Histochemical analysis of rat testicular glycoconjugates. 1. Subsets of N-linked saccharides in seminiferous tubules

Summary The distribution of N-linked glycans in rat testis has been probed using a panel of lectins derived fromGalanthus nivalis (snowdrop, GNA),Canavalia ensiformis (jack bean, Con A),Lens culinaris (lentil, LCA),Pisum sativum (garden pea, PSA) andPhaseolus vulgaris, erythro- and leucoagglutinins (kidney bean, ePHA and IPHA). Several classes of N-linked glycan were identified in the spermatogenic series, and during differentiation into spermatozoa they altered in both their pattern of distribution and relative abundance. A population of tetra-antennary, non-bisected, complex glycans, detected by IPHA, was lost during the transition from spermatogonia to spermatocytes, while high-mannose structures were acquired; these were most abundant in spermatocytes, as were bi- and tri-antennary complex, non-bisected glycans, the latter becoming increasingly abundant on acrosomes and spermatozoa. Their bisected counterparts were more generally expressed throughout spermatogenic cells, although marked localization onto acrosomes and nuclear caps was again seen. Transition from spermatocytes to spermatids involved mainly changes of the acrosomal granule and nuclear cap, which were carried through to the final stages of differentiation. Sertoli cell surfaces and cytoplasmic granules showed a high level of N-glycan expression.     

Histochemical analysis of rat testicular glycoconjugates. 2. Beta-galactosyl residues in O- and N-linked glycans in seminiferous tubules.

Summary Rat testes have been examined with a panel of lectins that bind specifically to oligosaccharide sequences having terminal or subterminal -galactosyl residues in O-linked glycans, or in the outer chains of complex N-linked glycans:Arachis hypogaea (peanut, AHA),Erythrina cristagalli (coral tree, ECA),Ricinus communis (castor bean, RCA120) andAbrus precatorius (jequirity bean, APA) agglutinins. Pretreatment of sections with neuraminidase, -galactosidase and removal of alkali-labile O-linked sequences by -elimination allowed the structure of these glycans to be further explored. In spermatogonia and spermatocytes there was little evidence of glycans terminating in -galactosyl residues, although these were present at non-reducing terminals as sialylgalactosides. The acrosome contained two subsets of O-linked glycans terminating in sialylgalactosides, while the nuclear cap showed at least two subsets of N-linked sialylgalactosyl as well as O-linked glycans. Spermatozoa exhibited minor changes in the pattern of glycosylation, although the overall pattern of -galactosyl expression was similar. Binding to Sertoli cells showed the presence of some unsubstituted -galactosyl terminals on O-linked glycans but few such N-linked residues, while terminal -galactosides were scanty in tubular basement membranes.     

A simple method of generating axenic derivatives of Dictyostelium strains

We describe a generally applicable method of adapting Dictyostelium from growth on a bacterial food source to axenic growth. Cells are initially selected by growth on a plastic substratum but subsequently acquire the ability to grow in suspension culture. These strains can be transformed efficiently by DNA-mediated gene transfer.     

pH dependency of the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Escherichia coli

The variation with pH of the kinetic parameters associated with the mutase and dehydrogenase reactions catalyzed by chorismate mutase-prephenate dehydrogenase has been determined with the aim of elucidating the role that ionizing amino acid residues play in binding and catalysis. The pH dependency of log V for the dehydrogenase reaction shows that the enzyme possesses a single ionizing group with a pK value of 6.5 that must be unprotonated for catalysis. This same group is observed in the V/Kprephenate, as well as in the V/KNAD, profile. The V/Kprephenate profile exhibits a second ionizing residue with a pK value of 8.4 that must be protonated for the binding of prephenate to the enzyme. For the mutase reaction, the V/Kchorismate profile indicates the presence of three ionizing residues at the active site. Two of these residues, with similar pK values of about 7, must be protonated, while the third, with a pK value of 6.3, must be unprotonated. It can be concluded that all three groups are concerned with the binding of chorismate to the enzyme since the maximum velocity of the mutase reaction is essentially independent of pH. This conclusion is confirmed by the finding that the Ki profile for the competitive inhibitor, (3-endo,8-exo)-8-hydroxy-2-oxabicyclo[3.3]non-6-ene-3,5-dicarboxylic acid, shows the same three ionizing groups as observed in the V/Kchorismate profile. By contrast, the Ki profile for carboxyethyldihydrobenzoate shows only one residue, with a pK value of 7.3, that must be protonated for binding of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parathyroid hormone related protein and hypercalcaemia in breast cancer.

OBJECTIVE--To see whether parathyroid hormone related protein has a humoral role in breast cancer. DESIGN--Plasma concentrations and tumour expression of parathyroid hormone related protein were determined (by two site immunoradiometric assay and immunohistochemistry respectively) in women with breast cancer and related to the presence of bone metastases and serum calcium concentrations. SUBJECTS--Plasma concentrations of parathyroid hormone related protein were measured in 57 women with early breast cancer without apparent bone metastases, 28 women with bone metastases, and 13 women with bone metastases and hypercalcaemia. Tissue positivity for parathyroid hormone related protein was determined retrospectively in 106 primary breast tumours from women without apparent bone metastases and 72 tumours from women with bone metastases, 25 of whom subsequently developed hypercalcaemia. RESULTS--Plasma parathyroid hormone related protein concentrations were detectable (greater than 0.23 pmol/l) in 12 (92%) of the 13 hypercalcaemic patients with bone metastases compared with 10 (36%) of the 28 normocalcaemic patients with bone metastases and five (9%) of the 57 normocalcaemic patients without bone metastases. Parathyroid hormone related protein concentrations were significantly higher in hypercalcaemic than normocalcaemic patients with bone metastases. Tumour staining was positive for parathyroid hormone related protein in 22 (88%) of the 25 primary breast cancers from patients with bone metastases. Tumour staining was positive for parathyroid hormone related protein in 22 (88%) of the 25 primary breast cancers from patients with bone metastases who later developed hypercalcaemia compared with 25 (53%) of the 47 from women in this group who remained normocalcaemic and 55 (52%) of the 106 early breast cancers from women without known metastases. CONCLUSION--Tumour derived parathyroid hormone related protein may have an important humoral role in hypercalcaemia associated with metastatic breast cancer.     

