Prevalence and genetic characterization of a Babesia microti-like species in the North American river otter (Lontra canadensis).

A 4.5-month-old, male, North American river otter (Lontra canadensis) from Athens-Clarke County, Georgia, USA being temporarily housed at a rehabilitation facility, presented with a three-day history of lethargy, anorexia, and severe anemia. Antemortem blood smears revealed intraerythrocytic piroplasms. Supportive care and antiparasitic treatments were initiated, but the animal died three days following presentation. Gross necropsy revealed yellow discoloration of all adipose tissue throughout the carcass and a mildly enlarged, diffusely yellow to pale orange liver. Microscopically, moderate, centrilobular hepatocellular degeneration and necrosis were observed, consistent with hypoxia secondary to apparent hemolytic anemia. Piroplasms were frequently observed in red blood cells in histologic sections. The nearly full-length 18S rRNA gene sequence (1588 bp) was identical to a previously described piroplasm from North American river otters from North Carolina. Phylogenetically, based on the 18S rRNA gene sequence, the otter Babesia sp. was in a sister group with a clade that included several strains of Babesia microti-like species including Babesia sp. from badgers (Meles meles), Babesia vulpes, and Babesia sp. from raccoons (Procyon lotor). To better understand the distribution and genetic variability of this Babesia species, otters from four states in the eastern U.S. and California were tested. Overall, 30 of 57 (53%) otters were positive for Babesia sp. None of four otters from California were positive, but prevalences in eastern states were generally high, 5/9 (55%) in Georgia, 7/14 (50%) in South Carolina, 10/17 (59%) in North Carolina, and 8/13 (62%) in Pennsylvania). Partial 18S rRNA gene sequences from all populations were identical to the clinical case sequence. No Babesia sensu stricto infections were detected. There were six unique COI sequences (937 bp) detected in 18 positive otters. The most common lineage (A) was detected in 12 of 18 (67%) samples from Georgia, North Carolina, South Carolina, and Pennsylvania. Lineage B was found in two otters and the remaining lineage types were found in single otters. These six lineages were 99-99.8% similar to each other and were < 88% similar to related parasites such as B. vulpes, B. microti-like species of raccoons, B. microti, and B. rodhaini. Phylogenetically, the Babesia sp. of otters grouped together in a well-supported clade separate from a sister group including B. vulpes from fox (Vulpes vulpes) and domestic dogs. In conclusion, this report demonstrates that this piroplasm is a potential pathogen of North American river otters and the parasite is widespread in otter populations in the eastern United States.

A gap approach to exploring quality of life in mental health.

Improving quality of life (QoL) is an important treatment outcome for the serious mentally ill. There is, however, a need for an instrument which both captures consumers own assessments and gives direct information for intervention. A useful approach is to define QoL as the gap between actual and ideal life circumstances, which is weighted by importance. In this paper we detail how we developed and evaluated a QoL instrument which follows this model. This instrument, the 'QoL-GAP', is based on self-appraised items within various life domains. For each item respondents firstly identify what they have (actual) and then what they would like (ideal). They then rate the item for its importance and make any comments. A weighted gap score for each item is subsequently derived from the ideal actual gap being weighted by the importance rating. This weighted gap score is then related to domain satisfaction ratings, while their average from each domain is related to overall satisfaction and well-being. We surveyed 120 individuals with a serious and enduring mental illness living in different types of residences, such as psychiatric hospitals, hostels, or their own homes, in a largely urban part of Queensland. Sixty-eight percent were males, and 92% had schizophrenia or related disorders. We found that our approach demonstrated good psychometric properties, and that the model-based predictions were borne out: weighted gap measures were consistently more strongly related to domain satisfaction than were the actual circumstances alone. While further work is being undertaken--in such matters as short-forms and further evaluation of the QoL-GAP in a longitudinal study--our results suggest that this 'gap' approach helps consumers state their own goals and give their opinions and so is particularly relevant for consumer-focused mental health delivery and research.

Immunocytochemical localization of actin and tubulin in the integument of land crab (Gecarcinus lateralis) and lobster (Homarus americanus).

