Immunohistochemical localization of carbonic anhydrase isoenzymes in the human male reproductive tract

The distribution of human carbonic anhydrase (HCA) isoenzymes I, II and VI in the human male reproductive tract was studied using specific antisera against affinity purified isoenzymes in conjunction with the peroxidase-antiperoxidase complex method. HCA VI-specific staining could not be demonstrated in any of the tissues studied, and HCA I was observed only in red blood cells. Immunostaining denoted HCA II in the epithelia of the seminal vesicle, ampulla of the ductus deferens and distal ductus deferens. Some cells in the epithelium of the corpus and cauda epididymidis also stained for HCA II. The staining for HCA II in the epithelium of the reproductive tract declined from the strongly positive seminal vesicle to the proximal part of the ductus deferens, which stained negatively. There were also HCA II-positive particles derived from the apical protrusions of the epithelium in the lumina of the seminal vesicle, ampulla of the ductus deferens and ductus deferens. The physiological role of HCA II is linked to the secretion of bicarbonate into the seminal plasma and thereby to the regulation of sperm motility and pH in the seminal plasma.     

A high activity carbonic anhydrase isoenzyme (CA II) is present in mammalian spermatozoa

Summary Human and rat spermatozoa were stained for different carbonic anhydrase (CA) isoenzymes using specific antisera to human CA I, II and VI in conjunction with the immunofluorescence technique. The spermatozoa of both species were found to contain only CA II, which was located principally in the postacrosomal region of the human spermatozoa and in the acrosomal cap region of the rat spermatozoa. The presence of CA II could be confirmed by immunoblotting, which revealed a 29 K polypeptide in both the human and rat spermatozoa. No CA I or VI-specific fluorescence could be detected in the spermatozoa of either species. The immunoblottings were also negative. The results show mammalian spermatozoa to contain the high activity carbonic anhydrase isoenzyme II. Its presence is probably linked to hydration of CO₂ produced by active energy metabolism and thereby to the maintaining of an adequate intraspermatozoal bicarbonate concentration as required for the maintenance of sperm motility.     

Immunohistochemical localization of carbonic anhydrase isoenzymes in the human male reproductive tract

Summary The distribution of human carbonic anhydrase (HCA) isoenzymes I, II and VI in the human male reproductive tract was studied using specific antisera against affinity purified isoenzymes in conjunction with the peroxidase-antiperoxidase complex method. HCA VI-specific staining could not be demonstrated in any of the tissues studied, and HCA I was observed only in red blood cells. Immunostaining denoted HCA II in the epithelia of the seminal vescle, ampulla of the ductus deferens and distal ductus deferens. Some cells in the epithelium of the corpus and cauda epididymidis also stained for HCA II. The staining for HCA II in the epithelium of the reproductive tract declined from the strongly positive seminal vesicle to the proximal part of the ductus deferens, which stained negatively. There were also HCA II-positive particles derived from the apical protrusions of the epithelium in the lumina of the seminal vesicle, ampulla of the ductus deferens and ductus deferens. The physiological role of HCA II is linked to the secretion of bicarbonate into the seminal plasma and thereby to the regulation of sperm motility and pH in the seminal plasma.     

A high activity carbonic anhydrase isoenzyme (CA II) is present in mammalian spermatozoa

Human and rat spermatozoa were stained for different carbonic anhydrase (CA) isoenzymes using specific antisera to human CA I, II and VI in conjunction with the immunofluorescence technique. The spermatozoa of both species were found to contain only CA II, which was located principally in the postacrosomal region of the human spermatozoa and in the acrosomal cap region of the rat spermatozoa. The presence of CA II could be confirmed by immunoblotting, which revealed a 29 K polypeptide in both the human and rat spermatozoa. No CA I or VI-specific fluorescence could be detected in the spermatozoa of either species. The immunoblottings were also negative. The results show mammalian spermatozoa to contain the high activity carbonic anhydrase isoenzyme II. Its presence is probably linked to hydration of CO2 produced by active energy metabolism and thereby to the maintaining of an adequate intraspermatozoal bicarbonate concentration as required for the maintenance of sperm motility.     

