The preparation of chloromethylketone analogues of amino acids: inhibition of leucine aminopeptidase

Chloromethyl ketone analogs of glycine, dl-leucine, dl-alanine, and d-alanine were prepared. The dl-leucine and dl-alanine analogs were potent reversible inhibitors of leucine aminopeptidase. The enzyme was not inhibited by the d-alanine analog.     

Insulin action in isolated fat cells. I. Effects of divalent cations on the stimulation by insulin of glucose uptake.

The effects of divalent cations, in particular Ca2+ and Mg2+, on glucose uptake by rat isolated fat cells in the presence and absence of insulin have been studied. EDTA (disodium salt) was used to deplete the bovine serum albumin present in the incubation medium of endogenous divalent cations prior to incubation with the cells, but was not present in the incubation medium during the incubation of the cells. The removal of Ca2+ and Mg2+ from the incubation medium did not affect the basal glucose uptake, but abolished the ability of insulin to stimulate glucose uptake by the cells. Addition of 25 microM MgCl2 or CaCl2 to the incubation medium restored a significant insulin stimulation, and this stimulation was maximal when 0.1 mM MgCl2 or CaCl2 had been added. SrCl2 and BaCl2 were also effective in restoring the insulin stimulation, but did not substitute fully for Ca2+ and Mg2+ in the incubation medium. Possible explanation for these observations are discussed.     

The nature of the carbohydrate binding module determines the catalytic efficiency of xylanase Z of Clostridium thermocellum

Xylanase Z of Clostridium thermocellum exists as a complex in the cellulosome with N-terminus feruloyl esterase, a carbohydrate binding module (CBM6) and a dockerin domain. To study the role of the binding modules on the activity of XynZ, different variants with the CBM6 attached to the catalytic domain at its C-terminal (XynZ-CB) and N-terminal (XynZ-BC), and the CBM22 attached at N-terminus (XynZ-B′C) were expressed in Escherichia coli at levels around 30% of the total cell proteins. The activities of XynZ-BC, XynZ-CB and XynZ-B′C were 4200, 4180 and 20,700 U μM⁻¹ against birchwood xylan, respectively. Substrate binding studies showed that in case of XynZ-BC and XynZ-CB the substrate birchwood xylan remaining unbound were 51 and 52%, respectively, whereas in the case of XynZ-B′C the substrate remaining unbound was 39% under the assay conditions used. The molecular docking studies showed that the binding site of CBM22 in XynZ-B′C is more exposed and thus available for substrate binding as compared to the tunnel shape binding pocket produced in XynZ-BC and thus hindering the substrate binding. The substrate binding data for the two constructs are in agreement with this explanation.     

n‐3 Fatty Acids Modulate T‐Cell Calcium Signaling in Obese Macrosomic Rats

Objective: We investigated the effects of a diet containing EPAX‐7010, rich in PUFAs such as eicosapentaenoic acid [20:5(n‐3)] and docosahexaenoic acid [22:6(n‐3)], i.e., a PUFA/EPAX regimen, on T‐cell activation in diabetic pregnant rats and their obese pups. Research Methods and Procedures: Mild hyperglycemia in pregnant rats was induced by intraperitoneal injection of streptozotocin on Day 5 of gestation. T‐cell blastogenesis was assayed by using ³H‐thymidine, whereas intracellular free calcium concentrations ([Ca²⁺]i) were measured by using Fura‐2 in diabetic pregnant rats and their obese offspring. Results: Concavalin‐A‐stimulated T‐cell proliferation was decreased in both pregnant diabetic rats and their obese pups as compared with control animals. Feeding the PUFA/EPAX diet restored T‐cell proliferation in both groups of animals. We also employed ionomycin, which at 50 nM opens calcium channels, and thapsigargin (TG), which recruits [Ca²⁺]i from endoplasmic reticulum pool. We observed that ionomycin‐induced increases in [Ca²⁺]i in T‐cells of diabetic mothers and obese offspring were greater than in those of control rats. Furthermore, feeding PUFA/EPAX diet diminished significantly the ionomycin‐evoked rise in [Ca²⁺]i in diabetic and obese animals. TG‐induced increases in [Ca²⁺]i in T‐cells of diabetic pregnant rats and their obese offspring were greater than in those of control rats. The feeding of the experimental diet significantly curtailed the TG‐evoked increases in [Ca²⁺]i in both diabetic and obese rats. Discussion: Together, these observations provide evidence that T‐cell activation and T‐cell calcium signaling are altered during gestational diabetes and macrosomia. Hence, dietary fish oils, particularly eicosapentaenoic acid and docosahexaenoic acid, may restore these T‐cell abnormalities.     

