Photoactivation of lysosomally sequestered sunitinib after angiostatic treatment causes vascular occlusion and enhances tumor growth inhibition.

The angiogenesis inhibitor sunitinib is a tyrosine kinase inhibitor that acts mainly on the VEGF and PDGF pathways. We have previously shown that sunitinib is sequestered in the lysosomes of exposed tumor and endothelial cells. This phenomenon is part of the drug-induced resistance observed in the clinic. Here, we demonstrate that when exposed to light, sequestered sunitinib causes immediate destruction of the lysosomes, resulting in the release of sunitinib and cell death. We hypothesized that this photoactivation of sunitinib could be used as a vaso-occlusive vascular-targeting approach to treating cancer. Spectral properties of sunitinib and its lysosomal accumulation were measured in vitro. The human A2780 ovarian carcinoma transplanted onto the chicken chorioallantoic membrane (CAM) and the Colo-26 colorectal carcinoma model in Balb/c mice were used to test the effects of administrating sunitinib and subsequently exposing tumor tissue to light. Tumors were subsequently resected and subject to immunohistochemical analysis. In A2780 ovarian carcinoma tumors, treatment with sunitinib+light resulted in immediate specific angio-occlusion, leading to a necrotic tumor mass 24 h after treatment. Tumor growth was inhibited by 70% as compared with the control group (**P<0.0001). Similar observations were made in the Colo-26 colorectal carcinoma, where light exposure of the sunitinib-treated mice inhibited tumor growth by 50% as compared with the control and by 25% as compared with sunitinib-only-treated tumors (N≥4; P=0.0002). Histology revealed that photoactivation of sunitinib resulted in a change in tumor vessel architecture. The current results suggest that the spectral properties of sunitinib can be exploited for application against certain cancer indications.

Changes of connexin43 expression in non-pregnant porcine myometrium correlate with progesterone concentration during oestrous cycle.

Connexin43 (Cx43) is a major protein of myometrial gap junctions. The number of Cx43 gap junctions increase dramatically with the onset of labour in association with development of synchronized uterine contractions. The formation of myometrial gap junctions follows an increase in the oestrogen to progesterone ratio indicating an important role of steroid hormones in regulating Cx43 expression at term. However, no relationship has been established between the expression of Cx43 in the non-pregnant myometrium and concentration of steroid hormones during the oestrous cycle. Here, we used immunofluorescence and Western blotting to analyse the expression of Cx43 gap junctions in the myometrium of pre-pubertal pigs (n = 7) and mature pigs at pre-ovulatory (n = 7), luteal (n = 5) and late luteal (n = 3) stages of the oestrous cycle. The number of Cx43 gap junctions calculated per 1 mm(2) of the myometrial section was low in pre-pubertal pigs and significantly higher (p < 0.022) in pre-ovulatory animals. In relation to pre-ovulatory animals the number of myometrial gap junctions was significantly lower (p < 0.019) at the luteal phase and correlated with significantly higher (p < 0.005) concentration of endogenous progesterone. Phosphorylated isoforms of Cx43 protein were expressed in the myometrium of pre-pubertal pigs and mature animals at pre-ovulatory and late luteal phases, while they were down regulated at the luteal stage. These results indicate that changes of Cx43 expression in the porcine myometrium during the oestrous cycle may be regulated by progesterone concentration and may contribute to the modulation of uterine motility.

Recent developments in tumor angiogenesis.

Angiogenesis is a developmental process that also plays the central role in adults during the female menstruation cycle, wound healing and neoplastic growth and metastasis. Ideally, blocking neovessel growth starves the developing tumor and induces tumor regression. Restricting the vascular ingrowth into the tumor might have adverse effect on drugs targeting the tumor. Nevertheless, anti-VEGF treatment of the neoplastic diseases when combined with chemotherapy significantly increases median survival in treated patients. This suggests alternative mechanisms of anti-angiogenesis therapy. A number of molecules that are in current clinical trials have been identified using angiogenesis models. However, current angiogenesis models have advantages and inconvenience and conclusion drawn upon their use should be interpreted with caution. Thus, it is necessary to optimize existing models and to develop new ones that take into account the complexity of the angiogenic process as it happens in many angiogenesis-related diseases and in particular in cancer.