Expression, purification, kinetic, and structural characterization of an alpha-class carbonic anhydrase from Aedes aegypti (AaCA1)

Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. The alpha-class CAs are found predominantly in vertebrates, but they are also expressed in insects like mosquitoes. Recently, an alpha-CA from the midgut of Aedes aegypti larvae (AaCA1) was identified, cloned, and subsequently shown to share high sequence homologous to human CA I (HCA I). This paper presents the bacterial expression, purification, and kinetic characterization of the soluble CA domain of AaCA1. The data show AaCA1 is a highly active CA that displays inhibition by methazolamide and ethoxzolamide with nM affinity. Additionally, a homology model of AaCA1, based on the crystal structure of HCA I, is presented and the overall structure, active site, and surface charge properties are compared to those of HCA I and II. Measurements of catalysis show that AaCA1 is more like HCA II in terms of proton transfer, but more similar to HCA I in terms of conversion of carbon dioxide to bicarbonate, and these differences are rationalized in terms of structure. These results also indicate that amino acid differences in the active site of AaCA1 compared to human CAs could be used to design specific CA inhibitors for the management of mosquito populations.     

Porphyromonas gingivalis within Placental Villous Mesenchyme and Umbilical Cord Stroma Is Associated with Adverse Pregnancy Outcome

Intrauterine presence of Porphyromonas gingivalis (Pg), a common oral pathobiont, is implicated in preterm birth. Our aim was to determine if the location of Pg within placental and/or umbilical cord sections was associated with a specific delivery diagnosis at preterm delivery (histologic chorioamnionitis, chorioamnionitis with funisitis, preeclampsia, and preeclampsia with HELLP-syndrome, small for gestational age). The prevalence and location of Pg within archived placental and umbilical cord specimens from preterm (25 to 32 weeks gestation) and term control cohorts were evaluated by immunofluorescent histology. Detection of Pg was performed blinded to pregnancy characteristics. Multivariate analyses were performed to evaluate independent effects of gestational age, being small for gestational age, specific preterm delivery diagnosis, antenatal steroids, and delivery mode, on the odds of having Pg in the preterm tissue. Within the preterm cohort, 49 of 97 (51%) placentas and 40 of 97 (41%) umbilical cord specimens were positive for Pg. Pg within the placenta was significantly associated with shorter gestation lengths (OR 0.63 (95%CI: 0.48–0.85; p = 0.002) per week) and delivery via caesarean section (OR 4.02 (95%CI: 1.15–14.04; p = 0.03), but not with histological chorioamnionitis or preeclampsia. However, the presence of Pg in the umbilical cord was significantly associated with preeclampsia: OR 6.73 (95%CI: 1.31–36.67; p = 0.02). In the term cohort, 2 of 35 (6%) placentas and no umbilical cord term specimens were positive for Pg. The location of Pg within the placenta was different between preterm and term groups in that Pg within the villous mesenchyme was only detected in the preterm cohort, whereas Pg associated with syncytiotrophoblasts was found in both preterm and term placentas. Taken together, our results suggest that the presence of Pg within the villous stroma or umbilical cord may be an important determinant in Pg-associated adverse pregnancy outcomes.     

Carbonic anhydrase isoenzymes II and I are present in the zona glomerulosa cells of the human adrenal gland

Human carbonic anhydrase isoenzymes I and II (HCA I and II) were purified from human erythrocytes by inhibitor affinity chromatography and ion-exchange chromatography. These isoenzymes were then located in the human adrenal gland using specific polyclonal antisera raised in rabbits and specific detection by immunohistochemical techniques. Both HCA II and I were located in the zona glomerulosa cells, although the staining for HCA I was faint. The cells of the zona fasciculata and the zona reticularis failed to stain with either antiserum. Control stainings with preimmune or anti-HCA VI sera were negative. The presence of HCA II and I in the zona glomerulosa cells may be linked to regulation of the biosynthesis or secretion of mineralocorticoids.     

