International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Progress to Date and Future Plans.

The International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice proposal (INHAND) has been operational since 2005. A Global Editorial Steering Committee manages the overall objectives of the project, and the development of harmonized terminology for each organ system is the responsibility of the Organ Working Groups, drawing upon experts from North America, Europe, and Japan. Great progress has been made with 9 systems published to date--respiratory, hepatobiliary, urinary, central/peripheral nervous systems, male reproductive and mammary, zymbals, clitoral, and preputial glands in Toxicologic Pathology and the integument and soft tissue and female reproductive in the Journal of Toxicologic Pathology as supplements and on a Web site--www.goReni.org. INHAND nomenclature guides offer diagnostic criteria and guidelines for recording lesions observed in rodent toxicity and carcinogenicity studies. The guides provide representative photomicrographs of morphologic changes, information regarding pathogenesis, and key references. The purpose of this brief communication is to provide an update on the progress of INHAND.

Annexin II is a major component of fusogenic endosomal vesicles.

We have used an in vitro assay to follow the proteins transferred from a donor to an acceptor upon fusion of early endosomes. The acceptor was a purified early endosomal fraction immunoisolated on beads and the donor was a metabolically-labeled early endosomal fraction in suspension. In the assay, both fractions were mixed in the presence of unlabeled cytosol, and then the beads were retrieved and washed. The donor proteins transferred to the acceptor were identified by two-dimensional gel electrophoresis and autoradiography. Approximately 50 major proteins were transferred and this transfer fulfilled all criteria established for endosome fusion in vitro. However, only a small subset of proteins was efficiently transferred, if donor endosomes were briefly sonicated to generate small (0.1 micron diam) vesicles before the assay. These include two acidic membrane proteins, and three alkaline peripheral proteins exposed on the cytoplasmic face of the membrane. Partial sequencing and Western blotting indicated that one of the latter components is annexin II, a protein known to mediate membrane-membrane interactions. Immunogold labeling of cryosections confirmed that annexin II is present on early endosomes in vivo. These data demonstrate that annexin II, together with the other four proteins we have identified, is a major component of fusogenic endosomal vesicles, suggesting that these proteins are involved in the binding and/or fusion process.

VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles.

In simple epithelial cells, apical and basolateral proteins are sorted into separate vesicular carriers before delivery to the appropriate plasma membrane domains. To dissect the putative sorting machinery, we have solubilized Golgi-derived transport vesicles with the detergent CHAPS and shown that an apical marker, influenza haemagglutinin (HA), formed a large complex together with several integral membrane proteins. Remarkably, a similar set of CHAPS-insoluble proteins was found after solubilization of a total cellular membrane fraction. This allowed the cloning of a cDNA encoding one protein of this complex, VIP21 (Vesicular Integral-membrane Protein of 21 kD). The transiently expressed protein appeared on the Golgi-apparatus, the plasma membrane and vesicular structures. We propose that VIP21 is a component of the molecular machinery of vesicular transport.

Involvement of the transmembrane protein p23 in biosynthetic protein transport.

Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/micron2 membrane surface area, or approximately 30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment. Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

Regulation of telomerase activity by the p53 family member p73.

The terminal ends of eukaryotic chromosomes, termed telomeres, progressively shorten during each round of cell division eventually leading cells into senescence. Tumor cells typically overcome this barrier to unlimited proliferation by activation of the human telomerase reverse transcriptase (hTERT) gene. In contrast, in most human somatic cells hTERT expression is tightly repressed by multiple tumor suppressors. Here, we studied the regulation of hTERT by the p53 family member p73. We show that forced expression of p73 or activation of endogenous p73 by E2F1 results in the downregulation of telomerase activity. Vice versa, siRNA-mediated knockdown of p73 induces hTERT expression. Responsiveness to p73 is conferred by Sp1 binding sites within the hTERT core promoter. In tumor cells, p73 isoforms lacking the transactivation domain (DeltaNp73) are frequently overexpressed and believed to function as oncogenes. We show that DeltaNp73 antagonizes the repressive effect of the proapoptotic p53 family members on hTERT expression and, in addition, induces hTERT expression in telomerase-negative cells by interfering with E2F-RB-mediated repression of the hTERT core promoter. These data provide evidence that the p73 gene functions as an important regulator of telomerase activity with implications for embryonic development, cellular differentiation and tumorigenesis.

Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy.

