Impaired stress-coping and fear extinction and abnormal corticolimbic morphology in serotonin transporter knock-out mice.

A lesser-expressing form of the human 5-HT transporter (5-HTT) gene has been associated with increased fear and anxiety and vulnerability to the effects of stress. These phenotypic abnormalities are linked to functional and anatomical disturbances in a neural pathway connecting the prefrontal cortex (PFC) and amygdala. Likewise, rodent and nonhuman primate studies indicate a major role for PFC and amygdala in the mediation of fear- and stress-related behaviors. We used a 5-HTT knock-out (KO) mouse to examine the effects of genetically driven loss of 5-HTT function for the following: (1) depression-related behavior in response to repeated stress, and pavlovian fear conditioning, extinction, and extinction recall; and (2) dendritic morphology and spine density of Golgi-stained pyramidal neurons in the infralimbic cortex (IL) and the basolateral amygdala (BLA). 5-HTT KO mice exhibited increased depressive-like immobility after repeated exposure to forced swim stress, compared with wild-type (WT) controls. Whereas fear conditioning and fear extinction was normal, 5-HTT KO mice exhibited a significant deficit in extinction recall. The apical dendritic branches of IL pyramidal neurons in 5-HTT KO mice were significantly increased in length relative to WT mice. Pyramidal neurons in BLA had normal dendritic morphology but significantly greater spine density in 5-HT KO mice compared with WT mice. Together, the present findings demonstrate a specific phenotypic profile of fear- and stress-related deficits in 5-HTT KO mice, accompanied by morphological abnormalities in two key neural loci. These data provide insight into the behavioral sequelae of loss of 5-HTT gene function and identify potential neural substrates underlying these phenotypes.

Tx1: a transposable element from Xenopus laevis with some unusual properties.

A family of transposable genetic elements in the genome of the frog, Xenopus laevis, is described. They are designated Tx1. Transposability of the elements was deduced by characterization of a chromosomal locus which is polymorphic for the presence or absence of a Tx1 element. Nucleotide sequence analysis suggested that Tx1 elements show target site specificity, as they are inserted at the pentanucleotide TTTAA in all four cases that were examined. The elements appear to have 19-base-pair (bp) inverted terminal repeats, and they are flanked by 4-bp target duplications (TTAA), although the possibility that they do not create target site duplications is discussed. Tx1 elements have several unusual characteristics: the central portion of each element is comprised of a variable number of two types of 393-bp repeating units; the rightmost 1,000 bp of the element contains separate regions potentially capable of forming bends, left-handed Z-form DNA, and alternative stem-loop structures. Comparisons among single frogs suggest that germ line transposition is relatively infrequent and that variations in numbers of internal repeats accumulate quite slowly at any locus.

Translational control of germ cell-expressed mRNA imposed by alternative splicing: opioid peptide gene expression in rat testis.

The three genes encoding the opioid peptide precursors (prodynorphin, proenkephalin, and proopiomelanocortin) are expressed in the rat testis. The sizes of the three opioid mRNAs in the testis differ from the sizes of the corresponding mRNAs in other rat tissues in which these genes are expressed. The smaller testicular proopiomelanocortin mRNA has previously been demonstrated to arise from alternative transcriptional initiation. In the present study, we found that the smaller testicular prodynorphin mRNA, expressed in Sertoli cells, results from alternative mRNA processing. Exon 2, which makes up 5' untranslated sequence, is removed from the mature transcript. Polysome analysis of brain and testis RNA indicates that the alteration of the prodynorphin leader sequence in the testis-specific transcript does not affect the efficiency of translation of this mRNA. The larger testicular proenkephalin transcript, expressed in developing germ cells, also results from alternative mRNA processing. Alternative acceptor site usage in the splicing of intron A results in a germ cell-specific proenkephalin transcript with a 491-nucleotide 5' untranslated leader sequence preceding the preproenkephalin-coding sequence. Polysome analysis indicates that this germ cell-specific proenkephalin mRNA is not efficiently translated. Mechanisms by which alternative mRNA splicing may serve to confer translational regulation upon the testicular proenkephalin transcript are discussed.

Composite transposable elements in the Xenopus laevis genome.