Two new mutations and a linkage map of Anopheles stephensi

Genetic analyses of two new mutations in Anopheles stephensi are presented. Spotless wings (sl) and 2nd-3rd costal spots fused (2-3f) have been mapped on chromosome 2, approximately 79.5 map units apart. A preliminary linkage map for this species also is presented.     

Surgical Sympathetic Denervation Increases α1Adrenoceptor-Mediated Accumulation of myo-Inositol Trisphosphate and Muscle Contraction in Rabbit Iris Dilator Smooth Muscle

Sympathetic denervation of the iris muscle produces increases in both the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) and in muscle contraction in response to norepinephrine (NE). To shed more light on the biochemical basis underlying this supersensitivity we investigated: the effects of NE on PIP2 breakdown, measured as myo-inositol trisphosphate (IP3) accumulation, and on muscle contraction in normal and denervated rabbit iris dilator; and the effects of denervation on selected biochemical properties of this muscle. The data obtained from these studies can be summarized as follows: The EC50 values (microM) for NE-induced IP3 accumulation in normal and denervated dilators were 14 and 3, respectively. This accumulation of IP3 was blocked by prazosin (1 microM). The EC50 values (microM) for NE-induced contraction for the normal and denervated muscles were 10 and 0.6, respectively. The NE-induced muscle contraction was blocked by prazosin (1 microM). The t1/2 values (s) for IP3 accumulation in normal and denervated muscles were 31 and 11, respectively, and for contraction the values were 19 and 9, respectively. Denervation increased significantly (15-18%) the basal labelling of phosphoinositides from myo-[3H]inositol, but not from 32P or [14C]arachidonic acid. Denervation had little effect on the activities of the enzymes involved in phosphoinositide metabolism. However, the activities of protein kinase C and Ca2+-ATPase increased in the denervated muscle. It is concluded that sympathetic denervation of the iris dilator renders the coupling between alpha1 receptors and PIP2 breakdown into IP3 and 1,2-diacylglycerol (DG) more efficient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of Gossypium species for resistance to cotton leaf curl Burewala virus

Cotton leaf curl disease (CLCuD), caused by cotton leaf curl Burewala virus (CLCuBV), has emerged as a major threat to cotton production in Pakistan. Resistance to CLCuBV was evaluated in cultivated and wild cotton genotypes representing six Gossypium species by visual symptom scoring and virus assessment using PCR tests. Considerable variation in responses was observed when using whitefly and graft transmission to inoculate Gossypium genotypes with CLCuBV in field and greenhouse experiments. Under field evaluation, all cultivated genotypes of Gossypium hirsutum and three genotypes of G. barbadense were susceptible. Eleven genotypes that represented six wild and cultivated Gossypium species were considered to be highly resistant as they were free from infection. Similar results were obtained when these genotypes were tested using whitefly transmission. To verify these findings, 132 cultivated and wild genotypes were tested by graft inoculation. All G. hirsutum genotypes (116 cultivated, 1 wild, 1 transgenic Coker-312 and 1 non-transgenic Coker-312), three G. barbadense genotypes and one G. thurberi genotype were highly susceptible and exhibited symptoms 9–12 days after grafting. Four genotypes of G. arboreum and one genotype of G. anomalum did not express symptoms but had a detectable level of virus. One genotype of G. herbaceum and three wild genotypes of G. hirsutum showed mild symptoms (severity indexes of 1–2) and exhibited delayed disease development. These genotypes were classified as moderately resistant to resistant. Resistant genotypes that were identified in this study will be useful sources for exploitation of breeding programmes aimed at developing CLCuBV-resistant varieties and increasing genetic diversity.     

Insulin action in isolated fat cells. II. Effects of divalent cations on stimulation by insulin of protein synthesis, on inhibition of lipolysis by insulin, and on the binding of 125I-labelled insulin to isolated fat cells.

The effects of ommission of Ca2+ and Mg2+ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca2+ + Mg2+)-free incubation medium incorporation of L-[14C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[14C]leucine incorporation only in the presence of added CaCl2 or MgCl2. Incubation of the cells in the (Ca2+ + Mg2+)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of 125I-labelled insulin to the fat cells was reduced in the absence of Ca2+ and Mg2+ but was not abolished, even in the presence of EDTA. Ca2+ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg2+, Sr2+ and Ba2+. Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.