Biomedical subjects

J C Brooks

Publications and source records attributed to J C Brooks.

At least 19 recordsLinked to original sources

PubMed2006

Chromosomal loci that influence oral nicotine consumption in C57BL/6J x C3H/HeJ F2 intercross mice.

Several studies have demonstrated that there are genetic influences on free-choice oral nicotine consumption in mice. In order to establish the genetic architecture that underlies individual differences in free-choice nicotine consumption, quantitative trait loci (QTL) mapping was used to identify chromosomal regions that influence free-choice nicotine consumption in male and female F(2) mice derived from a cross between C57BL/6J and C3H/HeJ mice. These two mouse strains were chosen not only because they differ significantly for oral nicotine consumption, but also because they are at or near phenotypic extremes for all measures of nicotine sensitivity that have been reported. A four-bottle choice paradigm was used to assess nicotine consumption over an 8-day period. The four bottles contained water or water supplemented with 25, 50 or 100 microg/ml of nicotine base. Using micrograms of nicotine consumed per milliliter of total fluid consumed per day as the nicotine consumption phenotype, four significant QTL were identified. The QTL with the largest LOD score was located on distal chromosome 1 (peak LOD score = 15.7). Other chromosomes with significant QTL include central chromosome 4 (peak LOD score = 4.1), proximal chromosome 7 (peak LOD score = 6.1) and distal chromosome 15 (peak LOD score = 4.8). These four QTL appear to be responsible for up to 62% of the phenotypic variance in oral nicotine consumption.

Administration, Oral↗

PubMed2006

Effects of feeding whole cottonseed and cottonseed products on performance and carcass characteristics of finishing beef cattle.

Three experiments were conducted to determine the effects of whole cottonseed or cottonseed products on performance and carcass characteristics of beef cattle. In Exp. 1, 120 beef steers (initial BW = 381 +/- 31.7 kg) were fed steam-flaked corn-based finishing diets with 10% (DM basis) basal roughage, and whole cottonseed or individual cottonseed components (cottonseed hulls, meal, and oil). Over the entire feeding period, ADG did not differ (P = 0.95), but DMI increased (P = 0.07) and G:F decreased (P = 0.06) for steers fed the cottonseed diets compared with the control diet. Dressing percent (P = 0.02) and marbling scores (P = 0.02) of carcasses from steers fed the cottonseed diets were less than for steers fed the control diet. In Exp. 2, 150 beef steers (initial BW = 364 +/- 9.9 kg) were used to determine the effects of whole cottonseed or pelleted cottonseed (PCS) on performance and carcass characteristics. Cattle were fed steam-flaked corn-based finishing diets in which whole cottonseed or PCS replaced all of the dietary roughage, supplemental fat, and supplemental natural protein of the control diet. Over the entire feeding period, steers fed the cottonseed diets had lower (P = 0.04) DMI and greater (P < 0.01) G:F than steers fed the control diet. Carcass characteristics did not differ (P = 0.16 to 0.96) among dietary treatments. In Exp. 3, 150 beef heifers (initial BW = 331 +/- 17.1 kg) were used to determine the effects of PCS or delinted, whole cottonseed (DLCS) on performance and carcass characteristics. Heifers were fed rolled corn-based finishing diets in which cottonseed replaced the dietary roughage, supplemental fat, and all or part of the supplemental natural protein of the control diet. Over the entire feeding period, ADG, DMI, and G:F of heifers fed the control diet did not differ (P = 0.19 to 0.80) from those of the cottonseed diets; however, heifers fed the diets containing PCS had greater ADG (P = 0.03) and G:F (P = 0.09) than heifers fed diets containing DLCS. Carcass characteristics of heifers fed the control diet did not differ (P > or = 0.28) from those fed the cottonseed diets. Heifers fed the diets containing PCS had greater (P < or = 0.03) HCW, dressing percent, and LM area than those fed DLCS. Based on our results, whole cottonseed, or products derived from processing whole cottonseed, can replace feedstuffs commonly used in beef cattle finishing diets with no adverse effects on animal performance or carcass characteristics.

Animal Feed↗

PubMed2003

Effects of growth implants on consumer perceptions of meat tenderness in beef steers.

