Comparative evaluation of fish larval preservation methods on microbiome profiles to aid in metagenomics research.

Applications of microbiome research through metagenomics promise to generate microbiome manipulation strategies for improved larval survival in aquaculture. However, existing lacunae on the effects of sample preservation methods in metagenome profiles hinder the successful application of this technique. In this context, four preservation methods were scrutinized to identify reliable methods for fish larval microbiome research. The results showed that a total of ten metagenomics metrics, including DNA yield, taxonomic and functional microbiome profiles, and diversity measures, were significantly (P < 0.05) influenced by the preservation method. Activity ranking based on the performance and reproducibility showed that three methods, namely immediate direct freezing, room temperature preservation in absolute ethanol, and preservation at - 20 °C in lysis, storage, and transportation buffer, could be recommended for larval microbiome research. Furthermore, as there was an apparent deviation of the microbiome profiles of ethanol preserved samples at room temperature, the other methods are preferred. Detailed analysis showed that this deviation was due to the bias towards Vibrionales and Rhodobacterales. The microbial taxa responsible for the dissimilarity across different methods were identified. Altogether, the paper sheds light on the preservation protocols of fish larval microbiome research for the first time. The results can help in cross-comparison of future and past larval microbiome studies. Furthermore, this is the first report on the activity ranking of preservation methods based on metagenomics metrics. Apart from methodological perspectives, the paper provides for the first time certain insights into larval microbial profiles of Rachycentron canadum, a potential marine aquaculture species. KEY POINTS: • First report on effects of preservation methods on fish larval microbiome profiles. • First report on activity ranking of preservation methods based on metagenomics metrics. • Storage methods influenced DNA yield, taxonomic and functional microbiome profiles.

Morphological and molecular characterization of Ceratomyxa xanthopteri n. sp. (Myxosporea: Ceratomyxidae) from the marine ornamental fish Acanthurus xanthopterus Valenciennes 1835 (Acanthuridae) off Vizhinjam coast, Kerala.

A new species of Ceratomyxa infecting the gallbladder of the marine ornamental fish Acanthurus xanthopterus collected from the Vizhinjam coast of Kerala is described. The parasite exhibited a prevalence of 100%. Mature spores recovered from the gallbladder were slightly crescentic with rounded lateral extremities and possessed convex anterior and slightly concave to straight posterior margins. Spore valves two, equal, joined by a straight and prominent suture. Myxospores measured 5.5 ± 0.6 µm in length and 15.9 ± 2.3 µm in thickness. Polar capsules two, equal, spherical, positioned anteriorly on either sides of the suture, 2.3 ± 0.2 µm long and 2.2 ± 0.2 µm wide. Polar filament with four to five coils, 21.2 ± 0.6 µm when extruded. Posterior angle 173.6 ± 5.2°. Early sporogonic stages and monosporic, disporic, and multisporic plasmodial stages were spherical to irregular in shape, with or without filopodia. Histopathologic analysis revealed that spores and developing stages were attached to the gallbladder wall as well as found free in the lumen. Morphologic and morphometric comparison of the present parasite with known species of Ceratomyxa indicated significant differences. In molecular and phylogenetic analyses, the present myxosporean revealed high divergence with related forms and occupied an independent position within the Ceratomyxa clade with high nodal support. Considering the morphological, morphometric, molecular, and phylogenetic dissimilarities with the previously described species of Ceratomyxa and the differences in host and geographic locations, the present species of myxosporean is treated as new and is named Ceratomyxa xanthopteri n. sp.

Assessment of gamma radiation and associated radiation hazards in coastal sediments of south east coast of Tamilnadu, India with statistical approach.

