Expression of collagen type I and type II in consecutive stages of human osteoarthritis

In normal hyaline cartilage the predominant collagen type is collagen type II along with its associated collagens, for example, types IX and XI, produced by normal chondrocytes. In contrast, investigations have demonstrated that in vitro a switch from collagen type II to collagen type I occurs. Some authors have detected collagen type I in osteoarthritic cartilage also in vivo, especially in late stages of osteoarthritis, while others have not. In the light of these diverging results, we have attempted to elucidate which type of collagen, type I and/or type II, is synthesized in the consecutive stages of human osteoarthritis. We performed in situ hybridization and immunohistochemistry with cartilage tissue samples from patients suffering from various stages of osteoarthritis. Furthermore, we quantitated our results on the gene expression of collagen type I and type II with the help of real-time PCR. We found that with the progression of the disease not only collagen type II, but also increasing amounts of collagen type I mRNA were produced. This supports the conclusion that collagen type I gradually becomes one of the factors involved in the pathogenesis of osteoarthritis.     

Preactivation of β-catenin in osteoblasts improves the osteoanabolic effect of PTH in type 1 diabetic mice

Type 1 diabetes (T1D) is correlated with osteopenia primarily due to low bone formation. Parathyroid hormone (PTH) is a known anabolic agent for bone, the anabolic effects of which are partially mediated through the Wnt/β-catenin signaling pathway. In the present study, we first determined the utility of intermittent PTH treatment in a streptozotocin-induced T1D mouse model. It was shown that the PTH-induced anabolic effects on bone mass and bone formation were attenuated in T1D mice compared with nondiabetic mice. Further, PTH treatment failed to activate β-catenin signaling in osteoblasts of T1D mice and was unable to improve osteoblast proliferation and differentiation. Next, the Col1–3.2 kb-CreERTM; β-cateninfx(ex3) mice were used to conditionally activate β-catenin in osteoblasts by injecting tamoxifen, and we addressed whether or not preactivation of β-catenin boosted the anabolic action of PTH on T1D-related bone loss. The results demonstrated that pretreatment with activation of osteoblastic β-catenin followed by PTH treatment outperformed PTH or β-catenin activation monotherapy and led to greatly improved bone structure, bone mass, and bone strength in this preclinical model of T1DM. Further analysis demonstrated that osteoblast proliferation and differentiation, as well as osteoprogenitors in the marrow, were all improved in the combination treatment group. These findings indicated a clear advantage of developing β-catenin as a target to improve the efficacy of PTH in the treatment of T1D-related osteopenia.     

Role of neuregulin-1β in dexamethasone-enhanced surfactant synthesis in fetal type II cells

It is well established that glucocorticoids elevate the production of fibroblast-pneumocyte factor (FPF), which induces type II cells to synthesize surfactant phospholipids. FPF, however, has not been identified and it is not clear whether it is a single factor or a complex mixture of factors. In this study it has been shown that, when lung fibroblasts are exposed to dexamethasone, the concentration of neuregulin-1β (NRG1β) in conditioned medium is elevated 2-fold (P < 0.05), even though NRG1β gene expression is unaffected. This, together with the finding that exposure of type II cells to NRG1β directly stimulates by 3-fold the rate of phospholipid synthesis (P < 0.05), suggests that NRG1β is a component of FPF that promotes lung development.     

A Gly238Ser substitution in the α2 chain of type I collagen results in osteogenesis imperfecta type III

In general, osteogenesis imperfecta (brittle bone disease) is caused by heterozygous mutations in the genes encoding the 1 or 2 chains of type I collagen (COL1A1 and COL1A2, respectively). In this study we screened these genes in a proband presenting with the severe form (type III) of osteogenesis imperfecta for mutations which might result in the phenotype. Single-strand conformation polymorphism mapping analysis was used to identify a region suspected of harbouring the mutation and subsequent sequence analysis revealed a heterozygous G to A transition in the 2(I) gene of type I collagen in the individual. The resulting substitution of the glycine at position 238 of the chain by serine is the most N-terminal yet reported for this chain.     

