Circulating microRNAs associate with diabetic nephropathy and systemic microvascular damage and normalize after simultaneous pancreas-kidney transplantation.

Because microvascular disease is one of the most important drivers of diabetic complications, early monitoring of microvascular integrity may be of clinical value. By assessing profiles of circulating microRNAs (miRNAs), known regulators of microvascular pathophysiology, in healthy controls and diabetic nephropathy (DN) patients before and after simultaneous pancreas-kidney transplantation (SPK), we aimed to identify differentially expressed miRNAs that associate with microvascular impairment. Following a pilot study, we selected 13 candidate miRNAs and determined their circulating levels in DN (n = 21), SPK-patients (n = 37), healthy controls (n = 19), type 1 diabetes mellitus patients (n = 15) and DN patients with a kidney transplant (n = 15). For validation of selected miRNAs, 14 DN patients were studied longitudinally up to 12 months after SPK. We demonstrated a direct association of miR-25, -27a, -126, -130b, -132, -152, -181a, -223, -320, -326, -340, -574-3p and -660 with DN. Of those, miR-25, -27a, -130b, -132, -152, -320, -326, -340, -574-3p and -660 normalized after SPK. Importantly, circulating levels of some of these miRNAs tightly associate with microvascular impairment as they relate to aberrant capillary tortuosity, angiopoietin-2/angiopoietin-1 ratios, circulating levels of soluble-thrombomodulin and insulin-like growth factor. Taken together, circulating miRNA profiles associate with DN and systemic microvascular damage, and might serve to identify individuals at risk of experiencing microvascular complications, as well as give insight into underlying pathologies.

An insertional mutant of Chlamydomonas reinhardtii with defective microtubule positioning.

cmu1-1 is a new mutation of Chlamydomonas reinhardtii that causes a change in cell shape due to an alteration of cytoplasmic microtubule organization. cmu1 mutant cells were first identified based on their altered cell shape. Unlike wild-type cells, which are ellipsoid, cmu1 cells tend to be either round or egg-shaped with the flagella extending from the narrow end of the cell. Electron microscopic comparison of mutant and wild-type cells indicated that microtubule distribution was altered in the mutant cells. Immunofluorescence microscopy using anti-beta-tubulin antibodies revealed that, in wild-type cells, microtubules arise from the anterior end of the cell in the region of the basal bodies, pass posteriorly subjacent to the plasma membrane, and terminate near the posterior end of the cell. In mutant cells, the microtubules also arise from the basal body region but then become disarrayed. They frequently curl back anteriorly or wrap around the equator of the cell; some microtubules also extend completely to the posterior end of the cell, then turn back toward the anterior end. No changes in the basal body region were detected by electron microscopy. Some cmu1 cells had multiple nuclei or an aberrant number of flagella, both of which may be due to defects in cell division, a process dependent upon microtubules. Thus, cmu1-1, which was generated by insertional mutagenesis and is tagged, appears to encode a protein that plays an essential role in the spatial organization of cytoplasmic microtubules involved in both interphase and mitotic functions.

ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.

A new mutant strain of Chlamydomonas, ptx1, has been identified which is defective in phototaxis. This strain swims with a rate and straightness of path comparable with that of wild-type cells, and retains the photoshock response. Thus, the mutation does not cause any gross defects in swimming ability or photoreception, and appears to be specific for phototaxis. Calcium is required for phototaxis in wild-type cells, and causes a concentration-dependent shift in flagellar dominance in reactivated, demembranated cell models. ptx1-reactivated models are defective in this calcium-dependent shift in flagellar dominance. This indicates that the mutation affects one or more components of the calcium-dependent axonemal regulatory system, and that this system mediates phototaxis. The reduction or absence of two 75-kD axonemal proteins correlates with the nonphototactic phenotype. Axonemal fractionation studies, and analysis of axonemes from mutant strains with known structural defects, failed to reveal the structural localization of the 75-kD proteins within the axoneme. The proteins are not components of the outer dynein arms, two of the three types of inner dynein arms, the radial spokes, or the central pair complex. Because changes in flagellar motility ultimately require the regulation of dynein activity, cell models from mutant strains defective in specific dynein arms were reactivated at various calcium concentrations. Mutants lacking the outer arms, or the I1 or I2 inner dynein arms, retain the wild-type calcium-dependent shift in flagellar dominance. Therefore, none of these arms are the sole mediators of phototaxis.

Transmembrane assemblage of the photoreceptor connecting cilium and motile cilium transition zone contain a common immunologic epitope.