The crustacean integument consists of the exoskeleton and underlying epithelium and associated tissues. The epithelium, which is composed of a single layer of cells, is responsible for the cyclical breakdown and synthesis of the exoskeleton associated with molting (ecdysis). During premolt (proecdysis) the epithelial cells lengthen and secrete the two outermost layers (epicuticle and exocuticle) of the new exoskeleton while partially degrading the two innermost layers (endocuticle and membranous layer) of the overlying old exoskeleton. This increased cellular activity is associated with increased protein synthesis and a change in cell shape from cuboidal to columnar. The cytoskeleton, composed of microfilaments (actin) and microtubules (tubulin), plays important roles in the intracellular organization and motility of eukaryotic cells. Immunoblot analysis shows that the land crab exoskeleton contains actin, tubulin, and actin-related proteins (Varadaraj et al. 1996. Gene 171:177-184). In the present study, immunocytochemistry of land crab and lobster integument showed that both proteins were localized in various cell types, including epithelia, connective tissue, tendinal cells, and blood vessels. Muscle immunostained for actin and myosin, but not for tubulin. The membranous layer of land crab (the other layers of the exoskeleton were not examined) and membranous layer and endocuticle of lobster also reacted specifically with anti-beta-actin and anti-alpha-tubulin monoclonal antibodies, but not with an anti-myosin heavy chain antibody. During proecdysis immunolabeling of the membranous layer decreased probably due to protein degradation. The staining intensity for actin and tubulin in the proecdysial epithelium was similar to that in the intermolt (anecdysial) epithelium, suggesting that there was a net accumulation of both proteins proportional to the increase in cellular volume. These results support the previous biochemical analyses and, more specifically, localize actin and tubulin in exoskeletal structures, suggesting that they may serve both intracellular and extracellular functions in crustaceans. J. Exp. Zool. 286:329-342, 2000.

Transient expression of the basic helix-loop-helix protein NSCL-2 in the mouse cerebellum during postnatal development.

The spatio-temporal expression of the basic helix-loop-helix transcription factor NSCL-2 was analyzed in the postnatal development of the murine brain by in situ hybridization. We found that NSCL-2 is transiently expressed in the cerebellum. NSCL-2 was found exclusively in the premigratory zone of the external granule layer where postmitotic neurons undergo initial stages of neuronal differentiation. NSCL-2 expression was not detected in mature neurons. This pattern of expression suggests that NSCL-2 is critical for the onset of neuronal differentiation, but not for the maintenance of the differentiated state.

Donation of heart valve tissue: seeking consent and meeting the needs of donor families.

OBJECTIVE: To report on the experiences of senior social workers in seeking consent for donation of heart valve tissue at coronial autopsies. METHODS: Retrospective review of the records of an allograft heart valve program at a tertiary referral hospital in Brisbane between 11 October 1990 and 11 October 1995. RESULTS: Of the 305 families who could be contacted after the sudden death of a family member, 247 consented to donation (81%) and 58 did not (19%). In the context of coronial enquiries, a sensitive assessment of the counselling needs of potential donor families is necessary. Difficulties in seeking consent include: the sudden nature of the death and lack of information about the prospective donor and his/her family; and the limited time available for tissue retrieval as well as the necessity, in most cases, to obtain consent for donation by telephone. The needs of these families included information on autopsy, transplantation and recipient outcome; grief counselling; emotional and practical support; and recognition of their contribution. CONCLUSION: In this successful tissue donation program, senior social workers have maximised the availability of heart valve tissue for donation, while respecting the needs and wishes of potential donor families and helping them with their loss through counselling and assistance. The program has been extended to the retrieval of eye and bone tissue at coronial autopsies in Queensland.

Changes in the structure and function of the multicatalytic proteinase (proteasome) during programmed cell death in the intersegmental muscles of the hawkmoth, Manduca sexta.

The intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta are a well-characterized model system for examining the biochemical changes that accompany programmed cell death during development. These giant muscles die during a 30-hr period in response to a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone. When the ISMs become committed to die, there are dramatic increases in both ubiquitin expression and ubiquitin-dependent proteolysis. Since the multicatalytic proteinase (MCP) is responsible for ATP/ubiquitin-dependent proteolysis in cells, we examined its composition and properties. The purified enzyme from whole larval integumentary tissues resembles MCPs isolated from other species with respect to subunit composition and general catalytic properties. However, when MCP was isolated from condemned ISMs, we observed an approximately ninefold increase in proteinase activity compared to MCP from precommitment muscles. This increase in proteolytic activity was correlated with an approximately eightfold increase in the absolute amounts of MCP protein as determined by Western blotting and densitometry. When purified MCP from condemned muscles was examined by two-dimensional polyacrylamide gel electrophoresis, four new subunits that were not detected in the precommitment muscles were present. Correlated with the addition of these new subunits was a dramatic increase in the levels of immunodetectable MCP throughout the cytoplasm and within the nuclei of dying muscles. These changes in MCP were regulated by the same hormonal signals that mediate cell death. These data are consistent with the hypothesis that when the ISMs become committed to die, more MCP accumulates in cells and new subunits are synthesized that change both the enzymatic properties and the conformation of MCP, which in turn participates in the dramatic proteolysis that accompanies cell death.

The multicatalytic proteinase (proteasome) of the hawkmoth, Manduca sexta: catalytic properties and immunological comparison with the lobster enzyme complex.

The proteasome plays a central role in ubiquitin-dependent and -independent proteolysis in eukaryotic cells. The hawkmoth proteasome was purified from larval body wall and characterized with respect to substrate specificity, sensitivity to protease inhibitors, and cross-reactivity with monoclonal antibodies (mAbs) raised against human placenta proteasome. Leupeptin selectively inhibited the trypsin-like activity (T-L) and N-ethylmaleimide inhibited both T-L and chymotrypsin-like activities, whereas 0.02% sodium dodecyl sulfate stimulated the peptidylglutamyl peptide hydrolase, branched-chain amino acid preferring, and caseinolytic activities 20-, 18-, and 3.8-fold, respectively. All four peptidase activities were inhibited by 3,4-dichloroisocoumarin. One-dimensional immunoblot analysis showed that the level and subunit composition of the proteasome varied between tissues. The relative levels of proteasome were high in intersegmental muscle and ovary, lower in Malpighian tubule, male accessory gland, and ventral nerve cord, and lowest in flight muscle and fat body. The tissues differed in the relative amount of a 41-kDa doublet; a 22-kDa subunit was present only in the male accessory gland. Two-dimensional polyacrylamide gel electrophoresis showed that the hawkmoth proteasome contained at least 26 subunits, compared with 28 subunits in lobster. Immunological analysis using four subunit-specific mAbs identified the putative homologs of the human zeta, C2, C3, and C8 alpha-type subunits in the hawkmoth and lobster enzymes. Two of the four mAbs reacted with three or more of the hawkmoth subunits and three of the mAbs reacted with two or more of the lobster subunits. In addition, two other mAbs that recognize epitopes shared by a number of alpha-type subunits indicated that at least 15 (lobster) or 16 (hawkmoth) subunits were alpha-type. These results suggest that much of the subunit complexity of the arthropod proteasomes is a consequence of extensive post-translational modifications.

Branched-chain-amino-acid-preferring peptidase activity of the lobster multicatalytic proteinase (proteasome) and the degradation of myofibrillar proteins.

The multicatalytic proteinase (MCP or proteasome) is a large proteolytic complex that contains at least five catalytic components: the trypsin-like, chymotrypsin-like, peptidylglutamyl-peptide hydrolase (PGPH), branched-chain-amino-acid-preferring (BrAAP) and small-neutral-amino-acid-preferring activities. We have shown that brief heating of the lobster muscle proteasome activates a proteolytic activity that degrades casein and myofibrillar proteins and is distinct from the trypsin-like, chymotrypsin-like and PGPH components. Here we identify the BrAAP activity as a catalytic component involved in the initial degradation of myofibrillar proteins in vitro. This conclusion is based on the following. (1) The BrAAP component was activated by heat-treatment, whereas the other four peptidase activities were not. (2) The BrAAP and proteolytic activities showed similar sensitivities to cations and protease inhibitors: both were inhibited by 3,4-dichloroisocoumarin, chymostatin, N-ethylmaleimide and Mg2+, but were not affected by leupeptin, phenylmethanesulphonyl fluoride or Li+. (3) The BrAAP activity was inhibited most strongly by casein substrates and troponin; conversely, the troponin-degrading activity was inhibited by the BrAAP substrate. Another significant finding was that incubation of the heat-activated MCP in the presence of chymostatin resulted in the limited cleavage of troponin-T2 (45 kDa) to two fragments of 41 and 42 kDa; this cleavage was completely suppressed by leupeptin. These results suggest that under certain conditions the trypsin-like component can cleave endogenous protein.