Acetylcholine receptor (AChR) supramolecular aggregates that have hitherto only been accessible to examination by electron microscopy were imaged with stimulated emission depletion (STED) fluorescence microscopy, providing resolution beyond limits of diffraction of classical wide-field or confocal microscopes. We examined a Chinese hamster ovary cell liner CHO-K1/A5, that stably expresses adult murine AChR. Whereas confocal microscopy displays AChR clusters as diffraction-limited dots of approximately 200 nm diameter, STED microscopy yields nanoclusters with a peak size distribution of approximately 55 nm. Utilizing this resolution, we show that cholesterol depletion by acute (30 min, 37 degrees C) exposure to methyl-beta-cyclodextrin alters the short and long range organization of AChR nanoclusters on the cell surface. In the short range, AChRs form larger nanoclusters, possibly related to the alteration of cholesterol-dependent protein-protein associations. Ripley's K-test on STED images reveals changes in nanocluster distribution on larger scales (0.5-3.5 microm), which possibly are related to the abolition of cytoskeletal physical barriers preventing the lateral diffusion of AChR nanoclusters.

Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol.

Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Although previous studies have shown that annexin II is involved in early endosome dynamics and organization, the precise biological role of the protein is unknown. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, however, that membrane-associated annexin II could be quantitatively released by low concentrations of the cholesterol-sequestering agents filipin and digitonin. Both treatments released an identical and limited set of proteins but had no effects on other membrane-associated proteins. Among the released proteins, we identified, in addition to annexin II itself, the cortical cytoskeletal proteins alpha-actinin, ezrin and moesin, and membrane-associated actin. Our biochemical and immunological observations indicate that these proteins are part of a complex containing annexin II and that stability of the complex is sensitive to cholesterol sequestering agents. Since annexin II is tightly membrane-associated in a cholesterol-dependent manner, and since it seems to interact physically with elements of the cortical actin cytoskeleton, we propose that the protein serves as interface between membranes containing high amounts of cholesterol and the actin cytoskeleton.

Purification and properties of the squalene-hopene cyclase from Rhodopseudomonas palustris, a purple non-sulfur bacterium producing hopanoids and tetrahymanol.

The squalene-hopene cyclase of the hopanoid- and tetrahymanol-producing Rhodopseudomonas palustris was released from the isolated membranes by CHAPS and purified to homogeneity by successive chromatography on DEAE Sephacel, Octyl Sepharose, and Blue Sepharose. The enzyme has a molecular weight of 70 kDa as determined by SDS-PAGE and an isoelectric point at about pH 5.0. The enzyme activity has a maximum at 30 degrees C and at pH 6.5. No production of tetrahymanol could be demonstrated by using either crude or purified cyclase preparations.

Structure of the anchor-domain of myristoylated and non-myristoylated HIV-1 Nef protein.

Negative factor (Nef) is a regulatory myristoylated protein of human immunodeficiency virus (HIV) that has a two-domain structure consisting of an anchor domain and a core domain separated by a specific cleavage site of the HIV proteases. For structural analysis, the HIV-1 Nef anchor domain (residues 2-57) was synthesized with a myristoylated and non-myristoylated N terminus. The structures of the two peptides were studied by1H NMR spectroscopy and a structural model was obtained by restrained molecular dynamic simulations. The non-myristoylated peptide does not have a unique, compactly folded structure but occurs in a relatively extended conformation. The only rather well-defined canonical secondary structure element is a short two-turn alpha-helix (H2) between Arg35 and Gly41. A tendency for another helical secondary structure element (H1) can be observed for the arginine-rich region (Arg17 to Arg22). Myristoylation of the N-terminal glycine residue leads to stabilization of both helices, H1 and H2. The first helix in the arginine-rich region is stabilized by the myristoylation and now contains residues Pro14 to Arg22. The second helix appears to be better defined and to contain more residues (Ala33 to Gly41) than in the absence of myristoylation. In addition, the hydrophobic N-terminal myristic acid residue interacts closely with the side-chain of Trp5 and thereby forms a loop with Gly2, Gly3 and Lys4 in the kink region. This interaction could possibly be disturbed by phosphorylation of a nearby serine residue, and modifiy the characteristic membrane interactions of the HIV-1 Nef anchor domain.

Psychotherapy in psychosomatic disorders.

Published controlled studies of various psychotherapeutic techniques in psychosomatic disorders with adequate designs have formed the basis for the following conclusions: Psychotherapeutic techniques are effective in some patients with psychosomatic disorders. Some psychosomatic disorders, for example, bronchial asthma, peptic ulcer, and migraine headaches are perhaps more amenable to psychotherapy than others, for example, hypertension and ulcerative colitis. There is evidence to suggest that there are differences between the effectiveness of various psychotherapeutic techniques; a few patients differ perhaps from the rest in that they are helped most by the technique that is less effective for the majority.

