Expression of transmembrane carbonic anhydrases IX and XII in ovarian tumours.

AIMS: Carbonic anhydrase (CA) isozymes IX and XII have been suggested to play a role in oncogenic processes. The aim of the present study was to investigate CA IX and XII expression in patients with ovarian tumours. METHODS AND RESULTS: A series of ovarian tumours was immunostained for CA IX and XII and the results were correlated with histopathological and clinical parameters. Most cases of borderline mucinous cystadenomas, mucinous cystadenocarcinomas and serous cystadenocarcinomas were moderately or strongly positive for CA IX. In malignant tumours, the staining was most prominent in hypoxic regions. Expression of CA XII was detected in all tumour categories, although the mean staining intensity was weaker than for CA IX in all groups except for clear cell carcinomas. CONCLUSIONS: The wide expression of CA IX and XII in ovarian tumours suggests that these isozymes could represent potential targets in ovarian cancer therapy. The expression pattern of CA IX suggests that it could also serve as a useful histopathological marker protein for hypoxia in malignant ovarian tumours.

Stepwise pH-gradient elution for the preparative separation of natural anthraquinones by multiple liquid-liquid partition.

Preparative-scale separation of substituted anthraquinones by multiple liquid-liquid partition was studied using isopropylmethyl ketone (IMK)/aqueous phosphate buffer (aq.) as the solvent system and the Hietala apparatus with 100 partition units as the partition equipment. The lower (aq.) phase was chosen as mobile, while the upper (IMK) phase remained stationary. Hence, the principle of stepwise pH-gradient elution could be utilized to separate the components in two complex mixtures of hydroxyanthraquinones and hydroxyanthraquinone carboxylic acids, isolated from the fungus Dermocybe sanguinea. In spite of the nonlinearity of the partition isotherms for these anthraquinones, implying deviations from the Nernst partition law, remarkable separations were achieved for the components in each mixture. Every anthraquinone carboxylic acid showed markedly irregular partition behavior, appearing in the effluent as two more or less resolved concentration zones. Such splitting was attributed to the formation of relatively stable sandwich-dimers, which were in a slow equilibrium with the monomers in the more nonpolar organic phase. At lower pH-values, only the polar monomers were distributed into the mobile aqueous phase and moved forward, whereas the nonpolar sandwich-dimers remained almost entirely in the stationary organic phase and lagged behind. When the pH of the mobile aqueous phase was raised high enough, the firmly linked dimers were monomerized and emerged from the apparatus as a second concentration profile. Intermolecular hydrogen bonding and pi-pi interaction between the pi-systems of two anthraquinone molecules in a parallel orientation were considered responsible for the nonlinear and markedly irregular partition behavior of the natural anthraquinones studied. The nonlinearity of the partition behavior of the hydroxyanthraquinones lacking the carboxyl group, appeared merely as excessive broadening of the experimental concentration profile, which impaired the resolution between the components only insignificantly. Thus, e.g. the main components, dermocybin and emodin, could both be obtained from Separation 1 in a purity of at least 99%.

Results of neurophysiologic evaluation in fecal incontinence.

PURPOSE: Several methods of neurophysiologic assessment exist in the investigation of patients with fecal incontinence. However, the clinical significance of the information gained is uncertain. The aim of this prospective study was to evaluate the results of pudendal nerve terminal motor latency and fiber density in relation to clinical variables and manometric measurements. METHODS: Seventy-two patients with fecal incontinence (63 women; mean age, 62; range, 24-81 years) responded to a bowel questionnaire and underwent anorectal manovolumetry, anal ultrasonography, defecography, and electromyography, including pudendal nerve terminal motor latency and fiber density. RESULTS: Pudendal neuropathy (pudendal nerve terminal motor latency > 2.5 ms) was found in 46 percent and increased fiber density (> 1.7) in 82 percent. Pudendal neuropathy and increased fiber density were most common in patients with rectal prolapse or intra-anal intussusception. No difference was seen concerning anal resting and incremental pressures, rectal compliance, rectal sensibility or severity of incontinence in patients with unilateral, bilateral, or marked (> 4 ms) pudendal neuropathy vs. patients with normal pudendal nerve terminal motor latency. In contrast, patients with increased fiber density had lower incremental pressures (P < 0.05) and stated decreased rectal sensibility (P < 0.05) compared with those with normal fiber density. These differences were most pronounced in patients with neurogenic or idiopathic incontinence. CONCLUSIONS: Pudendal neuropathy and increased fiber density are common in patients with fecal incontinence. Fiber density but not pudendal nerve terminal motor latency was correlated with clinical and manometric variables. The severity of nerve injury correlated with anal motor and sensory function in patients with neurogenic or idiopathic incontinence. The routine use of pudendal nerve terminal motor latency in the assessment of patients with fecal incontinence can be questioned.