Sodium dodecyl sulfate and heat induce two distinct forms of lobster muscle multicatalytic proteinase: the heat-activated form degrades myofibrillar proteins.

A multicatalytic proteinase (MCP) purified from lobster claw and abdominal muscles degrades a variety of peptide and protein substrates. The enzyme is activated by low concentrations (0.03%) of sodium dodecyl sulfate (SDS) and brief (1 min) heating at 60 degrees C. The lobster MCP can assume three stable and functionally distinct states in vitro; these are classified as the basal, heat-activated, and SDS-activated forms. The basal MCP possessed high trypsin-like peptidase activity and low chymotrypsin-like peptidase, peptidylglutamyl-peptide hydrolase, and caseinolytic activities; incubation of the basal form with SDS stimulated the peptidylglutamyl-hydrolase activity about 30-fold and inhibited the other three activities 80% to 100%. Heating the basal form stimulated caseinolytic activity about 6-fold with little effect on the peptidase activities. The heat-activated enzyme also degraded myosin, tropomyosin, troponin, and actin depolymerizing factor; alpha-actinin was resistant to proteolysis. Incubation of the heat-activated MCP with SDS inhibited the trypsin-like, chymotrypsin-like, and proteinase activities 95 to 100% and stimulated the peptidylglutamyl-hydrolase activity about 16-fold. Incubation of myosin with either the basal or the heat-activated forms in the presence of SDS generated identical proteolytic fragments of the myosin heavy chain, suggesting that SDS induced a third form that can be produced from either the basal or the heat-activated forms. The heat-activated form produced proteolytic fragments of myosin heavy chain different from those generated by either basal or heat-activated enzymes in the presence of SDS. Furthermore, 100 mM KCl stimulated the caseinolytic activity of the heat-activated form 24% and inhibited the trypsin-like and peptidylglutamyl-hydrolase activities 56 and 20%, respectively. These results, though indirect, suggest that heating induced a proteinase activity that was distinct from the three peptidase activities. Activation of the basal form with SDS was reversible, since precipitation of dodecyl sulfate with 100 mM KCl restored trypsin-like activity and inhibited peptidylglutamyl-hydrolase activity. In contrast, removal of dodecyl sulfate from the SDS-activated form that was derived from the heat-activated MCP induced its conversion to the basal form. Thus, although heat-activation was irreversible, the heat-activated form was converted back to the basal form via the SDS-activated form.

Three-dimensional architecture of identified cerebral neurosecretory cells in an insect.

The organization of identified neurosecretory cell groups in the larval brain of the tobacco hornworm, Manduca sexta, was investigated immunocytologically. Computer-assisted three-dimensional reconstruction was used to examine the architecture of the neurosecretory cell groups. The group III lateral neurosecretory cells (L-NSC-III) which produce the prothoracicotropic hormone are located dorsolaterally in the protocerebrum and extend axons medially that decussate to the contralateral lobe prior to exiting the brain through the nervi corporis cardiaci I + II. The group IIa2 medial neurosecretory cells (M-NSC IIa2) are located anteriorly in the medial dorsal protocerebrum. The axons of these cells also exit the brain via the contralateral nervi corporis cardiaci I + II. However, their axons traverse a different pathway through the brain from that of the L-NSC III axons. Each of the cell groups possesses elaborate dendrites with terminal varicosities. The dendrites can be classified into specific fields based upon their location and projection pattern within the brain. The dendrites for these two neurosecretory cell groups overlap in specific regions of the protocerebral neuropil. After the axons of these neurosecretory cells exit the brain through the retrocerebral nerve, they innervate the corpus allatum where they arborize to form neurohemal terminals in strikingly different patterns. The L-NSC III penetrate throughout the glandular structure and the M-NSC IIa2 terminals are restricted to the external sheath. A third group of cerebral neurosecretory cells, the ventromedial neurons (VM) which stain with the monoclonal antibody to prothoracicotropic hormone in Manduca, are located anteriorly in the medial region of the brain. The axons of these cells do not exit the brain to the retrocerebral complex, but rather pass through the circumesophageal connectives and ventral nerve cord. These neurons appear to be the same VM neurons that produce eclosion hormone. One dendritic field of the L-NSC III terminates in close apposition to the VM neurons. The distinct morphologies of these neurosecretory cell groups in relation to other cell groups and the distribution of neuropeptides within the neurons suggest that insect neurosecretory cells, like their vertebrate counterparts, may have multiple regulatory roles.

