Analysis of the biological and chemical characteristics of cabbage (Brassica oleracea var. Capitata L.) Fed with compost based on sugarcane residues.

The pandemic and the geopolitical conflict between Russia and Ukraine are events that have caused economic instability in Peru. Reason that was investigated on the analysis of the biological and chemical characteristics of cabbage fed with compost based on sugarcane residues. The objective was to analyze the physical, chemical and biological characteristics of cabbage from the adequate dose of compost based on sugarcane residues and distance between plants. It is based on the methodology applied with an experimental approach, for which the Completely Random Block Design with a 4x 2 factorial arrangement was used, which consisted of 3 blocks and 8 treatments that were the combination: F1 with 0, F2 with 8, T3 with 10 and F4 with 12 t/ha and spacing (D1) with 0.30 m between plants and 0.60 m between rows and (D2) with 0.35 m between plants and 0.60 m between rows. The physical characteristics of cabbage were evaluated and processed by analysis of variance, nutrient concentrations in leaves and stomatal density were analyzed. The results determined that T7 stood out in plant height with 41.88 cm, yield with 26.76 tn/ha and T6 in root length with 20.22 cm. In chemical analysis of leaves T1 stood out in nitrogen, phosphorus, potassium and T4 in calcium, magnesium and sulfur. In T7 stomata density with 977 stomata/mm2. It concludes that at an adequate dose and greater distance that T7 stands out in the concentration of nutrients that are within normal values and high density of stomata; Therefore, these characteristics influenced the optimal biochemical reactions, which obtained good development and yield that differed at 51.39% and 32.17% with respect to the controls T1 and T5.

A novel human opsin in the inner retina.

Here we report the identification of a novel human opsin, melanopsin, that is expressed in cells of the mammalian inner retina. The human melanopsin gene consists of 10 exons and is mapped to chromosome 10q22. This chromosomal localization and gene structure differs significantly from that of other human opsins that typically have four to seven exons. A survey of 26 anatomical sites indicates that, in humans, melanopsin is expressed only in the eye. In situ hybridization histochemistry shows that melanopsin expression is restricted to cells within the ganglion and amacrine cell layers of the primate and murine retinas. Notably, expression is not observed in retinal photoreceptor cells, the opsin-containing cells of the outer retina that initiate vision. The unique inner retinal localization of melanopsin suggests that it is not involved in image formation but rather may mediate nonvisual photoreceptive tasks, such as the regulation of circadian rhythms and the acute suppression of pineal melatonin. The anatomical distribution of melanopsin-positive retinal cells is similar to the pattern of cells known to project from the retina to the suprachiasmatic nuclei of the hypothalamus, a primary circadian pacemaker.

A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene.

A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens disease at the molecular level. zeta-Crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several zeta-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3' end of the coding region. This deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic zeta-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the zeta-crystallin gene disclosed a dinucleotide deletion of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered zeta-crystallin protein. This is the first time a genomic mutation in an enzyme/crystallin gene has been directly linked to a congenital cataract.

Human gene for the RNA polymerase II seventh subunit (hsRPB7): structure, expression and chromosomal localization.

The human gene for the seventh largest subunit of RNA polymerase II complex, hsRPB7 was cloned, sequenced and mapped. This complex is an integral part of the transcription-coupled DNA repair mechanism and has been shown to be involved in several human genetic diseases and implicated in many others. The hsRPB7 gene consists of 8 exons and spans approximately 5.1 kb. Southern blots of genomic and cloned DNA suggest that hsRPB7 is coded for by a single gene. Using human radiation hybrids and YACs, the gene was localized to 11q13.1, within 70 kb of marker D11S1765. The sequence of the 5' flanking region does not contain a TATA element, but does contain several Sp1 binding sites, an AP-1 site and a novel inverted polymorphic GATA tandem repeat. This novel GATA repeat can be used for linkage analysis. The hsRPB7 gene seems to be highly conserved among eukaryotic species, showing general sequence conservation to yeast and Drosophila. Northern blot analysis reveals a high degree of tissue-specific expression. For example, adult retina, brain and kidney exhibit a relatively high level of expression. A moderate level of expression is observed in heart, lung, testis, cornea, retinal pigmented epithelium/choroid and placenta with a lower level of expression in the uterus, small intestine and skeletal muscle. A very low level of expression was observed in stomach and liver. Comparison between four fetal and adult tissues also demonstrate a surprising level of developmental specificity. Expression in fetal retina is considerably lower than fetal brain but similar to adult retina.

