Vitamin D3 correlates inversely with systemic dendritic cell numbers and bone erosion in chronic rhinosinusitis with nasal polyps and allergic fungal rhinosinusitis.

Vitamin D(3) (VD(3) ) is a steroid hormone that regulates bone health and numerous aspects of immune function and may play a role in respiratory health. We hypothesized that T helper type 2 (Th2) disorders, chronic rhinosinusitis with nasal polyps (CRSwNP) and allergic fungal rhinosinusitis (AFRS) would have VD(3) deficiencies, resulting in increased mature dendritic cells (DCs) and bone erosion. We conducted a retrospective study examining VD(3) levels in patients with AFRS (n = 14), CRSwNP (n = 9), chronic rhinosinusitis without nasal polyps (CRSsNP) (n = 20) and cerebrospinal fluid leak repair (non-diseased controls) (n = 14) at time of surgery. Circulating immune cell levels were determined by immunostaining and flow cytometric analysis. Plasma VD(3) and immune regulatory factors (granulocyte-macrophage colony-stimulating factor and prostaglandin E(2) ) were measured by enzyme-linked immunosorbent assay. It was observed that CRSwNP and AFRS demonstrated increased circulating DCs, while chronic rhinosinusitis without nasal polyps displayed increased circulating macrophages. CRSwNP and AFRS were to found to have insufficient levels of VD(3) which correlated inversely with circulating numbers of mature DCs, DC regulatory factors and bone erosion. CRSsNP displayed no change in circulating DC numbers or VD(3) status compared to control, but did display increased numbers of circulating macrophages that was independent of VD(3) status. Lastly, VD(3) deficiency was associated with more severe bone erosion. Taken together, these results suggest support a role for VD(3) as a key player in the immunopathology of CRSwNP and AFRS.

RNA Editing in Plant Mitochondria: [alpha]-Phosphate Is Retained during C-to-U Conversion in mRNAs.

RNA editing in higher plant mitochondria frequently results in the post-transcriptional conversion of specific cytidine residues to uridine residues and infrequently results in the reverse conversion. The mechanisms by which this transition could occur are deamination or transamination of the amide at C-4 of cytosine, transglycosylation of the ribosyl residue, or deletion of a CMP residue and insertion of a UMP residue. Intact maize or petunia mitochondria were supplied with [alpha]-32P-CTP to radiolabel CMP residues in the nascent transcripts, and the fate of the [alpha]-phosphate was examined by digestion of the RNA to nucleotide monophosphates and analysis by two-dimensional chromatography. A small fraction of radioactivity comigrated with UMP on two different two-dimensional thin-layer chromatography systems, and the amount of radiolabeled UMP increased between l0-min and 2-hr incubations. The conversion of cytidine-to-uridine residues was detected in the highly edited mRNA fraction but was not detected in the rRNA fraction. Recovery of radiolabeled UMP residues suggests that the [alpha]-phosphate is retained during the editing reaction. These results are consistent with either deamination or transamination, or transglycosylation mechanisms for RNA editing.

Transcriptional and posttranscriptional regulation of maize mitochondrial gene expression.

Lysed maize mitochondria synthesize RNA in the presence of radioactive nucleoside triphosphates, and this assay was utilized to compare the rates of transcription of seven genes. The rates of incorporation varied over a 14-fold range, with the following rank order: 18S rRNA greater than 26S rRNA greater than atp1 greater than atp6 greater than atp9 greater than cob greater than cox3. The products of run-on transcription hybridized specifically to known transcribed regions and selectively to the antisense DNA strand; thus, the isolated run-on transcription system appears to be an accurate representation of endogenous transcription. Although there were small differences in gene copy abundance, these differences cannot account for the differences in apparent transcription rates; we conclude that promoter strength is the main determinant. Among the protein coding genes, incorporation was greatest for atp1. The most active transcription initiation site of this gene was characterized by hybridization with in vitro-capped RNA and by primer extension analyses. The DNA sequences at this and other transcription initiation sites that we have previously mapped were analyzed with respect to the apparent promoter strengths. We propose that two short sequence elements just upstream of initiation sites form at least a portion of the sequence requirements for a maize mitochondrial promoter. In addition to modulation at the level of transcription, steady-state abundance of protein-coding mRNAs varied over a 20-fold range and did not correlate with transcriptional activity. These observations suggest that posttranscriptional processes are important in the modulation of mRNA abundance.