Capillary density and capillary-to-fibre ratio in vastus lateralis muscle of untrained and trained men.

Muscle fibre profile area (Af), volume density (Vv), capillary-to-fibre ratio (CF) and number of capillaries per fibre square millimetre (CD) were determined from needle biopsies of vastus lateralis of twenty-four male volunteers (mean +/- SD: age 25.4+/-5.8 years, height 178.6+/-5.5 cm, body mass 72.1+/-7.7 kg) of different training background. Seven subjects were untrained students (group A), nine were national and sub-national level endurance athletes (group B) with the background of 7.8+/-2.9 years of specialised training, and eight subjects were sprint-power athletes (group C) with 12.8+/-8.7 years of specialised training. Muscle biopsies of vastus lateralis were analysed histochemically for mATPase. Capillaries were visualized and counted using CD31 antibodies against endothelial cells. There were significant differences in the Vv of type I and type II muscle fibres in both trained groups, B (51.8%; 25.6%) and C (50.5%; 26.4%). However, in untrained group A that was treated as a reference group, the difference between Vv of type I and type II fibres was less prominent, nevertheless statistically significant (42.1%; 35.1%). There was also a significant difference in CF: 1.9 in group A and 2.1 in groups B and C. The number of capillaries per mm2 (CD) was 245 (group A), 308 (group B) and 325 (group C). Significant differences (P<0.05) in CF and CD, were found only between group A (1.9; 245) and both groups of trained men, B and C (2.1; 308 and 325). However, endurance athletes (group B), such as long-distance runners, cyclists and cross country skiers, did not differ from the athletes representing short term, high power output sports (group C) such as ice hockey, karate, ski-jumping, volleyball, soccer and modern dance.

Characteristics of myosin profile in human vastus lateralis muscle in relation to training background.

Twenty-four male volunteers (mean +/- SD: age 25.4+/-5.8 years, height 178.6+/-5.5 cm, body mass 72.1+/-7.7 kg) of different training background were investigated and classified into three groups according to their physical activity and sport discipline: untrained students (group A), national and sub-national level endurance athletes (group B, 7.8+/-2.9 years of specialised training) and sprint-power athletes (group C, 12.8+/-8.7 years of specialised training). Muscle biopsies of vastus lateralis were analysed histochemically for mATPase and SDH activities, immunohistochemically for fast and slow myosin, and electrophoretically followed by Western immunoblotting for myosin heavy chain (MyHC) composition. Significant differences (P<0.05) regarding composition of muscle fibre types and myosin heavy chains were found only between groups A (41.7+/-1.6% of MyHCI, 40.8+/-4.0% of MyHCIIA and 17.5+/-4.0% of MyHCIIX) and B (64.3+/-0.8% of MyHCI, 34.0+/-1.4% of MyHCIIA and 1.7+/-1.4% of MyHCIIX) and groups A and C (59.6+/-1.6% of MyHCI, 37.2+/-1.3% of MyHCIIA and 3.2+/-1.3% of MyHCIIX). Unexpectedly, endurance athletes (group B) such as long-distance runners, cyclists and cross country skiers, did not differ from the athletes representing short term, high power output sports (group C) such as ice hockey, karate, ski-jumping, volleyball, soccer and modern dance. Furthermore, the relative amount of the fastest MyHCIIX isoform in vastus lateralis muscle was significantly lower in the athletes from group C than in students (group A). We conclude that the myosin profile in the athletes belonging to group C was unfavourable for their sport disciplines. This could be the reason why those athletes did not reach international level despite of several years of training.

Influence of the CBA genetic background on sperm morphology and fertilization efficiency in mice with a partial Y chromosome deletion.