NH tautomerism in the natural chlorin derivatives.

The NH tautomerism of five Mg-free chlorophyll a and b derivatives 2-6 was studied utilizing NMR spectroscopy and molecular modeling. The results from the dynamic NMR measurements of the chlorins revealed that substituent effects contribute crucially to the free energy of activation (DeltaG(double dagger)) in the NH tautomeric processes. An intermediate tautomer for the total tautomeric NH exchange in a chlorin was observed for the first time, when the (1)H NMR spectra of chlorin e(6) TME (3) and rhodin g(7) TME (4) (TME = trimethyl ester) were measured at lower temperatures. The lower energy barriers (DeltaG(1)(double dagger)) obtained for the formation of the intermediate tautomers of 3 and 4, assigned to the N(22)-H, N(24)-H trans-tautomer, were 10.8 and 10.6 kcal/mol, respectively. The energy barrier (DeltaG(2)(double dagger) value) for the total tautomeric NH exchange in the five chlorins was found to vary from 13.6 kcal/mol to values higher than 18 kcal/mol. The lowest DeltaG(2)(double dagger) value (13.6 kcal/mol) was obtained for rhodochlorin XV dimethyl ester (2), which was the only chlorophyll derivative lacking the C(15) substituent. In the case of chlorins 4 and 5, the steric crowding around the methoxycarbonylmethyl group at C(15) raised the DeltaG(2)(double dagger) activation free-energy to 17.1 kcal/mol. However, the highest energy barrier with DeltaG(2)(double dagger) > 18 kcal/mol was observed for the NH exchange of pyropheophorbide a methyl ester (6), possessing the macrocycle rigidifying isocyclic ring E. Our results demonstrate that the steric strain, arising either from the steric crowding around the bulky substituent at C(15) or the macrocycle rigidifying isocyclic ring E, slows down the NH tautomeric process. We suggest that deformations in the chlorin skeleton are closely connected to the NH tautomeric exchange and that the exchange occurs by a stepwise proton-transfer mechanism via a hydrogen bridge.

Two-dimensional TLC separation and mass spectrometric identification of anthraquinones isolated from the fungus Dermocybe sanguinea.

A new two-dimensional TLC technique was developed to separate substituted anthraquinones on silica plates using n-pentanol-pyridine-methanol (6:4:3, v/v/v) and toluene-ethyl acetate-ethanol-formic acid (10:8:1:2, v/v/v/v) as eluents. The good separation power of the new technique was demonstrated by applying it to the analysis of complex anthraquinone mixtures isolated from the Scandinavian Dermocybe sanguinea. Emodin, physcion, endocrocin, dermolutein, dermorubin, 5-chlorodermorubin, emodin-1-beta-D-glucopyranoside, dermocybin-1-beta-D-glucopyranoside and dermocybin, and five new, earlier in D. sanguinea unidentified compounds, 7-chloroemodin, 5,7-dichloroemodin, 5,7-dichloroendocrocin, 4-hydroxyaustrocorticone and austrocorticone, were separated and identified on the basis of Rf-values, UV/Vis spectra and mass spectra.

Enhanced retinal longwave sensitivity using a chlorophyll-derived photosensitiser in Malacosteus niger, a deep-sea dragon fish with far red bioluminescence.

Through partial bleaching of both visual pigment extracts and cell suspensions we show that the deep-sea stomiid Malacosteus niger, which produces far red bioluminescence, has two visual pigments within its retina which form a rhodopsin/porphyropsin pigment pair with lambda max values around 520 and 540 nm, but lacks the very longwave sensitive visual pigments (lambda max > 550 nm) observed in two other red light producing stomiids. The presence of only a single opsin gene in the M. niger genome was confirmed by molecular and cladistic analysis. To compensate for its apparently reduced longwave sensitivity compared to related species, the outer segments of M. niger contain additional pigments, which we identify as a mixture of defarnesylated and demetallated derivatives of bacteriochlorophylls c and d, that are used as a photosensitiser to enhance its sensitivity to longwave radiation.

Analysis of lipids in crude extracts by 13C nuclear magnetic resonance.

Phospho- and glycolipids with mono- or polyunsaturated fatty acid chains, free and acylated steroids, carotenes and carotenoids, chlorophylls, and related pheophytins were detected in crude extracts by NMR spectroscopy without any need for prior separation. A broad range of molecules belonging to different lipid classes could be identified in one-dimensional 13C NMR spectra obtained by applying a polarization transfer pulse sequence (DEPT 135 degrees). Directly detected or 1H-detected two-dimensional heterocorrelated NMR experiments were performed to facilitate the assignment of peaks arising from carotenoids, unsaturated fatty acid chains, and chlorophylls. 13C NMR data of crude lipid extracts from the macroalagae Ulva rigida and Fucus virsoides are shown to yield an informative overview of their lipid content. NMR is thus proposed as a simple, nonselective, and nondestructive technique for the first screening of the main lipid classes in complex lipid mixtures.