RNA editing intermediates of cox2 transcripts in maize mitochondria.

Eighteen cytidines are changed to uridines in the coding sequence of transcripts for cytochrome c oxidase subunit 2 (cox2) in maize mitochondria. The temporal relationship of editing and splicing was examined in cox2 transcripts by sequence analysis of spliced and unspliced cDNAs. Cloned cDNAs of unspliced cox2 transcripts ranged from clones with no edited nucleotides to completely edited forms, while spliced cDNAs were nearly completely edited. Incompletely edited transcripts in the nascent pool of unspliced transcripts represent intermediates of the editing process. These results indicate that editing proceeds without a strong directional bias and suggest that RNA editing is a posttranscriptional process.

Protein polymorphism generated by differential RNA editing of a plant mitochondrial rps12 gene.

The rps12 gene transcripts encoding mitochondrial ribosomal protein S12 are partially edited in petunia mitochondria. Different petunia lines were found vary in the extent of rps12 transcript editing. To test whether multiple forms of RPS12 proteins are produced in petunia mitochondria as a result of partial editing, we probed mitochondrial proteins with specific antibodies against edited and unedited forms of a 13-amino-acid RPS12 peptide spanning two amino acids affected by RNA editing. Both antibodies reacted with mitochondrial proteins at the expected size for RPS12 proteins. The amounts of unedited RPS12 protein in different petunia lines correlate with the abundance of unedited transcripts in these plants. Unedited rps12 translation products are also detected in other plant species, indicating that polymorphism in mitochondrial rps12 expression is widespread. Moreover, we show that RPS12 proteins recognized by both edited-specific and unedited-specific antibodies are present in a petunia mitochondrial ribosome fraction. These results demonstrate that partially edited transcripts can be translated and that the protein product can accumulate to detectable levels. Therefore, genes exhibiting incompletely edited transcripts can encode more than one gene product in plant mitochondria.

Addition of non-genomically encoded nucleotides to the 3'-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA.

The 3'-termini of maize mitochondrial RNAs were characterized by ligation of an anchor oligonucleotide, reverse transcription and amplification. DNA sequence analysis of cDNA clones for tRNA(Ser) and 18S rRNA confirmed the expected 3'-terminal nucleotides and demonstrated the accuracy and fidelity of the protocol. Analysis of cDNAs for rps12, cox2 and atp9 indicated that non-genomically encoded nucleotides were present at the 3'-terminus. rps12 cDNAs exhibited the highest degree of modification, with 94% of 35 cDNA clones analyzed containing one to four non-genomically encoded C or A residues; 83% of these cDNAs terminated with the trinucleotide CCA. DNA sequence and transcript mapping analyses demonstrated that four positions exhibited modified 3'-termini within a small region of the 3' flank of rps12 transcripts. These transcript termini represented low abundance, truncated forms of rps12 mRNAs which may be intermediates in degradation. cox2 mRNAs are also modified at a truncated position. Sixty percent of the cox2 cDNAs were modified with 1-5 nt that most frequently included A and C residues, but also included a few G and T residues. Non-genomically encoded nucleotides were detected in 27% of the atp9 cDNAs as a single C or A residue.

Etiologic factors in carcinoma of the prostate.

