Energy requirement of bovine spermatozoa for in vitro capacitation

The oxidative energy requirements of bovine spermatozoa capacitated with dilauroil-phosphatidylcholine liposomes (PC 12) and the effect of these liposomes on acrosome reaction necessary for in vitro fertilization were studied. Mitochondrial respiration was measured using 3 different substrates (pyruvate-lactate-glucose) and endogenous substrates. The samples were either treated with PC 12 or were left untreated and used as the control. A 2.8-fold increase in the consumption of oxygen was observed in the PC 12 treated spermatozoa in the presence of the 3 combined substrates (pyruvate-lactate-glucose). Respiration changes were not observed when the spermatozoa were capacitated with only 2 of the 3 substrates or with glucose alone. When endogenous substrates were used, the consumption of oxygen increased 1.7 times, and mitochondrial uncoupling was observed in the treated samples. The hypermotility characteristic of the capacitation process was not observed when glucose or endogenous substrates were used. When the percentage of intact acrosomes was determined using differential-interferential contrast (DIC) microscopy, it was found that in the presence of oxidative substrates there was a 26% decrease compared with that of the control sample. The proportion of reacted acrosomes was in the range of 41.3 to 49.6%, as measured by the chlortetracycline epifluorescence method in the presence of calcium ionophore A23187. Only 4% of the spermatozoa showed acrosome reaction with endogenous substrates. A higher percentage of fertilized oocytes were observed when the spermatozoa were capacitated in the presence of the 3 substrates (pyruvate-lactate-glucose), confirming that the success of in vitro fertilization depends on the energy conditions associated with the capacitation process. The results of these experiments indicate that the presence of oxidative energy is necessary to produce capacitation and the hyperactivation characteristic in frozen-thawed bovine spermatozoa treated with liposomes.     

Reactions of the haloalcohols HO(CH2)nCl (n = 2, 3) with platinum(II) - alkynyl and -alkyne complexes. X-ray crystal structure of trans-[Pt(Me) {C(OCH2CH2Cl) (CH2Ph)} (PPh3)2] [BF4]

The chloro complexes trans-[Pt(Me)(Cl)(PPh₃)₂], after treatment with AgBF₄, react with 1-alkynes HC---C---R in the presence of NEt₃ to afford the corresponding acetylide derivatives trans-[Pt(Me) (C---C---R) (PPh₃)₂] (R = p-tolyl (1), Ph (2), C(CH₃)₃ (3)). These complexes, with the exception of the t-butylacetylide complex, react with the chloroalcohols HO(CH₂)_nCl (n = 2, 3) in the presence of 1 equiv. of HBF₄ to afford the alkyl(chloroalkoxy)carbene complexes trans-[Pt(Me) {C[O(CH₂)_nCl](CH₂R) } (PPh₃)₂][BF₄] (R = p-tolyl, N = 2 (4), N = 3 (5); R=Ph, N = 2 (6)). A similar reaction of the bis(acetylide) complex trans-[Pt(C---C---Ph)₂(PMe₂Ph)₂] with 2 equiv. HBF₄ and 3-chloro-1-propanol affords trans-[Pt(C---CPh) {C(OCH₂CH₂CH₂Cl)(CH₂Ph) } (PMe₂Ph)₂][BF₄] (7). T alkyl(chloroalkoxy)-carbene complex trans-[Pt(Me) {C(OCH₂CH₂Cl)(CH₂Ph) } (PPh₃)₂][BF₄] (8) is formed by reaction of trans-[Pt(Me)(Cl)(PPh₃)₂], after treatment with AgBF₄ in HOCH₂CH₂Cl, with phenylacetylene in the presence of 1 equiv. of n-BuLi. The reaction of the dimer [Pt(Cl)(μ-Cl)(PMe₂Ph)]₂ with p-tolylacetylene and 3-chloro-1-propanol yields cis-[PtCl₂{C(OCH₂CH₂CH₂Cl)(CH₂C₆H₄-p-Me}(PMe₂Ph)] (9). The X-ray molecular structure of (8) has been determined. It crystallizes in the orthorhombic system, space group Pna2₁, with a = 11.785(2), B = 29.418(4), C = 15.409(3) Å, V = 4889(1) ų and Z = 4. The carbene ligand is perpendicular to the Pt(II) coordination plane; the PtC(carbene) bond distance is 2.01(1) Å and the short C(carbene)-O bond distance of 1.30(1) Å suggests extensive electronic delocalization within the Pt---C(carbene)---O moietry.     

The neutrophil migration induced by tumour necrosis factor alpha in mice is unaffected by glucocorticoids

Macrophages harvested from the peritoneal cavities of rats release a neutrophil chemotactic factor (MNCF) in response to stimulation with Gram-negative bacterial lipopolysaccharide (LPS). MNCF has been shown to be active in rats treated with dexamethasone, a glucocorticoid that usually inhibits the neutrophil migration induced in this species by interleukin (IL)-1, tumour necrosis factor alpha (TNFalpha), IL-8, C5a and leukotriene B(4) (LTB(4)). Here we report that macrophages harvested from peritoneal cavities of mice, and stimulated in vitro with LPS, also release a factor that induces neutrophil migration in dexamethasone-treated animals. This chemotactic activity was neutralized by the incubation of the LPS-stimulated macrophage supernatants with a purified polyclonal IgG anti-mouse TNFalpha. In addition, significant amounts of TNF were detected in the supernatants. The neutrophil migration induced by intraperitoneal administration of recombinant murine TNFalpha was also unaffected by pretreatment of the mice with dexamethasone. Moreover, neutrophil migration induced by intraperitoneal injection of LPS was completely blocked by pretreatment of the mice with a monoclonal antibody against murine TNFalpha. In conclusion, our results support the hypothesis that, in contrast to the role of TNF in rats (where it indirectly induces neutrophil migration), in mice, it may be an important mediator in the recruitment of neutrophils to inflammatory sites.     