Gamma spectroscopy was performed to determine the concentrations of 226Ra, 232Th and 40K in sediment samples collected from Periyakalapet to Parangaipettai, East coast of Tamilnadu. The activity concentrations were determined by direct counting using a hyper pure germanium (HPGe) detector inter phased with a multi channel analyzer (MCA). The average activity concentrations of the corresponding nuclides were 30.81 Bq kg-1 for 226Ra, 85.67 Bq kg-1 for 232Th and 425.72 Bq kg-1 for 40K. The average activity concentration of 232Th and 40K are slightly higher and 226Ra is lower than world average values. The radiation hazard indices namely Radium Equivalent Activity (Raeq) Absorbed Gamma Dose Rate (DR), Annual Effective Dose Rate (HR), Representative Level Index (RLI), Annual Gonadal Dose Equivalent (AGDE), Internal Hazard Index (Hin) and External Hazard index (Hex) are calculated and compared with the previously reported data. The extracted values are comparable to the recommended values and they all fall within the safety limits. Hence harmful radiation effects are not posed to the public and tourists going to the beaches for recreation or to the fishermen involved in their activities in the area as a result of the natural radioactivity of sediments. Multivariate Statistical analyses were carried out between the parameters obtained from the radioactivity to know the existing relations and to study the spatial distribution of radionuclides.

Histological profiling of gonads depicting protandrous hermaphroditism in Eleutheronema tetradactylum.

The fourfinger threadfin Eleutheronema tetradactylum is reported as a protandrous hermaphrodite from Australian waters, while being a gonochorist in reports from Singapore and India, with a single report of protandrous hermaphroditism from the latter. Histological analysis of gonads of fish from Indian waters confirms protandrous hermaphroditism in E. tetradactylum. The study was based on 480 fish examined from eight locations along the Indian coast. Mean total length (LT ) of male fish was 240 mm with the transition to female starting from 280 mm LT . Specimens confirmed as mature females were >380 mm LT .

Antioxidant, functional properties and amino acid composition of pepsin-derived protein hydrolysates from whole tilapia waste as influenced by pre-processing ice storage.

In recent years, hygienic handling of fishery waste is demanded owing to the fact that the fishery waste is an ideal raw material for the preparation of bioactive compounds. In the present study, the effect of pre-processing storage (at 4 ± 2 °C) of whole tilapia waste (WTW) on the properties of its protein hydrolysate derived using pepsin was evaluated. Fish protein hydrolysates (FPH) were prepared from 0, 24 and 48 h old ice stored WTW and designated as FPH-0, FPH-1, and FPH-2, respectively. Total amino acids, total essential amino acids and total hydrophobic amino acids of FPH samples increased with the storage period of raw material (WTW). Antioxidant activities such as DPPH (2, 2 diphynyl-1-picrylhydrazyl) free radical scavenging activity and ferric reducing power of FPH samples were dose dependent. FPH-0 had better antioxidant properties including linoleic acid peroxidation inhibition activity than FPH-1 and FPH-2. The DNA nicking assay revealed the protective effect of FPH preparations against Fenton's reaction mediated oxidative damage. FPH-2 had better emulsifying properties and foaming stability whereas the FPH-0 had relatively good foaming capacity. SDS-PAGE indicated the presence of peptides ranging from 116 to < 14.4 kDa in FPH-0 and less than 18 kDa in FPH-1 and FPH-2. The present study, clearly demonstrated that whole tilapia waste can effectively be converted to FPH and could be a potential ingredient in functional food and as a rich source of high-quality protein in animal feed formulations.

Emergence of carp edema virus in cultured ornamental koi carp, Cyprinus carpio koi, in India.

