Avasopasem manganese (GC4419) protects against cisplatin-induced chronic kidney disease: An exploratory analysis of renal metrics from a randomized phase 2b clinical trial in head and neck cancer patients.

Head and neck squamous cell carcinoma (HNSCC) patients treated with high-dose cisplatin concurrently with radiotherapy (hdCis-RT) commonly suffer kidney injury leading to acute and chronic kidney disease (AKD and CKD, respectively). We conducted a retrospective analysis of renal function and kidney injury-related plasma biomarkers in a subset of HNSCC subjects receiving hdCis-RT in a double-blinded, placebo-controlled clinical trial (NCT02508389) evaluating the superoxide dismutase mimetic, avasopasem manganese (AVA), an investigational new drug. We found that 90 mg AVA treatment prevented a significant reduction in estimated glomerular filtration rate (eGFR) three months as well as six and twelve months after treatment compared to 30 mg AVA and placebo. Moreover, AVA treatment may have allowed renal repair in the first 22 days following cisplatin treatment as evidenced by an increase in epithelial growth factor (EGF), known to aid in renal recovery. An upward trend was also observed in plasma iron homeostasis proteins including total iron (Fe-blood) and iron saturation (Fe-saturation) in the 90 mg AVA group versus placebo. These data support the hypothesis that treatment with 90 mg AVA mitigates cisplatin-induced CKD by inhibiting hdCis-induced renal changes and promoting renal recovery.

Anticariogenic Activity of Three Essential Oils from Brazilian Piperaceae.

The current trend toward using natural food additives, cosmetics, and medicines has motivated industries to substitute synthetic compounds for natural products. Essential oils (EOs) from medicinal plants are a well-known source of chemical compounds that display several interesting biological activities, including antimicrobial action. In this study, we investigated the antibacterial activity of EOs extracted from three Piperaceae species collected in the Brazilian Amazon region against a representative panel of cariogenic bacteria. The minimum inhibitory concentration (MIC) of the essential oils extracted from Peperomia pellucida (PP-EO), Piper marginatum (PM-EO), and Piper callosum (PC-EO) was determined against Streptococcus mutans, S. mitis, S. sanguinis, S. salivarius, S. sobrinus, Enterococcus faecalis, and Lactobacillus casei by using the microplate microdilution method. PM-EO, PC-EO, and PP-EO displayed antibacterial activity against all the tested cariogenic bacteria. PM-EO displayed the best inhibitory activity, with MIC values ranging from 50 to 500 µg/mL. The lowest MIC values were obtained for PM-EO against S. mitis (MIC = 75 µg/mL), Lactobacillus casei (MIC = 50 µg/mL), and S. mutans (MIC = 50 µg/mL). Gas chromatography mass spectrometry (GC-MS) analysis allowed the chemical composition of all the EOs to be identified. The main constituents of PM-EO, PC-EO, and PP-EO were 3,4-(methylenedioxy)propiophenone, α-pinene, and dillapiole, respectively. Finally, the compounds that were exclusively detected in PM-EO are highlighted. Our results suggest that PM-EO may be used in products for treating dental caries and periodontal diseases.

Chemical composition and insecticidal activity of the essential oils of Piper marginatum , Piper callosum and Vitex agnus-castus.

During grain storage, a considerable amount of product is lost because of insects, such as Zabrotes subfasciatus. Currently, to mitigate these risks, studies are searching for plants with potential for the control of agricultural pests, also known as botanical insecticides. In this study, the fumigant toxicity of the essential oils of Piper callosum (PC-EO), Piper marginatum (PM-EO) and Vitex agnus-castus (VA-EO) against Zabrotes subfasciatus was investigated. The essential oils of PC-EO, PM-EO and VA-EO were analysed by gas chromatography (GC-MS), and the major components were 3,4-methylenedioxypropiophenone (10.4%), bicyclogermacrene (10.1%) and germacrene D (9.9%) for PM-EO; safrol (29.3%) for PC-EO; and 1,8-cineol (23.8%) for VA-EO. In fumigation tests, VA-EO killed 100% Zabrotes subfasciatus at a concentration of 0.004 µL/L air after 24 h of treatment, whereas PC-EO and PM-EO at 0.01 µL/L air caused 100% Z. subfasciatus mortality after 48 h. The VA-EO sample provided the lowest LD50 after 24 h (0.17 µL/L air), followed by PC-EO (0.78 µL/L air) and PM-EO (1.17 µL/L air). These results demonstrate that the essential oils of these species can be an alternative to control pests in stored products. This is the first report of the fumigant potential of these species against Z. subfasciatus.