High-voltage electrophoretic separation of tryptophan metabolites of the kynurenine pathway.

An analytical separation method for tryptophan and its seven metabolites of the kynurenine pathway by high-voltage paper electrophoresis is presented. Anthranilic acid, 3-hydroxyanthranilic acid, kynurenic acid, kynurenine, nicotinic acid, quinolinic acid, xanthurenic acid and unmetabolized tryptophan are measured in urine. Using radioactive labelling and scintillation counting as a quantification method, the relative standard deviation varied from 3.5% to 14.4%, corresponding to kynurenine and nicotinic acid, respectively. The recovery of labelled tryptophan added to urine was 95%. An advantage of the electrophoretic method is the minor tailing of spots and, hence, a good resolution of the components. For the monovalent anions of the kynurenine pathway metabolites, a linear correlation (r = 0.9996) was found between the experimental relative electrophoretic mobility and the quantity M-2/3, where M is the molecular mass of the anion.

Polymer phase partition in the purification of cytochrome P-450 and cytochrome b5 from the yeast Brettanomyces anomalus.

About 0.5% of the total cellular protein in the yeast Brettanomyces anomalus is membrane-bound cytochrome P-450, when this yeast is grown in the presence of 5% glucose as the main carbon and energy source. A partial purification of cytochrome P-450 by phase partition is described. Breakdown of yeast cell walls with microbial enzyme preparations led to extensive losses of this hemoprotein. Instead, by a carefully controlled mechanical breakage as much as 50% of the total cellular cytochrome P-450 could be recovered. During the solubilization of cytochrome P-450 from the cell homogenate with Triton X-100, the protective agents dithiothreitol, EDTA, and butylated hydroxytoluene prevented major losses of the hemoprotein. Applying a three-phase partition system (polyethylene glycol-Ficoll-dextran) to the solubilized whole cell homogenate in the presence of 1 M sodium chloride, followed by a precipitation of the top "oily layer" with 25% polyethylene glycol, a 25- to 60-fold enrichment of cytochrome P-450 was obtained. This corresponds to a specific content of 0.8-2.2 nmol of cytochrome P-450 per milligram of protein. Cytochrome b5 enriched (41%) to the PEG-Ficoll interphase, and NADPH-cytochrome c reductase and "cytochromes P-420" to the Ficoll and dextran phases. The polymer phase partition system thus serves as an excellent initial purification step of cytochrome P-450 without a need for the preparation of the microsomal fraction. Another advantage of the method is that it allows the simultaneous partial purification of cytochrome b5.

Evolution of nerve conduction velocity in later childhood and adolescence.

Sensory and/or motor nerve conduction velocities (NCVs) were measured in median, radial, sural, and peroneal nerves of 54 healthy girls and 75 healthy boys aged 3-19 years. Independent of the nerve and sex, both motor and sensory NCVs increased in the upper extremities and decreased in the lower ones as a function of age/growth in length. NCV increased in the upper limbs on an average 3.17 m/sec in the boys and in the girls 1.47 m/sec per 10 years of life. As for the nerves of the lower extremities, NCV slowed down on the average 1.81 m/sec in the boys and in the girls 3.62 m/sec per 10 years of age. The change varied in different nerves. A highly significant one (P less than 0.001) was found in both the motor and sensory NCVs of peroneal nerve in the girls and in the sensory NCV of radial nerve in boys. On the average NCV was in most nerves faster in the girls than in boys of the same age. According to these results, changes occur in the function---and thus probably also in the morphology---of peripheral nerves even in later childhood and adolescence. These changes pertain to general maturation, growth in length of the limbs, and possibly also sex.

Intraneural stimulation as a method to study sympathetic function in the human skin.

Intraneural electrical stimulation of sympathetic postganglionic axons was made in human skin nerves combined with recordings of skin resistance and a photoelectric pulse plethysmogram within the innervation zone. Tungsten microelectrodes were used first to record multiunit sympathetic activity in sural or median nerves. After blocking the nerve with local anaesthetics proximal to the recording site the electrodes were then used for intraneural stimulation. Stimulation led to reduction of skin resistance which was frequency dependent. A short train of stimuli reduced skin resistance transiently and the response was potentiated by a single stimulus delivered up to 2.5 min. prior to the train. Vasoconstrictor responses did not always occur and were relatively independent of stimulation frequency. The method may be useful for physiological and pathophysiological studies of sympathetic neuroeffector transmission in man.

