Distribution and neurochemical characterization of protein kinase C-theta and -delta in the rodent hypothalamus.

PKC-theta (PKC-θ), a member of the novel protein kinase C family (nPKC), regulates a wide variety of functions in the periphery. However, its presence and role in the CNS has remained largely unknown. Recently, we demonstrated the presence of PKC-θ in the arcuate hypothalamic nucleus (ARC) and knockdown of PKC-θ from the ARC protected mice from developing diet-induced obesity. Another isoform of the nPKC group, PKC-delta (PKC-δ), is expressed in several non-hypothalamic brain sites including the thalamus and hippocampus. Although PKC-δ has been implicated in regulating hypothalamic glucose homeostasis, its distribution in the hypothalamus has not previously been described. In the current study, we used immunohistochemistry to examine the distribution of PKC-θ and -δ immunoreactivity in rat and mouse hypothalamus. We found PKC-θ immunoreactive neurons in several hypothalamic nuclei including the ARC, lateral hypothalamic area, perifornical area and tuberomammillary nucleus. PKC-δ immunoreactive neurons were found in the paraventricular and supraoptic nuclei. Double-label immunohistochemisty in mice expressing green fluorescent protein either with the long form of leptin receptor (LepR-b) or in orexin (ORX) neurons indicated that PKC-θ is highly colocalized in lateral hypothalamic ORX neurons but not in lateral hypothalamic LepR-b neurons. Double-label immunohistochemistry in oxytocin-enhanced yellow fluorescent protein mice or arginine vasopressin-enhanced green fluorescent protein (AVP-EGFP) transgenic rats revealed a high degree of colocalization of PKC-δ within paraventricular and supraoptic oxytocin neurons but not the vasopressinergic neurons. We conclude that PKC-θ and -δ are expressed in different hypothalamic neuronal populations.

Purification and properties of Arabidopsis thaliana type 1 protein phosphatase (PP1).

The Arabidopsis thaliana type 1 protein phosphatase (PP1) catalytic subunit was released from its endogenous regulatory subunits by ethanol precipitation and purified by anion exchange and microcystin affinity chromatography. The enzyme was identified by MALDI-TOF mass spectrometry from a tryptic digest of the purified protein as a mixture of PP1 isoforms (TOPP 1-6) indicating that at least 4-6 of the eight known PP1 proteins are expressed in sufficient quantities for purification from A. thaliana suspension cells. The enzyme had a final specific activity of 8950 mU/mg using glycogen phosphorylase a as substrate, had a subunit molecular mass of 35 kDa as determined by SDS-PAGE and behaved as a monomeric protein of approx. 39 kDa on Superose 12 gel filtration chromatography. Similar to the mammalian type 1 protein phosphatases, the A. thaliana enzyme was potently inhibited by Inhibitor-2 (IC(50)=0.65 nM), tautomycin (IC(50)=0.06 nM), microcystin-LR (IC(50)=0.01 nM), nodularin (IC(50)=0.035 nM), calyculin A (IC(50)=0.09 nM), okadaic acid (IC(50)=20 nM) and cantharidin (IC(50)=60 nM). The enzyme was also inhibited by fostriecin (IC(50)=22 microM), NaF (IC(50)=2.1 mM), Pi (IC(50)=9.5 mM), and PPi (IC(50)=0.07 mM). Purification of the free catalytic subunit allowed it to be used to probe protein phosphatase holoenzyme complexes that were enriched on Q-Sepharose and a microcystin-Sepharose affinity matrix and confirmed several proteins to be PP1 targeting subunits.

Multiple WW domains, but not the C2 domain, are required for inhibition of the epithelial Na+ channel by human Nedd4.

