Purification, crystallization and preliminary X-ray analysis of NtdA, a putative pyridoxal phosphate-dependent aminotransferase from Bacillus subtilis.

NtdA is a putative sugar aminotransferase that is required for the synthesis of 3,3'-neotrehalosadiamine. The enzyme was purified to homogeneity by means of Ni(2+)-affinity chromatography and was crystallized using the microbatch method. X-ray diffraction data were collected from a single crystal to 2.3 A resolution at the Canadian Light Source (CLS). The crystals belonged to space group P2(1), with unit-cell parameters a = 50.3, b = 106.7, c = 96.7 A, beta = 96.2 degrees, and contained two molecules per asymmetric unit.

Purification, crystallization and preliminary X-ray analysis of inositol dehydrogenase (IDH) from Bacillus subtilis.

Inositol dehydrogenase (IDH) is an enzyme that catalyses the NAD(+)-dependent oxidation of myo-inositol to scyllo-inosose. The enzyme has been purified to homogeneity by means of Ni(2+)-affinity chromatography and was crystallized in both native and selenomethionine (SeMet) labelled forms using the microbatch method. SAD X-ray diffraction data were collected to 2.0 A resolution from a SeMet-labelled crystal at the Advanced Photon Source (APS) and a MAD data set was collected to 1.75 A resolution at the Canadian Light Source (CLS); this is the first reported anomalous diffraction experiment from the CLS. The crystals belong to space group I222 and contain one molecule per asymmetric unit.

The role of the membrane-spanning domain sequence in glycoprotein-mediated membrane fusion.

The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events.

13-cis-retinoic acid with alpha-2a-interferon enhances radiation cytotoxicity in head and neck squamous cell carcinoma in vitro.

The treatment of locally advanced squamous cell carcinomas of the head and neck presents a challenge for oncologists. Radiation therapy alone fails to control many of these tumors. Chemotherapy added to radiation therapy has not clearly demonstrated an improvement in survival in the majority of trials reported to date. In this study, we have evaluated whether IFN-alpha-2a and/or 13-cis-retinoic acid (RA) enhance radiation cytotoxicity in a head and neck squamous cell carcinoma cell line (FaDu). Using a clonogenic cell survival assay, IFN-alpha-2a (1000 units/ml) or RA (1 microM) alone did not significantly enhance radiation cytotoxicity. The combination of the two agents, however, significantly increased the cytotoxicity of radiation against FaDu cells. The calculated survival fraction at 2 Gy was decreased from 0.649 with radiation alone to 0.477 when combined with the other two agents (P = 0.016), and the MID was decreased from 3.318 to 2.499 Gy (P = 0.028). A Phase I clinical trial to combine IFN-alpha-2a and/or RA in patients with unresectable head and neck cancer has been initiated.

The 1.2 A resolution structure of the Con A-dimannose complex.

The complex between concanavalin A (Con A) and alpha1-2 mannobiose (mannose alpha1-2 mannose) has been refined to 1.2 A resolution. This is the highest resolution structure reported for any sugar-lectin complex. As the native structure of Con A to 0.94 A resolution is already in the database, this gives us a unique opportunity to examine sugar-protein binding at high resolution. These data have allowed us to model a number of hydrogen atoms involved in the binding of the sugar to Con A, using the difference density map to place the hydrogen atoms. This map reveals the presence of the protonated form of Asp208 involved in binding. Asp208 is not protonated in the 0.94 A native structure. Our results clearly show that this residue is protonated and hydrogen bonds to the sugar. The structure accounts for the higher affinity of the alpha1-2 linked sugar when compared to other disaccharides. This structure identifies different interactions to those predicted by previous modelling studies. We believe that the additional data presented here will enable significant improvements to be made to the sugar-protein modelling algorithms.

Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold.

The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 A resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic two-fold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.

Effects of insulin and insulin-like growth factors on cultured human hair follicles: IGF-I at physiologic concentrations is an important regulator of hair follicle growth in vitro.