A disease outbreak was reported in adult koi, Cyprinus carpio koi, from a fish farm in Kerala, India, during June 2015. The clinical signs were observed only in recently introduced adult koi, and an existing population of fish did not show any clinical signs or mortality. Microscopic examination of wet mounts from the gills of affected koi revealed minor infestation of Dactylogyrus sp. in a few koi. In bacteriological studies, only opportunistic bacteria were isolated from the gills of affected fish. The histopathological examination of the affected fish revealed necrotic changes in gills and, importantly, virus particles were demonstrated in cytoplasm of gill epithelial cells in transmission electron microscopy. The tissue samples from affected koi were negative for common viruses reported from koi viz. cyprinid herpesvirus 3, spring viraemia of carp virus, koi ranavirus and red sea bream iridovirus in PCR screening. However, gill tissue from affected koi carp was positive for carp edema virus (CEV) in the first step of nested PCR, and sequencing of PCR amplicons confirmed infection with CEV. No cytopathic effect was observed in six fish cell lines following inoculation of filtered tissue homogenate prepared from gills of affected fish. In bioassay, the symptoms could be reproduced by inoculation of naive koi with filtrate from gill tissue homogenate of CEV-positive fish. Subsequently, screening of koi showing clinical signs similar to koi sleepy disease from different locations revealed that CEV infection was widespread. To our knowledge, this is the first report of infection with CEV in koi from India.

Morphological and Molecular Characterization of Two New Species of Ceratomyxa Thélohan, 1892 (Cnidaria: Myxosporea) from the Marine Ornamental Fish Zanclus cornutus (Linnaeus, 1758) off Lakshadweep Islands, Arabian Sea.

PURPOSE: The present study provides the complete morphological and molecular description of two new species of myxosporeans, Ceratomyxa zancli n. sp. and Ceratomyxa cornuti n. sp. infecting the gallbladder of Zanclus cornutus from the Lakshadweep Islands, Arabian Sea. METHODS: Zanclus cornutus were screened for the presence of myxosporeans, and the recovered myxospores were morphologically characterized using Nomarski Differential Interference Contrast (DIC) optics. The sequences of SSU rDNA were employed for molecular and phylogenetic studies. RESULTS: Both the parasites exhibited a prevalence of 21% each. C. zancli n. sp. is characterized by broadly cresentic myxospores with convex anterior and slightly concave to straight posterior margins and rounded ends. Spore valves two, unequal, measured 9.6 ± 0.7 µm × 25.2 ± 1.3 µm. Polar capsules two, unequal, spherical, measured 4 ± 0.6 µm × 3.5 ± 0.6 µm. Polar filament exceptionally long and arranged irregularly. Myxospores of C. cornuti n. sp. are elongated with convex anterior and slightly concave to straight posterior margins. Spore valves two, unequal, measured 7.00 ± 0.4 µm × 26.56 ± 1.8 µm. Polar capsules spherical, unequal, measured 3.52 ± 0.2 × 3.36 ± 0.35. Molecular analysis of C. zancli n. sp. (ON818297) and C. cornuti n. sp. (ON818298) resulted in 1469 and 1491 bp long SSU rDNA sequences, respectively. Molecularly C. zancli n. sp. is close to C. diplodae and C. barnesi with 91.39% similarity, while C. cornuti n. sp. appears closer to C. robertsthomsoni with 97.46% similarity. In phylogenetic analyses, C. zancli n. sp. branched separately within the Ceratomyxa clade while C. cornuti n. sp. clustered with C. robertsthomsoni and C. thalassomae. CONCLUSION: Based on the differences in morphological, morphometric, molecular, and phylogenetic characteristics, as well as differences in the host and geographic location, the above two species of myxosporeans are considered novel. The study forms the first report of a species of Ceratomyxa from Z. cornutus.

Full-length transcriptome from different life stages of cobia (Rachycentron canadum, Rachycentridae).

Cobia (Rachycentron canadum, Rachycentridae) is one of the prospective species for mariculture. The transcriptome-based study on cobia was hampered by an inadequate reference genome and a lack of full-length cDNAs. We used a long-read based sequencing technology (PacBio Sequel II Iso-Seq3 SMRT) to obtain complete transcriptome sequences from larvae, juveniles, and various tissues of adult cobia, and a single SMRTcell generated 99 gigabytes of data and 51,205,946,694 bases. A total of 8609435, 7441673 and 9140164 subreads were generated from the larval, juvenile, and adult sample pools, with mean sub-read lengths of 2109.9, 1988.2 and 1996.2 bp, respectively. All samples were combined to increase transcript recovery and clustered into 35661 high-quality reads. This is the first report on a full-length transcriptome from R. canadum. Our results illustrate a significant increase in the identified amount of cobia LncRNAs and alternatively spliced transcripts, which will help improve genome annotation. Furthermore, this information will be beneficial for nutrigenomics and functional studies on cobia and other commercially important mariculture species.

