Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.     

Psychosexual development of women with congenital adrenal hyperplasia

Women with congenital adrenal hyperplasia (CAH) (N = 31) and their unaffected sisters or female cousins (N = 15) participated in a study of psychosexual development. All participants were > or = 18 years of age (mean age, 25 years; range, 18-40). Comparisons were also made between the CAH women with the salt-wasting (SW) form of the disorder and those with simple virilization (SV). A psychosexual assessment protocol examined six variables: (1) sex assignment at birth (probands only); (2) recalled sex-typed behavior during childhood; (3) gender identity and gender role identification in adulthood; (4) relationship status; (5) sexual orientation in fantasy; and (6) sexual orientation in behavior. Salt-wasting status and sex assignment at birth were also ascertained for the CAH women who either refused to participate in the study (N = 10) or could not be traced (N = 13). Compared to the controls, the women with CAH recalled more cross-gender role behavior and less comfort with their sense of "femininity" during childhood. The two groups did not differ in degree of gender dysphoria in adulthood, although the probands showed more cross-gender role identification. Three of the nonparticipant probands were living, as adults, in the male social role (2 reared from birth as boys and 1 who changed from the female to the male social role during adolescence). The CAH women and the controls did not differ in relationship status (married/cohabiting vs. single). The CAH women had lower rates of exclusive heterosexual fantasy and fewer sexual experiences with men than the controls; however, the CAH women did not have more sexual experiences with women than the controls. Comparisons between the SW and SV revealed several differences: the SW were less likely to be assigned to the female sex at birth, recalled more cross-gender role behavior during childhood, were less likely to be married or cohabiting, and had lower rates of sexual experiences with men. The results were discussed in relation to the effects of prenatal androgens on psychosexual differentiation.     

Crystal growth in dental enamel: the role of amelogenins and albumin

Amelogenin-mineral interactions were investigated using an in vitro binding approach. Rat incisor enamel matrix proteins (mainly amelogenins) were dissolved in synthetic enamel fluid and allowed to equilibrate with deproteinised developing enamel crystals. The results showed that amlogenin proteins of 21, 23, 24, 26 and 27-kDa (corresponding to nascent and partially degraded amelogenins) were associated with the crystals whilst the lower Mr amelogenins (< 21 KDa) remained free in the synthetic enamel fluid. These data suggest the nascent and partially degraded amelogenins may interact with developing enamel crystals and could influence their growth. Albumin-mineral interactions were investigated by extracting developing rat incisor enamel with synthetic enamel fluid. Insoluble material (including the enamel crystals) was then further extracted with 0.1 M phosphate buffer (pH 7.4) to desorb any mineral bound proteins. Western blotting using anti-albumin antibodies showed that almost all of the albumin from the secretory stage enamel and a significant proportion of the albumin present in early transition stage was extractable in the synthetic enamel fluid. However, synthetic enamel fluid did not extract albumin from late transition or maturation stage tissue, which could only be removed following further extraction with phosphate buffer. Albumin degradation was apparent during the transition and maturation stages, where it is degraded and ultimately removed. This binding pattern may be related to amelogenin degradation and removal during the transition stage, permitting albumin access to the previously obscured crystal surfaces. That the secretory stage matrix appears to "protect" secretory stage crystals from albumin may be an important consideration in the aetiology of enamel hypoplasias (i.e. incomplete crystal growth) and when using dissociative extraction procedures for the identification of mineral bound proteins.     

