Glycerol as a partial replacement for lactose in milk replacer for young dairy calves.

Glycerol (glycerin) is increasingly available from biodiesel manufacture and edible oil refining and it has been used successfully in diets for chickens, pigs, and adult cattle; however, less information is available on its nutritional value in young calves. Our objective was to determine the effects on calf growth and health when glycerol replaced a portion of lactose in milk replacer. Holstein calves (12 male, 12 female) born at the University of Illinois dairy unit were assigned alternately to 1 of 2 treatments (24 calves total): control milk replacer or milk replacer supplemented with 15% glycerol in replacement of lactose. The experimental base milk replacer contained greater protein, fat, minerals, and vitamins so that when glycerol was added, the composition would be the same as that of the control, except that glycerol replaced some lactose. Calves were housed in individual hutches bedded with straw, and water was freely available. Starter was offered beginning on d 36. The amount of milk replacer offered was reduced by half on d 43, and calves were weaned at d 49. Calves were fed milk replacers twice daily from d 3 of life. Milk replacers contained 28% protein (all from whey proteins), 2.6% lysine, and 15% fat. Control milk replacer contained 40% lactose, and the glycerol milk replacer contained 25% lactose. Both replacers were reconstituted to 15% solids. Glycerol (liquid) was added to reconstituted base milk replacer at each feeding. During wk 1, milk replacers were fed at a rate of 0.25 Mcal/kg of metabolic body weight (BW) (about 1.5% of BW daily as powder, or approximately 675 g/d) and from wk 2 to 6 at 0.30 Mcal/kg of metabolic BW (about 2% of BW daily, or approximately 900 to 1,200 g/d). Measurements of BW and stature were made weekly through d 56. Calf BW and average daily gain through d 35 (0.66 vs. 0.65 kg/d for controls and glycerol, respectively) did not differ significantly between treatments. Stature measurements (withers height, body length, heart girth) and measures of health (fecal scores, medical treatments) did not differ between treatments. Under the conditions of this experiment, glycerol was an acceptable replacement for at least 37.5% of the total lactose in milk replacer (15% of the formula) if economically favorable.

Residual efficacy of three spinosad formulations against Psorophora columbiae larvae in small rice plots.

Experiments were conducted to determine the residual efficacy of both a briquette and a granular formulation (2 rates) of a numbered spinosad compound against Psorophora columbiae larvae in small rice plots. Comparisons were also made between the numbered compounds and labeled granular and briquette formulations of methoprene. Both rates of the spinosad granules had the highest initial efficacy (100% control), with the spinosad briquette being the least effective. However, 1 wk after treatment, the spinosad briquette was equally effective to both spinosad granules, suggesting a slow release in the briquette. The experimental briquette and the high rate of the experimental granules had the most residual activity, providing over 80% control for 33 days posttreatment. The granular methoprene formulation was the least residually effective compound, providing only 12% control at 7 days posttreatment.

Comparison of three residential mosquito traps against riceland mosquitoes in southeast Arkansas.

Field trials were conducted at 3 locations in Arkansas County, AR, to compare the effectiveness of 3 residential mosquito traps, the Stinger Mosquito Vacuum, the Mosquito Magnet Defender, and the Mosquito Deleto 2500 Active System, against riceland mosquitoes, specifically Anopheles quadrimaculatus and Psorophora columbiae. Both the Stinger Mosquito Vacuum and the Mosquito Deleto captured significantly more An. quadrimaculatus and total mosquitoes than did the Mosquito Magnet Defender. The Mosquito Deleto captured significantly more Ps. columbiae than did either of the other 2 traps.

Evaluation of linear array human papillomavirus genotyping using automatic optical imaging software.