Structural organization and chromosomal localization of the human ribosomal protein L9 gene.

The intron-containing gene for the human ribosomal protein L9 has been cloned, sequenced and localized. The gene is approximately 5.5 kb in length and contains 8 exons. Splice sites follow the AG/GT consensus rule. The message for human rpL9 is 712 nt in length and is detected in all tissues examined. In the adult, expression is highest in retina and liver while brain shows highest expression among the fetal tissues tested. The transcription start site contains an oligopyrimidine tract, TTCTTTCTT, similar to those found in other ribosomal protein genes. As in other previously characterized ribosomal protein genes, a TATA box is absent from the 5' flanking region but a number of elements recognized by common transcription factors are present including Sp1 sites, CACCC boxes, inverted CCAAT boxes, and GATA elements. Another possible element of interest in the rpL9 5' flanking region is RFX1 also found in the well characterized rat rpL30 promoter. The gene was mapped by fluorescent in situ hybridization to band 13p of chromosome 4. At least 8 possible pseudogenes are present in the human genome, one of which is on Xp. As assessed by Southern 'Zoo-blot' analysis and direct cDNA sequence comparison, the human ribosomal protein L9 gene, like other ribosomal protein genes, is highly conserved among mammals.

Cloning and characterization of a chick embryo cDNA and gene for IGF-binding protein-2.

We have isolated and characterized a cDNA for IGF-binding protein-2 (IGFBP-2) and its gene from the chick embryo. Using primers from a conserved region of the mammalian IGFBP-2 sequence, a cDNA clone (1.6 kb) was isolated from an embryonic day-18 chick retina cDNA library. Although the clone was truncated at the 5' end, the complete coding sequence was obtained from 5' rapid amplification of cDNA ends and genomic sequencing. The open reading frame encoded a 311 amino acid precursor protein which contains a putative 36 residue signal peptide. The mature 275 amino acid protein had a predicted M(r) of 33,500 and exhibited 71, 68, 68 and 66% identity to rat, bovine, ovine and human IGFBP-2 cDNA respectively, with conservation of all 18 cysteines. The cDNA contained an RGD peptide but lacked a putative ATP-binding motif. A single transcript of approximately 2.3 kb was present in embryonic day-15 eye, brain, skeletal muscle, heart and intestine, but was virtually absent from embryonic day-15 liver. The chicken IGFBP-2 gene spanned approximately 38 kb, consisted of four exons, and was similarly organized to that of the rat and human. Southern blot analysis of chicken genomic DNA suggested that it is encoded by a single gene. The sequence information from the avian IGFBP-2 should be of value in examining the role of IGFBP-2 in vertebrate development.

Osteonectin/SPARC secreted by RPE and localized to the outer plexiform layer of the monkey retina.

