Physical and functional interaction between Hck tyrosine kinase and guanine nucleotide exchange factor C3G results in apoptosis, which is independent of C3G catalytic domain

The hematopoietic cell kinase Hck is a Src family tyrosine kinase expressed in cells of myelomonocytic lineage, B lymphocytes, and embryonic stem cells. To study its role in signaling pathways we used the Hck-SH3 domain in protein interaction cloning and identified C3G, the guanine nucleotide exchange factor for Rap1 and R-Ras, as a protein that associated with Hck. This interaction was direct and was mediated partly through the proline-rich region of C3G. C3G could be co-immunoprecipitated with Hck from Cos-1 cells transfected with Hck and C3G. C3G was phosphorylated on tyrosine 504 in cells when coexpressed with Hck but not with a catalytically inactive mutant of Hck. Phosphorylation of endogenous C3G at Tyr-504 was increased by treatment of human myelomonocytic THP-1 cells with mercuric chloride, which is known to activate Hck tyrosine kinase specifically. Coexpression of Hck with C3G induced a high level of apoptosis in many cell lines by 30-42 h of transfection. Induction of apoptosis was not dependent on Tyr-504 phosphorylation or the catalytic domain of C3G but required the catalytic activity of Hck. Using dominant negative constructs of caspases we found that caspase-1, -8, and -9 are involved in this apoptotic pathway. These results suggest that C3G and Hck interact physically and functionally in vivo to activate kinase-dependent and caspase-mediated apoptosis, which is independent of catalytic domain of C3G.     

Fluorescence and nucleic acid binding properties of bovine leukemia virus nucleocapsid protein

Peptidyl-prolyl cis/trans isomerization, observed in the native state of an increasing number of proteins, is of considerable biological significance. The first evidence for an asymmetric transmission along the polypeptide chain of the structural effects of prolyl isomerization is now derived from the statistics of the C(alpha)/C(alpha)-atom distance distributions in the crystal structures of 848 non-homologous proteins. More detailed information on how isomerization affects segments adjacent to proline is obtained from crystal structures of proteins, that are more than 95% homologous, and that exhibit two different states of isomerization at a particular prolyl bond. The resulting 64 cases, which represent 3.8% of the database used, form pairs of coordinates which were analyzed for the existence of isomer-specific intramolecular nonbonded C(alpha)/C(alpha)-atom distances around the critical proline, and for the positional preferences for particular amino acids in the isomeric sequence segment. The probability that a native protein exhibits both prolyl isomers in the crystalline state increases in particular with a Pro at the third position N-terminal to the isomeric bond (-3 position), and with Ser, Gly and Asp at the position preceding the isomeric bond (-1 position). Structural alignment of matched pairs of isomeric proteins generates three classes with respect to position-specific distribution of C(alpha)-atom displacements around an isomeric proline imide bond. In the majority of cases the distribution of these intermolecular isomer-specific C(alpha)-atom distances shows a symmetric behavior for the N-terminal and C-terminal segment flanking the proline residue, and the magnitude did not exceed 1.3+/-0.6 A including the C(alpha) atoms in proximity to the prolyl bond. However, in the remaining 12 protein pairs the structural changes are unidirectional relative to the isomerizing bond whereby the magnitude of the isomer-specific effect exceeds 3.0+/-2.0 A even at positions remote to proline. Interestingly, the magnitude of the intramolecular isomer-specific C(alpha) atom displacements reveals a lever-arm amplification of the isomerization-mediated structural changes in a protein backbone. The observed backbone effects provide a structural basis for isomer-specific reactions of proline-containing polypeptides, and thus may play a role in biological recognition and regulation.     

Erythrocyte sodium and Na+, K(+)-ATPase activity in untreated hypertensives and their first degree relatives.

In this paper we report the erythrocyte sodium concentration and Na+, K(+)-ATPase activity in 86 untreated hypertensives and their 77 first degree relatives and also in sex and age matched controls. There was significant increase in erythrocyte sodium both in the hypertensives and their first degree relatives (p < 0.01), whereas Na+, K(+)-ATPase activity was significantly reduced in the study group when compared with controls. The possibility of using these parameters as genetic markers is suggested.     

Capsaicin formation in p-fluorophenylalanine resistant and normal cell cultures ofCapsicum frutescens and activity of phenylalanine ammonia lyase

Callus cultures ofCapsicum frutescens capable of producing a maximum of 53 μg capsaicin/g FW were exposed to various levels of p-fluorophenyialanine (PFP) at 100, 400, 1000 and 2000 μM to develop a resistant cell line that over produces capsaicin. After 15 days of culturing on media lacking PFP, cell lines resistant to 100, 400 and 1000 μM registered 18%, 34.5% and 45% increase in capsaicin content over normal cell line (cells not exposed to PFP). Capsaicin accumulation was inhibited in 2000 μM PFP resistant cell line. The profile of phenylalanine ammonia lyase (PAL), the key enzyme in pheny1propanoid pathway in resistant cell cultures was studied and compared with normal cell cultures to understand its role in capsaicin formation. Importantly increased production of capsaicin was obtained using PFP resistant cell lines. The activity profile of PAL had no correlation with capsaicin content in both control and PFP resistant cells.     

Expression and Immunolocalisation of the Snowdrop Lectin,GNA in Transgenic Rice Plants

Transgenic rice (Oryza sativa L.) plants generated through particle bombardment expressed high levels of an insecticidal protein (the snowdrop lectin, GNA) directed against sap-sucking insects. Engineered plants expressed GNA either constitutively or in a tissue specific manner, depending on the nature of the promoter used to drive expression of the gene. We used specific antibodies raised against GNA to localize its expression in phloem tissue in plants engineered with the rice sucrose synthase promoter driving GNA expression. We report here molecular, biochemical and immunological analyses for fifteen independently-derived transformants out of more than 200 plants we generated.     