Variations in biological behavior suggest that each carcinogenic human papillomavirus (HPV) type should be considered individually in etiologic studies. HPV genotyping assays might have clinical applications if they are approved for use by the FDA. A widely used genotyping assay is the Roche Linear Array HPV genotyping test (LA). We used LA to genotype the HPV isolates from cervical specimens from women with the full spectrum of cervical disease: cervical cancer, cervical intraepithelial neoplasia (CIN), and HPV infections. To explore the feasibility and value of the automated reading of the LA results, we custom-designed novel, optical imaging software that provides optical density measurements of LA bands. We compared unmagnified visual examination with the automated measurements. The two measurements were highly associated. By either method, the threshold between a negative and a positive result was fairly sharp, with a clear bimodal distribution. Visually, most positive results were judged to be strong or medium, with fewer equivocal results categorized as weak (9.5% of positive samples), very weak (6.5% of positive samples), or extremely weak (7.7% of positive samples). The automated measurements of the intensities were significantly associated with the strength of the visual categories (P < 0.001). At the extremes of the automated signal intensities (< or = 20 units or > or = 120 units), the bands were almost always categorized visually as negative and positive, respectively. In the equivocal zone (20 to 119 units), specimens were more increasingly likely to be judged to be visually positive as the number of other, definite infections on the same strip increased (P for trend < 0.001). Multiple, concurrent infections comprise > or = 25% of HPV infections; thus, any systematic visual tendency that influences their evaluation when the result is equivocal should be minimized. Therefore, automated reading is probably worth development if easy-to-calibrate hardware and software can be optimized.

Riceland mosquito management practices for Anopheles quadrimaculatus larvae.

Two separate but related studies were conducted regarding management of Anopheles quadrimaculatus larval populations in commercial rice fields near Cleveland, MS, in 2004. Study 1 was to evaluate the effectiveness of 2 treatments of aerially applied ultra-low volume applications of Bacillus thuringiensis var. israelensis (Bti) against An. quadrimaculatus larvae in dense, high-canopy mid- to late-season rice crop. Study 2 was to investigate the effect of preflood treatments of lambda-cyhalothrin (Karate), which is commonly used against rice water weevil (Lissorhoptrus oryzophilus), on An. quadrimaculatus larvae. Excellent initial, but short residual control (>99% control 1 day after treatment) was observed in the Bti-treated fields in both mid- and late-season rice. Little or no effect on mosquito larvae was observed in the lambda-cyhalothrin-treated fields. Results indicate that Bti can be effectively used by mosquito management personnel to control larval populations of An. quadrimaculatus in late-season rice fields; however, lambda-cyhalothrin did not effectively control larval An. quadrimaculatus when applied preflood to rice fields.

4-thiophenoxy-N-(3,4,5-trialkoxyphenyl)pyrimidine-2-amines as potent and selective inhibitors of the T-cell tyrosine kinase p56lck.

We have developed a series of 4-thiophenoxy-N-(3,4,5-trialkoxyphenyl) pyrimidine-2-amines as potent and selective inhibitors of p56lck tyrosine kinase activity. In particular, the most potent inhibitor shows cellular activity in T-cell receptor (TCR) stimulated models of cytokine release, which suggests an immunomodulatory role for this class of inhibitor.

CDP840. A prototype of a novel class of orally active anti-inflammatory phosphodiesterase 4 inhibitors.

The discovery, synthesis and biological activity of a series of triarylethane phosphodiesterase 4 inhibitors is described. Structure-activity relationship studies are presented for CDP840 (29), a potent, chiral, selective inhibitor of PDE 4 (IC(50) 4nM). CDP840 is non-emetic in the ferret at 30mgkg(-1) (po), active in models of inflammation and reverses ozone-induced bronchial hyperreactivity in the guinea pig.

Polymorphisms in the TNF-alpha and TNF-receptor genes in patients with coronary artery disease.

