Factors affecting the removal of low-molecular-weight fractions (LMWF) from egg yolk and seminal plasma in extended semen by dialysis: effect on post-thaw sperm survival

Three factors affecting dialysis of bovine semen were studied. These factors were (1) dialysis rates of egg yolk, seminal plasma, and glycerol, (2) temperature (37 degrees C, 5 degrees C, and while cooling from 37 to 5 degrees C), and (3) dialysis ratios between retentate and dialysate (1:1, 1:10, 1:20, 1:50, and 1:100). Ninety percent of the low-molecular-weight fraction (LMWF) from seminal plasma, egg yolk, and glycerol was removed from the retentate in a 2-hr period at 5 degrees C, and only slight changes were detected after the third hour of dialysis. Temperature affected dialysis and was faster at 37 degrees C. It was also found that a 1:20 dialysis ratio was sufficient to obtain 90% clearance of the LMWF. The effect of sperm dilution ratio, dialysis ratio, and exchange of the LMWF from egg yolk and/or seminal plasma for buffer systems was also studied. An improvement in post-thaw motility of spermatozoa (P less than 0.05) was obtained when the LMWF from both seminal plasma and egg yolk were replaced. A third experiment was conducted to study the effect of different combinations between the buffer systems, TEST and Na citrate, in the dialysate. The results indicated that a 1:1 combination of iso-osmotic solutions (320-325 mOsm/Kg, pH 7.0) between these two buffers, with 5% glycerol (v/v), yielded significant (P less than 0.05) sperm post-thaw motility as compared with the individual use of TEST-glycerol or Na citrate-glycerol. Dialyzed samples also yielded sperm post-thaw motility higher than that of the nondialyzed samples. Colloidal materials in the dialysate did not affect survival of spermatozoa.     

Cultivar and pH effects on competition for nodule sites between isolates of Rhizobium in beans

Two experiments were carried out to evaluate the effect of acidity on bean-Rhizobium competition for nodule sites. SevenPhaseolus vulgaris host cultivars differing in acid-pH tolerance were grown in sand culture, and irrigated using a sub-irrigation system and nutrient solutions of pH 4.5, 5.0, 5.5, and 6.0. A mixed inoculant of two antibiotically markedRhizobium leguminosarum bvphaseoli strains CIAT899 (acid-tolerant) and CIAT632 (acid-sensitive) was used. The acid-tolerant CIAT899 dominated CIAT632 in nodule occupancy across all cultivars and pH treatments. Although several of the varieties had previously been identified as PH-tolerant, and these cultivars performed better than those reported to be acid sensitive, all showed a marked increase in nodulation and plant development when the pH was raised from 4.5 to 6.0. The second experiment using a modified Leonard jar system varied the inoculation ratio between CIAT899 and UMR1116 (acid-sensitive, inefficient in N₂-fixation) and contrasted nodulation response for the bean varieties Preto 143 (pH-tolerant) and Negro Argel (pH-sensitive) at 3 pH treatments (4.5, 5.5, 6.5). There was a significant effect of host cultivar, ratio of inoculation, and pH on the percentage of nodule occupancy by each strain. At low pH CIAT899 had higher nodule occupancy than UM1116 in the variety Negro Argel but had the same percentage of nodulation when the variety was Preto 143. Increasing the cell concentration of UMR1116 produced more inefficient nodules at all treatment combinations and reduced plant growth for both cultivars used.     