Distribution of CA 125 in placental tissues

The presence of the tumor marker CA 125 was studied in different compartments of the human placenta. Levels of CA 125 in the cytosol of chorionic villi ranged from 27-17100 U/g (median 560 U/g). In the placental amnion and chorion concentrations ranged from 175-29000 U/g, median 1060 U/g and were not statistically different. In the umbilical cord values were significantly lower (range 44-7600 U/g; median 180 U/g). Maternal serum probes were above the upper limit of normal in all cases (range 48-500 U/ml; median 131 U/ml). Immunohistochemistry detected CA 125 exclusively within the amniotic cells of the placenta and the umbilical cord. This might be because CA 125 fixes more to insoluble structures in the amnion or because of contamination of chorionic villi with the underlying decidua.     

The human carbonic anhydrase isoenzymes I and II inhibitory effects of some hydroperoxides,alcohols, and acetates

The carbonic anhydrases (CAs, EC 4.2.1.1) represent a superfamily of widespread enzymes, which catalyze a crucial biochemical reaction, the reversible hydration of carbon dioxide to bicarbonate and protons. Human CA isoenzymes I and II (hCA I and hCA II) are ubiquitous cytosolic isoforms. In this study, a series of hydroperoxides, alcohols, and acetates were tested for the inhibition of the cytosolic hCA I and II isoenzymes. These compounds inhibited both hCA isozymes in the low nanomolar ranges. These compounds were good hCA I inhibitors (K_is in the range of 24.93–97.99?nM) and hCA II inhibitors (K_is in the range of 26.04–68.56?nM) compared to acetazolamide as CA inhibitor (K_i: 34.50?nM for hCA I and K_i: 28.93?nM for hCA II).     

Kinetic and crystallographic studies of the role of tyrosine 7 in the active site of human carbonic anhydrase II

The rate limiting step in catalysis of bicarbonate dehydration by human carbonic anhydrase II (HCA II) is an intramolecular proton transfer from His64 to the zinc-bound hydroxide. We have examined the role of Tyr7 using site-specific mutagenesis and measuring catalysis by the ¹⁸O exchange method using membrane inlet mass spectrometry. The side chain of Tyr7 in HCA II extends into the active-site cavity about 7 Å from the catalytic zinc atom. Replacement of Tyr7 with eight other amino acids had no effect on the interconversion of bicarbonate and CO₂, but in some cases caused enhancements in the rate constant of proton transfer by nearly 10-fold. The variant Y7I HCA II enhanced intramolecular proton transfer approximately twofold; its structure was determined by X-ray crystallography at 1.5 Å resolution. No changes were observed in the ordered solvent structure in the active-site cavity or in the conformation of the side chain of the proton shuttle His64. However, the first 11 residues of the amino-terminal chain in Y7I HCA II assumed an alternate conformation compared with the wild type. Differential scanning calorimetry showed variants at position 7 had a melting temperature approximately 8 °C lower than that of the wild type.     

Inflammatory and Haematological Markers in the Maternal,Umbilical Cord and Infant Circulation in Histological Chorioamnionitis

Background

The relationship between histological chorioamnionitis and haematological and biochemical markers in mothers and infants at delivery, and in infants postnatally, is incompletely characterised. These markers are widely used in the diagnosis of maternal and neonatal infection. Our objective was to investigate the effects of histological chorioamnionitis (HCA) on haematological and biochemical inflammatory markers in mothers and infants at delivery, and in infants post-delivery.

Methods

Two hundred and forty seven mothers, delivering 325 infants, were recruited at the only tertiary perinatal centre in Western Australia. Placentae were assessed for evidence of HCA using a semi-quantitative scoring system. Maternal high sensitivity C-reactive protein (hsCRP), procalcitonin, and umbilical cord hsCRP, procalcitonin, white cell count and absolute neutrophil count were measured at delivery. In infants where sepsis was clinically suspected, postnatal CRP, white cell count and absolute neutrophil count were measured up to 48 hours of age. The effect of HCA on maternal, cord and neonatal markers was evaluated by multivariable regression analysis.

