Activation State-Dependent Substrate Gating in Ca2+/Calmodulin-Dependent Protein Kinase II.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is highly concentrated in the brain where its activation by the Ca2+ sensor CaM, multivalent structure, and complex autoregulatory features make it an ideal translator of Ca2+ signals created by different patterns of neuronal activity. We provide direct evidence that graded levels of kinase activity and extent of T287 (T286α isoform) autophosphorylation drive changes in catalytic output and substrate selectivity. The catalytic domains of CaMKII phosphorylate purified PSDs much more effectively when tethered together in the holoenzyme versus individual subunits. Using multisubstrate SPOT arrays, high-affinity substrates are preferentially phosphorylated with limited subunit activity per holoenzyme, whereas multiple subunits or maximal subunit activation is required for intermediate- and low-affinity, weak substrates, respectively. Using a monomeric form of CaMKII to control T287 autophosphorylation, we demonstrate that increased Ca2+/CaM-dependent activity for all substrates tested, with the extent of weak, low-affinity substrate phosphorylation governed by the extent of T287 autophosphorylation. Our data suggest T287 autophosphorylation regulates substrate gating, an intrinsic property of the catalytic domain, which is amplified within the multivalent architecture of the CaMKII holoenzyme.

Pilonidal sinus laser-assisted closure (PiLAC) - a low-morbidity alternative to excision with excellent long-term outcomes.

INTRODUCTION: Recurrence after surgery for pilonidal sinus disease is a recognised problem and patients often re-present months after discharge. We routinely treat primary and recurrent pilonidal sinus disease with Pilonidal sinus Laser-Assisted Closure (PiLAC). Long-term outcomes following PiLAC surgery was examined following clinical and telephone review. METHODS: All patients undergoing PiLAC as a day-case between April 2016 and July 2019 were included. Patients were followed up in a nurse-led clinic until complete healing or recurrence. A prospective database and retrospective audit of notes combined with longer-term follow-up by telephone were used. RESULTS: A total of 35 patients underwent PiLAC, median age 28 (18-53 years), 28 males:7 females. A total of 28 patients had long-term (>60 days) follow-up, mean 407 days (range 67-887 days); 25/28 patients (89.3%) had healed with no recurrence on long-term follow-up. Of these 28 patients, 11 were first presentation of pilonidal disease and underwent PiLAC as their first treatment, with a 91% heal rate long term. A total of 15 patients had seton drainage prior to PiLAC, with a 93% heal rate versus no seton (83%). Fisher's exact test showed no significant difference between sex, new/recurrent pilonidal disease and seton placement (p>0.05). CONCLUSIONS: Healing after PiLAC for the treatment of primary and recurrent pilonidal sinus disease is preserved with excellent long-term outcomes. We recommend it as an alternative to surgical excision.

Characterization of ruminal dynamics in Holstein dairy cows during the periparturient period.

We used four pregnant Holstein cows to delineate ruminal adaptations as cows transitioned from one lactation to the next. Cows were fed typical diets through far-off and close-up dry periods and lactation. We measured ruminal characteristics on day 72 (late lactation), 51 (far-off dry), 23 and 9 (close-up dry) prepartum and on days 6, 20, 34, 48, 62, 76 and 90 postpartum (early lactation). Measurements included: ruminal fill (weight of actual contents), ruminal capacity (volume of rumen when fully filled), digestibilities and ruminal passage rates. Ruminal capacity tended to increase linearly during early lactation but was stable during dry and transition periods. Both total and liquid fill decreased linearly during the dry period, increased across parturition, and increased linearly through early lactation. Dry matter fill decreased as cows were fed the close-up diet at day 23 prepartum then increased near parturition and continued to increase across early lactation. Solid passage rate was greatest when cows were fed the close-up diet, and decreased throughout the transition period. In lactation, solid passage rate responded quadratically with peak at day 48 followed by decreases through day 90 postpartum. Liquid passage increased linearly across the transition period. Total tract organic matter digestibilities increased linearly over the dry period with significant increases prior to or immediately after parturition, then they remained relatively stable over early lactation until they increased at day 90. Fibre digestibilities demonstrated quadratic responses over early lactation, being higher on day 6 and day 90 than at other times. Starch digestibilities decreased linearly across both the dry and transition periods with decreases in lactation until day 62 followed by increases until day 90. High producing lactating dairy cows go through a multitude of ruminal adaptations, in terms of digestion, passage, capacity and fill, as they transition from one lactation to the next.

