A Preliminary Genetic Investigation of VOLVOX CARTERI.

A preliminary genetic analysis of a number of genetic variants of Volvox carteri f. nagariensis is presented. Techniques are outlined for mutagenesis of Volvox, isolation of mutants and routine genetic analysis. All of the mutants show simple Mendelian segregation patterns and have been tentatively placed in 14 linkage groups.

Effects of intrathecal thyrotropin-releasing hormone (protirelin) in refractory depressed patients.

BACKGROUND: Therapeutic effects of the tripeptide protirelin (thyrotropin-releasing hormone) have been postulated in the affective disorders, but direct assessment in humans has been hindered by poor blood-brain barrier permeability. METHODS: Eight medication-free inpatients with refractory depression received 500 micrograms of protirelin via a lumbar intrathecal injection and an identical sham lumbar puncture procedure, separated by 1 week, in a double-blind crossover design. RESULTS: Five of eight patients responded to intrathecal protirelin, defined as a 50% or greater reduction in an abbreviated Hamilton Rating Scale for Depression score. Suicidality also was reduced significantly (P < .05). Responses were rapid and clinically robust, but short-lived. CONCLUSION: Administration of protirelin by an intrathecal route induced a rapid improvement in mood and suicidality in these refractory depressed patients, supporting the hypothesis that thyrotropin-releasing hormone could be a positive modulator of mood.

Comparative antidepressant effects of intravenous and intrathecal thyrotropin-releasing hormone: confounding effects of tolerance and implications for therapeutics.

A significant amount of preclinical and human data indicate that thyrotropin-releasing hormone (TRH) has antidepressant effects. Although early studies showing these effects using intravenous TRH were not consistently replicated, it has been suggested that this could be explained by its poor blood-brain barrier penetration. For this reason we compared the antidepressant effect of intrathecal and intravenous TRH administered in a double-blind design to 2 treatment-refractory patients with bipolar II disorder. Each experienced a robust antidepressant response by both routes; subsequent open trials of intravenous TRH also were effective until apparent tolerance developed. Intrathecal TRH was readministered and both subjects again experienced robust antidepressant responses. These preliminary data suggest a differential mechanism of tolerance to the two routes of administration and raise the possibility that a subgroup of patients may be responsive to the antidepressant effects of TRH independent of its route of administration.

Lack of correlation between cerebrospinal fluid thyrotropin-releasing hormone (TRH) and TRH-stimulated thyroid-stimulating hormone in patients with depression.

BACKGROUND: It has been proposed that elevated central thyrotropin-releasing hormone (TRH) is associated with the blunted thyroid-stimulating hormone (TSH) response to TRH in patients with depression. Few studies have directly evaluated this relationship between central nervous system and peripheral endocrine systems in the same patient population. METHODS: 15 depressed patients (4 male, 11 female, 12 bipolar, and 3 unipolar) during a double-blind, medication-free period of at least 2 weeks duration, underwent a baseline lumbar puncture followed by a TRH stimulation test. Cerebrospinal fluid (CSF) TRH and serial serum TSH, free thyroxine, triiodothyronine, prolactin, and cortisol were measured. A blunted response to TRH was defined as a delta TSH less than 7 microU/mL. RESULTS: There was no significant difference in mean CSF TRH between "blunters" (2.82 +/- 1.36 pg/mL) and "non-blunters" (3.97 +/- 0.62 pg/mL, p = .40). There was no evidence of an inverse relationship between CSF TRH and baseline or delta TSH. There was no correlation between CSF TRH and the severity of depression or any other endocrine measure. CONCLUSIONS: These data are not consistent with the prediction of hypothalamic TRH hypersecretion and subsequent pituitary down-regulation in depression; however, CSF TRH may be from a nonparaventricular nucleus-hypothalamic source (i.e., limbic area, suprachiasmatic nucleus, brain stem-dorsal raphe) and thus, not necessarily related to peripheral neuroendocrine indices.

Inverse relationship of peripheral thyrotropin-stimulating hormone levels to brain activity in mood disorders.