Morphological and molecular description of a new species of Myxobolus (Myxosporea: Myxobolidae) infecting Planiliza macrolepis (Smith, 1846) from India.

The present paper describes a novel species of Myxobolus parasitizing the gill filaments of the largescale mullet, Planiliza macrolepis from Cochin backwaters, Kerala, India. The parasite develops in the gill filaments; plasmodia elongated, milky white, measured 1.37-2.18 (1.78 ± 0.35) mm × 0.07-0.12 (0.10 ± 0.02) mm in size. Mature myxospores ovoid in valvular view, biconvex in sutural view with smooth shell valves and measured 6.24-7.02 (6.63 ± 0.23) × 5.01-6.18 (5.68 ± 0.25) µm in size. Polar capsules equal, oval with pointed anterior ends, 3.07-3.58 (3.33 ± 0.12) × 1.68-2.42 (2.09 ± 0.18) µm in size. Polar filaments with 4 coils, measured 29.61 ± 4.75 µm in length when extruded. Sporoplasm binucleate with a rudimentary nucleus and a vacuole. A comparison with related Myxobolus species revealed significant morphological and morphometric differences. In BLASTN and genetic distance analysis, the present parasite showed high divergence with other myxosporean sequences, indicating its molecular uniqueness. In Maximum Likelihood and Bayesian Inference analysis, the present species stands out with M. ramadus as sister branch within the Myxobolus clade. In infected gill filaments, the plasmodia caused swelling/deformation, compression of lamellae and reduction in respiratory surface area. Three of 222 P. macrolepis screened were infected, indicating a prevalence of 1.3%. Considering the morphological, morphometric, molecular and phylogenetic differences with the previously described species of myxosporeans, along with the dissimilarities in host and geographical locations, the present parasite is treated as a new species and the name Myxobolus cochinensis n. sp. is proposed.

Mitogenomic architecture of the multivalent endemic black clam (Villorita cyprinoides) and its phylogenetic implications.

The Indian black clam Villorita cyprinoides (Family: Cyrenidae), an extractive commercially exploited species with aquaculture importance contributing more than 70% of clam fishery in India, is endemic to the Indian peninsula. Currently, there is very sparse information, especially on the molecular data of Villorita. The present study aims to provide a comprehensive knowledge of mitogenome architecture and assess the phylogenetic status of Cyrenidae. This has resulted in reporting the first complete mitogenome of V. cyprinoides using next-generation sequencing technology. The A+T circular mitogenome was 15,880 bp long, exhibiting 13 protein-coding genes (PCGs) including ATP8 (absent in several bivalves), 22 transfer RNA, and two ribosomal RNA genes residing in the heavy strand in a clockwise orientation and a gene order akin to Corbicula fluminea. The molecular phylogeny inferred from a concatenated multi-gene sequence [14 mitochondrial (12 PCGs, rrnS and rrnL) and two nuclear genes (Histone H3, 18S rRNA)] from 47 representative species of superorder Imparidentia, clustered V. cyprinoides and Cyrenid clams to a single clade supporting the monophyly of Cyrenidae. The subsequent mitochondrial gene order analysis substantiates the close relationship of V. cyprinoides and C. fluminea, analogous to phylogenetic output. The multilocus tree topology calibrated with verified fossil data deciphered the origin and diversification of Cyrenid clams during late Triassic-early Jurassic. The data derived from this study shall contribute remarkably for further insights on cryptic species identification, molecular characterization of bivalve mitogenomes and mitochondrial evolutionary history of genus Villorita. Moreover, complete mitogenome can aid in potential marker development for assessing the genetic health of black clam populations.

