Biomedical subjects

R E Campbell

Publications and source records attributed to R E Campbell.

At least 19 recordsLinked to original sources

PubMed2001

Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications.

Yellow mutants of the green fluorescent protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor protein-protein interactions. However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37 degrees C. Protein evolution in Escherichia coli has produced a new YFP named Citrine, in which the mutation Q69M confers a much lower pK(a) (5.7) than for previous YFPs, indifference to chloride, twice the photostability of previous YFPs, and much better expression at 37 degrees C and in organelles. The halide resistance is explained by a 2.2-A x-ray crystal structure of Citrine, showing that the methionine side chain fills what was once a large halide-binding cavity adjacent to the chromophore. Insertion of calmodulin within Citrine or fusion of cyan fluorescent protein, calmodulin, a calmodulin-binding peptide and Citrine has generated improved calcium indicators. These chimeras can be targeted to multiple cellular locations and have permitted the first single-cell imaging of free [Ca(2+)] in the Golgi. Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired and is particularly advantageous within genetically encoded fluorescent indicators of physiological signals.

Amino Acid Substitution↗

PubMed2001

Hypothalamic circuitry of neuropeptide Y regulation of neuroendocrine function and food intake via the Y5 receptor subtype.

Neuropeptide Y (NPY) displays diverse modes of action in the CNS including the modulation of feeding behavior, gonadotropin releasing hormone release, and stress responses. Many of the above physiological actions have been at least partially attributed to actions of NPY on the NPY Y5 receptor subtype. We utilized an antibody directed against the NPY Y5 receptor to characterize the distribution of this receptor in the rat brain. Using Western blot analysis, this antibody recognized a single major band at approximately 57 kD. To further verify the specificity of the antibody, animals were treated for 5 days with antisense oligonucleotides for the Y5 receptor. The antisense treatment significantly reduced food intake and body weight. Furthermore, the Y5 antibody detected a significant decrease in Y5 receptor protein. Y5-like immunoreactivity (-ir) was observed throughout the hypothalamus, thalamus, hippocampus and cortex. Double-label immunofluorescence demonstrated that Y5-ir was colocalized with the following neuronal phenotypes in the hypothalamus, gonadotropin-releasing hormone, neurophysins, corticotropin-releasing hormone, and gamma-amino butyric acid. In addition, functional interactions were demonstrated by the presence of close appositions of NPY fibers with Y5-ir expressing cells. The wide distribution of the Y5 receptor-ir, as well as the colocalization within specific neuronal populations, agrees with the distribution of the Y5 receptor mRNA and the known physiological roles of the NPY/Y5 system. The role of the NPY/Y5 receptor system as a mediator between signals of peripheral energy availability and reproductive neuroendocrine function is discussed.

Animals↗

PubMed2000

The structure of UDP-N-acetylglucosamine 2-epimerase reveals homology to phosphoglycosyl transferases.

Bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible epimerization at C-2 of UDP-N-acetylglucosamine (UDP-GlcNAc) and thereby provides bacteria with UDP-N-acetylmannosamine (UDP-ManNAc), the activated donor of ManNAc residues. ManNAc is critical for several processes in bacteria, including formation of the antiphagocytic capsular polysaccharide of pathogens such as Streptococcus pneumoniae types 19F and 19A. We have determined the X-ray structure (2.5 A) of UDP-GlcNAc 2-epimerase with bound UDP and identified a previously unsuspected structural homology with the enzymes glycogen phosphorylase and T4 phage beta-glucosyltransferase. The relationship to these phosphoglycosyl transferases is very intriguing in terms of possible similarities in the catalytic mechanisms. Specifically, this observation is consistent with the proposal that the UDP-GlcNAc 2-epimerase-catalyzed elimination and re-addition of UDP to the glycal intermediate may proceed through a transition state with significant oxocarbenium ion-like character. The homodimeric epimerase is composed of two similar alpha/beta/alpha sandwich domains with the active site located in the deep cleft at the domain interface. Comparison of the multiple copies in the asymmetric unit has revealed that the epimerase can undergo a 10 degrees interdomain rotation that is implicated in the regulatory mechanism. A structure-based sequence alignment has identified several basic residues in the active site that may be involved in the proton transfer at C-2 or stabilization of the proposed oxocarbenium ion-like transition state. This insight into the structure of the bacterial epimerase is applicable to the homologous N-terminal domain of the bifunctional mammalian UDP-GlcNAc "hydrolyzing" 2-epimerase/ManNAc kinase that catalyzes the rate-determining step in the sialic acid biosynthetic pathway.

Amino Acid Sequence↗

PubMed2000

The first structure of UDP-glucose dehydrogenase reveals the catalytic residues necessary for the two-fold oxidation.