Molecular studies of linkage group XIX of Chlamydomonas reinhardtii: evidence against a basal body location.

Linkage group XIX (also known as the UNI linkage group) in the green alga, Chlamydomonas reinhardtii, exhibits a number of unusual properties that have lead to the suggestion that it represents a basal body-associated chromosome. To begin a molecular analysis of this linkage group, we have identified DNA sequences from it and used them to determine the copy number of linkage group XIX within the cell. We find that linkage group XIX is present in the same copy number per cell as nuclear linkage groups in both haploid and diploid strains. We also find that the copy number of linkage group XIX is unchanged in mutants lacking basal bodies. We conclude that there is no convincing evidence that linkage group XIX localizes to the basal bodies of Chlamydomonas reinhardtii cells.

Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) participates in many processes including early developmental events, angiogenesis, wound healing, and maintenance of neuronal cell viability. A 130-kilodalton protein was isolated on the basis of its ability to specifically bind to bFGF. A complementary DNA clone was isolated with an oligonucleotide probe corresponding to determined amino acid sequences of tryptic peptide fragments of the purified protein. The putative bFGF receptor encoded by this complementary DNA is a transmembrane protein that contains three extracellular immunoglobulin-like domains, an unusual acidic region, and an intracellular tyrosine kinase domain. These domains are arranged in a pattern that is different from that of any growth factor receptor described.

Mechanism of insect resistance to the microbial insecticide Bacillus thuringiensis.

Receptor binding studies show that resistance of a laboratory-selected Plodia interpunctella strain to a Bacillus thuringiensis insecticidal crystal protein (ICP) is correlated with a 50-fold reduction in affinity of the membrane receptor for this protein. The strain is sensitive to a second type of ICP that apparently recognizes a different receptor. Understanding the mechanism of resistance will provide strategies to prevent or delay resistance and hence prolong the usefulness of B. thuringiensis ICPs as environmentally safe insecticides.

Effects of dry aging of bone-in and boneless strip loins using two aging processes for two aging times.

This experiment investigated the combined effects of two dry-aging methods (unpackaged and in a bag), two loin-cut styles (bone-in shell loins and boneless strip loins), and two aging times (21 and 28days) on the physical, chemical, sensory, and microbial properties of dry-aged beef. Sections from shell and strip loin were assigned randomly to be aged unpackaged or aged packaged in a bag with high moisture permeability. Weight losses increased with aging time. Shell loins lost more (P<0.05) weight during aging compared with strip loins; dry aging in a bag had less (P<0.05) weight loss than unpackaged aging. There were no differences (P>0.05) in any of the sensory traits between shell and strip loins or dry aging using a traditional method or in a bag. Dry aging in a bag creates positive effects on yields, no negative effects on product quality, and adds flexibility and control of the aging environment.

Signal transduction pathways that contribute to myeloid differentiation.

The production of mature, differentiated myeloid cells is regulated by the action of hematopoietic cytokines on progenitor cells in the bone marrow. Cytokines drive the process of myeloid differentiation by binding to specific cell-surface receptors in a stage- and lineage-specific manner. Following the binding of a cytokine to its cognate receptor, intracellular signal-transduction pathways become activated that facilitate the myeloid differentiation process. These intracellular signaling pathways may promote myelopoiesis by stimulating expansion of a progenitor pool, supporting cellular survival during the differentiation process, or by directly driving the phenotypic changes associated with differentiation. Ultimately, pathways that drive the differentiation process converge on myeloid transcription factors, including PU.1 and the C/EBP family, that are critical for differentiation to proceed. While much is known about the cytokines, cytokine receptors and transcription factors that regulate myeloid differentiation, less is known about the precise roles that specific signaling mediators play in promoting myeloid differentiation. Recently, however, the application of novel pharmacologic inhibitors, siRNA strategies, and transgenic and knockout models has begun to shed light on the involvement and function of signaling pathways in normal myeloid differentiation. This review will discuss the roles that key signaling pathways and mediators play in myeloid differentiation.