OBJECTIVE: The author's goal was to investigate relationships between peripheral thyroid hormone levels and cerebral blood flow (CBF) and cerebral glucose metabolism in affectively ill patients. METHOD: Medication-free inpatients with major depression or bipolar disorder were studied with oxygen-15 water and positron emission tomography (PET) to measure CBF (N = 19) or with [18F] fluorodeoxyglucose and PET to measure cerebral glucose metabolism (N = 29). Linear regression was used to correlate global CBF and cerebral glucose metabolism with serum thyrotropin-stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4), and free T4 concentrations. Statistical parametric mapping was used to correlate regional CBF and cerebral glucose metabolism with these thyroid indexes. Post hoc t tests were used to further explore the relationships between serum TSH and global CBF and cerebral glucose metabolism. RESULTS: Serum TSH was inversely related to both global and regional CBF and cerebral glucose metabolism. These relationships persisted in the cerebral glucose metabolism analysis and, to a lesser extent, in the CBF analysis after severity of depression had been controlled for. In contrast, no significant relationships were observed between T3, T4, or free T4 and global or regional CBF and cerebral glucose metabolism. CONCLUSIONS: These data suggest that peripheral TSH (putatively the best marker of thyroid status) is inversely related to global and regional CBF and cerebral glucose metabolism. These findings indicate relationships between thyroid and cerebral activity that could provide mechanistic hypotheses for thyroid contributions to primary and secondary mood disorders and the psychotropic effects of thyroid axis manipulations.

Clinical practice guidelines for bipolar disorder from the Department of Veterans Affairs.

BACKGROUND: For the last several years, the Department of Veterans Affairs (VA) has been involved in the development of practice guidelines for major medical, surgical, and mental disorders. This article describes the development and content of the VA-Clinical Practice Guidelines for Bipolar Disorder, which are available in their entirety on the Journal Web site (http://www. psychiatrist.com). METHOD: A multidisciplinary work group composed of content experts in the field of bipolar disorder and practitioners in general clinical practice was convened by the VA's Office of Performance and Quality and the Mental Health Strategic Health Group. The work group was instructed in algorithm development and methods of evidence evaluation. Draft guidelines were developed over the course of 6 months of meetings and conference calls, and that draft was then sent to nationally prominent content experts for final critique. RESULTS: The Bipolar Guidelines are part of the family of the VA Clinical Guidelines for Management of Persons with Psychosis and consist of explicit algorithms supplemented by annotations that explain the specific decision points and their basis in the scientific literature. The guidelines are organized into 5 modules: a Core Module for diagnosis and assignment to mood state plus 4 treatment modules (Manic/Hypomanic/Mixed Episode, Bipolar Depressive Episode, Rapid Cycling, and Bipolar Disorder With Psychotic Features). The modules specify particular diagnostic and treatment tasks at each step, including both somatotherapeutic and psychotherapeutic interventions. CONCLUSION: The VA Bipolar Guidelines are designed for easy clinical reference in decision making with individual patients, as well as for use as a scholarly reference tool. They also have utility in training activities and quality improvement programs.

The place of anticonvulsant therapy in bipolar illness.

With the increasing recognition of lithium's inadequacy as an acute and prophylactic treatment for many patients and subtypes of bipolar illness, the search for alternative agents has centered around the mood stabilizing anticonvulsants carbamazepine and valproate. In many instances, these drugs are effective alone or in combination with lithium in those patients less responsive to lithium monotherapy, including those with greater numbers of prior episodes, rapid-cycling, dysphoric mania, co-morbid substance abuse or other associated medical problems, and patients without a family history of bipolar illness in first-degree relatives. Nineteen double-blind studies utilizing a variety of designs suggest that carbamazepine, or its keto-congener oxcarbazepine, is effective in acute mania; six controlled studies report evidence of the efficacy of valproate in the treatment of acute mania as well. Fourteen controlled or partially controlled studies of prophylaxis suggest carbamazepine is also effective in preventing both manic and depressive episodes. valproate prophylaxis data, although based entirely on uncontrolled studies, appear equally promising. Thus, both drugs are widely used and are now recognized as major therapeutic tools for lithium-nonresponsive bipolar illness. The high-potency anticonvulsant benzodiazepines, clonazepam and lorazepam, are used adjunctively with lithium or the anticonvulsant mood stabilizers as substitutes or alternatives for neuroleptics in the treatment of manic breakthroughs. Preliminary controlled clinical trials suggest that the calcium channel blockers may have antimanic or mood-stabilizing effects in a subgroup of patients. A new series of anticonvulsants has just been FDA-approved and warrant clinical trials to determine their efficacy in acute and long-term treatment of mania and depression. Systematic exploration of the optimal use of lithium and the mood-stabilizing anticonvulsants alone and in combination, as well as with adjunctive antidepressants, is now required so that more definitive treatment recommendations for different types and stages of bipolar illness can be more strongly evidence based.