Insulin stimulated hair follicle growth in a dose-dependent manner over the range of 0.01 to 100 micrograms/ml. Maximum rates of hair follicle growth were observed when follicles were maintained in medium containing 10 micrograms/ml insulin, which is supraphysiologic. Hair follicles maintained in the absence of insulin or at physiologic levels showed premature entry into a catagen-like state. Insulin-like growth factor (IGF)-I and -II had no significant effect on hair follicle growth when maintained in the presence of 10 micrograms/ml insulin. However, in the absence of insulin, both IGF-I (0.01-100 ng/ml) and IGF-II (0.01-100 ng/ml) stimulated hair follicle growth in a dose-dependent manner. IGF-I was more potent than either insulin or IGF-II, stimulating maximum rates of hair follicle growth at 10 ng/ml, whereas IGF-II gave maximum stimulation at 100 ng/ml. The rates of hair follicle growth stimulated by 10 ng/ml IGF-I were identical to those stimulated by 10 micrograms/ml insulin. IGF-II (100 ng/ml), however, was unable to stimulate hair follicle growth to the same extent as insulin. Both IGF-I (10 ng/ml) and IGF-II (100 ng/ml) were more potent than insulin at preventing hair follicles from entering into a catagen-like state. Growth hormone had no effect on hair follicle growth or morphology in the absence of insulin. These data suggest that in vitro IGF-I may be an important physiologic regulator of hair growth and possibly the hair growth cycle. Moreover, the removal of insulin from tissue culture medium may be a useful method of generating large numbers of catagen hair follicles for further in vitro studies.

In the absence of streptomycin, minoxidil potentiates the mitogenic effects of fetal calf serum, insulin-like growth factor 1, and platelet-derived growth factor on NIH 3T3 fibroblasts in a K+ channel-dependent fashion.

There is considerable evidence to suggest that the opening of K+ channels plays an important role in stimulating mitogenesis. K+ channel blockers have been shown to inhibit mitogenesis in vitro, mitogens increase cytosolic membrane K+ channel permeability, K+ channel openers stimulate hair growth in vivo, and the Ras/Raf signal transduction pathway induces K+ channel activity. Paradoxically, however, K+ channel openers such as minoxidil have been reported in vitro not to modulate, or even to inhibit, mitogenesis in a range of cell types. Only untherapeutic concentrations have stimulated mitogenesis. These experiments, however, appear to have been carried out in the presence of aminoglycoside antibiotics, which inhibit potassium channel activity. We now report that in the absence of aminoglycoside antibiotics, minoxidil at 10 microg/ml (0.05 mM) causes a significant stimulation of proliferation of NIH 3T3 fibroblasts maintained over a 10-d period in 5% fetal calf serum-supplemented medium. Further, we show that in the presence of 100 microg streptomycin per ml, minoxidil at 10 microg/ml produces an initial inhibition of proliferation, which apparently confirms, in NIH 3T3 fibroblasts, that the inhibition of mitogenesis by minoxidil in the presence of streptomycin is an artifact. The potentiation of NIH 3T3 cell growth by minoxidil can be attributed to the opening of potassium channels, because the potassium channel blocker tolbutamide (5 mM) or combinations of the blockers tolbutamide (1 mM)/tetraethylammonium (2 mM) or glibenclamide (1 microM)/apamin (10 nM) block the minoxidil-induced stimulation of growth. We also demonstrate that minoxidil is able to significantly potentiate the mitogenic effects of both platelet-derived growth factor and insulin-like growth factor 1 on NIH 3T3 fibroblasts in the presence of CPSR-2 (a cytokine free serum substitute). Thus we have shown that minoxidil potentiates the mitogenic effects of fetal calf serum in vitro on NIH 3T3 fibroblasts by opening potassium channels and is also able to potentiate the mitogenic effects of the growth factors platelet-derived growth factor and insulin-like growth factor 1.

Early events in the neoplastic transformation of respiratory epithelium.

The complex process of epithelial carcinogenesis is composed of discrete biologic events including the early activation events of "initiation" and "promotion." For lung cancer, these events are only now being elucidated. Despite the identification of possible target genes and their mutations, the "initiation" events for lung cancer remain poorly understood. The identification of these "initiation" events is a crucial step toward the development of practical molecular markers for early detection of this disease. The reversible process of tumor promotion remains somewhat enigmatic but is a promising target for chemoprevention. A wide range of substances, including asbestos and various substances in cigarette smoke, behave as tumor promoters for lung cancer. They appear to promote tumor formation by inducing cellular proliferation mediated in part by growth factors. The intracellular signals these factors provide are ultimately translated into cellular growth via steps involving nuclear transcription factors. Early response genes such as the jun and fos gene family members encode such nuclear transcription factors which are expressed in lung cancer cells and primary bronchial epithelial cells. The expression of these transcription factors is highly responsive to stimulation by growth factors including serum, transforming growth factor, and gastrin-releasing peptide. A more thorough understanding of this process will allow the development of molecular and/or pharmacologic antagonists that can interfere with the biologic process of tumor promotion and therefore function as chemoprevention agents.

Desmoplastic small round cell tumour of unknown primary origin with lymph node and lung metastases: histological, cytological, ultrastructural, cytogenetic and molecular findings.

