Mutation analysis of feline Niemann-Pick C1 disease

Niemann-Pick C (NPC) disease is an autosomal recessive neurovisceral lysosomal storage disorder that results in defective intracellular transport of cholesterol. The major form of human NPC (NPC1) has been mapped to chromosome 18, the NPC1 gene (NPC1) has been sequenced and several mutations have been identified in NPC1 patients. A feline model of NPC has been characterized and is phenotypically, morphologically, and biochemically similar to human NPC1. Complementation studies using cultured fibroblasts from NPC affected cats and NPC1 affected humans support that the gene responsible for the NPC phenotype in this colony of cats is orthologous to human NPC1. Using human-based PCR primers, initial fragments of the feline NPC cDNA were amplified and sequenced. From these sequences, feline-specific PCR primers were generated and designed to amplify six overlapping bands that span the entire feline NPC1 open reading frame. A single base substitution (2864G-C) was identified in NPC1 affected cats. Obligate carriers are heterozygous at the same allele and a PCR-based assay was developed to identify the geneotype of all cats in the colony. The mutation results in an amino acid change from cysteine to serine (C955S). Several of the mutations identified in people occur in the same region. Marked similarity exists between the human and feline NPC1 cDNA sequences, and is greater than that between the human and murine NPC1 sequences. The human cDNA sequence predicts a 1278aa protein with a lysosomal targeting sequence, several trans-membrane domains and extensive homology with other known mediators of cholesterol homeostasis.     

Two new species of Eimeria Schneider 1875 (Apicomplexa: Eimeriidae) from the broad-toothed field mouse, Apodemus mystacinus Danford and Alston 1877 (Rodentia: Muridae) from Jordan

Coprological examination of 40 Apodemus mystacinus Danford and Alston 1877 from Jordan revealed oocysts of three species of genus Eimeria. Two species are described as new. Eimeria zuhairamri sp. n. has broadly ellipsoidal oocysts 29.6 (27.0–34.0)×23.3 (22.0–25.0) m with distinctly granulated wall and oocyst residuum. Endogenous development occurs in jejunum and ileum. Eimeria alorani sp. n. has oocysts 26.9 (23.0–29.0)×19.3 (18.0–22.0) m with smooth wall and absent residuum. Endogenous development is confined to the caecum. The third species, developing in jejunum, has oocysts morphologically indistinguishable from Eimeria uptoni. The identity of E. uptoni and the taxonomy of Eimeria of Apodemus are discussed.     

An improved method for the detection of HIV antigen in the blood of carriers

The measurement of HIV antigen levels in sera or plasma of HIV-infected individuals is critical for determining the existence of antigen or infectious virus before seroconversion and for prognosis. Pretreatment of sera or plasma of HIV carriers by heating at 70 degrees C for 10 min at an acidic pH enabled us to estimate antigens efficiently in immune complexes. This procedure will also be useful in determining antigen levels in HIV carriers more precisely.     

Comparison of Classical and Clozapine Treatment on Schizophrenia Using Positive and Negative Syndrome Scale of Schizophrenia (PANSS) and SPECT Imaging

Many neuroimaging studies of schizophrenia have shown abnormalities in the frontal cortex, limbic system, basal ganglia, temporal and parietal lobes. These findings are not specific or consistent enough to build up a coherent theory of the origin of the brain abnormality in schizophrenia. This paper describes a state-of-the-art approach of SPECT to correlate neuropsychological evaluation. PANSS scores and different brain focal abnormalities of two groups of patients receiving Clozapine and classical antipsychotic treatments were observed. A total of 20 drug-free patients, actively psychotic schizophrenic, were selected according to the DSM-IV criteria. Pre-Post-treatment was designed using PANSS and 99mTc- ECD-SPECT to assess regional Cerebral Blood Flow (rCBF). The results showed that after treatment, differences in PANSS scores were significant in both groups, with superior scores resulting from the Clozapine therapy. Results were supported by SPECT, which showed a greater improvement in the Clozapine group. Both positive and negative symptoms were improved with Clozapine as well. Before treatment, hypofrontality was the most common (85%) finding, whereas after treatment hypofrontality was mostly cleared. However, in some areas like temporal and caudate, hyperfrontality was induced. Negative symptoms showed linkage to hypofrontality in both groups before and after treatment, and both positive and negative symptoms were improved more with Clozapine therapy than with classical treatment.     

Antibodies against the extracellular enveloped virus B5R protein are mainly responsible for the EEV neutralizing capacity of vaccinia immune globulin

In the event of smallpox bioterrorism, widespread vaccination may be required. Vaccinia immune globulin (VIG) has been used to treat complications from the smallpox vaccine. While the potency of VIG was defined by its ability to neutralize intracellular mature virus, a second form of vaccinia called the extracellular enveloped virus (EEV) is critical for virus spread in the host. The B5R-protein is one of many EEV-specific proteins. Immunoprecipitation and ELISA revealed that VIG recognizes the B5R-protein. An EEV plaque-reduction assay using a recombinant vaccinia that lacks the majority of the extracellular domain of B5R showed that the ability of VIG to neutralize EEV is principally directed at B5R. In addition, absorbing out the anti-B5R antibody present in VIG through the addition of recombinant B5R protein abrogated VIG's ability to significantly neutralize wild-type EEV. This work demonstrates the prominent role of B5R as a target of EEV-neutralizing activity of human antibodies.     