Mutational analysis in longest known survivor of mucopolysaccharidosis type VII

Mucopolysaccharidosis VII (MPS VII) is an autosomal recessive disorder caused by the deficiency of beta-glucuronidase leading to the intralysosomal storage of heparan, dermatan, and chondroitin sulfate. Here, we report the identification of two novel missense mutations K350N and R577L in a 37-year-old patient with beta-glucuronidase deficiency and a relatively mild MPS VII phenotype. Expression of the K350N mutation in baby hamster kidney cells has revealed residual enzymatic activity and normal transport of the enzyme to the lysosome. However, expression of the R577L or the double mutant K350N/R577L results in rapid degradation of the enzyme in early biosynthetic compartments and a total loss of enzymatic activity. We attribute the mild phenotype to the residual catalytic activity provided by the K350N mutant. At the time of her death at the age of 37 years, this patient was the longest known survivor with MPS VII.     

Pattern of rise in subplasma membrane Ca concentration determines type of fusing insulin granules in pancreatic β cells

We simultaneously analyzed insulin granule fusion with insulin fused to green fluorescent protein and the subplasma membrane Ca²⁺ concentration ([Ca²⁺]_PM) with the Ca²⁺ indicator Fura Red in rat β cells by dual-color total internal reflection fluorescence microscopy. We found that rapid and marked elevation in [Ca²⁺]_PM caused insulin granule fusion mostly from previously docked granules during the high KCl-evoked release and high glucose-evoked first phase release. In contrast, the slow and sustained elevation in [Ca²⁺]_PM induced fusion from newcomers translocated from the internal pool during the low KCl-evoked release and glucose-evoked second phase release. These data suggest that the pattern of the [Ca²⁺]_PM rise directly determines the types of fusing granules.     

Is the origin of type 1 diabetes in the gut?

In type 1 diabetes, insulin-producing beta-cells in the pancreas are destroyed by immune-mediated mechanisms. The manifestation of the disease is preceded by the so-called pre-diabetic period that may last several years and is characterized by the appearance of circulating autoantibodies against beta-cell antigens. The role of the gut as a regulator of type 1 diabetes was suggested in animal studies, in which changes affecting the gut immune system modulated the incidence of diabetes. Dietary interventions, alterations in the intestinal microbiota and exposure to enteric pathogens, regulate the development of autoimmune diabetes in animal models. It has been demonstrated that these modulations affect the gut barrier mechanisms and intestinal immunity. Because the pancreas and the gut belong to the same intestinal immune system, the link between autoimmune diabetes and the gut is not unexpected. The gut hypothesis in the development of type 1 diabetes is also supported by the observations made in human type 1 diabetes. Early diet could modulate the development of beta-cell autoimmunity; weaning to hydrolysed casein formula decreased the risk of beta-cell autoimmunity by age 10 in the infants at genetic risk. Increased gut permeability, intestinal inflammation with impaired regulatory mechanisms and dysregulated oral tolerance have been observed in children with type 1 diabetes. The factors that contribute to these intestinal alterations are not known, but interest is focused on the microbial stimuli and function of innate immunity. It is likely that our microbial environment does not support the healthy maturation of the gut and tolerance in the gut, and this leads to the increasing type 1 diabetes as well as other immune-mediated diseases regulated by intestinal immune system. Thus, the interventions, aiming to prevent or treat type 1 diabetes in humans, should be targeting the gut immune system.     

The predominant role of IP₃ type 1 receptors in activation of store-operated Ca²+ entry in liver cells

Physiologically, hormone induced release of Ca2+ from intracellular stores occurs in response to inositol 1,4,5-trisphosphate (IP?) binding to its receptors expressed on the membranes of intracellular organelles, mainly endoplasmic reticulum. These IP? receptors act as channels, releasing Ca2+ into the cytoplasmic space where it is responsible for regulating a host of distinct cellular processes. The depletion of intracellular Ca2+ stores leads to activation of store-operated Ca2+ channels on the plasma membrane which replenishes lost Ca2+ and sustain Ca2+ signalling. There are three isoforms of IP? receptor, each exhibiting distinctive properties, however, little is known about the role of each isoform in the activation of store-operated Ca2+ entry. Recent evidence suggest that at least in some cell types the endoplasmic reticulum is not a homogeneous Ca2+ store, and there might be a sub-compartment specifically linked to the activation of store-operated Ca2+ channels, and Ca2+ release activated Ca2+ (CRAC) channel in particular. Furthermore, this sub-compartment might express only certain types of IP? receptor but not the others. Here we show that H4IIE liver cells express all three types of IP? receptor, but only type 1 and to a lesser extent type 3, but not type 2, participate in the activation of CRAC current (I(CRAC)), while type 1 and type 2, but not type 3, participate in observed Ca2+ release in response to receptor stimulation. Presented results suggest that in H4IIE rat liver cells the sub-compartment of intracellular Ca2+ store linked to the activation of I(CRAC) predominantly expresses type 1 IP? receptors.     