A staining method is presented by which azocarmine combines with spermine to form a poorly soluble red precipitate highlighted against the yellow cytoplasm of positive cells achieved by counterstaining with metanil yellow. In the prostate, the spermine-dye complex was abundant in normal acinar epithelial cells, moderate in the epithelial cells of hyperplastic glands, and moderate to absent in carcinoma cells. Spermine is evidently concerned with stabilizing the structure of desoxyribose nucleic acid and probably of proteins, notably several zinc metalloenzymes. Diagnosis and treatment of carcinoma of the prostate should include an adequate basic diet, retention of testes, hormonal treatment oriented toward testicular, pituitary and hypothalamic hormones; zinc--ordinary and radioactive; and utilization of spermine and precursors.

Neural cells in culture.

In vitro culture of neural cells (nerve sheath cells, melanoblasts, neuroblasts, astrocytes) for three to 20 months revealed a non-mitotic type of replication characterized by appearance between 30 and 120 days. Unipolar and bipolar neural cells, resulting from an initial phase of mitotic reproduction, developed multipolar and dendritic forms with large cell bodies and numerous primary and secondary processes with broad proximal ends exhibiting right-handed helices by phase contrast, differential interference electron microscopy and time lapse cinemicrophotography. The nuclei of the conspicuous cell bodies were quite large, one to three, and contained round, rod, ovid or comma nucleoli up to three or four per nucleus. After 30 days of culture, the nuclei of the cell bodies were notably quiescent with regard to mitotic proliferation, but nucleoli were active and mitochondria were numerous around the nuclei. The cytoplasm of the cell bodies often contained channels interpreted as cisternae of endoplasmic reticulum. The helices of the processes were interpreted as cytoplasmic pumps for conveying mitochondria into their distal ends. The slender distal ends became packed with rod and filamentous mitochondria, which then broke up into fine granules associated with the formation of new nuclei appearing in the preterminal and terminal ends of the processes. The preterminal or terminal unit--knob containing nucleus and distal end of process packed with mitochondrial dust--detached by was of a thread-like connection as a new cell, which entered into a new growth phase toward non-mitotic proliferation.

Mammary cancer in the dog: a study of 120 cases.

Of the 120 cases of mammary cancer occurring in 117 female dogs (15 spayed), 2 male dogs, and 1 dog of undetermined sex, 107 (nearly 90%) were observed in dogs 8 to 15 years old. Mammary tumors occurred in nearly 14% of 875 female dogs with neoplasms. Nearly 60% of 128 neoplasms were located in the 4th and 5th mammary glands. Of the 128 cancers in these 120 dogs, 85 were classified as duct carcinoma, 38 as lobular carcinoma, 3 as malignant mixed tumor, and 2 as duct and lobular carcinomas. Most duct carcinomas originated in the epithelial cells of ducts at all levels, and a few arose in previously benign duct papillomas. The lobular carcinomas arose in alveoli and developed into progressively larger lobules. A negative factor in the development of mammary cancer is ovariectomy before or shortly after the first estrous cycle in the dog and before the age of 40 in women. In both dog and man, aging is a positive factor in the development of mammary cancer. In women, other positive factors are nulliparity and inheritance; e.g., a high rate of breast cancer in close female relatives of Jewish extraction. An epidemiologic study of breast cancer in man and dog in high-risk countries(e.g., United States) and low-risk countries (e.g., Japan) is indicated.

PIP: 120 cases of canine mammary cancers were analyzed. 102 were surgical cases, 2 surgical and necropsy cases, and 16 necropsy cases. Of these, 117 were female dogs (15 spayed), 2 were male dogs and 1 was of undetermined sex. Tissues were fixed in 10% formalin and stained with hemotoxylin and eosin. Nearly 90% of mammary cancers occurred in dogs 8 to 15 years old. 128 mammary cancers in the 120 dogs consisted of 85 duct carcinomas; 38 lobular carcinomas; 3 malignant mixed tumors;, and 2 combined duct and lobular carcinomas. There was a lack of clear-cut predilection of any breed of dog in the U.S. for mammary cancer. Duct carcinomas originating in the epithelium at all levels of the duct system are more common than lobular carcinomas arising in the epithelial cells of alveoli proliferated in a progressively enlarging or burgeoning lobular pattern. Ovariectomy before or shortly after the first estrous cycle in the dog and before the age of 40 in women protects against the development of mammary cancer. Aging is a contributing factor in the development of mammary cancer, as are nulliparity and inheritance (e.g., high rate of breast cancer in close female relatives or Jewish extraction). Further study of the human and canine population of Japan and the U.S. should be done to determine the incidence and possible etiologic factors for mammary cancer in the human female and in the bitch.