The application of enzyme-linked immunosorbent assays (ELISA) to neuropeptides.

Procedures are presented for routine evaluation of antibody specificity, titre, and quantitation of antigen levels in tissue extracts without the use of radiolabeled probes. A colorimetric, enzyme-linked immunosorbent assay (ELISA) is described for general use with neuropeptides, using neurotensin as a primary example. These assays use rabbit anti-neurotensin immune serum which is colorimetrically identified after combination with an alkaline phosphatase-conjugated, affinity purified, goat anti-rabbit IgG and reaction with the chromogenic substrate, p-nitrophenyl phosphate. Because the principle of these methods can be adapted for use with various proteins and neuropeptides, they should find widespread applicability in neurobiology.

Psychosocial interventions for bipolar disorder.

The limitations of pharmacotherapy and the emergence of data supporting a role for psychosocial factors in the course of bipolar disorder have led to increased interest in the use of psychosocial interventions to improve outcomes. Although this area of study has suffered from a lack of systematic data, preliminary evidence suggests that the combined use of psychosocial interventions and medication is superior to pharmacologic treatment alone. Further research is necessary to identify and the psychosocial risk factors associated with bipolar disorder to design effective interventions to diminish their effects and improve outcome. The introduction of formal, manual-based psychotherapeutic interventions that include specific educational components has been particularly promising.

Drug interactions in psychopharmacology.

The importance of psychotropic drug interactions has become increasingly evident in recent years. Although drug interactions may lead to therapeutic benefits, they also may result in diminished efficacy of drug therapy or may cause toxic or life-threatening reactions. To avoid these unwanted effects, it is important for the clinician to be aware of the basic principles that govern drug interactions.

Evaluating the clinical significance of drug interactions: a systematic approach.

Remembering the myriad of psychotropic drug interactions is extremely difficult. Nevertheless, by applying a systematic approach, the clinician can often predict the occurrence and time course of such interactions. Several factors must be considered when assessing the potential consequences. Drug-related factors that increase the risk for clinically significant interactions include a low therapeutic index or narrow therapeutic window, a multiplicity of pharmacological actions, and inhibition or inducement of cytochrome P450 enzymes. Next, patient-related factors that can increase the risk for significant drug interactions should be considered. These include genetically based variations in drug-metabolizing capacity, as well as advanced age, underlying medical illness, and comorbid substance abuse. Finally, the literature should be carefully reviewed to as-certain the potential clinical relevance of available data. If a clinically significant drug interaction appears likely to occur, the patient's clinical status should be followed closely; therapeutic drug monitoring should be used if applicable and dosage adjustments made accordingly. Rational polypharmacy requires an understanding of the pharmacological principles governing drug interactions and a knowledge of the factors that increase the likelihood of clinically significant variations in drug action. This will allow the clinician to maximize beneficial effects while minimizing the risk of adverse events.

Resistance of Transgenic Prunus domestica to Plum Pox Virus Infection.

Transgenic plum trees (Prunus domestica) containing the plum pox potyvirus coat protein (PPV-CP) gene were inoculated with PPV by aphid feeding or chip budding. Infection was monitored by evaluation of virus symptoms, DAS-ELISA, and immunoblot assays. Based on observations and analyses over 3 years including two dormancy cycles, one out of five transgenic clones (C-5), was found to be resistant to infection whether inoculated by aphids or by chip budding. PPV could not be detected in any inoculated plants of the C-5 clone by immunoblot or immunocap-ture-reverse transcriptase-polymerase chain reaction assays. To our knowledge, this is the first P. domestica clone resistant to PPV infection produced by genetic engineering.

Genetics of methyl-accepting chemotaxis proteins in Escherichia coli: cheD mutations affect the structure and function of the Tsr transducer.