Bacterial UDP-glucose dehydrogenase (UDPGlcDH) is essential for formation of the antiphagocytic capsule that protects many virulent bacteria such as Streptococcus pyogenes andStreptococcus pneumoniae type 3 from the host's immune system. We have determined the X-ray structures of both native and Cys260Ser UDPGlcDH from S. pyogenes (74% similarity to S. pneumoniae) in ternary complexes with UDP-xylose/NAD(+) and UDP-glucuronic acid/NAD(H), respectively. The 402 residue homodimeric UDPGlcDH is composed of an N-terminal NAD(+) dinucleotide binding domain and a C-terminal UDP-sugar binding domain connected by a long (48 A) central alpha-helix. The first 290 residues of UDPGlcDH share structural homology with 6-phosphogluconate dehydrogenase, including conservation of an active site lysine and asparagine that are implicated in the enzyme mechanism. Also proposed to participate in the catalytic mechanism are a threonine and a glutamate that hydrogen bond to a conserved active site water molecule suitably positioned for general acid/base catalysis.

Amino Acid Sequence↗

PubMed2000

Neuropeptide Y Y5 receptor protein in the cortical/limbic system and brainstem of the rat: expression on gamma-aminobutyric acid and corticotropin-releasing hormone neurons.

Neuropeptide Y displays diverse modes of action in the CNS including the modulation of cortical/limbic function. Some of these physiological actions have been at least partially attributed to actions of neuropeptide Y on the Y5 receptor subtype. We utilized an antibody raised against the Y5 receptor to characterize the distribution of this receptor subtype in the rat cortical/limbic system and brainstem. Y5-like immunoreactivity was located primarily in neuronal cell bodies and proximal dendritic processes throughout the brain. In the cortex, Y5 immunoreactivity was limited to a subpopulation of small gamma-aminobutyric-acid interneurons (approximately 15 microm diameter) scattered throughout all cortical levels. Double label immunofluorescence was also used to demonstrate that all of the Y5 immunoreactive neurons in the cortex displayed intense corticotropin releasing hormone immunoreactivity. The most intense Y5 immunoreactive staining in the hippocampus was located in the pyramidal cell layer of the small CA2 subregion and the fasciola cinerea, with lower levels of staining in the hilar region of the dentate gyrus and CA3 subregion of the pyramidal cell layer. Nearly all of the Y5 immunoreactive neurons in the hilar region of the hippocampus displayed gamma-aminobutyric-acid immunoreactivity. In the brainstem, Y5 immunoreactivity was most intense in the Edinger-Westphal nucleus, locus coeruleus and the mesencephalic trigeminal nucleus. The present study provides neuroanatomical evidence for the possible sites of action of the neuropeptide Y/Y5 receptor system in the control of cortical/limbic function. The presence of Y5 immunoreactivity on cell bodies and proximal dendritic processes in specific regions of the hippocampus suggests that this receptor functions to modulate postsynaptic activity. These data also suggest that the neuropeptide Y/Y5 system may play a role in the modulation of a specific population of GABAergic neurons in the cortex, namely those that contain corticotropin-releasing hormone. The location of the Y5 receptor immunoreactivity fits with the known physiological actions of neuropeptide Y and this receptor.

Animals↗

PubMed1998

Molecular and immunologic characterization of group A bovine rotavirus field isolates with P8[11] spike protein.

Bovine rotavirus strain B223 is the North American prototype for group A P8[11] serotype/genotype rotaviruses. Rotaviruses with this serotype/genotype are a prevalent cause of neonatal calf diarrhea and have also been isolated from asymptomatic human infants. On the basis of deduced amino acid sequence of the outer capsid viral protein 4 (VP4), strain B223 lacks a cysteine at position 318 that is conserved among all other rotavirus strains. It has been speculated that this may result in the loss of disulfide bond and a change in the structure of the VP4 that may affect the infectivity and antigenicity of the virus. This paper describes partial sequences of the VP4 gene (nucleotides 613 to 1016 coding for amino acid positions 202 to 335) of 16 bovine rotavirus field isolates with P8[11] that were obtained from calves in Nebraska and Indiana. All the isolates lack a cystein at position 203 and had a conserved valine residue at position 318, thus indicating that the prototype strain B223 is representative of group A rotaviruses with P8[11] serotype/genotype.

Amino Acid Sequence↗

PubMed1997

Properties and kinetic analysis of UDP-glucose dehydrogenase from group A streptococci. Irreversible inhibition by UDP-chloroacetol.

UDP-glucuronic acid is used by many pathogenic bacteria in the construction of an antiphagocytic capsule that is required for virulence. The enzyme UDP-glucose dehydrogenase catalyzes the NAD+-dependent 2-fold oxidation of UDP-glucose and provides a source of the acid. In the present study the recombinant dehydrogenase from group A streptococci has been purified and found to be active as a monomer. The enzyme contains no chromophoric cofactors, and its activity is unaffected by the presence of EDTA or carbonyl-trapping reagents. Initial velocity and product inhibition kinetic patterns are consistent with a bi-uni-uni-bi ping-pong mechanism in which UDP-glucose is bound first and UDP-glucuronate is released last. UDP-xylose was found to be a competitive inhibitor (Ki, 2.7 microM) of the enzyme. The enzyme is irreversibly inactivated by uridine 5'-diphosphate-chloroacetol due to the alkylation of an active site cysteine thiol. The apparent second order rate constant for the inhibition (ki/Ki) was found to be 2 x 10(3) mM-1 min-1. Incubation with the truncated compound, chloroacetol phosphate, resulted in no detectable inactivation when tested under comparable conditions. This supports the notion that uridine 5'-diphosphate-chloroacetol is bound in the place of UDP-glucose and is not simply acting as a nonspecific alkylating agent.