Functional somatic symptoms and hypochondriasis. A survey of empirical studies.

Empirical studies suggest the following main conclusions: functional somatic symptoms are extremely common; a large proportion appear to be caused by physiologic activity and tend to be aggravated by emotion. Hypochondriacal patients misunderstand the nature and significance of these symptoms and believe that they are evidence of serious disease. Hypochondriasis can be a part of another syndrome, usually an affective one, or it can be a primary disorder. The prevalence differs between cultures and social classes. Constitutional factors, disease in the family in childhood, and previous disease predispose to hypochondriasis. Various stressors can be precipitating events. Selective perception of symptoms, motivated by fear of disease, and subsequent increase in anxiety with more somatic symptoms appear to be links in the vicious cycle of the hypochondriacal reaction. Psychotherapy as well as psychotropic drugs are effective in the treatment of functional somatic symptoms. There are no adequate controlled studies of psychotherapy in hypochondriasis, and the recommended treatments are based on studies with similar disorders. The prognosis of treated hypochondriasis is good in a substantial proportion of patients.

Hypochondriacal fears and beliefs in medical and law students.

We administered two validated scales of hypochondriacal concerns (the Illness Behavior Questionnaire and the Illness Attitude Scales) to 60 medical students and matched law students. Medical students took more precautions about their health and attended more to somatic symptoms, but the prevalence of hypochondriacal fears, beliefs, and attitudes did not differ significantly between the two groups. Five students (8.3%) in each group scored in the range of patients with hypochondriacal neurosis. Most of the students were free of these concerns. The prevalence of hypochondriacal concerns in medical students was substantially lower than the previously reported incidence over four years of study; this supports the previous observation that most of these reactions are short lived.

Anxiety in schizophrenia. The responses to chlordiazepoxide in an intensive design study.

Six anxious schizophrenic patients who were maintained with phenothiazines participated in a double-blind intensive design study of chlordiazepoxide and placebo for 12 weeks or longer. There were substantial differences between patients in their responses to chlordiazepoxide: two patients experienced significant and conspicuous relief of distress and reduction of typical schizophrenic symptoms. In another patient the differences, although statistically significant, were clinically less striking. In the three remaining patients no differences were observed between responses to the two treatments except that one patient was more depressed with chlordiazepoxide than with placebo. The findings suggest that there are at least two kinds of anxiety in schizophrenia. A few anxious schizophrenic patients apparently benefit more from a combination of a phenothiazine with chlordiazepoxide than from a phenothiazine alone.

Two dosages of imipramine in hospitalized endogenous and neurotic depressives.

Fifty-one newly hospitalized depressed patients participated in a double-blind comparison of two dosage levels of imipramine hydrochloride (150 mg vs 300 mg daily). Although some patients were suffering from neurotic depressions, they, together with the endogenous depressives, were a severely depressed group who required hospitalization. Improvement occurred with both dosage regimens, although a greater and more consistent improvement was noted in the 300-mg group than in the 150-mg group. There were a few differences between the response of the endogenous and that of the neurotic depressives, as assessed by the physician and self=rating scales. However, endogenous depressives who received 150 mg were overrepresented in the treatment failure group. A comparison of the response of deluded and nondeluded depressives indicated that the deluded patients responded less well than the nondeluded depressives, although half of the delusional group did respond to the treatment.

Dose-response study of N,N-dimethyltryptamine in humans. II. Subjective effects and preliminary results of a new rating scale.