Males of the B10.BR-Ydel mouse strain, with a deletion in the long arm of the Y chromosome, were backcrossed to CBA females to introduce the Ydel chromosome to the genetic background of the CBA mice. The CBA-Ydel males (sixth backcross generation) had similar symptoms to those previously described for B10.BR-Ydel males (deterioration of sperm quality and of efficiency of fertilization), but these effects were much less pronounced, showing a favourable influence of the CBA genetic background. The CBA-Ydel males produced only 12% severely misshapen spermatozoa, and mating with B10.BR females gave 100% successful fertilization. Although nearly all sperm heads were abnormal (92% versus 6% in control males), most of the spermatozoa (76%) had deformation only in the acrosomal part, that is, flat heads, which were not found in the control males. These abnormalities were analysed in detail. As shown by differential staining, the acrosomes of the spermatozoa with flat heads were deformed; 18% of these acrosomes looked damaged, and often contained a vesicle, which stained in a similar way to the acrosome but lacked the reaction for acrosomal proteinase. Electron microscopy of testis sections revealed that deformations appeared already in round spermatids as distortion of the acrosomal vesicle and asymmetrical position of the acrosomal granule; in many elongating spermatids the proximal end had a flat or concave shape, and the acrosomes contained a translucent vesicle. It is possible that the genes that are missing in the Yq deletion have some important regulatory function in the course of spermiogenesis, which may explain the various sperm defects observed in Y-del males.

Glycoconjugates of the intestinal goblet cells of four cyprinids.

The aim of this work was to show differences in the terminal and subterminal sugar composition of carbohydrate chains of glycoconjugates produced by the goblet cells of the intestines of four cyprinids. We analysed intestines of two herbivorous species--sneep and grass carp--and two omnivorous ones--chub and common carp. We compared four intestinal regions of every studied species. In every region, the presence of neutral and acidic glycoconjugates was confirmed. The smallest amount of acidic glycoconjugates was present in the second region of sneep intestine. Sulphated glycoconjugates were absent in the third and fourth region of chub intestine. Lectin histochemistry provided evidence for the presence of beta-D-galactose, a-N-acetylgalactosamine, beta-N-acetylglucosamine and sialic acids. Additionally, the occurrence of alpha-L-fucose in the goblet cells of chub, grass carp and sneep was confirmed. We tried to correlate the pattern of glycoconjugate glycosylation with feeding habits of the studied fishes.

Multiple connexins localized to individual gap-junctional plaques in human myometrial smooth muscle.

The synchronous contractions of the uterus in labour depend on electrical coupling of myometrial smooth muscle cells by gap junctions. In the human myometrium, gap junctions are scarce in the non-pregnant uterus, but become abundant at term in preparation for labour. We have previously demonstrated that in the human myometrium at term, three different gap-junctional proteins are expressed, connexins 43, 45, and 40. These connexins are known to have distinctive functional capacities in in vitro expression systems but whether, in the human myometrium in vivo, they are co-assembled into the same gap junction or form different types of gap junction has previously been unclear. By applying triple immunogold labelling to sections of Lowicryl-embedded tissue for electron microscopy, together with complementary immunoconfocal microscopy, we demonstrate here that connexins 43, 45, and 40 are commonly present as mixtures within the same gap-junctional plaque. While all gap junctions contain connexin43, the relative signal for each connexin type varies between individual junctions. The presence within single gap-junctional plaques of three different connexins, each with the potential for conferring distinctive channel properties, suggests an inherent versatility for modulation of smooth muscle cell intercellular communication properties during human parturition.

Differential effects of oncostatin M and leukaemia inhibitory factor expression in astrocytoma cells.