The epithelial Na+ channel (ENaC) absorbs Na+ across the apical membrane of epithelia. The activity of ENaC is controlled by its interaction with Nedd4; mutations that disrupt this interaction increase Na+ absorption, causing an inherited form of hypertension (Liddle's syndrome). Nedd4 contains an N-terminal C2 domain, a C-terminal ubiquitin ligase domain, and multiple WW domains. The C2 domain is thought to be involved in the Ca2+-dependent localization of Nedd4 at the cell surface. However, we found that the C2 domain was not required for human Nedd4 (hNedd4) to inhibit ENaC in both Xenopus oocytes and Fischer rat thyroid epithelia. Rather, hNedd4 lacking the C2 domain inhibited ENaC more potently than wild-type hNedd4. Earlier work indicated that the WW domains bind to PY motifs in the C terminus of ENaC. However, it is not known which WW domains mediate this interaction. Glutathione S-transferase-fusion proteins of WW domains 2-4 each bound to alpha, beta, and gammaENaC in vitro. The interactions were abolished by mutation of two residues. WW domain 3 (but not the other WW domains) was both necessary and sufficient for the binding of hNedd4 to alphaENaC. WW domain 3 was also required for the inhibition of ENaC by hNedd4; inhibition was nearly abolished when WW domain 3 was mutated. However, the interaction between ENaC and WW domain 3 alone was not sufficient for inhibition. Moreover, inhibition was decreased by mutation of WW domain 2 or WW domain 4. Thus, WW domains 2-4 each participate in the functional interaction between hNedd4 and ENaC in intact cells.

A randomized trial of integrated outpatient treatment for medically ill alcoholic men.

BACKGROUND: Medically ill alcoholics often do not respond to conventional alcoholism treatment or decline physician referrals. Integrated outpatient treatment (IOT), a new treatment specifically designed for this population, combines comprehensive medical care with alcoholism interventions. OBJECTIVE: To compare the efficacy of IOT with that of standard treatment approaches. METHODS: One hundred five male veterans with severe medical complications caused by alcoholism and recent drinking were randomly assigned to receive IOT or referral to standard alcoholism and medical treatment and were evaluated over 2 years. Integrated outpatient treatment patients received medical care and alcoholism interventions once or twice monthly. Patients in the control group were referred for alcoholism treatment, but few accepted. However, patients in the control group did engage in outpatient medical care. RESULTS: At baseline, the mean +/- SD age of the control group was 57.2 + 10.0 years, compared with 52.8 +/- 11.5 years in the IOT group (P= .04). The groups were well matched in other respects. The mean +/- SD number of visits over 2 years for the IOT patients was 42.2 +/- 29.1, compared with 17.4 +/- 15.6 for the control patients (P<.001); the frequency of hospital use was similar in both groups. After 2 years, 28 (74%) of 38 surviving IOT patients and 17 (47%) of 36 control patients were abstinent (P=.02). Nearly twice as many control patients (30% [n = 16]) as IOT patients (18% [n= 9]) died, but the results of Cox survival analysis were not significant. There were no differences in symptoms of alcohol dependence, quality of life, or life problems. The incremental cost of IOT was approximately $1100 per patient per year. CONCLUSIONS: Standard medical care alone was surprisingly effective in inducing abstinence in surviving medically ill alcoholics. Integrated outpatient treatment significantly increased both engagement and abstinence for a modest annual cost. Further refinement and testing of IOT is indicated.

Prostaglandin endoperoxide-H synthase-1 and -2 messenger ribonucleic acid levels in human amnion with spontaneous labor onset.

Increased prostaglandin (PG) production within the uterine compartment has a pivotal role in the processes leading to labor onset in women. Two PG endoperoxide-H synthase (PGHS) isoenzymes have been identified in a number of cell types. PGHS-1 is constitutively expressed in most cases, whereas PGHS-2 expression is rapidly induced by several agonists. The aims of this study were to determine the levels of PGHS-1 and PGHS-2 expression before and after spontaneous labor (SL) onset in the amnion and to assess the contribution of PGHS-1 and PGHS-2 to enzyme activity. We established and validated ribonuclease protection assays to quantify PGHS-1 and PGHS-2 messenger ribonucleic acid (mRNA) levels in the amnion. PGHS enzyme activity was measured with an established assay. The antisense RNA probes used in the protection assays were generated using human PGHS-1 and PGHS-2 complementary DNAs. These probes specifically detected the 2.8-kilobase mRNA of PGHS-1 and the 4.8-kilobase mRNA of PGHS-2 in amnion RNA samples on Northern blots. We measured mRNA levels in amnion from patients after SL at term and from patients not in labor undergoing elective cesarean section (CS) at term. PGHS-2 mRNA levels were markedly higher after SL compared to levels in CS amnion [5.18 +/- 1.08 (n = 16) and 2.27 +/- 0.50 (n = 15), densitometric units, respectively; P < 0.02], whereas there was no difference in PGHS-1 mRNA levels after labor compared with CS samples. PGHS-2 mRNA levels were also positively correlated with PGHS enzyme activity in 4 separate assays with a total of 25 patients (r = 0.65-0.88; P < 0.05). There was no correlation between PGHS-1 mRNA levels and enzyme activity. We conclude that PGHS-2 mRNA is present in human amnion; its levels are elevated after SL onset, and they are correlated with enzyme activity. The stimulation of PGHS activity at labor onset probably involves increased expression of PGHS-2. The expression of PGHS-1 does not change in association with labor in human amnion.