Dermatoglyphic studies in rheumatic heart disease

62 patients suffering from rheumatic heart disease were taken from Institute of Child Health, Niloufer Hospital, Hyderabad, for the present dermatoglyphic investigation. The finger, palm and toe prints were analysed to see if there was an association between rheumatic heart disease and any of the dermatoglyphic traits. The parameters which were significantly different from the controls are: (1) reduced frequency of arches on the finger tips in males and increased frequency of whorls in females; (2) increased frequency of patterns in the III interdigital area in males; (3) decreased d-t ridge count in females, and (4) higher incidence of multiple axial triradii in females.     

Optical analysis of RE3+ (RE = Eu,Tb):MgLa2V2O9 nano-phosphors

Red and green rare-earth ion (RE³⁺) (RE = Eu, Tb):MgLa₂V₂O₉ micro-powder phosphors were produced utilizing a standard solid-state chemical process. The X-ray diffraction examination performed on the phosphors showed that they were crystalline and had a monoclinic structure. The particles grouped together, as shown in the scanning electron microscopy (SEM) images. Powder phosphors were examined using a variety of spectroscopic techniques, including photoluminescence (PL), Fourier-transform infrared, and energy dispersive X-ray spectroscopy. Brilliant red emission at 615 nm (⁵D₀ → ⁷F₂) having an excitation wavelength (λ_exci) of 396 nm (⁷F₀ → ⁵L₆) and green emission at 545 nm (⁵D₄ → ⁷F₅) having an λ_exci = 316 nm (⁵D₄ → ⁷F₂) have both been seen in the emission spectra of Tb³⁺:MgLa₂V₂O₉ nano-phosphors. The emission mechanism that is raised in Eu³⁺:MgLa₂V₂O₉ and Tb³⁺:MgLa₂V₂O₉ powder phosphors has been explained in an energy level diagram.     

Evolutionary conservation of a germ cell-specific lamin persisting through mammalian spermiogenesis.

We had identified earlier a germ cell-specific lamin of 60 kDa in rat which is related to somatic lamin B. This polypeptide was shown to be the only major component organizing the lamina structure of round spermatids. In the present study, we find that this 60-kDa polypeptide persists in the testicular and epididymal sperms of rat. We also show, by indirect immunofluorescence studies, that the 60-kDa protein is antigenically conserved in the germ cells of grasshopper, rooster, and frog and in plant meiocytes. The distribution of fluorescence among the various germ cell populations shows that the antigen is located around the nuclear cortex of pre- and postmeiotic germ cells, while it is distributed all over the pachytene nuclei. The anti-60-kDa polyclonal antibodies also reacted with a 60-kDa polypeptide in the Western blot analysis of nuclear matrix proteins of grasshopper germ cells. The similar fluorescent localization pattern of the antigen observed in various eukaryotic species strongly suggests that this germ cell-specific lamin may play a very crucial role during meiotic prophase, particularly during homologous chromosome pairing and recombination.     

Functional characterization of N-terminal nucleotide binding domain (NBD-1) of a major ABC drug transporter Cdr1p of Candida albicans: uncommon but conserved Trp326 of Walker B is important for ATP binding

Using purified N-terminal NBD (NBD-512) domain of Cdr1p, a major multidrug extrusion pump of human pathogenic yeast Candida albicans, we show the relevance of the unique positioning of an atypical Trp326 residue. Similar to Cys193 in Walker A, Trp326 in the Walker B motif of Cdr1p is also a conserved feature of other fungal ATP Binding Cassette (ABC) transporters. By employing fluorescence spectroscopy, chemical modification, and site-directed mutagenesis, we demonstrate that of the five Trp residues in the NBD-512 domain, Trp326 alone is important for nucleotide binding and subsequent conformational changes within the domain. Furthermore, mutation of Trp326 to Ala results in an increased K(M) without appreciably affecting V(max) of ATPase activity. Thus, Trp326 in NBD-512 appears to be important for nucleotide binding and not for its hydrolysis. Additionally, the role of Trp326 in ATP binding is independent of the presence of the adjacent well-conserved Asp327 residue which, like Cys193, has a catalytic role in ATP hydrolysis. Considering that Trp326 of Cdr1p is a typical feature of fungal transporters alone, our study suggests that these ABC transporters may reflect mechanistic differences with regard to nucleotide binding and hydrolysis as compared to their counterparts of non-fungal origin.     

Electromyographic (EMG) Biofeedback in the Comprehensive Treatment of Central Pain and Ataxic Tremor Following Thalamic Stroke

Peripheral pain and ataxic tremor can appear suddenly following thalamic stroke and can significantly alter a patient's psychological, social, and physical functioning. The present paper reports the case of a 70-year-old Caucasian female who sustained an acute left posterior cerebral artery infarction involving the thalamus and left mesiotemporal regions. She subsequently developed Central Poststroke Pain and ataxic movement of her right arm and hand in addition to a significant right-side claudication. She was treated over 16 weeks (6 weeks of EMG biofeedback and 10 weeks of psychotherapy) with a combination of EMG biofeedback, progressive muscle relaxation, behavioral pain coping skills training, Forced Use Therapy, and Cognitive Behavioral Therap 7 years after her initial cerebral accident. The case demonstrates the utility of biofeedback when combined as part of a comprehensive treatment program to address the multiple complications associated with thalamic stroke.