The tsr gene specifies a methyl-accepting membrane protein involved in chemotaxis to serine and several repellent compounds. We have characterized a special class of tsr mutations designated cheD which alter the signaling properties of the Tsr transducer. Unlike tsr null mutants, cheD strains are generally nonchemotactic, dominant in complementation tests, and exhibit a pronounced counterclockwise bias in flagellar rotation. Several lines of evidence showed that cheD mutations were alleles of the tsr gene. First, cheD mutations were mapped into the same deletion segments as conventional tsr mutations. Second, restriction site analysis of the transducing phage deletions used to construct the genetic map demonstrated that the endpoints of the deletion segments fell within the tsr coding sequence. Third, a number of the cheD mutants synthesized Tsr proteins with slight changes in electrophoretic mobility, consistent with alterations in Tsr primary structure. These mutant proteins were able to undergo posttranslational deamidation and methylation reactions in the same manner as wild-type Tsr protein; however, the steady-state level of Tsr methylation in cheD strains was very high. The methylation state of the Tar protein, another species of methyl-accepting protein in Escherichia coli, was also higher than normal in cheD strains, suggesting that the aberrant Tsr transducer in cheD mutants has a generalized effect on the sensory adaptation system of the cell. These properties are consistent with the notion that the Tsr protein of cheD mutants is locked in an excitatory signaling mode that both activates the sensory adaptation system and drowns out chemotactic signals generated by other transducer species. Further study of cheD mutations thus promises to reveal valuable information about the functional architecture of the Tsr protein and how this transducer controls flagellar behavior.

Chemotaxis in Escherichia coli: construction and properties of lambda tsr transducing phage.

The tsr gene of Escherichia coli, located at approximately 99 min on the chromosomal map, encodes a methyl-accepting protein that serves as the chemoreceptor and signal transducer for chemotactic responses to serine and several repellents. To determine whether any other chemotaxis or motility genes were located in the tsr region, we constructed and characterized two lambda tsr transducing phages that each contain about 12 kilobases of chromosomal material adjacent to tsr. lambda tsr70 carries sequences from the promoter-proximal side of tsr; lambda tsr72 carries sequences from the promoter-distal side of tsr. Restriction maps of the bacterial inserts in these phages and Southern hybridization analyses of the bacterial chromosome indicated that the tsr gene is transcribed in the counterclockwise direction on the genetic map. Insert deletions were isolated in lambda tsr70 and transferred into the host chromosome to examine the null phenotype of tsr. All such strains exhibited wild-type swimming patterns and chemotactic responses to a variety of stimuli, but were specifically defective in serine taxis and other Tsr-mediated responses. In addition, UV programming experiments demonstrated that Tsr and several of its presumptive degradation products were the only bacterial proteins encoded by lambda tsr70 and lambda tsr72 that required host FlbB/FlaI function for expression. These findings indicate that there are probably no other chemotaxis-related genes in the tsr region. A series of tsr point mutations were isolated by propagating lambda tsr70 on a mutD host and used to construct a fine-structure map of the tsr locus. These mutations should prove valuable in exploring structure-function relationships in the Tsr transducer.

Comparison of Pch313 (pTOM13 Homolog) RNA Accumulation during Fruit Softening and Wounding of Two Phenotypically Different Peach Cultivars.

Pch313 was isolated as a cDNA whose RNA accumulated during the softening period of peach (Prunus persica L. Batsch) fruit development. To better understand the role of the gene, we compared the amount of pch313-related RNA detected during fruit softening and tissue wounding between cultivars with different softening characteristics. The cultivar that softened faster, "Bailey," had a significantly higher amount of pch313-related RNA accumulate during softening than the slower-softening cultivar, "Suncrest." Pch313 was sequenced and found to be related to a tomato fruit cDNA clone, pTOM13, which has been shown to encode the ethylene-forming enzyme. The derived amino acid sequence of pch313 is 74 to 83% identical to the pTOM13-related sequences. A pch313-3' noncoding region probe was used to demonstrate that pch313 is related to both a wound-induced RNA transcript and the major fruit-softening transcript. The relationship of pch313 RNA accumulation and ethylene evolution was examined upon wounding and appeared to be both tissue and cultivar specific. When leaves were wounded, more pch313-related RNA was detected in Bailey and the rate of ethylene evolved was also higher in Bailey. When fruits were wounded, the levels of ethylene evolved were nearly identical but Suncrest accumulated more pch313-related RNA. Southern analysis of the DNA indicated a small number of related genes.

Seasonal expression of a dehydrin gene in sibling deciduous and evergreen genotypes of peach (Prunus persica [L.] Batsch).