Desmoplastic small round cell tumour (DSRCT) is an extremely aggressive neoplasm belonging to the family of "small round blue cell tumours", which includes primitive neuroectodermal tumour (PNET), Wilms' tumour and Ewing's sarcoma. DSRCT is considered to be a neoplasm derived from a primitive cell. It is immunohistochemically reactive with epithelial, neuronal and mesenchymal cell markers, demonstrating divergent differentiation along three cell lines. Originally thought to arise from serosal surfaces, the tumour has recently been reported in the central nervous system and ovary. The tumour in this case is a neoplasm that meets the histological, immunohistochemical, cytological and cytogenetic criteria of DSRCT; it is not associated with serosal membranes, and it has supraclavicular and axillary lymph node deposits and multiple pulmonary and brain metastases. Tumour cells from our case show cytogenetic similarities with Ewing's sarcoma and PNET. Electron microscopic findings suggest similarities between DSRCT and Wilms' tumour. Cloning and sequencing of PCR products showed in-frame fusion of EWS exon 7 to WT1 exon 8.

Laboratory and field studies of an antigen capture ELISA for bluetongue virus.

An improved bluetongue antigen capture ELISA (BTACE) technique was evaluated for its ability to detect the full range of 24 bluetongue (BLU) serotypes. The BTACE detected all 24 serotypes in cell culture fluids, including eight serotypes where the representative strains originated from both Australia and also from the South African reference collection. The amount of infectious virus required to obtain a positive BTACE result varied between 100-1000 TCID50. This was approximately 10-fold more sensitive than the antigen capture test described previously (Hosseini, M., Hawkes, R.A., Kirkland, P.D., Dixon, R., 1998. J. Virol. Methods 75, 39-46.). The BTACE method was compared with conventional passage in cell culture to detect the presence of virus in the tissues of embryonated chicken eggs (ECEs) which had been inoculated intravenously with the blood of sheep and cattle infected experimentally with the eight Australian serotypes of BLU (1, 3, 9, 15, 16, 20, 21, and 23). The BTACE method was at least as sensitive as the conventional cell culture detecting virus in ECEs, obviating the need for prolonged cell culture passage to detect the virus. A comparison of the amount of antigen detected in different embryo tissues indicated that liver homogenates gave the highest positive to negative ratios in the BTACE and were selected as the specimen of choice. In studies of sheep infected with all 24 South African reference BLU serotypes this new BTACE was able to detect viraemia with all serotypes. Finally, the BTACE was validated in surveillance programs for BLU in both New South Wales, Australia and in Yunnan Province, People's Republic of China. Blood samples from sentinel cattle were inoculated into ECEs. Homogenised ECE livers were tested by BTACE and those positive were passaged subsequently in cell culture for virus isolation and identification. This protocol led to the efficient isolation of field isolates of many serotypes. The high sensitivity and broad reactivity of the method indicates that it should be valuable for BLU diagnosis and surveillance programs.

Whole hair follicle culture.

In this article the authors have reviewed the historical background behind the organ culture of whole hair follicles. The methods developed by the authors and others for the isolation and whole organ maintenance of hair follicles from both human and other species are described. How whole organ models have been used to further understanding of the biology of the hair follicle and how they may be used in the future are discussed.

Plasmid transfection and retroviral transduction of porcine muscle cells for cell-mediated gene transfer.

Cell-mediated gene transfer is a potential tool for studying muscle growth, but efficient genetic manipulation and implantation strategies have not been developed for pigs. The objectives of the present study were to determine methods for transient and stable incorporation of reporter genes into porcine muscle cells and to investigate their use for cell-mediated gene transfer in pigs. Porcine myoblasts and fibroblasts were isolated from muscle of 2-wk-old male pigs. Myogenic cell lines were identified using muscle-specific monoclonal antibodies, myotube fusion assays, and the presence of muscle-specific markers (MyoD and desmin). Four commercial cationic liposomes (lipofectAMINE, lipofectin, cellFECTIN, and DMRIE-C) were tested at different DNA:lipid ratios for their ability to transfect myoblasts and fibroblasts transiently with a luciferase reporter plasmid. LipofectAMINE resulted in the greatest (P < .01) transient luciferase activity for both cell types. Electroporation of cells for transient transfection resulted in less luciferase activity than cationic transfection. Stable transfections were conducted using a green fluorescence protein (GFP) reporter plasmid containing the neomycin resistance gene. LipofectAMINE transfection resulted in stable GFP expression in 1:16,000 myoblasts and 1:33,000 fibroblasts. Stable electroporation resulted in efficiencies that were significantly lower than established with cationic liposomes. Porcine cells were transduced with GFP using vesicular stomatitis virus glycoprotein G pseudotyped retrovirus and resulted in efficiencies of 1:1.2 for myoblasts and 1:1.1 for fibroblasts. These results show that cationic liposomes are superior to electroporation for transfection, but retroviral transduction produced stable reporter gene expression in > 80% of porcine muscle cells. Transduced GFP-positive cells were separated from GFP-negative cells by fluorescence-activated cell sorting and implanted into 2-wk-old male pigs. On d 4, implanted muscles were removed and subjected to immunodetection of GFP protein. Fibroblast implantation resulted in limited GFP expression within muscle, whereas myoblast implantation resulted in GFP within muscle fibers. This suggests that cell-mediated gene transfer is possible in porcine muscle and may be useful as an approach for studying muscle growth in pigs.