BACKGROUND: Coronary artery disease (CAD) is both multifactorial and polygenic in nature. Atheroma formation, the pathological hallmark of CAD, is an inflammatory process, with pro-inflammatory cytokines, such as tumour necrosis factor-alpha (TNF-alpha), having a major role in its pathogenesis. We have therefore investigated whether polymorphisms in the TNF-alpha (- 238 and - 308), TNF receptor 1 (position - 609 and + 10, intron 6) and TNF receptor 2 (position + 422, codon 198) genes show an association with CAD. MATERIALS AND METHODS: Patients with angiographically proven single vessel (n = 58) and multivessel (n = 122) CAD were compared to patients with angiographically proven normal coronary arteries (n = 79) and volunteers without clinical evidence of CAD (n = 250). Genotyping was performed by PCR-RFLP analysis. For the TNF-alpha polymorphisms, a meta-analysis of all published studies was also undertaken. RESULTS: No significant differences in allele or genotype frequencies were found between the normal coronary artery group or healthy volunteers and patients with CAD for any of the polymorphisms. There was also no difference in allele frequency between patients with single- and multivessel disease. For the - 308 and - 238 TNRalpha gene polymorphisms, a meta-analysis of our data and previously published studies failed to demonstrate any significant association with CAD. CONCLUSIONS: Polymorphisms in the TNF-alpha promoter region and TNF-receptor genes are not associated with the development of CAD.

Development of a recombinant cell-based system for the characterisation of phosphodiesterase 4 isoforms and evaluation of inhibitors.

We described the development of a recombinant cell-based system for the characterisation of phosphodiesterase (PDE) 4 isoforms and the evaluation of inhibitors. The Chinese hamster ovary (CHO) cell, which was found to have a low endogenous PDE4 background and no beta-adrenergic receptors (beta-AR), was transiently transfected with beta-AR and various PDE4 isoforms which were expressed as functionally coupled molecules. From correlations of elevation of adenosine 3',5'-cyclic monophosphate in situ and the inhibition of catalytic activity in vitro with the various PDE4 isoforms, it was apparent that PDE4A4, 4B2, 4C2, 4D2, and 4D3 all adopted a high-affinity binding conformation (i.e. expressed the high-affinity rolipram binding site) in the CHO cell, whereas PDE4A330 was expressed in a low-affinity conformation in situ. This gives the opportunity of using this system to screen and optimise inhibitors against a low-affinity conformation of PDE4 in situ and use a high-affinity conformation of PDE4 as a counterscreen, as inhibitor activity against this conformer has been linked with undesirable side effects. This system could also be utilised to screen inhibitors against various PDE4 isoforms in isolation against a low endogenous PDE background in situ for isoform-selective inhibitors.

Characterization of CHO-K1 cells stably expressing PDE-IV enzymes. Whole-cell cAMP determinations vs broken-cell enzymatic assays.

A CHO-K1 cell line stably expressing a recombinant full-length human PDE-IVa (HSPDE4A4B) enzyme was established under hygromycin B selection. Full-length expression of the protein was determined by Western blot analysis, which revealed the presence of a 125-kDa immunoreactive band using rabbit anti-PDE-IVa antibodies. The potency of inhibitor compounds was examined by their ability to increase cAMP in the whole-cell, and by their ability to inhibit cAMP hydrolysis in a 100,000 g supernatant (soluble enzyme preparation) obtained from the same cell line. Inhibition of the expressed PDE-IVa activity by selective PDE-IV inhibitors--(R) and (S)-rolipram, RS 14203, and CDP 840--at 100 nM substrate demonstrated that RS 14203 and CDP 840 were the most potent with IC50 = 9 nM, followed by (R)-rolipram (IC50 = 110 nM) and (S)-rolipram (IC50 = 420 nM). The rank order of potencies of the inhibitors in elevating cAMP in the whole-cell assay was quite different from that on the soluble enzyme. RS 14203 was still the most potent compound in elevating cAMP. Moreover, the relative rank order of potencies between CDP 840 and (R)-rolipram changed dramatically, such that (R)-rolipram was more potent than CDP 840 = (S)-rolipram. An apparent 30-fold stereoselectivity between (R)- and (S)-rolipram was also noted. The whole-cell rank order of potencies was also maintained when PKA activity ratios were measured in place of cAMP levels. The ability of the compounds to elevate cAMP in the stable CHO-K1 cells appeared to track better with the potency of the compounds against the high-affinity (Sr) conformer of the enzyme rather than the low-affinity catalytic state.