The effects of the production of two closely related cytokines, oncostatin M (OSM) and leukaemia inhibitory factor (LIF), by astrocytoma cells were investigated using the stable cell line human U373-MG, which expressed and secreted both biologically active polypeptides. The expression of LIF by these cells caused resistance to this cytokine due to loss of the LIF receptor (LIFR), from the cell surface, suggesting its retention. In contrast, cells expressing OSM were stimulated by this cytokine, utilizing an autocrine mechanism, and possessed receptors for OSM, but not LIF, on the cell surface. In these cells the continuous up-regulation of OSM-induced gene expression was found even though the Janus kinase-signal transducer and activator of transcription ('JAK/STAT') pathway was almost exhausted due to long-term autocrine stimulation of the cells by OSM. The amount of LIFR was down-regulated in both LIF- and OSM-producing cells and this effect was not found in wild-type U373-MG cells treated with externally added cytokines. To investigate the mechanism of autocrine stimulation by OSM we constructed a stable cell line expressing a form of OSM that is retained in the endoplasmic reticulum (ER). This biologically active cytokine was not secreted, but was localized in the ER. In addition, it did not stimulate the astrocytoma cells in an autocrine manner. We conclude that expression of LIF causes resistance of astrocytoma cells to this cytokine, whereas expression of OSM leads to autocrine stimulation.

Correlation of myofibrillar ATPase activity and myosin heavy chain content in ventricular and atrial myocardium of fish heart.

In the fish heart, ventricular and atrial muscles contain different isoforms of native myosin and myosin heavy chain (MyHC) but the significance of this diversity is still not known. We have analysed ventricular and atrial myocardium of six freshwater fish species (goldfish, roach, bream, rudd, perch and pike-perch) using histochemical staining for myofibrillar ATPase activity as well as non-denaturing and SDS gel electrophoreses for native myosin and MyHC content. In the range of fish species studied, the intensity of ATPase reaction was higher in the atrial myocardium than in the ventricular myocardium and the composition of native myosin isoforms differed between these two muscles. The MyHC content in the cardiac muscle showed some species-related differences. In the goldfish, both atrial and ventricular cardiac muscle contained electrophoretically similar MyHC. In the other fish species, however, the ventricular myocardium showed electrophoretically faster MyHC than that present in the atrial myocardium. These results indicate that there are consistent and characteristic species-related differences between the ventricular and atrial muscles at the level of ATPase staining and the type of MyHC expressed. The findings suggest that fish ventricular and atrial muscles may differ in their contractile properties.

Contractile elements of Lemna trisulca L. glycerinated cell models during chloroplast translocations.

Electron microscopy of Lemna glycerinated cell models depicts contractile elements during chloroplast translocations. One contractile element, the thin ectoplasmic layer, is < or = 0.4 microm thick, pressed against plasma membrane-cell wall. Thin ectoplasmic layer contains numerous oriented filaments and some appear to be actin and myosin. Another contractile element is the outer chloroplast membrane which envelops each chloroplast and joins or fuses with the thin ectoplasmic layer. Chloroplast interconnections are formed between two or more chloroplasts by outer chloroplast membranes; they enhance chloroplast communications, translocations, and molecular exchanges.

Electron probe X-ray microanalysis of cultured myogenic C2C12 cells with scanning and scanning transmission electron microscopy.

Heterogeneity of the elemental content of myogenic C2C12 cultured cells was studied by electron probe X-ray microanalysis (EPXMA) with scanning (SEM EPXMA) and scanning transmission electron microscopy (STEM EPXMA). The best plastic substrate for growing cells was Thermanox. For STEM EPXMA, a Formvar film coated with carbon was found to be suitable substrate. The cells examined by scanning transmission electron microscopy showed great heterogeneity in their elemental content in comparison with the cells examined in the scanning electron microscope despite of an almost identical preparation procedure for EPXMA. Nevertheless the K/Na ratios obtained from both methods of EPXMA were very close (4.1 and 4.3). We conclude that the observed discrepancy in the elemental content obtained by the two methods may be due to differences in instrumentation and this must be taken into account when planning a comparative study.

Connexin43 in porcine myocardium and non-pregnant myometrium.

It is generally accepted that connexin43 (Cx43) is a major constituent of heart and myometrial gap junctions. However, the presence of Cx43 gap junctions in non-pregnant myometrium is still poorly documented. Tissue sections of porcine heart and non-pregnant uterus and myometrial smooth muscle cell cultures were immunostained with monoclonal antibody against Cx43. In the heart, intensive immunostaining was confined to the intercalated discs as previously reported. In the non-pregnant uterus, punctuate immunostaining of Cx43 was seen throughout the myometrium along cell interfaces between myocytes. The expression of Cx43 was sustained in cultured smooth muscle cells isolated from non-pregnant myometrium. Western blotting has detected single isoform of Cx43 in both, cardiac and myometrial tissues. The electrophoretic mobility of porcine heart Cx43 was similar to that of myometrial isoform but different from the pattern of mobility of Cx43 of the rat heart. Hence, porcine myometrium may provide attractive model for studying cellular mechanisms triggering expression of gap junction protein in normal (non-pregnant) uterus.

