an initial lag of a few hours, the acid-soluble ribose and protein contents also rose to maxima at 40hr. No increase in DNA content occurred until at least 24hr. after treatment. 2. The RNA from immature rat uterus isolated at various times up to 6hr. after administration of oestradiol-17beta was labelled by injecting [(3)H]uridine and [(3)H]guanosine intraperitoneally 30min. before the animals were killed. It was fractionated on columns of kieselguhr coated with methylated serum albumin and the radioactivity in fractions corresponding to transfer RNA, 7s RNA, ribosomal RNA, Q(1)-RNA, Q(2)-RNA and DNA-like RNA was determined. 3. The radioactivity of the whole RNA increased steadily for 6hr. after hormone treatment. The earliest changes occurred in the Q(1)-RNA (ribosomal RNA precursor), whereas at longer time-intervals the radioactivity of the ribosomal RNA, 7s RNA and transfer RNA increased by four- to five-fold. The radioactivity of the DNA-like RNA increased by about 50%, but only at the longer time-intervals. 4. It is concluded that one of the earliest changes in response to oestradiol treatment is a major increase in synthesis of ribosomal RNA followed later by a similar increase in synthesis of transfer RNA and by a much smaller increase in synthesis of DNA-like RNA. The change in synthesis of ribosomal RNA in immature rat uterus may represent one of the most important responses to oestradiol treatment.     

Differential expression of secreted phosphoprotein 1 in response to estradiol-17β and in ovarian tumors in chickens

Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2-Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2-Robo2 signaling is also important for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that overexpression of Robo2 by microinjection in embryonic kidneys is an effective approach to study the function of Robo2.     

Immobilization of fungal nitrilase and bacterial amidase – two enzymes working in accord

A nitrilase from Aspergillus niger and an amidase from Rhodococcus erythropolis co-immobilized on a 1-mL Butyl Sepharose column were used for the hydrolysis of 4-cyanopyridine into isonicotinic acid. The former enzyme converted the nitrile into the acid:amide mixture (molar ratio ca. 3:1), while the latter enzyme hydrolyzed the amide by-product. Therefore, the ratio of amide in the total product decreased to about 5%. Sodium sulfate was used as a component of the elution buffer, as the commonly used ammonium sulfate (0.8 M) acted as an amidase inhibitor. The hydrolysis of 4-cyanopyridine by a nitrilase from F. solani gave isonicotinic acid and isonicotinamide at a molar ratio of about 98:2. When using this enzyme and the amidase immobilized on two columns operated in tandem, the percentage of isonicotinamide in total product decreased to <0.2%.     

Laboratory-scale production of lignin-degrading enzymes by free and entrapped cells ofPhanerochœte chrysosporium in a tubular air-lift bioreactor

The production of lignin-degrading enzymes by free and entrapped cells ofPhanerochœte chrysosporium in a tubular air-lift bioreactor was studied. Under optimized cultural conditions the production of lignin peroxidase by free cells, calcium-alginate-entrapped cells and scouring-mesh-entrapped cells was in a ratio of 520∶720∶950 mU/mL, while the production of manganese peroxidase was in a ratio of 350∶480∶620 mU/mL. The stability of the entrapped cells by fed-batch systems was highest after 3 feeding experiments which is similarly demonstrated in the repeated use of the preparations in batch system.     

Testicular in vitro conversion of progesterone to testosterone and androstenedione in 17α-hydroxylase deficiency

Incubations of [³H]-progesterone with testicular tissue obtained from a new case of male with 17α-hydroxylase deficiency were performed. The per cent conversion to androstenedione and testosterone was virtually absent when compared to that obtained from an identical incubation performed using testicular tissue from a normal male with cryptochordism. The findings provide an in vitro evidence in support of the existence of 17α-hydroxylase testicular defect in this disorder.     

Urea cycle enzymes in white blood cells—ii. Arginase activity in some types of human leukemia

• 1.1. The activity of the “Krebs-Henseleit-Cycle” enzymes was investigated in crude extracts of leucocytes of patients with chronic lymphocytic leukemia (CLL), chronic granulocytic leukemia (CML) and osteomyelosclerosis (OMSL).
• 2.2. The average arginase activity in 25 CLL leucocyte extracts is about 30 times lower than the value obtained previously from normal donors. In contrast, the arginase activities in 13 CML leucocyte extracts do not show significant differences to the extracts of normal donor leucocytes.
• 3.3. No significant activities of N-acetylglutamate dependent carbamoylphosphate-synthetase (CPS I), glutamine dependent carbamoylphosphate-synthetase (CPS II), ornithine-carbamoyltransferase (OCT) and arginine-synthetase-system (ASS) were found.

     

Activities of thiamine-dependent enzymes in two experimental models of thiamine-deficiency encephalopathy 2. α-ketoglutarate dehydrogenase

Chronic thiamine deprivation in the rat leads to selective neuropathological damage to pontine structures. Onset of neurological symptoms of thiamine deprivation (ataxia, loss of righting reflex) was accompanied by selective decreases (of the order of 30%) in the activity of -ketoglutarate dehydrogenase (KGDH) in lateral vestibular nucleus and hypothalamus. Enzyme activities were decreased to a lesser extent in medulla oblongata, striatum and hippocampus and were unchanged in other brain structures. No changes in KGDH occurred prior to the onset of neurological signs of thiamine deprivation. Administration of the central thiamine antagonist, pyrithiamine, results within 3 weeks in loss of righting reflex and convulsions and in more widespread neuropathological changes than those observed following thiamine deprivation. KGDH activities were found to be substantially diminished in all brain regions studied following pyrithiamine treatment with most severe changes occurring in brain regions found to be vulnerable to pyrithiamine (lateral vestibular nucleus, hypothalamus, midbrain, medullapons). In some cases, KGDH changes preceded the appearance of neurological symptoms of pyrithiamine treatment. Such decreases in KGDH may explain previous findings of region-selective changes in energy metabolism and of decreased synthesis of glucose-derived neurotransmitters (acetylcholine, GABA, glutamate) in pyrithiamine-treated rat brain. Thiamine administration to symptomatic pyrithiamine treated rats resulted in reversal of neurological signs of encephalopathy and in normalisation of defective KGDH activity in all brain regions. These findings suggest that the reversible neurological symptoms associated with Wernicke's Encephalopathy in man likely result from region-selective impairment of KGDH.