Role of Ca2+ in induction and secretion of dengue virus-induced cytokines

The present study was undertaken to investigate the role of calcium ions (Ca₂₊) in the induction and secretion of the dengue type 2 virus induced cytotoxic factor and the cytotoxin. This was done by using calcium channel blocking drugs such as verapamil, nifedipine or diltiazem hydrochloride. The production of cytotoxic factor was significantly reduced by treatment of dengue type 2 virus infected mice with verapamil. Similarly, a dosedependent inhibition of the secretion of cytotoxic factor was observed, when spleen cells of the virus-primed mice were treatedin vitro with the 3 calcium channel blockers. The production of cytotoxin by macrophages was abrogated by pretreatment with calcium channel blockers but had little effect on its secretion as shown by treatment of macrophages with verapamil at 1 h after the induction to later periods up to 18 h. The findings thus show that in the induction of both the cytokines Ca₂₊ plays a critical role; on the other hand it is required for the secretion of the cytotoxic factor but not for that of the cytotoxin.     

Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.     

Enhancement of selectivity in recognition of nucleic acids via chemical autoligation.

A new approach to increase the selectivity of interaction between oligonucleotide probes and target nucleic acids is described. In place of a single, relatively long oligonucleotide probe, two or three short oligomers terminated by thiophosphoryl and bromoacetamido groups are employed. Fast and efficient autoligation takes place when the oligomers hybridize in a contiguous mode to the same complementary strand such that a thiophosphoryl group on one strand and a bromoacetamido group on another are brought into proximity. A single nucleotide mismatch for the short probes leads to marked reduction in the rate of autoligation. The binding affinity of the product is close to that for a natural probe of the same length. This approach could have potential in oligonucleotide-based diagnostics, chemical amplification systems, and therapeutic applications.     

Epididymal histoplasmosis diagnosed by isolation ofHistoplasma capsulatum from semen

An autochthonous case of epididymal histoplasmosis masquerading as tuberculosis in a 55-year-old male patient is reported from India. It was diagnosed by culture ofHistoplasma capsulatum from semen and by demonstration of the fungus upon re-examination of epididymal biopsy sections previously misinterpreted as tuberculous granuloma. The patient's main complaints were painful epididymal swelling, occasional fever and cough. He was treated successfully by excision of epididymis and vas deferens combined with amphotericin B therapy. This is believed to be the first case of epididymal histoplasmosis to be reported outside the American continent and the fourth of its type reported in the English literature. The case is also noteworthy in thatH. capsulatum was isolated for the first time from semen, and it underlines the importance of mycological culture of semen specimens for diagnosis of genitourinary infections of obscure etiology.Presented at the XII Congress of the International Society for Human and Animal Mycology, Adelaide, Australia, March 13–18, 1994.     

Helix-forming tendencies of amino acids depend on the restrictions of side-chain rotamer conformations: crystal structure of the tripeptide GAI in two crystalline forms.

In our attempts to design crystalline alpha-helical peptides, we synthesized and crystallized GAI (C11H21N3O4) in two crystal forms, GAI1 and GAI2. Form 1 (GAI1) Gly-L-Ala-L-Ile (C11H21N3O4.3H2O) crystals are monoclinic, space group P2(1) with a = 8.171(2), b = 6.072(4), c = 16.443(4) A, beta = 101.24(2) degrees, V = 800 A3, Dc = 1.300 g cm-3 and Z = 2, R = 0.081 for 482 reflections. Form 2 (GAI2) Gly-L-Ala-L-Ile (C11H21N3O4.1/2H2O) is triclinic, space group P1 with a = 5.830(1), b = 8.832(2), c = 15.008(2) A, alpha = 102.88(1), beta = 101.16(2), gamma = 70.72(2) degrees, V = 705 A3, Z = 2, Dc = 1.264 g cm-3, R = 0.04 for 2582 reflections. GAI1 is isomorphous with GAV and forms a helix, whereas GAI2 does not. In GAI1, the tripeptide molecule is held in a near helical conformation by a water molecule that bridges the NH3+ and COO- groups, and acts as the fourth residue needed to complete the turn by forming two hydrogen bonds. Two other water molecules form intermolecular hydrogen bonds in stabilizing the helical structure so that the end result is a column of molecules that looks like an incipient alpha-helix. GAI2 imitates a cyclic peptide and traps a water molecule. The conformation angles chi 11 and chi 12 for the side chain are (-63.7 degrees, 171.1 degrees) for the helical GAI1, and (-65.1 degrees, 58.6 degrees) and (-65.0 degrees, 58.9 degrees) for the two independent nonhelical molecules in GAI2; in GAI1, both the C gamma atoms point away from the helix, whereas in GAI2 the C gamma atom with the g+ conformation points inward to the helix and causes sterical interaction with atoms in the adjacent peptide plane. From these results, it is clear that the helix-forming tendencies of amino acids correlate with the restrictions of side-chain rotamer conformations. Both the peptide units in GAI1 are trans and show significant deviation from planarity [omega 1 = -168(1) degrees; omega 2 = -171(1) degrees] whereas both the peptide units in both the molecules A and B in GAI2 do not show significant deviation from planarity [omega 1 = 179.3(3) degrees; omega 2 = -179.3(3) degrees for molecule A and omega 1 = 179.5(3) degrees; omega 2 = -179.4(3) degrees for molecule B], indicating that the peptide planes in these incipient alpha-helical peptides are considerably bent.