Immunocytochemical localization of monoamine oxidase type B in enterochromaffin-like cells of rat oxyntic mucosa

We used immunohistochemistry to identify the localization of monoamine oxidase type B (MAOB) in the rat oxyntic mucosa. At light microscopic levels, MAOB-immunopositive cells were mostly located in the basal half of the oxyntic mucosa. By a double-labeling immunofluorescence method, it was shown that MAOB immunoreactivity was localized in almost all of histidine decarboxylase (HDC)-positive cells. Only a few MAOB-positive cells were negative for HDC. At electron microscopic levels, immunohistochemical reaction products of MAOB were detected on the mitochondrial outer membranes in cells that showed morphological characteristics of enterochromaffin-like (ECL) cells. These findings indicate that ECL cells contain MAOB in the rat. We provide a hypothesis that MAOB is involved in the inactivation mechanism of histamine that is released from ECL cells and activates parietal cells to secrete gastric acid.     

Analysis of gene expression profile of peripheral ganglia in early stage type Ⅱ diabetic rats

Diabetic neuropathy (DN) is defined as the presence of symptoms and/or signs of peripheral nerve dysfunction in people with diabetes. The aim of this study is to screen differentially expressed genes in peripheral ganglia in early stage type Ⅱ experimental diabetic rats. We compared gene expression profiles of peripheral ganglia in type Ⅱ diabetic and nondiabetic rats based on Illumina? Sentrix? BeadChip arrays. The results showed that 158 out of a total of 12 604 known genes were significantly differentially expressed, including 87 up-regulated and 71 down-regulated genes, in diabetic rats compared with those in the nondiabetic rats. It is noted that some up-regulated genes are involved in the biological processes of neuronal cytoskeleton and motor proteins. In contrast, the down-regulated genes are associated with the response to virus\biotic stimulus\ other organism in diabetic rats. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the most significant pathway enriched in the changed gene set is metabolism (P < 0.001). These results indicated that metabolic changes in peripheral ganglia of diabetic rats could be induced by hyperglycemia. Hyperglycemia could change the expression of genes involved in neuronal cytoskeleton and motor proteins through immune inflammatory response, and then impair the structure and function of the peripheral ganglia.     

Heterogeneous distribution of type I nitric oxide synthase in pulmonary vasculature of ovine fetus

The nitric oxide/guanosine 3',5'-cyclic monophosphate pathway plays an essential role in mediating pulmonary vasodilation at birth. Small resistance arteries in the fetal lung are vessels of major significance in the regulation of pulmonary vascular tone. The present study is to determine that type I nitric oxide synthase (NOS-I) is present in ovine fetal pulmonary vasculature and that NOS-I is distributed heterogeneously in ovine fetal pulmonary circulation. We used reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and NOS-I immunohistochemistry to localize NOS-I in fetal sheep lungs and showed a colocalization for NADPH-d activity with NOS-I immunoreactivity. Strong NOS-I immunoreactivity was observed exclusively in the endothelium of the terminal bronchiole and respiratory bronchiole-associated arteries. As a comparison, adult sheep lung did not show positive immunoreactivity in the pulmonary endothelium. NOS-I was absent in the umbilical or systemic arteries from the ovine fetus, whereas abundant NOS-III immunoreactivity was present in these arteries. We conclude that NOS-I is present uniquely in the ovine fetal pulmonary circulation as opposed to the adult pulmonary or the fetal systemic circulation. NOS-I is distributed heterogeneously in the ovine pulmonary vasculature. We speculate that NOS-I plays an active role in the regulation of perinatal pulmonary circulation.     