Primary adenocarcinoma, signet-ring, and transitional cell carcinoma of the bladder with penile metastasis.

This article reports a rare case of primary signet-ring cell carcinoma of the bladder with transitional cell and adenocarcinoma variants and metachronous metastases to the penis and lungs. This combination of lesions has not previously been reported. Together, they portend widespread dissemination and an early demise as is frequently the case with signet-ring cell carcinomas arising in other organs such as the breast and gastrointestinal tract. The optimal therapeutic intervention for this myriad of neoplasms with metastasis to the penis has yet to be ascertained because of the rarity of the lesions. Early diagnosis and an aggressive surgical approach appear to offer the best chance for quality survival and possible cure.

Differential coupling of the formyl peptide receptor to adenylate cyclase and phospholipase C by the pertussis toxin-insensitive Gz protein.

In neutrophils, activation of receptors for the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) leads to changes in intracellular events such as phosphoinositide turnover and Ca2+ mobilization. Studies have shown that activation of the cloned fMLP receptor can also lead to inhibition of cyclic AMP (cAMP) accumulation [Lang, Boulay, Li and Wollheim (1993) EMBO J. 12, 2671-2679; Uhing, Gettys, Tomhave, Snyderman and Didsbury (1992) Biochem. Biophys. Res. Commun. 183, 1033-1039]. These responses are apparently mediated through pertussis toxin-sensitive Gi proteins. Since other chemotactic factor receptors can couple to multiple G proteins, we examined the ability of the fMLP receptor to utilize a pertussis toxin-insensitive G protein, Gz, in its signal transduction pathways. The human fMLP receptor was transiently expressed in 293 and Ltk- cells, and subsequently assayed for receptor-mediated inhibition of cAMP accumulation and stimulation of phosphoinositide-specific phospholipase C. In transfected 293 cells, fMLP inhibited choriogonadotropin-stimulated cAMP accumulation by 50% and the response could be abolished by pertussis toxin. Co-expression of the fMLP receptor with the alpha subunit of Gz rendered the fMLP response pertussis toxin-insensitive, indicating that the endogenous Gi proteins can be substituted efficiently by Gz. In contrast, Ltk- cells expressing the fMLP receptor were able to respond to fMLP with an increase in the production of inositol phosphates, but this response was completely abolished by pertussis toxin even in cells co-expressing the alpha subunit of Gz. Thus, although both signalling pathways appeared to utilize Gi-like proteins, Gz can only replace Gi in mediating inhibition of cAMP accumulation, and not in the stimulation of phospholipase C. Differential interaction with Gz might represent a novel mechanism by which fMLP receptors regulate intracellular events.

Stimulation of type II adenylyl cyclase by chemoattractant formyl peptide and C5a receptors.