Hematology and plasma chemistry values in free-ranging Humboldt penguins (Spheniscus humboldti) in Chile

To establish baseline hematologic and plasma biochemistry values in free-ranging Humboldt penguins (Spheniscus humboldti), heparinized blood samples were collected from 51 apparently healthy, adult Humboldt penguins residing at two colonies off the Chilean coast. Thirty samples were collected in April, 1992, from penguins inhabiting the Ex-islote de los Pájaros Niños in Algarrobo, Chile. In September, 1992, 21 samples were collected from birds inhabiting Isla de Cachagua, Chile. Hematologic values measured include packed cell volume, leucocyte count, leucocyte differential, and the presence of blood parasites. Plasma biochemistry values measured include glucose, blood urea nitrogen, creatinine, uric acid, calcium, inorganic phosphorous, sodium, potassium, chloride, total protein, albumin, globulin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, and creatine kinase. Only the mean values for chloride and for the number of eosinophils differed significantly between the two sample groups. No blood parasites were seen. © 1995 Wiley-Liss, Inc.     

Effect of a Brief CO(2) Exposure on Ethylene Production

Ethylene production and respiration by Granny Smith apples were inhibited by treatment with 20% CO₂ for 2 hours. A similar effect was observed in tissue slices when treated at either 0 or 25°C.

The inhibition continued even after an extended aeration period. There is also an inhibition of ethylene emission in tissue slices incubated with exogenous 1-aminocyclopropane-1-carboxylic acid (ACC).

In general, CO₂ treatment increased the ACC content of the tissue. These observations are consistent with the idea the action of CO₂ is directed toward the enzyme system responsible for the conversion of ACC into ethylene.

     

X-inactivation patterns in lymphocytes and skin fibroblasts of three cases of X-autosome translocations with abnormal phenotypes

Summary X-inactivation patterns were studied by replication analyses both in lymphocytes and skin fibroblasts of two patients carrying balanced X-autosome translocations, t(X;10)-(pter;q11) and t(X;17)(q11;q11), and one patient with an unbalanced translocation t(X;22)(p21;q11). Preferential late replication of the normal X chromosome was found in lymphocytes of both patients carrying balanced translocations and in skin fibroblasts of the patient carrying the translocation t(X;17). However, skin fibroblasts of the patient with a translocation t(X;10) showed preferential late replication of the abnormal der(X) chromosome with no spreading of late replication to the autosomal segment. In the case of unbalanced translocation t(X;22) there was preferential late replication of the der(X) chromosome both in lymphocytes and skin fibroblasts. The abnormal phenotype of the patients is discussed in relation to the observed X-inactivation patterns and the variability of the patterns in different tissues.     

Freeze-preservation of rice cells: A physiological study of freeze-thawed cells

Rice ( Oryza sativa L.) cells returning to in vitro culture after preservation at superlow temperature in liquid nitrogen are characterized by a number of physiological alterations. These include: reduction in respiration and glucose uptake, loss of intracellular potassium, decrease in the cellular level of key metabolites (ATP, glucose-6-phosphate and pyruvate) and fragility of protoplasts following the action of cell wall-degrading enzymes.
Nevertheless, cell growth resumes after a short lag phase (2–4 days) with an actual 70–100% cell survival, thus indicating that the observed damage is not lethal and can be repaired in a short time.     

Chemical modification of enzymes: Critical evaluation of the graphical correlation between residual enzyme activity and number of groups modified

In the study of chemical modification of enzymes and other biologically active proteins, plots of fractional residual activity as a function of number of groups modified per enzyme molecule are often used to establish a correlation between the chemical modification and enzyme inactivation reactions and to determine the stoichiometry of the modification reaction. This paper presents a critical examination of the underlying theoretical framework of such graphs. Whereas these plots are usually presented as linear functions, it is shown here that the general equation describing the relationship between inactivation and modification contains an exponential term; therefore, in the general case, the plot is actually a curve. It is suggested that caution be exercised in the interpretation of such plots and that equations such as those derived in the text be used to fit theoretical curves to the data, in order to maximize the information gained from chemical modification experiments.     

Somatic embryogenesis in suspension cultures of Gossypium klotzschianum anderss

Somatic embryoids differentiated in suspension cultures of G. klotzschianum after 3–4 weeks of culture in a liquid medium containing glutamine (optimally, 10–15 mM). Embryogenesis occurred after a preculture of callus on a medium containing 10 mg/l of the cytokinin, 2iP. The embryoids had meristematic regions, a well formed epidermis, and formed roots and vestigial leaves. Asparagine was much less effective than glutamine in promoting embryoid differentiation. The presence of 2,4-D in the medium resulted in increased vigor of the suspension cultures and subsequently in the formation of many embryoids, but does not seem to be necessary for somatic embryogenesis in cotton.Technical Article 14646 from the Texas Agricultural Experiment Station