The photoreceptor connecting cilium bears a unique transmembrane assemblage which stably links cell surface glycoconjugates with the underlying axonemal cytoskeleton. Structural similarities between the photoreceptor connecting cilium and the transition zone of motile cilia suggests that this assemblage may also be present in motile cilia. Using a subcellular fraction enriched in detergent-extracted photoreceptor axonemes, three high molecular mass glycoconjugates (425, 600, and 700 kD) were previously identified as potential components of the assemblage. Through oligosaccharide characterization and binding of a specific monoclonal antibody, we have verified the localization of the 425 kD glycoconjugate to the transmembrane assemblage. Binding of the lectin peanut agglutinin (PNA) to the 425 kD glycoconjugate on nitrocellulose blots, and to isolated detergent-extracted axonemes, was assessed following treatment with the enzymes neuraminidase and O-glycanase. Changes in binding to the 425 kD glycoconjugate precisely paralleled changes in binding to intact axonemes, supporting the hypothesis that the 425 kD glycoconjugate is a component of the transmembrane assemblage. Furthermore, the results suggest that the 425 kD glycoconjugate contains sialated galactose-N-acetylgalactosamine oligosaccharides which are O-linked to the protein backbone. To directly assess the distribution of the 425 kD glycoconjugate, we produced a monoclonal antibody directed against this glycoconjugate. The antibody, K26, recognizes only the 425 kD on transblots of the axoneme fraction. K26 immunoreactivity of intact axonemes is identical to that seen by PNA staining. K26 staining of isolated photoreceptors and whole retina is uniquely localized to the region of the connecting cilium. Thus, in the photoreceptor, the 425 kD is not only a component of the transmembrane assemblage but is also completely restricted to the connecting cilium. Based on morphological similarities, the photoreceptor connecting cilium is thought to be homologous to the transition zone of the motile cilium. As such, we have stained oviduct epithelium with the K26 monoclonal antibody. Immunoreactivity is restricted to the region of the transition zone at the base of motile cilia.

Cytoskeletal-membrane interactions: a stable interaction between cell surface glycoconjugates and doublet microtubules of the photoreceptor connecting cilium.

The ciliary base is marked by a transition zone in which Y-shaped cross-linkers extend from doublet microtubules to the plasma membrane. Our goal was to investigate the hypothesis that the cross-linkers form a stable interaction between membrane or cell surface components and the underlying microtubule cytoskeleton. We have combined Triton X-100 extraction with lectin cytochemistry in the photoreceptor sensory cilium to investigate the relationship between cell surface glycoconjugates and the underlying cytoskeleton, and to identify the cell surface components involved. Wheat germ agglutinin (WGA) binds heavily to the cell surface in the region of the Y-shaped cross-linkers of the neonatal rat photoreceptor cilium. WGA binding is not removed by prior digestion with neuraminidase and succinyl-WGA also binds the proximal cilium, suggesting a predominance of N-acetylglucosamine containing glycoconjugates. Extraction of the photoreceptor plasma membrane with Triton X-100 removes the lipid bilayer, leaving the Y-shaped crosslinkers associated with the axoneme. WGA-binding sites are found at the distal ends of the crosslinkers after Triton X-100 extraction, indicating that the microtubule-membrane cross-linkers retain both a transmembrane and a cell surface component after removal of the lipid bilayer. To identify glycoconjugate components of the cross-linkers we used a subcellular fraction enriched in axonemes from adult bovine retinas. Isolated, detergent-extracted bovine axonemes show WGA binding at the distal ends of the cross-linkers similar to that seen in the neonatal rat. Proteins of the axoneme fraction were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. WGA labeling of the nitrocellulose transblots reveals three glycoconjugates, all of molecular mass greater than 400 kD. The major WGA-binding glycoconjugate has an apparent molecular mass of approximately 600 kD and is insensitive to prior digestion with neuraminidase. This glycoconjugate may correspond to the dominant WGA-binding component seen in cytochemical experiments.

Carbohydrate metabolism and the temporal occurrence of a serotonin-like indole in the male and female reproductive tract and its relationship with PGF and infertility: a review.

Both in the male and in the female reproductive tract glucose can be converted via either the pentose pathway or the sorbitol pathway. It is shown that a disturbed carbohydrate metabolism can lead to infertility, i.e. in the cow and in the bull semen. Evidence is provided that the serotonin-like indole which occurs in the protein-complex and is liberated, can exert effects on the uterine endometrium comparable to those caused by serotonin. It is suggested that the indole liberated can cause ischaemia, followed by regression of the endometrium. When this occurs in repeat breeder cows, exogenous PGF given in mid-cycle on a suitable day, may restore the endometrium so that the cow can again become pregnant. The possibility is mentioned that in humans the free indole might cause regression of the endometrium and some distress symptoms, but thereafter endogenous PGF does increase the vascular permeability resulting finally in bleeding.