High-frequency toneburst-evoked ABR latency-intensity functions in sensorineural hearing-impaired humans.

The latency-intensity functions (LIFs) of ABRs elicited by high-frequency (8, 10, 12, and 14 kHz) toneburst stimuli were evaluated in 20 subjects with confirmed 'moderate' high-frequency sensorineural hearing loss. Wave V results from clicks and tonebursts revealed all intra- and intersession data to be reliable (p > 0.05). Linear regression curves were highly significant (p < or = 0.0001), indicating linear relationships for all stimuli analyzed. Comparisons between the linear regression curves from a previously reported normal-hearing subject group and this sensorineural hearing-impaired group showed no significant differences. This study demonstrated that tonebursts at 8, 10, and 12 kHz evoked ABRs which decreased in latency as a function of increasing intensity and that these LIFs were consistent and orderly (14 kHz was not determinable). These results will contribute information to facilitate the establishment of change criteria used to predict change in hearing during treatment with ototoxic medications.

High-frequency tone burst-evoked ABR latency-intensity functions.

High-frequency tone burst stimuli (8, 10, 12, and 14 kHz) have been developed and demonstrated to provide reliable and valid auditory brainstem responses (ABRs) in normal-hearing subjects. In this study, latency-intensity functions (LIFs) were determined using these stimuli in 14 normal-hearing individuals. Significant shifts in response latency occurred as a function of stimulus intensity for all tone burst frequencies. For each 10 dB shift in intensity, latency shifts for waves I and V were statistically significant except for one isolated instance. LIF slopes were comparable between frequencies, ranging from 0.020 to 0.030 msec/dB. These normal LIFs for high-frequency tone burst-evoked ABRs suggest the degree of response latency change that might be expected from, for example, progressive hearing loss due to ototoxic insult, although these phenomena may not be directly related.

Early detection of ototoxicity using high-frequency, tone-burst-evoked auditory brainstem responses.

Subjects receiving treatment with ototoxic agents were evaluated concurrently with conventional and high-frequency (> or = 8 kHz) behavioral threshold measures and with ABR to click and to 8, 10, 12, and 14 kHz tone-burst stimuli. Behavioral threshold data revealed ototoxic change in 51 percent of ears evaluated. Of these ears demonstrating behavioral change, 90 percent revealed concurrent ABR changes. If only ABR monitoring with high-frequency tone-burst stimuli had been used, 87 percent of allears showing behavioral change would have been identified. Three fourths of these would have been identified from wave V responses, with 87 percent identified from the two highest frequencies tested for each individual. This research suggests that behavioral change is reflected accurately in the ABR, that high-frequency tone bursts will identify a majority of initial ototoxic changes, and that monitoring hearing with high-frequency, tone-burst-evoked ABRs during treatment with potentially ototoxic agents is significantly more effective than click-evoked ABRs for early detection of ototoxicity.

Comparing laboratory and portable tone-burst auditory brain-stem-response (ABR) systems for monitoring high-frequency (> or = 8 kHz) auditory function.