Technetium-99m labelling of the anti-tumour antibody PR1A3 by photoactivation

Irradiation of antibody with ultraviolet light leads to reduction of disulphide bonds. Thus irradiation can be used to generate free thiols prior to direct labelling of antibody with technetium-99m, and has a potential advantage over methods using chemical reducing agents such as mercaptoethanol or tin, in that no purification step is needed to remove excess reducing agent. We have used the photoactivation method developed by Sykes et al. to label the anti-tumour antibody PR1A3 with 99mTc. The antibody was irradiated at 300 nm using a Rayonet photochemical reactor with eight RMR3000 lamps. In a typical experiment, the antibody solution was injected into a nitrogen-filled borosilicate glass vial and purged with nitrogen. A degassed solution containing stannous fluoride and methylene diphosphonate was then added to the antibody and the vial was irradiated. Following the irradiation, [99mTc]pertechnetate was injected into the vial and the reaction mixture was incubated for 30 min at room temperature before being analysed by size-exclusion high-pressure liquid chromatography and instant thin-layer chromatography. Labelling yields greater than 95% were obtained using antibody concentrations ranging from 0.5mg/ml to 5mg/ml. Irradiation times as short as 5 min and tin to antibody ratios in the range between 11 and 32 microg tin per mg antibody gave high labelling yields. Labelling yields greater than 95% were obtained after storage of the photoactivated antibody at -70 degrees C for several weeks. The stability of the 99mTc-labelled photoactivated PR1A3 was similar to that of 99mTc-labelled mercaptoethanol-reduced PR1A3. The mean immunoreactive fraction was 77% for the photoactivation-labelled PR1A3, compared to 93% for PR1A3 labelled by mercaptoethanol reduction. Biodistribution studies were carried out using 99mTc-photoactivation-labelled PR1A3 or PR1A3 labelled by mercaptoethanol reduction in Balb/c mice and in nude mice with MKN-45 human tumour xenografts. There was no significant difference in tumour uptake between the mice that received photoactivated PR1A3 and those that received mercaptoethanol-reduced PR1A3. There was also no significant difference in uptake in most organs in Balb/c mice; however, the photoactivated antibody cleared more rapidly from the blood, and whole-body clearance was also faster for the photoactivated PR1A3. In conclusion, the photoactivation technique provides a very convenient "one-pot" method for labelling antibodies with 99mTc.     

Cloning of human cDNA encoding a novel heptahelix receptor expressed in Burkitt's lymphoma and widely distributed in brain and peripheral tissues

Using PCR with degenerate primers and screening of a human B-cell lymphoblast cDNA library, a full-length cDNA encoding a 375-amino-acid protein was isolated. It contains seven regions of hydrophobic amino acids probably representing membrane-spanning domains of a novel heptahelix receptor, tentatively named CMKRL2. It shows nearly 30% overall identity with the high-affinity IL8 receptor and similar degree of homology with other chemoattractant receptors, including the "fusin" coreceptors for HIV1. Measurements of various transduction pathways following application of a panel of chemokines to transfected cells failed to evoke any reproducible response. Although the natural ligand for CMKRL2 could, thus, not be identified, receptor expression in spleen and lymph nodes as well as in Burkitt's lymphoma (irrespective of EBV status) supports a functional role in activated B-cells. Receptor message was ubiquitously distributed in normal peripheral tissues and CNS, suggesting that CMKRL2 is expressed in widespread cell populations, such as macrophages and neuroglia.     

Evaluation of amplitude-based sorting algorithm to reduce lung tumor blurring in PET images using 4D NCAT phantom

PURPOSE: develop and validate a PET sorting algorithm based on the respiratory amplitude to correct for abnormal respiratory cycles. METHOD AND MATERIALS: using the 4D NCAT phantom model, 3D PET images were simulated in lung and other structures at different times within a respiratory cycle and noise was added. To validate the amplitude binning algorithm, NCAT phantom was used to simulate one case of five different respiratory periods and another case of five respiratory periods alone with five respiratory amplitudes. Comparison was performed for gated and un-gated images and for the new amplitude binning algorithm with the time binning algorithm by calculating the mean number of counts in the ROI (region of interest). RESULTS: an average of 8.87+/-5.10% improvement was reported for total 16 tumors with different tumor sizes and different T/B (tumor to background) ratios using the new sorting algorithm. As both the T/B ratio and tumor size decreases, image degradation due to respiration increases. The greater benefit for smaller diameter tumor and lower T/B ratio indicates a potential improvement in detecting more problematic tumors.     

A Dialectal Chinese Speech Recognition Framework

A framework for dialectal Chinese speech recognition is proposed and studied, in which a relatively small dialectal Chinese (or in other words Chinese influenced by the native dialect) speech corpus and dialect-related knowledge are adopted to transform a standard Chinese (or Putonghua, abbreviated as PTH) speech recognizer into a dialectal Chinese speech recognizer. Two kinds of knowledge sources are explored: one is expert knowledge and the other is a small dialectal Chinese corpus. These knowledge sources provide information at four levels: phonetic level, lexicon level, language level, and acoustic decoder level. This paper takes Wu dialectal Chinese (WDC) as an example target language. The goal is to establish a WDC speech recognizer from an existing PTH speech recognizer based on the Initial-Final structure of the Chinese language and a study of how dialectal Chinese speakers speak Putonghua. The authors propose to use context-independent PTH-IF mappings (where IF means either a Chinese Initial or a Chinese Final), context-independent WDC-IF mappings, and syllable-dependent WDC-IF mappings (obtained from either experts or data), and combine them with the supervised maximum likelihood linear regression (MLLR) acoustic model adaptation method. To reduce the size of the multi-pronunciation lexicon introduced by the IF mappings, which might also enlarge the lexicon confusion and hence lead to the performance degradation, a Multi-Pronunciation Expansion (MPE) method based on the accumulated uni-gram probability (AUP) is proposed. In addition, some commonly used WDC words are selected and added to the lexicon. Compared with the original PTH speech recognizer, the resulting WDC speech recognizer achieves 10-18% absolute Character Error Rate (CER) reduction when recognizing WDC, with only a 0.62% CER increase when recognizing PTH. The proposed framework and methods are expected to work not only for Wu dialectal Chinese but also for other dialectal Chinese languages and even other languages.     