Chromatography, Gel↗

PubMed1996

Yields, chemical composition, and value of beef shank tissues obtained using Baader processing.

This experiment was designed to determine yield of meat and sinew from beef shanks processed with a Baader desinewing machine and to determine whether this process added value to a beef carcass. Baader desinewing machines use belt pressure against a rotating, perforated steel drum to separate tissues. Boneless beef shanks had 9.8% fat and 14.1 mg/g of collagen. Using the Baader with a 5-mm drum, the first pass lean yield was 73.3% and had fat reduced to 7.1% and collagen to 10.5 mg/g. Second-pass lean yield through the 5-mm drum was 19.6% and had 16.1% fat and 13.8 mg/g of collagen, leaving 6.7% separated sinew. Using a 3-mm drum reduced first-pass lean yield to 66.1% and reduced fat content to 5.8%. Second-pass lean yield, using 3- and 5-mm drums, was 26.1% and had 18.6% fat and 27.8 mg/g of collagen with 6.8% sinew. Desinewed lean is worth more than whole shanks. Furthermore, 95% lean is worth more than 90% lean, and the sinew also has a salvage value. Upgrading shanks with this desinewing device can increase the value of a beef carcass by $2.01 using a 5-mm drum or by $3.20 using both 3- and 5-mm drums.

Animals↗

PubMed1996

Influence of steroid implants and concentrate feeding on performance and carcass composition of cull beef cows.

Sixty nonpregnant, mature beef cows were used to evaluate the influence of steroid implants on performance and carcass composition of beef cows fed a high-concentrate diet. Twelve nonfed cows were slaughtered at 0 d. Remaining cows were stratified by weight and randomly assigned to an implant treatment and feeding period (28 or 56 d). Treatments included 1) no implant (control), 2) a 200-mg trenbolone acetate (TBA) implant, 3) a 200-mg testosterone propionate +20 mg of estradiol benzoate (TEB) implant, or 4) both implants (TBA + TEB). As days on feed increased, hot carcass weight (HCW), carcass soft tissue (CST) weight, adjusted fat thickness (AFT), longissimus muscle area (LMA), percentage of CST, and percentage of CST lipid increased (P < .05) and percentage of CST crude protein and moisture decreased (P < .05). Cows fed for 56 d accumulated (P < .05) more kilograms of lean than cows fed for 28 d, and cows fed for 28 d tended (P = .08) to have more kilograms of lean than nonfed cows. Cows fed for 56 d had (P < .05) higher dressing percentages than nonfed cows or cows fed for 28 d, and nonfed cows had (P < .05) higher dressing percentages than cows fed for 28 d. Implanted cows compared with control cows had (P < .05) heavier final weights, increased gains, improved feed efficiency, heavier hot carcass weights, larger LMA, higher cutability yield grades, compositionally leaner CST (higher percentage of moisture and protein and lower percentage of lipid), and more kilograms of lean. In addition, double-implanted (TBA + TEB) cows had (P < .05) more kilograms of CST than control and TBA-implanted cows. Furthermore, cows implanted with TBA had (P < .05) lower dressing percentages than TEB-implanted cows, less AFT than controls, and fewer kilograms of lipid than cows on the other three treatments. Full feeding of thin beef cows for 28 or 56 d increased carcass weight through an increase in CST lean as well as fat (lipid). The live performance and accretion of lean during realimentation can be enhanced by using anabolic implants.

Animals↗

PubMed1994

Giant marginal ulcer.

Marginal ulcer is a well-known complication of gastroenterostomy. It occurs in 3% of patients post-Billroth II subtotal gastrectomy; it occurs in less than 1% if truncal vagotomy is included but in up to 30% of patients with gastroenterostomy without vagotomy. These ulcers occur at the anastomosis, but always on the jejunal side, and are known to develop complications of their own--e.g., intractable pain; hemorrhage, obstruction, perforation, and fistula formation. Prior to the advent of upper-GI endoscopy the main method of diagnosis was by history and upper GI series but the accuracy of the upper-Gi series was about 50% or less. Now that upper-GI endoscopy is available, the accuracy of diagnosis is 95% or better. Since truncal vagotomy has been widely adopted as an integral part of gastric surgery--e.g., antrectomy, hemigastrectomy, subtotal gastrectomy, and gastroenterostomy--the incidence of marginal ulcer has declined. The use of cimetidine, ranitidine, famotidine, omeprazole, sucralfate, and antacids has improved the medical management of duodenal ulcer to such a degree that in recent years there is much less need for surgical intervention and thus the incidence of marginal ulcer has declined even more. In addition, the H-2 blockers and omeprazole can be used in patients with marginal ulcer and achieve healing; therefore complications that so frequently required surgical intervention are much less frequent.(ABSTRACT TRUNCATED AT 250 WORDS)

Anastomosis, Surgical↗

PubMed1994