High-frequency (8-20 kHz) hearing sensitivity is of special interest because of its early warning potential for ototoxicity. Many ill patients, however, are unable to respond behaviorally to auditory test procedures. To objectively monitor high-frequency auditory function in these patients, laboratory instrumentation to evoke the auditory brain-stem response (ABR) with high-frequency (8-14 kHz) tone-burst stimuli was developed and documented. To provide evaluation at bedside, a portable high-frequency tone-burst generator was developed to elicit the ABR. Combined with a portable signal averager, this system was validated by comparison with the laboratory system. Thirty-five normal-hearing subjects were used to compare ABRs to high-frequency tone bursts from each system. Analysis of responses to tone bursts revealed no significant mean latency differences, and no significant intersession reliability differences between systems. These results confirm that the portable system is comparable to the laboratory system in obtaining reliable high-frequency tone-burst responses.

Reliability of evoked responses to high-frequency (8-14 kHz) tone bursts.

Instrumentation to evaluate the auditory brainstem response to high-frequency (8-14 kHz) tone bursts has been developed in the Auditory Research Laboratory, Portland, Oregon VA Medical Center. This system is intended to monitor the audition of patients receiving ototoxic drugs who are unresponsive to behavioral test procedures. The reliability of responses obtained with the high-frequency tone-burst system was studied in 30 normal ears. Intrasubject variability of intersession data from response waves I, III, and V to tone bursts of frequencies 8, 10, 12, and 14 kHz was not significantly different from click response variability. The results of this study demonstrate the reliability of the ABR to these high-frequency tone-burst stimuli. This technique may provide early identification of hearing loss in unresponsive subjects receiving treatment with potentially ototoxic agents, thus allowing alternative treatments to minimize or prevent communicative handicap.

A microcomputer-based system for processing 31-phosphorus nuclear magnetic resonance spectra from studies of cardiac metabolism in immature hearts.

We designed an interactive microcomputer-based digital data processing system for analysis of 31-phosphorus nuclear magnetic resonance (31P-NMR) spectra from studies of cardiac metabolism in immature and neonatal hearts. This system included a digitizing tablet (Kurta Series Two), a microcomputer (IBM PC XT) and a graphics plotter (Hewlett-Packard 7470A) used in conjunction with a Nicolet 1280 NMR signal processing computer. We obtained 31P spectra from isolated perfused rabbit hearts with a Nicolet NT-200 4.7 Tesla superconducting NMR spectrometer operated in the pulsed Fourier transform mode. The small size of the hearts resulted in increased noise in spectra and demanded comparison of methods used to quantitate changes in inorganic phosphorus, phosphocreatine and ATP during ischemic stress. We performed microcomputer operations and interfacing functions with a software package written in BASIC. This system simplified documentation, data filing and statistical data processing. Our microcomputer system displayed and made hard copies of digitized spectra and results of analyses. Errors in data entry were rectified directly with this program. Consistent data reduction improved the precision of the physiological results and reduced the influence of noise on 31P spectra from neonatal hearts weighing about 0.5 g. The system flexibility extends its application to NMR spectra analysis for other in vivo organ systems, and signal processing in other biological research.

Antibody responses in protein-energy restricted beef cows and their cold stressed progeny.

Antibody titers were measured in serum and colostral whey of pregnant beef cows immunized with tetanus toxoid and chicken red blood cells while being fed diets either restricted or nonrestricted in protein and/or metabolizable energy during the last 150 days of gestation. Serum antibody titers were also measured in the colostrum-fed, cold and noncold stressed progeny that were actively immunized with dinitrophenol conjugated to keyhole limpet hemocyanin. In general, there were no major or sustained differences in humoral immune responses to injection of tetanus toxoid or chicken red blood cells between cows fed diets that were adequate or restricted in protein or metabolizable energy. In the few cases where serum antibody titers to tetanus toxoid or chicken red blood cells differed (P less than 0.05) between adequately fed or restricted cows, the differences were no greater than twofold. Anti-chicken red blood cell titers were uniformly low (P less than 0.05) by a magnitude of two to threefold in colostral whey of cows restricted in protein and/or metabolizable energy when compared to titers in cows fed adequate amounts of protein and metabolizable energy. With one exception, neither maternal dietary restriction nor cold exposure had a major effect on the ability of the calves to absorb antitetanus toxoid and chicken red blood cell antibodies from colostrum. The humoral immune responses of all calves to injection of keyhole limpet hemocyanin and dinitrophenol were similar in magnitude.

Effects of prepartum protein restriction in the beef cow on immunoglobin content in blood and colostral whey and subsequent immunoglobin absorption by the neonatal calf.