Suggestibility of the child witness: a historical review and synthesis

The field of children's testimony is in turmoil, but a resolution to seemingly intractable debates now appears attainable. In this review, we place the current disagreement in historical context and describe psychological and legal views of child witnesses held by scholars since the turn of the 20th century. Although there has been consistent interest in children's suggestibility over the past century, the past 15 years have been the most active in terms of the number of published studies and novel theorizing about the causal mechanisms that underpin the observed findings. A synthesis of this research posits three "families" of factors--cognitive, social, and biological--that must be considered if one is to understand seemingly contradictory interpretations of the findings. We conclude that there are reliable age differences in suggestibility but that even very young children are capable of recalling much that is forensically relevant. Findings are discussed in terms of the role of expert witnesses.     

Identification of two distinct properties of class II major histocompatibility complex-associated peptides

We have examined the interactions of various peptides with the mouse class II major histocompatibility complex molecule I-Ak. The peptides were derived from the model protein hen egg white lysozyme (HEL). The immunodominant peptide of HEL is a 10-mer, residues 52-61. Our previous work established that this sequence contains the key residues for binding and presentation to T cells. Now we show that the binding of this 10-mer sequence resulted in complexes of I-Ak and peptide that, in SDS/PAGE (without boiling the protein), rapidly dissociated from the component alpha and beta chains. The binding interactions were studied in vitro, by incubating purified I-Ak and radiolabeled peptide, or ex vivo, by using antigen-presenting cells incubated with peptides. Peptides with additional residues at either the amino or carboxyl terminus behaved dramatically differently. Complexes of I-Ak with the longer peptides were stable to SDS/PAGE. Very few amino acid additions result in the change from unstable to stable complexes. The important issue here is that when cultured with HEL, antigen-presenting cells selected the HEL peptides containing the 52-61 sequences that favored stability [Nelson, C. A., Roof, R. W., McCourt, D. W. & Unanue, E. R. (1992) Proc. Natl., Acad. Sci. USA 89, 7380-7383]. Also, from other studies, such sequences correlate with a high immunogenicity of the peptide. We conclude that there are structural features of peptides that change the stability of the class II molecule and that are independent of the "core" peptide seen by the T cells.     

Pancreatic xanthomatous neuropathy associated with hyperlipidemia: a cause of abdominal pain mimicking chronic pancreatitis

"Hyperlipidemic crisis" is a term used to describe episodic abdominal pain in patients with hyperlipidemia. The morphologic correlates of this phenomenon have not been investigated and the etiology of the disorder is uncertain. We report a unique histologic finding in the pancreas of a 34-year-old woman with a 17-year history of episodic abdominal pain, sometimes accompanied by hyperamylasemia. At the age of 18 years, grossly elevated cholesterol and triglyceride levels were documented and type V hyperlipidemia was diagnosed. At the age of 34 years, subtotal pancreatectomy was performed for intractable abdominal pain. Histologic examination identified an increased number of enlarged pancreatic nerves that were infiltrated by foamy macrophages and encircled by fibrous tissue; endoneurial infiltration with macrophages occasionally split nerves into individual fascicles. Otherwise, the pancreas had only minimal fibrosis, nesidioblastosis, mucinous metaplasia of some pancreatic ducts, and scattered small collections of chronic inflammatory cells, subtle features suggesting very mild chronic pancreatitis. We propose that this novel xanthomatous neuropathy mimicked pancreatitis and was one of the underlying pathophysiologic mechanisms of abdominal pain in this patient. Further studies are necessary to document the prevalence of this new entity in patients with hyperlipidemia and to correlate its occurrence with "hyperlipidemic crisis" in those individuals.