Non-fertility in cows: treatment with PGF and investigation of uterine biopsies.

Experiments with seventeen 'repeat breeders' confirmed that the carbohydrate composition, the hormonal pattern, the cholesterol content and the occurrence of indole metabolites, such as blue fluorescing protein, in cervical mucus can be used as parameters for the fertilizing capacity of these cows. After careful rectal examination 25 mg PGF2 alpha was administered to thirteen 'repeat breeders' on that day of the luteal phase when next to the corpus luteum no follicle was palpable; ten of them became pregnant or were fertilized. Uterine biopsies were taken from eight 'normal' cows and from eight 'repeat breeders', simultaneously with cervical mucus samples. The biochemical composition of the biopsies was comparable to that of the mucus samples. The taking of two biopsies produced injurious consequences for the animals, perceptible in oestrous behaviour. Investigation of cervical mucus samples is therefore preferred to uterine biopsies for the examination of sterility in cows.

Non-fertility in cows: influence of PGF, PMSG followed by PGF, progesterone and light.

An injection of PGF-2 alpha into cows at D + 10, D + 12, or at D + 14-15 showed several effects: regression of the corpus luteum in all cases 24 h after the injection, endometrium affected especially at D + 12, and initiation of formation of sorbitol when progesterone was present or of glucuronic acid when oestrogen occurred. Suitable parameters for selecting cows for superovulation, next to a rectal investigation, seem to be a biochemical investigation of cervical mucus on the occurrence of carbohydrates and of abnormal indole- and cholesterol-metabolites. In 'repeat breeders' several deviations were found: a delayed regression of the corpus luteum, a delayed atresia of follicles, a disturbed indole- and carbohydrate-metabolism and no conversion of DHA into oestrogen. Endogenous prostaglandins are apparently not sufficiently effective in a number of these cows and so treatment with PGF, eventually preceded by PMSG, and placing under a fluorescent lamp seemed to be beneficial in six out of eight 'repeat breeders'.

Cyclic changes in hormones, carbohydrates and indole metabolism in cervical mucus of normal, fertilizing cows and the relationship with non-fertility.

In the cervical mucus of 'normal' cows the cholesterol content was very low at D--0; at D + 12 it was about fifty times as high; then it decreased to about half its value at D + 17 and then to the low value at D--0. Usually small amounts of oestrogen, testosterone, pregnanedione and progesterone were found at D--0; on D + 12 more pregnanedione was found and less oestrogen, while at D + 17 more oestrogen occurred and less pregnanedione. The fructose and glucose content was very low at D--0; then it increased until D + 12, when glucose was dominant, while at D + 17 it had decreased and rather large amounts of glucuronic acid and of sorbitol occurred. On D + 12 a blue fluorescing indole metabolite was sometimes found. Deviations from these patterns, were found particularly in the winter months, and coincided with lowered fertility. Indole metabolism was stronger in the winter months than in the summer months and occurred more in cows than in heifers.

Non-fertility in cows with an abnormal indole metabolism and disturbed carbohydrate composition in the cervical mucus: a preliminary report.

Cervical mucus samples of the cow appear to provide information on the probability of becoming pregnant. In six 'repeat breeders' a strong indole metabolism was found in the cervix. When luteal tissue was decreasing and follicles increasing, compounds were found indicating possible exfoliation of the endometrium, which in dogs is a normal process in late metoestrus. Exfoliation of the endometrium was actually demonstrated in two cows after slaughter. It can delay the onset of oestrus for several days and can disturb implantation of an embryo. The carbohydrate composition of the cervical mucus was also different from that in normal cows. Treatment with PMSG and PGF resulted only twice in the formation of glucuronic acid and sorbitol, which are seemingly necessary for the receipt of spermatozoa. A disturbed carbohydrate composition and an abnormal indole metabolism, which is easily detectable in the cervical mucus, may prevent or disturb fertilization in cows.

Extraction of pineal and uterine tissue at different pH values: a preliminary report on the occurrence of a few groups of compounds in both tissues.