The effects of 2-hydroxy-4-methylthio-butanoic acid supplementation on the rumen microbial population and duodenal flow of microbial nitrogen.

Four multiparous, lactating Holstein cows (average DIM 169.5 ± 20.5 d), fitted with ruminal and duodenal cannulas, were used in a 4 × 4 Latin square with a 2 × 2 factorial arrangement of treatments to investigate the effects of 2-hydroxy-4-methylthio-butanoic acid (HMTBA) when fed with diets differing in metabolizable protein (MP) supply and equal levels of crude protein on milk production and composition, rumen microbial activity, duodenal protein flow, and rumen bacterial community composition in vivo and in vitro. Experimental periods were 28 d in length. Cows were housed in individual tie stalls and were randomly assigned to 4 dietary treatments: low MP or high MP, supplemented with or without 25 g of HMTBA, which was top-dressed once daily at 0930 h. No interactions were observed between HMTBA and level of dietary MP, with the exception of ruminal acetate-to-propionate ratio. Milk yield was not affected by treatment and averaged 23.8 ± 2.06 kg/d. There was a tendency for increased milk protein percent in cows receiving low MP diets, averaging 3.30 ± 0.09% and 3.21 ± 0.09% for low MP and high MP, respectively. The total-tract apparent digestibility of organic matter, neutral detergent fiber, and nitrogen were greater in cows consuming the low MP diet. Rumen pH was lower in cows consuming high MP diets as well as in those consuming HMTBA. Rumen ammonia concentrations tended to be greater in cows consuming HMTBA, and volatile fatty acid concentrations were greater in cows consuming HMTBA. Duodenal dry matter flow, nitrogen flow, and microbial nitrogen flow did not differ between treatments. The bacterial community structure of cows receiving HMTBA was not affected at the phylum level. The relative abundance of bacterial phyla in vivo differed when compared with in vitro conditions for Firmicutes, Bacteroidetes, Proteobacteria, TM7, Tenericutes, Spirochaetes, SR1, and Verrucomicrobia.

Chemical Composition and Antibacterial Activity of the Essential Oil of Vitex agnus-castus L. (Lamiaceae).

Abnormal multiplication of oral bacteria causes dental caries and dental plaque. These diseases continue to be major public health concerns worldwide, mainly in developing countries. In this study, the chemical composition and antimicrobial activity of the essential oil of Vitex agnus-castus leaves (VAC‒EO) collected in the North of Brazil against a representative panel of cariogenic bacteria were investigated. The antimicrobial activity of VAC-EO was evaluated in terms of its minimum inhibitory concentration (MIC) values by using the broth microdilution method in 96-well microplates. The chemical constituents of VAC-EO were identified by gas chromatography (GC‒FID) and gas chromatography‒mass spectrometry (GC‒MS). VAC‒EO displayed some activity against all the investigated oral pathogens; MIC values ranged from 15.6 to 200 µg/mL. VAC-EO had promising activity against Streptococcus mutans (MIC= 15.6 µg/mL), Lactobacillus casei (MIC= 15.6 µg/mL), and Streptococcus mitis (MIC= 31.2 µg/mL). The compounds 1,8-cineole (23.8%), (E)-ß-farnesene (14.6%), (E)-caryophyllene (12.5%), sabinene (11.4%), and α-terpinyl acetate (7.7%) were the major chemical constituents of VAC‒EO. VAC-EO displays antimicrobial activity against cariogenic bacteria. The efficacy of VAC-EO against S. mutans is noteworthy and should be further investigated.