Ultrastructure of the domestic tom cat (Felis domestica ) and tiger (Panthera tigris altaica ) spermatozoa

The ultrastructure of spermatozoa from the domestic tom cat and the Siberian tiger was studied. Semen was collected from anesthetized tom cats and Siberian tigers by electroejaculation. Spermatozoa were fixed and processed for examination by transmission electron microscopy. The principle differences between the spermatozoa from the two species were the head shape, mitochondrial organization in the neck area and structure of the fibrous sheath. Tom cat spermatozoa had an elongated oval-shaped head, while tiger spermatozoa had a more rounded head shape. Circularly oriented mitochondria in the neck area, near the proximal centriole, were frequently observed in tiger cells but rarely observed in tom cat cells. The semicircular ribs of tom cat spermatozoa were larger than the ribs of tiger spermatozoa. Also, the dense fibers (Numbers 3 and 8) of the corresponding microtubule doublets were fused or connected to the longitudinal columns in tiger spermatozoa but showed only occasional attachment in tom cat spermatozoa. These differences could influence results when the tom cat is used as a model for studying tiger semen.     

Purification of a class C A-type beta-lactamase from a derepressed strain of Enterobacter cloacae. Comparison of the wild-type and mutant enzyme with those from strains P99, 208 and GN7471.

Enterobacter cloacae strain 5822 expresses low levels of a class C beta-lactamase which can be induced 100-fold by imipenem. Mutants that constitutively express high levels of beta-lactamase can be selected on aztreonam or cefotaxime. The beta-lactamase from one such mutant (5822M2) has been purified to homogeneity and compared on the basis of subunit Mr, pI, substrate specificity, inhibitor sensitivity and immunological cross-reactivity with the enzyme from strains P99, GN7471 and 208, which have been studied previously. The enzyme from strain 5822M2 is clearly related to these other forms and is of the A-type according to the criteria of Seeberg, Tolxdorff-Neutzling & Wiedemann [Antimicrob. Agents Chemother. (1983) 23, 918-925]. The enzyme from the wild-type strain (5822) is shown to be identical to that found in the depressed strain (5822M2), indicating that the mutation is in a regulatory gene. A detailed analysis of the kinetics of the enzyme from strain 5822M2 shows that all of the beta-lactams studied are substrates and that a mechanism involving the formation of an acyl-enzyme is probably applicable in every case. The substrates however can clearly be grouped into two classes, i.e. 'good' substrates with kcat. values of 80-1200 s-1 and 'poor' substrates/good inhibitors with kcat. values of 0.009-0.00007 s-1. The permeability barrier to aztreonam is 4-fold less in the derepressed strain when compared with the wild-type strain. This is associated with significant changes in the expression of outer membrane porins. The observed resistance in the derepressed mutant appears to be linked to the elevated levels of beta-lactamase (3000-fold) rather than to the modest changes in the permeability barrier.     

Association of pigmentary anomalies with chromosomal and genetic mosaicism and chimerism.

We have evaluated eight patients with pigmentary anomalies reminiscent of incontinentia pigmenti or hypomelanosis of Ito. All demonstrated abnormal lymphocyte karyotypes with chromosomal mosaicism in lymphocytes and/or skin fibroblasts. In seven the skin was darkly pigmented, and in all of these seven cases the abnormal pigmentation followed Blaschko lines. The literature contains at least 36 similar examples of an association between pigmentary anomalies and chromosomal mosaicism, as well as five examples of an association with chimerism. The pigmentary anomalies are pleomorphic, and the chromosomal anomalies involve autosomes and sex chromosomes. The pigmentation patterns are reminiscent of the archetypal paradigm seen in allophenic mice and demonstrate the clonal origin of melanoblasts from neural crest precursors. Patients with anomalous skin pigmentation, particularly when it follows a pattern of Blaschko lines, should be appropriately evaluated for a possible association with chromosomal or genetic mosaicism or chimerism.     