A Strain Device Imposing Dynamic and Uniform Equi-Biaxial Strain to Cultured Cells

The objective of this study is to design a new apparatus to allow the control of the magnitude and frequency of dynamic stretch applied uniformly to cells cultured on a silicon elastic membrane. The apparatus is designed to produce equi-biaxial dynamic stretches with area changes ranging from 0% to 55% and frequencies ranging from 0 to 2 Hz. Homogeneous finite strain analysis using triangles of markers was performed to compute the symmetric two-dimensional Lagrangian strain tensor on the membrane. Measurements of strain in both static and dynamic conditions showed that the shear component of the strain tensor (E_rc) was near zero, and that there was no significant difference between radial (E_rr) and circumferential (E_cc) components, indicating the attainment of equi-biaxial strain. Bovine aortic endothelial cells were transiently transfected with a chimeric construct in which the luciferase reporter is driven by TPA-responsive elements (TRE). The transfected cells cultured on the membrane were stretched. The luciferase activity increased significantly only when the cells were stretched by 15% or more in area. Cells in different locations of the membrane showed similar induction of luciferase activities, confirming that strain is uniform and equi-biaxial across the membrane. © 1998 Biomedical Engineering Society. PAC98: 8780+s, 8745-k, 8722-q     

The role of COX-2 in angiogenesis and rheumatoid arthritis

Recent evidence suggests that cyclooxygenase (COX)-2 is a mediator of angiogenesis, and COX-2 activity is known to be upregulated in the rheumatoid arthritis (RA) synovium. We examined whether mediation of angiogenesis by COX-2 was occuring in cells of the RA synovium and in microvascular endothelial cells (ECs) that are similar to those found in the RA synovium. We demonstrate that rofecoxib, a selective COX-2 inhibitor, acts directly on human dermal microvascular ECs (HMVECs) to inhibit their chemotactic and tube forming ability. Likewise, pretreatment of HMVECs with rofecoxib significantly inhibited their ability to form tubes induced by conditioned media (CM) of activated RA synovial fibroblasts. When RA synovial fibroblasts were pretreated with rofecoxib for 16 h and then stimulated with interleukin (IL)-1beta, their CM induced significantly less HMVEC tube formation when compared with CM from vehicle-treated RA synovial fibroblasts. ELISAs performed on activated RA fibroblast CM for known proangiogenic factors demonstrated a significant reduction in bFGF, in addition to the expected decrease in PGE(2). Our studies suggest that COX-2-induced angiogenic activity is an active mechanism within diseased synovium and may provide an additional rationale for the use of COX-2 inhibitors in RA.     

Influence of age on iminodipropionitrile-induced vestibular and neurobehavioral toxicities in rats

A direct association between aging and drug-induced dyskinesia has been reported by several investigators. Iminiodipropionitrile (IDPN), a prototype nitrile compound produces a motor syndrome in rodents, which resembles neuroleptic drug induced dyskinesia. In this investigation attempt has been made to study the effect of age on IDPN induced vestibular hair cell degeneration and resulting dyskinetic syndrome. Male Wistar rats aged 3, 6 and 12 weeks received IDPN in the doses of 0, 200 and 400 mg/kg, intraperitoneally for 3 consecutive days. IDPN-induced dyskinesia was assessed using a behavioral testing battery on days 3, 4, 5, 6, 7, 14, 21 and 28. The rats were sacrificed on day 28; temporal bones were excised for vestibular histopathology and sera were collected for measuring the indices of oxidative stress (glutathione and conjugated dienes). IDPN in the dose of 200 mg/kg produced dyskinesia in 12 weeks old rats, but failed to do so in 3 and 6 weeks old rats. The high dose of IDPN (400 mg/kg) caused dyskinesia in all age groups, however, its onset and severity were age-dependent. Older rats showed an early onset and significantly high incidence of dyskinesia as compared to younger rats. The susceptibility of rats to IDPN-induced behavioral deficits was proportional to oxidative stress and degeneration of sensory hair cells in the crista ampullaris.     

Correlation between the number of apoptotic cells and expression of the apoptosis-related antigens Fas, Ley and bcl-2 protein in non-Hodgkin's lymphomas

The relationship between the number of apoptotic cells and the expression of apoptosis-related antigens was examined In 56 cases of non-Hodgkin's lymphomas and in 10 cases of reactive hyperplastic lymph nodes (RHL). Apoptosis was visually quantified by the in situ end-labeling (ISEL) method, and the expression of Fas, Le^y antigens and bcl-2 protein was examined by Immunohistochemistry. The expression of Le^y antigen was observed in germinal centers of RHL and 45% of non-Hodgkin's lymphomas. The apoptotic cell count (AC) in follicular lymphomas was significantly less than that in diffuse lymphomas. The distribution pattern of apoptotic cells In follicular lymphomas was inverse to that in RHL. In follicular lymphomas, AC was lower in follicles than in inter-follicular areas. In contrast, AC was higher in follicles than in Interfollicular areas in RHL. Le^y antigen-positive lymphomas showed a significantly higher AC than the negative cases. The Fas antigen-positive lymphomas showed a higher AC than the negative cases. However, AC in bcl-2 protein-positive and negative cases was not significantly different. These results suggest that Le^y and Fas antigens appear to be involved in the apoptotic tendency of tumor cells in non-Hodgkin's lymphomas, whereas bcl-2 does not necessarily.