Differential effects of oleic acid, sodium dodecyl sulfate, and protease inhibitors on the endopeptidase activities of the lobster multicatalytic proteinase.

1. Lobster muscles contain a latent multicatalytic proteinase; heating at 60 degrees C for 1-2 min converts the latent form to a heat-activated form with enhanced proteolytic activity. Both forms have three endopeptidase activities, which are classified as the trypsin-like, chymotrypsin-like, and peptidylglutamylpeptide bond hydrolyzing activities. 2. Sulfhydryl reagents (mersalyl acid, N-ethylmaleimide, hemin, iodoacetamide, and p-chloromercurisulfonic acid), benzamidine, and chloromethyl ketones inhibited all three activities of the heat-activated form. Leupeptin and antipain inhibited only the trypsin-like activity, while the chymotrypsin-like activity was the most sensitive to diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, aprotinin, and soybean trypsin inhibitor. Pepstatin and L-trans-epoxysuccinylpeptides had little effect on the peptidase activities. 3. Sodium dodecyl sulfate and oleic acid preferentially activated the peptidylglutamyl-peptide hydrolyzing activity of the latent form, whereas N-ethylmaleimide stimulated both the trypsin-like and peptidylglutamyl-peptide hydrolases. These results suggest that the lobster enzyme is an atypical serine proteinase.

T-cell-mediated suppression of Epstein-Barr virus-induced B lymphocyte activation in multiple sclerosis.

Significantly reduced levels of T-cell-mediated "suppression" of numbers of Epstein-Barr virus (EBV)-activated B lymphocytes which secrete IgG or IgM were found in preparations of mononuclear cells (MNC) from peripheral blood of 28 patients with multiple sclerosis (MS) compared with 28 healthy subjects, all seropositive for EBV. A statistically significant correlation was not found between the suppression of EBV-induced activation of B lymphocytes and either EBV-specific T-cell-mediated cytotoxicity or numbers of T8-positive cells.

Viral antibody titers. Comparison in patients with multiple sclerosis and rheumatoid arthritis.

Higher titers of antibodies to measles virus envelope antigens, hemolysin and hemagglutinin, Epstein-Barr virus (EBV) capsid antigen and nuclear antigen, and rubella virus hemagglutinin were demonstrated in serum samples of patients with multiple sclerosis and rheumatoid arthritis than in age- and sex-matched control subjects. A significant correlation was observed between antibodies to measles and rubella viruses both in patients with multiple sclerosis and rheumatoid arthritis, but such a correlation was not observed between antibodies to EBV and measles or rubella viruses. Whether elevated levels of antibodies to EBV are due to reactivation of the virus, or elevated levels of antibodies to all the enveloped viruses result from cross-reactions between viruses and host tissue, or perhaps reflect defects in immunoregulation, needs further investigation.

Diversity of autoantibodies in patients with antimitochondrial antibody and their diagnostic value.

A retrospective analysis of clinical and laboratory features of 102 patients, whose sera contained antibody to mitochondria, showed that primary biliary cirrhosis was diagnosed in 50% of them. Immunofluorescence showed that the sera of the patients with primary biliary cirrhosis all had the M2 antimitochondrial antibody staining pattern. A new staining pattern, designated M2(1), which could be mistaken for the M2 pattern, was not found in any patients with either primary biliary cirrhosis or chronic active hepatitis. Other serological variables such as antibody to mitochondria in IgM class, to multiple nuclear dots, and to the XR antigen, were associated with primary biliary cirrhosis, and taken in association with antimitochondrial antibody of M2 type, contribute to the diagnosis of the disease.