Members of two related families of transposable elements, Tx1 and Tx2, were isolated from the genome of Xenopus laevis and characterized. In both families, two versions of the elements were found. The smaller version in each family (Tx1d and Tx2d) consisted largely of two types of 400-base-pair tandem internal repeats. These elements had discrete ends and short inverted terminal repeats characteristic of mobile DNAs that are presumed to move via DNA intermediates, e.g., Drosophila P and maize Ac elements. The longer versions (Tx1c and Tx2c) differed from Tx1d and Tx2d by the presence of a 6.9-kilobase-pair internal segment that included two long open reading frames (ORFs). ORF1 had one cysteine-plus-histidine-rich sequence of the type found in retroviral gag proteins. ORF2 showed more substantial homology to retroviral pol genes and particularly to the analogs of pol found in a subclass of mobile DNAs that are supposed retrotransposons, such as mammalian long interspersed repetitive sequences, Drosophila I factors, silkworm R1 elements, and trypanosome Ingi elements. Thus, the Tx1 elements present a paradox by exhibiting features of two classes of mobile DNAs that are thought to have very different modes of transposition. Two possible resolutions are considered: (i) the composite versions are actually made up of two independent elements, one of the retrotransposon class, which has a high degree of specificity for insertion into a target within the other, P-like element; and (ii) the composite elements are intact, autonomous mobile DNAs, in which the pol-like gene product collaborates with the terminal inverted repeats to cause transposition of the entire unit.

Isolated clusters of paired tandemly repeated sequences in the Xenopus laevis genome.

There exist in the Xenopus laevis genome clusters of tandemly repeated DNA sequences, consisting of two types of 393-base-pair repeating unit. Each such cluster contains several units of one of these paired tandem repeats (PTR-1), followed by several units of the other repeat (PTR-2). The number of repeats of each type is variable from cluster to cluster and averages about seven of each type per cluster. Every cluster has ca. 1,000 base pairs of common left flanking sequence (adjacent to the PTR-1 repeats) and 1,000 base pairs of common right flanking sequence (adjacent to the PTR-2 repeats). Beyond these common flanks, the DNA sequences are different in the eight cloned genomic fragments we have studied. Thus, the hundreds of PTR clusters in the genome are dispersed at apparently unrelated sites. Nucleotide sequences of representative PTR-1 and PTR-2 repeats are 64% homologous. These sequences do not reveal an obvious function. However, the related species X. mulleri and X. borealis have sequences homologous to PTR-1 and PTR-2, which show the same repeat lengths and genomic organization. This evolutionary conservation suggests positive selection for the clusters. Maintenance of these sequences at dispersed sites imposes constraints on possible mechanisms of concerted evolution.

Effect of N-methyl-d-aspartate receptor blockade on plasticity of frontal cortex after cholinergic deafferentation in rat.

Cholinergic projections from the nucleus basalis play a critical role in cortical plasticity. For instance, cholinergic deafferentation increases dendritic spine density and expression of the GluR1 subunit of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor in frontal cortex. Acetylcholine modulates glutamatergic activity in cortex, and the N-methyl-d-aspartate subtype of glutamate receptor plays a role in many forms of synaptic plasticity. To assess whether N-methyl-d-aspartate receptors mediate the increase in GluR1 and spine density resulting from cholinergic deafferentation, we examined the effect of N-methyl-d-aspartate receptor blockade on nucleus basalis lesion-induced upregulation of GluR1 and dendritic spines. Rats received unilateral sham or 192 IgG saporin lesions of the nucleus basalis. Half of the rats in each group were treated with the N-methyl-d-aspartate antagonist MK-801 or phosphate-buffered saline. Two weeks later, brains were processed for either immunohistochemical staining of the GluR1 subunit or Golgi histology. In layer II-III of frontal cortex, neuronal GluR1 expression was assessed using an unbiased stereological technique, and spine density was assessed on basilar branches of pyramidal neurons. GluR1 expression was increased after nucleus basalis lesion, but this increase was prevented with MK-801. Similarly, nucleus basalis-lesioned animals had significantly higher spine densities, and this effect was also prevented by treatment with MK-801. Thus, N-methyl-d-aspartate receptor blockade prevented both GluR1 and spine density upregulation following cholinergic deafferentation, suggesting that these effects are N-methyl-d-aspartate receptor-mediated.

Lipid metabolite profiles and milk production for Holstein and Jersey cows fed rumen-protected choline during the periparturient period.