Plant folk medicines for gastrointestinal disorders among the main tribes of Sonora, Mexico.

This paper describes the herbal remedies used by ethnic groups from Sonora, Mexico, for treatment of gastrointestinal diseases. Twelve types of these illnesses are cured using 85 different species which belong to 38 families. Thirty nine spp. are used to treat diarrhea, 28 for stomach-ache, 12 for constipation, 9 for intestinal parasites, 6 for indigestion, 3 for stomach or intestinal cancer, 3 for stomach inflammation and only 1 to treat gastrointestinal sicknesses, ulcers, gastritis, colitis and colic. Regarding the use of species of plant per ethnic group the following was observed: Mayo 47; Seri, 27; Yaqui, 13; Guarijio, 12, Pima, 5 and Papago, 3. The plants are used by two or more tribes, for the same or different illness but always related to the gastrointestinal system.

Leveraging Genomics for Head and Neck Cancer Treatment.

The genomic landscape of head and neck squamous cell carcinoma (HNSCC) has been recently elucidated. Key epigenetic and genetic characteristics of this cancer have been reported and substantiated in multiple data sets, including those distinctive to the growing subset of human papilloma virus (HPV)-associated tumors. This increased understanding of the molecular underpinnings of HNSCC has not resulted in new approaches to treatment. Three Food and Drug Administration-approved molecular targeting agents are currently available to treat recurrent/metastatic disease, but these have exhibited efficacy only in subsets of HNSCC patients, and thus surgery, chemotherapy, and/or radiation remain as standard approaches. The lack of predictive biomarkers to any therapy represents an obstacle to achieving the promise of precision medicine. This review aims to familiarize the reader with current insights into the HNSCC genomic landscape, discuss the currently approved and promising molecular targeting agents under exploration in laboratories and clinics, and consider precision medicine approaches to HNSCC.

The human fibroblast growth factor receptor genes: a common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain.

To determine the mechanisms by which multiple forms of fibroblast growth factor (FGF) receptors are generated, we have mapped the arrangement of exons and introns in the human FGF receptor 1 (FGFR 1) gene (flg). We found three alternative exons encoding a portion of the third immunoglobulin (Ig)-like domain of the receptor. One of these alternatives encodes a sequence that is part of a secreted form of FGFR 1. The other two encode sequences that are likely part of transmembrane forms of FGFR 1. One of these forms has not been previously reported in published cDNAs. Also, we have determined the structural organization of a portion of the human FGFR 2 gene (bek) and found a similar arrangement of alternative exons for the third Ig-like domain. The arrangement of these genes suggests that there are conserved mechanisms governing the expression of secreted FGF receptors as well as the expression of at least two distinct membrane-spanning forms of the FGF receptors. The diverse forms appear to be generated by alternative splicing of mRNA and selective use of polyadenylation signals.

Diverse forms of a receptor for acidic and basic fibroblast growth factors.

We recently reported the isolation of a chicken cDNA clone encoding a basic fibroblast growth factor (FGF) receptor that has three immunoglobulinlike domains in the extracellular region. We have now identified four unique human cDNA clones encoding previously unknown FGF receptor variants which contain only two immunoglobulinlike domains. Two of the human clones encode membrane-spanning receptors, and two encode putative secreted forms. Both the three- and two-immunoglobulinlike-domain forms mediate biological responsiveness to acidic and basic FGF. Thus, the first immunoglobulinlike domain of the three-domain form may have a function other than binding of acidic and basic FGF.

Differential splicing in the extracellular region of fibroblast growth factor receptor 1 generates receptor variants with different ligand-binding specificities.