Extraction of pineal glands has been performed at different pH values. After extraction at pH +/- 4.9, 'porphyrin'-containing protein did not occur in the extracts, while at pH +/- 6.3 these proteins had passed over partly into the extract; at pH +/- 6.8 the proteins could no longer be detected in the residues, but a partial breakdown had taken place. The activity of pineal extracts depends on the method of extraction. In the watery extracts (pH +/- 6.3) both of pineal and of uterine tissue the presence could be shown among others of: porphyrins bound to protein, the indole compound 'bufotenine' also bound to protein, 'kynurenine', a 'basic peptide' containing amines and 'flavoproteins'. The hypothesis is proposed that in uterine endometrium, tryptophan can be metabolized in at least two pathways, one leading to kynurenine and one to bufotenine. Formation of bufotenine, probably a physiologically important compound, might be influenced by the oestrous cycle and conversely.

Some biochemical parameters for qualification of bull semen.

Since the macroscopical and microscopical examination of bull semen does not in all cases appear to give conclusive indications concerning the fertilizing capacity, an investigation of some biochemical parameters was undertaken. It was shown that the following biochemical examination could furnish some more information about the quality of the semen: (1) Determination of the phenylalanine- alpha-ketoglutarate transminase activity both in the seminal plasma and in the whole semen. A high transminase activity in the plasma points to leakage out of the spermatozoa, thus indicating an affection of the spermatozoa. In that case larger amounts of basic amino acids and of leucine were often found in the seminal plasma than would normally be observed. (2) Gas chromatographic examination of the steroids occurring both in the seminal plasma and in the spermatozoa. In a number of infertile bulls small peaks of progesterone were found together with those of other, unidentified compounds. (3) Investigation of the carbohydrate metabolism in the semen. In a number of fertile bulls the following deviations were found: a. the presence of quite a lot of fructose in the spermatozoa; b. the presence of glucuronic acid and/of other foreign compounds in the spermatozoa or in the plasma; c. an elevated pH in the seminal plasma some 2 to 3 hours after ejaculation; d. a low phenylalanine- alpha-ketoglutarate transaminase activity in the whole semen (the spermatozoa had been disrupted by freezing). (4) Investigation of the presence of reducing aldehydes (glycoladehyde and glyceraldehyde) inside the spermatozoa. The determinations mentioned under 1, 3c and 3d can easily be carried out in the laboratories of each District Animal Health Service and of the A.I. stations; the same is true of the occurrence of amino acids in the seminal plasma and of fructose and aldehydes inside the spermatozoa if facilities are available for electrophoresis and chromatography.

Cyclical changes in the carbohydrate composition of dog endometrium.

In the first part of prooestrus glucuronic acid could not be detected, but small amounts of glucose, fructose, inositol and sorbitol were found. When progesterone was formed a small amount of glucuronic acid could be observed and sorbitol had disappeared. During the first weeks of metoestrus the amounts of fructose and glucose increased gradually, but those of glucuronic acid remained low. When more progesterone and pregnanedione were found in the endometrium the amounts of glucuronic acid began to increase and the highest values were reached in anoestrus. During the last days of anoestrus a sudden change in the carbohydrate composition of the endometrium occurred: glucuronic acid and fructose could no longer be detected, the amounts of glucose and inositol decreased and sorbitol was found. These findings show that next to the cyclical ovarian events, there is a cyclical change in te carbohydrate composition of dog endometrium.

Effects of sheep pineal fractions on the activity of male rat hypothalami in vitro.

High molecular weight substances could be isolated from sheep pineals with the "Bensinger" extraction method, followed by ultrafiltration of the waterlayer through different diaflomembranes. Two of the pineal fractions, XM100R and PM30R, stimulate the gonadotropin releasing activity of the medial basal hypothalamus (MBH). In experiments in which comparable pineal fractions were incubated without MBH and without pituitary and injected in immature mice no effect was detectable. All experiments in which a similar amount of rat cerebral cortex was used for incubation with pineal fractions did not show any activity.

Some biochemical parameters for qualification of bull semen.