Choline is important for assembly of very low density lipoproteins to export triglyceride from liver; however, studies to assess the effect of rumen-protected choline (RPC) supplementation on blood lipid metabolites in periparturient dairy cows have not been conducted. Thirty-two multiparous Holstein and 10 multiparous Jersey cows were randomly assigned to control or RPC treatments. A close-up diet was fed from approximately 3 wk before parturition through parturition, followed by a lactation diet from parturition through 49 d postpartum. For RPC, diets were top-dressed once daily with 60 g of a RPC product (25% choline as choline chloride) from 21 d before expected parturition through 21 d postpartum. Treatment did not affect dry matter intake either prepartum (12.0 vs. 12.1 kg/d for RPC and control, respectively) or during the first 3 wk postpartum (14.8 vs. 15.7 kg/d, respectively). Daily yields of 3.5% fat-corrected milk (39.4 vs. 37.4 kg/d), fat (1.46 vs. 1.38 kg/d), and protein (1.09 vs. 1.05 kg/d) did not differ statistically by treatment (RPC vs. control, respectively). Jersey cows in the control group had lower concentrations of nonesterified fatty acids and beta-hydroxybutyrate in plasma during d 1 to 10 postpartum than did other breed and treatment combinations. Cows fed RPC tended to have greater serum triglycerides prepartum (17.0 vs. 14.7 mg/dL) and lower plasma phospholipid at parturition (65.2 vs. 78.1 mg/dL) than control cows. Treatment did not affect cholesterol and phospholipid at other time points, but concentrations followed patterns of dry matter intake pre- and postpartum. Cows were in moderate body condition score (mean = 3.3) at the start of the study and did not lose excessive condition by 3 wk postpartum (mean body condition score loss = 0.5); therefore, cows might not have been at great risk for hepatic lipid accumulation. Additionally, calculated Met balance was negative postpartum; supplemental RPC might not have spared enough Met to produce a physiological benefit. More research is needed to determine how choline affects prevention or alleviation of fatty liver syndrome and to confirm potential differences between Holstein and Jersey cows.

Human chorionic gonadotropin regulates expression of the proenkephalin gene in adult rat Leydig cells.

The gene encoding the proenkephalin precursor is expressed in both progenitor spermatogenic cells and somatic cells in the rat testis. The 1750-nucleotide (nt) germ cell-specific proenkephalin mRNA is the predominant form detected in the adult rat. The 1400-nt somatic cell form has previously been shown to be expressed in cultured Sertoli cells derived from sexually immature rats, and FSH treatment increases expression of the proenkephalin gene in that experimental system. In the present report we demonstrate that the 1400-nt proenkephalin transcript is present in a freshly isolated, enriched preparation of adult rat Leydig cells. This gene continues to be expressed when the Leydig cells are maintained in primary culture. Treatment of cultured Leydig cells with hCG or a cAMP analog leads to a rapid increase in proenkephalin-mRNA levels. The expression of the proenkephalin gene throughout rat development in the two major somatic cell types of the testis further suggests that proenkephalin-derived peptides play an important role in modulating testicular function. The gene encoding another of the opioid peptide precursors, POMC, has previously been reported to be expressed in rat Leydig cells. We were unable to detect POMC transcripts in cultured Leydig cells. POMC mRNA is abundant in a germ cell-enriched cell fraction.

A spermatozoa-associated factor regulates proenkephalin gene expression in the rat epididymis.

The gene encoding the opioid peptide precursor preproenkephalin is expressed at high levels in the initial segment of the adult rat epididymis. Expression is localized to principal cells, the secretory epithelial cells lining the epididymal duct. During development, epididymal proenkephalin mRNA levels show a pronounced increase at about 44 days of age, coincident with the initial entry of spermatozoa into the epididymal lumen. Hypophysectomy leads to a 60-fold decrease in epididymal proenkephalin mRNA levels. Testosterone replacement can prevent this decline in a manner consistent with an effect upon spermatogenesis. Castration studies demonstrate that a gonadal factor other than testosterone directly regulates epididymal proenkephalin expression, and the results of efferent duct ligation suggest that this factor must be supplied through an intact connection of the testis and epididymis. Proenkephalin mRNA levels in the epididymis correlate with the decline and reappearance of spermatozoa induced by the alkylating agent busulphan. Thus, the developmental profile of proenkephalin expression, coupled with the results of both surgical and pharmacological manipulations of the reproductive tract, indicate that spermatozoa, or a spermatozoa-associated factor, regulate proenkephalin gene expression in the epididymis.

