Quaternary structure and geminate recombination in hemoglobin: flow-flash studies on alpha 2CO beta 2 and alpha 2 beta 2CO.

The kinetics of geminate recombination for the diliganded species alpha 2CO beta 2 and alpha 2 beta 2CO of human hemoglobin were studied using flash photolysis. The unstable diliganded species were generated just before photolysis by chemical reduction in a continuous flow reactor from the more stable valency hybrids alpha 2CO beta 2+ and alpha 2+ beta 2CO, which could be prepared by high pressure liquid chromatography. Before the flash photolysis studies, the hybrids had been characterized by double-mixing stopped-flow kinetics experiments. At pH 6.0 in the presence of inositol hexaphosphate (IHP) both of the diliganded species show second order kinetics for overall addition of a third CO that is clearly characteristic of the T state (l' = 1-2 x 10(5) M-1 s-1), whereas at higher pH and in the absence of IHP they show combination rates characteristic of an R state. The kinetics of geminate recombination following photolysis of a bound CO, however, showed little dependence on pH and IHP concentration. This surprising observation is explained on the basis that the kinetics of geminate recombination of CO primarily depends on the tertiary structure of the ligand binding site, which apparently does not differ much between the R state and the liganded T state formed on adding IHP in this system. Since this explanation requires distinguishing different tertiary structures within a particular quaternary structure, it amounts to a contradiction to the two-state allosteric model.     

Role of protein phosphorylation in activation of interferon-stimulated gene factors.

Possible involvement of protein phosphorylation in interferon (IFN)-mediated activation of IFN-stimulated gene factor 3 (ISGF3) was investigated. For this purpose, in vivo experiments with specific inhibitors of protein kinases and in vitro experiments with protein phosphatases were carried out. In HeLaM cells, 2-aminopurine, an inhibitor of double-stranded RNA-dependent protein kinase, blocked the induction of ISGF3 gamma subunit but not the activation of ISGF3 alpha subunit. A series of experiments using combinations of protein and RNA synthesis inhibitors and 2-aminopurine indicated that the block elicited by 2-aminopurine was at the level of ISGF3 gamma mRNA synthesis. Activation of ISGF3 alpha, although insensitive to 2-aminopurine, was completely blocked by 10 nM staurosporine, an inhibitor of protein kinase C. On the other hand, even 500 nM staurosporine did not block the induction of ISGF3 gamma. Incubation of cytoplasmic or nuclear extracts of IFN-treated HeLaM cells in vitro with alkaline phosphatase completely eliminated their ability to form the ISGF3 complex but not the ISGF1 complex. Treatment with acid phosphatase, on the other hand, changed the electrophoretic mobility of the ISGF3 complex but did not obliterate it. Complementation experiments revealed that ISGF3 alpha was the alkaline phosphatase-sensitive component of the complex. These results suggest that a protein kinase C-mediated phosphorylation step is involved in ISGF3 alpha activation and a 2-aminopurine-sensitive component is involved in ISGF3 gamma mRNA induction.     

Binding of sperm to somatic cells via HLA-DR. Modulation by sulfated carbohydrates.

We have shown previously that human sperm bind and enter leukocytes expressing surface HLA class II molecules. In the present study, mutant B lymphoblastoid cells and HLA-DR-transfected murine 3T3 fibroblasts are used to confirm that HLA class II molecules are somatic cell receptors for sperm. Further, for isolated HLA-DR expressed on murine cells, we show that sperm receptor activity requires the presence of sulfated carbohydrates. As carriers of multiple HLA-DR binding ligands, sperm may 1) mimic the target cell-activating effects of anti-DR antibody and 2) bind HIV through CD4-like or alternate receptors. By these or other mechanisms, sperm/somatic cell interactions in the female reproductive tract may affect fertility and potentiate the sexual transmission of AIDS.     

