Location and characterization of autonomously replicating sequences from chromosome VI of Saccharomyces cerevisiae.

We have reported the isolation of linking clones of HindIII and EcoRI fragments, altogether spanning a 230-kb continuous stretch of chromosome VI. The presence or absence of autonomously replicating sequence (ARS) activities in all of these fragments has been determined by using ARS searching vectors containing CEN4. Nine ARS fragments were identified, and their positions were mapped on the chromosome. Structures essential for and/or stimulative to ARS activity were determined for the ARS fragments by deletions and mutations. The organization of functional elements composed of core and stimulative sequences was found to be variable. Single core sequences were identified in eight of nine ARSs. The remaining ARS (ARS603) essential element is composed of two core-like sequences. The lengths of 3'- and 5'-flanking stimulative sequences required for the full activity of ARSs varied from ARS to ARS. Five ARSs required more than 100 bp of the 3'-flanking sequence as stimulative sequences, while not more than 79 bp of the 3' sequence was required by the other three ARSs. In addition, five ARSs had stimulative sequences varying from 127 to 312 bp in the 5'-flanking region of the core sequence. In general, these stimulative activities were correlated with low local delta Gs of unwinding, suggesting that the low local delta G of an ARS is an important element for determining the efficiency of initiation of replication of ARS plasmids.     

Benzimidazole bearing oxadiazole and triazolo-thiadiazoles nucleus: Design and synthesis as anticancer agents

Two new series of benzimidazole bearing oxadiazole[1-(1H-benzo[d]imidazol-2-yl)-3-(5-substituted-1,3,4-oxadiazol-2-yl)propan-1-ones (4a-l)] and triazolo-thiadiazoles[1-(1H-benzo[d]imidazol-2-yl)-3-(6-(substituted)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)propan-1-one (7a-e)] have been synthesized successfully from4-(1H-benzo[d]imidazol-2-yl)-4-oxobutanehydrazide (3) with an aim to produce promising anticancer agents. In vitro anticancer activities of synthesized compounds were screened at the National Cancer Institute (NCI), USA, according to their applied protocol against full NCI 60 human cell lines panel; results showed good to remarkable anticancer activity. Among them, compound (4j, NCS: 761980) exhibited significant growth inhibition and further screened at 10-fold dilutions of five different concentrations (0.01, 0.1, 1, 10 and 100μM) with GI(50) values ranging from 0.49 to 48.0μM and found superior for the non-small cell lung cancer cell lines like HOP-92 (GI(50) 0.49, TGI 19.9,LC(50) >100 and Log(10)GI(50) -6.30, Log(10)TGI -4.70, Log(10)LC(50) >-4.00).     

SPECIAL PAPER: HAPLOIDS FROM POLLEN GRAINS—RETROSPECT AND PROSPECT

The production of large numbers of haploid plants from pollen grains in aseptic culture of anthers or isolated pollen grains has now been demonstrated in many angiosperms. It is a technique which holds much promise for creating desired variability by mutations for biochemical and applied genetics and for producing pure lines for crop improvement. This paper reviews the research on the production of haploids from pollen grains. The response is dependent on a variety of factors such as media formulation (especially the hormone component), genotype and physiological status of the donor plant, developmental stage of the pollen grain, anther culture environment and anther wall itself—all of which influence greatly the successful production of haploid plants. Although still in its infancy, the culture of isolated pollen is seen as a promising line of further research as it has the potential of offering several advantages over the culture of whole anthers.     