Anabolic steroid implants are routinely used to increase growth performance and profitability; however, there are concerns that the use of implants, particularly those containing trenbolone acetate, may have detrimental effects on carcass quality and beef tenderness. Thus, the objectives of the current study were to determine the effects of various commonly used implant regimens on shear force values, sensory properties, and consumer satisfaction of beef top loin steaks from cattle of Bos indicus influence. Cattle were supplied by producers that agreed to provide sire and dam information in exchange for carcass and sensory data. Steers (n = 2,748) were assigned randomly to one of three implant treatments (12/sire; four steers from each sire were placed into each treatment group): 1) unimplanted controls (n = 1,368); 2) Synovex-S followed by another Synovex-S (n = 660); or 3) Synovex-S followed by Revalor-S (n = 720). Steaks sampled after 3, 7, and 14 d of aging indicated that unimplanted cattle had lower (P < 0.05) Warner-Bratzler Shear force values than those from implanted animals. No differences (P > 0.05) in shear force values were found between the two treatments or the control groups for steaks sampled following a 21-d aging period. Steaks from implanted animals sampled after 3, 7, and 14 d aging were rated lower (P < 0.05) for initial and sustained trained sensory panel tenderness scores. Consumers failed to detect any differences in steak samples related to implant treatment after 7 and 14 d of aging. Consumer education level and family income did not affect overall acceptability (P > 0.10 and 0.18, respectively) or tenderness acceptability (P > 0.11 and 0.68, respectively); however, consumers with postgraduate degrees recorded lower (P < 0.05) overall quality, beef flavor, juiciness, and tenderness scores than consumers in all other education classifications. Additionally, family income had no effect on overall quality (P > 0.21), beef flavor (P > 0.28), juiciness (P > 0.58), or tenderness (P > 0.45) scores. Results indicate that using a moderate implant program in Bos indicus-influenced cattle has no detrimental effects on beef tenderness and consumer acceptability.

Animals↗

PubMed2002

National Beef Quality Audit-2000: survey of targeted cattle and carcass characteristics related to quality, quantity, and value of fed steers and heifers.

The National Beef Quality Audit-2000 was conducted to assess the current status ofthe quality and consistency of U.S. fed steers and heifers. Between May and November 2000, survey teams assessed hide condition (n = 43,415 cattle for color, brands, mud/manure), bruises (n = 43,595 carcasses), offal and carcass condemnation (n = 8,588 cattle), and carcass quality and yield information (n = 9,396 carcasses) in 30 U.S. beef packing plants. Hide colors were black (45.1%), red (31.0%), yellow (8.0%), Holstein (5.7%), gray (4.0%), white (3.2%), brown (1.7%), and brindle (1.3%). Brand frequencies were no (49.3%), one (46.2%), and two or more (4.4%), and brands were located on the butt (36.3%), side (13.7%), and shoulder (3.6%). Most cattle had no (18.0%) or a small amount (55.8%) of mud/manure on their hides, and they had no (77.3%) horns. Most carcasses (53.3%) were not bruised, 30.9% had one bruise, and 15.8% had multiple bruises. Bruise location and incidence were round (14.9%), loin (25.9%), rib (19.4%), chuck (28.2%), and brisket, flank, and plate (11.6%). Condemnation item and incidence were liver (30.3%), lungs (13.8%), tripe (11.6%), heads (6.2%), tongues (7.0%), and carcasses (0.1%). Carcass evaluation revealed these traits and frequencies: steer (67.9%), heifer (31.8%), and bullock (0.3%) sex-classes; dark-cutters (2.3%); A (96.6%), B (2.5%), and C or older (0.9%) overall maturities; and native (90.1%), dairy-type (6.9%), and Bos indicus (3.0%) breed-types. Mean USDA yield grade traits were USDA yield grade (3.0), carcass weight (356.9 kg), adjusted fat thickness (1.2 cm), longissimus muscle area (84.5 cm2), and kidney, pelvic, and heart fat (2.4%). USDA yield grades were Yield Grade 1 (12.2%), Yield Grade 2 (37.4%), Yield Grade 3 (38.6%), Yield Grade 4 (10.4%), and Yield Grade 5 (1.3%). Mean USDA quality grade traits were USDA quality grade (Select85), marbling score (Small23), overall maturity (A66), lean maturity (A65), and skeletal maturity (A67). Marbling score distribution was Slightly Abundant or higher (2.3%), Moderate (4.8%), Modest (13.1%), Small (33.3%), Slight (43.3%), and Traces (3.4%). USDA quality grades were Prime (2.0%), Choice (49.1%), Select (42.3%), Standard (5.6%), and Commercial, Utility, Cutter, and Canner (0.9%). This information will help the beef industry measure progress compared to the past two surveys and will provide a benchmark for future educational and research activities.