The capacity of N-formylmethionyl-leucyl-phenylalanine (fMLP) and C5a receptors to regulate type II adenylyl cyclase was examined in transient transfection studies. Coexpression of either one of the chemoattractant receptors with type II adenylyl cyclase in human embryonic kidney 293 cells allowed the corresponding chemotactic factor to stimulate cAMP accumulation in a dose-dependent manner. The chemoattractant-induced stimulation of type II adenylyl cyclase was absolutely dependent on the presence of GTP-bound alpha subunit of GS, as revealed by the coexpression of alpha s-Q227L, a constitutively activated mutant of alpha s. Stimulation of type II adenylyl cyclase by either fMLP or C5a was mediated via pertussis toxin-sensitive Gi-like proteins, because the response was abrogated by the toxin. The ability of Gz (a pertussis toxin-insensitive G protein that can couple to a number of Gi-linked receptors) to replace Gi in chemoattractant-induced stimulation of type II adenylyl cyclase was examined. The chemoattractant-induced response became insensitive to pertussis toxin upon coexpression of the alpha subunit of Gz. Interestingly, coexpression of alpha z significantly enhanced the chemotactic factor-stimulated type II adenylyl cyclase activities. When other G protein alpha subunits were tested under similar experimental conditions, all three forms of alpha 1 and alpha o1 were able to potentiate the fMLP response to various extents, whereas alpha q and alpha t slightly inhibited the fMLP response. The alpha subunit-mediated potentiation of the type II adenylyl cyclase response appears to reflect a productive coupling between alpha subunits and the fMLP receptor, because such enhancements were not seen with the constitutively activated alpha subunit mutants. Coexpression of the constitutively activated mutants of alpha z, alpha q, alpha 01, and alpha i1-3 neither enhanced nor inhibited the fMLP-stimulated cAMP accumulation. These results indicated that the observed enhancement of type II adenylyl cyclase responses was dependent on the ability of the wild-type alpha subunits to functionally interact with the fMLP receptor and that the fMLP receptor can couple to Gi1-3, Gz, and Go1 but not to Gs, Gq, or Gt.

The human chemoattractant complement C5a receptor inhibits cyclic AMP accumulation through Gi and Gz proteins.

The human C5a receptor is known to signal through Gi proteins. The ability of the cloned C5a receptor to inhibit adenylyl cyclase or to stimulate phospholipase C through Gi proteins was examined in transfected cells. Activation of recombinant C5a receptors resulted in the stimulation of phospholipase C in Ltk- cells and inhibition of adenylyl cyclase in 293 cells. Pertussis toxin potently abolished both responses indicating the involvement of Gi proteins. Previous studies have shown that Gi-mediated inhibition of adenylyl cyclase can be similarly regulated by the pertussis toxin-insensitive GZ. In 293 cells co-transfected with the alpha subunit of GZ, the C5a-mediated inhibition of cAMP accumulation became pertussis toxin-resistant, signifying functional coupling between the C5a receptor and GZ. However, GZ cannot substitute for Gi in the C5a-induced stimulation of phospholipase C or inhibition of adenylyl cyclase in Ltk- cells.

Early treatment of Class III incisor relationship using the chincap appliance.

Twenty-three patients (10 male, 13 female), with Class III relationships of the incisor teeth at the mixed dentition stage, were treated with the chincap appliance associated, where appropriate, with an upper removable appliance to procline upper incisors and to free the occlusion. Cephalometric analyses at the beginning and at the end of active treatment were compared with those in a carefully matched untreated control group selected from the Belfast Growth Study. At the beginning of treatment patients differed significantly from the controls in the SNB was larger, ANB was negative, Wits' analysis was negative, and the intermaxillary angle significantly reduced. The overjet was reversed, the lower incisors were ahead of A-Po, and the upper lip was far behind the E line. Significant changes brought about by the treatment were improvement in the Wits' relationship (but there was no significant change in the ANB angle), increases in the intermaxillary angle and lower facial height, improvement in the overjet, proclination of upper incisors and retroclination of lower incisors, backward movement of the lower incisors in relation to A-Po, and backward movement of the lower lip. The efficacy of the chincap appliance can be attributed to retroclination of lower incisors and downward movement of the mandible which may improve the jaw relationship without affecting the ANB angle. There were marked changes in the lip posture.

Studies of the thermal inactivation of cardiac adenylyl cyclase: evidence for a conformational change in the reaction mechanism.