Analytical validation of a modified turbidimetric assay to screen sulphur oxidizing bacteria.

Conventional turbidimetric assay for sulphate determination was modified to 100 times lesser reaction volume on a convenient format using microtitre plate based platform, targeting routine microbiological applications to screen sulphur oxidizing bacteria (SOB) cultures. The modified assay was linear up to 1500 mg/L of sulphate concentration, which is about 37.5 times more than that of conventional assay. Upon regression analysis, linear equation y = 1.243× + 0.011 was obtained having R2 value of 0.998. The modified assay was fully validated in terms of precision, limit of detection (LOD), limit of quantification (LOQ), sensitivity, selectivity and robustness to assure the reliability during final applications. LOD and LOQ were found as 7.4 mg/L and 24.8 mg/L of sulphate concentration respectively. Further, accuracy of the assay over routine SOB screening media components was tested, and proved as reliable and suitable for the intended application.

Comparison of observed body retention of uranium in natural condition in an average Indian adult with the values predicted by the ICRP biokinetic model.

The daily intake of natural uranium and its contents in the lungs, skeleton, liver and kidney of an Indian adult population group was estimated using radiochemical neutron activation analysis (RNAA). These data on daily intake (through inhalation and ingestion) were used to compute the uranium contents in the lungs and other systemic organs such as the skeleton, liver and kidney, using the new human respiratory tract model (HRTM) and the new biokinetic model of uranium. The theoretically computed uranium contents in the lungs, skeleton, liver and kidney of an average Indian adult are 1.16, 1.96, 0.07 and 0.04 microg, respectively, and the corresponding experimentally measured values are 1.23 (1.76), 2.92 (2.5), 0.07 (1.76) and 0.19 (1.47) microg in an urban population group living in Mumbai. The values given in parentheses are geometric standard deviation (GSD). It is seen that the measured uranium contents in the lungs, skeleton and liver agree very well with the corresponding computed values, but the measured value for the kidney is observed to be on the higher side of the computed value. However, in view of many uncertainties, the overall agreement between the measured and the computed values can be considered to be good. Therefore, the result from this study can be taken as a validation of the new biokinetic model of uranium in Indian conditions.

Mitochondrial signatures revealed panmixia in Lutjanus argentimaculatus (Forsskål 1775).

Mangrove red snapper, Lutjanus argentimaculatus is a commercially important fish. The genetic stock structure of L. argentimaculatus from Indian waters was identified using mitochondrial ATPase 6 and ATPase 8, and cytochrome b (Cytb) genes. A 842 bp region of ATPase 6/8 genes and 1105 bp region of Cytb gene were amplified in 120 samples from six different locations along the Indian coast and obtained 58 and 66 haplotypes, respectively. The high haplotype and low nucleotide diversity values along with mismatch distribution, Tajima's D and Fu's Fs analysis suggested a genetic bottleneck events or founder effect, with subsequent population expansion in L. argentimaculatus. Coefficient of genetic differentiation (FST) values was low and nonsignificant for both ATPase 6/8 gene and Cytb genes indicating low genetic differentiation in L. argentimaculatus which can be managed as a unit stock in Indian waters.

Lipoprotein-proteoglycan complexes induce continued cholesteryl ester accumulation in foam cells from rabbit atherosclerotic lesions.