Characterization of the rat prodynorphin gene.

The structure of full-length rat prodynorphin cDNA and the corresponding gene has been determined. The 2400 base rat prodynorphin mRNA is encoded by four exons. Exons 1 and 2 encode the majority of the 5'-untranslated sequence, while exons 3 and 4 contain the translated region; the entire 3' -untranslated region is contained on exon 4 as well. RNase protection studies, in which a genomic DNA fragment was used to generate a cRNA hybridization probe, have determined the major transcriptional initiation site for both brain and testicular prodynorphin mRNA. Transient expression of transfected fusion genes containing the 5'-flanking DNA of the rat prodynorphin gene linked to the structural sequence of a reporter gene has been used to identify specific genomic DNA fragments from the prodynorphin gene locus which are capable of acting as transcriptional promoters. Multiple regions of genomic DNA appear to have transcriptional promoter activity when introduced into various eukaryotic cell lines.

Calcitonin-secreting cells of the thyroid express an extracellular calcium receptor gene.

Calcitonin (CT) secretion by parafollicular cells of the thyroid (C cells) is regulated by small changes in the concentration of extracellular calcium ([Ca2+]e). Elevation of [Ca2+]e elicits a rise in the C cell cytoplasmic calcium concentration and stimulates CT release. The molecular entity through which C cells detect changes in [Ca2+]e and modulate hormone secretion is unknown. Recently, an extracellular calcium-sensing receptor (CaR) complementary DNA was isolated from bovine parathyroid gland. To assess whether parathyroid cells and C cells use similar mechanisms to detect changes in ambient Ca2+, rat, human, and sheep C cells were examined for expression of the parathyroid CaR or a related receptor isoform. Reverse transcription-polymerase chain reaction analysis identified CaR transcripts in rat and human thyroid gland. Northern blot analysis demonstrated CaR messenger RNA (mRNA) in rat thyroid gland, a human medullary thyroid carcinoma (MTC) isolate, and a highly enriched preparation of sheep C cells. Rat MTC 44-2 cells, a cell line responsive to changes in [Ca2+]e, express abundant levels of CaR mRNA. Human TT cells, a C cell line lacking the extracellular calcium-sensing function, have undetectable levels of CaR mRNA by Northern blot analysis. Western blot analysis, using antiserum specific to the parathyroid CaR, detected CaR protein in rMTC 44-2, but not TT cells. Immunostaining of both dispersed sheep C cells and rat thyroid gland sections identified C cell-specific expression of the CaR protein, and in situ hybridization analysis confirmed the C cell-specific expression of CaR mRNA in the intact rat thyroid. The nucleotide sequence of the coding region of the rMTC 44-2 CaR transcripts was found to encode the same CaR protein as that expressed in the parathyroid and kidney. The results demonstrate that C cells express the same extracellular calcium-sensing receptor that is found in parathyroid and kidney, and the presence of this receptor protein in C cell lines correlates with the extracellular calcium-sensing function. This CaR is likely to represent the primary molecular entity through which C cells detect changes in [Ca2+]e and control CT release, suggesting that activation of the same receptor can either stimulate or inhibit hormone secretion in different cell types.

Clustered inactivating mutations and benign polymorphisms of the calcium receptor gene in familial benign hypocalciuric hypercalcemia suggest receptor functional domains.

The predominant variety of familial benign hypocalciuric hypercalcemia (FBHH) is FBHH(3q), which is associated with presumed inactivating mutations of the cell surface calcium receptor (CaR) gene on chromosome 3q13.3-q21. We sought mutations of the CaR gene in FBHH by direct sequencing of PCR-amplified genomic DNA from 14 affected families: 8 mapped to 3q13, 1 mapped to chromosome 19p, and 5 unmapped. We sequenced the entire coding region of the gene (exons 2-7) in one or two affected members of each family and found six point mutations that altered one amino acid, cosegregated with hypercalcemia, and were absent in more than 100 unaffected persons. Four mutations were unique (S53P, D215G, S657Y, and P748R), and two had been reported previously (P55L and R185Q). Of four mutant CaR proteins expressed in Xenopus oocytes, three were deficient in extracellular Ca2+-induced signaling. No CaR mutations were found in eight families, including the one mapped to chromosome 19p. Three benign polymorphisms occurred in the COOH-terminal region of the CaR protein in 10%, 15%, and 30% of more than 100 unaffected persons. Thus, FBHH-causing CaR mutations were clustered in the NH2-terminal extracellular and membrane-spanning regions of the receptor protein. We suggest that these are important functional domains, probably for calcium binding and signal transduction, respectively. Finally, mutations in regulatory or intronic regions of the CaR gene may also underlie many cases of FBHH.