Summary Since the macroscopical and microscopical examination of bull semen does not in all cases appear to give conclusive indications concerning the fertilizing capacity, an investigation of some biochemical parameters was undertaken. It was shown that the following biochemical examination could furnish some more information about the quality of the semen: 1. Determination of the phenylalanine- α-ketoglutarate transaminase activity both in the seminal plasma and in the whole semen. A high transaminase activity in the plasma points to leakage out of the spermatozoa, thus indicating an affection of the spermatozoa. In that case larger amounts of basic amino acids and of leucine were often found in the seminal plasma than would normally be observed. 2. Gas chromatographic examination of the steroids occurring both in the seminal plasma and in the spermatozoa. In a number of infertile bulls small peaks of progesterone were found together with those of other, unidentified compounds. 3. Investigation of the carbohydrate metabolism in the semen. In a number of fertile bulls the following deviations were found: a. the presence of quite a lot of fructose in the spermatozoa; b. the presence of glucuronic acid and / of other foreign compounds in the spermatozoa or in the plasma; c. an elevated pH in the seminal plasma some 2 to 3 hours after ejaculation; d. a low phenylalanine-α -ketoglutarate transaminase activity in the whole semen (the spermatozoa had been disrupted by freezing). 4. Investigation of the presence of reducing aldehydes (glycolaldehyde and glyceraldehyde) inside the spermatozoa. The determinations mentioned under 1, 3c and 3d can easily be carried out in the laboratories of each District Animal Health Service and of the A.I. stations; the same is true of the occurrence of amino acids in the seminal plasma and of fructose and aldehydes inside the spermatozoa if facilities are available for electrophoresis and chromatography.

The influence of chlormadinone acetate treatment on the concentration of some steroids in the blood, on the ovarian activity, and on the sexual behaviour of the mare.

The effect of orally administered chlormadinone acetate (CAP), 10 mg daily for a period of 16 days, was investigated in the case of four mares with an irregular oestrous pattern accompanied by low ovarian activity (group 1), four mares which did not show oestrous symptoms at all and which had also low ovarian activity (group 2), and two ovariectomized and two ovario-hysterectomized mares (group 3). In all mares of group 1 and in the two ovariectomized mares of group 3 oestrus symptoms became apparent during treatment. Two mares of group 2 came into heat 8 and 11 days after the cessation of treatment. The two other mares of group 2 and the ovario-hysterectomized mares of group 3 showed no oestrous symptoms at all. In all except the two ovario-hysterectomized mares the progesterone concentration was found to increase in the peripheral blood from less than 1 ng/ml up to at least 4-6 ng/ml. In the mares which came into oestrus this progesterone increase occurred about three days before symptoms of heat became apparent. It is assumed that the uterus played an important role in the increase of the progesterone concentration in the blood during or after CAP treatment. A similar rise of the progesterone level was also found in the ovariectomized mares, indicating that this progesterone did not originate in the ovaries.

Some steroids in the blood of mares suffering from endometritis and post-partum mares as compared to normal cycling mares.

It appeared that in mares suffering from endometritis the concentrations of pregnenolone, of the compound "5.4", and of progesterone in the blood during the first 2 days of the heat period were significantly higher than in normal cycling mares, in which the concentrations of all steroids were less than or equal to 1 ng/ml. In five endometritis mares the "5.4" and progesterone concentrations decreased after the first days of the heat period, and at about two days before ovulation the values were comparable to those of normal cycling mares. Eight mares suffering from a severe endometritis did not ovulate, their oestrous period was prolonged, and the follicles became atretic. Possibly the relatively high progesterone content found in these mares, caused the reduction of the follicular growth, and this might explain the prolonged oestrous period which is observed in many mares suffering from endometritis. Often we observed in post-partum mares the same changes in concentration as in endometritis mares. An elevated level of these steroids was sometimes found in the blood a few days after foaling and, in four of eight post-partum mares, during the foal heat. Most probably this was caused by infection or stimulation of the uterus. The hypothesis is advanced that progesterone originating in the uterus can influence ovarian activity and that it may be one of the factors responsible for the anoestrous periods which can occur in mares with endometritis and in post-partum mares.

The influence of taking uterine biopsies on the concentration of some steroids in the blood and in the uterus, on the ovarian activity, and on the sexual behaviour of the mare.

It has been shown that stimulation of the uterus of mares by the daily taking of biopsies can result in the occurrence of oestrous symptoms. This is accompanied by some follicular growth and a progesterone content in the blood often higher than 1 ng/ml. The following observations suggest that this progesterone originates in the uterus and not in the ovaries: (1) no active corpora lutea appeared to be present in the ovaries after ovariectomy, (2) ovariectomized mares showed the same oestrous symptoms in similar experiments and even mating took place, (3) in the uterine biopsies the concentration of the compound referred to as "5.4", which is assumed to be easily convertible into progesterone, had already increased considerably in the second biopsy, (4) administration of stilboestrol reduced the rise of the progesterone level in the uterine biopsies as well as in the blood. The absence of oestradiol-17 beta in the ovarian follicles and the fact that ovariectomized mares also come into heat suggest that oestrogens cannot be held responsible for the oestrous symptoms in these mares. Our experiments demonstrate that the uterus can be involved in sexual behaviour and the formation of steroids.