PURPOSE: Osteonectin/SPARC is a secreted protein that has been implicated in ocular disease. Deletion of osteonectin/SPARC causes age-onset cataract in mice and the cataractous human lens has increased expression of osteonectin/SPARC. In this study, the expression and localization of osteonectin/SPARC in the monkey retina were determined as was secretion by cultured human retinal pigment epithelial (RPE) cells. METHODS: Adult Rhesus monkey eyes (Macaca mulatta) were dissected, and 5-mm macula and peripheral retina punches were obtained. Supernatants were collected from cultured human RPE cells. Subcellular fractionation of whole monkey retina was also performed. Osteonectin/SPARC expression and/or secretion was monitored by Northern and Western blot analyses, and localization was determined by immunocytochemistry. RESULTS: Outside of the retina osteonectin/SPARC mRNA is broadly expressed in many human tissues. Northern blot analysis shows that in the retina osteonectin/SPARC is expressed almost exclusively by the macular RPE/choroid. Western blot analysis revealed osteonectin/SPARC in both the macula and the peripheral neural retina but only in trace amounts in the RPE/choroid. In subcellular fractions of the whole retina, osteonectin/SPARC was detected, mainly in the soluble fraction but also in the membrane and nuclear fractions. Immunohistochemical analysis localized osteonectin/SPARC specifically to the outer plexiform layer. Western blot analysis of conditioned medium from human RPE cells cultured on porous substrates indicated that osteonectin/SPARC is secreted in large amounts from both the apical and basal sides of the RPE. CONCLUSIONS: Collectively these data provide evidence that osteonectin/SPARC is synthesized in the macular RPE, secreted, and subsequently transported to the outer plexiform layer. The expression pattern of osteonectin/SPARC in the subcellular retinal fractions is consistent with a soluble protein that is transported and internalized.

Novel mutations in the XLRS1 gene may be caused by early Okazaki fragment sequence replacement.

PURPOSE: To determine whether two families diagnosed with X-linked retinoschisis contained mutations in the XLRS1 gene. METHODS: DNA from the patients was obtained from blood lymphocytes using commercially available kits. Single-strand conformation assay was performed in an electrophoresis apparatus using 10% acrylamide TBE gels at 10 degrees C. The gels were stained with SYB green II and were scanned in a phosphoimager. DNA was sequenced using an automated fluorescence sequencer. RESULTS: A deletion that eliminates exon 2 was found in one family. An abnormal sequence replacement in exon 4 was found in the other family. Both mutations have severe effects in the coding region by inserting premature stop codons. CONCLUSIONS: Both of the families have mutations in the XLRS1 gene. One of these mutations points to a novel mechanism. The mutation is caused by a replacement of 17 bp of a normal sequence with 20 bp of a sequence originating from two different places in the antisense strand. This suggests that early Okazaki fragments were incorporated into the sense strand of exon 4, replacing the normal sequence.

Differential temporal and spatial expression of insulin-like growth factor binding protein-2 in developing chick ocular tissues.

PURPOSE: To determine the developmental expression and localization of mRNA for insulin-like growth factor binding protein-2 (IGFBP-2), a major binding protein of IGF-I and IGF-II, in ocular tissues of the embryonic and early posthatched chick. METHODS: In situ hybridization and northern blot analysis were used to analyze the cellular origin and relative expression of IGFBP-2 mRNA in ocular tissues. RESULTS: Wholemount in situ hybridization reveals that, as early as 3.5 days of embryonic development (E3.5), IGFBP-2 mRNA is already expressed in many areas of the embryo, including surface ectoderm, certain regions of the brain, pharyngeal clefts, somites, and limb buds. In the eye, IGFBP-2 mRNA is expressed only in the presumptive corneal epithelium at this time. By E6, IGFBP-2 mRNA expression is present in both the corneal epithelium and endothelium. By E12, IGFBP-2 mRNA is detected clearly in the corneal stroma as well as in several other ocular structures, such as the sclera, eyelid, and ciliary body. In the neural retina, a low, diffuse expression of IGFBP-2 mRNA is found at E6, which becomes more localized to the nuclear layers by E12. Northern blot analysis confirms that a high level of IGFBP-2 expression is present in the cornea and sclera by E8 to E12. A high level of IGFBP-2 mRNA expression, however, is not observed in the retina until E18. At posthatch day 2 (P2), northern blot analyses of ocular tissues reveal that the cornea contains the highest ocular level of IGFBP-2 mRNA expression, a value equal to that of brain and liver. CONCLUSIONS: The early appearance, along with differential temporal and spatial expression of IGFBP-2 mRNA in developing ocular tissues, suggests a role for IGFBP-2 in the regulation of growth and differentiation of several ocular tissues, including the cornea, sclera, and retina.

Structural and comparative analysis of the mouse gene for pigment epithelium-derived factor (PEDF).