Related alphaN- and epsilonN-methyltransferases methylate the large and small subunits of Rubisco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is methylated at the ax amino position of the N-terminal methionyl residue of the processed and assembled form of the small subunit (SS), and is also methylated in some species at the epsilon-amino group of lysine-14 in the large subunit (LS). The gene (rbcMT-S) and cDNAs for the SS alphaN-methyltransferase (SSMT) from spinach (Spinach oleracea) have been cloned, sequenced, and expressed. The gene is closely related to a previously characterized LS methyltransferase (Rubisco LSMT) cDNA from pea (Rubisco LSMT) and a Rubisco LSMT gene from tobacco. Sequence analysis of the cDNA and transcript mapping experiments demonstrate that the rbcMT-S pre-mRNAs experience alternative 3' splice site selection, such that mRNAs for a long form with a four amino acid insertion and a short form are expressed at approximately equal abundance. The coding sequence of spinach SSMT includes a putative targeting presequence with sequence identity at a plastid processing site. A N-terminal truncated form of spinach SSMT was expressed and purified from E. coli cells. Both long and short forms of the cDNAs were shown to catalyze methylation of the a amine of the N-terminal methionine of the SS of Rubisco.

Parenteral nutrition program in a major cancer center.

In this article, formulation ordering, manufacturing procedures, and the quality control techniques utilized by the parenteral nutrition program at Memorial Sloan-Kettering Cancer Center are described. The cancer patient's metabolic status frequently changes; therefore, individualized prescriptions are used for all parenteral nutrition solutions. Due to the cancer patient's high risk of infection, related to disease-induced or iatrogenic immunosuppression, strict attention to aseptic procedures is required, including personnel wearing disposable gowns, head and shoe coverings, face masks, and surgical gloves. Specific emphasis has been placed on maximal cost effectiveness in manufacturing protocols. Preparation of solutions is, however, only one component of the role played by the pharmacists on the nutrition team. We describe the clinical responsibilities which are of paramount importance, since medications and other treatment modalities can markedly alter nutritional status and electrolyte balance. Monitoring the medications a patient receives and carefully watching a patient's laboratory results are important functions of the pharmacist. For the cancer patient, particular emphasis must be placed on nutritional and metabolic aberrations caused by antibiotics, corticosteroids, diuretics, antineoplastic agents, and narcotic analgesics. Radiation therapy can result in serious physiologic and nutritional effects. An innovative patient profile for monitoring a cancer patient receiving parenteral nutrition is introduced. The pharmacists are also involved in several teaching programs and a new outpatient program for administration of I.V. antibiotics to selected home total parenteral nutrition patients.

A survey of epithelial inclusions in the ovarian cortex of 470 patients.

The ovaries of 470 patients obtained for various indications at operation revealed 178 with 193 ovarian cysts and tumors - including 63 serous, 23 endometrial, and 27 mucinous - as well as 103 with tubal (serous), 35 with endometrial, and 2 with cervical (mucinous) epithelial inclusions. A very high frequency of transformation of mucinous epithelial inclusions into cysts and tumors contrasted with about 40% similar transformation of serous and endometrial inclusions into cysts and tumors.

Pathogenesis of teratoid tumors of the ovary and testis.