The association between asthma drugs and severe life-threatening attacks.

OBJECTIVES: To measure the association between asthma drugs and death or ICU admission due to asthma (severe life-threatening attack of asthma [SLTA]), and to assess the possibility that these associations may not be causal but due to the prescription of these drugs to patients with more severe disease (confounding). DESIGN: Retrospective cohort study of 655 asthmatics who attended an emergency department in 1986 to 1987 followed till death or May 1989. METHODS: Outcome events were death or ICU admission due to asthma (SLTA). All hospital attendances were identified and patients classified at each according to drug exposure and a wide variety of measures of asthma severity. Incidence rates were computed as total outcome events divided by person-time contributed for each subject classified according to drug use and asthma severity. Rate ratio (RR) estimates for severe asthma outcomes associated with use as compared to nonuse of asthma drugs were calculated. Severity markers were identified and used to adjust the crude RR estimates. RESULTS: One hundred five SLTAs (15 deaths, 90 ICU admissions) occurred in 66 patients. Like inhaled fenoterol, oral beta-agonists, theophylline, cromolyn, inhaled steroids, and oral steroids were all associated with an increased risk of SLTA. When adjusted progressively for measures of severity, these increased risks became insignificant except for cromolyn. CONCLUSION: Unadjusted RR estimates for severe asthma events comparing exposure to a particular drug with nonuse are overestimates due to confounding. Control with two severity markers (hospital admission in the last year, use of oral corticosteroid at the time of previous admission) removes some confounding but control for additional severity markers not available in previous studies reduces the effect estimates further. These results suggest that the problem of confounding is substantial in nonrandomized epidemiologic studies of asthma drugs. Previous studies reporting RR estimates are likely to be confounded.

The effect of adding ipratropium bromide to salbutamol in the treatment of acute asthma: a pooled analysis of three trials.

OBJECTIVE: To assess the effect on FEV1 and clinical outcomes of adding ipratropium bromide to salbutamol in the treatment of acute asthma. METHODS: We conducted a pooled analysis of three randomized double-blinded clinical trials conducted in the United States, Canada, and New Zealand. The studies enrolled 1,064 patients aged 18 to 55 years who presented at the emergency department with acute asthma. Patients were randomized to treatment with a combination of nebulized 2.5 mg salbutamol plus 0.5 mg ipratropium bromide, or 2.5 mg salbutamol alone. Medications were administered at baseline and, in the US study, at 45 min. FEV1 was measured at baseline, 45 min, and 90 min. Patients were followed up for 48 h after hospital discharge for occurrence of asthma exacerbation and hospitalization. RESULTS: Treatment groups were comparable at baseline. Of the 1,064 patients randomized, 1,015 patients (95%) remained in the study for measurement at 45 min, and 961 patients (90%) completed the final measurement at 90 min. Comparison of overall improvement in FEV1 at 45 min indicated a better response for patients receiving combination therapy (mean difference=43 mL, 95% confidence interval [CI]=-20, 107). The distribution of change in FEV1 was skewed by a small number of patients with extreme values (38 of 1,064=3.6%) that may have been due to unreliable lung function testing. Removing these outliers produced a larger and more precise estimate of effect (mean difference=55 mL, 95% CI=2,107). Because the distribution was skewed, we performed nonparametric analyses that showed evidence of a beneficial effect of combination therapy. The difference between median values at 45 min is 40 mL (Wilcoxon p value=0.03). In addition, 4.9% (95% CI=-1%, 11%) more patients in the combination group achieved at least 20% of their potential improvement, as measured by the difference between their baseline FEV1 and their predicted FEV1. Patients receiving combination therapy had lower risk for each of three clinical outcomes: the need for additional treatment (relative risk [RR]=0.92, 95% CI=0.84, 1.0), risk of asthma exacerbation (RR=0.84, 95% CI=0.67, 1.04), and risk of hospitalization (RR=0.80, 95% CI=0.61, 1.06). CONCLUSION: Adding ipratropium bromide to salbutamol in the treatment of acute asthma produces a small improvement in lung function, and reduces the risk of the need for additional treatment, subsequent asthma exacerbations, and hospitalizations. These apparent benefits of adding ipratropium bromide were independent of the amount of beta-agonist that had been used earlier in the attack, and possibly related to a recent upper respiratory tract infection. Confirmatory studies are needed, especially for clinical outcomes.