BACKGROUND: Validation of animal models of hallucinogenic drugs' subjective effects requires human data. Previous human studies used varied groups of subjects and assessment methods. Rating scales for hallucinogen effects emphasized psychodynamic principles or the drugs' dysphoric properties. We describe the subjective effects of graded doses of N,N-dimethyltryptamine (DMT), an endogenous hallucinogen and drug of abuse, in a group of experienced hallucinogen users. We also present preliminary data from a new rating scale for these effects. METHODS: Twelve highly motivated volunteers received two doses (0.04 and 0.4 mg/kg) of intravenous (IV) dimethyltryptamine fumarate "nonblind," before entering a double-blind, saline placebo-controlled, randomized study using four doses of IV DMT. Subjects were carefully interviewed after resolution of drug effects, providing thorough and systematic descriptions of DMT's effects. They also were administered a new instrument, the Hallucinogen Rating Scale (HRS). The HRS was drafted from interviews obtained from an independent sample of 19 experienced DMT users, and modified during early stages of the study. RESULTS: Psychological effects of IV DMT began almost immediately after administration, peaked at 90 to 120 seconds, and were almost completely resolved by 30 minutes. This time course paralleled DMT blood levels previously described. Hallucinogenic effects were seen after 0.2 and 0.4 mg/kg of dimethyltryptamine fumarate, and included a rapidly moving, brightly colored visual display of images. Auditory effects were less common. "Loss of control," associated with a brief, but overwhelming "rush," led to a dissociated state, where euphoria alternated or coexisted with anxiety. These effects completely replaced subjects' previously ongoing mental experience and were more vivid and compelling than dreams or waking awareness. Lower doses, 0.1 and 0.05 mg/kg, were primarily affective and somaesthetic, while 0.1 mg/kg elicited the least desirable effects. Clustering of HRS items, using either a clinical, mental status method or principal components factor analysis provided better resolution of dose effects than did the biological variables described previously. CONCLUSIONS: These clinical and preliminary quantitative data provide bases for further psychopharmacologic characterization of DMT's properties in humans. They also may be used to compare the effects of other agents affecting relevant brain receptors in volunteer and psychiatric populations.

Prodromal symptoms in affective disorders.

OBJECTIVE: The aim of this paper was to review the clinical and conceptual implications of the studies investigating prodromal symptoms of mania, depression, and panic disorder. METHOD: Twenty-four studies specifically addressing the issue of prodromal symptoms in mood and anxiety disorders were selected by computer search (Medline) and manual search of Index Medicus and the psychiatric literature. RESULTS: Most of the studies have described a prodromal phase in the development of mania, depression, and panic attacks. CONCLUSIONS: The appearance of prodromal symptoms may precede the full syndrome by weeks or months; if these symptoms are detected, recurrences of affective disorders (bipolar illness, unipolar depression, panic disorder) could be treated earlier and perhaps more effectively. DSM-III has emphasized the traditional clinical syndromes and cross-sectional descriptions. Appraisal of prodromal and residual phases may complement this approach. The longitudinal study of prodromes, the fully developed disorder, and residual states calls for an assessment of personality, neurotic traits, and their interaction in the evolution of affective disorders.

Reduction of distress in hyperprolactinemia with bromocriptine.

In a double-blind placebo-controlled crossover study of bromocriptine in eight hyperprolactinemic patients, self-rated distress decreased and well-being increased parallel with the fall in prolactin levels; for the majority of measures the differences were significant.

Changes in chronic nightmares after one session of desensitization or rehearsal instructions.

OBJECTIVE: The purpose of this study was to examine the effects of one session of instructions on the frequency of chronic nightmares and on self-rated distress. METHOD: Twenty-eight volunteers with chronic nightmares (mean duration = 19 years) were treated with either one session of desensitization with instructions on how to practice this treatment or with one session of instructions to change the nightmare and how to rehearse the new version. The authors administered four scales of the SCL-90 and the corresponding scales of the Symptom Questionnaire. RESULTS: At 7-month follow-up of 23 patients, there was a significant reduction in the frequency of nightmares and significant decreases in self-rated depression, anxiety, and hostility. There were no significant differences between the effects of the two types of treatment. In four patients, whose mean duration of nightmares was 23 years, the nightmares ceased. CONCLUSIONS: The results of this preliminary study suggests that the instructions given to the patients reduced the frequency of their chronic nightmares and decreased their self-rated distress.

Hyperprolactinemia, distress, and hostility.

The scores of 14 women with hyperprolactinemia on the Symptom Rating Test and the Symptom Questionnaire were compared with those of nonpsychotic women attending a psychiatric clinic, women attending a family practice clinic, and female nonpatient employees. The scores of the hyperprolactinemic women were similar to those of the psychiatric patients. Hyperprolactinemic patients were significantly more hostile, depressed, and anxious and had more feelings of inadequacy than family practice patients and nonpatient employees. The authors recommend measuring the serum prolactin levels of women with depression, hostility, anxiety, and symptoms or signs suggestive of hyperprolactinemia.

Prolactin, cortisol, and antidepressant treatment.

In four depressed patients with abnormal dexamethasone suppression test results before treatment, plasma prolactin levels significantly increased after successful amitriptyline therapy. Such an increase did not take place in five depressed patients with normal dexamethasone suppression test findings.