Characterization of gp120 and its single-chain derivatives, gp120-CD4D12 and gp120-M9: implications for targeting the CD4i epitope in human immunodeficiency virus vaccine design

Single-chain derivatives of JRFL gp120 linked to the first two domains of human CD4 (gp120-CD4D12) or to the CD4 miniprotein analog CD4M9 (gp120-M9), have been constructed. Biacore studies revealed that gp120-CD4D12 and gp120-M9 bound to antibody 17b with dissociation constants of 0.8 and 25 nM, respectively, at pH 7.0, while gp120 alone did not bind. The binding of gp120-CD4D12 to 17b is not affected by the addition of excess soluble CD4D12, while the binding of gp120-M9 is enhanced. This finding indicates that the M9 component of the single chain interacts relatively weakly with gp120 and can be displaced by soluble CD4D12. Immunogenicity studies of gp120, gp120-CD4D12, and gp120-M9 were carried out with guinea pigs. All three molecules were highly immunogenic. The resulting antisera were examined for neutralizing activities against various human immunodeficiency virus type 1 isolates. Broadly neutralizing activity was observed only with sera generated against gp120-CD4D12. These antisera were depleted of anti-CD4D12 antibodies by being passed over a column containing immobilized CD4D12. The depleted sera showed a loss of broadly neutralizing activity. Sera that were affinity purified over a column containing immobilized gp120-M9 also lacked such neutralizing activity. This finding suggests that the broadly neutralizing response observed is exclusively due to anti-CD4 antibodies. Competition experiments showed that only antisera generated against gp120-CD4D12 competed with the CD4i antibody 17b and that this activity was not affected by depletion of anti-CD4 antibodies. The data indicate that although antibodies targeting the CD4i epitope were generated by the gp120-CD4D12 immunogen, these antibodies were nonneutralizing.     

Physiological responses to single versus double stepping pattern of ascending the stairs

The aim of this study was to compare the physiological responses and energy cost between two ascending patterns, the single-step (SS) and the double-step (DS), in climbing a public staircase. In the SS pattern, a person climbs one step at a time whilst in the double-step (DS) pattern, the individual traverses two steps in a single stride. Advocates of each stepping pattern claimed that their type of ascent is physically more taxing and expends more calories. Thirty subjects (10 males and 20 females) climbed a typical 11-storey flat (each step height of 0.15 m, a total of 180 steps and a vertical displacement of 27.0 m). The subjects climbed using either the SS pattern at a tempo of 100 steps x min(-1) or the DS pattern at 50 steps x min(-1). The prescribed stepping frequencies ensured that an equal amount of total work was performed between the SS and DS patterns. The climbing patterns were performed in random order. Physiological measures during the last 30 s of the climbs were used in the comparative analysis. The results showed that ventilation, oxygen uptake and heart rate values were significantly higher (all p < 0.01) in the SS as compared to the DS pattern. However, the caloric expenditure during the SS pattern was calculated to be only marginally higher than the DS pattern. In conclusion, ascending with the SS pattern led to significantly higher physiological responses compared to the DS pattern. The higher calorie expended with the SS compared to the DS pattern was deemed to be of little practical significance.     

Design and synthesis of potent pyridazine inhibitors of p38 MAP kinase

Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibition of LPS-induced TNF-alpha production in mice and exhibited good efficacy in the rat collagen induced arthritis model.     

Mouse model of male germ cell apoptosis in response to a lack of hormonal stimulation

As a prerequisite for studies using mutant mice, we established a mouse model for induction of male germ cell apoptosis after deprivation of gonadotropins and intratesticular testosterone (T). We employed a potent long acting gonadotropin-releasing hormone antagonist (GnRH-A), acyline, alone or in combination with an antiandrogen, flutamide for effective induction of germ cell apoptosis in mice. Combined treatment with continuous release of acyline (3 mg/kg BW/day) with flutamide (in the form of sc pellets of 25 mg) resulted in almost the same level of suppression of spermatogenesis, as judged by testis weight and by germ cell apoptotic index, in 2 weeks as that reported for rats after treatment with 1.25 mg/kg BW Nal-Glu GnRH-A for the same time period. Within the study paradigm, the maximum suppression of spermatogenesis occurred after a single sc injection of high (20 mg/kg BW) dose of acyline with flutamide. The combined treatment resulted in complete absence of elongated spermatids. Germ cell counts at stages VII-VIII showed a significant (P < 0.05) reduction in the number of preleptotene (27.1%) and pachytene spermatocytes (81.9%), and round spermatids (96.6%) in acyline + flutamide group in comparison with controls. In fact, treatment with a single high (20 mg/kg BW) dose of acyline combined with flutamide in mice achieved same or greater level of suppression, measured by germ cell counts at stages VII-VIII, in two weeks when compared with those reported after daily treatment with Nal-Glu GnRH-A for 4 weeks in rats. Both plasma and testicular T levels were markedly suppressed after administration of acyline alone either by miniosmotic pump or by a single sc injection. Addition of flutamide to acyline had no discernible effect on plasma or intratesticular T levels when compared with acyline alone. These results demonstrate that optimum suppression of spermatogenesis through increased germ cell death is only possible in mice if total abolition of androgen action is achieved and further emphasize the usefulness of acyline + flutamide treated mice as a suitable model system to study hormonal regulation of testicular germ cell apoptosis.     