Spirometry in primary care practice: the importance of quality assurance and the impact of spirometry workshops.

OBJECTIVE: To determine the quality of spirometry performed in primary care practice and to assess the impact of formal training. DESIGN: Randomized, controlled prospective interventional study. SETTING: Primary care practice, Auckland City, New Zealand. PARTICIPANTS: Thirty randomly selected primary care practices randomized to "trained" or "usual" groups. One doctor and one practice nurse were nominated to participate from each practice. INTERVENTIONS: "Trained" was defined as participation in an "initial" spirometry workshop at week 0 and a "maintenance of standards" workshop at week 12. "Usual" was defined as no formal training until week 12, when participants they attended the same "initial" workshop provided for the trained group. The study duration was 16 weeks. Each practice was provided with a spirometer to be used at their clinical discretion. MEASUREMENTS AND RESULTS: Spirometry data were uploaded weekly and analyzed using American Thoracic Society (ATS) criteria for acceptability and reproducibility. The workshops were assessed objectively with practical and written assessments, confirming a significant training effect. However, analysis of spirometry performed in clinical practice by the trained practitioners revealed three acceptable blows in only 18.9% of patient tests. In comparison, 5.1% of patient tests performed by the usual practitioners had three acceptable blows (p<0.0001). Only 13.5% of patient tests in the trained group and 3.4% in the usual group (p<0.0001) satisfied full acceptability and reproducibility criteria. However, 33.1% and 12.5% of patient tests in the trained and usual groups, respectively (p<0.0001), achieved at least two acceptable blows, the minimum requirement. Nonacceptability was largely ascribable to failure to satisfy end-of-test criteria; a blow of at least 6 s. Visual inspection of the results of these blows as registered on the spirometer for the presence of a plateau on the volume-time curve suggests that < 15% were acceptable. CONCLUSIONS: Although a significant training effect was demonstrated, the quality of the spirometry performed in clinical practice did not generally satisfy full ATS criteria for acceptability and reproducibility. Further study would be required to determine the clinical impact. However, the ATS guidelines allow for the use of data from unacceptable or nonreproducible maneuvers at the discretion of the interpreter. Since most of the failures were end-of-test related, the FEV1 levels are likely to be valid. Our results serve to emphasize the importance of effective training and quality assurance programs to the provision of successful spirometry in primary care practice.

Differential patterns of regulated gene expression in the adult rat epididymis.

Specialization among the principal epithelial cells of the epididymal tubule is documented following the analysis of transcriptional activity of four distinct species of mRNA. In situ histochemical analysis revealed a unique pattern of expression for each transcript. This observation supports the concept that region-specific patterns of transcriptional expression along the epididymal tubule serve as the major molecular basis underlying region-specific patterns of luminal proteins within the tubule. Additionally, multiple testicular factors appear to regulate expression of these mRNAs. The transcript encoding peptidyl-prolyl cis-trans isomerase is constitutively expressed. Those encoding the major secretory proteins, protein B/C and protein D/E, are directly regulated by testicular androgen. That encoding the opioid peptide precursor, proenkephalin, is regulated by a non-androgen testicular factor(s), specifically, spermatozoa or a spermatozoa-related factor. Thus, a complex array of nuclear events and signals received by the principal cells serve to determine the transcriptional status of genes expressed within this epididymal cell type.

Conus peptides: novel probes for nicotinic acetylcholine receptor structure and function.