Chemical Composition and Antibacterial Activity of the Essential Oil of Vitex agnus-castus L. (Lamiaceae)

ABSTRACT Abnormal multiplication of oral bacteria causes dental caries and dental plaque. These diseases continue to be major public health concerns worldwide, mainly in developing countries. In this study, the chemical composition and antimicrobial activity of the essential oil of Vitex agnus-castus leaves (VAC‒EO) collected in the North of Brazil against a representative panel of cariogenic bacteria were investigated. The antimicrobial activity of VAC-EO was evaluated in terms of its minimum inhibitory concentration (MIC) values by using the broth microdilution method in 96-well microplates. The chemical constituents of VAC-EO were identified by gas chromatography (GC‒FID) and gas chromatography‒mass spectrometry (GC‒MS). VAC‒EO displayed some activity against all the investigated oral pathogens; MIC values ranged from 15.6 to 200 μg/mL. VAC-EO had promising activity against Streptococcus mutans (MIC= 15.6 μg/mL), Lactobacillus casei (MIC= 15.6 μg/mL), and Streptococcus mitis (MIC= 31.2 μg/mL). The compounds 1,8-cineole (23.8%), (E)-β-farnesene (14.6%), (E)-caryophyllene (12.5%), sabinene (11.4%), and α-terpinyl acetate (7.7%) were the major chemical constituents of VAC‒EO. VAC-EO displays antimicrobial activity against cariogenic bacteria. The efficacy of VAC-EO against S. mutans is noteworthy and should be further investigated.

Effect of phosphorus on arsenic uptake and metabolism in rice cultivars differing in phosphorus use efficiency and response.

A hydroponic experiment was carried out to investigate the effect of phosphorus (P) nutrition on arsenic (As) uptake and translocation within the seedlings of rice cultivars. The experiment occurred in three stages: I 5 days of acclimatization (nutritive solution); II 10 days under P (0.0 and 0.09 mM) and As (0.0 and 100 mM) treatments; III 5 days under recovery. The As exposure had significant effect reducing dry weights of shoots or roots, resulted in elevated concentrations of As in shoot tissues. BR-IRGA 409 showed the highest susceptibility to As in biomass production and root system parameters regardless the P level. This cultivar showed contrasting responses of As translocation to shoot tissue dependent on P levels, with the highest As concentration under low P and lowest under normal P levels. P nutrition was most striking on plants recovery for all cultivars under As exposure. Clearer separation of cultivars for phosphorus use efficiency (PUE) occurred at lower shoot P contents, that was, at higher levels of P deficiency stress. IRGA 424 showed higher PUE as compared to the others cultivars. Our results go some way to understanding the role of P nutrition in controlling the effects of As in rice shoots.

Effect of phosphorus on arsenic uptake and metabolism in rice cultivars differing in phosphorus use efficiency and response

ABSTRACT A hydroponic experiment was carried out to investigate the effect of phosphorus (P) nutrition on arsenic (As) uptake and translocation within the seedlings of rice cultivars. The experiment occurred in three stages: I 5 days of acclimatization (nutritive solution); II 10 days under P (0.0 and 0.09 mM) and As (0.0 and 100 mM) treatments; III 5 days under recovery. The As exposure had significant effect reducing dry weights of shoots or roots, resulted in elevated concentrations of As in shoot tissues. BR-IRGA 409 showed the highest susceptibility to As in biomass production and root system parameters regardless the P level. This cultivar showed contrasting responses of As translocation to shoot tissue dependent on P levels, with the highest As concentration under low P and lowest under normal P levels. P nutrition was most striking on plants recovery for all cultivars under As exposure. Clearer separation of cultivars for phosphorus use efficiency (PUE) occurred at lower shoot P contents, that was, at higher levels of P deficiency stress. IRGA 424 showed higher PUE as compared to the others cultivars. Our results go some way to understanding the role of P nutrition in controlling the effects of As in rice shoots.

Swimming exercise and diphenyl diselenide-supplemented diet affect the serum levels of pro- and anti-inflammatory cytokines differently depending on the age of rats.