We studied the metabolism of lipoprotein-proteoglycan complexes by macrophage-derived foam cells. Foam cells were isolated from atherosclerotic rabbit aortas. ApoB-lipoprotein-proteoglycan complex was isolated from human aorta fibrous plaque lesions and LDL-proteoglycan complex was formed in vitro. Both in vitro and in vivo complexes stimulated cholesteryl ester synthesis in foam cells by a dose-dependent, saturable process that resulted in the intracellular accumulation of cholesteryl ester. Stimulation of cholesteryl ester synthesis was linear with time over a 32-h period. Polyinosinic acid inhibited the stimulation of cholesteryl ester synthesis by the complexes by 32-37%, whereas cytochalasin D only produced a 6-16% inhibition. Foam cells degraded 125I-LDL-proteoglycan complex and 125I-acetyl LDL in a saturable, dose-dependent manner. Excess unlabeled acetyl-LDL inhibited the degradation of 125I-LDL-proteoglycan complex by 52%, while LDL had no effect. Similarly, excess unlabeled complex suppressed the degradation of 125I-acetyl-LDL by 48%. Foam cells degraded 125I-methyl-LDL-proteoglycan complex to the same extent as 125I-LDL-proteoglycan complex. These results show that foam cells from atherosclerotic lesions metabolize lipoprotein-proteoglycan complexes predominantly via receptor-mediated endocytosis and consequently continue to accumulate intracellular cholesteryl ester.

Assessment of Natural Radioactivity and Radiological Hazards in Brick Samples Used in Tiruvannamalali District, Tamilnadu, India, With a Statistical Approach.

The activity concentration of Ra, Th and K in brick samples used in Tiruvannamalai District of Tamilnadu has been determined using gamma ray spectrometry. The activity concentration ranges from BDL to 16.02 Bq kg, 17.86 to 120.19 Bq kg, 240.09 to 481.35 Bq kg for Ra, Th, and K, respectively. The concentration of these radionuclides is compared with reported data from other countries. The radium equivalent activity (Raeq), absorbed gamma dose rate (DR), annual effective dose rate (HR), annual gonadal dose equivalent (AGDE), criteria formula (CF), representative level index (RLI), activity utilization index (AUI), gamma index (Iγ), alpha index (Iα), the external hazard (Hex), and internal hazard (Hin) indices are calculated for the measured samples to assess the radiation hazards due to the use of these materials in the construction of dwellings. Multivariate statistical techniques (Pearson correlation, principal component analysis and cluster analysis) are used to study the relation between radionuclides and radiation hazards. The treatment of 14 radioactive variables sampled at 32 bricks by the factor and cluster analyses provided a possible interpretation of the collective data. The spatial distribution pattern of radionuclides has been depicted through the Kriging method using MapInfo software.

Micronutrient Composition of 35 Food Fishes from India and Their Significance in Human Nutrition.

The micronutrients (vitamins and minerals) are required in small amounts but are essential for health, development, and growth. Micronutrient deficiencies, which affect over two billion people around the globe, are the leading cause of many ailments including mental retardation, preventable blindness, and death during childbirth. Fish is an important dietary source of micronutrients and plays important role in human nutrition. In the present investigation, micronutrient composition of 35 food fishes (includes both finfishes and shellfishes) was investigated from varying aquatic habitats. Macrominerals (Na, K, Ca, Mg) and trace elements (Fe, Cu, Zn, Mn, Se) were determined by either atomic absorption spectroscopy (AAS) or inductively coupled plasma mass spectrometry (ICP-MS)/atomic emission spectrometry (ICP-AES). Phosphorus content was determined either spectrophotometrically or by ICP-AES. Fat-soluble vitamins (A, D, E, K) were analyzed by high-performance liquid chromatography (HPLC). The analysis showed that, in general, the marine fishes were rich in sodium and potassium; small indigenous fishes (SIFs) in calcium, iron, and manganese; coldwater fishes in selenium; and the brackishwater fishes in phosphorous. The marine fishes Sardinella longiceps and Epinephelus spp. and the SIFs were rich in all fat-soluble vitamins. All these recommendations were made according to the potential contribution (daily value %) of the species to the recommended daily allowance (RDA). Information on the micronutrients generated would enhance the utility of fish in both community and clinical nutrition.

DHA and EPA Content and Fatty Acid Profile of 39 Food Fishes from India.