Based upon a representative sample of testicular tumors studied at the Armed Forces Institute of Pathology, several testicular and ovarian tumors observed in Denver, pertinent papers in the literature, and the singular thesis of Chevassu on tumors of the testis, the pathogenesis of such neoplasms is elaborated. The findings are philosophical, speculative, and established. Man is a multicellular individual to be regarded as a vehicle for the transmission of unicellular organisms or germ cells from one generation to the next. These cells remain distinct from somatic and trophoblastic cells. The mature human female not only tolerates the normal expression of the fertilized ovum during pregnancy (sex cells, blastoderm, and trophoblast) but also seems capable of greater differentiation of immature somatic cells resulting from parthenogenesis of one or more ova into cells of the three germ layers, as well as the suppression of the growth of neoplastic sex cells and trophoblast cells, with benign cystic teratoma as the most common culmination. The preponderance of malignant teratoid tumors before sexual maturity is a corollary. In contrast, the human male is not equipped with organizers postulated for the human female and thus is unable to differentiate malignant immature somatic cells, the most common cancerous element in testicular tumors. The explanation for such neoplasms must be on the basis of segregation of such cells and abnormal spermatogonia or less often trophoblastic cells in the embryo, with later expression as neoplastic cells, since spermatogonia and progeny are unable to form a new individual. To paraphrase Wilms, the statement may be made that malignant testicular and ovarian tumors of teratoid type are related, despite their different microscopic appearance, to a common form. They differ only in the quality, not in the quantity, of the different tissues comprising them. These tumors contain neoplastic blastodermic cells and differentiated cells of the three germ layers, neoplastic sex cells, and neoplastic trophoblastic cells. The cells of these tumors and the tissues they form resemble very nearly the tissues of the human embryo with nonaxial formation of alimentary and respiratory structures in many instances. The notable frequency of variably differentiated neural elements in the teratoids tumors of the ovary is in sharp contrast to their uncommon occurrence in like tumors of the testis. Dysgenesis of the ovaries and the testes of testicular feminization syndrome should be regarded as likely soil for the development of teratoid tumors.

Properties of a Membrane-bound Phosphatase from the Thylakoids of Spinach Chloroplasts.

A 3-phosphoglycerate phosphatase activity of about 2 micromoles per minute per milligram chlorophyll is associated with the thylakoid membranes of spinach chloroplasts. The K(m) for 3-phosphoglycerate is 3 millimolar. The enzyme can be solubilized from thylakoid membranes by treatment with 0.33 molar MgCl(2) or sodium deoxycholate. The activity is not stimulated by sulfhydryl reagents or the addition of 10 millimolar MgCl(2). The enzymic activity is insensitive to ethylenediaminetetraacetate. The pH optimum is broad, between 5.5 to 7.5. Although the substrate specificity is broad, 3-phosphoglycerate is the best substrate of those tested at neutral pH. However, p-nitrophenyl phosphate was a more effective substrate at pH 5.5. The enzyme exhibits the general characteristics of an acid phosphatase.

Distribution of maize mitochondrial transcripts in polysomal RNA: evidence for non-selectivity in recruitment of mRNAs.

The distribution of maize mitochondrial transcripts in polysomal RNA fractions obtained from root tissue, shoot tissue, or isolated intact mitochondria was analyzed. The distribution of cox3 transcripts that differ in 5' untranslated RNA sequence was similar in total polysomal and total mitochondrial RNA fractions, suggesting that 5' heterogeneity does not affect recruitment of transcripts into the polysomal RNA. The distribution of spliced and unspliced cox2 transcripts was also analyzed in polysomes from total tissue or isolated mitochondria, and both precursor and mature mRNAs were present in the high-molecular-weight RNA fraction. These results suggest that ribosomal association with mitochondrial transcripts is not selective.

Developmental- and tissue-specificity of RNA editing in mitochondria of suspension-cultured maize cells and seedlings.

C to U editing of apt9, nad3, and cox2 mRNAs was investigated in maize seedlings at various developmental stages as well as in suspension-cultured cells. Heterogeneity of mRNAs that result from incomplete editing was analyzed for each gene and from five tissues or developmental conditions. The editing status of approximately 30 cDNA clones was determined by digestion with a restriction enzyme that discriminates between unedited and edited DNA sequences. The atp9 and spliced cox2 cDNAs were essentially completely edited in all samples examined. Analysis of three editing sites of nad3 cDNAs indicated that incompletely edited cDNAs were detected in all tissues and treatments with a temporal increase in the overall editing status, from 50% at 3 days to about 75% at 7 days. These results indicate that incompletely edited mRNAs are prevalent for some plant mitochondrial genes, and can change with developmental or growth conditions.