Self-transcription: RNA polymerase transcription of its own genes, and its role in cellular differentiation and cell cycling.

The concept of RNA polymerase self-transcription, where eukaryotic RNA polymerase II and prokaryotic holoenzyme are responsible for transcription of their own genes, would give these enzymes a unique role in the cellular transcriptional and translational machinery. This self-transcriptional ability would equip a cell with an exquisite mechanism of autogenous regulation for the appearance of these transcriptional units. Such a mechanism could allow layering of other transcriptional control pathways upon the RNA polymerase self-transcriptional pathway, thus forming a complex array of mechanisms to regulate transcription of genes concerned with cellular differentiation and cell cycling. It is proposed that RNA polymerase self-transcription is the central control point of gene expression in cellular differentiation and for cell cycling, thus fulfilling the role of an intrinsic biological clock.

Receptor interactions through phosphorylation and methylation pathways in bacterial chemotaxis.

The effects of messages initiated by one receptor on the covalent modification of a second receptor were studied by use of a technique for rapidly separating the receptors. Methylation of the bacterial-chemotactic serine receptor increases as a result of aspartate binding to the aspartate receptor. The aspartate-induced methylation on the serine receptor is absent in a strain that lacks cheA and cheW genes and is not the result of physical interaction, such as the formation of heterodimers between the aspartate and serine receptors, or of alterations in the affinity of the serine receptor for the methyltransferase and the methylesterase. Serine-induced methylation of the serine receptor did not require cheA and cheW. A model is presented in which the receptor methylation level depends on the combination of (i) a ligand-induced conformational change on the receptor substrate of the methylation enzymes and (ii) an indirect cytoplasmic signal that operates through the methylesterase.

The GTPase superfamily: conserved structure and molecular mechanism.

GTPases are conserved molecular switches, built according to a common structural design. Rapidly accruing knowledge of individual GTPases--crystal structures, biochemical properties, or results of molecular genetic experiments--support and generate hypotheses relating structure to function in other members of the diverse family of GTPases.

Roles of methylation and phosphorylation in the bacterial sensing system.

Chemotaxis is an intriguing model system for the study of second-messenger pathways. One of the puzzles of second-messenger pathways in eukaryotic cells has been that many of these pathways interact, with one pathway either desensitizing or sensitizing an alternate messenger pathway. The chemotaxis system offered a particularly interesting chance to analyze such systems, because one of them is a methylation pathway and the other a phosphorylation pathway. The above description indicates that these two pathways interact with each other in a highly sophisticated way to produce an extremely important survival system. The stimulus on a receptor activates an excitation system that operates through phosphorylation, or the inhibition of phosphorylation, depending on whether the stimulus is a repellent or an attractant. That system ultimately generates or inhibits phosphorylation of a small peptide, the CheY protein, which apparently is the response regulator. The instant that this fast excitation is generated by a change in gradient, the change in conformation of the protein sets in motion a second process, i.e., the adaptation. That is a device which, over a longer period of time, has two functions: It serves as the comparator, which allows the comparison of the past with the present, essential for deletion of a gradient; it also sets in motion the reset to zero, so that the bacterium will not be overwhelmed by any one stimulus but can use all of its receptors to optimize its environment. These two systems by themselves are adequate for chemotaxis, but there is a further elegent complexity: The excitation system feeds back into the adaptation system to produce an asymmetry in the responses. The reason for that asymmetry is that the bacterium wishes to travel in the wrong direction only long enough to produce a detectable signal that it is migrating incorrectly. It wishes to keep swimming in the positive direction as long as the signals indicate that the direction is favorable. Hence, the feedback between the phosphorylation and methylation system involves further fine tuning.