Animals↗

PubMed2001

A proton magnetic resonance spectroscopy study of age-related changes in frontal lobe metabolite concentrations.

Ageing is associated with reduction of grey matter volume and it is reported that the frontal lobes are preferentially affected. We have applied quantitative magnetic resonance spectroscopy (MRS), incorporating measurement of brain tissue water content and metabolite T(2) relaxation times, to determine absolute concentrations of the putative neuronal marker N-acetylaspartate (NAA), creatine (Cr) and choline (Cho) compounds in the frontal lobe of 50 male subjects aged between 20 and 70 years (10 per decade). The fractional brain water content (beta(MR)) did not change significantly as a function of age (r = 0.07, P = 0.65) and had a mean value of 81% (CV = 2%). The concentration (in millimoles per litre brain tissue) of NAA decreased significantly with age (r = -0.42, P = 0.003), with an overall decrease of 12% between the third and seventh decades. The concentrations of Cr and Cho did not change significantly with age. The interpretation of the age-dependent decrease in NAA concentration as reflecting either a reduction in neuronal volume, number or function is discussed.

Adult↗

PubMed2001

Effects of supplemental vitamin D3 on feed intake, carcass characteristics, tenderness, and muscle properties of beef steers.

Research was conducted to determine the effects of supplemental dietary vitamin D3 on DMI, carcass traits, Warner Bratzler shear (WBS) force, calpastatin activity, plasma minerals, pH (0, 3, 12, and 24 h after slaughter), water-holding capacity (WHC), and sensory characteristics of three muscles. Pre-slaughter vitamin D3 treatments included no supplemental vitamin D3, 6 x 106 IU (MIU) of vitamin D3 for 4 d, or 6 MIU of vitamin D3 for 6 d. Cattle were slaughtered and carcasses were chilled for 48 h before removal of steaks from the longissimus, gluteus medius, and biceps femoris muscles. Steaks were aged at 2 degrees C for 7, 14, or 21 d before cooking to a final internal temperature of 70 degrees C for WBS and sensory panel analysis. Dry matter intake was lower for steers supplemented with vitamin D3 for 4 or 6 d. Live and carcass weights were lower (P < 0.05) in steers supplemented with vitamin D3. Supplementing 6 MIU/6 d of vitamin D3 decreased (P < 0.05) WBS values of gluteus steaks (pooled over aging times). Longissimus steaks from steers supplemented with vitamin D3 for 6 d had lower (P < 0.05) WBS force values than these steaks from control steers or steers fed vitamin D3 for 4 d at 7 d postmortem. Biceps femoris steaks from steers receiving vitamin D3 for 4 d had higher WBS values than steaks from control steers at 14 and 21 d postmortem. Feeding vitamin D3 at 6 MIU for 6 d decreased (P < 0.05) the percentage of steaks that had WBS values > or = 3.86 kg for all steaks. Feeding vitamin D3 had no effect on palatability traits evaluated by trained panelists. Blood Ca concentrations were greater (P < 0.05) when vitamin D3 was fed and with increased vitamin D3 feeding time. Feeding vitamin D3 for 6 d (vs 4 d) delayed pH decline for all muscle types after 0, 3, and 12 h postmortem. Water-holding capacity was increased (P > 0.02) after 0 h, 24 h, and 21 d postmortem when vitamin D3 was fed and was greater at 0 and 24 h if vitamin D3 was fed for 6 d rather than 4 d. These data suggest that supplementing 6 MIU of vitamin D3 will decrease DMI and improve beef tenderness through increased blood plasma Ca concentrations and WHC.

Animals↗

PubMed2001

Vitamin-mineral supplementation and accelerated chilling effects on quality of pork from pigs that are monomutant or noncarriers of the halothane gene.