Pathogenesis of deficient serum α1-antitrypsin in the type ZZ homozygote

Why is the serum α ₁ -antitrypsin activity in the type ZZ (deficient) individual only one-tenth that of the type MM (normal) individual? To determine whether the explanation is simply decreased concentration or also decreased specific activity, the specific activity was determined for ZZ and MM sera. Four ZZ sera had a mean specific activity of 0.665±0.170 sd mg of trypsin inhibited per milliliter of serum and 20 MM sera had a mean specific activity of 0.573±0.157 sd. Hence the lower activity of ZZ serum is due entirely to its lower concentration of α ₁ -antitrypsin. This lower concentration, in turn, could be due to decreased entry into or increased removal from the circulation. Increased removal in ZZ could be due to an increased intrinsic lability or to an accelerated removal mechanism. At acid pH (4.6–6.6) and at elevated temperatures (54–58 C), the activity of ZZ sera was not more labile than that of MM sera. An accelerated removal mechanism may also be unlikely in view of the reported finding that labeled α ₁ -antitrypsin had the same removal rate when injected into an MM or a ZZ individual. If the lower concentration is not due to increased removal, it must be due to decreased entry into the circulation. This could be the result of a decreased rate of synthesis or secretion. The fact that antitrypsin accumulates in the hepatic parenchymal cells of the ZZ individual favors a defect in hepatic secretion.     

Structural analysis of protein–protein interactions in type I polyketide synthases

Abstract

Polyketide synthases (PKSs) are responsible for synthesizing a myriad of natural products with agricultural, medicinal relevance. The PKSs consist of multiple functional domains of which each can catalyze a specified chemical reaction leading to the synthesis of polyketides. Biochemical studies showed that protein–substrate and protein–protein interactions play crucial roles in these complex regio-/stereo-selective biochemical processes. Recent developments on X-ray crystallography and protein NMR techniques have allowed us to understand the biosynthetic mechanism of these enzymes from their structures. These structural studies have facilitated the elucidation of the sequence–function relationship of PKSs and will ultimately contribute to the prediction of product structure. This review will focus on the current knowledge of type I PKS structures and the protein–protein interactions in this system.     

The role of Cas8?in type?I CRISPR interference

CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use ‘cascade’ [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5–Cas7–crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA.     

Biosynthesis of biphenyls and benzophenones – Evolution of benzoic acid-specific type III polyketide synthases in plants

Type III polyketide synthases (PKSs) generate a diverse array of secondary metabolites by varying the starter substrate, the number of condensation reactions, and the mechanism of ring closure. Among the starter substrates used, benzoyl-CoA is a rare starter molecule. Biphenyl synthase (BIS) and benzophenone synthase (BPS) catalyze the formation of identical linear tetraketide intermediates from benzoyl-CoA and three molecules of malonyl-CoA but use alternative intramolecular cyclization reactions to form 3,5-dihydroxybiphenyl and 2,4,6-trihydroxybenzophenone, respectively. In a phylogenetic tree, BIS and BPS group together closely, indicating that they arise from a relatively recent functional diversification of a common ancestral gene. The functionally diverse PKSs, which include BIS and BPS, and the ubiquitously distributed chalcone synthases (CHSs) form separate clusters, which originate from a gene duplication event prior to the speciation of the angiosperms. BIS is the key enzyme of biphenyl metabolism. Biphenyls and the related dibenzofurans are the phytoalexins of the Maloideae. This subfamily of the Rosaceae includes a number of economically important fruit trees, such as apple and pear. When incubated with ortho-hydroxybenzoyl (salicoyl)-CoA, BIS catalyzes a single decarboxylative condensation with malonyl-CoA to form 4-hydroxycoumarin. A well-known anticoagulant derivative of this enzymatic product is dicoumarol. Elicitor-treated cell cultures of Sorbus aucuparia also formed 4-hydroxycoumarin when fed with the N-acetylcysteamine thioester of salicylic acid (salicoyl-NAC). BPS is the key enzyme of benzophenone metabolism. Polyprenylated benzophenone derivatives with bridged polycyclic skeletons are widely distributed in the Clusiaceae (Guttiferae). Xanthones are regioselectively cyclized benzophenone derivatives. BPS was converted into a functional phenylpyrone synthase (PPS) by a single amino acid substitution in the initiation/elongation cavity. The functional behavior of this Thr135Leu mutant was rationalized by homology modeling. The intermediate triketide may be redirected into a smaller pocket in the active site cavity, resulting in phenylpyrone formation by lactonization.     