Results

The median gestational age was 34 weeks and HCA was present in 26 of 247 (10.5%) placentae. Mothers whose pregnancies were complicated by HCA had higher hsCRP (median 26 (range 2–107) versus 5.6 (0–108) mg/L; P<0.001). Histological chorioamnionitis was associated with higher umbilical cord hsCRP (75^th percentile 2.91 mg/L (range 0–63.9) versus 75^th percentile 0 mg/L (0–45.6); P<0.001) and procalcitonin (median 0.293 (range 0.05–27.37) versus median 0.064 (range 0.01–5.24) ug/L; P<0.001), with a sustained increase in neonatal absolute neutrophil count (median 4.5 (0.1–26.4)×10⁹/L versus 3.0 (0.1–17.8)×10⁹/L), and CRP up to 48 hours post-partum (median 10 versus 6.5 mg/L) (P<0.05 for each).

Conclusion

Histological chorioamnionitis is associated with modest systemic inflammation in maternal and cord blood. These systemic changes may increase postnatally, potentially undermining their utility in the diagnosis of early-onset neonatal infection.     

Ubiquitin-Mediated Stress Response in a Rat Model of Brain Transient Ischemia/Hypoxia

Ubiquitin (Ub) is a small 76-residue protein, involved in intracellular protein degradation through a specific ATP-dependent system, which uses Ub as a tag to label proteins committed to be hydrolyzed by a specific 26 S protease. PGP-9.5 is another important component of the Ub system, i.e. a neuron-specific carboxyl-terminal hydrolase, which recycles Ub from Ub-polypeptide complexes. We have investigated the expression of Ub and PGP-9.5 in rat hippocampal neurons in an early phase of reperfusion in a model of transient global brain ischemia/hypoxia (bilateral occlusion of common carotid arteries for 10 min accompanied by mild hypoxia—15% O₂—for 20 min), by means of immunohistochemical methods using light and electron microscopy. The intensity of Ub and PGP-9.5 immunoreactivity was evaluated by image analysis. We have detected a marked increase of Ub immunoreactivity (UIR) in neurons of CA1, CA2, CA3, CA4, and dentate gyrus subfields 1 hr after ischemia/hypoxia (but not after hypoxia only), statistically significant as confirmed by image analysis. Such increase in immunoreactivity in ischemic/hypoxic rats was localized essentially in the nuclei of hippocampal neurons. There were no changes in PGP-9.5 immunoreactivity. The data suggest that in the present model of rat brain ischemia/hypoxia Ub is involved in the neuronal stress response.     

1H‐indazole molecules reduced the activity of human erythrocytes carbonic anhydrase I and II isoenzymes

Carbonic anhydrase (CA) is an important metabolic enzyme family closely related to many physiological and pathological processes. Currently, carbonic anhydrase inhibitors are the target molecules in the treatment and diagnosis of many diseases. In present study, we investigated the inhibitory effects of some indazole molecules on the CA‐I and CA‐II isoenzymes isolated from human erythrocytes. We showed that human CA‐I and CA‐II activities were reduced by of some indazoles at low concentrations. IC₅₀ values, K_i constants, and inhibition types for each indazole molecule were determined. The indazoles showed K_i constants in a range of 0.383 ± 0.021 to 2.317 ± 0.644 mM, 0.409 ± 0.083 to 3.030 ± 0.711 mM against CA‐I and CA‐II, respectively. Each indazole molecule exhibited a noncompetitive inhibition effect. Bromine‐ and chlorine‐bonded indazoles were found to be more potent inhibitory effects on carbonic anhydrase isoenzymes. In conclusion, we conclude that these results may be useful in the synthesis of carbonic anhydrase inhibitors.     

Mitochondrial carbonic anhydrase in the nervous system: expression in neuronal and glial cells

Carbonic anhydrase (CA) V is a mitochondrial enzyme that has been reported in several tissues of the gastrointestinal tract. In liver, it participates in ureagenesis and gluconeogenesis by providing bicarbonate ions for two other mitochondrial enzymes: carbamyl phosphate synthetase I and pyruvate carboxylase. This study presents evidence of immunohistochemical localization of CA V in the rodent nervous tissue. Polyclonal rabbit antisera against a polypeptide of 17 C-terminal amino acids of rat CA V and against purified recombinant mouse isozyme were used in western blotting and immunoperoxidase stainings. Immunohistochemistry showed that CA V is expressed in astrocytes and neurons but not in oligodendrocytes, which are rich in CA II, or capillary endothelial cells, which express CA IV on their plasma face. The specificity of the immunohistochemical results was confirmed by western blotting, which identified a major 30-kDa polypeptide band of CA V in mouse cerebral cortex, hippocampus, cerebellum, spinal cord, and sciatic nerve. The expression of CA V in astrocytes and neurons suggests that this isozyme has a cell-specific, physiological role in the nervous system. In astrocytes, CA V may play an important role in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase. The neuronal CA V could be involved in the regulation of the intramitochondrial calcium level, thus contributing to the stability of the intracellular calcium concentration. CA V may also participate in bicarbonate ion-induced GABA responses by regulating the bicarbonate homeostasis in neurons, and its inhibition could be the basis of some neurotropic effects of carbonic anhydrase inhibitors.     