We examined the effects of vitamin and mineral supplementation of the finishing diet on growth and accelerated chilling of carcasses on carcass and muscle traits of halothane gene carrier and noncarrier pigs. Barrows and gilts that were either monomutants (MON, n = 49) or noncarriers (NON, n = 28) of the halothane gene were fed a standard finishing diet until they reached 86 kg. They then were randomly assigned to one of four finishing diets formulated to contain 11 IU/kg vitamin E (0), 311 IU/kg vitamin E plus additional vitamins and minerals (300), 611 IU/kg vitamin E plus additional vitamins and minerals (600), or 911 IU/kg vitamin E plus additional vitamins and minerals (900) until they were slaughtered (118 kg). Alternating carcass sides were assigned either a normal chilling procedure (NC, 4 degrees C for 24 h) or an accelerated chilling procedure (AC, -20 degrees C for 1.5 h and then 4 degrees C for 22.5 h). Supplementing vitamin E in the finishing diet increased (P < 0.05) the concentration of vitamin E in the longissimus muscle. Supplementing vitamin E in the diets of MON pigs did not affect color, firmness, or cooking losses of loins or color and firmness of hams. For the NON genotype, increasing the level of vitamin E in the diet decreased (P < 0.05) the percentage of PSE loins and hams. Color and firmness scores of the gluteus medius and longissimus muscles were improved 0.4 unit (P < 0.005) by AC compared with NC of carcasses. Loin chop juiciness and flavor were improved (P < 0.05) in the MON genotype for AC compared to NC. Accelerated chilling reduced (P < 0.05) the percentage of PSE loins from 38 to 17% and PSE hams from 32 to 10% for the MON genotype, but percentage of PSE was not affected (P > 0.05) by chilling treatment for the NON genotype. No interaction between diet and chill treatments existed for muscle quality traits (P > 0.05). Supplementing finishing diets of NON pigs with at least 600 IU/kg vitamin E, in addition to other vitamins and minerals, or accelerated chilling of MON carcasses can reduce the incidence of PSE pork.

Animals↗

PubMed2000

Proton magnetic resonance spectroscopy and morphometry of the hippocampus in chronic fatigue syndrome.

Seven patients with chronic fatigue syndrome (CFS) were matched with ten healthy control subjects of similar age. Hippocampal volume, obtained from magnetic resonance images using an unbiased method, showed no difference between the two groups, whereas proton magnetic resonance spectroscopy showed a significantly reduced concentration of N-acetylaspartate in the right hippocampus of CFS patients (p = 0.005).

Adult↗

PubMed2000

National Beef Tenderness Survey-1998.

Fifty-six retail stores representing 15 retail chains and 14 foodservice facilities in eight U.S. cities were sampled to determine the tenderness of beef steaks at retail and foodservice levels based on Warner-Bratzler shear (WBS) values and consumer evaluation panels. Retail consumer panels were conducted at five universities. Each retail and foodservice steak was evaluated using 10-point scales. Steaks were divided into the following quality groups for statistical analysis: Prime, Top Choice, Choice, Select, and Lean or No Roll. Quality group had no effect on WBS values of retail clod, chuck roll, top round, bottom round, eye of round, top loin, top sirloin, or ribeye steaks but did (P < .05) affect values for the T-bone/porterhouse. The percentages of retail top round, eye of round, and bottom round steaks with a WBS force > 3.9 kg were 39.6, 55.9, and 68.0, respectively. Foodservice ribeye, top loin, and top sirloin steaks had WBS values less than 3.4 kg for all quality groups, with Prime ribeye steaks having lower (P < .05) WBS values than ribeyes from the other quality groups. With the exception of the retail ribeye steak, quality group did not affect consumer sensory ratings of retail and foodservice steaks. Average postfabrication aging times were 32 d for foodservice subprimals and 19 d for retail cut subprimals. These data indicate that improvements in the tenderness of retail cuts from the round are needed. Finally, quality group had little or no effect on consumer sensory evaluations and WBS values of retail and foodservice steaks used in this study.

Animals↗

PubMed1999

Magnetic resonance imaging-based compartmentation and its application to measuring metabolite concentrations in the frontal lobe.