3-Phenylpropanoic Acid Improves the Affinity of Ruminococcus albus for Cellulose in Continuous Culture

A continuous-culture device, adapted for use with solid substrates, was used to evaluate the effects of 3-phenylpropanoic acid (PPA) upon the ability of the South African strain Ruminococcus albus Ce63 to ferment cellulose. Steady states of fermentation were established with a dilution rate of 0.17 h⁻¹, and the extent and volumetric rates of cellulose fermentation were determined over four consecutive days. When the growth medium contained no additions (control), 25 μM phenylacetate alone, 25 μM PPA alone, or 25 μM each of phenylacetate and PPA, the extent of cellulose hydrolysis was determined to be 41.1, 35.7, 90.2, and 86.9%, respectively, and the volumetric rate of cellulose hydrolysis was 103.0, 97.9, 215.5, and 230.4 mg liter⁻¹ h⁻¹, respectively. To evaluate the effect of PPA availability on affinity for cellulose, the values for dilution rate and extent of cellulose hydrolysis were used in combination with values for maximum specific growth rate determined from previous studies of growth rates and kinetics of cellulose hydrolysis. The findings support the contention that PPA maintains a competitive advantage for R. albus when grown in a dynamic, fiber-rich environment.     

The cincinnati lipid research clinic family study: Familial determinants of plasma uric acid

Summary Commingling analysis of plasma uric acid levels in a random sample of 160 nuclear families supports the hypothesis that there is a mixture of three distributions. Assuming one, two, and three components in the underlying distribution, we obtained the corresponding p-values (for power transformation) as 0.059, 1.040, and 1.643, respectively. Path analysis with p=0.059 gives genetic (h ²) and cultural (c ²) heritabilities as 0.256 and 0.199, without much support for intergenerational differences, assortative mating, or maternal effects. Complex segregation analysis with p=0.059 supports multifactorial inheritance, consistent with the findings of Gulbrandsen et al. (1979) and Morton (1979) in other populations. This study also fails to support a major locus hypothesis, contrary to earlier reports.This work was supported in part by N.I.H. and N.I.M.H. Grants GM 28719, and MH 31302, and by contract NO-1-HV-2-2914L from the National Heart, Lung, and blood Institute (Lipid Research Clinic's Program), General Clinical Research Center, and the CLINFO center Grant RR-00068-19     

Non-Markov negative correlation between interspike intervals in mammalian sympathetic efferent discharges

The background discharge of sympathetic efferent fibres in the hypogastric and splanchnic nerves of the cat was analyzed. Stationary discharges were renewal or had significant negative first order serial correlation coefficients. Negatively correlated discharges were non-Markov and the post-spike depression persisted for up to 5 s, covering the same time course as the prolonged inhibitory phenomena of sympathetic reflexes.     

The role of male accessory-gland substance on female reproduction with some observations of spermatogenesis in the stable fly

The bulbous tubular portion of the median ejaculatory duct functions as the accessory gland in the male stable fly, Stomoxys calcitrans. The gland started to enlarge after emergence when the fly was fed on sugar-water or blood. Implants of accessory glands from sugar-water or blood-fed males were effective in stimulating oviposition in virgin females. The injection into females of accessory-gland extracts from males fed blood prevented insemination; this extract was effective in concentration as low as 0.25 gland per female. Accessory-gland extracts from sugar-water fed males were only partially effective in preventing female insemination. However, the accessory-gland extracts of male stable flies had little effect on insemination when injected into three other species of female dipterans.Spermatogenesis was completed by the time of emergence, or shortly thereafter, a process independent of blood feeding. Three types of spermatids were identified, forming a continuum of spermiogenesis stages. Fat, fusiform spermatozoa elongated to become thin, elongate and mature spermatozoa.     

ICR-191 and ethyl methanesulfonate induced mutagenesis at the immunoglobulin locus in the Y5606 cultured myeloma cell line

The Y5606 mouse tumor synthesizing an IgG3, lambda immunoglobulin (Ig) was adapted to continuous growth in tissue culture. The spontaneous mutation rate at the Ig locus (approximately 3 X 10(-5)/cell/generation) in this cell line was found to be less than that in other cultured mouse myeloma lines. Treatment with either ICR-191 or ethyl methanesulfonate (EMS) increased the mutation rate approx. 100-fold. Spontaneous and ICR-191 induced mutants were synthetic variants that is they synthesized either heavy (H) or light (L) chains alone instead of the H and L chains synthesized by the parent. Following EMS treatment assembly variants which were synthesizing structurally altered H chains were isolated in addition to synthetic variants. The assembly variants appear to be a unique consequence of EMS mutagenesis.