Effect of progesterone and oestradiol on expression of connexin43 in cultured human myometrium cells.

In human myometrium, the formation of gap junctions at various stages of labour and in correlation with the concentration of progesterone and oestradiol in maternal blood was described previously by electron microscopy and laser confocal microscopy of immunohistochemically stained myometrial sections. The present investigation focused on the effect of continuous exposure of isolated myometrial tissue to progesterone and oestradiol on the number of gap junction plaques in human myometrium cells in vitro. The presence of gap junctions was evaluated by immunocytochemistry with antibodies against gap junction protein, connexin43 (Cx43). Human myometrial cells were isolated from biopsies obtained from term pregnant women who had an elective caesarean operation in the 37th or 40th week of pregnancy. The dispersed myometrial cells that were obtained by limited enzymatic digestion of the myometrial samples were maintained in monolayer culture for 1, 3 and 6 days in the presence of medium that contained foetal bovine serum and the steroids at different concentration. In primary culture, as well as after several passages, the characteristics of these cells were morphologically and biochemically similar to those of smooth muscle cells and myometrial tissue. The obtained results showed that the cells in culture responded synchronously to the increased concentrations of oestradiol/progesterone mixtures. The number of gap junctions increased significantly on days 1, 3 and 6 in culture and showed positive correlation (p < 0.05) with the cell number when the concentration of oestradiol was raised to 1 microgram/mL in the progesterone ratio (1.0 microgram/0.5 microgram/mL). No significant correlation, however, in connexin43 gap junction number versus cell number was observed between the six experimental groups treated with progesterone only.

Different methods of tissue preparation for X-ray microanalysis combined with scanning electron microscopy based on zebra fish, Brachydanio rerio muscle fibres.

Four preparation methods for electron probe X-ray microanalysis (EPXMA) of muscle fibres were compared: (1) dissection, freezing in LN2 (liquid nitrogen), cutting in cryostat, freeze-drying and analysis; (2) dissection, immersing in Tissue-Tek, freezing in LN2, cutting in cryostat, the following steps as in method 1; (3) dissection, freezing in LN2, freeze-drying, separation of fibres into groups; (4) dissection, cutting into 2 mm thick slices by razor blade, freezing in LN2, following steps as in method 1. The contents of Na, Cl, and K as well as K/Na, Cl/K, ratios were taken as criteria of good preservation of muscle fibres. The best results were obtained by method 1. Good morphological preservation can be routinely observed in sections prepared by methods 3 and 4. However, the diffusible elements and K/Na, Cl/K ratios were not retained at relatively constant level among the individual samples. Fibres prepared by methods 1 and 3 showed, despite of freezing artefacts, high K/Na, and low Cl/K ratio. After method 1, high level of the elemental contents was retained, and it did not differ significantly among the samples, showing relatively low standard deviation values.

Binding of Zn(2+) to the plasma protein inter-alpha-inhibitor.

Inter-alpha-inhibitor (IalphaI) is a serum protein consisting of a chondroitin-sulfate-containing protein of 25 kDa (bikunin) and two other polypeptides of 75-80 kDa (heavy chains 1 and 2). The physiological function of IalphaI is unclear but recent results suggest that it is required for the formation of the extracellular matrix of certain cell types and that it has anti-inflammatory activity. It was previously reported that IalphaI isolated from serum contains bound Zn(2+), but details of this binding are lacking. Using equilibrium dialysis, we have found that when the free Zn(2+) concentration is raised from 0.3 to 50 micromol/L, the number of bound ions increases from 0.1 to 7. The concentration of free Zn(2+) in plasma is in the nanomolar range; our results therefore suggest that inter-alpha-inhibitor does not contain stoichiometric amounts of zinc ions under normal in vivo conditions.