The increase in the inflammatory process is one of the main factors that contribute to aging. The aim of this study was to investigate the effects of a diphenyl diselenide (PhSe)2-supplemented diet (1p.p.m., 4weeks) and swimming exercise (3% of body weight, 20min per day, 4weeks) on the serum levels of cytokines in Wistar rats of different ages. The results demonstrated an increase in the levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNFα and INFγ) and a decrease in the levels of IL-10, an anti-inflammatory cytokine, with age. In middle-age rats, the swimming exercise and (PhSe)2-supplemented diet decreased serum levels of pro-inflammatory cytokines and increased the levels of IL-10. By contrast, in old rats the swimming exercise protocol increased the serum levels of pro-inflammatory cytokines and decreased the levels IL-10. Diet supplemented with (PhSe)2 did not alter the serum levels of cytokines in old rats. Middle-age and old rats subjected to swimming exercise and supplemented with (PhSe)2 in the diet had a decrease in the serum levels of pro-inflammatory cytokines and an increase in the levels of IL-10. This study demonstrated that swimming exercise and (PhSe)2-supplemented diet affect the serum levels of pro- and anti-inflammatory cytokines differently depending on the age of rats. (PhSe)2 supplemented in the diet had an anti-inflammatory effect, similar to that of induced by swimming exercise, in middle-age rats and reversed the pro-inflammatory effects of swimming exercise in old rats.

Melatonin improves insulin sensitivity independently of weight loss in old obese rats.

In aged rats, insulin signaling pathway (ISP) is impaired in tissues that play a pivotal role in glucose homeostasis, such as liver, skeletal muscle, and adipose tissue. Moreover, the aging process is also associated with obesity and reduction in melatonin synthesis from the pineal gland and other organs. The aim of the present work was to evaluate, in male old obese Wistar rats, the effect of melatonin supplementation in the ISP, analyzing the total protein amount and the phosphorylated status (immunoprecipitation and immunoblotting) of the insulin cascade components in the rat hypothalamus, liver, skeletal muscle, and periepididymal adipose tissue. Melatonin was administered in the drinking water for 8- and 12 wk during the night period. Food and water intake and fasting blood glucose remained unchanged. The insulin sensitivity presented a 2.1-fold increase both after 8- and 12 wk of melatonin supplementation. Animals supplemented with melatonin for 12 wk also presented a reduction in body mass. The acute insulin-induced phosphorylation of the analyzed ISP proteins increased 1.3- and 2.3-fold after 8- and 12 wk of melatonin supplementation. The total protein content of the insulin receptor (IR) and the IR substrates (IRS-1, 2) remained unchanged in all investigated tissues, except for the 2-fold increase in the total amount of IRS-1 in the periepididymal adipose tissue. Therefore, the known age-related melatonin synthesis reduction may also be involved in the development of insulin resistance and the adequate supplementation could be an important alternative for the prevention of insulin signaling impairment in aged organisms.

Flavonoids bearing an O-arabinofuranosyl-(1-->3)-rhamnoside moiety from Cladocolea micrantha: inhibitory effect on human melanoma cells.

Eleven known triterpenes (alpha-amyrin, beta-amyrin, lupeol, and their respective acetates, 3-O-acetyl derivatives of betulinic, oleanolic, and ursolic acids, cycloartenol, and tirucall-7,24-dienol), two new flavonols presenting an uncommon interglycosidic O-(1-->3) linkage (kaempferol 3-O-alpha-L-arabinofuranosyl(1-->3)-alpha-L-rhamnoside and quercetin 3-O-alpha-L-arabinofuranosyl-(1-->3)-alpha-L-rhamnoside), beta-sitosterol, stigmasterol, quercetin, and gallic acid were isolated from the Amazonian medicinal mistletoe, Cladocolea micrantha Kuijt (Loranthaceae). Their structures were established by spectral methods and eventual chromatographic comparisons. The quercetin derivative was not cytotoxic to MV3 human melanoma cells, but was able, when administered at 1 microg/mL, to promote a twofold inhibition of the migration of the cells through the transwell system when compared with paclitaxel at 5 microM.

Potassium intake and risk of incident type 2 diabetes mellitus: the Coronary Artery Risk Development in Young Adults (CARDIA) Study.