A cDNA library was created from cold-acclimated bark tissue of peach and selectively probed using an antibody directed against the lysine-rich consensus region of dehydrin proteins. Several clones were thus obtained which had a high degree of sequence similarity to other dehydrin genes. Northern analysis, using clone 5a, indicated that a 1.8 kb transcript was seasonally expressed in sibling deciduous and evergreen genotypes of peach, and also inducible by water deficit in cv. Rio Oso Gem. The evergreen and deciduous genotypes differ significantly in both their ability to cold-acclimate and in the seasonal expression of the dehydrin transcript and protein. In both genotypes, the transcript was maximally expressed during winter and undetectable in May-July. The evergreen genotype (less cold-tolerant), however, displayed transcript accumulation which lagged behind and declined sooner than in the deciduous genotype. Protein expression was similar to transcript expression, however, protein expression in the evergreen genotype lagged considerably behind transcript accumulation in the fall. This indicates that several levels of regulation of dehydrin proteins may exist during cold acclimation. A genomic clone (G10a) was isolated which contained the full-length dehydrin gene, designated ppdhn1. The peach dehydrin gene encodes 472 amino acids with a predicted size of 50,020 Da. The encoded protein (PCA60) contains nine of the lysine-rich repeats characteristic of dehydrins and two DEYGNP motifs at the amino acid terminus. A genomic blot, probed with clone 5a under stringent conditions, indicated that one or two highly homologous genes are present in peach, whereas an additional member was detected under low-stringency conditions. It is suggested that several members of the dehydrin gene family may exist in peach that vary in their relation to ppdhn1.

Isolation and sequencing of cDNA clones encoding ethylene-induced putative peroxidases from cucumber cotyledons.

A cDNA library from ethephon-treated cucumber cotyledons (Cucumis sativus L. cv. Poinsett 76) was constructed. Two cDNA clones encoding putative peroxidases were isolated by means of a synthetic probe based on a partial amino acid sequence of a 33 kDa cationic peroxidase that had been previously shown to be induced by ethylene. DNA sequencing indicates that the two clones were derived from two closely related RNA species that are related to published plant peroxidase sequences. Southern analysis indicates that there are 1-5 copies in a haploid genome of a gene homologous to the cDNA clones. The deduced amino acid sequences are homologous with a tobacco (55% sequence identity), a horseradish (53%), a turnip (45%), and a potato (41%) peroxidase. The cloned sequences do not encode the 33 kDa peroxidase from which the original synthetic probe was been derived, but rather other putative peroxidases. An increase in the level of mRNA is evident by 3 hours after ethephon or ethylene treatment and plateaus by 15 hours.

Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene.

Plum hypocotyl slices were transformed with the coat protein (CP) gene of plum pox virus (PPV-CP) following cocultivation with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPVCP-33. This binary vector carries the PPV-CP gene construct, as well as the chimeric neomycin phosphotransferase and ß-glucuronidase genes. Integration and expression of the transferred genes into regenerated plum plants was verified through kan resistance, GUS assays, and PCR amplification of the PPV-CP gene. Twenty-two transgenic clones were identified from approximately 1800 hypocotyl slices. DNA, mRNA, and protein analyses of five transgenic plants confirmed the integration of the engineered CP gene, the accumulation of CP mRNA and of PPV-CP-immunoreactive protein. CP mRNA levels ranged from high to undetectable levels, apparently correlated with gene structure, as indicated by DNA blot analysis. Western analysis showed that transgenic plants produced amounts of CP which generally correlated with amounts of detected mRNA.

The emerging role of cytochrome P450 3A in psychopharmacology.

Recent advances in molecular pharmacology have allowed the characterization of the specific isoforms that mediate the metabolism of various medications. This information can be integrated with older clinical observations to begin to develop specific mechanistic and predictive models of psychotropic drug interactions. The polymorphic cytochrome P450 2D6 has gained much attention, because competition for this isoform is responsible for serotonin reuptake inhibitor-induced increases in tricyclic antidepressant concentrations in plasma. However, the cytochrome P450 3A subfamily and the 3A3 and 3A4 isoforms (CYP3A3/4) in particular are becoming increasingly important in psychopharmacology as a result of their central involvement in the metabolism of a wide range of steroids and medications, including antidepressants, benzodiazepines, calcium channel blockers, and carbamazepine. The inhibition of CYP3A3/4 by medications such as certain newer antidepressants, calcium channel blockers, and antibiotics can increase the concentrations of CYP3A3/4 substrates, yielding toxicity. The induction of CYP3A3/4 by medications such as carbamazepine can decrease the concentrations of CYP3A3/4 substrates, yielding inefficiency. Thus, knowledge of the substrates, inhibitors, and inducers of CYP3A3/ and other cytochrome P450 isoforms may help clinicians to anticipate and avoid pharmacokinetic drug interactions and improve rational prescribing practices.