Asymmetric function of peripheral nerves in children with cerebral palsy.

Nerve conduction velocities (NCVs) in both motor and sensory nerves as well as nerve action potentials (NAPs) of sensory nerves were measured bilaterally in 24 children with cerebral palsy. The NAP amplitude and both sensory and motor NCV were on the average higher on the intact or less affected side. The NCV side difference was still present after the temperature asymmetry of the limbs had been taken into account by calculating the temperature-corrected NCV values, and was statistically significant for motor NCV in N. peroneus and for sensory NCV in N. suralis; this could neither be explained on the basis of spasticity nor by the length difference of the extremities. No correlation of NCV asymmetry with the degree of atrophy seemed to exist.

Thermoregulatory and rhythm-generating mechanisms governing the sudomotor and vasoconstrictor outflow in human cutaneous nerves.

1. Recordings of multiunit sympathetic activity were made from human nerve fascicles supplying hairy and glabrous skin of the extremities in healthy subjects exposed to different ambient temperatures. Sudomotor and vasomotor events accompanying the neural activity were monitored by simultaneous recordings of electrodermal and pulse plethysmographic events (Pleth) in the neural innervation zones. 2. By exposing the subject to warm (43 degrees C) or cold (15 degrees C) environments, it was possible to obtain a selective activation of either the sudomotor or the vasoconstrictor neural system, respectively, with suppression of spontaneous activity in the other system. 3. Bursts of both vasoconstrictor and sudomotor nerve activity were found to occur at certain preferred intervals which were integer multiples of a period of about 0 . 6 sec (100 cycles/min). With high sudomotor or vasoconstrictor tone the 100 cycles/min rhythm was prominent but with decreasing tone slower subharmonic rhythms prevailed. Respiratory rhythms were also discerned as well as slower rhythms attributable to oscillatory tendencies in thermoregulatory servos. 4. Vasoconstrictor bursts had longer mean duration than sudomotor bursts, a finding attributed to a slower conduction velocity of vasoconstrictor as compared to sudomotor impulses. 5. With increasing incidence of bursts transient electrodermal or plethysmographic responses following individual bursts merged, and thus the fast neural rhythms were not discernible in either the electrodermal or Pleth traces. Given increments in firing rate of nerves produced less additional vasoconstriction at high than at low firing rates. The rhythm generating mechanisms may help to restrict rates of individual fibres to the low range which provides high gain in the neuroeffector transfer functions.

Regional similarities and differences in thermoregulatory vaso- and sudomotor tone.

1. Skin nerve sympathetic activity was recorded simultaneously from the following pairs of nerves: left and right median, median and peroneal, left and right peroneal, posterior cutaneous antebrachial and superficial radial, posterior cutaneous antebrachial and median. The recordings were performed on healthy subjects exposed to different ambient temperatures. Electrodermal responses and pulse plethysmograms were recorded from the neural innervation zones. 2. Vasoconstriction impulse bursts recorded simultaneously from the median and peroneal nerves during exposure to a cold environment showed a striking similarity with respect to the timing and strength of individual bursts. A similar strong correlation was observed also among sudomotor bursts recorded simultaneously from the posterior cutaneous antebrachial and superficial radial nerve during exposure to a warm environment. 3. On some occasions, such as during exposure to a moderately warm environment or emotional stress, a temporal correlation was also observed between vasoconstrictor bursts recorded from the median and sudomotor bursts recorded simultaneously from the posterior cutaneous antebrachial nerve. 4. The double nerve recordings provided evidence that in the distal glabrous skin areas reflex thermoregulatory functions are mainly executed via vasoconstrictor fibres whereas sudomotor fibres are brought into action only at relatively high temperature. On the contrary, in the hairy skin on the dorsal side of forearm and hand reflex thermoregulation is to a large extent executed via sudomotor fibres.

Thin-layer chromatography of chlorophylls and their derivatives on cellulose layers.

A method for the separation and identification of chlorophyll a and b, pheophytin a and b, pheophorbide a and b, chlorin e6, rhodin g7, and the corresponding chlorophyll a' and b' derivatives, as well as the saponification products of pheophorbide a and b, by thin-layer chromatography on commercial cellulose layers on the micro-scale has been developed. Two solvent systems were used: light petroleum (b.p.60-80 degrees)-pyridine (9:1, v/v) and n-heptane-pyridine (7:3, v/v). The former was suitable for chlorophylls, pheophytins and pheophorbides, and the latter for pheophorbides, chlorin, rhodin and their esters. The separation of the derivatives was good and no chemical alteration of the derivatives could be observed. The method is rapid and easy to use and is therefore suitable for checking the purity of derivatives during the preparation of chlorophylls and their derivatives.