AIMS/HYPOTHESIS: Serum potassium has been found to be a significant predictor of diabetes risk, but the effect of dietary potassium on diabetes risk is not clear. We sought to determine if dietary potassium is associated with risk of incident type 2 diabetes in young adults. METHODS: We used data from the Coronary Artery Risk Development in Young Adults (CARDIA) Study. Potassium intake was measured by (1) an average of three 24 h urinary potassium collections at the 5-year study visit, and (2) the CARDIA dietary assessment instrument at baseline. Incident type 2 diabetes cases were ascertained on the basis of use of diabetes medication and laboratory measurements. Analyses were adjusted for relevant confounders including intake of fruit and vegetables and other dietary factors. RESULTS: Of 1,066 participants with urinary potassium measurements, 99 (9.3%) developed diabetes over 15 years of follow-up. In multivariate models, adults in the lowest urinary potassium quintile were more than twice as likely to develop diabetes as their counterparts in the highest quintile (HR 2.45; 95% CI 1.08, 5.59). Of 4,754 participants with dietary history measurements, 373 (7.8%) developed diabetes over 20 years of follow-up. In multivariate models, African-Americans had a significantly increased risk of diabetes with lower potassium intake, which was not found in whites. CONCLUSIONS/INTERPRETATION: Low dietary potassium is associated with increased risk of incident diabetes in African-Americans. Randomised clinical trials are needed to determine if potassium supplementation, from either dietary or pharmacological sources, could reduce the risk of diabetes, particularly in higher-risk populations.

Pathological laughing and crying : epidemiology, pathophysiology and treatment.

Pathological laughing and crying (PLC) is characterized by frequent, brief, intense paroxysms of uncontrollable crying and/or laughing due to a neurological disorder. When sufficiently frequent and severe, PLC may interfere with the performance of activities of daily living, interpersonal functioning, or both, and is a source of distress for affected patients and their families. PLC is also often misunderstood by patients and their families, and is under-recognized by the clinicians caring for patients with this disorder. However, this syndrome is easily recognized when understood properly and is highly responsive to treatment with a variety of pharmacological agents. This review aims to facilitate the diagnosis and treatment of patients with PLC, and begins by providing definitions of mood and affect that will help clinicians distinguish between mood disorders, such as major depression and mania, and disorders of affect, such as PLC. In addition, the various terms used to describe this syndrome are reviewed and a recommendation for the use of the term PLC is made. The core clinical features of PLC are also presented and the epidemiology of this syndrome is reviewed. A discussion of the pathophysiology of PLC, including the neuroanatomic and neurochemical bases, is provided. Finally, the evaluation and treatment of patients with PLC is described. Based on the pathophysiology of PLC and on a detailed review of published treatment studies, SSRIs are recommended as first-line pharmacotherapy for this disorder. When SSRIs are ineffective or poorly tolerated, other treatment options, including TCAs, noradrenergic reuptake inhibitors, novel antidepressants, dopaminergic agents and uncompetitive NMDA receptor antagonists may be useful second-line treatments.

Treatment of pathologic laughing and crying.

Pathologic laughing and crying (PLC) denotes paroxysms of involuntary and uncontrollable crying and/or laughing resulting from neurologic illnesses. These paroxysms of affect are often provoked by nonsentimental stimuli; even when the inciting stimulus is sentimentally meaningful, the intensity of the affective response is excessive. The crying and/or laughing of PLC are variably accompanied by episode-congruent subjective emotional feelings. In unusual cases, episode-related feelings are of a valence contradictory to the expressed affect (ie, feeling happy while crying, or vice versa). PLC does not bear a predictable relationship to the prevailing mood of the patient, and the occurrence of such episodes does not produce a sustained mood disturbance. Therefore, patients with PLC must not be misunderstood as "depressed" or "manic" solely on the basis of their frequent episodic crying or laughing. In rare circumstances, PLC or PLC-like symptoms may be the presenting symptom of a neurologic illness. In such circumstances, a prompt and thorough diagnostic evaluation for that neurologic illness should be undertaken before initiating treatment for PLC. Selective serotonin reuptake inhibitors (SSRIs) are efficacious, safe, and well-tolerated treatments for PLC and are recommended as first-line treatments for this condition. Tricyclic antidepressants, dextromethorphan/quinidine, or dopaminergic agents may be useful alternative treatments in patients in whom SSRIs are ineffective or poorly tolerated. Education and supportive therapy may help patients and families mitigate the social isolation and embarrassment that PLC episodes frequently produce.