We have cloned a genomic region of the murine fibroblast growth factor (FGF) receptor 1 (FGFR1) gene that includes three alternative exons for the third immunoglobulinlike domain in the extracellular region of the receptor. The mRNA of one of these splice variants encodes a secreted receptor that lacks transmembrane and cytoplasmic sequences as well as a portion of the third immunoglobulinlike domain. Highest levels of mRNA encoding this variant were found in brain, skeletal muscle, and skin. We expressed this form of FGFR1 in CHO cells and showed that the recombinant secreted protein binds acidic FGF. We also discovered a novel alternative exon in the third immunoglobulinlike domain that encodes part of a transmembrane FGFR1 mRNA. This exon is highly homologous to the corresponding region of the keratinocyte growth factor receptor. Transcripts including this exon were present at highest levels in the skin. We cloned an FGFR1 cDNA which includes this exon and expressed this receptor variant in L6 rat skeletal muscle myoblasts. The new receptor variant had a 50-fold-lower affinity for basic FGF than does the published FGFR1 variant, whereas both forms of receptor bound acidic FGF with high affinity. These results show that the third immunoglobulinlike domain plays an important role in determining the binding specificities for different FGFs. Our data provide the first evidence that differential splicing in the extracellular region of a receptor gene generates receptor variants with different ligand-binding specificities.

Efficacy of lactic acid salts and sodium acetate on ground beef colour stability and metmyoglobin-reducing activity.

This study examined two concentrations (0.6 and 1.0mol) of three lactic acid salts (calcium lactate, CaL; potassium lactate, KL; and sodium lactate, NaL), with and without 0.01mol sodium acetate (n=3 replications), for effects on ground beef colour stability and metmyoglobin-reducing activity (MRA). Ground beef with CaL was least colour stable (P<0.05). Increasing CaL and NaL concentration decreased (P<0.05) colour stability. Ground beef with acetate only was most colour stable (P<0.05), but it did not result in more MRA (P>0.05) than control ground beef. Including both lactate and acetate was not as effective (P>0.05) in increasing colour stability as acetate alone. In general, both KL levels were equal (P>0.05) to the lower NaL concentration, and all three were superior in colour stability (P<0.05) to CaL and the higher NaL concentration. More MRA was generated by including lactates (P<0.05); KL and NaL had more MRA than CaL (P<0.05). However, these increases in MRA did not result in improved colour stability. Overall, adding KL to ground beef would not increase ground beef colour stability over adding nothing, but CaL and high levels of NaL would decrease colour stability. Using 0.01mol sodium acetate maximized ground beef colour stability.

Effects of ascorbic and citric acid on beef lumbar vertebrae marrow colour.

Citric acid was evaluated as a way of improving ascorbic acid's ability to stabilize beef lumbar vertebrae colour in high-oxygen packaging (MAP; 80% O(2)/20% CO(2)). Vertebrae were treated with citric acid (1%, 3%, or 10%), ascorbic acid (1%, 3%, or 10%), or a combination of both. Citric acid demonstrated no positive effects (P>0.05), compared with ascorbic acid, which inhibited (P<0.05) discolouration throughout the 7d display. Although ascorbic acid inhibited discolouration (visual colour and a(∗); P<0.05), 3% and 10% ascorbic acid were most effective. However, if vertebrae are displayed for less than 7d, there may be no significant colour-stabilizing advantages associated with increasing ascorbic acid from 3 to 10%. The significant oxidizing effects of citric acid at 10% were reversed (P<0.05) by ascorbic acid. Combining citric and ascorbic acid had no synergistic affect (P>0.05) on vertebrae colour.

Comparison of ascorbic acid and sodium erythorbate: Effects on the 24h display colour of beef lumbar vertebrae and longissimus lumborum packaged in high-oxygen modified atmospheres.

Sodium erythorbate and ascorbic acid were compared as a means to stabilize surface colour of bone-in beef steaks in high-oxygen modified atmosphere (80% oxygen and 20% carbon dioxide). Bone-in strip loins (n=8) were fabricated into 1.9-cm thick steaks, of which both the lumbar vertebrae and longissimus lumborum were topically treated with either ascorbic acid or sodium erythorbate (0, 0.05, 0.1, 0.5, 1.0, or 1.5%, wt/wt basis). Colour (L(∗)a(∗)b(∗)) was evaluated before treatment and 24h after packaging (display at 1°C). Sodium erythorbate was as effective as ascorbic acid for inhibiting vertebrae discolouration (P>0.05). Either reducing agent at 0.5, 1.0, or 1.5% improved (P<0.05) vertebrae redness (compared with 0%, 0.05% and 0.1%). No detrimental effects on muscle colour were observed. When selecting antioxidants intended for bone-in beef steaks displayed in high-oxygen packaging, sodium erythorbate may be a cost effective substitute for ascorbic acid.