Docosahexaenoic acid (DHA) is the principal constituent of a variety of cells especially the brain neurons and retinal cells and plays important role in fetal brain development, development of motor skills, and visual acuity in infants, lipid metabolism, and cognitive support and along with eicosapentaenoic acid (EPA) it plays important role in preventing atherosclerosis, dementia, rheumatoid arthritis, Alzheimer's disease, and so forth. Being an essential nutrient, it is to be obtained through diet and therefore searching for affordable sources of these ω-3 polyunsaturated fatty acids (PUFA) is important for consumer guidance and dietary counseling. Fish is an important source of PUFA and has unique advantage that there are many food fish species available and consumers have a wide choice owing to availability and affordability. The Indian subcontinent harbors a rich fish biodiversity which markedly varies in their nutrient composition. Here we report the DHA and EPA content and fatty acid profile of 39 important food fishes (including finfishes, shellfishes, and edible molluscs from both marine water and freshwater) from India. The study showed that fishes Tenualosa ilisha, Sardinella longiceps, Nemipterus japonicus, and Anabas testudineus are rich sources of DHA and EPA. Promotion of these species as DHA rich species would enhance their utility in public health nutrition.

Endothelial cell-conditioned medium modulates the synthesis and structure of proteoglycans in vascular smooth muscle cells.

We studied the effect of bovine endothelial cell-conditioned medium on proteoglycan synthesis by bovine aorta smooth muscle cells. Confluent cultures were incubated with [35S]sulfate, [3H]glucosamine or [3H]serine in medium alone (control), or medium that had been conditioned on confluent endothelial cells. Metabolically labelled proteoglycans secreted into the culture medium and associated with the cell layer were quantified. During a 24 h incubation, endothelial cell-conditioned medium increased [35S]sulfate and [3H]glucosamine incorporation into medium and cell-layer proteoglycans by 59% and 95%, respectively, above controls. [3H]Serine incorporation into proteoglycan core protein was increased by 150%. The effect of endothelial cell-conditioned medium on [35S]sulfate incorporation was concentration dependent. The stimulatory effects of the conditioned medium were abolished by cycloheximide and actinomycin D, inhibitors of protein synthesis and transcription, respectively. Endothelial cell-conditioned medium caused no significant change in the degradation or secretion of proteoglycans, indicating that the increase in proteoglycans was due to increased de novo synthesis. TGF-beta neutralizing antibody inhibited 22% of the stimulatory effect of the conditioned medium, suggesting that part of the stimulation was mediated by TGF-beta. Ion-exchange chromatography of [35S]proteoglycans in the culture medium of smooth muscle cells yielded two major peaks at 0.52 and 0.57 M NaCl in both control and experimental cultures. In both cases the second peak, which represented approx. 80% of the total radioactivity, contained isomeric chondroitin sulfate proteoglycan with chondroitin sulfate and dermatan sulfate accounting for 90% and 10% of the isomers, respectively. The isomeric chondroitin sulfate proteoglycan was fractionated by hydrodynamic size on Sepharose CL-4B, resulting in three fractions (A, B and C). Analytical column chromatography of fractions A and B on Sepharose CL-2B demonstrated that proteoglycans from cultures incubated with endothelial cell-conditioned medium were larger in size than those from control cultures (M(r) fraction A, 1700,000, compared with 1200,000 M(r); fraction B, 540,000, compared with 390,000). The molecular weights of the core proteins were unchanged. The larger size of proteoglycan A in cultures exposed to endothelial cell-conditioned medium was due to an increase in both the glycosaminoglycan chain number (29 compared to 25) and molecular mass (M(r) 52,000, compared to 40,000). The hydrodynamic size of the glycosaminoglycans in proteoglycan B of control and experimental cultures was identical (M(r) 40,000). Therefore, the increase in the molecular mass of this proteoglycan was attributable to an increase in glycosaminoglycan chain number (12 compared to 9).(ABSTRACT TRUNCATED AT 400 WORDS)

Studies on the mechanism of uptake of low density lipoprotein-proteoglycan complex in macrophages.