Energy intakes of children with Crohn's disease treated with enteral nutrition as primary therapy.

BACKGROUND: Enteral nutrition (EN) is widely used and is effective in the treatment of children with Crohn's disease given as an exclusive feed for 6-8 weeks. Current dietetic practice during EN is to recommend an energy intake based on estimated average requirement (EAR) for energy for age. AIMS: To examine factors affecting energy intake and weight gain during EN in relation to disease site and nutritional status. METHODS: A retrospective cohort study examining energy intake and weight gain during the exclusive EN feeding period in 40 patients newly diagnosed with Crohn's in relation to EAR, nutritional status, disease site. RESULTS: All patients improved clinically and gained weight during EN with improvement in the CRP as a marker of the systemic inflammatory response. Energy intake was higher than EAR in 82% (33/40 patients), with the median 117.5% of EAR. Weight gain correlated with body mass index standard deviation score (P = 0.001) at start of treatment, but not energy intake or CRP. CONCLUSION: Estimated average requirement underestimates energy intakes in most children with newly diagnosed Crohn's disease. During EN, an energy intake in the range of 100-149% (median 117.5%) EAR for energy for age may be required. Energy balance studies in children with active disease are required.

Pharmaceutical interventions for emotionalism after stroke.

BACKGROUND: Antidepressants may be useful in the treatment of abnormal crying associated with stroke. OBJECTIVES: To determine whether pharmaceutical treatment reduces the frequency of emotional displays in people who suffer from emotionalism after stroke. SEARCH STRATEGY: We searched the Cochrane Stroke Group Trials Register (last searched June 2003). In addition we searched the following electronic databases: Cochrane Central Register of Controlled Trials (The Cochrane Library, Issue 3 2002), MEDLINE (1966 to September 2002), EMBASE (1980 to September 2002), CINAHL (1982 to September 2002), PsychINFO (1967 to September 2002), Applied Science and Technology Plus (1986 to September 2002), Arts and Humanities Index (1991 to September 2002), Biological Abstracts (1969 to September 2002), General Science Plus (1994 to September 2002), Science Citation Index (1992 to September 2002), Social Sciences Citation Index (1991 to September 2002), and Sociofile (1974 to September 2002). We searched reference lists from relevant articles and textbooks, and contacted authors of known studies and pharmaceutical companies who manufacture psychotropic medications. SELECTION CRITERIA: Randomised and quasi-randomised controlled trials, comparing psychotropic medication to placebo, in people with stroke and emotionalism (also known as emotional lability or pathological crying and laughing). DATA COLLECTION AND ANALYSIS: Data were obtained on people who no longer met criteria for emotionalism, as defined in studies, and on reduction in frequency of crying at the end of treatment. Data were not pooled because of the multiplicity of definitions and outcome measures. MAIN RESULTS: Five trials involving 103 participants were included. Four trials showed large effects of treatment: 50% reduction in emotionalism, improvements (reduction) in the frequency of compulsive laughter, and lower (better) scores on the Pathological Laughter and Crying scale. The confidence intervals were wide, however, indicating that treatment may have had only a small positive effect, or even a small negative effect (in one trial). Subgroup analysis was not performed due to the multiple methods of assessment of emotionalism within and between trials. Only one study systematically recorded and reported adverse events; no discernible difference was seen between groups. Participants allocated active treatment were more likely to leave early from trials. REVIEWERS' CONCLUSIONS: Antidepressants can reduce the frequency and severity of crying or laughing episodes. The effect do not seem specific to one drug or class of drugs. However, our conclusions must be qualified by several methodological deficiencies in the studies. More reliable data are required before recommendations can be made about the treatment of post-stroke emotionalism.

Early embryonic death of glutamate carboxypeptidase II (NAALADase) homozygous mutants.