Evaluation of strain competitiveness in Rhizobium leguminosarum bv phaseoli using a nod + fix− natural mutant

Competition from native soil rhizobia is likely to be an important factor limiting Phaseolus vulgaris L. inoculant response in Latin America. We used UMR 1116, a nod ⁺ fix^– natural mutant of Rhizobium leguminosarum bv phaseoli strain CC511, as a reference strain to study competition for nodulation sites in this species. When P. vulgaris cv Carioca was planted in soils containing different proportions of UMR 1116 and the effective and competitive strain UMR 1899, UMR 1116 occupied more than 50% of the nodules at all inoculant ratios tested, though increasing the proportion of UMR 1899 in the inoculant did enhance the number and percentage of effective nodules and plant dry weight. Sixty two strains of bean rhizobia were tested in competition with UMR 1116. An inoculant ratio of 1:1 was used, with all strains applied to the soil rather than to seeds. Strains varied in the number and percentage of effective nodules produced in competition with UMR 1116, and in plant dry weight, and there was a strong correlation between variation in each of these traits and plant N accumulation. Seven of the strains (UMR 1073, 1084, 1102, 1125, 1165, 1378 and 1384) were identified as both superior in competitive ability and active in N₂ fixation. Site of placement of the inoculant and ambient temperature influenced strain response.Journal paper 16736, Agricultural Experiment Station, University of Minnesota, St. Paul, MN 55108, USA     

Susceptibility of autologous target cells to lysis by lymphokine-activated effectors from interferon-α-treated chronic myelogenous leukaemia patients

Summary Chronic myelogenous leukemia (CML) patients in chronic phase display compromised lymphokine-activated killer (LAK) cell induction, which is partly restored after therapy with interferon . However, the relative resistance of the leukemic cells from these patients to autologous or allogeneic LAK lysis is not affected by this treatment. In an attempt to render CML cells more susceptible to lysis or cytostasis, they were precultured in serum-free medium with or without recombinant growth factors. In eight patients studied, interleukin-3 (IL-3) significantly enhanced the spontaneous short-term (6-day) proliferation of CML cells, with retention of ability to form colonies in methylcellulose. Culture in either medium alone or IL-3 led to a significant enrichment of CD14⁺ and CD33⁺ cells but to a reduction in CD34⁺ cells. In contrast, culture of the same cells in IL-2 (to generate autologous LAK activity) resulted in a loss of CD14⁺ and CD33⁺ as well as CD34⁺ cells but in a significant increase in CD3⁺ and CD56⁺ cells. Despite similarities in their phenotypes, IL-3 cultured cells but not those cultured in medium alone acquired susceptibility to lysis by the IL-2-cultured autologous LAK cells. These results may have significance for the design of novel combination immunotherapy in CML.This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 120)     

The achondroplasia gene is not linked to the locus for neurofibromatosis 1 on chromosome 17

Summary We have investigated genetic linkage of von Recklinghausen neurofibromatosis (NF1) and achondroplasia (ACH) using chromosome-17 markers that are known to be linked to NF1. Physical proximity of the two loci was suggested by the report of a patient with mental retardation and the de novo occurrence of both NF1 and ACH. Since the chance of de novo occurrence of these two disorders in one individual is 1 in 600 million, this suggested a chromosomal deletion as a single unifying molecular event and also that the ACH and NF1 loci might be physically close. To test this, we performed linkage analysis on a three-generation family with ACH. We used seven DNA probes that are tightly linked to the NF1 locus, including DNA sequences that are known to flank the NF1 locus on the centromeric and telomeric side. We detected two recombinants between the ACH trait and markers flanking the NF1 locus. In one recombinant, the flanking markers themselves were nonrecombinant. Multi-point linkage analysis excluded the ACH locus from a region surrounding the NF1 locus that spans more than 15cM (lod score < -2). Therefore, analysis of this ACH pedigree suggests that the ACH locus is not linked to the NF1 locus on chromosome 17.     

The role of carrier ATPases and neuromediators in neurological disorders

Physiological and biochemical processes which take place in the nervous system at stroke and neurotrauma are reviewed, and the experience of using low doses of steroid derivatives with piridamole in the treatment of central nervous system (CNS) disorders is summarized. ATPases (including Na,K-ATPase) are reported to play an important role in CNS functioning, the correlation between Na,K-ATPase activity and the extent of CNS injury is revealed. The use of NMR-spectroscopy method for investigation of brain and spinal cord condition in vivo is suggested.