Localization of hepatocyte growth factor activator inhibitor type 1 in Langhans' cells of human placenta

Activation of hepatocyte growth factor (HGF) is a crucial limiting step in HGF-induced signaling pathway. The HGF activator inhibitor type 1 (HAI-1) was identified as a potent inhibitor of HGF activator (HGFA), a serine proteinase that is responsible for the activation of HGF in vivo. HAI-1 is an integral membrane Kunitz-type serine proteinase inhibitor, and its mRNA has been reported to be most abundant in the placenta. In this report, specific antibody to HAI-1 was used in an immunohistochemical procedure to determine the localization of HAI-I in human placenta. HAI-1 was expressed in cytotrophoblasts (Langhans' cells) of the double-layered trophoblastic epithelium of chorionic villi tissue, and syncytiotrophoblasts were almost negative. On the other hand, extravillous trophoblasts of cytotrophoblastic columns showed markedly decreased immunoreactivity, and those infiltrating into the superficial decidua membrane of early placenta were hardly stainable. The amnionic epithelial cells were also immunostained intensely. The presence of HAI-1 mRNA was also confirmed in a cultured human cytotrophoblastic cell line. In addition to HAI-1, low but distinct expression of HGFA mRNA was observed in the placenta tissue and cultured cytotrophoblasts by using a sensitive RT-PCR method. Since HGF plays an essential role in the placenta development, expression of HAI-1 and HGFA may have an important regulatory role in the placenta. The localization of HAI-I in the proliferating trophoblastic stem cells (Langhans' cells), but not in syncytiotrophoblasts and extravillous trophoblasts, suggest a possible role of HAI-1 in the proliferation of trophoblasts.     

Two unusual types of syndactyly in the same family; Cenani-Lenz type and "new" type versus severe type I syndactyly?

Cenani-Lenz syndactyly is a very rare syndrome where the syndactyly is totally disorganized with abnormal development of pattern formation of the hand. We report here an additional case of Cenani-Lenz syndactylism in a woman who has congenital cataract and an unusual type of duplication of big toes not described so far. She had a half cousin who had an unusual new type or severe type I syndactyly. It is not clear whether these two types of syndactyly present in this family may be coincidental or not.     

Immunocytochemical localization of 5α-reductase type 1 in the prostate of normal and castrated rats

We immunohistochemically studied the localization of 5-reductase type 1 in combination with androgen receptor (AR) expression in individual lobes of the prostates of intact and castrated rats. In the normal rat prostate, 5-reductase was localized in the cytoplasm of most epithelial cells in the ventral, dorsal, and lateral type 1 (L1) lobes. Epithelial cells of lateral type 2 (L2) lobes were negative for 5-reductase. AR was present in the nuclei of all epithelial and stromal cells throughout the prostate. The number of 5-reductase-immunoreactive cells rapidly decreased in the ventral and L1 lobes after castration, whereas many positive cells remained in the dorsal lobe even at 4 weeks after castration. AR immunostaining was lost in the ventral, dorsal, and L1 lobes at 1 week after castration, but remained in the L2 lobe of 4-week-castrated rats. Electron microscopic immunocytochemistry showed that 5-reductase was exclusively localized in the rough endoplasmic reticulum membranes and that there were no distinct structural differences between the positively and negatively stained epithelial cells. These findings suggested that the expression of 5-reductase type 1 in the epithelial cell is heterogeneous within and among the individual lobes of the rat prostate, and does not correspond to AR expression.     

A new type of anvil in the Acheulian of Gesher Benot Ya‘aqov,Israel

We report here on the identification and characterization of thin basalt anvils, a newly discovered component of the Acheulian lithic inventory of Gesher Benot Ya‘aqov (GBY). These tools are an addition to the array of percussive tools (percussors, pitted stones and anvils) made of basalt, flint and limestone. The thin anvils were selected from particularly compact, horizontally fissured zones of basalt flows. This type of fissuring produces a natural geometry of thick and thin slabs. Hominins at GBY had multiple acquisition strategies, including the selection of thick slabs for the production of giant cores and cobbles for percussors. The selection of thin slabs was carried out according to yet another independent and targeted plan. The thinness of the anvils dictated a particular range of functions. The use of the anvils is well documented on their surfaces and edges. Two main types of damage are identified: those resulting from activities carried out on the surfaces of the anvils and those resulting from unintentional forceful blows (accidents de travaille). Percussive activities that may have been associated with the thin anvils include nut cracking and the processing of meat and bones, as well as plants.