Synthesis and inhibitory properties of some carbamates on carbonic anhydrase and acetylcholine esterase

A series of carbamate derivatives were synthesized and their carbonic anhydrase I and II isoenzymes and acetylcholinesterase enzyme (AChE) inhibitory effects were investigated. All carbamates were synthesized from the corresponding carboxylic acids via the Curtius reactions of the acids with diphenyl phosphoryl azide followed by addition of benzyl alcohol. The carbamates were determined to be very good inhibitors against for AChE and hCA I, and II isoenzymes. AChE inhibition was determined in the range 0.209–0.291?nM. On the other hand, tacrine, which is used in the treatment of Alzheimer’s disease possessed lower inhibition effect (K_i: 0.398?nM). Also, hCA I and II isoenzymes were effectively inhibited by the carbamates, with inhibition constants (K_i) in the range of 4.49–5.61?nM for hCA I, and 4.94–7.66?nM for hCA II, respectively. Acetazolamide, which was clinically used carbonic anhydrase (CA) inhibitor demonstrated K_i values of 281.33?nM for hCA I and 9.07?nM for hCA II. The results clearly showed that AChE and both CA isoenzymes were effectively inhibited by carbamates at the low nanomolar levels.     

Ultrastructural study of neurotensin immunoreactivity in the superficial laminae of the dorsal horn of the rat

Neurotensin immunoreactivity was identified in cell bodies, dendrites, spines, axons, terminals and varicosities in superficial laminae of rat spinal cord with the electron microscope. Unlabeled terminals synapsed with neurotensin-immunoreactive cell bodies, dendrites and spines. Presynaptic terminals contained round or pleomorphic vesicles and generally made symmetrical contacts with medium-sized neurotensin-containing dendrites in outer lamina II, and asymmetrical or symmetrical contacts with large and small dendrites and spines in inner lamina II. Neurotensin immunoreactive axons were unmyelinated, and their terminals were presynaptic to unlabeled dendrites and spines in laminae I and II. Terminals contained small, round, clear vesciles (31 nm) and occasional large granular vesicles (78 nm). Contacts in outer lamina II were evenly distributed among dendrites of various sizes and spines, whereas the majority of labeled terminals in inner lamina II made contacts onto small dendrites and spines. These findings indicate that neurotensin effects in rat spinal cord are mediated by axodendritic synapses, and that neurotensin cells at the inner and outer borders of lamina II contact dendrites of efferent neurons or other interneurons in the dorsal horn.     

Tissue and cellular distribution of the extended family of protein kinase C isoenzymes

Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.     

Collagen of umbilical cord vein and its alterations in pre-eclampsia

The state of the vascular system of the mother and of placenta is known to exert a great influence on intrauterinal development of the fetus. Pre-eclampsia is the most common pathological syndrome connected with pregnancy. Since collagen is one of the main constituents of the vessel wall a comparison was made with collagen content and its molecular polymorphism in umbilical cord veins of newborns from healthy and pre-eclamptic mothers. It was found that umbilical cord veins of newborns from mothers with pre-eclampsia contained 18% less collagen than those of the newborns from normal pregnancies. This decrease was accompanied by a slight decrease of collagen solubility, but all its types (I, II, IV, V and VI) were present. However, the umbilical vein wall of newborns from mothers with pre-eclampsia contained relatively less of type I and more of type III collagen than the normal umbilical cord. These differences may be connected with a disturbance of blood flow in fetus of a woman with pre-eclampsia.