Partial volume mixing of water compartments within a spectroscopy voxel (e.g. cerebrospinal fluid within a "brain" voxel) may, if not corrected for, lead to underestimation of brain metabolite concentrations. To correct for this source of bias, a new imaging-based method of compartmentation analysis is presented. Brain water, cerebrospinal fluid and solid matter content were obtained from proton density- and T2-weighted images of the brain and an external standard in 10 healthy young males (21 to 30 years), and results compared with a previously-described technique based on spectroscopy. Mean (SD) fractional water content (betaMR) of the 2 x 2 x 2 cm3 voxel in the frontal lobes was 0.79 (0.03) by imaging, slightly but significantly (p = 0.03) smaller than the value of 0.83 (0.03) obtained by spectroscopy. From water-suppressed spectra recorded at five echo times, using betaMR determined by imaging, the T2-corrected concentrations of compounds containing N-acetylaspartate, creatine, choline and myo-inositol were 10.6 (1.0), 8.0 (0.9), 1.6 (0.3) and 3.7 (0.7) mmol.l(-1) of brain, respectively. Imaging-based compartmentation is a rapid and straightforward technique, and can be performed on standard MR systems.

Adult↗

PubMed1999

Noncardiogenic pulmonary edema immediately following rapid protamine administration.

OBJECTIVE: To report the case of a rare, potentially preventable, immediate noncardiogenic pulmonary edema reaction to the rapid administration of protamine during coronary artery bypass graft (CABG) surgery. CASE SUMMARY: A 74-year-old white man was administered a 250-mg bolus of protamine sulfate toward the end of CABG surgery to reverse the heparin anticoagulation. Immediately following the administration of protamine, oxygen saturation declined, pink frothy sputum was suctioned from the trachea, and 1500 mL of serous fluid was removed from the airway. The patient was stabilized, but the surgeons were unable to close his chest because of the profound edema. Chest closure occurred on hospital day 6, with discharge from the intensive care unit on hospital day 28. DISCUSSION: Noncardiogenic pulmonary edema is a rare adverse event that occurs in 0.2% of cardiopulmonary bypass patients, with mortality rates approaching 30%. Complement activation or direct pharmacologic release of histamine by high concentrations of protamine is the suspected cause. High concentrations of protamine in the lungs may directly release histamine, with significant vasodilating effects. CONCLUSIONS: Immediate reversal of heparin anticoagulation with protamine is necessary to control bleeding; however, rapid protamine injection can be associated with life-threatening pulmonary edema. Slower, cautious administration and accurate calculation of protamine doses may prevent such an event.

Aged↗

PubMed1998

Absolute metabolite quantification by in vivo NMR spectroscopy: II. A multicentre trial of protocols for in vivo localised proton studies of human brain.

We have performed a multicentre trial to assess the performance of three techniques for absolute quantification of cerebral metabolites using in vivo proton nuclear magnetic resonance (NMR). The techniques included were 1) an internal water standard method, 2) an external standard method based on phantom replacement, and 3) a more sophisticated method incorporating elements of both the internal and external standard approaches, together with compartmental analysis of brain water. Only the internal water standard technique could be readily implemented at all participating sites and gave acceptable precision and interlaboratory reproducibility. This method was insensitive to many of the experimental factors affecting the performance of the alternative techniques, including effects related to loading, standing waves and B1 inhomogeneities; and practical issues of phantom positioning, user expertise and examination duration. However, the internal water standard method assumes a value for the concentration of NMR-visible water within the spectroscopic volume of interest. In general, it is necessary to modify this assumed concentration on the basis of the grey matter, white matter and cerebrospinal fluid (CSF) content of the volume, and the NMR-visible water content of the grey and white matter fractions. Combining data from 11 sites, the concentrations of the principal NMR-visible metabolites in the brains of healthy subjects (age range 20-35 years) determined using the internal water standard method were (mean+/-SD): [NAA]=10.0+/-3.4 mM (n=53), [tCho]=1.9+/-1.0 mM (n=51), [Cr + PCr]=6.5+/-3.7 mM (n=51). Evidence of system instability and other sources of error at some participating sites reinforces the need for rigorous quality assurance in quantitative spectroscopy.

Adult↗

PubMed1997

Rechargeability of fluoride releasing pit and fissure sealants and restorative resin composites.