Conus is a genus of predatory marine snails that uses venom to capture prey. Among the neurotoxins widely utilized by the cone snails are the alpha-conotoxins which are disulfide-rich peptides that target muscle or neuronal subtypes of nicotinic acetylcholine receptors. The small size and receptor subtype specificity of these peptides make them particularly useful for characterizing both native and heterologously expressed nicotinic receptors. In this report, we demonstrate that alpha-conotoxin MII potently blocks beta3-containing neuronal nicotinic receptors. Furthermore, initial evidence suggests that subpopulations of alpha3beta2beta3-containing receptors are differentially sensitive to alpha-conotoxin MII. Thus, alpha-conotoxin MII promises to be a useful tool for studying neuronal nicotinic receptors containing the beta3 subunit.

The clinical utility of arterialized earlobe capillary blood in the assessment of patients for long-term oxygen therapy.

The prescription of long-term oxygen (LTOT) is underpinned by the measurement of arterial PO2, generally obtained by radial artery puncture. This test is commonly associated with patient discomfort and a test that is reliable, well-tolerated and non-invasive would be advantageous. Cutaneous oximetry has not proved sufficiently accurate. Arterialized earlobe capillary sampling has been proposed, with some authors stating that it is under-utilized. However, to date studies have yielded conflicting results and the clinical utility remains uncertain. Our regional oxygen service based at a specialist respiratory hospital undertook a prospective study of consecutive patients with chronic respiratory disease undergoing assessment for LTOT. Simultaneous radial artery and arterialized earlobe sampling was performed. Rigorous steps were taken to ensure optimal arterialization of the earlobe samples. Agreement between arterial and arterialized PO2 and PCO2 was compared using the Bland-Altman method. One hundred patients were studied. Procedural difficulties (insufficient sample or air in sample) were similar for both procedures, however clotting occurred more frequently in arterialized earlobe samples. Sixty-four sample pairs were available for comparison. The bias and limits of agreement between arterialized and arterial PO2 were wide, mean (+/- 2 SD), -048 (-2.05-1.09) kPa. The bias and limits of agreement for PCO2 were smaller. Using the absolute criterion (arterial PO2 < 7.3 kPa), 9/55 (16%) patients would receive oxygen inappropriately based on the arterialized earlobe sample. Conversely, no patients would have been denied LTOT. Radial artery puncture gave rise to significantly greater discomfort (P < 0.0001) and level of concern (P < 0.0001). Patient preference strongly favoured arterialized earlobe sampling. However, despite rigorous attention to arterialization earlobe sampling was insufficiently accurate to replace radial artery puncture in the prescription of LTOT.

An evaluation of short-term oxygen therapy: the prescription of oxygen to patients with chronic lung disease hypoxic at discharge from hospital.

The provision of domiciliary oxygen to patients hypoxic at hospital discharge has been termed short-term oxygen therapy (STOT). This practice appears widespread, although there is a paucity of literature and no evidence-based guidelines. We undertook this audit to examine the prescription of STOT and determine the proportion fulfilling for long-term oxygen therapy (LTOT) 2 months post-discharge. STOT was defined prospectively: resting PaO2 < or = 7.3 kPa (55 mmHg) or PaO2 between 7.3 and 8.0 kPa (60 mmHg) with any of the following: clinical evidence of cor pulmonale (pedal oedema or jugular venous distension), ECG evidence of pulmonale, echocardiogram evidence of pulmonary hypertension, haematocrit > 0.55 (adapted directly from LTOT criteria). Patients were evaluated for LTOT 2 months post-discharge when clinically stable on optimal medical management. All referrals to the Auckland Regional Oxygen Service between July 1998 and 1999 were systematically reviewed. The majority 289/405 (71%) of new referrals were for the prescription of STOT/LTOT in patients with chronic lung disease: 160/289 (55%) derived from hospitalized patients with the majority 130 (81%) fulfilling criteria for STOT, median age 73, range 24-96 years. Mean hospital stay was 10.2 days. Two months after discharge 22/127 (17%) of STOT patients had died, comparable with 4/22 (18%) not fulfilling criteria for STOT. A total of 123 patients were assessed for LTOT at 2 months; 76 (62%) fulfilled criteria for LTOT. The prescription of oxygen at hospital discharge represented a considerable proportion of our referral load. There was a high mortality in the 2-month follow-up period. A significant proportion of STOT patients did not subsequently fulfill criteria for LTOT. Further prospective studies are required in order to develop evidence-based guidelines.