Glutamate carboxypeptidase II (EC 3.4.17.21) catalyzes the hydrolysis (Km = 0.2 microM) of the neuropeptide N-acetylaspartylglutamate to yield N-acetylaspartate and glutamate and also serves as a high-affinity folate hydrolase in the gut, cleaving the polyglutamate chain to permit the absorption of folate. N-acetylaspartylglutamate is an agonist at the mGluR3 metabotropic receptor and a source of extracellular glutamate through hydrolysis by glutamate carboxypeptidase II. Given the important role of glutamate in brain development and function, we were interested in the effects of a null mutation of glutamate carboxypeptidase II that would potentiate the effects of N-acetylaspartylglutamate. The PGK-Neomycin cassette was inserted to delete exons 9 and 10, which we previously demonstrated encode for the zinc ligand domain essential for enzyme activity. Successful germline transmission was obtained from chimeras derived from embryonic stem cells with the targeted mutation of glutamate carboxypeptidase II. Homozygous null mutants did not survive beyond embryonic day 8. Folate supplementation of the heterozygous mothers did not rescue the homozygous embryos. Mice heterozygous for the null mutation appeared grossly normal and expressed both mutated and wild-type mRNA but the activity of glutamate carboxypeptidase II is comparable to the wild-type mice. The results indicate that the expression of glutamate carboxypeptidase II is upregulated when one allele is inactivated and that its activity is essential for early embryogenesis.

The neurochemical characterisation of hypothalamic pathways projecting polysynaptically to brown adipose tissue in the rat.

The identification of leptin and a range of novel anorectic and orexigenic peptides has focussed attention on the neural circuitry involved in the genesis of food intake and the reflex control of thermogenesis. Here, the neurotropic virus pseudorabies has been utilised in conjunction with the immunocytochemical localisation of a variety of neuroactive peptides and receptors to better define the pathways in the rat hypothalamus directed polysynaptically to the major thermogenic endpoint, brown adipose tissue. Infected neurones were detected initially in the stellate ganglion, then in the spinal cord followed by the appearance of third-order premotor neurones in the brainstem and hypothalamus. Within the hypothalamus these were present in the paraventricular nucleus, lateral hypothalamus, perifornical region, and retrochiasmatic nucleus. At slightly longer survival times virus-infected neurones appeared in the arcuate nucleus and dorsomedial hypothalamus. Neurones in the retrochiasmatic nucleus and in the adjacent lateral arcuate nucleus which project to the brown adipose tissue express cocaine- and amphetamine-regulated transcript, pro-opiomelanocortin and leptin receptors. Neurones in the lateral hypothalamus, a site traditionally associated with the promotion of feeding, project to brown adipose tissue and large numbers of these contained melanin-concentrating hormone and orexin A and B. These data provide part of an anatomical framework which subserves the regulation of energy expenditure.

WAKs: cell wall-associated kinases linking the cytoplasm to the extracellular matrix.

There are only a few proteins identified at the cell surface that could directly regulate plant cell wall functions. The cell wall-associated kinases (WAKs) of angiosperms physically link the plasma membrane to the carbohydrate matrix and are unique in that they have the potential to directly signal cellular events through their cytoplasmic kinase domain. In Arabidopsis there are five WAKs and each has a cytoplasmic serine/threonine protein kinase domain, spans the plasma membrane, and extends a domain into the cell wall. The WAK extracellular domain is variable among the five isoforms, and collectively the family is expressed in most vegetative tissues. WAK1 and WAK2 are the most ubiquitously and abundantly expressed of the five tandemly arrayed genes, and their messages are present in vegetative meristems, junctions of organ types, and areas of cell expansion. They are also induced by pathogen infection and wounding. Recent experiments demonstrate that antisense WAK expression leads to a reduction in WAK protein levels and the loss of cell expansion. A large amount of WAK is covalently linked to pectin, and most WAK that is bound to pectin is also phosphorylated. In addition, one WAK isoform binds to a secreted glycine-rich protein (GRP). The data support a model where WAK is bound to GRP as a phosphorylated kinase, and also binds to pectin. How WAKs are involved in signaling from the pectin extracellular matrix in coordination with GRPs will be key to our understanding of the cell wall's role in cell growth.