Inhibition of sodium/proton exchange by a Rab-GTPase-activating protein regulates endosomal traffic in yeast

Endosomal Na+/H+ exchangers are important for salt and osmotolerance, vacuolar pH regulation, and endosomal trafficking. We show that the C terminus of yeast Nhx1 interacts with Gyp6, a GTPase-activating protein for the Ypt/Rab family of GTPases, and that Gyp6 colocalizes with Nhx1 in the endosomal/prevacuolar compartment (PVC). The gyp6 null mutant exhibits novel phenotypes consistent with loss of negative regulation of Nhx1, including increased tolerance to hygromycin, increased vacuolar pH, and decreased plasma membrane potential. In contrast, overexpression of Gyp6 increases sensitivity to hygromycin, decreases vacuolar pH, and results in a slight missorting of vacuolar carboxypeptidase Y to the cell surface. We conclude that Gyp6 is a negative regulator of Nhx1-dependent trafficking out of the PVC. Taken together with its GTPase-activating protein-dependent role as a negative regulator of Ypt6-mediated retrograde traffic to the Golgi, we propose that Gyp6 coordinates upstream and downstream events in the PVC to Golgi pathway. Our findings provide a possible molecular link between intraendosomal pH and regulation of vesicular trafficking.     

Early-onset and robust cerebral microvascular accumulation of amyloid beta-protein in transgenic mice expressing low levels of a vasculotropic Dutch/Iowa mutant form of amyloid beta-protein precursor

Cerebrovascular deposition of amyloid beta-protein (Abeta) is a common pathological feature of Alzheimer's disease and related disorders. In particular, the Dutch E22Q and Iowa D23N mutations in Abeta cause familial cerebrovascular amyloidosis with abundant diffuse amyloid plaque deposits. Both of these charge-altering mutations enhance the fibrillogenic and pathogenic properties of Abeta in vitro. Here, we describe the generation of several transgenic mouse lines (Tg-SwDI) expressing human neuronal Abeta precursor protein (AbetaPP) harboring the Swedish K670N/M671L and vasculotropic Dutch/Iowa E693Q/D694N mutations under the control of the mouse Thy1.2 promoter. Tg-SwDI mice expressed transgenic human AbetaPP only in the brain, but at levels below those of endogenous mouse AbetaPP. Despite the paucity of human AbetaPP expression, quantitative enzyme-linked immunosorbent assay measurements revealed that Tg-SwDI mice developed early-onset and robust accumulation of Abeta in the brain with high association with isolated cerebral microvessels. Tg-SwDI mice exhibited striking perivascular/vascular Abeta deposits that markedly increased with age. The vascular Abeta accumulations were fibrillar, exhibiting strong thioflavin S staining, and occasionally presented signs of microhemorrhage. In addition, numerous largely diffuse, plaque-like structures were observed starting at 3 months of age. In vivo transport studies demonstrated that Dutch/Iowa mutant Abeta was more readily retained in the brain compared with wild-type Abeta. These results with Tg-SwDI mice demonstrate that overexpression of human AbetaPP is not required for early-onset and robust accumulation of both vascular and parenchymal Abeta in mouse brain.