PURPOSE: To evaluate the effect of exposure of 1.23% acidulated phosphate fluoride gel on fluoride release from fluoride releasing pit and fissure sealants and restorative resin composites. MATERIALS AND METHODS: Specimens of four fluoride releasing pit and fissure sealants (Helioseal F, Ultraseal XT, Seal Rite-Low Viscosity and Seal Rite-Regular Viscosity) and two fluoride releasing restorative resin composites (Tetric and Heliomolar Radiopaque) were prepared using a Teflon mold. The specimens were divided into two groups. The experimental group was exposed to 1.23% acidulated phosphate fluoride gel and the control group was exposed to 1.23% acidulated phosphate gel without fluoride. The specimens were stored in deionized water and the fluoride concentration of the solution was measured every 24 hours. On day 7, the discs were exposed to the respective gel for 4 minutes and then rinsed in a continuous stream of deionized water for 30 seconds. The fluoride concentration was then measured for another 7 days. RESULTS: There was a significant increase after the fluoride gel treatment in the amount of fluoride released from all of the materials studied. When the cumulative amount of fluoride released was evaluated, Tetric, Heliomolar radiopaque, Helioseal F, and Ultraseal XT released significantly (P < 0.01) more fluoride than the respective control groups. There was no significant difference between groups from day 8 to day 14 for Seal Rite-Regular Viscosity and Seal Rite-Low Viscosity.

Acidulated Phosphate Fluoride↗

PubMed1995

Amylase secretion by cultured porcine parotid cells.

The similarity of porcine and human physiology and the availability of slaughterhouse tissues suggests the use of porcine parotid cells as a model for amylase secretion. A procedure is described for the isolation of porcine parotid cells by collagenase-P/dispase digestion of the tissue. The preparation consisted of individual cells and small aggregates that were maintained in primary culture, during which the cells formed aggregates that firmly attached to the plastic substrate. The amylase content of the cultured cells remained adequate for assay of secretory activity during culture for one week after isolation. Depending upon variations in experimental treatments, the cultured cells secreted approx. 35-65% of cellular amylase in response to a carbachol challenge. The cells were slightly responsive to long exposures to isoproterenol, and were unresponsive to nicotine, elevated extracellular K+ or substance P. Secretion induced by carbachol required extracellular Ca2+, was inhibited by atropine and occurred with a nearly linear response over a 30-min period. The Ca2+ ionophore A23187 was also a potent secretagogue for amylase secretion, producing levels of secretion equal to that induced by carbachol. The ease of preparation and the retention of amylase during primary culture suggests that the preparation will be useful in studies on muscarinic receptor-mediated control of amylase secretion.

Amylases↗

PubMed1994

The inhibitory effect of cyclophosphamide-induced MAC-1+ natural suppressor cells on IL-2 and IL-4 utilization in MLR.

Treatment of adult mice with high doses of the immunosuppressive drug cyclophosphamide (CY) induces transient splenic natural suppressor (NS) cell activity mediated largely by cells bearing the MAC-1+ cell-surface marker. Here we show that culture supernatants from mixed lymphocyte reactions (MLR) suppressed by MAC-1+ NS cells exhibit decreased IL-2 and IL-4 activity in bioassays for these lymphokines. However, inhibition of MLR was maximal whether the regulatory cells were added at initiation of culture or 24 hr postinitiation, suggesting that inhibition of lymphokine synthesis is not likely to be the reason for diminished lymphocyte proliferation, since these particular lymphokine genes are known to be transcribed and expressed during the first 12 hr of culture. Furthermore, flow cytofluorometric analysis demonstrated that the presence of MAC-1+ NS cells did not alter the percentage of lymphokine-producing CD4+ T cells in MLR. IL-2 receptor (p55) expression was also normal in suppressed MLR. The addition of exogenous IL-2 and/or IL-4 to MLR failed to reverse the inhibitory effect of MAC-1+ NS cells on lymphocyte proliferation, indicating that these regulatory cells block the utilization of these lymphokines in MLR. The inhibitory effect of MAC-1+ NS cells on lymphocyte proliferation in MLR is dependent on interferon-gamma, since NS activity was dramatically decreased in the presence of neutralizing antibodies to interferon-gamma. MAC-1+ NS cell-induced suppression of MLR was also diminished in the presence of indomethacin, suggesting that prostaglandins play a role in this NS system.