Particle--lamella complexes in a case of human benign prostate hyperplasia: brief communication.

Particle--lamella complexes (PLC's), described for the first time, were found in glandular epithelial cells of the hyperplastic prostate tissues from a patient with transitional cell carcinoma of the urinary bladder. PLC's observed in this patient were similar to those seen in human hematopoietic neoplastic cells. They showed cylindroid forms and were composed of concentrically arranged lamellae and particles found in rows between these lamellae. PLC is closely related to rough endoplasmic reticulum (RER), and some PLC's were completely surrounded by RER. Particles approximately 25--30 nm in diameter were similar to ribosomes in size, shape, and electron density; lamellae approximately 10 nm thick appeared circular in cross sections and lamellar in longitudinal sections. Although the nature and function of PLC's are as yet unknown, the present observation indicated that PLC's are not a characteristic structure restricted to malignant tumors of hematopoietic origin.

Virus-like particles in a case of human prostate carcinoma.

Two morphologically different types of intracisternal virus-like particles were observed electron microscopically in a biopsy specimen of human prostate cancer. Particles of one type were 150-200 nm in diameter and contained either an electron-dense core or two concentric inner layers. Particles of the other type were smaller, 80-100 nm in diameter, and appeared mostly in filamentous or chainlike formation. Both types of particles and budding were observed in endoplasmic cavities of epithelial tumor cells. The particles had ultrastructural characteristics that suggested a viral nature but were different from the known type B, type C, or type H (hamster type R) virus particles. This was the first election microscopic observation in prostate cancer of virus-like particles similar to those previously reported in a case of human breast carcinoma.

Superiority of an acid-labile daunorubicin-monoclonal antibody immunoconjugate compared to free drug.

We conjugated the chemotherapy agent daunorubicin to the anti-T-cell monoclonal antibody T101 using an active ester intermediate of the acid-labile linker cis-aconitate anhydride. By converting carbohydrate hydroxyl groups on the antibody to amines prior to conjugation, average drug to antibody ratios of 25:1 were achieved with retention of cytotoxicity and only minimal loss of immunoreactivity. The pH sensitivity of the linkage was confirmed. The preparation was cytotoxic for antigen-bearing cells but not antigen-negative cells, even up to 48-h incubation in vitro. Specific cytotoxicity was apparently mediated through the endocytosis of the intact T101 immunoconjugate and the release of the active drug in the lysosomal compartment. Athymic mice bearing human tumor xenografts who received a single injection of the immunoconjugate had less tumor growth and more tumor regressions than animals receiving antibody alone, drug alone, or a mixture of drug plus antibody. This approach appears promising for further investigation.

Inhibition of human T-cell tumor growth by T101-ricin A-chain in an athymic mouse model.

An immunotoxin prepared with the pan-T-cell, anti-CD5, antibody T101, and purified ricin A-chain (RTA) was selectively cytotoxic in vitro, inactivating protein synthesis in the human T-cell line MOLT-4 but not in the human B-cell line 8392. Modulation studies showed that the immunoconjugate was more rapidly cleared from the cell surface than unconjugated T101. Preclinical evaluation of T101-RTA was conducted in a human T-cell, athymic mouse model (Dillman et al., Cancer Res., 45:5632-5336, 1985). Tumor-bearing mice received single i.p. injections of saline, T101, UPC-10 (irrelevant IgG2a), unconjugated RTA, an irrelevant conjugate, UPC-10-RTA, a mixture of T101 plus RTA, or T101-RTA. T101-RTA was the most effective reagent. Thirty animals given injections of 33 micrograms of T101 showed reductions in tumor growth (compared to tumor growth in animals receiving phosphate-buffered saline) but no complete regressions. No decrease in tumor growth was observed with UPC-10. Animals given 12 micrograms of free RTA exhibited reduced tumor growth but only one complete regression was observed; similar results were obtained with mice given 45 micrograms of UPC-10-RTA or a mixture of 33 micrograms of T101 plus 12 micrograms of RTA. Eleven complete regressions and 18 partial regressions were produced in the 46 animals given injections of 45 micrograms of T101-RTA and tumor growth was almost completely blocked. No toxicity was observed in any experimental arm. These results suggest that T101-RTA may be administered safely and with significant antitumor effect.