X-ray microanalysis of myotomal muscle in the sunbleak, Leucaspius delineatus (Heckel) and goldfish, Carassius auratus gibelio.

The functioning of a group of muscle fibres as a tissue that performs a well characterized type of contraction (slow or fast) depends on their biochemical and structural organization that is already well established. The biochemical and structural diversities between three types of fish muscle fibres found also a reflection in the content of light elements. The present work demonstrates significant differences in the content of diffusible elements (Cl, K, Na, and Mg) and bound elements (P and S) between the muscle fibres types. In general all muscle fibre types of goldfish (Carassius auratus gibelio) that belongs to stationary slow-swimming fish has lower K/Na ratios than those in all three fibre types of fast swimming sunbleak (Leucaspius delineatus).

Immunohistochemical analysis of connexin26 and 43 expression in the mouse alimentary tract.

The spatial pattern of connexins26 (Cx26) and 43 (Cx43) expressions were investigated in the mouse digestive tract by immunocytochemistry. High levels of connexin43 in the epithelium of the oesophagus, non-glandular part of the stomach, and the circular layer of duodenal and ilea muscularis externa were detected. Cx26 was expressed in stratum granulosum of oesophagal folds and in the non-glandular part of the stomach. A low level of immunoreactivity of Cx43 was observed in the circular, and very low in the longitudinal layer of the muscularis externa in the stomach and colon. No immunoreactivity for Cx26 and Cx43 was found in the entire muscularis externa of the oesophagus or in the longitudinal muscle layers of the duodenum and ileum.

Antiserum against connexin32 fragment reacts with a 32-kD protein localised in gap junctions of mouse and rat liver, endometrium and in the fish heart.

The expression of various connexins so far identified is metabolically and developmentally regulated. Examples include uterine endometrium where the expression of gap junction protein, connexin32 (Cx32) is regulated by steroid hormones. In this study we attempted to synthesise a short peptide which matches the portion of the amino acid sequence of the Cx32. Cx32 has primary structure predicted from the nucleotide sequence of cDNA clone. A fragment of Cx32 molecule was synthesised to produce anti-peptide antibody for detecting gap junction protein in mouse and rat liver and endometrium. The 12-peptide, plus Abu residue that corresponds to residues 108-119 (LEGHGDPLHLEE-Abu) of the rat Cx32 (283-mer) was synthesised by the solid phase method. Antibodies against this peptide were raised in rabbits, screened for reactivity and specificity using dot blot assays [15] and used for immunocytochemical staining at the light and at electron microscope levels. The antibodies also reacted with fish heart myocardium.

Identification of two further gap-junctional proteins, connexin40 and connexin45, in human myometrial smooth muscle cells at term.

The powerful synchronous contractions of the uterus in labor depend on electrical coupling of myometrial smooth muscle cells by gap junctions. In humans and other mammals, gap junctions are scarce in the myometrium of the non-pregnant uterus, but become abundant at term and/or with the onset of labor. Previous work has shown that the gap-junctional protein (connexin) expressed by human myometrial smooth muscle cells is connexin43, the same connexin type that predominates in cardiac muscle. Here we show that two further gap junctional proteins, connexin40 and connexin45, are expressed by the myometrial smooth muscle cells of the human uterus at term. Transcripts encoding the human isoforms of these connexins were demonstrated by Northern blot analysis, and immunoconfocal microscopy enabled precise localization of the corresponding proteins to punctate contact points (i.e., gap junctions) between interacting smooth muscle cells. Double labeling demonstrated that, while some fluorescent spots comprise only connexin43, both connexin40 and connexin45 predominantly colocalize to connexin43-positive fluorescent spots. Triple labeling revealed that where all three connexin types were expressed, they frequently localized to the same gap junction spot. As gap-junctional channels composed of different connexin types have been demonstrated in vitro to have different functional properties, multiple connexin expression may contribute to modulation of gap junction function in human myometrial smooth muscle cells in vivo.