Protein intake of first-calf beef heifers was restricted during the last 100 days of gestation, and the effects on passive transfer of colostral immunoglobins from the cow to the neonatal calf were examined. There were no significant correlations between concentration of immunoglobins (IgM, IgG1 and IgG2) in the sera or colostrum of the cow and prenatal crude protein consumption (.52 to .98 kg crude protein/day). Absorption of certain colostral immunoglobins (IgG1, and IgG2) by the calf were positively correlated (P less than .01) at 12, 18, 24 and 36 hr after birth to the maternal crude protein consumption. Colostrum was collected from the first milkings of pluriparous dairy cows, and then freeze-dried, mixed and reconstituted to be equivalent to 1 liter of colostrum. Mean IgG1 concentrations for the high and low protein groups were 6.02 +/- .90 and .78 +/- .15 mg . ml-1 (P less than .01), respectively. No relationship (P greater than .05) was found between the concentration of IgM in calf sera and daily crude protein intake of the dam. These data indicate that there was a selective decrease in absorption of IgG1 and IgG2 in calves from heifers fed low protein prenatal diets.

Effects of maternal nutritional restriction and cold stress on young calves: absorption of colostral immunoglobulins.

Fifty-seven newborn calves delivered from heifers fed rations either adequate or restricted in protein or metabolizable energy were housed in cold (1 C) or normothermic (21 C) environmental chambers for 3 days to determine the effects of maternal nutritional stress and cold exposure on ability of the animals to absorb colostral immunoglobulins (Ig). In general, the serum Ig concentrations in the newborn calves from dams fed rations restricted in protein or metabolizable energy and the concentrations in sera of the respective calves from dams fed adequate protein or metabolizable energy were similar throughout the 3-day period of observation. Likewise, the serum Ig concentrations in the cold-exposed calves were similar to those in the calves kept at normothermic temperature. An exponential increase in mean serum concentrations of IgM, IgG1, and IgG2 occurred between 3 and 6 hours of age; the values continued to increase, but at a slower rate after 6 hours and reached a peak by 12 hours of age. Then, IgM and IgG2 decreased until 48 hours, after which time they increased, but IgG1 varied only slightly from the peak at 12 hours and then increased. With one exception, data indicated that neither the maternal dietary restrictions or the cold exposure imposed on the calves caused significant (P greater than 0.05) differences in absorption of colostral Ig when compared with that of the respective calves from dams fed adequate diet or the calves kept at normothermic temperature.

Total complement hemolytic activity of colostral whey and sera from dairy cows.

Two methods were used to clarify bovine colostrum for quantitation of total complement (C') hemolytic activity in the whey. The renin precipitation method produced whey which tended to have higher C' hemolytic activity (5.0 +/- 3.4 CH50 units) than did whey prepared by ultracentrifugation (2.5 +/- 2.6 CH50 units) although this difference was not significant. No correlation was found between levels of complement in the sera of cows at parturition and in the colostral whey from the same animal. Total complement activity was not detected in milk taken a 96 hours or one month after parturition. This is apparently the first report of quantitation of total C' hemolytic activity in bovine colostrum.

Immunoglobulin levels in serum and colostral whey or protein-metabolisable energy restricted beef cows.

Aberdeen Angus cows were fed adequate diets or diets restricted in protein and, or metabolisable energy for the last 156 days of gestation to determine effects of nutritional restriction on concentrations of immunoglobulins in serum and colostral whey. There were no significant interactions between the effects of low protein and metabolisable energy on immunoglobulin concentrations. Thus, observed differences in immunoglobulin concentrations between the restricted and adequate dietary groups were attributed to the main effects of treatment. Low protein or metabolisable energy had little overall effect on serum IgM concentrations although levels began to decrease sooner in gestation in restricted animals than in those fed adequate diets. Concentrations of IgG1 in serum of all animals were similar and a precipitous decrease in concentration was noted at about 240 days of gestation and this decrease continued until parturition. Serum IgG2 concentrations increased in all animals as parturition approached. Immunoglobulin concentrations in colostral whey were either similar to or tended to be slightly higher in dietary restricted animals than in animals fed adequate diets although the differences were not significant.