Animals↗

PubMed1992

Thiophosphorylated proteins in chromaffin cells are chromaffin vesicle matrix proteins.

Bovine adrenal chromaffin cells were incubated with inorganic thiophosphate, using a protocol similar to experiments with inorganic phosphate, in order to determine the source of previously observed thiophosphoproteins. Incubation of cultured cells with [35S]thiophosphate resulted in its incorporation into cell constituents within 2 min. SDS-PAGE of the treated cells showed incorporation of label into a broad 97-121 kDa band that was evident after 5 min of treatment and increased progressively to the 40 min exposure limit. Monolayers of chronically treated cells were fractionated into subcellular constituents. The only particulate fraction containing radiolabelled proteins was the chromaffin vesicle fraction. Two-dimensional electrophoresis of the treated cells and isolated chromaffin vesicles showed a majority of proteins in the acidic region of the first dimension gel. A fluorogram of the gel revealed two regions of radiolabelled proteins at acidic and neutral regions of the 2-D gel. These were within the boundaries of the 97-121 kDa band. The thiophosphorylated proteins were released as soluble proteins upon osmotic or freeze-thaw lysis of the vesicles. Chromaffin vesicles isolated from either cultured cells or adrenal medulla tissue were energized by 2 mM ATP but not by the analog adenosine 5'-O-(3-thiotriphosphate). The 97-121 kDa proteins in intact or lysed vesicles prepared from adrenal medulla tissue were not thiophosphorylated by either inorganic thiophosphate or adenosine 5'-O-(3-thiotriphosphate) in the presence or absence of energization by ATP. Nearly complete loss of radiolabel from matrix proteins treated with chondroitinase ABC suggests that it is a component of vesicle proteolgycans.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1992

Inorganic thiophosphate effects on chromaffin cell structure and function.

The effect was determined of replacing medium inorganic phosphate with thiophosphate on the structure and function of cultured bovine chromaffin cells. Cell cultures were incubated in normal medium containing fetal bovine serum, phosphate free medium or similar medium supplemented with inorganic phosphate or thiophosphate. In contrast to the other media, cells cultured with thiophosphate medium for 3-4 days showed seriously compromised structure and functions. The cells lost 75% of their catecholamine content and their ability to secrete remaining catecholamines in response to nicotine stimulation. Radiolabelled thiophosphate was rapidly taken up by the cells and, in long-term experiments, was incorporated largely into a 97-121 kDa protein band on SDS-PAGE. Additional minor bands were found to a lesser, variable extent. Transmission electron micrographs of cells treated with thiophosphate showed extensive depletion of chromaffin vesicles and disruption of mitochondria, suggesting that the functional damage noted with these cells could be associated with damage to mitochondria. Analysis of general cell metabolic activity by conversion of the dye (3-[3,4-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide) to its formazan derivative indicated increased metabolic activity at early stages of exposure to thiophosphate followed by a decline with continued exposure, supporting the argument for an overall depression of cell metabolism. Uptake of the dye neutral red, which is avidly accumulated by chromaffin cells, was also reduced for cells exposed to thiophosphate. The data suggest that thiophosphate enters chromaffin cells and disrupts energy dependent cell functions, including catecholamine storage and secretion.

Animals↗

PubMed1989

Ultrastructural demonstration of exocytosis in the intact rat adrenal medulla.

Evidence is presented for morphological proof of exocytosis in the rat adrenal medulla in situ. Techniques were modified to allow perfusion of the intact adrenal gland with secretagogues (or electrical stimulation) followed by tannic acid. Unstimulated specimens demonstrated exocytotic (omega-shaped) profiles filled with flocculent material. This flocculation was also seen in the intercellular space. Stimulation of the adrenal medulla also resulted in the appearance of exocytotic profiles and an accumulation of the flocculent mass. This was often most evident in the subendothelial space. This is the first demonstration of exocytosis in the rat adrenal medulla by electron microscopy. The techniques used in this study will be useful for studying the pathway of secretory products of the adrenal chromaffin cell before they enter the vascular system.

Adrenal Medulla↗