Protection of tryptic-sensitive sites in the large subunit of ribulosebisphosphate carboxylase/oxygenase by catalysis.

Limited tryptic proteolysis of spinach (Spinacia oleracea) ribulose bisphosphate carboxylase/oxygenase (ribulose-P(2) carboxylase) resulted in the ordered release of two adjacent N-terminal peptides from the large subunit, and an irreversible, partial inactivation of catalysis. The two peptides were identified as the N-terminal tryptic peptide (acetylated Pro-3 to Lys-8) and the penultimate tryptic peptide (Ala-9 to Lys-14). Kinetic comparison of hydrolysis at Lys-8 and Lys-14, enzyme inactivation, and changes in the molecular weight of the large subunit, indicated that proteolysis at Lys-14 correlated with inactivation, while proteolysis at Lys-8 occurred much more rapidly. Thus, enzyme inactivation is primarily the result of proteolysis at Lys-14. Proteolysis of ribulose-P(2) carboxylase under catalytic conditions (in the presence of CO(2), Mg(2+), and ribulose-P(2)) also resulted in ordered release of these tryptic peptides; however, the rate of proteolysis at lysyl residues 8 and 14 was reduced to approximately one-third of the rate of proteolysis of these lysyl residues under noncatalytic conditions (in the presence of CO(2) and Mg(2+) only). The protection of these lysyl residues from proteolysis under catalytic conditions could reflect conformational changes in the N-terminal domain of the large subunit which occur during the catalytic cycle.

Identification of a 4.5S-like ribonucleoprotein in maize mitochondria.

Escherichia coli has a ribonucleoprotein complex that is composed of a 114 nucleotide 4.5S RNA and a 48 kDa polypeptide (P48) that has been demonstrated to function in translation and in the secretion of periplasmic polypeptides. A small RNA of approximately 220 nucleotides has been identified in maize mitochondria that includes sequence identity with the highly conserved domain of the bacterial 4.5S RNA. The transcript is mitochondrially encoded and maps to a region upstream of the gene for ATP synthase subunit I. The mitochondrial 4.5S-like RNA has 15 nucleotides of sequence identity with the highly conserved region of the bacterial 4.5S RNA. Sucrose density gradient centrifugation of a maize mitochondrial lysate demonstrated that the 4.5S RNA is a component of a high molecular weight complex under native conditions, and could be disrupted by phenol. Anti-P48 immune serum immuno-precipitated a mitochondrial protein of approximately 48 kDa, and RNA gel blot analysis of the immunoprecipitation reaction indicated that the 4.5S-like RNA co-immuno-precipitated with the 48 kDa polypeptide. The mitochondrial 4.5S ribonucleoprotein complex could function in translation or protein targeting.

Heat stress results in incomplete C-to-U editing of maize chloroplast mRNAs and correlates with changes in chloroplast transcription rate.

The editing status rps14 and rpl20 mRNAs decreased rapidly from nearly 100% edited to about 30% edited when maize plants were shifted from 20 degrees C to 37 degrees C. A decrease in the extent of editing was easily detected within 2 h and decreased to a steady-state level of about 40% C-to-U conversion at 37 degrees C. In contrast, the editing status of these chloroplast mRNAs increased relatively slowly after plants were shifted from 37 degrees C to 20 degrees C. Chloroplasts isolated from maize plants which were grown at 20 degrees C and then shifted to 37 degrees C for 24 h were 5-10 times more transcriptionally active than chloroplasts isolated from maize plants grown continuously at 20 degrees C. Thus, the high transcription rate at 37 degrees C may establish a kinetic condition where the rate of transcription exceeds the capacity of the editing apparatus and results in incomplete editing.