Membrane bound cardiac adenylyl cyclase was shown to undergo a spontaneous and irreversible thermal inactivation with a t1/2 of approximately 10 min. The loss of activity could not be explained by the action of endogenous proteases. Repeated freeze-thaw of membrane preparations resulted in a much increased rate of thermal inactivation (t1/2 = approx. 2 min). ATP, adenylimidodiphosphate, ADP, and PPi protected the enzyme from thermal inactivation with dissociation constants (Kd) of 193, 5.04, 84.4, and 6.3 microM, respectively. 5'-AMP and cyclic AMP were ineffective as protectors at concentrations as high as 3 mM. Activators of adenylyl cyclase such as Mn2+, forskolin, 5-guanylylimidodiphosphate, and NaF and 9 mM Mg2+ protected against thermal inactivation with Kd of 16.8 microM, 8.81 microM, 0.23 microM and 1.04 mM, respectively. Mg2+ alone was without effect. Thermal inactivation was first order under all conditions tested. Arrhenius plots of the rate constants for inactivation vs temperature were linear. The increased stability of ligand bound adenylyl cyclase was shown to be associated with an increased free energy of activation (delta G 0). These data provide evidence for the existence of two distinct conformations of cardiac adenylyl cyclase based on different susceptibilities to thermal inactivation. These enzyme conformations, termed E1 and E2, may be important reaction intermediates. The thermal stability of E1 was highly influenced by the enzyme's membrane lipid environment. The formation of E2 from E1 was enhanced by interaction with substrate, PPi, activators of adenylyl cyclase, and by interaction with dissociated stimulatory guanine nucleotide binding protein-alpha beta gamma heterotrimers.

Identification and partial characterization of a retinal pigment epithelial membrane receptor for plasma retinol-binding protein.

We have developed a membrane binding assay by which we have been able to characterize the interaction between 125I-labeled retinol-binding protein and its receptor in microsome fractions derived from retinal pigment epithelial cells. The binding of retinol-binding protein to the membranes was fast, with a dissociation constant in the range of 31-72 nM, and maximum binding occurred at neutral pH. Receptor binding sites were also found in microsome fractions of liver and kidney, whereas lung and muscle contained few, if any. Chemical cross-linking of retinol-binding protein to the microsomal membranes yielded a major molecular complex of Mr 86,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein responsible for binding of retinol-binding protein was identified as a Mr 63,000 protein using a label transfer cross-linking technique. Further characterization demonstrated that the receptor for retinol-binding protein is a terminally glycosylated membrane protein noncovalently associated with a high molecular weight complex.

Na+/Ca2+ exchange activity is increased in Alzheimer's disease brain tissues.

These studies were performed to determine the changes that occur in Na+/Ca2+ exchange activity in Alzheimer's disease (AD) brain tissues. Cerebral plasma membrane vesicles were purified by sucrose density gradient centrifugation from frozen postmortem hippocampal/temporal cortex tissue slices derived from age matched brains of normal, AD and non-Alzheimer dementia (NAD) origin (autopsy confirmed). Membrane marker assays (Na/K ATPase, muscarinic receptor, cytochrome c oxidase) revealed no change in membrane purity across different preparations. Thin-section electron microscopy revealed predominantly intact unilamellar vesicles. Vesicles were preincubated for 15 min (37 degrees C) in buffer containing 132 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 10 mM glucose and 10 mM HEPES (pH 7.4). Ca2+ uptake was initiated by diluting vesicles 20-fold with buffer containing either 132 mM NaCl or 132 mM choline chloride and 45CaCl2 then terminated by addition of 200 microM LaCl3 and rapid filtration. Ca2+ content increased rapidly at first and then maintained a steady plateau for up to 5 min. When the Ca2+ ionophore A23187 (10 microM) with 100 microM EGTA was added after 4 min, Ca2+ content was reduced to 10% of its original value. Ruthenium red (10 microM) had no effect on Ca2+ content. Na(+)-dependent Ca2+ uptake (Ca2+ content measured in choline chloride minus that measured in NaCl) was increased in AD brains as evidenced by both an increase in the initial rise in Ca2+ content and in elevated values of peak plateau Ca2+ content.(ABSTRACT TRUNCATED AT 250 WORDS)