PURPOSE: Pigment epithelium-derived factor (PEDF), a neurotrophic protein, is a member of the serine protease inhibitor supergene family. PEDF promotes both neuronal phenotype in cultured Y79 cells and neuronal survival in cultured cerebellar granulocytes. The purpose of this study was to clone the mouse PEDF gene and to determine its structure and levels of expression in different tissues. METHODS: The mouse PEDF cDNA was cloned from a mouse liver cDNA library using human PEDF cDNA as a probe. The mouse PEDF gene was cloned from a mouse ES genomic P1 library. DNA sequencing was performed using a PE-Applied Biosystems model 373 automated fluorescent sequencer. RESULTS: The mouse PEDF cDNA is 1461 bp in length and contains an open reading frame of 417 amino acids. The mouse PEDF gene spans approximately 13 kb and, like the human, it is fragmented into 8 exons. The splice sites follow the AG/GT consensus rule. Southern blot analysis indicates that the mouse genome contains only one gene for PEDF. Northern blot analysis shows the presence of the PEDF transcript in a broad range of adult mouse tissues with liver showing the highest level of expression. CONCLUSIONS: The mouse and human PEDF promoters share overall 27% similarity but are nearly identical between mouse +86 to -166 and human +102 to -96. The present study will allow us to move from in vitro experiments to in vivo studies through the development of a "knock-out" mouse model.

Organization, evolutionary conservation, expression and unusual Alu density of the human gene for pigment epithelium-derived factor, a unique neurotrophic serpin.

PEDF is a neurotrophic serpin that promotes a neuronal phenotype and augments neuronal cell survival. The isolation, sequence and structural analysis of the human PEDF gene and its promoter along with its evolutionary conservation and expression in human tissues are now described. The gene spans approximately 16 kb and is divided among 8 exons and 7 introns, the junctions of which conform to the AG/GT consensus rule. PEDF appears to fall into the ovalbumin/PAI-2 subgrouping of serpins and is structurally far different from GDN/PN-1, the only other neurotrophic serpin reported to date. The immediate 5'-flanking region is dominated by a dense cluster of Alu repeats in which are embedded several promoter consensus sequences. A CAAT box is present at -43. The putative promoter region is also far different from that reported for GDN/PN-1. Comparable hybridization signals of 23 kb EcoRI fragments containing the PEDF gene are observed by Southern blot analysis in all primate, mammal and avian species examined; conservation is particularly evident among the primates. Northern blot analysis confirms the presence of the PEDF transcript in a broad range of human fetal and adult tissues including almost all brain areas examined, underscoring differences with GDN/PN-1 which, in the adult brain, is only expressed in glia and a subset of neurons.

Natural melatonin 'knockdown' in C57BL/6J mice: rare mechanism truncates serotonin N-acetyltransferase.

Pineal melatonin synthesis (serotonin --> N-acetylserotonin --> melatonin) is severely compromised in most inbred strains of mice, in many cases because serotonin is not acetylated by serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT). We have found that in the C57BL/6J strain, AANAT mRNA encodes a severely truncated AANAT protein, because a pseudo-exon containing a stop codon is spliced in. This is the first identification of a natural mutation which knocks down melatonin synthesis. The decrease in melatonin signaling may have been a selective factor in the development of laboratory strains of mice because melatonin can inhibit reproduction and modify circadian rhythmicity.

Gene structure, localization and role in oxidative stress of methionine sulfoxide reductase A (MSRA) in the monkey retina.