Earlier, we (Vijayagopal, P. et al. (1988) Biochim. Biophys. Acta 960, 210) showed that mouse peritoneal macrophages metabolize low density lipoprotein (LDL)-proteoglycan complex by a receptor pathway distinct from the acetyl-LDL receptor. Further studies were conducted to probe further into the mechanism of LDL-proteoglycan complex uptake by macrophages. Both 125I-methyl-LDL-proteoglycan complex and 125I-LDL-proteoglycan complex were taken up and degraded by the cells to the same extent. Similarly, the ability of these ligands to stimulate cholesteryl ester synthesis was also indistinguishable. These results rule out the possibility of apoB,E receptor involvement in the uptake of LDL-proteoglycan complex in macrophages. Sodium fluoride, cytochalasin D and aggregated LDL inhibited degradation of the complex by 24%, 26% and 28%, respectively, indicating that phagocytosis is only a minor pathway for the uptake. Both binding and degradation of the complex were not inhibited by excess hyaluronic acid suggesting that ligand recognition was not through hyaluronic acid binding sites. As compared to acetyl-LDL, the cellular degradation of LDL-proteoglycan complex was retarded. Macrophages exhibited a rapid stimulation of [3H]inositol trisphosphate (IP3) release and diacylglycerol production when incubated with LDL-proteoglycan complex. Furthermore, pertussis toxin produced a 62% inhibition of LDL-proteoglycan complex mediated IP3 release, suggesting that LDL-proteoglycan complex metabolism in macrophages is dependent upon the G-protein coupled signal transduction mechanism. These results show that receptor mediated endocytosis plays a major role in the metabolism of LDL-proteoglycan complex in macrophages.

Interaction of a high-affinity heparin subfraction with low-density lipoprotein stimulates cholesteryl ester accumulation in mouse macrophages.

A high-affinity heparin subfraction accounting for 8% of whole heparin from bovine lung was isolated by low-density lipoprotein (LDL)-affinity chromatography. When compared to whole heparin, the high-affinity subfraction was relatively higher in molecular weight (11,000 vs. 17,000) and contained more iduronyl sulfate as hexuronic acid (76% vs. 86%), N-sulfate ester (0.75 vs. 0.96 mol/mol hexosamine), and O-sulfate ester (1.51 vs. 1.68 mol/mol hexosamine). Although both heparin preparations formed insoluble complexes with LDL quantitatively in the presence of 30 mM Ca2+, the concentrations of NaCl required for 50% reduction in maximal insoluble complex formation was markedly higher with high-affinity subfraction (0.55 M vs. 0.04 M). When compared to complex of 125I-LDL and whole heparin (H-125I-LDL), complex of 125I-LDL and high-affinity heparin subfraction (HAH-125I-LDL) produced marked increase in the degradation of lipoproteins by macrophages (7-fold vs. 1.4-fold over native LDL, after 5 h incubation) as well as cellular cholesteryl ester synthesis (16.7-fold vs. 2.2-fold over native LDL, after 18 h incubation) and content (36-fold vs. 2.7-fold over native LDL, after 48 h incubation). After a 5 h incubation, macrophages accumulated 2.3-fold more cell-associated radioactivity from HAH-125I-LDL complex than from [125I]acetyl-LDL. While unlabeled HAH-LDL complex produced a dose-dependent inhibition of the degradation of labeled complex, native unlabeled LDL did not elicit any effect even at a 20-fold excess concentration. Unlabeled particulate LDL aggregate competed for 33% of degradation of labeled complex; however, cytochalasin D, known inhibitor of phagocytosis, did not effectively inhibit the degradation of labeled complex. Unlabeled acetyl-LDL produced a partial (33%) inhibition of the degradation of labeled complex. These results indicate that (1) the interaction of high-affinity heparin subfraction with LDL leads to scavenger receptor mediated endocytosis of the lipoprotein, and stimulation of cholesteryl ester synthesis and accumulation in the macrophages; and (2) with respect to macrophage recognition and uptake, HAH-LDL complex was similar but not identical to acetyl-LDL. These observations may have implications for atherogenesis, because both mast cells and endothelial cells can synthesize heparin in the arterial wall.