Natural killer cell-mediated lysis of herpes simplex virus-infected fibroblasts: inability to detect soluble factors that contribute to lysis

We investigated the role of soluble factors in natural killer (NK) cell-mediated lysis of herpes simplex virus (HSV)-infected cells. Supernatants generated by incubating human peripheral blood mononuclear cells with HSV-infected human fibroblasts contained tumor necrosis factor (TNF) and lysed uninfected U937 cells, but not HSV-infected fibroblasts. U937 cells, but not HSV-infected fibroblasts, were lysed when exposed to recombinant TNF (rTNF) for 18 hr. NK cell-mediated lysis of HSV-infected fibroblasts was not inhibited by addition of anti-TNF or anti-lymphotoxin (LT) antibodies to cytotoxicity assays. Thus, a role for soluble factors, and in particular TNF and LT, in NK cell-mediated lysis of HSV-infected cells could not be demonstrated.     

Are “multiple cross-multiple pollen hybrids’ an answer for productive populations in Brassica campestris var. ‘brown sarson’?

Summary A set of complex crosses with multiple crosses as female parents were made using multiple pollen in turnip rape (Brassica campestris L.). These multiple cross — multiple pollen hybrids (mucromphs) were evaluated for a large number of quantitative characters including yield. New methods were proposed to study such genetic material in depth so as to formulate suitable strategies to breed for attractive seed yield.Part of the Ph. D. Thesis of junior author submitted to Indian Agricultural Research Institute, New Delhi     

Purification and properties of nuclear and cytoplasmic DNA polymerases from JLS-V9 cells.

Four DNA polymerases, two enzymes from the nucleus and two from the cytoplasm, were purified 2000- to 7000-fold from continuous mouse cell-line (JLS-V9), by sequential column chromatography. Each of these polymerases require all the deoxynucleoside-5′-triphosphates in order to synthesize DNA, using activated DNA as a primer-template, and can copy the ribonucleotide strand of hybrid templates, but their rate of efficiency varies. The molecular weights of these DNA polymerases range from 35,000 to 160,000, as estimated by Sephadex column chromatography. Three out of the four DNA polymerases are probably a single polypeptide chain, since they have a single major band in polyacrylamide gel electrophoresis as well as one enzymatically active peak in guanidine hydrochloride gel filtration. The highly purified preparation of the high molecular weight cytoplasmic DNA polymerase contains two major bands in sodium dodecyl sulfate polyacrylamide gel electrophoresis and two enzymatically active peaks in guanidine hydrochloride gel filtration.     

Male‐specific alterations in structure of isolation call sequences of mouse pups with 16p11.2 deletion

16p11.2 deletion is one of the most common gene copy variations that increases the susceptibility to autism and other neurodevelopmental disorders. This syndrome leads to developmental delays, including speech impairment and delays in expressive language and communication skills. To study developmental impairment of vocal communication associated with 16p11.2 deletion syndrome, we used the 16p11.2del mouse model and performed an analysis of pup isolation calls (PICs). The earliest PICs at postnatal day 5 from 16p11.2del pups were found altered in a male‐specific fashion relative to wild‐type (WT) pups. Analysis of sequences of ultrasonic vocalizations (USVs) emitted by pups using mutual information between syllables at different positions in the USV spectrograms showed that dependencies exist between syllables in WT mice of both sexes. The order of syllables was not random; syllables were emitted in an ordered fashion. The structure observed in the WT pups was identified and the pattern of syllable sequences was considered typical for the mouse line. However, typical patterns were totally absent in the 16p11.2del male pups, showing on average random syllable sequences, while the 16p11.2del female pups had dependencies similar to the WT pups. Thus, we found that PICs were reduced in number in male 16p11.2 pups and their vocalizations lack the syllable sequence order emitted by WT males and females and 16p11.2 females. Therefore, our study is the first to reveal sex‐specific perinatal communication impairment in a mouse model of 16p11.2 deletion and applies a novel, more granular method of analysing the structure of USVs.     

Bacopasaponins with cytotoxic activity against human breast cancer cells in vitro

Molecular Biology Reports - Globally, breast cancer is a serious concern that exhibits a persistent rise in its incidence and related mortality even after significant advancement in the field of...