MSRA (EC 1.8.4.6) is a member of the methionine sulfoxide reductase family that can reduce methionine sulfoxide (MetO) in proteins. This repair function has been shown to protect cells against oxidative damage. In this study we have assembled the complete gene structure of msrA and identified the presence of two distinct putative promoters that generate three different transcripts. These transcripts were cloned by 5'RACE and code for three MSRA isoforms with different N-termini. The different forms of MSRA target to distinct intracellular regions. The main MSRA transcript (msrA1) had been previously shown to target the mitochondria. MsrA2 and 3 originate from a second promoter and target the cytosol and nuclei. In the monkey retina msrA message was detected mainly in the macular RPE-choroid region while its activity was measured mainly in the soluble fractions of fractionated neural retina and RPE-choroid. The MSRA protein is found throughout the retina but is especially abundant at the photoreceptor synapses, ganglion and Müller cells. Interestingly, MSRA was not detected in the mitochondria of the photoreceptor inner segments. The RPE in the peripheral retina shows very low levels of expression but the RPE in the macular region is strongly labeled. Targeted silencing of msrA message rendered cultured RPE cells more sensitive to oxidative damage suggesting a role for MSRA in RPE protection against oxidative stress. Collectively these data suggest MSRA may play an important role in protecting macular RPE from oxidative damage.

A 42,000-Da protein in rabbit tissues and in a glycogen synthase preparation cross-reacts with antibodies to glycogenin.

Rabbit skeletal muscle glycogen previously has been shown to be covalently bound to a 40,000-Da protein ("glycogenin") via a novel glucosyl-tyrosine linkage [I.R. Rodriguez and W.J. Whelan (1985) Biochem. Biophys. Res. Commun. 132, 829-836]. Antibodies raised against rabbit skeletal muscle glycogenin cross-react with a similar protein present in rabbit heart and liver glycogens, as well as with a 42,000-Da "acceptor protein" present in high-speed supernatants of rabbit muscle, heart, retina, and liver. This 42,000-Da protein incorporates [U-14C]Glc when an ammonium sulfate fraction prepared from the tissue supernatants is incubated with UDP-[U-14C]Glc. The [U-14C]Glc incorporated can be removed quantitatively by treatment with amylolytic enzymes, indicating that the [U-14C]Glc incorporation represents elongation of a preexisting glucan attached to the acceptor protein. Furthermore, a commercial preparation of rabbit skeletal muscle glycogen synthase contains this 42,000-Da protein. We propose that the 42,000-Da protein represents the free form of glycogenin in tissues, with its covalently attached glucan chain(s) providing a "primed" elongation site for glycogen synthesis.

Glycogenesis in the amphibian retina: in vitro conversion of [2-3H]mannose to [3H]glucose and subsequent incorporation into glycogen.

We previously demonstrated by light and electron microscopic autoradiography that Xenopus retinas incubated with [3H]mannose exhibit tunicamycin-insensitive radiolabeling of glycogen storage compartments, especially cone parabaloids. In the present study, we utilized biochemical methods to evaluate the identity of the material presumed to be [3H]glycogen in Xenopus retinas obtained from eyecups incubated under similar conditions. A crude glycogen-containing fraction was isolated, solubilized with 8 M urea, and purified by Sepharose CL-4B column chromatography. The retinal glycogen was hydrolyzed either chemically or with specific amylolytic enzymes, followed by Sephacryl S-200 column chromatography and HPLC of the hydrolysis products. Under the conditions employed, [3H]glycogen represented at least 10% of the total radiolabeled macromolecules. Hydrolysis of the [3H]glycogen released all of the radiolabel in the form of [3H]glucose, not [3H]mannose, which indicated that direct incorporation of [3H]mannose into glycogen had not occurred. [3H]Glucose was distributed throughout the glycogen molecule, not just in the outer tiers, which indicated that de novo glycogenesis had occurred. Furthermore, enzymatic isomerization of the glycogen-derived [3H]glucose with glucose isomerase yielded fructose with retention of tritium. This demonstrated that positions other than the C-2 carbon of glucose were radiolabeled. Analysis of the medium after several hours of incubation revealed the presence of 3H2O as the major radiolabeled compound. These results support the conclusion that the in vitro incorporation of [2-3H]mannose into retinal glycogen involves initial catabolism of the radiolabeled substrate and subsequent reincorporation of the label via gluconeogenesis into precursors utilized for de novo glycogenesis.

A novel glycosyl-amino acid linkage: rabbit-muscle glycogen is covalently linked to a protein via tyrosine.

A recent review summarizes our identification in rabbit-muscle glycogen of a protein that resists all attempts at removal by means that should displace non-covalently bound protein [Kennedy et al. (1985) In Membranes and Muscle (Berman, M.C., Gevers, W. and Opie, L.H. eds.) pp. 65-84, ICSU Press/IRL Press, Oxford]. Here we confirm that the glycogen is covalently bonded to the protein and report that the attachment is via a novel glycosidic linkage involving the hydroxyl group of tyrosine.

Cloning of a novel member of the reticulon gene family (RTN3): gene structure and chromosomal localization to 11q13.

A novel member of the neuron-specific protein (NSP) or newly named reticulon (RTN) gene family was isolated during a subtraction cloning between macula and peripheral retina. The mRNA for this NSP/RTN-like gene is approximately threefold more abundant in macula than in peripheral retina. The cDNA is 2527 bp long and contains an open reading frame of 236 amino acids. The deduced peptide shows a strong similarity to the NSP/RTN and tropomyosin-like gene families but it is clearly a novel member. The gene contains seven exons and spans more than 15 kb. The gene was localized to chromosome 11q13 between markers D11S4535 and D11S4627 using somatic cell hybrid panels. Southern blot analysis identified the presence of a pseudogene(s) that was subsequently localized to chromosome 4. Multitissue Northern blot analysis found this gene to be widely expressed in human tissues with the highest expression in the brain. We are calling this gene RTN3 to reflect the newly proposed nomenclature.

Characterization and function of pig intestinal sucrase-isomaltase and its separate subunits.

Papain-solubilized pig intestinal sucrase-isomaltase was purified to homogeneity in a four-step process with a yield of 50%. The substrate specificities of the two enzyme activities were studied together and separately after inactivation or inhibition of one of the activities. Michaelis constants, maximum velocities and time courses of hydrolysis of several substrates, in particular alpha-limit dextrins, were used to characterize this complex of alpha-glucosidases. The participation of the enzyme complex in the hydrolysis of alpha-limit dextrins and more generally in pathways for starch breakdown in the pig digestive tract is examined and discussed.

Chromosomal localization of the human heme oxygenase genes: heme oxygenase-1 (HMOX1) maps to chromosome 22q12 and heme oxygenase-2 (HMOX2) maps to chromosome 16p13.3.

Heme oxygenase catalyzes the oxidation of heme to biliverdin, the precursor of the bile pigment bilirubin, and carbon monoxide, a putative neurotransmitter. We have employed polymerase chain reaction and fluorescence in situ hybridization to determine the chromosome localization of the genes coding for the two known heme oxygenase isozymes. Heme oxygenase-1 (HMOX1), the inducible form, was localized to human chromosome 22q12, while heme oxygenase-2 (HMOX2), the constitutive form, was localized to chromosome 16p13.3.

Structural analysis of the human hydroxyindole-O-methyltransferase gene. Presence of two distinct promoters.

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the last step in the metabolic pathway that synthesizes the hormone melatonin. We have found HIOMT mRNA present in small amounts in human retina and in relatively high abundance in the pineal gland. Two distinct 5' ends were found in human retina using a solid-phase 5'-rapid amplification of cDNA ends technique. The two 5' regions appear to originate from two distinct putative promoters. Although many similarities exist between the two promoters, they contain distinctive elements. Putative promoter A, for example, contains a recently discovered photoreceptor-conserved element (PCE-1, CAATTAAG) at -27 not found in promoter B, while promoter B contains an Ap1 site (ATGAGTCAA) at -166 and an octamer site (ATGCAAT) at -59 not found in promoter A. The HIOMT messages are also alternatively spliced in between exons 6 and 8, generating three distinct messages. One of the alternatively spliced messages contains a line-1 repetitive element that is spliced into the mRNA precisely as exon 6. Importantly, the downstream open reading frame is not altered by any of these splicing combinations. The gene is approximately 35 kilobases long containing either 9 or 10 exons (including the line-1 element) depending on which promoter is active. All of the splice sites follow the GT/AG rule. The dual promoters and opportunities for alternative splicing suggest a variety of mechanisms for control of HIOMT expression and biological activity in different tissues not previously recognized.